U.S. patent application number 11/016110 was filed with the patent office on 2005-06-23 for treatment of disorders associated with elevated blood glucose or blood pressure.
Invention is credited to DeNichilo, Mark, Riser, Bruce L..
Application Number | 20050136502 11/016110 |
Document ID | / |
Family ID | 26796145 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050136502 |
Kind Code |
A1 |
Riser, Bruce L. ; et
al. |
June 23, 2005 |
Treatment of disorders associated with elevated blood glucose or
blood pressure
Abstract
The present invention relates to methods for treating or
delaying the onset of pathologies associated with elevated blood
glucose and/or elevated blood pressure. The methods are directed to
modulating, regulating, or inhibiting the expression or activity of
Connective Tissue Growth Factor (CTGF) or fragments thereof.
Inventors: |
Riser, Bruce L.; (Marshall,
MI) ; DeNichilo, Mark; (Daly City, CA) |
Correspondence
Address: |
FIBROGEN, INC.
INTELLECTUAL PROPERTY DEPARTMENT
225 GATEWAY BOULEVARD
SOUTH SAN FRANCISCO
CA
94080
US
|
Family ID: |
26796145 |
Appl. No.: |
11/016110 |
Filed: |
December 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11016110 |
Dec 16, 2004 |
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10687479 |
Oct 16, 2003 |
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10687479 |
Oct 16, 2003 |
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09392024 |
Sep 8, 1999 |
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60099471 |
Sep 8, 1998 |
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60112855 |
Dec 16, 1998 |
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Current U.S.
Class: |
435/14 ;
435/6.14; 435/7.2; 514/47; 536/26.1 |
Current CPC
Class: |
A61P 7/04 20180101; C07K
16/22 20130101; A61P 19/04 20180101; A61K 38/00 20130101; A61K
39/395 20130101; A61P 13/12 20180101; C07K 2317/73 20130101; A61P
9/12 20180101; A61P 3/10 20180101; A61K 39/395 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
435/014 ;
536/026.1; 514/047; 435/007.2; 435/006 |
International
Class: |
G01N 033/53; C12Q
001/54; A61K 031/7076; C07H 019/04; C12Q 001/68; G01N 033/567 |
Claims
What is claimed is:
1. A method of identifying an agent that modulates CTGF induction
by glucose, the method comprising (a) culturing a first cell
population in media containing high glucose; (b) culturing a second
cell population in media containing high glucose and an agent; (c)
measuring CTGF level in the culture media from the first and second
cell populations; and (d) comparing CTGF level in the culture media
from the second cell population to CTGF level in the culture media
from the first cell population, wherein a difference in CTGF level
between the second and first cell populations is indicative of an
agent that modulates CTGF induction by glucose.
2. The method of claim 1, wherein the glucose concentration in the
media containing high glucose is at least about 20 mM.
3. The method of claim 1, wherein the glucose concentration in the
media containing high glucose is at least about 35 mM.
4. The method of claim 1, wherein the first and second cell
populations are of the same cell type and are fibroblasts.
5. The method of claim 4, wherein the fibroblasts are kidney
mesangial cells.
6. An agent that modulates CTGF induction by glucose, wherein the
agent is identified by the method of claim 1.
7. The agent of claim 6, wherein the agent additionally increases
cyclic nucleotide level in a cell.
8. A method of reducing expression of CTGF in a subject having
elevated blood glucose, the method comprising administering the
agent of claim 6 to a subject having or at risk for having high
blood glucose.
9. A method of identifying an agent that modulates CTGF induction
associated with mechanical stress, the method comprising (a)
culturing a first cell population in the absence of agent under
conditions that place the cells under increased mechanical stress;
(b) culturing a second cell population in the presence of agent
under conditions that place the cells under increased mechanical
stress; (c) measuring CTGF level in the culture media from first
and second cell populations; and (d) comparing CTGF level in the
culture media from the second cell population to CTGF level in the
culture media from the first cell population, wherein a difference
in CTGF level between the second and first cell populations is
indicative of an agent that modulates CTGF induction associated
with mechanical stress.
10. The method of claim 9, wherein mechanical stress is cyclic
stretching of cells up to and including 19% maximum elongation.
