U.S. patent application number 13/876859 was filed with the patent office on 2014-03-20 for methods for predicting the progression and treating a chronic kidney disease in a patient.
The applicant listed for this patent is Martine Burtin, Khalil El Karoui, Clement Nguyen, Fabiola Terzi, Amandine Viau. Invention is credited to Martine Burtin, Khalil El Karoui, Clement Nguyen, Fabiola Terzi, Amandine Viau.
Application Number | 20140079769 13/876859 |
Document ID | / |
Family ID | 44514192 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140079769 |
Kind Code |
A1 |
Terzi; Fabiola ; et
al. |
March 20, 2014 |
METHODS FOR PREDICTING THE PROGRESSION AND TREATING A CHRONIC
KIDNEY DISEASE IN A PATIENT
Abstract
The present invention relates to a method for predicting the
progression of chronic kidney disease (CKD) in a patient and also
to an inhibitor of NGAL gene expression or an NGAL antagonist for
use in the prevention or the treatment of CKD.
Inventors: |
Terzi; Fabiola; (Paris,
FR) ; Viau; Amandine; (Paris, FR) ; Nguyen;
Clement; (Paris, FR) ; Burtin; Martine;
(Paris, FR) ; El Karoui; Khalil; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Terzi; Fabiola
Viau; Amandine
Nguyen; Clement
Burtin; Martine
El Karoui; Khalil |
Paris
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR
FR |
|
|
Family ID: |
44514192 |
Appl. No.: |
13/876859 |
Filed: |
October 3, 2011 |
PCT Filed: |
October 3, 2011 |
PCT NO: |
PCT/EP2011/067236 |
371 Date: |
September 5, 2013 |
Current U.S.
Class: |
424/450 ;
424/172.1; 435/6.11; 435/6.12; 435/7.9; 436/501; 514/44A;
514/44R |
Current CPC
Class: |
C12N 15/1135 20130101;
G01N 2800/347 20130101; C07K 2317/76 20130101; C12N 2310/122
20130101; C12N 2310/14 20130101; C12Q 1/6883 20130101; A61K 9/0019
20130101; C12N 2310/531 20130101; C12N 2310/16 20130101; C12Q
2600/118 20130101; G01N 33/6893 20130101; C07K 16/2803 20130101;
G01N 2800/56 20130101 |
Class at
Publication: |
424/450 ;
435/6.12; 436/501; 514/44.A; 514/44.R; 424/172.1; 435/6.11;
435/7.9 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 2010 |
EP |
10306077.8 |
Claims
1. A method for predicting the progression of chronic kidney
disease (CKD) or for monitoring CKD therapy in a patient,
comprising the following steps: a. providing a biological sample
from said patient suffering from CKD, b. determining the expression
level of Neutrophil Gelatinase-Associated Lipocalin (NGAL) gene,
and c. correlating the expression level of NGAL gene with the
prediction of the progression of CKD.
2. The method according to claim 1, wherein said expression level
of NGAL gene is compared to a predetermined value.
3. The method according to claim 1, wherein said expression level
of NGAL gene is determined by determining the quantity of NGAL mRNA
and said biological sample is a cell or tissue sample.
4. The method according to claim 3, wherein said tissue sample is a
kidney biopsy.
5. The method according to claim 1, wherein said expression level
of NGAL gene is determined by measuring the concentration of NGAL
protein in a biological sample obtained from said patient.
6. The method according to claim 5, wherein said biological sample
is a blood sample, a serum sample, a plasma sample or a urine
sample.
7. The method according to claim 5, wherein the concentration of
NGAL protein is measured by immunoassay.
8. The method according to claim 1, wherein the CKD is selected in
the group consisting of polycystic kidney disease
glomerulonephritis, interstitial nephritis, nephropathy and
obstructive uropathy.
9-13. (canceled)
14. The method of claim 8, wherein said polycystic kidney disease
is Autosomal Dominant Polycystic Kidney Disease (ADPKD) or
Autosomal Recessive Polycystic Kidney Disease (ARPKD).
15. A method of preventing or treating chronic kidney disease (CKD)
in a patient in need thereof, comprising administering to said
patient an inhibitor of Neutrophil Gelatinase-Associated Lipocalin
(NGAL) gene expression.
16. The method of claim 15, wherein said inhibitor is selected from
the group consisting of antisense RNA or DNA molecules, small
inhibitory RNAs (siRNAs), short hairpin RNA and ribozymes.
17. A method of preventing or treating chronic kidney disease (CKD)
in a patient in need thereof, comprising administering to said
patient an antagonist of Neutrophil Gelatinase-Associated Lipocalin
(NGAL) gene expression.
18. The method of claim 17, wherein said antagonist is an antibody
or an aptamer directed against NGAL.
19. A pharmaceutical composition comprising an inhibitor of
Neutrophil Gelatinase-Associated Lipocalin (NGAL) gene expression
selected from the group consisting of antisense RNA or DNA
molecules, small inhibitory RNAs (siRNAs), short hairpin RNA and
ribozymes; or an NGAL antagonist selected from an antibody and an
aptamer directed against NGAL; and a pharmaceutically acceptable
vehicle.
20. The pharmaceutical composition of claim 19, wherein said
pharmaceutical composition is formulated: for parenteral
administration, as a solid tablet, as a liposomal formulation, or
as a time release capsule.
21. The pharmaceutical composition of claim 20, wherein said
parenteral administration is via intravenous or intramuscular
injection.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for predicting the
progression of chronic kidney disease (CKD) in a patient and also
to an inhibitor of NGAL gene expression or an NGAL antagonist for
use in the prevention or the treatment of CKD.
BACKGROUND OF THE INVENTION
[0002] Regardless of the initial insult, human chronic kidney
disease (CKD) is characterized by progressive destruction of the
renal parenchyma and the loss of functional nephrons which
ultimately lead to end stage renal failure (ESRF). CKD represents a
worldwide concern: in the USA, 102,567 patients began dialysis in
2003 (341 patients/year per/million) (1), and similar rates were
found in developing countries and in particular ethnic groups (2).
However, these numbers are a small fraction of the millions of
patients who are thought to have some degree of renal impairment.
In the United States the prevalence of chronically reduced kidney
function is 11% of adults (3). Understanding the pathophysiology of
CKD progression is, therefore, a key challenge for medical
planning.
[0003] The mechanisms of CKD progression are poorly understood. It
has been shown that reduction of the number of functional nephrons
triggers molecular and cellular events promoting compensatory
growth of the remaining ones (4). In some cases, this compensatory
process becomes pathological with the development of renal lesions
and ESRF. Although the pathophysiology of compensation and
progression is complex, unregulated proliferation of glomerular,
tubular and interstitial cells may promote the development of
glomerulosclerosis, tubular cysts, and interstitial fibrosis (5-7).
The molecular programs that control this cascade of events are
largely unknown.
[0004] Attempts to dissect the molecular basis of CKD have been
facilitated by the development of several experimental models of
renal deterioration. Among these, the remnant kidney model is a
mainstay, since nephron reduction characterizes the evolution of
most human CKD. Consequently, this model recapitulates many
features of human CKD, including hypertension, proteinuria,
glomerular and tubulointerstitial lesions. Over the last fifty
years, this model has led to the discovery of critical pathways
and, more importantly, to the design of therapeutic strategies to
slow down the progression of CKD, such as the widely clinically
used renin-angiotensin inhibitors (8).
[0005] More recently, studies in different mouse strains have
highlighted the importance of genetic factors in the evolution of
experimental nephron reduction (9-11). We previously showed that
the course and extent of renal lesions following nephron reduction
vary significantly between two mouse strains: whereas the FVB/N
mice develop severe lesions, the (C57BL/6.times.DBA2)F1 (hereafter
denoted B6D2F1) undergoes compensation alone (12). Moreover, we
observed that the development of renal lesions paralleled the
extent of cell proliferation (12). In fact, once the compensatory
growth is achieved, a second wave of cell proliferation occurs only
in the FVB/N strain.
[0006] There is a need in the art for a reliable biomarker which
allows the prediction of the progression of CKD in particular in
human patients suffering from said disease as well as relevant
treatments for preventing or treating CKD.
SUMMARY OF THE INVENTION
[0007] The present invention thus relates to a method for
predicting the progression of chronic kidney disease (CKD) or for
monitoring CKD therapy in a patient,
[0008] comprising the following steps:
[0009] a. providing a biological sample from said patient suffering
from CKD,
[0010] b. determining the expression level of Neutrophil
Gelatinase-Associated Lipocalin (NGAL) gene, and
[0011] c. correlating the expression level of NGAL gene with the
prediction of the progression of CKD.
[0012] The present invention also relates to an inhibitor of NGAL
gene expression for use in the prevention or the treatment of
CKD.
[0013] The present invention further relates to an NGAL antagonist
for use in the prevention or the treatment of CKD.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0014] Throughout the specification, several terms are employed and
are defined in the following paragraphs.
