U.S. patent application number 15/295746 was filed with the patent office on 2017-02-02 for methods and compositions for diagnosis and prognosis of renal injury and renal failure.
The applicant listed for this patent is ASTUTE MEDICAL, INC.. Invention is credited to Joseph Anderberg, Jeff Gray, Paul McPherson, Kevin Nakamura.
Application Number | 20170030927 15/295746 |
Document ID | / |
Family ID | 42153314 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170030927 |
Kind Code |
A1 |
Anderberg; Joseph ; et
al. |
February 2, 2017 |
METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PROGNOSIS OF RENAL
INJURY AND RENAL FAILURE
Abstract
The present invention relates to methods and compositions for
monitoring, diagnosis, prognosis, and determination of treatment
regimens in subjects suffering from or suspected of having a renal
injury. In particular, the invention relates to using assays that
detect one or more markers selected from the group consisting of
Clusterin, Heart-type fatty acid binding protein, Hepatocyte growth
factor, Interferon gamma, Interleukin-12 subunit beta,
Interleukin-16, Interleukin-2, 72 kDa type IV collagenase, Matrix
metalloproteinase-9, Midkine, and Serum amyloid P-component as
diagnostic and prognostic biomarkers in renal injuries.
Inventors: |
Anderberg; Joseph;
(Encinitas, CA) ; Gray; Jeff; (Solana Beach,
CA) ; McPherson; Paul; (Encinitas, CA) ;
Nakamura; Kevin; (Cardiff by the Sea, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASTUTE MEDICAL, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
42153314 |
Appl. No.: |
15/295746 |
Filed: |
October 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14673353 |
Mar 30, 2015 |
9470695 |
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15295746 |
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13128392 |
Jun 9, 2011 |
8993250 |
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PCT/US2006/063906 |
Nov 10, 2009 |
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14673353 |
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61117168 |
Nov 22, 2008 |
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61115049 |
Nov 15, 2008 |
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61113074 |
Nov 10, 2008 |
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61117160 |
Nov 22, 2008 |
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61117158 |
Nov 22, 2008 |
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61117162 |
Nov 22, 2008 |
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61117163 |
Nov 22, 2008 |
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61117180 |
Nov 22, 2008 |
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61117151 |
Nov 22, 2008 |
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61117161 |
Nov 22, 2008 |
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61117178 |
Nov 22, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/775 20130101;
G01N 2333/57 20130101; G01N 2333/4753 20130101; G01N 2333/96486
20130101; G01N 2333/475 20130101; G01N 2333/5446 20130101; G01N
2333/4709 20130101; G01N 2333/4704 20130101; G01N 33/6893 20130101;
G01N 2800/347 20130101; G01N 2333/5434 20130101; G16B 40/00
20190201; G01N 2333/96494 20130101; G01N 2333/55 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for evaluating renal status in a subject, comprising:
performing one or more assays configured to detect a kidney injury
marker selected from the group consisting of Clusterin, Heart-type
fatty acid binding protein, Hepatocyte growth factor, Interferon
gamma, Interleukin-12 subunit beta, Interleukin-16, Interleukin-2,
72 kDa type IV collagenase, Matrix metalloproteinase-9, Midkine,
and Serum amyloid P-component by introducing the urine sample
obtained from the subject into an assay instrument which (i)
contacts the urine sample with one or more antibodies which
specifically bind for detection the biomarker(s) which are assayed,
and (ii) generates one or more assay results indicative of binding
of each biomarker which is assayed to a respective antibody to
provide one or more assay results; using the one or more assay
results, or one or more values derived therefrom, to assign a
probability that a future acute renal injury will occur within 48
hours of the time the urine sample is obtained to the subject,
wherein the assignment is made by comparing the assay result or
value derived therefrom to a predetermined threshold value selected
in a population study to separate the population of individuals in
the population study into a first subpopulation which is at an
increased probability for acute renal failure within 48 hours
relative to a second subpopulation; and if the one or more assay
results, or one or more values derived therefrom, assign the
subject into the first subpopulation, treating the subject by one
or more of initiating renal replacement therapy, withdrawing
delivery of compounds that are known to be damaging to the kidney,
delaying or avoiding procedures that are known to be damaging to
the kidney, and modifying diuretic administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/673,353, filed Mar. 30, 2015, now U.S. Pat.
No. 9,470,695, issued Oct. 18, 2016, which claims benefit of
priority to U.S. patent application Ser. No. 13/128,392, filed Jun.
9, 2011, now U.S. Pat. No. 8,993,250, issued Mar. 31, 2015, which
is a U.S. national phase application of International Application
No. PCT/US2009/063906, filed Nov. 10, 2009, which designated the
U.S. and claims the benefit of priority to U.S. Provisional Patent
Application 61/117,168 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/115,049 filed Nov. 15, 2008; U.S. Provisional Patent
Application 61/113,074 filed Nov. 10, 2008; U.S. Provisional Patent
Application 61/117,160 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,158 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,162 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,163 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,180 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,178 filed Nov. 22, 2008; U.S. Provisional Patent
Application 61/117,151 filed Nov. 22, 2008; and U.S. Provisional
Patent Application 61/117,161 filed Nov. 22, 2008, each of which is
hereby incorporated in its entirety including all tables, figures,
and claims.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Oct. 17,
2016, is named AST6900CT SeqListing.txt, and is 38 kilobytes in
size.
BACKGROUND OF THE INVENTION
[0003] The following discussion of the background of the invention
is merely provided to aid the reader in understanding the invention
and is not admitted to describe or constitute prior art to the
present invention.
[0004] The kidney is responsible for water and solute excretion
from the body. Its functions include maintenance of acid-base
balance, regulation of electrolyte concentrations, control of blood
volume, and regulation of blood pressure. As such, loss of kidney
function through injury and/or disease results in substantial
morbidity and mortality. A detailed discussion of renal injuries is
provided in Harrison's Principles of Internal Medicine, 17.sup.th
Ed., McGraw Hill, New York, pages 1741-1830, which are hereby
incorporated by reference in their entirety. Renal disease and/or
injury may be acute or chronic. Acute and chronic kidney disease
are described as follows (from Current Medical Diagnosis &
Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815,
which are hereby incorporated by reference in their entirety):
"Acute renal failure is worsening of renal function over hours to
days, resulting in the retention of nitrogenous wastes (such as
urea nitrogen) and creatinine in the blood. Retention of these
substances is called azotemia. Chronic renal failure (chronic
kidney disease) results from an abnormal loss of renal function
over months to years".
[0005] Acute renal failure (ARF, also known as acute kidney injury,
or AKI) is an abrupt (typically detected within about 48 hours to 1
week) reduction in glomerular filtration. This loss of filtration
capacity results in retention of nitrogenous (urea and creatinine)
and non-nitrogenous waste products that are normally excreted by
the kidney, a reduction in urine output, or both. It is reported
that ARF complicates about 5% of hospital admissions, 4-15% of
cardiopulmonary bypass surgeries, and up to 30% of intensive care
admissions. ARF may be categorized as prerenal, intrinsic renal, or
postrenal in causation. Intrinsic renal disease can be further
divided into glomerular, tubular, interstitial, and vascular
abnormalities. Major causes of ARF are described in the following
table, which is adapted from the Merck Manual, 17.sup.th ed.,
Chapter 222, and which is hereby incorporated by reference in their
entirety:
TABLE-US-00001 Type Risk Factors Prerenal ECF volume depletion
Excessive diuresis, hemorrhage, GI losses, loss of intravascular
fluid into the extravascular space (due to ascites, peritonitis,
pancreatitis, or burns), loss of skin and mucus membranes, renal
salt- and water-wasting states Low cardiac output Cardiomyopathy,
MI, cardiac tamponade, pulmonary embolism, pulmonary hypertension,
positive-pressure mechanical ventilation Low systemic vascular
Septic shock, liver failure, antihypertensive drugs resistance
Increased renal vascular NSAIDs, cyclosporines, tacrolimus,
hypercalcemia, resistance anaphylaxis, anesthetics, renal artery
obstruction, renal vein thrombosis, sepsis, hepatorenal syndrome
Decreased efferent ACE inhibitors or angiotensin II receptor
blockers arteriolar tone (leading to decreased GFR from reduced
glomerular transcapillary pressure, especially in patients with
bilateral renal artery stenosis) Intrinsic Renal Acute tubular
injury Ischemia (prolonged or severe prerenal state): surgery,
hemorrhage, arterial or venous obstruction; Toxins: NSAIDs,
cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene
glycol, hemoglobin, myoglobin, ifosfamide, heavy metals,
methotrexate, radiopaque contrast agents, streptozotocin Acute
glomerulonephritis ANCA-associated: Crescentic glomerulonephritis,
polyarteritis nodosa, Wegener's granulomatosis; Anti- GBM
glomerulonephritis: Goodpasture's syndrome; Immune-complex: Lupus
glomerulonephritis, postinfectious glomerulonephritis,
cryoglobulinemic glomerulonephritis Acute tubulointerstitial Drug
reaction (eg, .beta.-lactams, NSAIDs, sulfonamides, nephritis
ciprofloxacin, thiazide diuretics, furosemide, phenytoin,
allopurinol, pyelonephritis, papillary necrosis Acute vascular
Vasculitis, malignant hypertension, thrombotic nephropathy
microangiopathies, scleroderma, atheroembolism Infiltrative
diseases Lymphoma, sarcoidosis, leukemia Postrenal Tubular
precipitation Uric acid (tumor lysis), sulfonamides, triamterene,
acyclovir, indinavir, methotrexate, ethylene glycol ingestion,
myeloma protein, myoglobin Ureteral obstruction Intrinsic: Calculi,
clots, sloughed renal tissue, fungus ball, edema, malignancy,
congenital defects; Extrinsic: Malignancy, retroperitoneal
fibrosis, ureteral trauma during surgery or high impact injury
Bladder obstruction Mechanical: Benign prostatic hyperplasia,
prostate cancer, bladder cancer, urethral strictures, phimosis,
paraphimosis, urethral valves, obstructed indwelling urinary
catheter; Neurogenic: Anticholinergic drugs, upper or lower motor
neuron lesion
[0006] In the case of ischemic ARF, the course of the disease may
be divided into four phases. During an initiation phase, which
lasts hours to days, reduced perfusion of the kidney is evolving
into injury. Glomerular ultrafiltration reduces, the flow of
filtrate is reduced due to debris within the tubules, and back
leakage of filtrate through injured epithelium occurs. Renal injury
can be mediated during this phase by reperfusion of the kidney.
Initiation is followed by an extension phase which is characterized
by continued ischemic injury and inflammation and may involve
endothelial damage and vascular congestion. During the maintenance
phase, lasting from 1 to 2 weeks, renal cell injury occurs, and
glomerular filtration and urine output reaches a minimum. A
recovery phase can follow in which the renal epithelium is repaired
and GFR gradually recovers. Despite this, the survival rate of
subjects with ARF may be as low as about 60%.
[0007] Acute kidney injury caused by radiocontrast agents (also
called contrast media) and other nephrotoxins such as cyclosporine,
antibiotics including aminoglycosides and anticancer drugs such as
cisplatin manifests over a period of days to about a week. Contrast
induced nephropathy (CIN, which is AKI caused by radiocontrast
agents) is thought to be caused by intrarenal vasoconstriction
(leading to ischemic injury) and from the generation of reactive
oxygen species that are directly toxic to renal tubular epithelial
cells. CIN classically presents as an acute (onset within 24-48 h)
but reversible (peak 3-5 days, resolution within 1 week) rise in
blood urea nitrogen and serum creatinine.
[0008] A commonly reported criteria for defining and detecting AKI
is an abrupt (typically within about 2-7 days or within a period of
hospitalization) elevation of serum creatinine. Although the use of
serum creatinine elevation to define and detect AKI is well
established, the magnitude of the serum creatinine elevation and
the time over which it is measured to define AKI varies
considerably among publications. Traditionally, relatively large
increases in serum creatinine such as 100%, 200%, an increase of at
least 100% to a value over 2 mg/dL and other definitions were used
to define AKI. However, the recent trend has been towards using
smaller serum creatinine rises to define AKI. The relationship
between serum creatinine rise, AKI and the associated health risks
are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens
14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370,
2005, which, with the references listed therein, are hereby
incorporated by reference in their entirety. As described in these
publications, acute worsening renal function (AKI) and increased
risk of death and other detrimental outcomes are now known to be
associated with very small increases in serum creatinine. These
increases may be determined as a relative (percent) value or a
nominal value. Relative increases in serum creatinine as small as
20% from the pre-injury value have been reported to indicate
acutely worsening renal function (AKI) and increased health risk,
but the more commonly reported value to define AKI and increased
health risk is a relative increase of at least 25%. Nominal
increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have
been reported to indicate worsening renal function and increased
risk of death. Various time periods for the serum creatinine to
rise to these threshold values have been used to define AKI, for
example, ranging from 2 days, 3 days, 7 days, or a variable period
defined as the time the patient is in the hospital or intensive
care unit. These studies indicate there is not a particular
threshold serum creatinine rise (or time period for the rise) for
worsening renal function or AKI, but rather a continuous increase
in risk with increasing magnitude of serum creatinine rise.
[0009] One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605,
2004, hereby incorporated by reference in its entirety)
investigated both increases and decreases in serum creatinine.
Patients with a mild fall in serum creatinine of -0.1 to -0.3 mg/dL
following heart surgery had the lowest mortality rate. Patients
with a larger fall in serum creatinine (more than or equal to -0.4
mg/dL) or any increase in serum creatinine had a larger mortality
rate. These findings caused the authors to conclude that even very
subtle changes in renal function (as detected by small creatinine
changes within 48 hours of surgery) seriously effect patient's
outcomes. In an effort to reach consensus on a unified
classification system for using serum creatinine to define AKI in
clinical trials and in clinical practice, Bellomo et al., Crit
Care. 8(4):R204-12, 2004, which is hereby incorporated by reference
in its entirety, proposes the following classifications for
stratifying AKI patients: [0010] "Risk": serum creatinine increased
1.5 fold from baseline OR urine production of <0.5 ml/kg body
weight/hr for 6 hours; [0011] "Injury": serum creatinine increased
2.0 fold from baseline OR urine production <0.5 ml/kg/hr for 12
h; [0012] "Failure": serum creatinine increased 3.0 fold from
baseline OR creatinine >355 .mu.mol/l (with a rise of >44) or
urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12
hours; And included two clinical outcomes: [0013] "Loss":
persistent need for renal replacement therapy for more than four
weeks. [0014] "ESRD": end stage renal disease--the need for
dialysis for more than 3 months. These criteria are called the
RIFLE criteria, which provide a useful clinical tool to classify
renal status. As discussed in Kellum, Crit. Care Med. 36: S141-45,
2008 and Ricci et al., Kidney Int. 73, 538-546, 2008, each hereby
incorporated by reference in its entirety, the RIFLE criteria
provide a uniform definition of AKI which has been validated in
numerous studies.
[0015] More recently, Mehta et al., Crit. Care 11:R31
(doi:10.1186.cc5713), 2007, hereby incorporated by reference in its
entirety, proposes the following similar classifications for
stratifying AKI patients, which have been modified from RIFLE:
[0016] "Stage I": increase in serum creatinine of more than or
equal to 0.3 mg/dL (.gtoreq.26.4 .mu.mol/L) or increase to more
than or equal to 150% (1.5-fold) from baseline OR urine output less
than 0.5 mL/kg per hour for more than 6 hours; [0017] "Stage II":
increase in serum creatinine to more than 200% (>2-fold) from
baseline OR urine output less than 0.5 mL/kg per hour for more than
12 hours; [0018] "Stage III": increase in serum creatinine to more
than 300% (>3-fold) from baseline OR serum creatinine
.gtoreq.354 .mu.mol/L accompanied by an acute increase of at least
44 .mu.mol/L OR urine output less than 0.3 mL/kg per hour for 24
hours or anuria for 12 hours.
[0019] The CIN Consensus Working Panel (McCollough et al, Rev
Cardiovasc Med. 2006;7(4):177-197, hereby incorporated by reference
in its entirety) uses a serum creatinine rise of 25% to define
Contrast induced nephropathy (which is a type of AKI). Although
various groups propose slightly different criteria for using serum
creatinine to detect AKI, the consensus is that small changes in
serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to
detect AKI (worsening renal function) and that the magnitude of the
serum creatinine change is an indicator of the severity of the AKI
and mortality risk.
[0020] Although serial measurement of serum creatinine over a
period of days is an accepted method of detecting and diagnosing
AKI and is considered one of the most important tools to evaluate
AKI patients, serum creatinine is generally regarded to have
several limitations in the diagnosis, assessment and monitoring of
AKI patients. The time period for serum creatinine to rise to
values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for
AKI can be 48 hours or longer depending on the definition used.
Since cellular injury in AKI can occur over a period of hours,
serum creatinine elevations detected at 48 hours or longer can be a
late indicator of injury, and relying on serum creatinine can thus
delay diagnosis of AKI. Furthermore, serum creatinine is not a good
indicator of the exact kidney status and treatment needs during the
most acute phases of AKI when kidney function is changing rapidly.
Some patients with AKI will recover fully, some will need dialysis
(either short term or long term) and some will have other
detrimental outcomes including death, major adverse cardiac events
and chronic kidney disease. Because serum creatinine is a marker of
filtration rate, it does not differentiate between the causes of
AKI (pre-renal, intrinsic renal, post-renal obstruction,
atheroembolic, etc.) or the category or location of injury in
intrinsic renal disease (for example, tubular, glomerular or
interstitial in origin). Urine output is similarly limited, Knowing
these things can be of vital importance in managing and treating
patients with AKI.
[0021] These limitations underscore the need for better methods to
detect and assess AKI, particularly in the early and subclinical
stages, but also in later stages when recovery and repair of the
kidney can occur. Furthermore, there is a need to better identify
patients who are at risk of having an AKI.