11. The method of claim 9, wherein the first and second cell
populations are of the same cell type and are fibroblasts.
12. The method of claim 11, wherein the fibroblasts are kidney
mesangial cells.
13. An agent that modulates CTGF induction associated with
mechanical stress, wherein the agent is identified by the method of
claim 9.
14. A method of reducing expression of CTGF in a subject having
elevated blood pressure, the method comprising administering the
agent of claim 13 to a subject having elevated blood pressure.
15. A method of treating or delaying onset of a disorder associated
with elevated blood glucose, the method comprising administering to
a subject in need an effective amount of an agent that modulates,
regulates, or inhibits the expression or activity of CTGF or
fragments thereof.
16. The method of claim 15, wherein the subject has or is at risk
of having hyperglycemia.
17. The method of claim 15, wherein the agent is an antibody that
specifically binds to CTGF or fragments thereof.
18. The method of claim 15, wherein the agent is an antisense
oligonucleotide that specifically binds to a polynucleotide
sequence encoding CTGF.
19. The method of claim 15, wherein the agent is a small
molecule.
20. The method of claim 19, wherein the agent additionally
increases cyclic nucleotide levels in a cell.
21. The method of claim 15, wherein the disorder is diabetes.
22. The method of claim 15, wherein the disorder is a renal
disorder.
23. The method of claim 22, wherein the renal disorder is selected
from the group consisting of glomerulosclerosis,
glomerulonephritis, and diabetic nephropathy.
24. The method of claim 15, wherein the disorder is an ocular
disorder.
25. The method of claim 24, wherein the ocular disorder is diabetic
retinopathy.
26. A method of treating or delaying onset of a disorder associated
with elevated blood pressure, the method comprising administering
to a subject in need an effective amount of an agent that
modulates, regulates, or inhibits the expression or activity of
CTGF or fragments thereof.
27. The method of claim 26, wherein the agent is an antibody that
specifically binds to CTGF or fragments thereof.
28. The method of claim 26, wherein the agent is an antisense
oligonucleotide that specifically binds to a polynucleotide
sequence encoding CTGF.
29. The method of claim 26, wherein the agent is a small
molecule.
30. The method of claim 29, wherein the agent additionally
increases cyclic nucleotide levels in a cell.
31. The method of claim 26, wherein the disorder is diabetes.
32. The method of claim 26, wherein the disorder is a renal
disorder.
33. The method of claim 32, wherein the renal disorder is selected
from glomerulosclerosis, glomerulonephritis, and diabetic
nephropathy.
34. The method of claim 26, wherein the disorder is an ocular
disorder.
35. The method of claim 34, wherein the ocular disorder is diabetic
retinopathy.
36. A method of treating or delaying onset of a disorder associated
with renal hypertension, the method comprising administering to a
subject in need an effective amount of an agent that modulates,
regulates, or inhibits the expression or activity of CTGF or
fragments thereof.
37. The method of claim 36, wherein the agent is an antibody that
specifically binds to CTGF or fragments thereof.
38. The method of claim 36, wherein the agent is an antisense
oligonucleotide that specifically binds to a polynucleotide
sequence encoding CTGF.
39. The method of claim 36, wherein the agent is a small
molecule.
40. The method of claim 39, wherein the agent additionally
increases cyclic nucleotide levels in a cell.
41. The method of claim 36, wherein the disorder is diabetes.
42. The method of claim 36, wherein the disorder is a renal
disorder.
43. The method of claim 42, wherein the renal disorder is selected
from glomerulosclerosis, glomerulonephritis, and diabetic
nephropathy.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/687,479, filed Oct. 16, 2003, which is a
continuation of U.S. patent application Ser. No. 09/392,024, filed
on Sep. 8, 1999; and claims the benefit of U.S. Provisional
Application Ser. No. 60/099,471, filed on Sep. 8, 1998; and U.S.
Provisional Application Ser. No. 60/112,855, filed on Dec. 16,
1998; all of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the role of Connective
Tissue Growth Factor (CTGF) in the production of extracellular
matrix. More specifically, the invention relates to methods of
detecting, preventing, and treating kidney fibrosis and other
conditions associated with overproduction of the extracellular
matrix by targeting CTGF.