[0015] As used herein, the terms "Lipocalin 2" (Lcn2) or "NGAL"
have their general meaning in the art and refer to the Neutrophil
Gelatinase-Associated Lipocalin as described in Schmidt-Ott K M. et
al. (2007). NGAL can be from any source, but typically is a
mammalian (e.g., human and non-human primate) NGAL, particularly a
human NGAL. The term "NGAL gene" refers to any nucleotide sequence
encoding the NGAL mRNA and protein, such as a genomic DNA sequence
and any naturally occurring NGAL and variants and modified forms
thereof. It can also encompass artificial sequences such as cDNA
encoding the NGAL mRNA and protein. An exemplary human native NGAL
nucleotide sequence is provided in GenBank database under accession
number NM.sub.--005564. The term "NGAL mRNA" has its general
meaning in the art and refers to the messenger RNA which is
synthesized upon expression of the NGAL gene. The term "NGAL
protein" refers to the amino acid sequence resulting from the
expression of the NGAL gene, and any naturally occurring NGAL and
variants and modified forms thereof. An exemplary human native NGAL
amino acid sequence is provided in GenPept database under accession
number NP.sub.--005555. NGAL is a glycoprotein and was originally
identified as a neutrophil specific granule component and a member
of the lipocalin family of proteins. The protein was shown to exist
both as a 25-kDa monomer and a 45-kDa disulfide-linked homodimer,
and it may also be covalently complexed with neutrophil gelatinase
(also known as matrix metalloproteinase 9, MMP-9) via an
intermolecular disulphide bridge as a 135-kDa heterodimeric
form.
[0016] An "inhibitor of gene expression" refers to a natural or
synthetic compound that has a biological effect to inhibit or
significantly reduce the expression of a gene. Thus, an "inhibitor
of NGAL gene expression" refers to a natural or synthetic compound
that has a biological effect to inhibit or significantly reduce the
expression of the gene encoding for NGAL.
[0017] The term "NGAL antagonist" refers to a compound, natural or
not, which has the capability to inhibit (partly or totally) the
biological activity of the NGAL protein. The scope of the present
invention includes all those NGAL antagonists now known and those
NGAL antagonists to be discovered in the future. This term includes
anti-NGAL antibody.
[0018] The term "anti-NGAL antibody" refers to an antibody or a
fragment thereof which recognizes NGAL.
[0019] The term "chronic kidney disease" (CKD) has its general
meaning in the art and is used to classify numerous conditions that
affect the kidney, destruction of the renal parenchyma and the loss
of functional nephrons. CKD include polycystic kidney disease
(Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal
Recessive Polycystic Kidney Disease (ARPKD), glomerulonephritis,
interstitial nephritis, nephropathy and obstructive uropathy.
[0020] As used herein, the term "predetermined value" refers to the
amount of NGAL in biological samples obtained from the general
population or from a selected population of subjects. For example,
the selected population may be comprised of apparently healthy
subjects, such as individuals who have not previously had any sign
or symptoms indicating the presence of chronic kidney disease
(CKD). In another example, the predetermined value may be of the
amount of NGAL obtained from subjects having an established CKD.
The predetermined value can be a threshold value, or a range. The
predetermined value can be established based upon comparative
measurements between apparently healthy subjects and subjects with
established CKD.
[0021] As used herein, the term "patient" denotes a mammal, such as
a rodent, a feline, a canine, and a primate. Preferably, a patient
according to the invention is a human.
Predictive Methods of the Invention
[0022] The present invention relates to a method for predicting the
progression of chronic kidney disease (CKD) or for monitoring CKD
therapy in a patient,
[0023] comprising the following steps:
[0024] a. providing a biological sample from said patient suffering
from CKD,
[0025] b. determining the expression level of Neutrophil
Gelatinase-Associated Lipocalin (NGAL) gene, and
[0026] c. correlating the expression level of NGAL gene with the
prediction of the progression of CKD.
[0027] In one embodiment, the present invention relates to a method
for predicting the progression of chronic kidney disease (CKD) or
for monitoring CKD therapy in a patient comprising determining the
quantity of mRNA encoding NGAL in a cell or tissue sample obtained
from said patient.
[0028] In a particular embodiment, the tissue sample is a kidney
biopsy.
[0029] Determination of the expression level of a gene can be
performed by a variety of techniques. Generally, the expression
level as determined is a relative expression level.
[0030] More preferably, the determination comprises contacting the
sample with selective reagents such as probes, primers or ligands,
and thereby detecting the presence, or measuring the amount of
nucleic acids of interest originally in the sample.
[0031] In a preferred embodiment, the expression level may be
determined by determining the quantity of mRNA.
[0032] Methods for determining the quantity of mRNA are well known
in the art. For example the nucleic acid contained in the samples
(e.g., cell or tissue prepared from the patient) is first extracted
according to standard methods, for example using lytic enzymes or
chemical solutions or extracted by nucleic-acid-binding resins
following the manufacturer's instructions. The extracted mRNA is
then detected by hybridization (e.g., Northern blot analysis)
and/or amplification (e.g., RT-PCR). In a preferred embodiment, the
expression level of the NGAL gene is determined by RT-PCR,
preferably quantitative or semi-quantitative RT-PCR, even more
preferably real-time quantitative or semi-quantitative RT-PCR.
[0033] Other methods of amplification include ligase chain reaction
(LCR), transcription-mediated amplification (TMA), strand
displacement amplification (SDA) and nucleic acid sequence based
amplification (NASBA).
[0034] Nucleic acids having at least 10 nucleotides and exhibiting
sequence complementarity or homology to the mRNA of interest herein
find utility as hybridization probes or amplification primers. It
is understood that such nucleic acids need not be identical, but
are typically at least about 80% identical to the homologous region
of comparable size, more preferably 85% identical and even more
preferably 90-95% identical. In certain embodiments, it will be
advantageous to use nucleic acids in combination with appropriate
means, such as a detectable label, for detecting hybridization. A
wide variety of appropriate indicators are known in the art
including, fluorescent, radioactive, enzymatic or other ligands
(e.g. avidin/biotin).
[0035] Probes typically comprise single-stranded nucleic acids of
between 10 to 1000 nucleotides in length, for instance of between
10 and 800, more preferably of between 15 and 700, typically of
between 20 and 500. Primers typically are shorter single-stranded
nucleic acids, of between 10 to 25 nucleotides in length, designed
to perfectly or almost perfectly match a nucleic acid of interest,
to be amplified. The probes and primers are "specific" to the
nucleic acids they hybridize to, i.e. they preferably hybridize
under high stringency hybridization conditions (corresponding to
the highest melting temperature Tm, e.g., 50% formamide, 5.times.
or 6.times.SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
[0036] The nucleic acid primers or probes used in the above
amplification and detection method may be assembled as a kit. Such
a kit includes consensus primers and molecular probes. A preferred
kit also includes the components necessary to determine if
amplification has occurred. The kit may also include, for example,
PCR buffers and enzymes; positive control sequences, reaction
control primers; and instructions for amplifying and detecting the
specific sequences.
[0037] In another embodiment, the present invention relates to a
method for predicting the progression of CKD or for monitoring CKD
therapy in a patient comprising measuring the concentration of NGAL
protein in a biological sample obtained from said patient.
[0038] In a particular embodiment, the concentration of the NGAL
protein is measured in a blood sample, a plasma sample, a serum
sample or a urine sample obtained from said patient.
[0039] In still another embodiment, the methods of the invention
comprise contacting the biological sample with a binding partner
capable of selectively interacting with the NGAL protein present in
the biological sample. The binding partner may be an antibody that
may be polyclonal or monoclonal, preferably monoclonal. In another
embodiment, the binding partner may be an aptamer.
[0040] Polyclonal antibodies of the invention or a fragment thereof
can be raised according to known methods by administering the
appropriate antigen or epitope to a host animal selected, e.g.,
from pigs, cows, horses, rabbits, goats, sheep, and mice, among
others. Various adjuvants known in the art can be used to enhance
antibody production. Although antibodies useful in practicing the
invention can be polyclonal, monoclonal antibodies are
preferred.
[0041] Monoclonal antibodies of the invention or a fragment thereof
can be prepared and isolated using any technique that provides for
the production of antibody molecules by continuous cell lines in
culture. Techniques for production and isolation include but are
not limited to the hybridoma technique originally described by
Kohler and Milstein (1975); the human B-cell hybridoma technique
(Cote et al., 1983); and the EBV-hybridoma technique (Cole et al.
1985).
[0042] Alternatively, techniques described for the production of
single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be
adapted to produce anti-NGAL, single chain antibodies. Antibodies
useful in practicing the present invention also include anti-NGAL
fragments including but not limited to F(ab')2 fragments, which can
be generated by pepsin digestion of an intact antibody molecule,
and Fab fragments, which can be generated by reducing the disulfide
bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv
expression libraries can be constructed to allow rapid
identification of fragments having the desired specificity to NGAL.
For example, phage display of antibodies may be used. In such a
method, single-chain Fv (scFv) or Fab fragments are expressed on
the surface of a suitable bacteriophage, e.g., M13. Briefly, spleen
cells of a suitable host, e.g., mouse, that has been immunized with
a protein are removed. The coding regions of the VL and VH chains
are obtained from those cells that are producing the desired
antibody against the protein. These coding regions are then fused
to a terminus of a phage sequence. Once the phage is inserted into
a suitable carrier, e.g., bacteria, the phage displays the antibody
fragment. Phage display of antibodies may also be provided by
combinatorial methods known to those skilled in the art. Antibody
fragments displayed by a phage may then be used as part of an
immunoassay.