BRIEF SUMMARY OF THE INVENTION
[0022] It is an object of the invention to provide methods and
compositions for evaluating renal function in a subject. As
described herein, measurement of one or more markers selected from
the group consisting of Clusterin, Heart-type fatty acid binding
protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12
subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV
collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid
P-component (collectively referred to herein as "kidney injury
markers, and individually as a "kidney injury marker") can be used
for diagnosis, prognosis, risk stratification, staging, monitoring,
categorizing and determination of further diagnosis and treatment
regimens in subjects suffering or at risk of suffering from an
injury to renal function, reduced renal function, and/or acute
renal failure (also called acute kidney injury).
[0023] These kidney injury markers may be used, individually or in
panels comprising a plurality of kidney injury markers, for risk
stratification (that is, to identify subjects at risk for a future
injury to renal function, for future progression to reduced renal
function, for future progression to ARF, for future improvement in
renal function, etc.); for diagnosis of existing disease (that is,
to identify subjects who have suffered an injury to renal function,
who have progressed to reduced renal function, who have progressed
to ARF, etc.); for monitoring for deterioration or improvement of
renal function; and for predicting a future medical outcome, such
as improved or worsening renal function, a decreased or increased
mortality risk, a decreased or increased risk that a subject will
require renal replacement therapy (i.e., hemodialysis, peritoneal
dialysis, hemofiltration, and/or renal transplantation, a decreased
or increased risk that a subject will recover from an injury to
renal function, a decreased or increased risk that a subject will
recover from ARF, a decreased or increased risk that a subject will
progress to end stage renal disease, a decreased or increased risk
that a subject will progress to chronic renal failure, a decreased
or increased risk that a subject will suffer rejection of a
transplanted kidney, etc.
[0024] In a first aspect, the present invention relates to methods
for evaluating renal status in a subject. These methods comprise
performing an assay method that is configured to detect one or more
kidney injury markers of the present invention in a body fluid
sample obtained from the subject. The assay result(s), for example
a measured concentration of one or more markers selected from the
group consisting of Clusterin, Heart-type fatty acid binding
protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12
subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV
collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid
P-component is/are then correlated to the renal status of the
subject. This correlation to renal status may include correlating
the assay result(s) to one or more of risk stratification,
diagnosis, prognosis, staging, classifying and monitoring of the
subject as described herein. Thus, the present invention utilizes
one or more kidney injury markers of the present invention for the
evaluation of renal injury.
[0025] In certain embodiments, the methods for evaluating renal
status described herein are methods for risk stratification of the
subject; that is, assigning a likelihood of one or more future
changes in renal status to the subject. In these embodiments, the
assay result(s) is/are correlated to one or more such future
changes. The following are preferred risk stratification
embodiments.
[0026] In preferred risk stratification embodiments, these methods
comprise determining a subject's risk for a future injury to renal
function, and the assay result(s) is/are correlated to a likelihood
of such a future injury to renal function. For example, the
measured concentration(s) may each be compared to a threshold
value. For a "positive going" kidney injury marker, an increased
likelihood of suffering a future injury to renal function is
assigned to the subject when the measured concentration is above
the threshold, relative to a likelihood assigned when the measured
concentration is below the threshold. For a "negative going" kidney
injury marker, an increased likelihood of suffering a future injury
to renal function is assigned to the subject when the measured
concentration is below the threshold, relative to a likelihood
assigned when the measured concentration is above the
threshold.
[0027] In other preferred risk stratification embodiments, these
methods comprise determining a subject's risk for future reduced
renal function, and the assay result(s) is/are correlated to a
likelihood of such reduced renal function. For example, the
measured concentrations may each be compared to a threshold value.
For a "positive going" kidney injury marker, an increased
likelihood of suffering a future reduced renal function is assigned
to the subject when the measured concentration is above the
threshold, relative to a likelihood assigned when the measured
concentration is below the threshold. For a "negative going" kidney
injury marker, an increased likelihood of future reduced renal
function is assigned to the subject when the measured concentration
is below the threshold, relative to a likelihood assigned when the
measured concentration is above the threshold.
[0028] In still other preferred risk stratification embodiments,
these methods comprise determining a subject's likelihood for a
future improvement in renal function, and the assay result(s)
is/are correlated to a likelihood of such a future improvement in
renal function. For example, the measured concentration(s) may each
be compared to a threshold value. For a "positive going" kidney
injury marker, an increased likelihood of a future improvement in
renal function is assigned to the subject when the measured
concentration is below the threshold, relative to a likelihood
assigned when the measured concentration is above the threshold.
For a "negative going" kidney injury marker, an increased
likelihood of a future improvement in renal function is assigned to
the subject when the measured concentration is above the threshold,
relative to a likelihood assigned when the measured concentration
is below the threshold.
[0029] In yet other preferred risk stratification embodiments,
these methods comprise determining a subject's risk for progression
to ARF, and the result(s) is/are correlated to a likelihood of such
progression to ARF. For example, the measured concentration(s) may
each be compared to a threshold value. For a "positive going"
kidney injury marker, an increased likelihood of progression to ARF
is assigned to the subject when the measured concentration is above
the threshold, relative to a likelihood assigned when the measured
concentration is below the threshold. For a "negative going" kidney
injury marker, an increased likelihood of progression to ARF is
assigned to the subject when the measured concentration is below
the threshold, relative to a likelihood assigned when the measured
concentration is above the threshold.
[0030] And in other preferred risk stratification embodiments,
these methods comprise determining a subject's outcome risk, and
the assay result(s) is/are correlated to a likelihood of the
occurrence of a clinical outcome related to a renal injury suffered
by the subject. For example, the measured concentration(s) may each
be compared to a threshold value. For a "positive going" kidney
injury marker, an increased likelihood of one or more of: acute
kidney injury, progression to a worsening stage of AKI, mortality,
a requirement for renal replacement therapy, a requirement for
withdrawal of renal toxins, end stage renal disease, heart failure,
stroke, myocardial infarction, progression to chronic kidney
disease, etc., is assigned to the subject when the measured
concentration is above the threshold, relative to a likelihood
assigned when the measured concentration is below the threshold.
For a "negative going" kidney injury marker, an increased
likelihood of one or more of: acute kidney injury, progression to a
worsening stage of AKI, mortality, a requirement for renal
replacement therapy, a requirement for withdrawal of renal toxins,
end stage renal disease, heart failure, stroke, myocardial
infarction, progression to chronic kidney disease, etc., is
assigned to the subject when the measured concentration is below
the threshold, relative to a likelihood assigned when the measured
concentration is above the threshold.
[0031] In such risk stratification embodiments, preferably the
likelihood or risk assigned is that an event of interest is more or
less likely to occur within 180 days of the time at which the body
fluid sample is obtained from the subject. In particularly
preferred embodiments, the likelihood or risk assigned relates to
an event of interest occurring within a shorter time period such as
18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days,
14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24
hours, 12 hours, or less. A risk at 0 hours of the time at which
the body fluid sample is obtained from the subject is equivalent to
diagnosis of a current condition.
[0032] In preferred risk stratification embodiments, the subject is
selected for risk stratification based on the pre-existence in the
subject of one or more known risk factors for prerenal, intrinsic
renal, or postrenal ARF. For example, a subject undergoing or
having undergone major vascular surgery, coronary artery bypass, or
other cardiac surgery; a subject having pre-existing congestive
heart failure, preeclampsia, eclampsia, diabetes mellitus,
hypertension, coronary artery disease, proteinuria, renal
insufficiency, glomerular filtration below the normal range,
cirrhosis, serum creatinine above the normal range, or sepsis; or a
subject exposed to NSAIDs, cyclosporines, tacrolimus,
aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin,
ifosfamide, heavy metals, methotrexate, radiopaque contrast agents,
or streptozotocin are all preferred subjects for monitoring risks
according to the methods described herein. This list is not meant
to be limiting. By "pre-existence" in this context is meant that
the risk factor exists at the time the body fluid sample is
obtained from the subject. In particularly preferred embodiments, a
subject is chosen for risk stratification based on an existing
diagnosis of injury to renal function, reduced renal function, or
ARF.
[0033] In other embodiments, the methods for evaluating renal
status described herein are methods for diagnosing a renal injury
in the subject; that is, assessing whether or not a subject has
suffered from an injury to renal function, reduced renal function,
or ARF. In these embodiments, the assay result(s), for example a
measured concentration of one or more markers selected from the
group consisting of Clusterin, Heart-type fatty acid binding
protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12
subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV
collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid
P-component is/are correlated to the occurrence or nonoccurrence of
a change in renal status. The following are preferred diagnostic
embodiments.
[0034] In preferred diagnostic embodiments, these methods comprise
diagnosing the occurrence or nonoccurrence of an injury to renal
function, and the assay result(s) is/are correlated to the
occurrence or nonoccurrence of such an injury. For example, each of
the measured concentration(s) may be compared to a threshold value.
For a positive going marker, an increased likelihood of the
occurrence of an injury to renal function is assigned to the
subject when the measured concentration is above the threshold
(relative to the likelihood assigned when the measured
concentration is below the threshold); alternatively, when the
measured concentration is below the threshold, an increased
likelihood of the nonoccurrence of an injury to renal function may
be assigned to the subject (relative to the likelihood assigned
when the measured concentration is above the threshold). For a
negative going marker, an increased likelihood of the occurrence of
an injury to renal function is assigned to the subject when the
measured concentration is below the threshold (relative to the
likelihood assigned when the measured concentration is above the
threshold); alternatively, when the measured concentration is above
the threshold, an increased likelihood of the nonoccurrence of an
injury to renal function may be assigned to the subject (relative
to the likelihood assigned when the measured concentration is below
the threshold).
[0035] In other preferred diagnostic embodiments, these methods
comprise diagnosing the occurrence or nonoccurrence of reduced
renal function, and the assay result(s) is/are correlated to the
occurrence or nonoccurrence of an injury causing reduced renal
function. For example, each of the measured concentration(s) may be
compared to a threshold value. For a positive going marker, an
increased likelihood of the occurrence of an injury causing reduced
renal function is assigned to the subject when the measured
concentration is above the threshold (relative to the likelihood
assigned when the measured concentration is below the threshold);
alternatively, when the measured concentration is below the
threshold, an increased likelihood of the nonoccurrence of an
injury causing reduced renal function may be assigned to the
subject (relative to the likelihood assigned when the measured
concentration is above the threshold). For a negative going marker,
an increased likelihood of the occurrence of an injury causing
reduced renal function is assigned to the subject when the measured
concentration is below the threshold (relative to the likelihood
assigned when the measured concentration is above the threshold);
alternatively, when the measured concentration is above the
threshold, an increased likelihood of the nonoccurrence of an
injury causing reduced renal function may be assigned to the
subject (relative to the likelihood assigned when the measured
concentration is below the threshold).
[0036] In yet other preferred diagnostic embodiments, these methods
comprise diagnosing the occurrence or nonoccurrence of ARF, and the
assay result(s) is/are correlated to the occurrence or
nonoccurrence of an injury causing ARF. For example, each of the
measured concentration(s) may be compared to a threshold value. For
a positive going marker, an increased likelihood of the occurrence
of ARF is assigned to the subject when the measured concentration
is above the threshold (relative to the likelihood assigned when
the measured concentration is below the threshold); alternatively,
when the measured concentration is below the threshold, an
increased likelihood of the nonoccurrence of ARF may be assigned to
the subject (relative to the likelihood assigned when the measured
concentration is above the threshold). For a negative going marker,
an increased likelihood of the occurrence of ARF is assigned to the
subject when the measured concentration is below the threshold
(relative to the likelihood assigned when the measured
concentration is above the threshold); alternatively, when the
measured concentration is above the threshold, an increased
likelihood of the nonoccurrence of ARF may be assigned to the
subject (relative to the likelihood assigned when the measured
concentration is below the threshold).
[0037] In still other preferred diagnostic embodiments, these
methods comprise diagnosing a subject as being in need of renal
replacement therapy, and the assay result(s) is/are correlated to a
need for renal replacement therapy. For example, each of the
measured concentration(s) may be compared to a threshold value. For
a positive going marker, an increased likelihood of the occurrence
of an injury creating a need for renal replacement therapy is
assigned to the subject when the measured concentration is above
the threshold (relative to the likelihood assigned when the
measured concentration is below the threshold); alternatively, when
the measured concentration is below the threshold, an increased
likelihood of the nonoccurrence of an injury creating a need for
renal replacement therapy may be assigned to the subject (relative
to the likelihood assigned when the measured concentration is above
the threshold). For a negative going marker, an increased
likelihood of the occurrence of an injury creating a need for renal
replacement therapy is assigned to the subject when the measured
concentration is below the threshold (relative to the likelihood
assigned when the measured concentration is above the threshold);
alternatively, when the measured concentration is above the
threshold, an increased likelihood of the nonoccurrence of an
injury creating a need for renal replacement therapy may be
assigned to the subject (relative to the likelihood assigned when
the measured concentration is below the threshold).
[0038] In still other preferred diagnostic embodiments, these
methods comprise diagnosing a subject as being in need of renal
transplantation, and the assay result(s0 is/are correlated to a
need for renal transplantation. For example, each of the measured
concentration(s) may be compared to a threshold value. For a
positive going marker, an increased likelihood of the occurrence of
an injury creating a need for renal transplantation is assigned to
the subject when the measured concentration is above the threshold
(relative to the likelihood assigned when the measured
concentration is below the threshold); alternatively, when the
measured concentration is below the threshold, an increased
likelihood of the nonoccurrence of an injury creating a need for
renal transplantation may be assigned to the subject (relative to
the likelihood assigned when the measured concentration is above
the threshold). For a negative going marker, an increased
likelihood of the occurrence of an injury creating a need for renal
transplantation is assigned to the subject when the measured
concentration is below the threshold (relative to the likelihood
assigned when the measured concentration is above the threshold);
alternatively, when the measured concentration is above the
threshold, an increased likelihood of the nonoccurrence of an
injury creating a need for renal transplantation may be assigned to
the subject (relative to the likelihood assigned when the measured
concentration is below the threshold).
[0039] In still other embodiments, the methods for evaluating renal
status described herein are methods for monitoring a renal injury
in the subject; that is, assessing whether or not renal function is
improving or worsening in a subject who has suffered from an injury
to renal function, reduced renal function, or ARF. In these
embodiments, the assay result(s), for example a measured
concentration of one or more markers selected from the group
consisting of Clusterin, Heart-type fatty acid binding protein,
Hepatocyte growth factor, Interferon gamma, Interleukin-12 subunit
beta, Interleukin-16, Interleukin-2, 72 kDa type IV collagenase,
Matrix metalloproteinase-9, Midkine, and Serum amyloid P-component
is/are correlated to the occurrence or nonoccurrence of a change in
renal status. The following are preferred monitoring
embodiments.
[0040] In preferred monitoring embodiments, these methods comprise
monitoring renal status in a subject suffering from an injury to
renal function, and the assay result(s) is/are correlated to the
occurrence or nonoccurrence of a change in renal status in the
subject. For example, the measured concentration(s) may be compared
to a threshold value. For a positive going marker, when the
measured concentration is above the threshold, a worsening of renal
function may be assigned to the subject; alternatively, when the
measured concentration is below the threshold, an improvement of
renal function may be assigned to the subject. For a negative going
marker, when the measured concentration is below the threshold, a
worsening of renal function may be assigned to the subject;
alternatively, when the measured concentration is above the
threshold, an improvement of renal function may be assigned to the
subject.
[0041] In other preferred monitoring embodiments, these methods
comprise monitoring renal status in a subject suffering from
reduced renal function, and the assay result(s) is/are correlated
to the occurrence or nonoccurrence of a change in renal status in
the subject. For example, the measured concentration(s) may be
compared to a threshold value. For a positive going marker, when
the measured concentration is above the threshold, a worsening of
renal function may be assigned to the subject; alternatively, when
the measured concentration is below the threshold, an improvement
of renal function may be assigned to the subject. For a negative
going marker, when the measured concentration is below the
threshold, a worsening of renal function may be assigned to the
subject; alternatively, when the measured concentration is above
the threshold, an improvement of renal function may be assigned to
the subject.
[0042] In yet other preferred monitoring embodiments, these methods
comprise monitoring renal status in a subject suffering from acute
renal failure, and the assay result(s) is/are correlated to the
occurrence or nonoccurrence of a change in renal status in the
subject. For example, the measured concentration(s) may be compared
to a threshold value. For a positive going marker, when the
measured concentration is above the threshold, a worsening of renal
function may be assigned to the subject; alternatively, when the
measured concentration is below the threshold, an improvement of
renal function may be assigned to the subject. For a negative going
marker, when the measured concentration is below the threshold, a
worsening of renal function may be assigned to the subject;
alternatively, when the measured concentration is above the
threshold, an improvement of renal function may be assigned to the
subject.
[0043] In other additional preferred monitoring embodiments, these
methods comprise monitoring renal status in a subject at risk of an
injury to renal function due to the pre-existence of one or more
known risk factors for prerenal, intrinsic renal, or postrenal ARF,
and the assay result(s) is/are correlated to the occurrence or
nonoccurrence of a change in renal status in the subject. For
example, the measured concentration(s) may be compared to a
threshold value. For a positive going marker, when the measured
concentration is above the threshold, a worsening of renal function
may be assigned to the subject; alternatively, when the measured
concentration is below the threshold, an improvement of renal
function may be assigned to the subject. For a negative going
marker, when the measured concentration is below the threshold, a
worsening of renal function may be assigned to the subject;
alternatively, when the measured concentration is above the
threshold, an improvement of renal function may be assigned to the
subject.
[0044] In still other embodiments, the methods for evaluating renal
status described herein are methods for classifying a renal injury
in the subject; that is, determining whether a renal injury in a
subject is prerenal, intrinsic renal, or postrenal; and/or further
subdividing these classes into subclasses such as acute tubular
injury, acute glomerulonephritis acute tubulointerstitial
nephritis, acute vascular nephropathy, or infiltrative disease;
and/or assigning a likelihood that a subject will progress to a
particular RIFLE stage. In these embodiments, the assay result(s),
for example a measured concentration of one or more markers
selected from the group consisting of Clusterin, Heart-type fatty
acid binding protein, Hepatocyte growth factor, Interferon gamma,
Interleukin-12 subunit beta, Interleukin-16, Interleukin-2, 72 kDa
type IV collagenase, Matrix metalloproteinase-9, Midkine, and Serum
amyloid P-component is/are correlated to a particular class and/or
subclass. The following are preferred classification
embodiments.