BACKGROUND OF THE INVENTION
[0003] Kidney Diseases And Disorders. The kidney functions to
separate waste products from the blood, regulate acid
concentration, and maintain water balance. Kidneys control the
levels of various compounds in the blood, such as hydrogen, sodium,
potassium, and silicon, and eliminate waste in the form of urine.
Any degradation in kidney function can interfere with the body's
ability to adequately remove metabolic products from the blood, and
can disrupt the body's electrolyte balance. In its most severe
forms, degradation or impairment of kidney function can be
fatal.
[0004] A number of conditions can lead to chronic renal failure, a
decline in kidney function over time. For example, such conditions
as hypertension, diabetes, congestive heart failure, lupus, and
sickle cell anemia have been associated with renal failure. Acute
disease processes and injuries can trigger a more immediate decline
in kidney function.
[0005] It is thus well understood that individuals with diabetes,
hypertension, inflammatory and autoimmune diseases, and other
disorders are at risk for altered and progressive loss of kidney
function characterized by, for example, reduced glomerular
filtration, albuminuria, proteinuria, and progressive renal
insufficiency. More than half of the total number of kidney
disorders initiate kidney fibrosis. Fibrosis involves altered
formation or production of fibrous tissue, and can result in the
overproduction and increased deposition of extracellular matrix
components.
[0006] The extracellular matrix (ECM) is a complex network of
various glycoproteins, polysaccharides, and other macromolecules
secreted from a cell into extracellular space. The ECM provides a
supportive framework, directly influencing various cellular
characteristics, including shape, motility, strength, flexibility,
and adhesion. In fibrosis, overproduction and increased deposition
of ECM materials can result in thickening and malformation of
various membranous and cellular components, reducing local
flexibility and surface area of the affected site, and impairing a
number of bodily processes.
[0007] Kidney fibrosis is a common pathway in the progression of
various forms of renal injury. Kidney fibrosis typically spreads by
enlisting previously undamaged regions of the kidney. As normal
filtration processes decline, function of surviving tissue and of
various regions of the kidney is systematically destroyed. Kidney
fibrosis can be manifested as a diffuse thickening of kidney
membranous components, the accumulation and expansion leading to a
loss of filtration surface area and a corresponding disruption in
the body's electrolyte composition and acid-base balance.
[0008] Fibrosis of the kidney is observed in a number of
conditions, including, for example, diabetic, autoimmune, and
transplant nephropathy; hypertension; and certain forms of
glomerular injury or disease. Diabetes mellitus (diabetes) is a
complex disease that affects several hundred million people
worldwide. Diabetes is characterized by hyperglycemia or elevated
levels of glucose in the blood. Glucose cannot enter the body's
cells to be utilized and therefore remains in the blood in high
concentrations. When the blood glucose level exceeds the
reabsorptive capacity of the renal tubules, glucose is excreted in
the urine. Diabetes produces a number of debilitating and
life-threatening complications.
[0009] Progressive nephropathy is one of the most frequent and
serious complications of diabetes. See, e.g., Hans-Henrik et al.,
1988, Diabetic Nephropathy: The Second World Conference on Diabetes
Research, New Frontiers. The Juvenile Diabetes Foundation
International, pp. 28-33. A hallmark of diabetic nephropathy, and
of renal sclerosis due to other forms of renal injury, is early
expansion of the glomerular mesangium, largely due to increased
accumulation of ECM proteins such as collagen types I and IV,
fibronectin, and laminin. See, e.g., Mauer et al., 1984, J Clin
Invest 74:1143-1155; Bruneval et al., 1985, Human Pathol 16:477484.
This pathological deposition results in impaired filtration,
leading to renal failure, a condition requiring transplantation or
life-long dialysis. Current therapies slow but do not arrest or
reverse the progressive loss of kidney function. Predominant causal
factors identified to date also include hyperglycemia, glomerular
hypertension, and abnormal cytokine environments. Tuttle, et al.,
1991, N Engl J Med 324:1626-1632; The Diabetes Control
Complications Trial Research Group, 1993, N Engl J Med 329:977-986;
Hostetter et al., 1981, Kidney Int 19:410-415; Anderson et al.,
1985, J. Clin. Invest 76;612-619; Border et al., 1993, Am J Kidney
Dis 22:105-113.