[0043] Monoclonal antibodies for NGAL are described, for example,
in Kjeldsen et al., (1996). Examples of commercially available
monoclonal antibodies for NGAL include those obtained from the
Antibody Shop, Copenhagen, Denmark, as HYB-211-01, HYB-211-02, and
NYB-211-05. Typically, HYB-211-01 and HYB-211-02 can be used with
NGAL in both its reduced and unreduced forms. NGAL antibodies can
also be purchased from R&D Systems under reference AF1857.
[0044] In another embodiment, the binding partner may be an
aptamer. Aptamers are a class of molecule that represents an
alternative to antibodies in term of molecular recognition.
Aptamers are oligonucleotide or oligopeptide sequences with the
capacity to recognize virtually any class of target molecules with
high affinity and specificity. Such ligands may be isolated through
Systematic Evolution of Ligands by EXponential enrichment (SELEX)
of a random sequence library, as described in Tuerk C. 1997. The
random sequence library is obtainable by combinatorial chemical
synthesis of DNA. In this library, each member is a linear
oligomer, eventually chemically modified, of a unique sequence.
Possible modifications, uses and advantages of this class of
molecules have been reviewed in Jayasena S. D., 1999. Peptide
aptamers consist of conformationally constrained antibody variable
regions displayed by a platform protein, such as E. coli
Thioredoxin A, that are selected from combinatorial libraries by
two hybrid methods (Colas et al., 1996).
[0045] The binding partners of the invention such as antibodies or
aptamers, may be labelled with a detectable molecule or substance,
such as a fluorescent molecule, a radioactive molecule or any
others labels known in the art. Labels are known in the art that
generally provide (either directly or indirectly) a signal. As used
herein, the term "labelled", with regard to the antibody or
aptamer, is intended to encompass direct labelling of the antibody
or aptamer by coupling (i.e., physically linking) a detectable
substance, such as a radioactive agent or a fluorophore (e.g.
fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or
Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect
labelling of the probe or antibody by reactivity with a detectable
substance. An antibody or aptamer of the invention may be labelled
with a radioactive molecule by any method known in the art. For
example radioactive molecules include but are not limited
radioactive atom for scintigraphic studies such as I123, I124,
In111, Re186, Re188.
[0046] The aforementioned assays generally involve the binding of
the binding partner (ie. antibody or aptamer) to a solid support.
Solid supports which can be used in the practice of the invention
include substrates such as nitrocellulose (e.g., in membrane or
microtiter well form); polyvinylchloride (e.g., sheets or
microtiter wells); polystyrene latex (e.g., beads or microtiter
plates); polyvinylidine fluoride; diazotized paper; nylon
membranes; activated beads, magnetically responsive beads, and the
like.
[0047] The concentration of the NGAL protein may be measured by
using standard immunodiagnostic techniques, including immunoassays
such as competition, direct reaction, or sandwich type assays. Such
assays include, but are not limited to, agglutination tests;
enzyme-labelled and mediated immunoassays, such as ELISAs;
biotin/avidin type assays; radioimmunoassays;
immunoelectrophoresis; immunoprecipitation.
[0048] In a particular embodiment, the concentration of the NGAL
protein is measured by immunoassay.
[0049] More particularly, an ELISA method can be used, wherein the
wells of a microtiter plate are coated with a set of anti-NGAL
antibodies. A biological sample containing or suspected of
containing NGAL is then added to the coated wells. After a period
of incubation sufficient to allow the formation of antibody-antigen
complexes, the plate(s) can be washed to remove unbound moieties
and a detectably labelled secondary binding molecule added. The
secondary binding molecule is allowed to react with any captured
sample marker protein, the plate washed and the presence of the
secondary binding molecule detected using methods well known in the
art.
[0050] Suitable ELISA methods for the detection of NGAL were
described in Kjeldsen et al. (1996), Mishra J. et al. (2005) and
Wang et al. (2007). A sandwich enzyme immunoassay for the detection
of NGAL was described by Blaser J. et al. (1995). A
radioimmunoassay for the detection of NGAL was described by Xu S Y.
et al. (1994).
[0051] ELISA kits for detecting NGAL are commercially available
from Antibody Shop (Grusbakken 8 DK-2820 Gentofte--Denmark) under
the reference KIT 036 or KIT 037, from R&D Systems Europe
(Lille--France) under the reference DLCN20 and from MBL
International, Woburn, Mass. 01801, USA) under reference
CY-8070.
[0052] Measuring the concentration of the NGAL protein (with or
without immunoassay-based methods) may also include separation of
the compounds: centrifugation based on the compound's molecular
weight; electrophoresis based on mass and charge; HPLC based on
hydrophobicity; size exclusion chromatography based on size; and
solid-phase affinity based on the compound's affinity for the
particular solid-phase that is used. Once separated, NGAL may be
identified based on the known "separation profile" e.g., retention
time, for that compound and measured using standard techniques.
[0053] Alternatively, the separated compounds may be detected and
measured by, for example, a mass spectrometer.
[0054] In one embodiment, the method of the invention further may
comprise a step of comparing the concentration of the NGAL protein
with a predetermined threshold value. Said comparison is indicative
of the progression of CKD in the patient or the responsiveness of
the patient to the treatment against CKD.
Therapeutic Methods and Uses
[0055] The invention provides methods and compositions (e.g.
pharmaceutical compositions) for use in the prevention or the
treatment of chronic kidney disease (CKD) in a patient.
[0056] Accordingly, in one aspect the present invention relates to
an inhibitor of NGAL gene expression for use in the prevention or
the treatment of CKD.
[0057] Inhibitors of NGAL gene expression for use in the present
invention may be based on anti-sense oligonucleotide constructs.
Anti-sense oligonucleotides, including anti-sense RNA molecules and
anti-sense DNA molecules, would act to directly block the
translation of NGAL mRNA by binding thereto and thus preventing
protein translation or increasing mRNA degradation, thus decreasing
the level of NGAL, and thus activity, in a cell. For example,
antisense oligonucleotides of at least about 15 bases and
complementary to unique regions of the mRNA transcript sequence
encoding NGAL can be synthesized, e.g., by conventional
phosphodiester techniques and administered by e.g., intravenous
injection or infusion. Methods for using antisense techniques for
specifically inhibiting gene expression of genes whose sequence is
known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135;
6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and
5,981,732).
[0058] Small inhibitory RNAs (siRNAs) may also function as
inhibitors of NGAL gene expression for use in the present
invention. NGAL gene expression can be reduced by contacting a
subject or cell with a small double stranded RNA (dsRNA), or a
vector or construct causing the production of a small double
stranded RNA, such that NGAL gene expression is specifically
inhibited (i.e. RNA interference or RNAi). Methods for selecting an
appropriate dsRNA or dsRNA-encoding vector are well known in the
art for genes whose sequence is known (e.g. see Tuschl, T. et al.
(1999); Elbashir, S. M. et al. (2001); Hannon, G J. (2002);
McManus, M T. et al. (2002); Brummelkamp, T R. et al. (2002); U.S.
Pat. Nos. 6,573,099 and 6,506,559; and International Patent
Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). Short
hairpin RNA (shRNA) may also function as inhibitors of NGAL gene
expression for use in the present invention.
[0059] In one embodiment, the sequence of the shRNA targeting NGAL
(Lcn2) is represented by SEQ ID NO: 1.
[0060] In another embodiment, the sequence of the shRNA targeting
NGAL (Lcn2) is represented by SEQ ID NO: 2.
[0061] Ribozymes may also function as inhibitors of NGAL gene
expression for use in the present invention. Ribozymes are
enzymatic RNA molecules capable of catalyzing the specific cleavage
of RNA. The mechanism of ribozyme action involves sequence specific
hybridization of the ribozyme molecule to complementary target RNA,
followed by endonucleolytic cleavage. Engineered hairpin or
hammerhead motif ribozyme molecules that specifically and
efficiently catalyze endonucleolytic cleavage of NGAL mRNA
sequences are thereby useful within the scope of the present
invention. Specific ribozyme cleavage sites within any potential
RNA target are initially identified by scanning the target molecule
for ribozyme cleavage sites, which typically include the following
sequences, GUA, GUU, and GUC. Once identified, short RNA sequences
of between about 15 and 20 ribonucleotides corresponding to the
region of the target gene containing the cleavage site can be
evaluated for predicted structural features, such as secondary
structure, that can render the oligonucleotide sequence unsuitable.
The suitability of candidate targets can also be evaluated by
testing their accessibility to hybridization with complementary
oligonucleotides, using, e.g., ribonuclease protection assays.
[0062] Both antisense oligonucleotides and ribozymes useful as
inhibitors of NGAL gene expression can be prepared by known
methods. These include techniques for chemical synthesis such as,
e.g., by solid phase phosphoramadite chemical synthesis.
Alternatively, anti-sense RNA molecules can be generated by in
vitro or in vivo transcription of DNA sequences encoding the RNA
molecule. Such DNA sequences can be incorporated into a wide
variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters. Various
modifications to the oligonucleotides of the invention can be
introduced as a means of increasing intracellular stability and
half-life. Possible modifications include but are not limited to
the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or
the use of phosphorothioate or 2'-O-methyl rather than
phosphodiesterase linkages within the oligonucleotide backbone.