[0045] In preferred classification embodiments, these methods
comprise determining whether a renal injury in a subject is
prerenal, intrinsic renal, or postrenal; and/or further subdividing
these classes into subclasses such as acute tubular injury, acute
glomerulonephritis acute tubulointerstitial nephritis, acute
vascular nephropathy, or infiltrative disease; and/or assigning a
likelihood that a subject will progress to a particular RIFLE
stage, and the assay result(s) is/are correlated to the injury
classification for the subject. For example, the measured
concentration may be compared to a threshold value, and when the
measured concentration is above the threshold, a particular
classification is assigned; alternatively, when the measured
concentration is below the threshold, a different classification
may be assigned to the subject.
[0046] A variety of methods may be used by the skilled artisan to
arrive at a desired threshold value for use in these methods. For
example, the threshold value may be determined from a population of
normal subjects by selecting a concentration representing the
75.sup.th, 85.sup.th, 90.sup.th, 95.sup.th, or 99.sup.th percentile
of a kidney injury marker measured in such normal subjects.
Alternatively, the threshold value may be determined from a
"diseased" population of subjects, e.g., those suffering from an
injury or having a predisposition for an injury (e.g., progression
to ARF or some other clinical outcome such as death, dialysis,
renal transplantation, etc.), by selecting a concentration
representing the 75.sup.th, 85.sup.th, 90.sup.th, 95.sup.th, or
99.sup.th percentile of a kidney injury marker measured in such
subjects. In another alternative, the threshold value may be
determined from a prior measurement of a kidney injury marker in
the same subject; that is, a temporal change in the level of a
kidney injury marker in the subject may be used to assign risk to
the subject.
[0047] The foregoing discussion is not meant to imply, however,
that the kidney injury markers of the present invention must be
compared to corresponding individual thresholds. Methods for
combining assay results can comprise the use of multivariate
logistical regression, loglinear modeling, neural network analysis,
n-of-m analysis, decision tree analysis, calculating ratios of
markers, etc. This list is not meant to be limiting. In these
methods, a composite result which is determined by combining
individual markers may be treated as if it is itself a marker; that
is, a threshold may be determined for the composite result as
described herein for individual markers, and the composite result
for an individual patient compared to this threshold.
[0048] The ability of a particular test to distinguish two
populations can be established using ROC analysis. For example, ROC
curves established from a "first" subpopulation which is
predisposed to one or more future changes in renal status, and a
"second" subpopulation which is not so predisposed can be used to
calculate a ROC curve, and the area under the curve provides a
measure of the quality of the test. Preferably, the tests described
herein provide a ROC curve area greater than 0.5, preferably at
least 0.6, more preferably 0.7, still more preferably at least 0.8,
even more preferably at least 0.9, and most preferably at least
0.95.
[0049] In certain aspects, the measured concentration of one or
more kidney injury markers, or a composite of such markers, may be
treated as continuous variables. For example, any particular
concentration can be converted into a corresponding probability of
a future reduction in renal function for the subject, the
occurrence of an injury, a classification, etc. In yet another
alternative, a threshold that can provide an acceptable level of
specificity and sensitivity in separating a population of subjects
into "bins" such as a "first" subpopulation (e.g., which is
predisposed to one or more future changes in renal status, the
occurrence of an injury, a classification, etc.) and a "second"
subpopulation which is not so predisposed. A threshold value is
selected to separate this first and second population by one or
more of the following measures of test accuracy: [0050] an odds
ratio greater than 1, preferably at least about 2 or more or about
0.5 or less, more preferably at least about 3 or more or about 0.33
or less, still more preferably at least about 4 or more or about
0.25 or less, even more preferably at least about 5 or more or
about 0.2 or less, and most preferably at least about 10 or more or
about 0.1 or less; [0051] a specificity of greater than 0.5,
preferably at least about 0.6, more preferably at least about 0.7,
still more preferably at least about 0.8, even more preferably at
least about 0.9 and most preferably at least about 0.95, with a
corresponding sensitivity greater than 0.2, preferably greater than
about 0.3, more preferably greater than about 0.4, still more
preferably at least about 0.5, even more preferably about 0.6, yet
more preferably greater than about 0.7, still more preferably
greater than about 0.8, more preferably greater than about 0.9, and
most preferably greater than about 0.95; [0052] a sensitivity of
greater than 0.5, preferably at least about 0.6, more preferably at
least about 0.7, still more preferably at least about 0.8, even
more preferably at least about 0.9 and most preferably at least
about 0.95, with a corresponding specificity greater than 0.2,
preferably greater than about 0.3, more preferably greater than
about 0.4, still more preferably at least about 0.5, even more
preferably about 0.6, yet more preferably greater than about 0.7,
still more preferably greater than about 0.8, more preferably
greater than about 0.9, and most preferably greater than about
0.95; [0053] at least about 75% sensitivity, combined with at least
about 75% specificity; [0054] a positive likelihood ratio
(calculated as sensitivity/(1-specificity)) of greater than 1, at
least about 2, more preferably at least about 3, still more
preferably at least about 5, and most preferably at least about 10;
or [0055] a negative likelihood ratio (calculated as
(1-sensitivity)/specificity) of less than 1, less than or equal to
about 0.5, more preferably less than or equal to about 0.3, and
most preferably less than or equal to about 0.1. [0056] The term
"about" in the context of any of the above measurements refers to
+/-5% of a given measurement.
[0057] Multiple thresholds may also be used to assess renal status
in a subject. For example, a "first" subpopulation which is
predisposed to one or more future changes in renal status, the
occurrence of an injury, a classification, etc., and a "second"
subpopulation which is not so predisposed can be combined into a
single group. This group is then subdivided into three or more
equal parts (known as tertiles, quartiles, quintiles, etc.,
depending on the number of subdivisions). An odds ratio is assigned
to subjects based on which subdivision they fall into. If one
considers a tertile, the lowest or highest tertile can be used as a
reference for comparison of the other subdivisions. This reference
subdivision is assigned an odds ratio of 1. The second tertile is
assigned an odds ratio that is relative to that first tertile. That
is, someone in the second tertile might be 3 times more likely to
suffer one or more future changes in renal status in comparison to
someone in the first tertile. The third tertile is also assigned an
odds ratio that is relative to that first tertile.
[0058] In certain embodiments, the assay method is an immunoassay.
Antibodies for use in such assays will specifically bind a full
length kidney injury marker of interest, and may also bind one or
more polypeptides that are "related" thereto, as that term is
defined hereinafter. Numerous immunoassay formats are known to
those of skill in the art. Preferred body fluid samples are
selected from the group consisting of urine, blood, serum, saliva,
tears, and plasma.
[0059] The foregoing method steps should not be interpreted to mean
that the kidney injury marker assay result(s) is/are used in
isolation in the methods described herein. Rather, additional
variables or other clinical indicia may be included in the methods
described herein. For example, a risk stratification, diagnostic,
classification, monitoring, etc. method may combine the assay
result(s) with one or more variables measured for the subject
selected from the group consisting of demographic information
(e.g., weight, sex, age, race), medical history (e.g., family
history, type of surgery, pre-existing disease such as aneurism,
congestive heart failure, preeclampsia, eclampsia, diabetes
mellitus, hypertension, coronary artery disease, proteinuria, renal
insufficiency, or sepsis, type of toxin exposure such as NSAIDs,
cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene
glycol, hemoglobin, myoglobin, ifosfamide, heavy metals,
methotrexate, radiopaque contrast agents, or streptozotocin),
clinical variables (e.g., blood pressure, temperature, respiration
rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score
for UA/NSTEMI, Framingham Risk Score), a glomerular filtration
rate, an estimated glomerular filtration rate, a urine production
rate, a serum or plasma creatinine concentration, a urine
creatinine concentration, a fractional excretion of sodium, a urine
sodium concentration, a urine creatinine to serum or plasma
creatinine ratio, a urine specific gravity, a urine osmolality, a
urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to
creatnine ratio, a renal failure index calculated as urine
sodium/(urine creatinine/plasma creatinine), a serum or plasma
neutrophil gelatinase (NGAL) concentration, a urine NGAL
concentration, a serum or plasma cystatin C concentration, a serum
or plasma cardiac troponin concentration, a serum or plasma BNP
concentration, a serum or plasma NTproBNP concentration, and a
serum or plasma proBNP concentration. Other measures of renal
function which may be combined with one or more kidney injury
marker assay result(s) are described hereinafter and in Harrison's
Principles of Internal Medicine, 17.sup.th Ed., McGraw Hill, New
York, pages 1741-1830, and Current Medical Diagnosis &
Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815,
each of which are hereby incorporated by reference in their
entirety.
[0060] When more than one marker is measured, the individual
markers may be measured in samples obtained at the same time, or
may be determined from samples obtained at different (e.g., an
earlier or later) times. The individual markers may also be
measured on the same or different body fluid samples. For example,
one kidney injury marker may be measured in a serum or plasma
sample and another kidney injury marker may be measured in a urine
sample. In addition, assignment of a likelihood may combine an
individual kidney injury marker assay result with temporal changes
in one or more additional variables.
[0061] In various related aspects, the present invention also
relates to devices and kits for performing the methods described
herein. Suitable kits comprise reagents sufficient for performing
an assay for at least one of the described kidney injury markers,
together with instructions for performing the described threshold
comparisons.
[0062] In certain embodiments, reagents for performing such assays
are provided in an assay device, and such assay devices may be
included in such a kit. Preferred reagents can comprise one or more
solid phase antibodies, the solid phase antibody comprising
antibody that detects the intended biomarker target(s) bound to a
solid support. In the case of sandwich immunoassays, such reagents
can also include one or more detectably labeled antibodies, the
detectably labeled antibody comprising antibody that detects the
intended biomarker target(s) bound to a detectable label.
Additional optional elements that may be provided as part of an
assay device are described hereinafter.
[0063] Detectable labels may include molecules that are themselves
detectable (e.g., fluorescent moieties, electrochemical labels, ed
l (electrochemical luminescence) labels, metal chelates, colloidal
metal particles, etc.) as well as molecules that may be indirectly
detected by production of a detectable reaction product (e.g.,
enzymes such as horseradish peroxidase, alkaline phosphatase, etc.)
or through the use of a specific binding molecule which itself may
be detectable (e.g., a labeled antibody that binds to the second
antibody, biotin, digoxigenin, maltose, oligohistidine,
2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
[0064] Generation of a signal from the signal development element
can be performed using various optical, acoustical, and
electrochemical methods well known in the art. Examples of
detection modes include fluorescence, radiochemical detection,
reflectance, absorbance, amperometry, conductance, impedance,
interferometry, ellipsometry, etc. In certain of these methods, the
solid phase antibody is coupled to a transducer (e.g., a
diffraction grating, electrochemical sensor, etc.) for generation
of a signal, while in others, a signal is generated by a transducer
that is spatially separate from the solid phase antibody (e.g., a
fluorometer that employs an excitation light source and an optical
detector). This list is not meant to be limiting. Antibody-based
biosensors may also be employed to determine the presence or amount
of analytes that optionally eliminate the need for a labeled
molecule.
BRIEF DESCRIPTION OF THE FIGURES
[0065] FIG. 1 provides data tables determined in accordance with
Example 6 for the comparison of marker levels in urine samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0) and in urine samples collected from subjects at 0, 24
hours, and 48 hours prior to reaching stage R, I or F in Cohort 2.
Tables provide descriptive statistics, AUC analysis, and
sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0066] FIG. 2 provides data tables determined in accordance with
Example 7 for the comparison of marker levels in urine samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0 or R) and in urine samples collected from subjects at 0, 24
hours, and 48 hours prior to reaching stage I or F in Cohort 2.
Tables provide descriptive statistics, AUC analysis, and
sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0067] FIG. 3 provides data tables determined in accordance with
Example 8 for the comparison of marker levels in urine samples
collected for Cohort 1 (patients that reached, but did not progress
beyond, RIFLE stage R) and in urine samples collected from subjects
at 0, 24 hours, and 48 hours prior to reaching stage I or F in
Cohort 2. Tables provide descriptive statistics, AUC analysis, and
sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0068] FIG. 4 provides data tables determined in accordance with
Example 9 for the comparison of marker levels in urine samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0) and in urine samples collected from subjects at 0, 24
hours, and 48 hours prior to reaching stage F in Cohort 2. Tables
provide descriptive statistics, AUC analysis, and sensitivity,
specificity and odds ratio calculations at various threshold
(cutoff) levels for the various markers.
[0069] FIG. 5 provides data tables determined in accordance with
Example 6 for the comparison of marker levels in plasma samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0) and in plasma samples collected from subjects at 0, 24
hours, and 48 hours prior to reaching stage R, I or F in Cohort 2.
Tables provide descriptive statistics, AUC analysis, and
sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0070] FIG. 6 provides data tables determined in accordance with
Example 7 for the comparison of marker levels in plasma samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0 or R) and in plasma samples collected from subjects at 0,
24 hours, and 48 hours prior to reaching stage I or F in Cohort 2.
Tables provide descriptive statistics, AUC analysis, and
sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0071] FIG. 7 provides data tables determined in accordance with
Example 8 for the comparison of marker levels in plasma samples
collected for Cohort 1 (patients that reached, but did not progress
beyond, RIFLE stage R) and in plasma samples collected from
subjects at 0, 24 hours, and 48 hours prior to reaching stage I or
F in Cohort 2. Tables provide descriptive statistics, AUC analysis,
and sensitivity, specificity and odds ratio calculations at various
threshold (cutoff) levels for the various markers.
[0072] FIG. 8 provides data tables determined in accordance with
Example 9 for the comparison of marker levels in plasma samples
collected for Cohort 1 (patients that did not progress beyond RIFLE
stage 0) and in plasma samples collected from subjects at 0, 24
hours, and 48 hours prior to reaching stage F in Cohort 2. Tables
provide descriptive statistics, AUC analysis, and sensitivity,
specificity and odds ratio calculations at various threshold
(cutoff) levels for the various markers.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present invention relates to methods and compositions
for diagnosis, differential diagnosis, risk stratification,
monitoring, classifying and determination of treatment regimens in
subjects suffering or at risk of suffering from injury to renal
function, reduced renal function and/or acute renal failure through
measurement of one or more kidney injury markers. In various
embodiments, a measured concentration of one or more markers
selected from the group consisting of Clusterin, Heart-type fatty
acid binding protein, Hepatocyte growth factor, Interferon gamma,
Interleukin-12 subunit beta, Interleukin-16, Interleukin-2, 72 kDa
type IV collagenase, Matrix metalloproteinase-9, Midkine, and Serum
amyloid P-component, or one or more markers related thereto, are
correlated to the renal status of the subject.
[0074] For purposes of this document, the following definitions
apply: As used herein, an "injury to renal function" is an abrupt
(within 14 days, preferably within 7 days, more preferably within
72 hours, and still more preferably within 48 hours) measurable
reduction in a measure of renal function. Such an injury may be
identified, for example, by a decrease in glomerular filtration
rate or estimated GFR, a reduction in urine output, an increase in
serum creatinine, an increase in serum cystatin C, a requirement
for renal replacement therapy, etc. "Improvement in Renal Function"
is an abrupt (within 14 days, preferably within 7 days, more
preferably within 72 hours, and still more preferably within 48
hours) measurable increase in a measure of renal function.
Preferred methods for measuring and/or estimating GFR are described
hereinafter.
[0075] As used herein, "reduced renal function" is an abrupt
(within 14 days, preferably within 7 days, more preferably within
72 hours, and still more preferably within 48 hours) reduction in
kidney function identified by an absolute increase in serum
creatinine of greater than or equal to 0.1 mg/dL (.gtoreq.8.8
.mu.mol/L), a percentage increase in serum creatinine of greater
than or equal to 20% (1.2-fold from baseline), or a reduction in
urine output (documented oliguria of less than 0.5 ml/kg per
hour).
[0076] As used herein, "acute renal failure" or "ARF" is an abrupt
(within 14 days, preferably within 7 days, more preferably within
72 hours, and still more preferably within 48 hours) reduction in
kidney function identified by an absolute increase in serum
creatinine of greater than or equal to 0.3 mg/dl (.gtoreq.26.4
.mu.mol/l), a percentage increase in serum creatinine of greater
than or equal to 50% (1.5-fold from baseline), or a reduction in
urine output (documented oliguria of less than 0.5 ml/kg per hour
for at least 6 hours). This term is synonymous with "acute kidney
injury" or "AKI."
[0077] In this regard, the skilled artisan will understand that the
signals obtained from an immunoassay are a direct result of
complexes formed between one or more antibodies and the target
biomolecule (i.e., the analyte) and polypeptides containing the
necessary epitope(s) to which the antibodies bind. While such
assays may detect the full length biomarker and the assay result be
expressed as a concentration of a biomarker of interest, the signal
from the assay is actually a result of all such "immunoreactive"
polypeptides present in the sample. Expression of biomarkers may
also be determined by means other than immunoassays, including
protein measurements (such as dot blots, western blots,
chromatographic methods, mass spectrometry, etc.) and nucleic acid
measurements (mRNA quatitation). This list is not meant to be
limiting.
[0078] As used herein, the term "Clusterin" refers to one or more
polypeptides present in a biological sample that are derived from
the Clusterin precursor (Swiss-Prot P10909 (SEQ ID NO: 1)).