[0010] Hyperglycemia may be damaging, in great part as increased
concentrations of glucose stimulate ECM accumulation by mesangial
cells. See, e.g., Ayo et al., 1990, Am. J. Pathol. 136:1339-1348;
Heneda et al., 1991, Diabetologia 34:190-200; Nahman et al., 1992,
Kidney Int 41:396402; Cortes et al., 1997, Kidney Int. 51:57-68. As
shown by Davies et al., 1992, Kidney Intl. 41:671-678, mesangial
cells are largely responsible for mesangial matrix synthesis in
situ. It has further been determined that the effect of glucose on
mesangial cell matrix production is linked to increased glucose
transport and utilization. Helig et al., 1995, J. Clin. Invest.
96:1802-1814. Moreover, Ziyadeh et al., 1994, J Clin Invest.
93:536-542, have shown the involvement of secreted soluble
mediators on mesangial cell matrix production.
[0011] Renal hypertension, which can appear as a secondary
manifestation of kidney disease in diabetic patients, can also
result from other diseases or disorders, including long-standing
hypertension. Secondary hypertension can be caused by virtually any
impairment in renal function. A greater understanding of the
pathogenic mechanisms for hypertension-induced ECM deposition is
developing. For example, in diabetes, an early impairment of normal
blood pressure dampening occurs at the glomerular afferent
arteriole, resulting in the exposure of glomerular capillaries to
large moment-to-moment variations in systemic blood pressure.
Hayashi et al., 1992, J Am Soc Nephrol 2:1578-1586; Bidani et al.,
1993, Am J Physiol 265:F391-F398. Due to the elasticity of the
glomerulus, increased capillary pressure produces expansion of
glomerular structure, resulting in augmentation of the mechanical
strain imposed on the mesangial cells. Riser et al., 1992, J Clin
Invest 90:1932-1943; Kriz et al., Kidney Int Suppl 30:S2-S9. In
addition, when cultured mesangial cells are subjected to cyclic
strain, the mesangial cells respond by increasing the synthesis and
accumulation of collagen types I and IV, fibronectin, and laminin.
Riser et al., 1992, supra. While increased glomerular pressure is
common in diabetes, it is not limited to this disease, and is
present in other forms of progressive renal disorders, including,
for example, certain forms of glomerular nephritis and hypertrophy.
See, e.g., Cortes et al., 1997, Kidney Int 51:57-68.
[0012] Kidney fibrosis and associated renal impairment are thus
present in the progression of various diseases and disorders,
including diabetes and hypertension, and methods of treating kidney
fibrosis are thus greatly desired.
[0013] Transforming Growth Factor .beta.(TGF-.beta.). The few
studies conducted to date regarding the physiological implications
of renal disorders and diseases, and, in particular, those due to
diabetes, have focused on the role of transforming growth
factor-.beta. (TGF-.beta.) in developing methods for targeting
overproduction (increased synthesis and accumulation) of
extracellular matrix components. The role of cytokine imbalance in
initiating and/or perpetuating glomerular matrix expansion has been
explored in experimental nephropathy studies involving TGF-.beta..
See, e.g., Sharma et al., Seminars In Nephrology 1:116-129.
Glomerular TGF-.beta. activity is increased in both human and
experimental diabetic glomerulosclerosis. See, e.g., Yamamoto et
al., 1993, Proc Natl Acad Sci 90:1814-1818; Sharma et al., 1994, Am
J Physiol 267:F1094-F1101; Shankland et al., 1994, Kidney Int
46:430-442. The exposure of cultured mesangial cells or glomeruli
to TGF-.beta. results in increased ECM production. See, e.g.,
Bollineni et al., 1993, Diabetes 42:1673-1677. In vivo induction of
glomerular matrix accumulation following transfection and
overexpression of the TGF-.beta. gene in rat kidney has been
demonstrated by, for example, Isaka et al., J Clin Invest
92:2597-2601.