[0063] Antisense oligonucleotides siRNAs and ribozymes of the
invention may be delivered in vivo alone or in association with a
vector. In its broadest sense, a "vector" is any vehicle capable of
facilitating the transfer of the antisense oligonucleotide siRNA or
ribozyme nucleic acid to the cells and preferably cells expressing
NGAL. Preferably, the vector transports the nucleic acid to cells
with reduced degradation relative to the extent of degradation that
would result in the absence of the vector. In general, the vectors
useful in the invention include, but are not limited to, plasmids,
phagemids, viruses, other vehicles derived from viral or bacterial
sources that have been manipulated by the insertion or
incorporation of the antisense oligonucleotide siRNA or ribozyme
nucleic acid sequences. Viral vectors are a preferred type of
vector and include, but are not limited to nucleic acid sequences
from the following viruses: retrovirus, such as moloney murine
leukemia virus, harvey murine sarcoma virus, murine mammary tumor
virus, and rouse sarcoma virus; adenovirus, adeno-associated virus;
SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma
viruses; herpes virus; vaccinia virus; polio virus; and RNA virus
such as a retrovirus. One can readily employ other vectors not
named but known to the art.
[0064] Preferred viral vectors are based on non-cytopathic
eukaryotic viruses in which non-essential genes have been replaced
with the gene of interest. Non-cytopathic viruses include
retroviruses (e.g., lentivirus), the life cycle of which involves
reverse transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. Most useful are those
retroviruses that are replication-deficient (i.e., capable of
directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, 1990 and in Murry, 1991).
[0065] Preferred viruses for certain applications are the
adeno-viruses and adeno-associated viruses, which are
double-stranded DNA viruses that have already been approved for
human use in gene therapy. The adeno-associated virus can be
engineered to be replication deficient and is capable of infecting
a wide range of cell types and species. It further has advantages
such as, heat and lipid solvent stability; high transduction
frequencies in cells of diverse lineages, including hemopoietic
cells; and lack of superinfection inhibition thus allowing multiple
series of transductions. Reportedly, the adeno-associated virus can
integrate into human cellular DNA in a site-specific manner,
thereby minimizing the possibility of insertional mutagenesis and
variability of inserted gene expression characteristic of
retroviral infection. In addition, wild-type adeno-associated virus
infections have been followed in tissue culture for greater than
100 passages in the absence of selective pressure, implying that
the adeno-associated virus genomic integration is a relatively
stable event. The adeno-associated virus can also function in an
extrachromosomal fashion.
[0066] Other vectors include plasmid vectors. Plasmid vectors have
been extensively described in the art and are well known to those
of skill in the art. See e.g. Sambrook et al., 1989. In the last
few years, plasmid vectors have been used as DNA vaccines for
delivering antigen-encoding genes to cells in vivo. They are
particularly advantageous for this because they do not have the
same safety concerns as with many of the viral vectors. These
plasmids, however, having a promoter compatible with the host cell,
can express a peptide from a gene operatively encoded within the
plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19,
pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to
those of ordinary skill in the art. Additionally, plasmids may be
custom designed using restriction enzymes and ligation reactions to
remove and add specific fragments of DNA. Plasmids may be delivered
by a variety of parenteral, mucosal and topical routes. For
example, the DNA plasmid can be injected by intramuscular,
intradermal, subcutaneous, or other routes. It may also be
administered by intranasal sprays or drops, rectal suppository and
orally. It may also be administered into the epidermis or a mucosal
surface using a gene-gun. The plasmids may be given in an aqueous
solution, dried onto gold particles or in association with another
DNA delivery system including but not limited to liposomes,
dendrimers, cochleate and microencap sulation.
[0067] In another aspect, the present invention relates to an NGAL
antagonist for use in the prevention or the treatment of CKD.
[0068] In one embodiment the NGAL antagonist may consist in an
antibody (the term including antibody fragment) that can block NGAL
activity.
[0069] Antibodies directed against the NGAL can be raised according
to known methods by administering the appropriate antigen or
epitope to a host animal selected, e.g., from pigs, cows, horses,
rabbits, goats, sheep, and mice, among others. Various adjuvants
known in the art can be used to enhance antibody production.
Although antibodies useful in practicing the invention can be
polyclonal, monoclonal antibodies are preferred. Monoclonal
antibodies against NGAL can be prepared and isolated using any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. Techniques for production and
isolation include but are not limited to the hybridoma technique
originally described by Kohler and Milstein (1975); the human
B-cell hybridoma technique (Cote et al., 1983); and the
EBV-hybridoma technique (Cole et al. 1985). Alternatively,
techniques described for the production of single chain antibodies
(see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce
anti-NGAL single chain antibodies. NGAL antagonists useful in
practicing the present invention also include anti-NGAL antibody
fragments including but not limited to F(ab').sub.2 fragments,
which can be generated by pepsin digestion of an intact antibody
molecule, and Fab fragments, which can be generated by reducing the
disulfide bridges of the F(ab').sub.2 fragments. Alternatively, Fab
and/or scFv expression libraries can be constructed to allow rapid
identification of fragments having the desired specificity to
NGAL.
[0070] Humanized anti-NGAL antibodies and antibody fragments
therefrom can also be prepared according to known techniques.
"Humanized antibodies" are forms of non-human (e.g., rodent)
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region (CDRs) of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Methods for making humanized antibodies are
described, for example, by Winter (U.S. Pat. No. 5,225,539) and
Boss (Celltech, U.S. Pat. No. 4,816,397).
[0071] Then after raising antibodies directed against the NGAL as
above described, the skilled man in the art can easily select those
blocking NGAL activity.
[0072] In another embodiment the NGAL antagonist is an aptamer
directed against NGAL. Aptamers are a class of molecule that
represents an alternative to antibodies in term of molecular
recognition. Aptamers are oligonucleotide or oligopeptide sequences
with the capacity to recognize virtually any class of target
molecules with high affinity and specificity. Such ligands may be
isolated through Systematic Evolution of Ligands by EXponential
enrichment (SELEX) of a random sequence library, as described in
Tuerk C. and Gold L., 1990. The random sequence library is
obtainable by combinatorial chemical synthesis of DNA. In this
library, each member is a linear oligomer, eventually chemically
modified, of a unique sequence. Possible modifications, uses and
advantages of this class of molecules have been reviewed in
Jayasena S. D., 1999. Peptide aptamers consists of a
conformationally constrained antibody variable region displayed by
a platform protein, such as E. coli Thioredoxin A that are selected
from combinatorial libraries by two hybrid methods (Colas et al.,
1996). Then after raising aptamers directed against the NGAL as
above described, the skilled man in the art can easily select those
blocking NGAL activity.
[0073] In still another embodiment, the NGAL antagonist may be a
low molecular weight antagonist, e.g. a small organic molecule. The
term "small organic molecule" refers to a molecule of a size
comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da.
[0074] The inhibitor NGAL gene expression or the NGAL antagonist
may be administered in the form of a pharmaceutical composition.
Preferably, said inhibitor or antagonist is administered in a
therapeutically effective amount.
[0075] By a "therapeutically effective amount" is meant a
sufficient amount of the NGAL antagonist or inhibitor to treat
and/or to prevent chronic kidney disease (CKD) at a reasonable
benefit/risk ratio applicable to any medical treatment.
[0076] It will be understood that the total daily usage of the
compounds and compositions of the present invention will be decided
by the attending physician within the scope of sound medical
judgment. The specific therapeutically effective dose level for any
particular patient will depend upon a variety of factors including
the disorder being treated and the severity of the disorder;
activity of the specific compound employed; the specific
composition employed, the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific polypeptide employed; and like
factors well known in the medical arts. For example, it is well
within the skill of the art to start doses of the compound at
levels lower than those required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved. However, the daily dosage of the products may
be varied over a wide range from 0.01 to 1,000 mg per adult per
day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5,
1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the
active ingredient for the symptomatic adjustment of the dosage to
the patient to be treated. A medicament typically contains from
about 0.01 mg to about 500 mg of the active ingredient, preferably
from 1 mg to about 100 mg of the active ingredient. An effective
amount of the drug is ordinarily supplied at a dosage level from
0.0002 mg/kg to about 20 mg/kg of body weight per day, especially
from about 0.001 mg/kg to 7 mg/kg of body weight per day.
Pharmaceutical Compositions
[0077] The inhibitor of NGAL gene expression or the NGAL antagonist
for use in the prevention or the treatment of chronic kidney
disease (CKD) as defined above may be combined with
pharmaceutically acceptable excipients, and optionally
sustained-release matrices, such as biodegradable polymers, to form
therapeutic compositions.
[0078] In the pharmaceutical compositions of the present invention,
the active principle, alone or in combination with another active
principle, can be administered in a unit administration form, as a
mixture with conventional pharmaceutical supports, to animals and
human beings. Suitable unit administration forms comprise
oral-route forms such as tablets, gel capsules, powders, granules
and oral suspensions or solutions, sublingual and buccal
administration forms, aerosols, implants, subcutaneous,
transdermal, topical, intraperitoneal, intramuscular, intravenous,
subdermal, transdermal, intrathecal and intranasal administration
forms and rectal administration forms.