TABLE-US-00002 10 20 30 40 MMKTLLLFVG LLLTWESGQV LGDQTVSDNE
LQEMSNQGSK 50 60 70 80 YVNKEIQNAV NGVKQIKTLI EKTNEERKTL LSNLEEAKKK
90 100 110 120 KEDALNETRE SETKLKELPG VCNETMMALW EECKPCLKQT 130 140
150 160 CMKFYARVCR SGSGLVGRQL EEFLNQSSPF YFWMNGDRID 170 180 190 200
SLLENDRQQT HMLDVMQDHF SRASSIIDEL FQDRFFTREP 210 220 230 240
QDTYHYLPFS LPHRRPHFFF PKSRIVRSLM PFSPYEPLNF 250 260 270 280
HAMFQPFLEM IHEAQQAMDI HFHSPAFQHP PTEFIREGDD 290 300 310 320
DRTVCREIRH NSTGCLRMKD QCDKCREILS VDCSTNNPSQ 330 340 350 360
AKLRRELDES LQVAERLTRK YNELLKSYQW KMLNTSSLLE 370 380 390 400
QLNEQFNWVS RLANLTQGED QYYLRVTTVA SHTSDSDVPS 410 420 430 440
GVTEVVVKLF DSDPITVTVP VEVSRKNPKF METVAEKALQ EYRKKHREE
[0079] The following domains have been identified in Clusterin:
TABLE-US-00003 Residues Length Domain ID 1-22 22 Signal peptide
23-449 427 Clusterin 23-227 205 Clusterin beta chain 228-449 222
Clusterin alpha chain
[0080] As used herein, the term "Heart-type fatty acid-binding
protein" refers to one or more polypeptides present in a biological
sample that are derived from the Heart-type fatty acid-binding
protein precursor (Swiss-Prot P05413 (SEQ ID NO: 2)).
TABLE-US-00004 10 20 30 40 MVDAFLGTWK LVDSKNFDDY MKSLGVGFAT
RQVASMTKPT 50 60 70 80 TIIEKNGDIL TLKTHSTFKN TEISFKLGVE FDETTADDRK
90 100 110 120 VKSIVTLDGG KLVHLQKWDG QETTLVRELI DGKLILTLTH 130
GTAVCTRTYE KEA
[0081] The following domains have been identified in Heart-type
fatty acid-binding protein:
TABLE-US-00005 Residues Length Domain ID 1 1 Initiator methionine
2-133 132 Heart-type fatty acid-binding protein
[0082] As used herein, the term "Hepatocyte growth factor" refers
to one or more polypeptides present in a biological sample that are
derived from the Hepatocyte growth factor precursor (Swiss-Prot
P14210 (SEQ ID NO: 3)).
TABLE-US-00006 10 20 30 40 MWVTKLLPAL LLQHVLLHLL LLPIAIPYAE
GQRKRRNTIH 50 60 70 80 EFKKSAKTTL IKIDPALKIK TKKVNTADQC ANRCTRNKGL
90 100 110 120 PFTCKAFVFD KARKQCLWFP FNSMSSGVKK EFGHEFDLYE 130 140
150 160 NKDYIRNCII GKGRSYKGTV SITKSGIKCQ PWSSMIPHEH 170 180 190 200
SFLPSSYRGK DLQENYCRNP RGEEGGPWCF TSNPEVRYEV 210 220 230 240
CDIPQCSEVE CMTCNGESYR GLMDHTESGK ICQRWDHQTP 250 260 270 280
HRHKFLPERY PDKGFDDNYC RNPDGQPRPW CYTLDPHTRW 290 300 310 320
EYCAIKTCAD NTMNDTDVPL ETTECIQGQG EGYRGTVNTI 330 340 350 360
WNGIPCQRWD SQYPHEHDMT PENFKCKDLR ENYCRNPDGS 370 380 390 400
ESPWCFTTDP NIRVGYCSQI PNCDMSHGQD CYRGNGKNYM 410 420 430 440
GNLSQTRSGL TCSMWDKNME DLHRHIFWEP DASKLNENYC 450 460 470 480
RNPDDDAHGP WCYTGNPLIP WDYCPISRCE GDTTPTIVNL 490 500 510 520
DHPVISCAKT KQLRVVNGIP TRTNIGWMVS LRYRNKHICG 530 540 550 560
GSLIKESWVL TARQCFPSRD LKDYEAWLGI HDVHGRGDEK 570 580 590 600
CKQVLNVSQL VYGPEGSDLV LMKLARPAVL DDFVSTIDLP 610 620 630 640
NYGCTIPEKT SCSVYGWGYT GLINYDGLLR VAHLYIMGNE 650 660 670 680
KCSQHHRGKV TLNESEICAG AEKIGSGPCE GDYGGPLVCE 690 700 710 720
QHKMRMVLGV IVPGRGCAIP NRPGIFVRVA YYAKWIHKII LTYKVPQS
[0083] The following domains have been identified in Hepatocyte
growth factor:
TABLE-US-00007 Residues Length Domain ID 1-31 31 signal sequence
32-494 463 Hepatocyte growth factor alpha chain 495-728 234
Hepatocyte growth factor beta chain
[0084] As used herein, the term "Interferon gamma" refers to one or
more polypeptides present in a biological sample that are derived
from the Interferon gamma precursor (Swiss-Prot P01579 (SEQ ID NO:
4)).
TABLE-US-00008 10 20 30 40 MKYTSYILAF QLCIVLGSLG CYCQDPYVKE
AENLKKYFNA 50 60 70 80 GHSDVADNGT LFLGILKNWK EESDRKIMQS QIVSFYFKLF
90 100 110 120 KNFKDDQSIQ KSVETIKEDM NVKFFNSNKK KRDDFEKLTN 130 140
150 160 YSVTDLNVQR KAIHELIQVM AELSPAAKTG KRKRSQMLFR GRRASQ
[0085] The following domains have been identified in Interferon
gamma:
TABLE-US-00009 Residues Length Domain ID 1-23 23 Signal peptide
24-161 138 Interferon gamma 162-166 5 Propeptide
[0086] As used herein, the term "Interleukin-12 subunit beta" (also
known as "Interleukin-12 p40") refers to one or more polypeptides
present in a biological sample that are derived from the
Interleukin-12 subunit beta precursor (Swiss-Prot P29460 (SEQ ID
NO: 5)).
TABLE-US-00010 10 20 30 40 MCHQQLVISW FSLVFLASPL VAIWELKKDV
YVVELDWYPD 50 60 70 80 APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK
90 100 110 120 EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ 130 140
150 160 KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSR 170 180 190 200
GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP 210 220 230 240
AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN 250 260 270 280
LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK 290 300 310 320
SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS EWASVPCS
[0087] The following domains have been identified in Interleukin-12
subunit beta:
TABLE-US-00011 Residues Length Domain ID 1-22 22 Signal peptide
23-328 306 Interleukin-12 subunit beta
[0088] As used herein, the term "Interleukin-16" refers to one or
more polypeptides present in a biological sample that are derived
from the Interleukin-16 precursor (Swiss-Prot Q14005 (SEQ ID NO:
6)).
TABLE-US-00012 10 20 30 40 MDYSFDTTAE DPWVRISDCI KNLFSPIMSE
NHGHMPLQPN 50 60 70 80 ASLNEEEGTQ GHPDGTPPKL DTANGTPKVY KSADSSTVKK
90 100 110 120 GPPVAPKPAW FRQSLKGLRN RASDPRGLPD PALSTQPAPA 130 140
150 160 SREHLGSHIR ASSSSSSIRQ RISSFETFGS SQLPDKGAQR 170 180 190 200
LSLQPSSGEA AKPLGKHEEG RFSGLLGRGA APTLVPQQPE 210 220 230 240
QVLSSGSPAA SEARDPGVSE SPPPGRQPNQ KTLPPGPDPL 250 260 270 280
LRLLSTQAEE SQGPVLKMPS QRARSFPLTR SQSCETKLLD 290 300 310 320
EKTSKLYSIS SQVSSAVMKS LLCLPSSISC AQTPCIPKEG 330 340 350 360
ASPTSSSNED SAANGSAETS ALDTGFSLNL SELREYTEGL 370 380 390 400
TEAKEDDDGD HSSLQSGQSV ISLLSSEELK KLIEEVKVLD 410 420 430 440
EATLKQLDGI HVTILHKEEG AGLGFSLAGG ADLENKVITV 450 460 470 480
HRVFPNGLAS QEGTIQKGNE VLSINGKSLK GTTHHDALAI 490 500 510 520
LRQAREPRQA VIVTRKLTPE AMPDLNSSTD SAASASAASD 530 540 550 560
VSVESTAEAT VCTVTLEKMS AGLGFSLEGG KGSLHGDKPL 570 580 590 600
TINRIFKGAA SEQSETVQPG DEILQLGGTA MQGLTRFEAW 610 620 630 NIIKALPDGP
VTIVIRRKSL QSKETTAAGD S 1-1332 1332 Pro-interleukin-16 1212-1332
121 Interleukin-16
[0089] As used herein, the term "Interleukin-2" refers to one or
more polypeptides present in a biological sample that are derived
from the Interleukin-2 precursor (Swiss-Prot P60568 (SEQ ID NO:
7)).
TABLE-US-00013 10 20 30 40 MYRMQLLSCI ALSLALVTNS APTSSSTKKT
QLQLEHLLLD 50 60 70 80 LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE
90 100 110 120 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE 130 140
150 TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT
[0090] The following domains have been identified in
Interleukin-2:
TABLE-US-00014 Residues Length Domain ID 1-20 20 Signal peptide
21-153 133 Interleukin-2
[0091] As used herein, the term "72 kDa type IV collagenase" (also
known as "Matrix metalloproteinase-2") refers to one or more
polypeptides present in a biological sample that are derived from
the 72 kDa type IV collagenase precursor (Swiss-Prot P08253 (SEQ ID
NO: 8)).
TABLE-US-00015 10 20 30 40 MEALMARGAL TGPLRALCLL GCLLSHAAAA
PSPIIKFPGD 50 60 70 80 VAPKTDKELA VQYLNTFYGC PKESCNLFVL KDTLKKMQKF
90 100 110 120 FGLPQTGDLD QNTIETMRKP RCGNPDVANY NFFPRKPKWD 130 140
150 160 KNQITYRIIG YTPDLDPETV DDAFARAFQV WSDVTPLRFS 170 180 190 200
RIHDGEADIM INFGRWEHGD GYPFDGKDGL LAHAFAPGTG 210 220 230 240
VGGDSHFDDD ELWTLGEGQV VRVKYGNADG EYCKFPFLFN 250 260 270 280
GKEYNSCTDT GRSDGFLWCS TTYNFEKDGK YGFCPHEALF 290 300 310 320
TMGGNAEGQP CKFPFRFQGT SYDSCTTEGR TDGYRWCGTT 330 340 350 360
EDYDRDKKYG FCPETAMSTV GGNSEGAPCV FPFTFLGNKY 370 380 390 400
ESCTSAGRSD GKMWCATTAN YDDDRKWGFC PDQGYSLFLV 410 420 430 440
AAHEFGHAMG LEHSQDPGAL MAPIYTYTKN FRLSQDDIKG 450 460 470 480
IQELYGASPD IDLGTGPTPT LGPVTPEICK QDIVFDGIAQ 490 500 510 520
IRGEIFFFKD RFIWRIVTPR DKPMGPLLVA TFWPELPEKI 530 540 550 560
DAVYEAPQEE KAVFFAGNEY WIYSASTLER GYPKPLTSLG 570 580 590 600
LPPDVQRVDA AFNWSKNKKT YIFAGDKFWR YNEVKKKMDP 610 620 630 640
GFPKLIADAW NAIPDNLDAV VDLQGGGHSY FFKGAYYLKL 650 660 ENQSLKSVKF
GSIKSDWLGC
[0092] The following domains have been identified in 72 kDa type IV
collagenase:
TABLE-US-00016 Residues Length Domain ID 1-29 29 Signal peptide
30-109 90 Activation peptide 110-660 551 72 kDa type IV
collagenase
[0093] As used herein, the term "Matrix metalloproteinase-9" refers
to one or more polypeptides present in a biological sample that are
derived from the Matrix metalloproteinase-9 precursor (Swiss-Prot
P14780 (SEQ ID NO: 9)).
TABLE-US-00017 10 20 30 40 MSLWQPLVLV LLVLGCCFAA PRQRQSTLVL
FPGDLRTNLT 50 60 70 80 DRQLAEEYLY RYGYTRVAEM RGESKSLGPA LLLLQKQLSL
90 100 110 120 PETGELDSAT LKAMRTPRCG VPDLGRFQTF EGDLKWHHHN 130 140
150 160 ITYWIQNYSE DLPRAVIDDA FARAFALWSA VTPLTFTRVY 170 180 190 200
SRDADIVIQF GVAEHGDGYP FDGKDGLLAH AFPPGPGIQG 210 220 230 240
DAHFDDDELW SLGKGVVVPT RFGNADGAAC HFPFIFEGRS 250 260 270 280
YSACTTDGRS DGLPWCSTTA NYDTDDRFGF CPSERLYTQD 290 300 310 320
GNADGKPCQF PFIFQGQSYS ACTTDGRSDG YRWCATTANY 330 340 350 360
DRDKLFGFCP TRADSTVMGG NSAGELCVFP FTFLGKEYST 370 380 390 400
CTSEGRGDGR LWCATTSNFD SDKKWGFCPD QGYSLFLVAA 410 420 430 440
HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR 450 460 470 480
HLYGPRPEPE PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER 490 500 510 520
PTAGPTGPPS AGPTGPPTAG PSTATTVPLS PVDDACNVNI 530 540 550 560
FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP QGPFLIADKW 570 580 590 600
PALPRKLDSV FEEPLSKKLF FFSGRQVWVY TGASVLGPRR 610 620 630 640
LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ 650 660 670 680
MVDPRSASEV DRMFPGVPLD THDVFQYREK AYFCQDRFYW 690 700 RVSSRSELNQ
VDQVGYVTYD ILQCPED
[0094] The following domains have been identified in Matrix
metalloproteinase-9:
TABLE-US-00018 Residues Length Domain ID 1-19 19 Signal peptide
20-93 74 Activation peptide 107-707 601 Matrix metalloproteinase-9
82 kDa form
[0095] In addition, a 67 kDa form of Matrix metalloproteinase-9 has
been identified.
[0096] As used herein, the term "Midkine" refers to one or more
polypeptides present in a biological sample that are derived from
the Midkine precursor (Swiss-Prot P21741 (SEQ ID NO: 10)).
TABLE-US-00019 10 20 30 40 MQHRGFLLLT LLALLALTSA VAKKKDKVKK
GGPGSECAEW 50 60 70 80 AWGPCTPSSK DCGVGFREGT CGAQTQRIRC RVPCNWKKEF
90 100 110 120 GADCKYKFEN WGACDGGTGT KVRQGTLKKA RYNAQCQETI 130 140
RVTKPCTPKT KAKAKAKKGK GKD
[0097] The following domains have been identified in Midkine:
TABLE-US-00020 Residues Length Domain ID 1-20 20 Signal peptide
21-143 123 Midkine
[0098] As used herein, the term "Serum amyloid P-component" refers
to one or more polypeptides present in a biological sample that are
derived from the Serum amyloid P-component precursor (Swiss-Prot
P02743 (SEQ ID NO: 11)).
TABLE-US-00021 10 20 30 40 MNKPLLWISV LTSLLEAFAH TDLSGKVFVF
PRESVTDHVN 50 60 70 80 LITPLEKPLQ NFTLCFRAYS DLSRAYSLFS YNTQGRDNEL
90 100 110 120 LVYKERVGEY SLYIGRHKVT SKVIEKFPAP VHICVSWESS 130 140
150 160 SGIAEFWING TPLVKKGLRQ GYFVEAQPKI VLGQEQDSYG 170 180 190 200
GKFDRSQSFV GEIGDLYMWD SVLPPENILS AYQGTPLPAN 210 220 ILDWQALNYE
IRGYVIIKPL VWV
[0099] The following domains have been identified in Serum amyloid
P-component:
TABLE-US-00022 Residues Length Domain ID 1-19 19 Signal peptide
20-223 204 Serum amyloid P-component 20-222 203 Serum amyloid
P-component (1-203)
[0100] As used herein, the term "relating a signal to the presence
or amount" of an analyte reflects this understanding. Assay signals
are typically related to the presence or amount of an analyte
through the use of a standard curve calculated using known
concentrations of the analyte of interest. As the term is used
herein, an assay is "configured to detect" an analyte if an assay
can generate a detectable signal indicative of the presence or
amount of a physiologically relevant concentration of the analyte.
Because an antibody epitope is on the order of 8 amino acids, an
immunoassay configured to detect a marker of interest will also
detect polypeptides related to the marker sequence, so long as
those polypeptides contain the epitope(s) necessary to bind to the
antibody or antibodies used in the assay. The term "related marker"
as used herein with regard to a biomarker such as one of the kidney
injury markers described herein refers to one or more fragments,
variants, etc., of a particular marker or its biosynthetic parent
that may be detected as a surrogate for the marker itself or as
independent biomarkers. The term also refers to one or more
polypeptides present in a biological sample that are derived from
the biomarker precursor complexed to additional species, such as
binding proteins, receptors, heparin, lipids, sugars, etc.
[0101] The term "positive going" marker as that term is used herein
refer to a marker that is determined to be elevated in subjects
suffering from a disease or condition, relative to subjects not
suffering from that disease or condition. The term "negative going"
marker as that term is used herein refer to a marker that is
determined to be reduced in subjects suffering from a disease or
condition, relative to subjects not suffering from that disease or
condition.
[0102] The term "subject" as used herein refers to a human or
non-human organism. Thus, the methods and compositions described
herein are applicable to both human and veterinary disease.
Further, while a subject is preferably a living organism, the
invention described herein may be used in post-mortem analysis as
well. Preferred subjects are humans, and most preferably
"patients," which as used herein refers to living humans that are
receiving medical care for a disease or condition. This includes
persons with no defined illness who are being investigated for
signs of pathology.
[0103] Preferably, an analyte is measured in a sample. Such a
sample may be obtained from a subject, or may be obtained from
biological materials intended to be provided to the subject. For
example, a sample may be obtained from a kidney being evaluated for
possible transplantation into a subject, and an analyte measurement
used to evaluate the kidney for preexisting damage. Preferred
samples are body fluid samples.