[0014] In addition, neutralization studies have shown that
anti-TGF-.beta. antibody mitigates the enhanced glomerular ECM gene
expression that occurs in experimental glomerulonephritis and
diabetes. Border et al., 1990, Nature 346:371-374; Sharma et al.,
1996, Diabetes 45:522-530. The sustained overexpression of
glomerular TGF-.beta. in diabetes may be the result of a mesangial
cellular response to both increased glucose levels and
hypertension. It has been reported that exposure of mesangial cells
to increased concentrations of glucose in the medium stimulates the
synthesis and release of TGF-.beta.1, as well as the increased
binding of TGF-.beta. to specific receptors. Ziyadeh et al., 1994,
J Clin Invest 93:536-542; Riser et al., 1998, J Am Soc Nephrol
9:827-836; Riser et al., 1999, Kidney Int 56:428439. It has also
been reported that mechanical force selectively stimulates the
production, release, and activation of TGF-.beta.1, as well as the
increased expression of TGF-.beta. receptors. Riser et al., 1996,
Am J Path 148:1915-1923.
[0015] In vitro neutralization studies of TGF-.beta. demonstrated a
significant reduction of collagen synthesis induced in mesangial
cells by increased glucose levels. See, e.g., Sharma et al., 1996,
supra; Ziyadeh et al., 1994, supra. Studies have also shown a
virtual elimination of collagen accumulation resulting from cyclic
stretching in the presence of excess glucose. Riser et al., 1997,
supra. TGF-.beta. stimulates the proliferation of mesangial cells
in vitro and in vivo, and may induce in these replicating cells
overproduction and increased deposition of ECM characteristic of
various renal disorders, including proliferative disorders such as
glomerular nephritis. See, e.g., Border et al., 1990, Nature
346:371-374; Habershroh et al., 1993, Am J Physiol 264:F199-205. As
a result of these findings, intense efforts have been directed
toward reducing TGF-.beta. availability and binding as a means of
mitigating matrix accumulation. However, the ubiquitous nature and
pluripotent functions of TGF-.beta., including tumor suppression
and the multiple levels of regulation, raise questions concerning
both the feasibility and the safety of its long-term inhibition.
See, e.g., Brattain et al., 1996, Curr Opin Oncol 8:49-53;
Franklin, 1997, Int J Biochem Cell Biol 29:79-89.
[0016] Therefore, a method for treating or preventing ECM
overproduction or increased deposition, without interfering with
the ubiquitous function of TGF-.beta., is needed.
[0017] Connective Tissue Growth Factor (CTGF). CTGF is a peptide
that may act downstream of TGF-.beta. to regulate matrix
accumulation. This novel growth factor has been reported and
described previously. See, e.g., U.S. Pat. No. 5,408,040; Bradham
et al., 1991, J Cell Biol 114:1285-1294. CTGF is characterized as a
polypeptide which exists as a monomer with a molecular weight of
approximately 36 to 38 kD. CTGF has been shown to be one of seven
cysteine-rich secreted proteins belonging to the CCN family, which
includes CTGF, cyr-61, and nov. Oemar et al., 1997, Arterioscler
Thromb Vasc Biol 17(8):1483-1489. CTGF is an immediate early
response gene that codes for a protein consisting of four modules
and one signal peptide. Oemar et al., 1997, supra. The four modules
are: 1) an insulin-like growth factor (IGF) binding domain, 2) a
von Willebrand factor type C repeat most likely involved in
oligomerization, 3) a thrombospondin type 1 repeat believed to be
involved in binding to the ECM, and 4) a C-terminal module which
may be involved in receptor binding. Recent reports suggest that
certain fragments of the whole CTGF protein possess CTGF activity.
See, e.g., Brigstock, et al., 1997, J Biol Chem
272(32):20275-20282. Human, mouse, and rat CTGF are highly
conserved with greater than 90% amino acid homology and a molecular
weight of about 38 kD. It was recently shown that the promoter of
CTGF contains a novel TGF-.beta. responsive element. Grotendorst et
al., 1996, Cell Growth Differ 7:469-480.