[0079] Preferably, the pharmaceutical compositions contain vehicles
which are pharmaceutically acceptable for a formulation capable of
being injected. These may be in particular isotonic, sterile,
saline solutions (monosodium or disodium phosphate, sodium,
potassium, calcium or magnesium chloride and the like or mixtures
of such salts), or dry, especially freeze-dried compositions which
upon addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0080] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0081] Solutions comprising compounds of the invention as free base
or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0082] The inhibitor of NGAL gene expression or the NGAL antagonist
of the invention can be formulated into a composition in a neutral
or salt form. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0083] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0084] Sterile injectable solutions are prepared by incorporating
the active polypeptides in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0085] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0086] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual
subject.
[0087] The inhibitor of NGAL gene expression or the NGAL antagonist
of the invention may be formulated within a therapeutic mixture to
comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1
milligrams, or about 0.1 to 1.0 or even about 10 milligrams per
dose or so. Multiple doses can also be administered.
[0088] In addition to the compounds of the invention formulated for
parenteral administration, such as intravenous or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.
tablets or other solids for oral administration; liposomal
formulations; time release capsules; and any other form currently
used.
[0089] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
EXAMPLE
[0090] Material & Methods
[0091] Animals:
[0092] Mice used for these studies were FVB/N, C57BL/6 and
C57BL/6.times.DBA2/F1 (B6D2F1) (Charles River), mutant jck bearing
a Nek8 mutation (Jackson Laboratories), transgenic EGFR-M
expressing a dominant negative isoform of EGFR under the control of
kidney-specific type 1 g-glutamyl transpeptidase promoter (26) and
Lcn2-/- mice (19). Lcn2-/- mice on FVB/N genetic background were
obtained using a marker-assisted speed congenic strategy.
Ninety-three microsatellite markers spanning each autosomal
chromosome (average distance of 14.2 cM) were used to discriminate
C57BL/6 and FVB/N alleles (http://www.cidr.jhmi.edu/mouse).
Heterozygous C57BL/6 Lcn2+/- mice were bred with heterozygous jck
mice to obtain double-homozygous transgenic Lcn2-/-/jck mice. All
experiments were performed on 9-week-old females, except for jck
mice that were studied 3 weeks after birth. Animals were fed ad
libitum and housed at constant ambient temperature in a 12-hour
light cycle. Animal procedures were approved by the Departmental
Director of "Services Veterinaires de la Prefecture de Police de
Paris" and by the ethical committee of the Paris Descartes
University.
[0093] Mice were subjected to 75% nephrectomy (Nx) or
sham-operation (controls), as previously described (26). After
surgery, mice were fed a defined diet containing 30% casein and
0.5% sodium. Several groups of mice were investigated in
complementary studies. For microarray studies, 6 and 9 mice from
each strain were subjected to either sham-operation or Nx,
respectively. For Lcn2 time course analysis, 5-6 sham-operated and
4-8 Nx mice were studied at each time point. Transgenic studies
employed EGFR-M or Lcn2-/- mice and wild-type (WT) littermates; for
each group, 4-6 mice were subjected to sham-operation and 10-16
mice to nephron reduction. For iron chelation experiments, 5
sham-operated and 6 Nx mice were injected with 100 mg/kg/d
Desferroxamine (DFO, Sigma) by subcutaneous osmotic mini-pumps
(2004, Alzet) for 2 months. For hypoxyprobe experiments, 6
sham-operated and 6 Nx mice were injected intraperitoneally with 60
mg/kg Pimonidazole (Chemicon) 2 hours before sacrifice.
Post-ischemic kidneys (2 hours renal pedicle clamping) were used as
positive hypoxic controls.
[0094] Mice were sacrificed 2 months after surgery. In addition,
for Lcn2 time course study, mice were also sacrificed at 4 and 6
weeks after surgery. One week before sacrifice, blood pressure was
recorded in both sham-operated (n=3) and subtotally nephrectomized
(n=6) awake Lcn2+/+ and Lcn2-/- mice for 2 consecutive days, using
tail-cuff plethysmography and PowerLab/4SP software (AD
Instruments). Urine samples were also collected using metabolic
cages from 6 mice of each experimental group over the course of 24
hours. At the time of sacrifice, the kidney was removed for
morphological, protein and mRNA studies.
[0095] Clinical Samples:
[0096] The study was conducted on 87 subjects with autosomal
dominant polycystic kidney disease (ADPKD) (40 M, 47 F; mean age
52.4 years; range 24.7-79.2 years). The mean serum creatinine level
of patients was 252.+-.169.9 mmol/l and the eGFR (assessed using
MDRD formula (53)) was 33.+-.20 ml/min/1.73 m2. 76 over the 87
patients were hypertensive under treatment. The decline of renal
function was evaluated retrospectively over 6 years, then patients
were divided into two groups: slow progressors (eGFR decline<4.5
ml/min/1.73 m2 per year, mean=2.4.+-.0.1, n=52) or fast progressors
(eGFR decline>4.5 ml/min/1.73 m2 per year, mean=6.0.+-.0.2,
n=35).
[0097] Kidneys from patients with ADPKD (n=9), oligomeganephronia
(n=11) and IgA nephropathy (n=12) were analyzed for LCN2
expression. Normal kidneys not used for transplantation or
tumor-free pole of kidneys removed for carcinoma were used as
controls (n=9).
[0098] This protocol was approved by the Hospital Plan for Clinical
Research (PHRC) program of the French Ministry of Health. Informed
consent was obtained before enrollment.
[0099] Cells:
[0100] For siRNA transfection experiments, transient inactivation
of Hif-1.alpha. expression in mIMCD-3 cells was obtained using
siRNA SMARTpool.RTM. from Dharmacon according to manufacturer's
recommendations. Cells were transfected with siRNA (100 nM) using
DharmaFECT.RTM.4 siRNA Transfection Reagent (Thermo Fisher
Scientific). Eight hours after transfection, cells were serum
starved for 12 hours and then treated with 40 ng/mL EGF (R&D
systems) in serum-deprived medium for 48 hours.
[0101] For shRNA transfections, mIMCD-3 cells were stably
transfected with pSuppressor Retro vector (Imgenex) containing a
shRNA for Lcn2 or a scramble oligonucleotide (Dharmacon). The Lcn2
shRNA sequence contains either the cloning nucleotides
5'-ggaaatatgcacaggtatc-3' (SEQ ID NO: 1) or
5'-gctactggatcagaacatt-3' (SEQ ID NO: 2) followed by a 9-base loop
and the inverted cloning sequence. In the scramble sequence, the
cloning sequence is replaced by 5'-gagcgtaccagattaaagt-3' (SEQ ID
NO: 3) or 5'-gattcgaccagacatgtat-3' (SEQ ID NO: 4). Cells stably
transfected were maintained in DMEM/HamF12 medium containing 10%
FBS.
[0102] For EGF experiments, cells were serum-starved for 18 hours
and then treated with 40 ng/mL EGF in serum-deprived medium for
24-96 hours. Cells were collected at 24 hours for Lcn2 assay and
apoptosis experiments and at 24-96 hours for proliferation
experiments.
[0103] cDNA Microarray:
[0104] RNAs were obtained from whole kidneys of 9 Nx mice from each
strain using RNeasy Midi kit (Qiagen) according to the
manufacturer's protocol. RNAs were reverse-transcribed and labeled
with either cyanine Cy-3 or Cy-5. FVB/N Cy3-cDNAs and B6D2F1
Cy5-cDNAs (and conversely FVB/N Cy5- and B6D2F1 Cy3-cDNAs) were
co-hybridized on mouse cDNA microarrays containing 5579 cDNAs
including expressed sequence tags (Genopole.RTM.). Preparations of
RNAs, cDNAs and hybridization were performed according to the
Genopole.RTM. protocol
(http://www.genopole.org/html/en/home/index.php). Six arrays were
hybridized. For each array, the RNAs from 3 mice were pooled.
Hybridized microarrays were scanned and images were analyzed using
Genepix Pro 4.0 software by the Genopole.RTM. microarray
facility.
[0105] Real-Time RT-PCR:
[0106] Lcn2 mRNA was detected in mouse kidneys and mIMCD-3 cells by
real-time RT-PCR using an ABI PRISM 7700 Sequence Detection system
(Applied Biosystems). Gapdh and Sdha were used as the normalization
controls in kidneys and cells, respectively.
[0107] Renal Function and Morphology:
[0108] For mice samples, proteinuria and blood urea nitrogen (BUN)
were measured using an Olympus multiparametric analyzer
(Instrumentation Laboratory), whereas serum creatinine was
evaluated by high performance liquid chromatography (HPLC). For
human samples, creatininuria and albuminuria were measured using a
Hitachi 917 analyzer (Roche Diagnostics).
[0109] Kidneys were fixed in 4% paraformaldehyde, paraffin
embedded, and 4-1 .mu.m sections were stained with PAS, Masson's
trichrome, H&E, picro-sirius red. Ferric iron deposits were
evidenced using Prussian blue staining according to Perls reaction.
The degree of glomerular and interstitial lesions was evaluated
using semiquantitative score methodology as previously described
(7). The degree of tubular lesions was automatically quantified
using a Nikon digital camera Dx/m/1200 and Lucia software
(Laboratory Imaging Ltd). Ten randomly selected microscopic fields
(X200) were scored. For jck mice, all the section was automatically
quantified at magnification X100. The tubular score was expressed
as the ratio between the tubular dilation surface and the total
section area.