[0104] The term "body fluid sample" as used herein refers to a
sample of bodily fluid obtained for the purpose of diagnosis,
prognosis, classification or evaluation of a subject of interest,
such as a patient or transplant donor. In certain embodiments, such
a sample may be obtained for the purpose of determining the outcome
of an ongoing condition or the effect of a treatment regimen on a
condition. Preferred body fluid samples include blood, serum,
plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural
effusions. In addition, one of skill in the art would realize that
certain body fluid samples would be more readily analyzed following
a fractionation or purification procedure, for example, separation
of whole blood into serum or plasma components.
[0105] The term "diagnosis" as used herein refers to methods by
which the skilled artisan can estimate and/or determine the
probability ("a likelihood") of whether or not a patient is
suffering from a given disease or condition. In the case of the
present invention, "diagnosis" includes using the results of an
assay, most preferably an immunoassay, for a kidney injury marker
of the present invention, optionally together with other clinical
characteristics, to arrive at a diagnosis (that is, the occurrence
or nonoccurrence) of an acute renal injury or ARF for the subject
from which a sample was obtained and assayed. That such a diagnosis
is "determined" is not meant to imply that the diagnosis is 100%
accurate. Many biomarkers are indicative of multiple conditions.
The skilled clinician does not use biomarker results in an
informational vacuum, but rather test results are used together
with other clinical indicia to arrive at a diagnosis. Thus, a
measured biomarker level on one side of a predetermined diagnostic
threshold indicates a greater likelihood of the occurrence of
disease in the subject relative to a measured level on the other
side of the predetermined diagnostic threshold.
[0106] Similarly, a prognostic risk signals a probability ("a
likelihood") that a given course or outcome will occur. A level or
a change in level of a prognostic indicator, which in turn is
associated with an increased probability of morbidity (e.g.,
worsening renal function, future ARF, or death) is referred to as
being "indicative of an increased likelihood" of an adverse outcome
in a patient.
[0107] Marker Assays
[0108] In general, immunoassays involve contacting a sample
containing or suspected of containing a biomarker of interest with
at least one antibody that specifically binds to the biomarker. A
signal is then generated indicative of the presence or amount of
complexes formed by the binding of polypeptides in the sample to
the antibody. The signal is then related to the presence or amount
of the biomarker in the sample. Numerous methods and devices are
well known to the skilled artisan for the detection and analysis of
biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855;
6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527;
5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The
Immunoassay Handbook, David Wild, ed. Stockton Press, New York,
1994, each of which is hereby incorporated by reference in its
entirety, including all tables, figures and claims.
[0109] The assay devices and methods known in the art can utilize
labeled molecules in various sandwich, competitive, or
non-competitive assay formats, to generate a signal that is related
to the presence or amount of the biomarker of interest. Suitable
assay formats also include chromatographic, mass spectrographic,
and protein "blotting" methods. Additionally, certain methods and
devices, such as bio sensors and optical immunoassays, may be
employed to determine the presence or amount of analytes without
the need for a labeled molecule. See, e.g., U.S. Pat. Nos.
5,631,171; and 5,955,377, each of which is hereby incorporated by
reference in its entirety, including all tables, figures and
claims. One skilled in the art also recognizes that robotic
instrumentation including but not limited to Beckman ACCESS.RTM.,
Abbott AXSYM.RTM., Roche ELECSYS.RTM., Dade Behring STRATUS.RTM.
systems are among the immunoassay analyzers that are capable of
performing immunoassays. But any suitable immunoassay may be
utilized, for example, enzyme-linked immunoassays (ELISA),
radioimmunoassays (RIAs), competitive binding assays, and the
like.
[0110] Antibodies or other polypeptides may be immobilized onto a
variety of solid supports for use in assays. Solid phases that may
be used to immobilize specific binding members include include
those developed and/or used as solid phases in solid phase binding
assays. Examples of suitable solid phases include membrane filters,
cellulose-based papers, beads (including polymeric, latex and
paramagnetic particles), glass, silicon wafers, microparticles,
nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and
multiple-well plates. An assay strip could be prepared by coating
the antibody or a plurality of antibodies in an array on solid
support. This strip could then be dipped into the test sample and
then processed quickly through washes and detection steps to
generate a measurable signal, such as a colored spot. Antibodies or
other polypeptides may be bound to specific zones of assay devices
either by conjugating directly to an assay device surface, or by
indirect binding. In an example of the later case, antibodies or
other polypeptides may be immobilized on particles or other solid
supports, and that solid support immobilized to the device
surface.
[0111] Biological assays require methods for detection, and one of
the most common methods for quantitation of results is to conjugate
a detectable label to a protein or nucleic acid that has affinity
for one of the components in the biological system being studied.
Detectable labels may include molecules that are themselves
detectable (e.g., fluorescent moieties, electrochemical labels,
metal chelates, etc.) as well as molecules that may be indirectly
detected by production of a detectable reaction product (e.g.,
enzymes such as horseradish peroxidase, alkaline phosphatase, etc.)
or by a specific binding molecule which itself may be detectable
(e.g., biotin, digoxigenin, maltose, oligohistidine,
2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
[0112] Preparation of solid phases and detectable label conjugates
often comprise the use of chemical cross-linkers. Cross-linking
reagents contain at least two reactive groups, and are divided
generally into homofunctional cross-linkers (containing identical
reactive groups) and heterofunctional cross-linkers (containing
non-identical reactive groups). Homobifunctional cross-linkers that
couple through amines, sulfhydryls or react non-specifically are
available from many commercial sources. Maleimides, alkyl and aryl
halides, alpha-haloacyls and pyridyl disulfides are thiol reactive
groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls
react with sulfhydryls to form thiol ether bonds, while pyridyl
disulfides react with sulfhydryls to produce mixed disulfides. The
pyridyl disulfide product is cleavable. Imidoesters are also very
useful for protein-protein cross-links. A variety of
heterobifunctional cross-linkers, each combining different
attributes for successful conjugation, are commercially
available.
[0113] In certain aspects, the present invention provides kits for
the analysis of the described kidney injury markers. The kit
comprises reagents for the analysis of at least one test sample
which comprise at least one antibody that a kidney injury marker.
The kit can also include devices and instructions for performing
one or more of the diagnostic and/or prognostic correlations
described herein. Preferred kits will comprise an antibody pair for
performing a sandwich assay, or a labeled species for performing a
competitive assay, for the analyte. Preferably, an antibody pair
comprises a first antibody conjugated to a solid phase and a second
antibody conjugated to a detectable label, wherein each of the
first and second antibodies that bind a kidney injury marker. Most
preferably each of the antibodies are monoclonal antibodies. The
instructions for use of the kit and performing the correlations can
be in the form of labeling, which refers to any written or recorded
material that is attached to, or otherwise accompanies a kit at any
time during its manufacture, transport, sale or use. For example,
the term labeling encompasses advertising leaflets and brochures,
packaging materials, instructions, audio or video cassettes,
computer discs, as well as writing imprinted directly on kits.
[0114] Antibodies
[0115] The term "antibody" as used herein refers to a peptide or
polypeptide derived from, modeled after or substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, capable of specifically binding an antigen or epitope.
See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed.,
Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods
175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
The term antibody includes antigen-binding portions, i.e., "antigen
binding sites," (e.g., fragments, subsequences, complementarity
determining regions (CDRs)) that retain capacity to bind antigen,
including (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the VH and
CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains
of a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Single chain
antibodies are also included by reference in the term
"antibody."
[0116] Antibodies used in the immunoassays described herein
preferably specifically bind to a kidney injury marker of the
present invention. The term "specifically binds" is not intended to
indicate that an antibody binds exclusively to its intended target
since, as noted above, an antibody binds to any polypeptide
displaying the epitope(s) to which the antibody binds. Rather, an
antibody "specifically binds" if its affinity for its intended
target is about 5-fold greater when compared to its affinity for a
non-target molecule which does not display the appropriate
epitope(s). Preferably the affinity of the antibody will be at
least about 5 fold, preferably 10 fold, more preferably 25-fold,
even more preferably 50-fold, and most preferably 100-fold or more,
greater for a target molecule than its affinity for a non-target
molecule. In preferred embodiments, Preferred antibodies bind with
affinities of at least about 10.sup.7 M.sup.-1, and preferably
between about 10.sup.8 M.sup.-1 to about 10.sup.9 M.sup.-1, about
10.sup.9 M.sup.-1 to about 10.sup.10 M.sup.-1, or about 10.sup.10
M.sup.-1 to about 10.sup.12M.sup.-1 .
[0117] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on
(k.sub.off is the dissociation rate constant, K.sub.on is the
association rate constant and K.sub.d is the equilibrium constant).
Affinity can be determined at equilibrium by measuring the fraction
bound (r) of labeled ligand at various concentrations (c). The data
are graphed using the Scatchard equation: r/c=K(n-r): where r=moles
of bound ligand/mole of receptor at equilibrium; c=free ligand
concentration at equilibrium; K=equilibrium association constant;
and n=number of ligand binding sites per receptor molecule. By
graphical analysis, r/c is plotted on the Y-axis versus r on the
X-axis, thus producing a Scatchard plot. Antibody affinity
measurement by Scatchard analysis is well known in the art. See,
e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and
Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
[0118] The term "epitope" refers to an antigenic determinant
capable of specific binding to an antibody. Epitopes usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents.
[0119] Numerous publications discuss the use of phage display
technology to produce and screen libraries of polypeptides for
binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl.
Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249,
404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner
et al., U.S. Pat. No. 5,571,698. A basic concept of phage display
methods is the establishment of a physical association between DNA
encoding a polypeptide to be screened and the polypeptide. This
physical association is provided by the phage particle, which
displays a polypeptide as part of a capsid enclosing the phage
genome which encodes the polypeptide. The establishment of a
physical association between polypeptides and their genetic
material allows simultaneous mass screening of very large numbers
of phage bearing different polypeptides. Phage displaying a
polypeptide with affinity to a target bind to the target and these
phage are enriched by affinity screening to the target. The
identity of polypeptides displayed from these phage can be
determined from their respective genomes. Using these methods a
polypeptide identified as having a binding affinity for a desired
target can then be synthesized in bulk by conventional means. See,
e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its
entirety, including all tables, figures, and claims.
[0120] The antibodies that are generated by these methods may then
be selected by first screening for affinity and specificity with
the purified polypeptide of interest and, if required, comparing
the results to the affinity and specificity of the antibodies with
polypeptides that are desired to be excluded from binding. The
screening procedure can involve immobilization of the purified
polypeptides in separate wells of microtiter plates. The solution
containing a potential antibody or groups of antibodies is then
placed into the respective microtiter wells and incubated for about
30 min to 2 h. The microtiter wells are then washed and a labeled
secondary antibody (for example, an anti-mouse antibody conjugated
to alkaline phosphatase if the raised antibodies are mouse
antibodies) is added to the wells and incubated for about 30 min
and then washed. Substrate is added to the wells and a color
reaction will appear where antibody to the immobilized
polypeptide(s) are present.
[0121] The antibodies so identified may then be further analyzed
for affinity and specificity in the assay design selected. In the
development of immunoassays for a target protein, the purified
target protein acts as a standard with which to judge the
sensitivity and specificity of the immunoassay using the antibodies
that have been selected. Because the binding affinity of various
antibodies may differ; certain antibody pairs (e.g., in sandwich
assays) may interfere with one another sterically, etc., assay
performance of an antibody may be a more important measure than
absolute affinity and specificity of an antibody.
Assay Correlations
[0122] The term "correlating" as used herein in reference to the
use of biomarkers refers to comparing the presence or amount of the
biomarker(s) in a patient to its presence or amount in persons
known to suffer from, or known to be at risk of, a given condition;
or in persons known to be free of a given condition. Often, this
takes the form of comparing an assay result in the form of a
biomarker concentration to a predetermined threshold selected to be
indicative of the occurrence or nonoccurrence of a disease or the
likelihood of some future outcome.
[0123] Selecting a diagnostic threshold involves, among other
things, consideration of the probability of disease, distribution
of true and false diagnoses at different test thresholds, and
estimates of the consequences of treatment (or a failure to treat)
based on the diagnosis. For example, when considering administering
a specific therapy which is highly efficacious and has a low level
of risk, few tests are needed because clinicians can accept
substantial diagnostic uncertainty. On the other hand, in
situations where treatment options are less effective and more
risky, clinicians often need a higher degree of diagnostic
certainty. Thus, cost/benefit analysis is involved in selecting a
diagnostic threshold.
[0124] Suitable thresholds may be determined in a variety of ways.
For example, one recommended diagnostic threshold for the diagnosis
of acute myocardial infarction using cardiac troponin is the
975.sup.th percentile of the concentration seen in a normal
population. Another method may be to look at serial samples from
the same patient, where a prior "baseline" result is used to
monitor for temporal changes in a biomarker level.
[0125] Population studies may also be used to select a decision
threshold. Reciever Operating Characteristic ("ROC") arose from the
field of signal dectection therory developed during World War II
for the analysis of radar images, and ROC analysis is often used to
select a threshold able to best distinguish a "diseased"
subpopulation from a "nondiseased" subpopulation. A false positive
in this case occurs when the person tests positive, but actually
does not have the disease. A false negative, on the other hand,
occurs when the person tests negative, suggesting they are healthy,
when they actually do have the disease. To draw a ROC curve, the
true positive rate (TPR) and false positive rate (FPR) are
determined as the decision threshold is varied continuously. Since
TPR is equivalent with sensitivity and FPR is equal to
1-specificity, the ROC graph is sometimes called the sensitivity vs
(1-specificity) plot. A perfect test will have an area under the
ROC curve of 1.0; a random test will have an area of 0.5. A
threshold is selected to provide an acceptable level of specificity
and sensitivity.
[0126] In this context, "diseased" is meant to refer to a
population having one characteristic (the presence of a disease or
condition or the occurrence of some outcome) and "nondiseased" is
meant to refer to a population lacking the characteristic. While a
single decision threshold is the simplest application of such a
method, multiple decision thresholds may be used. For example,
below a first threshold, the absence of disease may be assigned
with relatively high confidence, and above a second threshold the
presence of disease may also be assigned with relatively high
confidence. Between the two thresholds may be considered
indeterminate. This is meant to be exemplary in nature only.
[0127] In addition to threshold comparisons, other methods for
correlating assay results to a patient classification (occurrence
or nonoccurrence of disease, likelihood of an outcome, etc.)
include decision trees, rule sets, Bayesian methods, and neural
network methods. These methods can produce probability values
representing the degree to which a subject belongs to one
classification out of a plurality of classifications.
[0128] Measures of test accuracy may be obtained as described in
Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to
determine the effectiveness of a given biomarker. These measures
include sensitivity and specificity, predictive values, likelihood
ratios, diagnostic odds ratios, and ROC curve areas. The area under
the curve ("AUC") of a ROC plot is equal to the probability that a
classifier will rank a randomly chosen positive instance higher
than a randomly chosen negative one. The area under the ROC curve
may be thought of as equivalent to the Mann-Whitney U test, which
tests for the median difference between scores obtained in the two
groups considered if the groups are of continuous data, or to the
Wilcoxon test of ranks.
[0129] As discussed above, suitable tests may exhibit one or more
of the following results on these various measures: a specificity
of greater than 0.5, preferably at least 0.6, more preferably at
least 0.7, still more preferably at least 0.8, even more preferably
at least 0.9 and most preferably at least 0.95, with a
corresponding sensitivity greater than 0.2, preferably greater than
0.3, more preferably greater than 0.4, still more preferably at
least 0.5, even more preferably 0.6, yet more preferably greater
than 0.7, still more preferably greater than 0.8, more preferably
greater than 0.9, and most preferably greater than 0.95; a
sensitivity of greater than 0.5, preferably at least 0.6, more
preferably at least 0.7, still more preferably at least 0.8, even
more preferably at least 0.9 and most preferably at least 0.95,
with a corresponding specificity greater than 0.2, preferably
greater than 0.3, more preferably greater than 0.4, still more
preferably at least 0.5, even more preferably 0.6, yet more
preferably greater than 0.7, still more preferably greater than
0.8, more preferably greater than 0.9, and most preferably greater
than 0.95; at least 75% sensitivity, combined with at least 75%
specificity; a ROC curve area of greater than 0.5, preferably at
least 0.6, more preferably 0.7, still more preferably at least 0.8,
even more preferably at least 0.9, and most preferably at least
0.95; an odds ratio different from 1, preferably at least about 2
or more or about 0.5 or less, more preferably at least about 3 or
more or about 0.33 or less, still more preferably at least about 4
or more or about 0.25 or less, even more preferably at least about
5 or more or about 0.2 or less, and most preferably at least about
10 or more or about 0.1 or less; a positive likelihood ratio
(calculated as sensitivity/(1-specificity)) of greater than 1, at
least 2, more preferably at least 3, still more preferably at least
5, and most preferably at least 10; and or a negative likelihood
ratio (calculated as (1-sensitivity)/specificity) of less than 1,
less than or equal to 0.5, more preferably less than or equal to
0.3, and most preferably less than or equal to 0.1
[0130] Additional clinical indicia may be combined with the kidney
injury marker assay result(s) of the present invention. These
include other biomarkers related to renal status. Examples include
the following, which recite the common biomarker name, followed by
the Swiss-Prot entry number for that biomarker or its parent: Actin
(P68133); Adenosine deaminase binding protein (DPP4, P27487);
Alpha-1-acid glycoprotein 1 (P02763); Alpha-1-microglobulin
(P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797);
Annexin A2 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin
(P61679); Beta-galactosidase (P16278); BMP-7 (P18075); Brain
natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860);
Calcium-binding protein Beta (S100-beta, P04271); Carbonic
anhydrase (Q16790); Casein Kinase 2 (P68400); Cathepsin B (P07858);
Ceruloplasmin (P00450); Clusterin (P10909); Complement C3 (P01024);
Cysteine-rich protein (CYR61, O00622); Cytochrome C (P99999);
Epidermal growth factor (EGF, P01133); Endothelin-1 (P05305);
Exosomal Fetuin-A (P02765); Fatty acid-binding protein, heart
(FABP3, P05413); Fatty acid-binding protein, liver (P07148);
Ferritin (light chain, P02793; heavy chain P02794);
Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341);
Growth Hormone (P01241); Hepatocyte growth factor (P14210);
Insulin-like growth factor I (P01343); Immunoglobulin G;
Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma
(P01308); Lysozyme (P61626); Interleukin-lalpha (P01583);
Interleukin-2 (P60568); Interleukin-4 (P60568); Interleukin-9
(P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225);
Interleukin-16 (Q14005); Ll cell adhesion molecule (P32004);
Lactate dehydrogenase (P00338); Leucine Aminopeptidase (P28838);
Meprin A-alpha subunit (Q16819); Meprin A-beta subunit (Q16820);
Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2 (P08253); MMP-9
(P14780); Netrin-1 (O95631); Neutral endopeptidase (P08473);
Osteopontin (P10451); Renal papillary antigen 1 (RPA1); Renal
papillary antigen 2 (RPA2); Retinol binding protein (P09455);
Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum
Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform
(NHE3, P48764); Spermidine/spermine N1-acetyltransferase (P21673);
TGF-Beta1 (P01137); Transferrin (P02787); Trefoil factor 3 (TFF3,
Q07654); Toll-Like protein 4 (O00206); Total protein;
Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin
(Tamm-Horsfall protein, P07911).