[0110] In Situ Hybridization:
[0111] In situ hybridization was carried out on 8-1 .mu.m sections
of paraffin-embedded mouse kidneys using digoxigenin-labeled
riboprobe corresponding to the nucleotides 80 to 641 of the mouse
Lcn2 sequence (NM.sub.--008491). Riboprobe was synthesized using
reagents from Roche, according to the manufacturer's
instructions.
[0112] Western Blot:
[0113] Western blot were performed as previously described (12)
using either a goat antibody to mouse Lcn2 (R&D systems) at
1:1,000 in 1% milk/TBST or a rabbit antibody to mouse Hif-1.alpha.
or Hif-2.alpha. (Novus Biologicals) at 1:500 and 1:200 respectively
in 5% milk/TBST followed by either a rabbit horseradish
peroxidase-conjugated anti-goat antibody at 1:10,000 (Dako) or a
donkey horseradish peroxidase-conjugated anti-rabbit antibody at
1:2,000 (Amersham). Mouse monoclonal .alpha.-tubulin antibody
(Sigma-Aldrich) was used as control. Protein extracts from kidneys
of Lcn2-/- mice were used to confirm antibody specificity.
[0114] Immunohistochemistry:
[0115] For mouse samples, 4-.mu.m sections of paraffin-embedded
kidneys were incubated with a goat anti-mouse Lcn2 antibody
(R&D systems) at 1:300, followed by a rabbit anti-goat
biotinylated antibody (Dako) at 1:200. Biotinylated antibodies were
detected using HRP-labeled streptavidin (Dako) at 1:500 and
3-3'-diamino-benzidine-tetrahydrochloride (DAB) revelation.
[0116] For colocalization experiments, Lotus Tetragonolobus Lectin
(LTL) was detected using a biotinylated-LTL (Vector) at 1:50,
followed by a HRP-labeled streptavidin at 1:500. For Tamm-Horsfall
staining, mouse kidney sections were incubated with a goat
anti-Tamm-Horsfall antibody (Biogenesis) diluted 1:200, followed by
a biotinylated goat antibody (DAKO) at 1:500 and a HRP-labeled
streptavidin at 1:500. For Aquaporin 2 staining, sections were
incubated with a rabbit anti-aquaporin 2 antibody (SIGMA) 1:400,
followed by a donkey HRP-conjugated anti-rabbit antibody (Amersham)
at 1:300. Staining was revealed by DAB.
[0117] For hypoxyprobe staining, 4-.mu.m sections of
paraffin-embedded kidneys were treated with pronase 0.01%, then
incubated with an anti-hypoxyprobe adducuts antibody (Chemicon)
1:200, followed by a biotinylated mouse antibody (DAKO) at 1:500, a
HRP-labeled streptavidin at 1:500 and DAB revelation.
[0118] For human samples, 4-.mu.m sections of paraffin-embedded
kidneys were incubated with a goat anti-human LCN2 antibody
(R&D systems) at 1:100, followed by a HRP-labeled rabbit
anti-goat antibody (Dako) at 1:100 and DAB revelation.
[0119] Cell Proliferation Assay:
[0120] Proliferative cells were detected in mouse kidney using
proliferating cell nuclear antigen (PCNA) or Ki-67 immunostaining.
For PCNA staining, 4-.mu.m sections of paraffin-embedded kidneys
were incubated with a mouse anti-PCNA antibody (DAKO) at 1:50,
followed by a sheep HRP-conjugated anti-mouse antibody (Amersham)
at 1:100. For Ki-67 staining, 4-.mu.m kidney sections were
incubated with a mouse anti-human Ki-67 (BD Pharmingen), followed
by a biotinylated mouse antibody (Vector) at 1:400 and a
HRP-labeled streptavidin at 1:1,000. Staining was revealed by DAB.
The tubular proliferation index (PI) was calculated as the number
of PCNA (or Ki-67)-positive nuclei for the total number of tubular
nuclei in 10 randomly selected fields. The glomerular proliferation
index was calculated as the number of glomeruli with at least one
PCNA-positive nuclei for the total number of glomeruli. In vitro,
proliferation was evaluated by counting the cell number or by using
CellTiter 96.RTM. AQueous Cell Proliferation Reagent (Promega)
according to the manufacturer's instructions.
[0121] Apoptosis Assay:
[0122] Apoptosis was detected in 4-.mu.m sections of
paraffin-embedded kidneys by TUNEL assay using the In Situ Cell
Death Detection kit (Roche) according to the manufacturer's
protocol. The number of apoptotic cells was determined as the
number of TUNEL-positive nuclei per tubule in 20 randomly selected
fields. The glomerular apoptotic index was calculated as the number
of glomeruli with at least one TUNEL-positive nuclei for the total
number of glomeruli. In vitro, apoptotic cells were detected by
DAPI staining and the apoptotic index was calculated as the number
of apoptotic-positive nuclei for the total number of nuclei in 10
randomly selected fields.
[0123] Measurement of Urinary LCN2:
[0124] Fresh urine was collected with protease inhibitors,
centrifuged at 2,000 rpm at 4.degree. C. for 5 minutes and the
supernatant was removed and stored at -80.degree. C. LCN2 was
measured using ELISA (AntibodyShop). Specimens, standards and
reagents were prepared according to manufacturer's instructions.
LCN2 levels were expressed as nanograms per milligram of
creatinine. All experiments were performed in duplicate.
[0125] Data Analysis and Statistics:
[0126] Data were expressed as means.+-.SEM. Differences between the
experimental groups were evaluated using ANOVA, followed when
significant (P<0.05) by the Tukey-Kramer test. When only two
groups were compared, Mann-Whitney or Wilcoxon tests were used. The
Pearson's correlation coefficient was used to test correlation
between variables. For microarray experiments, results are
expressed as a Log 2 of the ratio Cy5/Cy3. Genes with a
false-discovery rate (FDR) <0.05 (using the Benjamini-Hochberg
procedure) and a fold change (FC) >1.5 were considered
significant. The statistical analysis was performed using Graph
Prism Software.
[0127] Results
[0128] Gene Profiling:
[0129] To elucidate the molecular pathways of CKD progression, we
performed unbiased profiling of gene expression in remnant kidneys
of two mouse strains that react differently to nephron reduction.
Using microarrays containing 5579 cDNAs, we found 70 genes whose
expression levels differed significantly two months after nephron
reduction (P<0.05). Among these transcripts, 44 were
up-regulated and 26 were down-regulated in damaged FVB/N kidneys as
compared with well-preserved kidneys from B6D2F1. Grouping these
results by gene ontology category, we observed a range of functions
for the 70 transcripts, although many of the down-regulated mRNA
(38%) regulated metabolic processes. The gene undergoing maximal
transcriptional induction (9.95 fold-change, P=0.008) in the FVB/N
lesion-prone strain was Lipocalin 2 (Lcn2 or neutrophil
gelatinase-associated lipocalin, NGAL; also known as siderocalin,
24p3 or uterocalin).
[0130] Lcn2 Correlates with Lesion Progression in Mouse and Human
with CKD:
[0131] Lcn2 is a member of the lipocalin superfamily (13), a family
of proteins that transport hydrophobic molecules such as retinoids,
fatty acids and organic chelators of iron (14). Real-time RT-PCR
confirmed that Lcn2 mRNA increased 10-fold two months after nephron
reduction in FVB/N but not in B6D2F1 mice, while it was almost
undetectable in control animals. In situ hybridization and
immunohistochemistry corroborated these observations and showed a
marked increase of Lcn2 mRNA and protein in damaged kidneys of
FVB/N mice. Lcn2 was predominantly found in proximal tubules and in
a few ascending limbs of Henle's loops and collecting ducts. High
magnification revealed that Lcn2 was mainly located in cytoplasmic
granules at the subapical zone. By combining in situ hybridization
and immunohistochemistry on serial sections, we found that a
proportion of Lcn2 must have derived from the glomerular filtrate
since in some proximal tubules Lcn2 mRNA was negative while
anti-Lcn2 staining was markedly positive (in situ- and antibody+).
On the other hand, the majority of proximal epithelia that had
undergone dilation and cystic transformation displayed both Lcn2
message and antibody staining (in situ+ and antibody+), indicating
not only endocytosis of filtered protein but ongoing local
synthesis and secretion of Lcn2. Renal Lcn2 mRNA and protein levels
correlated with the intensity of tubular damage (r=0.87, P<0.001
and r=0.74, P<0.01, for mRNA and protein, respectively). In
addition, we observed that renal Lcn2 protein content significantly
correlated with Lcn2 excretion (r=0.99, P<0.01), implicating the
kidney as the major source of urinary Lcn2.