[0131] For purposes of risk stratification, Adiponectin (Q15848);
Alkaline phosphatase (P05186); Aminopeptidase N (P15144);
CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of FIFO
ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa
(alpha-glutathione-S-transferase, P08263); GSTpi
(Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1
(P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane
protein 1 (Itm1, P46977); Interleukin-6 (P05231); Interleukin-8
(P10145); Interleukin-18 (Q14116); IP-10 (10 kDa
interferon-gamma-induced protein, P02778); IRPR (IFRD1, O00458);
Isovaleryl-CoA dehydrogenase (IVD, P26440); I-TAC/CXCL11 (O14625);
Keratin 19 (P08727); Kim-1 (Hepatitis A virus cellular receptor 1,
O43656); L-arginine:glycine amidinotransferase (P50440); Leptin
(P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500); MIG
(Gamma-interferon-induced monokine Q07325); MIP-la (P10147); MIP-3a
(P78556); MIP-1beta (P13236); MIP-1d (Q16663); NAG
(N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter
(OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356);
Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98)
(P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab
GDI-beta (P50395); Renal kallikrein (Q86U61); RT1.B-1 (alpha) chain
of the integral membrane protein (Q5Y7A8); Soluble tumor necrosis
factor receptor superfamily member 1A (sTNFR-I, P19438); Soluble
tumor necrosis factor receptor superfamily member 1B (sTNFR-II,
P20333); Tissue inhibitor of metalloproteinases 3 (TIMP-3, P35625);
uPAR (Q03405) may be combined with the kidney injury marker assay
result(s) of the present invention.
[0132] Other clinical indicia which may be combined with the kidney
injury marker assay result(s) of the present invention includes
demographic information (e.g., weight, sex, age, race), medical
history (e.g., family history, type of surgery, pre-existing
disease such as aneurism, congestive heart failure, preeclampsia,
eclampsia, diabetes mellitus, hypertension, coronary artery
disease, proteinuria, renal insufficiency, or sepsis, type of toxin
exposure such as NSAIDs, cyclosporines, tacrolimus,
aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin,
ifosfamide, heavy metals, methotrexate, radiopaque contrast agents,
or streptozotocin), clinical variables (e.g., blood pressure,
temperature, respiration rate), risk scores (APACHE score, PREDICT
score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a
urine total protein measurement, a glomerular filtration rate, an
estimated glomerular filtration rate, a urine production rate, a
serum or plasma creatinine concentration, a renal papillary antigen
1 (RPA1) measurement; a renal papillary antigen 2 (RPA2)
measurement; a urine creatinine concentration, a fractional
excretion of sodium, a urine sodium concentration, a urine
creatinine to serum or plasma creatinine ratio, a urine specific
gravity, a urine osmolality, a urine urea nitrogen to plasma urea
nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal
failure index calculated as urine sodium/(urine creatinine/plasma
creatinine). Other measures of renal function which may be combined
with the kidney injury marker assay result(s) are described
hereinafter and in Harrison's Principles of Internal Medicine,
17.sup.th Ed., McGraw Hill, New York, pages 1741-1830, and Current
Medical Diagnosis & Treatment 2008, 47.sup.th Ed, McGraw Hill,
New York, pages 785-815, each of which are hereby incorporated by
reference in their entirety.
[0133] Combining assay results/clinical indicia in this manner can
comprise the use of multivariate logistical regression, loglinear
modeling, neural network analysis, n-of-m analysis, decision tree
analysis, etc. This list is not meant to be limiting.
[0134] Diagnosis of Acute Renal Failure
[0135] As noted above, the terms "acute renal (or kidney) injury"
and "acute renal (or kidney) failure" as used herein are defined in
part in terms of changes in serum creatinine from a baseline value.
Most definitions of ARF have common elements, including the use of
serum creatinine and, often, urine output. Patients may present
with renal dysfunction without an available baseline measure of
renal function for use in this comparison. In such an event, one
may estimate a baseline serum creatinine value by assuming the
patient initially had a normal GFR. Glomerular filtration rate
(GFR) is the volume of fluid filtered from the renal (kidney)
glomerular capillaries into the Bowman's capsule per unit time.
Glomerular filtration rate (GFR) can be calculated by measuring any
chemical that has a steady level in the blood, and is freely
filtered but neither reabsorbed nor secreted by the kidneys. GFR is
typically expressed in units of ml/min:
GFR = Urine Concentration .times. Urine Flow Plasma Concentration
##EQU00001##
[0136] By normalizing the GFR to the body surface area, a GFR of
approximately 75-100 ml/min per 1.73 m.sup.2 can be assumed. The
rate therefore measured is the quantity of the substance in the
urine that originated from a calculable volume of blood.
[0137] There are several different techniques used to calculate or
estimate the glomerular filtration rate (GFR or eGFR). In clinical
practice, however, creatinine clearance is used to measure GFR.
Creatinine is produced naturally by the body (creatinine is a
metabolite of creatine, which is found in muscle). It is freely
filtered by the glomerulus, but also actively secreted by the renal
tubules in very small amounts such that creatinine clearance
overestimates actual GFR by 10-20%. This margin of error is
acceptable considering the ease with which creatinine clearance is
measured.
[0138] Creatinine clearance (CCr) can be calculated if values for
creatinine's urine concentration (U.sub.Cr), urine flow rate (V),
and creatinine's plasma concentration (P.sub.Cr) are known. Since
the product of urine concentration and urine flow rate yields
creatinine's excretion rate, creatinine clearance is also said to
be its excretion rate (U.sub.Cr.times.V) divided by its plasma
concentration. This is commonly represented mathematically as:
C Cr = U Cr .times. V P Cr ##EQU00002##
[0139] Commonly a 24 hour urine collection is undertaken, from
empty-bladder one morning to the contents of the bladder the
following morning, with a comparative blood test then taken:
C Cr = U Cr .times. 24 - hour volume P Cr .times. 24 .times. 60
mins ##EQU00003##
[0140] To allow comparison of results between people of different
sizes, the CCr is often corrected for the body surface area (BSA)
and expressed compared to the average sized man as ml/min/1.73 m2.
While most adults have a BSA that approaches 1.7 (1.6-1.9),
extremely obese or slim patients should have their CCr corrected
for their actual BSA:
C Cr - corrected = C Cr .times. 1.73 BSA ##EQU00004##
[0141] The accuracy of a creatinine clearance measurement (even
when collection is complete) is limited because as glomerular
filtration rate (GFR) falls creatinine secretion is increased, and
thus the rise in serum creatinine is less. Thus, creatinine
excretion is much greater than the filtered load, resulting in a
potentially large overestimation of the GFR (as much as a twofold
difference). However, for clinical purposes it is important to
determine whether renal function is stable or getting worse or
better. This is often determined by monitoring serum creatinine
alone. Like creatinine clearance, the serum creatinine will not be
an accurate reflection of GFR in the non-steady-state condition of
ARF. Nonetheless, the degree to which serum creatinine changes from
baseline will reflect the change in GFR. Serum creatinine is
readily and easily measured and it is specific for renal
function.
[0142] For purposes of determining urine output on a Urine output
on a mL/kg/hr basis, hourly urine collection and measurement is
adequate. In the case where, for example, only a cumulative 24-h
output was available and no patient weights are provided, minor
modifications of the RIFLE urine output criteria have been
described. For example, Bagshaw et al., Nephrol. Dial. Transplant.
23: 1203-1210, 2008, assumes an average patient weight of 70 kg,
and patients are assigned a RIFLE classification based on the
following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h
(Failure).
[0143] Selecting a Treatment Regimen
[0144] Once a diagnosis is obtained, the clinician can readily
select a treatment regimen that is compatible with the diagnosis,
such as initiating renal replacement therapy, withdrawing delivery
of compounds that are known to be damaging to the kidney, kidney
transplantation, delaying or avoiding procedures that are known to
be damaging to the kidney, modifying diuretic administration,
initiating goal directed therapy, etc. The skilled artisan is aware
of appropriate treatments for numerous diseases discussed in
relation to the methods of diagnosis described herein. See, e.g.,
Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research
Laboratories, Whitehouse Station, N.J., 1999. In addition, since
the methods and compositions described herein provide prognostic
information, the markers of the present invention may be used to
monitor a course of treatment. For example, improved or worsened
prognostic state may indicate that a particular treatment is or is
not efficacious.
[0145] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The examples provided herein are representative of preferred
embodiments, are exemplary, and are not intended as limitations on
the scope of the invention.
EXAMPLE 1
Contrast-Induced Nephropathy Sample Collection
[0146] The objective of this sample collection study is to collect
samples of plasma and urine and clinical data from patients before
and after receiving intravascular contrast media. Approximately 250
adults undergoing radiographic/angiographic procedures involving
intravascular administration of iodinated contrast media are
enrolled. To be enrolled in the study, each patient must meet all
of the following inclusion criteria and none of the following
exclusion criteria:
Inclusion Criteria
[0147] males and females 18 years of age or older; [0148]
undergoing a radiographic/angiographic procedure (such as a CT scan
or coronary intervention) involving the intravascular
administration of contrast media; [0149] expected to be
hospitalized for at least 48 hours after contrast administration.
[0150] able and willing to provide written informed consent for
study participation and to comply with all study procedures.
Exclusion Criteria
[0150] [0151] renal transplant recipients; [0152] acutely worsening
renal function prior to the contrast procedure; [0153] already
receiving dialysis (either acute or chronic) or in imminent need of
dialysis at enrollment; [0154] expected to undergo a major surgical
procedure (such as involving cardiopulmonary bypass) or an
additional imaging procedure with contrast media with significant
risk for further renal insult within the 48 hrs following contrast
administration; [0155] participation in an interventional clinical
study with an experimental therapy within the previous 30 days;
[0156] known infection with human immunodeficiency virus (HIV) or a
hepatitis virus.
[0157] Immediately prior to the first contrast administration (and
after any pre-procedure hydration), an EDTA anti-coagulated blood
sample (10 mL) and a urine sample (10 mL) are collected from each
patient. Blood and urine samples are then collected at 4 (.+-.0.5),
8 (.+-.1), 24 (.+-.2) 48 (.+-.2), and 72 (.+-.2) hrs following the
last administration of contrast media during the index contrast
procedure. Blood is collected via direct venipuncture or via other
available venous access, such as an existing femoral sheath,
central venous line, peripheral intravenous line or hep-lock. These
study blood samples are processed to plasma at the clinical site,
frozen and shipped to Astute Medical, Inc., San Diego, Calif. The
study urine samples are frozen and shipped to Astute Medical,
Inc.
[0158] Serum creatinine is assessed at the site immediately prior
to the first contrast administration (after any pre-procedure
hydration) and at 4 (.+-.0.5), 8 (.+-.1), 24 (.+-.2) and 48
(.+-.2)), and 72 (.+-.2) hours following the last administration of
contrast (ideally at the same time as the study samples are
obtained). In addition, each patient's status is evaluated through
day 30 with regard to additional serum and urine creatinine
measurements, a need for dialysis, hospitalization status, and
adverse clinical outcomes (including mortality).
[0159] Prior to contrast administration, each patient is assigned a
risk based on the following assessment: systolic blood pressure
<80 mm Hg=5 points; intra-arterial balloon pump=5 points;
congestive heart failure (Class III-IV or history of pulmonary
edema)=5 points; age >75 yrs=4 points; hematocrit level <39%
for men, <35% for women=3 points; diabetes=3 points; contrast
media volume=1 point for each 100 mL; serum creatinine level
>1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m.sup.2=2
points, 20-40 mL/min/1.73 m.sup.2=4 points, <20 mL/min/1.73
m.sup.2=6 points. The risks assigned are as follows: risk for CIN
and dialysis: 5 or less total points=risk of CIN-7.5%, risk of
dialysis-0.04%; 6-10 total points=risk of CIN-14%, risk of
dialysis-0.12%; 11-16 total points=risk of CIN-26.1%, risk of
dialysis-1.09%; >16 total points=risk of CIN=57.3%, risk of
dialysis-12.8%.
EXAMPLE 2
Cardiac Surgery Sample Collection
[0160] The objective of this sample collection study is to collect
samples of plasma and urine and clinical data from patients before
and after undergoing cardiovascular surgery, a procedure known to
be potentially damaging to kidney function. Approximately 900
adults undergoing such surgery are enrolled. To be enrolled in the
study, each patient must meet all of the following inclusion
criteria and none of the following exclusion criteria:
Inclusion Criteria
[0161] males and females 18 years of age or older; [0162]
undergoing cardiovascular surgery; [0163] Toronto/Ottawa Predictive
Risk Index for Renal Replacement risk score of at least 2
(Wijeysundera et al., JAMA 297: 1801-9, 2007); and [0164] able and
willing to provide written informed consent for study participation
and to comply with all study procedures.
Exclusion Criteria
[0164] [0165] known pregnancy; [0166] previous renal
transplantation; [0167] acutely worsening renal function prior to
enrollment (e.g., any category of RIFLE criteria); [0168] already
receiving dialysis (either acute or chronic) or in imminent need of
dialysis at enrollment; [0169] currently enrolled in another
clinical study or expected to be enrolled in another clinical study
within 7 days of cardiac surgery that involves drug infusion or a
therapeutic intervention for AKI; [0170] known infection with human
immunodeficiency virus (HIV) or a hepatitis virus.
[0171] Within 3 hours prior to the first incision (and after any
pre-procedure hydration), an EDTA anti-coagulated blood sample (10
mL), whole blood (3 mL), and a urine sample (35 mL) are collected
from each patient. Blood and urine samples are then collected at 3
(.+-.0.5), 6 (.+-.0.5), 12 (.+-.1), 24 (.+-.2) and 48 (.+-.2) hrs
following the procedure and then daily on days 3 through 7 if the
subject remains in the hospital. Blood is collected via direct
venipuncture or via other available venous access, such as an
existing femoral sheath, central venous line, peripheral
intravenous line or hep-lock. These study blood samples are frozen
and shipped to Astute Medical, Inc., San Diego, Calif. The study
urine samples are frozen and shipped to Astute Medical, Inc.
EXAMPLE 3
Acutely Ill Subject Sample Collection
[0172] The objective of this study is to collect samples from
acutely ill patients. Approximately 900 adults expected to be in
the ICU for at least 48 hours will be enrolled. To be enrolled in
the study, each patient must meet all of the following inclusion
criteria and none of the following exclusion criteria:
Inclusion Criteria
[0173] males and females 18 years of age or older; [0174] Study
population 1: approximately 300 patients that have at least one of:
[0175] shock (SBP <90 mmHg and/or need for vasopressor support
to maintain MAP >60 mmHg and/or documented drop in SBP of at
least 40 mmHg); and [0176] sepsis; [0177] Study population 2:
approximately 300 patients that have at least one of: [0178] IV
antibiotics ordered in computerized physician order entry (CPOE)
within 24 hours of enrollment; [0179] contrast media exposure
within 24 hours of enrollment; [0180] increased Intra-Abdominal
Pressure with acute decompensated heart failure; and [0181] severe
trauma as the primary reason for ICU admission and likely to be
hospitalized in the ICU for 48 hours after enrollment; [0182] Study
population 3: approximately 300 patients [0183] expected to be
hospitalized through acute care setting (ICU or ED) with a known
risk factor for acute renal injury (e.g. sepsis, hypotension/shock
(Shock=systolic BP <90 mmHg and/or the need for vasopressor
support to maintain a MAP >60 mmHg and/or a documented drop in
SBP>40 mmHg), major trauma, hemorrhage, or major surgery);
and/or expected to be hospitalized to the ICU for at least 24 hours
after enrollment.
Exclusion Criteria
[0183] [0184] known pregnancy; [0185] institutionalized
individuals; [0186] previous renal transplantation; [0187] known
acutely worsening renal function prior to enrollment (e.g., any
category of RIFLE criteria); [0188] received dialysis (either acute
or chronic) within 5 days prior to enrollment or in imminent need
of dialysis at the time of enrollment; [0189] known infection with
human immunodeficiency virus (HIV) or a hepatitis virus; [0190]
meets only the SBP <90 mmHg inclusion criterion set forth above,
and does not have shock in the attending physician's or principal
investigator's opinion.
[0191] After providing informed consent, an EDTA anti-coagulated
blood sample (10 mL) and a urine sample (25-30 mL) are collected
from each patient. Blood and urine samples are then collected at 4
(.+-.0.5) and 8 (.+-.1) hours after contrast administration (if
applicable); at 12 (.+-.1), 24 (.+-.2), and 48 (.+-.2) hours after
enrollment, and thereafter daily up to day 7 to day 14 while the
subject is hospitalized. Blood is collected via direct venipuncture
or via other available venous access, such as an existing femoral
sheath, central venous line, peripheral intravenous line or
hep-lock. These study blood samples are processed to plasma at the
clinical site, frozen and shipped to Astute Medical, Inc., San
Diego, Calif. The study urine samples are frozen and shipped to
Astute Medical, Inc.
EXAMPLE 4
Immunoassay Format
[0192] Analytes are is measured using standard sandwich enzyme
immunoassay techniques. A first antibody which binds the analyte is
immobilized in wells of a 96 well polystyrene microplate. Analyte
standards and test samples are pipetted into the appropriate wells
and any analyte present is bound by the immobilized antibody. After
washing away any unbound substances, a horseradish
peroxidase-conjugated second antibody which binds the analyte is
added to the wells, thereby forming sandwich complexes with the
analyte (if present) and the first antibody. Following a wash to
remove any unbound antibody-enzyme reagent, a substrate solution
comprising tetramethylbenzidine and hydrogen peroxide is added to
the wells. Color develops in proportion to the amount of analyte
present in the sample. The color development is stopped and the
intensity of the color is measured at 540 nm or 570 nm. An analyte
concentration is assigned to the test sample by comparison to a
standard curve determined from the analyte standards.
[0193] Concentrations are expressed in the following examples as
follows: Clusterin ng/mL, Heart-type fatty acid binding protein
ng/mL, Hepatocyte growth factor pg/mL, Interferon gamma pg/mL,
Interleukin-12 subunit beta pg/mL, Interleukin-16 pg/mL,
Interleukin-2 pg/mL, 72 kDa type IV collagenase ng/mL, Matrix
metalloproteinase-9 pg/mL (urine) and ng/mL (plasma), Midkine
ng/mL, and Serum amyloid P-component ng/mL.
EXAMPLE 5
Apparently Healthy Donor and Chronic Disease Patient Samples
[0194] Human urine samples from donors with no known chronic or
acute disease ("Apparently Healthy Donors") were purchased from two
vendors (Golden West Biologicals, Inc., 27625 Commerce Center Dr.,
Temecula, Calif. 92590 and Virginia Medical Research, Inc., 915
First Colonial Rd., Virginia Beach, Va. 23454). The urine samples
were shipped and stored frozen at less than -20.degree. C. The
vendors supplied demographic information for the individual donors
including gender, race (Black/White), smoking status and age.
[0195] Human urine samples from donors with various chronic
diseases ("Chronic Disease Patients") including congestive heart
failure, coronary artery disease, chronic kidney disease, chronic
obstructive pulmonary disease, diabetes mellitus and hypertension
were purchased from Virginia Medical Research, Inc., 915 First
Colonial Rd., Virginia Beach, Va. 23454. The urine samples were
shipped and stored frozen at less than -20 degrees centigrade. The
vendor provided a case report form for each individual donor with
age, gender, race (Black/White), smoking status and alcohol use,
height, weight, chronic disease(s) diagnosis, current medications
and previous surgeries.
EXAMPLE 6
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stage 0
[0196] Patients from the intensive care unit (ICU) were classified
by kidney status as non-injury (0), risk of injury (R), injury (I),
and failure (F) according to the maximum stage reached within 7
days of enrollment as determined by the RIFLE criteria.
[0197] Two cohorts were defined as (Cohort 1) patients that did not
progress beyond stage 0, and (Cohort 2) patients that reached stage
R, I, or F within 10 days. To address normal marker fluctuations
that occur within patients at the ICU and thereby assess utility
for monitoring AKI status, marker levels were measured in urine
samples collected for Cohort 1. Marker concentrations were measured
in urine samples collected from a subject at 0, 24 hours, and 48
hours prior to reaching stage R, I or F in Cohort 2. In the
following tables, the time "prior max stage" represents the time at
which a sample is collected, relative to the time a particular
patient reaches the lowest disease stage as defined for that
cohort, binned into three groups which are +/-12 hours. For
example, 24 hr prior for this example (0 vs R, I, F) would mean 24
hr (+/-12 hours) prior to reaching stage R (or I if no sample at R,
or F if no sample at R or I).
[0198] Each marker was measured by standard immunoassay methods
using commercially available assay reagents. A receiver operating
characteristic (ROC) curve was generated for each marker and the
area under each ROC curve (AUC) was determined. Patients in Cohort
2 were also separated according to the reason for adjudication to
stage R, I, or F as being based on serum creatinine measurements
(sCr), being based on urine output (UO), or being based on either
serum creatinine measurements or urine output. That is, for those
patients adjudicated to stage R, I, or F on the basis of serum
creatinine measurements alone, the stage 0 cohort may have included
patients adjudicated to stage R, I, or F on the basis of urine
output; for those patients adjudicated to stage R, I, or F on the
basis of urine output alone, the stage 0 cohort may have included
patients adjudicated to stage R, I, or F on the basis of serum
creatinine measurements; and for those patients adjudicated to
stage R, I, or F on the basis of serum creatinine measurements or
urine output, the stage 0 cohort contains only patients in stage 0
for both serum creatinine measurements and urine output. Also, for
those patients adjudicated to stage R, I, or F on the basis of
serum creatinine measurements or urine output, the adjudication
method which yielded the most severe RIFLE stage was used.
[0199] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F)
was determined using ROC analysis. SE is the standard error of the
AUC, n is the number of sample or individual patients ("pts," as
indicated). Standard errors were calculated as described in Hanley,
J. A., and McNeil, B. J., The meaning and use of the area under a
receiver operating characteristic (ROC) curve. Radiology (1982)
143: 29-36; p values were calculated with a two-tailed Z-test. An
AUC <0.5 is indicative of a negative going marker for the
comparison, and an AUC >0.5 is indicative of a positive going
marker for the comparison.
[0200] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0201] The results of these three analyses for various markers of
the present invention are presented in FIG. 1.
EXAMPLE 7
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stages 0 and R
[0202] Patients were classified and analyzed as described in
Example 6. However, patients that reached stage R but did not
progress to stage I or F were grouped with patients from non-injury
stage 0 in Cohort 1. Cohort 2 in this example included only
patients that progressed to stage I or F. Marker concentrations in
urine samples were included for Cohort 1. Marker concentrations in
urine samples collected within 0, 24, and 48 hours of reaching
stage I or F were included for Cohort 2.
[0203] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F)
was determined using ROC analysis.
[0204] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0205] The results of these three analyses for various markers of
the present invention are presented in FIG. 2.
EXAMPLE 8
Kidney Injury Markers for Evaluating Renal Status in Oatients
Progressing from Stage R to Stages I and F
[0206] Patients were classified and analyzed as described in
Example 6, but only those patients that reached Stage R were
included in this example. Cohort 1 contained patients that reached
stage R but did not progress to stage I or F within 10 days, and
Cohort 2 included only patients that progressed to stage I or F.
Marker concentrations in urine samples collected within 12 hours of
reaching stage R were included in the analysis for both Cohort 1
and 2.
[0207] The ability to distinguish cohort 1 (subjects remaining in
RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was
determined using ROC analysis.
[0208] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0209] The results of these three analyses for various markers of
the present invention are presented in FIG. 3.
EXAMPLE 9
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stage 0
[0210] Patients were classified and analyzed as described in
Example 6. However, patients that reached stage R or I but did not
progress to stage F were eliminated from the analysis. Patients
from non-injury stage 0 are included in Cohort 1. Cohort 2 in this
example included only patients that progressed to stage F. The
maximum marker concentrations in urine samples were included for
each patient in Cohort 1. The maximum marker concentrations in
urine samples collected within 0, 24, and 48 hours of reaching
stage F were included for each patient in Cohort 2.
[0211] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F)
was determined using ROC analysis.
[0212] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0213] The results of these three analyses for various markers of
the present invention are presented in FIG. 4.
Example 10
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stage 0
[0214] Patients from the intensive care unit (ICU) were classified
by kidney status as non-injury (0), risk of injury (R), injury (I),
and failure (F) according to the maximum stage reached within 7
days of enrollment as determined by the RIFLE criteria.
[0215] Two cohorts were defined as (Cohort 1) patients that did not
progress beyond stage 0, and (Cohort 2) patients that reached stage
R, I, or F within 10 days. To address normal marker fluctuations
that occur within patients at the ICU and thereby assess utility
for monitoring AKI status, marker levels were measured in the
plasma component of blood samples collected for Cohort 1. Marker
concentrations were measured in the plasma component of blood
samples collected from a subject at 0, 24 hours, and 48 hours prior
to reaching stage R, I or F in Cohort 2. In the following tables,
the time "prior max stage" represents the time at which a sample is
collected, relative to the time a particular patient reaches the
lowest disease stage as defined for that cohort, binned into three
groups which are +/-12 hours. For example, 24 hr prior for this
example (0 vs R, I, F) would mean 24 hr (+/-12 hours) prior to
reaching stage R (or I if no sample at R, or F if no sample at R or
I).
[0216] Each marker was measured by standard immunoassay methods
using commercially available assay reagents. A receiver operating
characteristic (ROC) curve was generated for each marker and the
area under each ROC curve (AUC) was determined. Patients in Cohort
2 were also separated according to the reason for adjudication to
stage R, I, or F as being based on serum creatinine measurements
(sCr), being based on urine output (UO), or being based on either
serum creatinine measurements or urine output. That is, for those
patients adjudicated to stage R, I, or F on the basis of serum
creatinine measurements alone, the stage 0 cohort may have included
patients adjudicated to stage R, I, or F on the basis of urine
output; for those patients adjudicated to stage R, I, or F on the
basis of urine output alone, the stage 0 cohort may have included
patients adjudicated to stage R, I, or F on the basis of serum
creatinine measurements; and for those patients adjudicated to
stage R, I, or F on the basis of serum creatinine measurements or
urine output, the stage 0 cohort contains only patients in stage 0
for both serum creatinine measurements and urine output. Also, for
those patients adjudicated to stage R, I, or F on the basis of
serum creatinine measurements or urine output, the adjudication
method which yielded the most severe RIFLE stage was used.
[0217] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F)
was determined using ROC analysis. SE is the standard error of the
AUC, n is the number of sample or individual patients ("pts," as
indicated). Standard errors were calculated as described in Hanley,
J. A., and McNeil, B. J., The meaning and use of the area under a
receiver operating characteristic (ROC) curve. Radiology (1982)
143: 29-36; p values were calculated with a two-tailed Z-test. An
AUC <0.5 is indicative of a negative going marker for the
comparison, and an AUC >0.5 is indicative of a positive going
marker for the comparison.
[0218] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0219] The results of these three analyses for various markers of
the present invention are presented in FIG. 5.
EXAMPLE 11
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stages 0 and R
[0220] Patients were classified and analyzed as described in
Example 10. However, patients that reached stage R but did not
progress to stage I or F were grouped with patients from non-injury
stage 0 in Cohort 1. Cohort 2 in this example included only
patients that progressed to stage I or F. Marker concentrations in
the plasma component of blood samples were included for Cohort 1.
Marker concentrations in the plasma component of blood samples
collected within 0, 24, and 48 hours of reaching stage I or F were
included for Cohort 2.
[0221] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F)
was determined using ROC analysis.
[0222] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0223] The results of these three analyses for various markers of
the present invention are presented in FIG. 6.
EXAMPLE 12
Kidney Injury Markers for Evaluating Renal Status in Patients
Progressing from Stage R to Stages I and F
[0224] Patients were classified and analyzed as described in
Example 10, but only those patients that reached Stage R were
included in this example. Cohort 1 contained patients that reached
stage R but did not progress to stage I or F within 10 days, and
Cohort 2 included only patients that progressed to stage I or F.
Marker concentrations in the plasma component of blood samples
collected within 12 hours of reaching stage R were included in the
analysis for both Cohort 1 and 2.
[0225] The ability to distinguish cohort 1 (subjects remaining in
RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was
determined using ROC analysis.
[0226] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0227] The results of these three analyses for various markers of
the present invention are presented in FIG. 7.
EXAMPLE 13
Kidney Injury Markers for Evaluating Renal Status in Patients at
RIFLE Stage 0
[0228] Patients were classified and analyzed as described in
Example 10. However, patients that reached stage R or I but did not
progress to stage F were eliminated from the analysis. Patients
from non-injury stage 0 are included in Cohort 1. Cohort 2 in this
example included only patients that progressed to stage F. The
maximum marker concentrations in the plasma component of blood
samples were included from each patient in Cohort 1. The maximum
marker concentrations in the plasma component of blood samples
collected within 0, 24, and 48 hours of reaching stage F were
included from each patient in Cohort 2.
[0229] The ability to distinguish cohort 1 (subjects remaining in
RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F)
was determined using ROC analysis.
[0230] Various threshold (or "cutoff") concentrations were
selected, and the associated sensitivity and specificity for
distinguishing cohort 1 from cohort 2 were determined. OR is the
odds ratio calculated for the particular cutoff concentration, and
95% CI is the confidence interval for the odds ratio.
[0231] The results of these three analyses for various markers of
the present invention are presented in FIG. 8.
[0232] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention. The examples provided herein are representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Modifications therein
and other uses will occur to those skilled in the art. These
modifications are encompassed within the spirit of the invention
and are defined by the scope of the claims.