[0132] A careful time course analysis of Lcn2 expression and renal
morphology revealed that the increase of both Lcn2 mRNA and protein
levels preceded the development of renal lesions 4 weeks after
nephron reduction. Moreover, we confirmed that Lcn2 upregulation
was associated with the progressive development of tubular
dilations in another experimental model of CKD, the jck (juvenile
cystic kidney) mice. Of note, these mice develop a form of
polycystic kidney disease similar to the human autosomal dominant
polycystic kidney disease (ADPKD) (15). Lastly, in patients with
ADPKD who are similar to our model in displaying severe and
progressive tubular dilations, LCN2 immunoreactivity was markedly
increased, particularly in cysts. Urinary LCN2 was most prominent
in fast progressors towards ESRF rather than in slow progressors
(496.+-.146 versus 152.+-.52 ng/mg creatinine, P<0.01) and it
inversely correlated with residual eGFR (r=-0.77, P<0.0001) and
microalbuminuria (r=0.72, P<0.0001). Interestingly, LCN2
expression was also increased in renal tubules of kidneys from
patients with either congenital nephron deficit, a pathological
condition very close to nephron reduction, or IgA nephropathy, the
most common primary form of CKD. Our findings in mice and humans
together with recent works (16-18) suggested that Lcn2 might
participate in the pathogenesis of cysts and CKD.
[0133] Lcn2 Gene Inactivation Prevents Lesion Development and Cyst
Formation:
[0134] To determine the role of Lcn2 in progressive CKD, we
performed 75% nephron reduction (Nx) in Lcn2.sup.-/- mice (19). To
this end, we first introduced the Lcn2 mutated allele in the
lesion-prone (FVB/N) background. The Lcn2.sup.-/- FVB/N mice
reproduced normally and had no apparent phenotype under
physiological conditions (data not shown). As expected, two months
after nephron reduction, wild-type mice developed severe renal
lesions, mainly comprising glomerulosclerosis, tubular atrophy and
cystic dilation, mild interstitial fibrosis and multifocal
mononuclear cell infiltration. However, the frequency and severity
of renal lesions were dramatically reduced in Lcn2.sup.-/- mice.
Quantification showed that Lcn2.sup.-/- mice had considerably fewer
glomerular, tubular and interstitial lesions as compared with
wild-type littermates. Notably, there were less tubular dilations
and no cysts in Lcn2.sup.-/- mice. Consistently, renal function was
better preserved in Lcn2.sup.-/- mice as compared to wild-type
littermates, two months after nephron reduction. Serum creatinine
and blood urea nitrogen were 5.+-.0.5, 18.+-.2.6 and 11.+-.0.6
.mu.mol/l (P<0.01) and 29.+-.1, 109.+-.15 and 65.+-.4 mg/dl
(P<0.01) in control, Nx Lcn2.sup.+/+ and Nx Lcn2.sup.-/-,
respectively. As expected, mean arterial blood pressure
significantly increased in wild-type mice as compared to control
animals (135.+-.7.5 and 116.+-.3.4 mm Hg, P<0.05) two months
after nephron reduction. The increase was of same magnitude in
Lcn2.sup.-/- mice (143.+-.2.2 mm Hg). Development of renal lesions
was accompanied by severe proteinuria in wild-type mice
(6.16.+-.1.21 versus 0.003.+-.0.001 mg/day, in Nx and control mice,
respectively, P<0.001), whereas proteinuria was substantially
decreased in Lcn2.sup.-/- animals (3.30.+-.1.03 mg/day, P<0.05).
Of note, Lcn2 inactivation did not change the course of nephron
reduction in lesion-resistant C57BL/6 mice.
[0135] To confirm the beneficial effect of Lcn2 gene inactivation
in renal deterioration and cyst formation, we bred Lcn2.sup.-/-
mice with the jck mice. Notably, the severity of renal lesions was
substantially reduced in double mutant jck/Lcn2.sup.-/- mice.
Quantification showed that the score of tubular dilation was
significantly lower in double mutant mice as compared to jck
littermates three weeks after birth. Collectively, these results
demonstrated that Lcn2 is an effector of renal damage during CKD
progression.
[0136] Iron Accumulation Does not Account for Progressive Renal
Dysfunction:
[0137] We next aimed at elucidating the mechanisms underlying the
lesion promoting effect of Lcn2. Lcn2 might act through iron
mobilization (20). In fact, abnormal levels of iron accumulate in
kidneys during CKD, where it may participate in the deterioration
process (21, 22). Perls staining confirmed that iron content
increased in damaged tubules two months after nephron reduction.
However, iron accumulation was similar in remnant kidneys of
Lcn2.sup.-/- mice as compared with wild-type littermates. More
importantly, chelation of iron by desferroxamine (DFO) unexpectedly
worsened renal disease in FVB/N mice. In particular, tubular
dilations were more severe and diffuse in mice treated with DFO two
months after nephron reduction. Notably, Lcn2 mRNA and protein
expression were dramatically increased in kidneys of DFO-treated
animals as compared with vehicle-treated counterparts.
Proliferation of tubular cells was also significantly enhanced two
months after nephron reduction in DFO-treated mice. Hence, whereas
iron deposited in the proximal tubules does not account for renal
deterioration in our model, the experiments with DFO clearly show
that manipulating Lcn2 levels is tightly correlated with
hyperproliferation and progressive damage.
[0138] Lcn2 is a Target of EGFR Signaling:
[0139] It is known that cell proliferation contributes to the
development of renal lesions, and particularly to cystogenesis
(23). Previous studies have suggested that Lcn2 can be induced by a
number of growth factors that stimulate tubular cell proliferation
(24). Among these, Epidermal Growth Factor Receptor (EGFR) is of
particular interest, since it is critical in the evolution of CKD
(25). We therefore hypothesized that Lcn2 could act downstream of
EGFR and mediate its growth effects. To investigate this
hypothesis, we first treated renal tubular mIMCD-3 cells with EGF.
Western blot analysis revealed that Lcn2 protein levels were
markedly increased after addition of EGF. Quantitative RT-PCR
showed that Lcn2 mRNA levels paralleled the increase of the protein
in EGF-treated cells, indicating that Lcn2 gene is
transcriptionally regulated by EGFR. To validate these findings in
vivo, we took advantage of a line of transgenic mice that
overexpresses a dominant negative EGFR isoform (EGFR-M) selectively
in proximal tubular cells (26). Inhibition of EGFR prevented the
increase of Lcn2 mRNA in remnant kidneys of transgenic mice, two
months after nephron reduction. Consistently, the severity of renal
lesions was substantially reduced in EGFR-M mice as compared with
wild-type littermates.
[0140] Hif-1.alpha. is a Critical Intermediate between EGFR and
Lcn2:
[0141] We next tried to identify the factors that account for Lcn2
transcription upon EGFR activation. The observation that DFO
dramatically stimulated Lcn2 expression after nephron reduction
suggested that hypoxia inducible factors (HIF) might play a role.
In fact, by inhibiting Fe.sup.2+-dependent prolyl hydroxylases, DFO
stabilizes Hif-1.alpha. and Hif-2a (27). Interestingly, our results
showed that Hif-1.alpha. protein levels increased in damaged
kidneys of FVB/N mice two months after nephron reduction. Since
previous studies have shown that hypoxia may develop in damaged
kidneys in CKD (28), we analyzed renal oxygenation two months after
nephron reduction. Pimonidazole hypoxia probe failed to detect any
positive tubules in remnant kidneys of FVB/N mice, with the
exception of those located in the surgical scars, demonstrating
that hypoxia did not account for Hif-1.alpha. overexpression in our
experimental model of CKD. In vitro experiments confirmed that EGF
stimulated Hif-1.alpha. expression in renal mIMCD-3 cells. In fact,
Hif-1.alpha. protein levels markedly increased upon EGF
stimulation. Hif-1.alpha. mRNA levels, determined by real time
RT-PCR, changed neither in vivo after nephron reduction nor, in
vitro upon EGF treatment (data not shown), suggesting that
Hif-1.alpha. is induced via a post-transcriptional mechanism. In
addition, we observed that the increase of Hif-1.alpha. was
specific, since the expression of Hif-2a changed neither in remnant
kidneys, nor in EGF-stimulated cells. More importantly, we showed
that Hif-1.alpha. silencing by siRNA partially inhibited Lcn2
expression either in basal condition and, mainly, upon EGF
stimulation in mIMCD-3 cell lines, indicating that Hif-1.alpha. is
a critical intermediate in EGFR-induced Lcn2 overexpression.
[0142] Lcn2 Mediates the Proliferative Effect of EGFR:
[0143] To next investigate if Lcn2 mediated the mitogenic effect of
EGFR, we established mIMCD-3 cell lines expressing Lcn2 shRNAs.
Quantitative RT-PCR and western blots revealed that Lcn2 mRNA was
depleted by 96% whereas the protein was undetectable in
Lcn2-silenced cells. Interestingly, Lcn2 silencing completely
abolished cell proliferation after the addition of EGF at different
experimental time points. Similar results were obtained by using
different clones and a second shRNA targeting Lcn2 (data not
shown). We found consistent results in our mouse model in vivo. In
fact, Lcn2 gene deletion prevented the increase of tubular cell
proliferation two months after nephron reduction, as reflected in
significantly lower PCNA-positive tubular cells in remnant kidneys
of Lcn2.sup.-/- mice as compared with wild-type littermates.
Notably, Lcn2 gene inactivation did not inhibit the increase of
cell proliferation in glomeruli. These results were confirmed using
an antibody directed against Ki-67, a protein selectively expressed
in proliferating cells. Thus, it appears that Lcn2 is an essential
mediator of the mitogenic effect of EGF in renal tubular cells.
[0144] The Dual Effect of Lcn2 Inactivation on Apoptosis:
[0145] Tubular growth reflects the balance between cell
proliferation and cell loss by apoptosis. Both EGFR and Lcn2 have
been implicated in the control of apoptosis (25, 29). TUNEL
analysis revealed an increase of apoptosis in both tubules and
glomeruli of wild-type mice as compared with control animals two
months after nephron reduction. The number of TUNEL-positive cells
was significantly reduced in Lcn2.sup.-/- mice in both glomerular
and tubular structures. However, Lcn2 silencing did not
significantly affect the number of apoptotic tubular mIMCD-3 cells,
regardless of the presence of EGF.
[0146] Discussion:
[0147] Unbiased profiling analyses offer a powerful approach to
uncover critical mediators and dissect novel molecular networks of
complex biological processes such as CKD progression. By combining
experimental models of CKD, mice from different genetic backgrounds
with microarray analyses, we have established a pivotal role for
Lcn2 in regulating the progression of CKD and cyst formation.
Furthermore, we have defined an important pathophysiological
mechanism by which Lcn2 mediates the mitogenic effect of EGFR,
consistent with its role in cell proliferation in cystogenesis.
Inhibition of this pathway by Lcn2 gene inactivation or by the
expression of a dominant negative EGFR isoform prevented lesion
development in the transgenic mice. Conversely, overexpression of
Lcn2 significantly correlated with hyperproliferation and CKD
progression in both mice and humans. We have further identified
Hif-1.alpha. as a crucial intermediate between EGFR and
Lcn2-upregulation. Collectively, these results elucidate a novel
molecular pathway of CKD progression and show that Lcn2 acts as a
growth-promoting factor whose overexpression identifies patients
with rapid CKD progression.
[0148] Lcn2, like all members of the lipocalin superfamily, binds
hydrophobic ligands; the ligand is thought to define the function
of the protein. Lcn2 binds enterochelin (20), parabactin (20) and
carboxymycobactin (30), which are siderophores produced by bacteria
for the purpose of binding iron. The siderophore-chelating property
of Lcn2 renders it a bacteriostatic agent (20). Consistently, Lcn2
mutant mice have a profound defect in the defense against E. coli
(19, 31) and M. tuberculosis (32). Nonetheless, Lcn2 expression
dramatically increases in several aseptic pathological conditions
such as cancers (33), inflammatory diseases (34) or acute kidney
injury (24), suggesting that Lcn2 may have other functions. To
date, its non-infectious activities have focused on its effects on
cell proliferation and/or apoptosis (24), but proof of these in a
physiological setting in vivo has been lacking. Even in the case of
acute kidney injury, a disease which is related to CKD, it remains
unclear whether Lcn2 is a critical mediator of tubular changes
(19). Hence, our work is the first clear demonstration that in vivo
Lcn2 has a critical function in a pathological condition other than
infection, namely serving as a growth regulator which mediates CKD
progression. Our findings in mice and humans may be generally
applicable to many forms of CKD, because Lcn2 is also expressed in
obstructive uropathy (16), diabetic nephropathy (16), and in
damaged kidneys of patients with IgA nephropathy (17) or
HIV-associated nephropathy (18).
[0149] Our study shows that Lcn2, which is induced by EGFR,
controls aberrant growth of renal tubules and cysts. Notably, we
demonstrated that Lcn2 gene inactivation inhibited proliferation of
tubular cells which led to a marked decrease of cyst formation in
mice. Consistently, we identified cystic tubular epithelia as the
major source of Lcn2 production. These data suggest that Lcn2 might
act as a tubulogenic factor that controls cell growth. This is
supported by additional evidence: first, Lcn2 induced tubular
development in in vitro assays in the rat (35); second, Lcn2
infusion favored tubular regeneration after ischemic injury in mice
(36); third, high Lcn2 levels were associated with higher cystic
growth in humans (37). This property was not limited to mammalian
cells: Lpr-1, a newly identified lipocalin family member,
controlled unicellular tube development in the excretory system of
Caenorhabditis Elegans (38). Our data also suggest that Lcn2 may
modulate tubular shape by controlling both cell proliferation and
apoptosis. In fact, the beneficial effect of Lcn2 gene inactivation
in mutant mice was accompanied by a decrease of tubular apoptosis,
consistent with a previous observation in proximal tubule lacking
Pkdl, a cystic disease-associated gene (39). However, this effect
may be indirect since Lcn2 silencing in vitro did not affect the
number of apoptotic tubular cells. Whether the growth promoting
effect of Lcn2 results by the binding of Lcn2 to a unique receptor,
thus inducing a signaling cascade, or alternatively by iron
mobilization, as suggested by the DFO experiments, remains to be
elucidated.
[0150] In the present study we observed that Lcn2 gene inactivation
protected from glomerulosclerosis and interstitial fibrosis after
nephron reduction, despite the fact that Lcn2 was expressed only by
tubules. The mechanism for this observation remains unknown. It may
result from the perfusion and filtration of serum Lcn2 which we
found by immunostaining in the tubules. Alternatively, since
injuries to tubular cells, i.e. proteinuria, result in the
expression of tubular cytokines and growth factors that ultimately
lead to mesangial cell proliferation and matrix synthesis (40), it
is tempting to evoke a cross-talk between tubular and surrounding
renal cells. Studies in transgenic mice strongly support this idea.
For example, it has been observed that mice that overexpressed VEGF
selectively in tubules developed interstitial fibrosis and
glomerular disease (41). And, we have previously showed that the
overexpression of a dominant negative isoform of EGFR in proximal
tubules prevented the development of glomerular and interstitial
lesions after nephron reduction (26). On the other hand, it has
been shown that interstitial scarring resulted in the loss of
microvessels which, in turn, impacted the adjacent unaffected
glomeruli (42). It is worthy to note that the synthesis of
paracrine mediators may increase in proliferating tubular cells
(43). Hence, we speculate that, by inhibiting tubular cell
proliferation, Lcn2 might protect glomeruli and interstitium from
lesions development.
[0151] Activation of EGFR has been implicated in the evolution of
CKD. Overexpression of an active EGFR form, the c-erb-B2 receptor,
induces tubular hyperplasia and the development of renal cysts in
transgenic mice (44). Conversely, expression of a dominant negative
EGFR isoform inhibits cell proliferation leading to reduced tubular
dilations after nephron reduction (26). Other genetic and
pharmacological approaches have confirmed the key role of EGFR and
cell proliferation in polycystic kidney diseases (45, 46), and
overexpression and mislocalization of EGFR was observed in cystic
epithelia of jck mice (15). On the other hand, we have previously
established that EGFR acts as a central integrator of angiotensin
II, a potent mediator of CKD (47). While the exact molecular
networks that mediate the deleterious effect of EGFR during CKD
have not been yet elucidated, our data point to Lcn2 as the crucial
transcriptional target of EGFR during cyst formation and
glomerulosclerosis. It is worthy to note that a very recent study
showed that Lcn2 is also required for c-erb-B2 receptor signaling
in breast cancer (48). In addition, our data show that Hif-1.alpha.
is a critical intermediate between EGFR and Lcn2, consistent with
the finding that Lcn2 is up-regulated in most pathological
conditions characterized by hypoxia, such as ischemia or cancers
(24, 33). Whether Hif-1.alpha. is more largely involved in the
control of Lcn2 gene expression requires further
investigations.
[0152] Clinical studies have suggested that urinary Lcn2 excretion
might mark patients with the most severe clinical course (49), but
whether Lcn2 is simply a marker of tubular damage or a key mediator
of the deterioration process has been unknown. Our data now show a
direct relation between Lcn2 expression and disease progression and
provide the first demonstration that Lcn2 is instrumental in CKD.
CKD is a progressive disease and there are many possible medical
interventions over its course if the disease is recognized and
treated in a timely manner. Current biomarkers of CKD progression,
i.e. creatinine or albuminuria, have their limitations in this goal
(50). An ideal biomarker should reflect tissue pathology, act as a
critical component of disease and be easily detectable by
non-invasive approaches. By showing that Lcn2 unites these
characteristics, we have provided strong evidence for the use of
this molecule as a candidate biomarker of CKD progression.
[0153] In conclusion, we have uncovered a novel function of Lcn2
and highlighted its crucial role in the pathogenesis of progressive
CKD. This is the first demonstration in vivo that Lcn2 acts as a
growth regulator by mediating the mitogenic effect of EGFR
signaling. Moreover, we have identified Lcn2 as one of the key
effectors of renal damage and cystogenesis and one of the most
promising biomarkers of CKD progression, ready for study in large
patient cohorts. We suspect that our findings will be critical in
other pathological conditions that are also characterized by
aberrant growth, such as cancers which demonstrate both EGFR
activation and intensive Lcn2 expression (51, 52).
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Sequence CWU 1
1
4119DNAArtificialSynthetic Lcn2 shRNA sequence 1ggaaatatgc
acaggtatc 19219DNAArtificialSynthetic Lcn2 shRNA sequence
2gctactggat cagaacatt 19319DNAArtificialSynthetic shRNA scramble
sequence 3gagcgtacca gattaaagt 19419DNAArtificialSynthetic shRNA
scramble sequence 4gattcgacca gacatgtat 19
* * * * *
References