[0233] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0234] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0235] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0236] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
111449PRTHomo sapiens 1Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu
Leu Leu Thr Trp Glu 1 5 10 15 Ser Gly Gln Val Leu Gly Asp Gln Thr
Val Ser Asp Asn Glu Leu Gln 20 25 30 Glu Met Ser Asn Gln Gly Ser
Lys Tyr Val Asn Lys Glu Ile Gln Asn 35 40 45 Ala Val Asn Gly Val
Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn 50 55 60 Glu Glu Arg
Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys 65 70 75 80 Lys
Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys 85 90
95 Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
100 105 110 Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala
Arg Val 115 120 125 Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu
Glu Glu Phe Leu 130 135 140 Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met
Asn Gly Asp Arg Ile Asp 145 150 155 160 Ser Leu Leu Glu Asn Asp Arg
Gln Gln Thr His Met Leu Asp Val Met 165 170 175 Gln Asp His Phe Ser
Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln 180 185 190 Asp Arg Phe
Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro 195 200 205 Phe
Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg 210 215
220 Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
225 230 235 240 His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu
Ala Gln Gln 245 250 255 Ala Met Asp Ile His Phe His Ser Pro Ala Phe
Gln His Pro Pro Thr 260 265 270 Glu Phe Ile Arg Glu Gly Asp Asp Asp
Arg Thr Val Cys Arg Glu Ile 275 280 285 Arg His Asn Ser Thr Gly Cys
Leu Arg Met Lys Asp Gln Cys Asp Lys 290 295 300 Cys Arg Glu Ile Leu
Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln 305 310 315 320 Ala Lys
Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg 325 330 335
Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met 340
345 350 Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn
Trp 355 360 365 Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln
Tyr Tyr Leu 370 375 380 Arg Val Thr Thr Val Ala Ser His Thr Ser Asp
Ser Asp Val Pro Ser 385 390 395 400 Gly Val Thr Glu Val Val Val Lys
Leu Phe Asp Ser Asp Pro Ile Thr 405 410 415 Val Thr Val Pro Val Glu
Val Ser Arg Lys Asn Pro Lys Phe Met Glu 420 425 430 Thr Val Ala Glu
Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu 435 440 445 Glu
2133PRTHomo sapiens 2Met Val Asp Ala Phe Leu Gly Thr Trp Lys Leu
Val Asp Ser Lys Asn 1 5 10 15 Phe Asp Asp Tyr Met Lys Ser Leu Gly
Val Gly Phe Ala Thr Arg Gln 20 25 30 Val Ala Ser Met Thr Lys Pro
Thr Thr Ile Ile Glu Lys Asn Gly Asp 35 40 45 Ile Leu Thr Leu Lys
Thr His Ser Thr Phe Lys Asn Thr Glu Ile Ser 50 55 60 Phe Lys Leu
Gly Val Glu Phe Asp Glu Thr Thr Ala Asp Asp Arg Lys 65 70 75 80 Val
Lys Ser Ile Val Thr Leu Asp Gly Gly Lys Leu Val His Leu Gln 85 90
95 Lys Trp Asp Gly Gln Glu Thr Thr Leu Val Arg Glu Leu Ile Asp Gly
100 105 110 Lys Leu Ile Leu Thr Leu Thr His Gly Thr Ala Val Cys Thr
Arg Thr 115 120 125 Tyr Glu Lys Glu Ala 130 3728PRTHomo sapiens
3Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1
5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly
Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser
Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile
Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg
Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe
Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe
Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu
Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile
Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135
140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln
Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro
Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys
Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cys Asn
Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly
Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg
His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255
Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260
265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr
Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu
Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly
Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg
Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu
Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn
Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro
Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380
Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385
390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met
Trp Asp 405 410 415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp
Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn
Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn
Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu
Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro
Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn
Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505
510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp
515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys
Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg
Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln
Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys
Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile
Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser
Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr
Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630
635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser
Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys
Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys
Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys
Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val Arg Val Ala Tyr
Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys
Val Pro Gln Ser 725 4166PRTHomo sapiens 4Met Lys Tyr Thr Ser Tyr
Ile Leu Ala Phe Gln Leu Cys Ile Val Leu 1 5 10 15 Gly Ser Leu Gly
Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu 20 25 30 Asn Leu
Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn 35 40 45
Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp 50
55 60 Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu
Phe 65 70 75 80 Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val
Glu Thr Ile 85 90 95 Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser
Asn Lys Lys Lys Arg 100 105 110 Asp Asp Phe Glu Lys Leu Thr Asn Tyr
Ser Val Thr Asp Leu Asn Val 115 120 125 Gln Arg Lys Ala Ile His Glu
Leu Ile Gln Val Met Ala Glu Leu Ser 130 135 140 Pro Ala Ala Lys Thr
Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg 145 150 155 160 Gly Arg
Arg Ala Ser Gln 165 5328PRTHomo sapiens 5Met Cys His Gln Gln Leu
Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu
Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu
Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50
55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val
Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly
Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys
Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys
Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn
Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser
Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180
185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro
Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu
Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro
Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn
Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln
Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe
Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305
310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 6631PRTHomo sapiens
6Met Asp Tyr Ser Phe Asp Thr Thr Ala Glu Asp Pro Trp Val Arg Ile 1
5 10 15 Ser Asp Cys Ile Lys Asn Leu Phe Ser Pro Ile Met Ser Glu Asn
His 20 25 30 Gly His Met Pro Leu Gln Pro Asn Ala Ser Leu Asn Glu
Glu Glu Gly 35 40 45 Thr Gln Gly His Pro Asp Gly Thr Pro Pro Lys
Leu Asp Thr Ala Asn 50 55 60 Gly Thr Pro Lys Val Tyr Lys Ser Ala
Asp Ser Ser Thr Val Lys Lys 65 70 75 80 Gly Pro Pro Val Ala Pro Lys
Pro Ala Trp Phe Arg Gln Ser Leu Lys 85 90 95 Gly Leu Arg Asn Arg
Ala Ser Asp Pro Arg Gly Leu Pro Asp Pro Ala 100 105 110 Leu Ser Thr
Gln Pro Ala Pro Ala Ser Arg Glu His Leu Gly Ser His 115 120 125 Ile
Arg Ala Ser Ser Ser Ser Ser Ser Ile Arg Gln Arg Ile Ser Ser 130 135
140 Phe Glu Thr Phe Gly Ser Ser Gln Leu Pro Asp Lys Gly Ala Gln Arg
145 150 155 160 Leu Ser Leu Gln Pro Ser Ser Gly Glu Ala Ala Lys Pro
Leu Gly Lys 165 170 175 His Glu Glu Gly Arg Phe Ser Gly Leu Leu Gly
Arg Gly Ala Ala Pro 180 185 190 Thr Leu Val Pro Gln Gln Pro Glu Gln
Val Leu Ser Ser Gly Ser Pro 195 200 205 Ala Ala Ser Glu Ala Arg Asp
Pro Gly Val Ser Glu Ser Pro Pro Pro 210 215 220 Gly Arg Gln Pro Asn
Gln Lys Thr Leu Pro Pro Gly Pro Asp Pro Leu 225 230 235 240 Leu Arg
Leu Leu Ser Thr Gln Ala Glu Glu Ser Gln Gly Pro Val Leu 245 250 255
Lys Met Pro Ser Gln Arg Ala Arg Ser Phe Pro Leu Thr Arg Ser Gln 260
265 270 Ser Cys Glu Thr Lys Leu Leu Asp Glu Lys Thr Ser Lys Leu Tyr
Ser 275 280 285 Ile Ser Ser Gln Val Ser Ser Ala Val Met Lys Ser Leu
Leu Cys Leu 290 295 300 Pro Ser Ser Ile Ser Cys Ala Gln Thr Pro Cys
Ile Pro Lys Glu Gly 305 310 315 320 Ala Ser Pro Thr Ser Ser Ser Asn
Glu Asp Ser Ala Ala Asn Gly Ser 325 330 335 Ala Glu Thr Ser Ala Leu
Asp Thr Gly Phe Ser Leu Asn Leu Ser Glu 340 345 350 Leu Arg Glu Tyr
Thr Glu Gly Leu Thr Glu Ala Lys Glu Asp Asp Asp 355 360 365 Gly Asp
His Ser Ser Leu Gln Ser Gly Gln Ser Val Ile Ser Leu Leu 370 375 380
Ser Ser Glu Glu Leu Lys Lys Leu Ile Glu Glu Val Lys Val Leu Asp 385
390 395 400 Glu Ala Thr Leu Lys Gln Leu Asp Gly Ile His Val Thr Ile
Leu His 405 410 415 Lys Glu Glu Gly Ala Gly Leu Gly Phe Ser Leu Ala
Gly Gly Ala Asp 420 425 430 Leu Glu Asn Lys Val Ile Thr Val His Arg
Val Phe Pro Asn Gly Leu 435 440 445 Ala Ser Gln Glu Gly Thr Ile Gln
Lys Gly Asn Glu Val Leu Ser Ile 450 455 460 Asn Gly Lys Ser Leu Lys
Gly Thr Thr His His Asp Ala Leu Ala Ile 465 470 475 480 Leu Arg Gln
Ala Arg Glu Pro Arg Gln Ala Val Ile Val Thr Arg Lys
485 490 495 Leu Thr Pro Glu Ala Met Pro Asp Leu Asn Ser Ser Thr Asp
Ser Ala 500 505 510 Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu
Ser Thr Ala Glu 515 520 525 Ala Thr Val Cys Thr Val Thr Leu Glu Lys
Met Ser Ala Gly Leu Gly 530 535 540 Phe Ser Leu Glu Gly Gly Lys Gly
Ser Leu His Gly Asp Lys Pro Leu 545 550 555 560 Thr Ile Asn Arg Ile
Phe Lys Gly Ala Ala Ser Glu Gln Ser Glu Thr 565 570 575 Val Gln Pro
Gly Asp Glu Ile Leu Gln Leu Gly Gly Thr Ala Met Gln 580 585 590 Gly
Leu Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala Leu Pro Asp 595 600
605 Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gln Ser Lys Glu
610 615 620 Thr Thr Ala Ala Gly Asp Ser 625 630 7153PRTHomo sapiens
7Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1
5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln
Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu
Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met
Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu
Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu
Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg
Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp
Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135
140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 8660PRTHomo sapiens
8Met Glu Ala Leu Met Ala Arg Gly Ala Leu Thr Gly Pro Leu Arg Ala 1
5 10 15 Leu Cys Leu Leu Gly Cys Leu Leu Ser His Ala Ala Ala Ala Pro
Ser 20 25 30 Pro Ile Ile Lys Phe Pro Gly Asp Val Ala Pro Lys Thr
Asp Lys Glu 35 40 45 Leu Ala Val Gln Tyr Leu Asn Thr Phe Tyr Gly
Cys Pro Lys Glu Ser 50 55 60 Cys Asn Leu Phe Val Leu Lys Asp Thr
Leu Lys Lys Met Gln Lys Phe 65 70 75 80 Phe Gly Leu Pro Gln Thr Gly
Asp Leu Asp Gln Asn Thr Ile Glu Thr 85 90 95 Met Arg Lys Pro Arg
Cys Gly Asn Pro Asp Val Ala Asn Tyr Asn Phe 100 105 110 Phe Pro Arg
Lys Pro Lys Trp Asp Lys Asn Gln Ile Thr Tyr Arg Ile 115 120 125 Ile
Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp Asp Ala Phe 130 135
140 Ala Arg Ala Phe Gln Val Trp Ser Asp Val Thr Pro Leu Arg Phe Ser
145 150 155 160 Arg Ile His Asp Gly Glu Ala Asp Ile Met Ile Asn Phe
Gly Arg Trp 165 170 175 Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys
Asp Gly Leu Leu Ala 180 185 190 His Ala Phe Ala Pro Gly Thr Gly Val
Gly Gly Asp Ser His Phe Asp 195 200 205 Asp Asp Glu Leu Trp Thr Leu
Gly Glu Gly Gln Val Val Arg Val Lys 210 215 220 Tyr Gly Asn Ala Asp
Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe Asn 225 230 235 240 Gly Lys
Glu Tyr Asn Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly Phe 245 250 255
Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr Gly 260
265 270 Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Glu
Gly 275 280 285 Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser
Tyr Asp Ser 290 295 300 Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg
Trp Cys Gly Thr Thr 305 310 315 320 Glu Asp Tyr Asp Arg Asp Lys Lys
Tyr Gly Phe Cys Pro Glu Thr Ala 325 330 335 Met Ser Thr Val Gly Gly
Asn Ser Glu Gly Ala Pro Cys Val Phe Pro 340 345 350 Phe Thr Phe Leu
Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly Arg 355 360 365 Ser Asp
Gly Lys Met Trp Cys Ala Thr Thr Ala Asn Tyr Asp Asp Asp 370 375 380
Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val 385
390 395 400 Ala Ala His Glu Phe Gly His Ala Met Gly Leu Glu His Ser
Gln Asp 405 410 415 Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr
Lys Asn Phe Arg 420 425 430 Leu Ser Gln Asp Asp Ile Lys Gly Ile Gln
Glu Leu Tyr Gly Ala Ser 435 440 445 Pro Asp Ile Asp Leu Gly Thr Gly
Pro Thr Pro Thr Leu Gly Pro Val 450 455 460 Thr Pro Glu Ile Cys Lys
Gln Asp Ile Val Phe Asp Gly Ile Ala Gln 465 470 475 480 Ile Arg Gly
Glu Ile Phe Phe Phe Lys Asp Arg Phe Ile Trp Arg Thr 485 490 495 Val
Thr Pro Arg Asp Lys Pro Met Gly Pro Leu Leu Val Ala Thr Phe 500 505
510 Trp Pro Glu Leu Pro Glu Lys Ile Asp Ala Val Tyr Glu Ala Pro Gln
515 520 525 Glu Glu Lys Ala Val Phe Phe Ala Gly Asn Glu Tyr Trp Ile
Tyr Ser 530 535 540 Ala Ser Thr Leu Glu Arg Gly Tyr Pro Lys Pro Leu
Thr Ser Leu Gly 545 550 555 560 Leu Pro Pro Asp Val Gln Arg Val Asp
Ala Ala Phe Asn Trp Ser Lys 565 570 575 Asn Lys Lys Thr Tyr Ile Phe
Ala Gly Asp Lys Phe Trp Arg Tyr Asn 580 585 590 Glu Val Lys Lys Lys
Met Asp Pro Gly Phe Pro Lys Leu Ile Ala Asp 595 600 605 Ala Trp Asn
Ala Ile Pro Asp Asn Leu Asp Ala Val Val Asp Leu Gln 610 615 620 Gly
Gly Gly His Ser Tyr Phe Phe Lys Gly Ala Tyr Tyr Leu Lys Leu 625 630
635 640 Glu Asn Gln Ser Leu Lys Ser Val Lys Phe Gly Ser Ile Lys Ser
Asp 645 650 655 Trp Leu Gly Cys 660 9707PRTHomo sapiens 9Met Ser
Leu Trp Gln Pro Leu Val Leu Val Leu Leu Val Leu Gly Cys 1 5 10 15
Cys Phe Ala Ala Pro Arg Gln Arg Gln Ser Thr Leu Val Leu Phe Pro 20
25 30 Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln Leu Ala Glu Glu
Tyr 35 40 45 Leu Tyr Arg Tyr Gly Tyr Thr Arg Val Ala Glu Met Arg
Gly Glu Ser 50 55 60 Lys Ser Leu Gly Pro Ala Leu Leu Leu Leu Gln
Lys Gln Leu Ser Leu 65 70 75 80 Pro Glu Thr Gly Glu Leu Asp Ser Ala
Thr Leu Lys Ala Met Arg Thr 85 90 95 Pro Arg Cys Gly Val Pro Asp
Leu Gly Arg Phe Gln Thr Phe Glu Gly 100 105 110 Asp Leu Lys Trp His
His His Asn Ile Thr Tyr Trp Ile Gln Asn Tyr 115 120 125 Ser Glu Asp
Leu Pro Arg Ala Val Ile Asp Asp Ala Phe Ala Arg Ala 130 135 140 Phe
Ala Leu Trp Ser Ala Val Thr Pro Leu Thr Phe Thr Arg Val Tyr 145 150
155 160 Ser Arg Asp Ala Asp Ile Val Ile Gln Phe Gly Val Ala Glu His
Gly 165 170 175 Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly Leu Leu Ala
His Ala Phe 180 185 190 Pro Pro Gly Pro Gly Ile Gln Gly Asp Ala His
Phe Asp Asp Asp Glu 195 200 205 Leu Trp Ser Leu Gly Lys Gly Val Val
Val Pro Thr Arg Phe Gly Asn 210 215 220 Ala Asp Gly Ala Ala Cys His
Phe Pro Phe Ile Phe Glu Gly Arg Ser 225 230 235 240 Tyr Ser Ala Cys
Thr Thr Asp Gly Arg Ser Asp Gly Leu Pro Trp Cys 245 250 255 Ser Thr
Thr Ala Asn Tyr Asp Thr Asp Asp Arg Phe Gly Phe Cys Pro 260 265 270
Ser Glu Arg Leu Tyr Thr Gln Asp Gly Asn Ala Asp Gly Lys Pro Cys 275
280 285 Gln Phe Pro Phe Ile Phe Gln Gly Gln Ser Tyr Ser Ala Cys Thr
Thr 290 295 300 Asp Gly Arg Ser Asp Gly Tyr Arg Trp Cys Ala Thr Thr
Ala Asn Tyr 305 310 315 320 Asp Arg Asp Lys Leu Phe Gly Phe Cys Pro
Thr Arg Ala Asp Ser Thr 325 330 335 Val Met Gly Gly Asn Ser Ala Gly
Glu Leu Cys Val Phe Pro Phe Thr 340 345 350 Phe Leu Gly Lys Glu Tyr
Ser Thr Cys Thr Ser Glu Gly Arg Gly Asp 355 360 365 Gly Arg Leu Trp
Cys Ala Thr Thr Ser Asn Phe Asp Ser Asp Lys Lys 370 375 380 Trp Gly
Phe Cys Pro Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala 385 390 395
400 His Glu Phe Gly His Ala Leu Gly Leu Asp His Ser Ser Val Pro Glu
405 410 415 Ala Leu Met Tyr Pro Met Tyr Arg Phe Thr Glu Gly Pro Pro
Leu His 420 425 430 Lys Asp Asp Val Asn Gly Ile Arg His Leu Tyr Gly
Pro Arg Pro Glu 435 440 445 Pro Glu Pro Arg Pro Pro Thr Thr Thr Thr
Pro Gln Pro Thr Ala Pro 450 455 460 Pro Thr Val Cys Pro Thr Gly Pro
Pro Thr Val His Pro Ser Glu Arg 465 470 475 480 Pro Thr Ala Gly Pro
Thr Gly Pro Pro Ser Ala Gly Pro Thr Gly Pro 485 490 495 Pro Thr Ala
Gly Pro Ser Thr Ala Thr Thr Val Pro Leu Ser Pro Val 500 505 510 Asp
Asp Ala Cys Asn Val Asn Ile Phe Asp Ala Ile Ala Glu Ile Gly 515 520
525 Asn Gln Leu Tyr Leu Phe Lys Asp Gly Lys Tyr Trp Arg Phe Ser Glu
530 535 540 Gly Arg Gly Ser Arg Pro Gln Gly Pro Phe Leu Ile Ala Asp
Lys Trp 545 550 555 560 Pro Ala Leu Pro Arg Lys Leu Asp Ser Val Phe
Glu Glu Pro Leu Ser 565 570 575 Lys Lys Leu Phe Phe Phe Ser Gly Arg
Gln Val Trp Val Tyr Thr Gly 580 585 590 Ala Ser Val Leu Gly Pro Arg
Arg Leu Asp Lys Leu Gly Leu Gly Ala 595 600 605 Asp Val Ala Gln Val
Thr Gly Ala Leu Arg Ser Gly Arg Gly Lys Met 610 615 620 Leu Leu Phe
Ser Gly Arg Arg Leu Trp Arg Phe Asp Val Lys Ala Gln 625 630 635 640
Met Val Asp Pro Arg Ser Ala Ser Glu Val Asp Arg Met Phe Pro Gly 645
650 655 Val Pro Leu Asp Thr His Asp Val Phe Gln Tyr Arg Glu Lys Ala
Tyr 660 665 670 Phe Cys Gln Asp Arg Phe Tyr Trp Arg Val Ser Ser Arg
Ser Glu Leu 675 680 685 Asn Gln Val Asp Gln Val Gly Tyr Val Thr Tyr
Asp Ile Leu Gln Cys 690 695 700 Pro Glu Asp 705 10143PRTHomo
sapiens 10Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu Leu Ala Leu
Leu Ala 1 5 10 15 Leu Thr Ser Ala Val Ala Lys Lys Lys Asp Lys Val
Lys Lys Gly Gly 20 25 30 Pro Gly Ser Glu Cys Ala Glu Trp Ala Trp
Gly Pro Cys Thr Pro Ser 35 40 45 Ser Lys Asp Cys Gly Val Gly Phe
Arg Glu Gly Thr Cys Gly Ala Gln 50 55 60 Thr Gln Arg Ile Arg Cys
Arg Val Pro Cys Asn Trp Lys Lys Glu Phe 65 70 75 80 Gly Ala Asp Cys
Lys Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90 95 Gly Thr
Gly Thr Lys Val Arg Gln Gly Thr Leu Lys Lys Ala Arg Tyr 100 105 110
Asn Ala Gln Cys Gln Glu Thr Ile Arg Val Thr Lys Pro Cys Thr Pro 115
120 125 Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys Gly Lys Asp
130 135 140 11223PRTHomo sapiens 11Met Asn Lys Pro Leu Leu Trp Ile
Ser Val Leu Thr Ser Leu Leu Glu 1 5 10 15 Ala Phe Ala His Thr Asp
Leu Ser Gly Lys Val Phe Val Phe Pro Arg 20 25 30 Glu Ser Val Thr
Asp His Val Asn Leu Ile Thr Pro Leu Glu Lys Pro 35 40 45 Leu Gln
Asn Phe Thr Leu Cys Phe Arg Ala Tyr Ser Asp Leu Ser Arg 50 55 60
Ala Tyr Ser Leu Phe Ser Tyr Asn Thr Gln Gly Arg Asp Asn Glu Leu 65
70 75 80 Leu Val Tyr Lys Glu Arg Val Gly Glu Tyr Ser Leu Tyr Ile
Gly Arg 85 90 95 His Lys Val Thr Ser Lys Val Ile Glu Lys Phe Pro
Ala Pro Val His 100 105 110 Ile Cys Val Ser Trp Glu Ser Ser Ser Gly
Ile Ala Glu Phe Trp Ile 115 120 125 Asn Gly Thr Pro Leu Val Lys Lys
Gly Leu Arg Gln Gly Tyr Phe Val 130 135 140 Glu Ala Gln Pro Lys Ile
Val Leu Gly Gln Glu Gln Asp Ser Tyr Gly 145 150 155 160 Gly Lys Phe
Asp Arg Ser Gln Ser Phe Val Gly Glu Ile Gly Asp Leu 165 170 175 Tyr
Met Trp Asp Ser Val Leu Pro Pro Glu Asn Ile Leu Ser Ala Tyr 180 185
190 Gln Gly Thr Pro Leu Pro Ala Asn Ile Leu Asp Trp Gln Ala Leu Asn
195 200 205 Tyr Glu Ile Arg Gly Tyr Val Ile Ile Lys Pro Leu Val Trp
Val 210 215 220
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