[0018] It appears that CTGF may be an important prosclerotic
molecule in both skin fibrosis and cardiac atherosclerosis. For
example, CTGF mRNA is expressed by fibroblasts in the lesions of
patients with systemic sclerosis, keloids, and localized
scleroderma, while there is no coresponding expression in adjacent
normal skin. See, e.g., Igarashi et al., 1995, J Invest Dermatol
105:280-284; Igarashi et al., 1996, J Invest Dermatol 106:729-733.
Cultured normal human skin fibroblasts respond to TGF-.beta. but
not to platelet-derived growth factor (PDGF), epidermal growth
factor (EGF), or basic fibroblast growth factor (bFGF), by
increasing levels of CTGF mRNA and CTGF protein. Igarashi et al.,
1993, Mol Biol Cell 4:637-645. Fibroblasts from lesions of
scleroderma show increased mitogenesis to TGF-.beta. and produce
greater amounts of CTGF than do normal fibroblasts. Kikuche et al.,
1995, J Invest Dermatol 105:128-132. Recombinant human CTGF
injected under the skin of NIH Swiss mice induces the same rapid
and dramatic increase in connective tissue cells and ECM as occurs
with TGF-.beta. treatment, whereas PDGF and EGF have little or no
effect on granulation. Frazier et al., 1996, J Invest Dermatol
107:404-411. Cultured vascular smooth muscle cells are also
stimulated by TGF-.beta. to produce CTGF. In heart disease
patients, CTGF mRNA is expressed at levels 50- to 100-fold higher
in atherosclerotic plaques than in normal arteries. Oemar et al.,
1997, Circulation 95(4):831-839.
[0019] In spite of mounting evidence implicating CTGF as a causal
factor in skin fibrosis and cardiac atherosclerosis, very little is
known of its expression in, for example, renal sclerosis or
diabetes. It has been shown, using an in vitro model of calcium
oxalate nephrolithasis, that monkey kidney epithelial cells respond
to calcium oxalate by upregulating the CTGF gene along with other
genes involved in matrix turnover. Hammes et al., 1995, Kidney Int
48:501-509. A similar response occurs in cultured renal epithelial
cells following mechanical wounding. See, e.g., Pawar et al., 1995,
J Cell Physiol 165:556-565. Most recently, CTGF mRNA was found in
biopsies from normal human kidneys. A qualitative assessment
indicated that, in a limited number of cases, CTGF expression was
increased in the tissues of patients with severe mesangial
proliferative lesions of crescentic glomerulonephritis, focal and
segmented glomerulosclerosis, and, in three cases, diabetic
glomerulosclerosis. Ito et al., 1998, Kidney Int 53:853-861. The
research, relying only on data obtained from biopsies, did not
include quantitative results or any measurement of CTGF protein
levels. Further, no connection between CTGF mRNA levels and the
production and deposition of ECM, and no quantitative method for
detecting renal disorders or diseases, including diabetes,
involving a determination of CTGF levels in samples, and did not
identify CTGF-expressing cells.
[0020] The role of CTGF in kidney diseases is thus unclear, and
there has been no research to date has shown that CTGF is causally
related to ECM overproduction and increased deposition and to
fibrosis in the kidney.
[0021] Diagnostics and Early-Stage Detection. Kidney failure is a
serious condition requiring extreme treatment such as hemodialysis
or transplantation. Early-stage detection and/or prevention of any
deviation from normal kidney pathology and function could minimize
the risk of a subject's developing a more serious condition.
Hypertension, for example, might be undetectable by a patient in
early stages, but can be deadly if not identified, monitored, and
treated. In addition, in some diseases, such as, for example,
diabetes, less invasive and disruptive and more affordable means of
treatment, such as dietary modification, are effective only at
early stages. Therefore, there is a critical need for effective and
reliable methods of diagnosis that permit early stage detection,
and corresponding prevention, of renal complications.
[0022] For example, kidney failure resulting from progressive
glomerulosclerosis is the leading cause of morbidity and mortality
among patients with type I, or juvenile, diabetes mellitus. See,
e.g., Dorman et al., 1984, Diabetes 33:271-276; Anderson et al.,
1983, Diabetologia 25:496-501. Current therapy with
angiotensin-converting enzyme (ACE) inhibitors, the drug class of
choice, effectively slows the progression of disease. See, e.g.,
Lewis et al., 1993, N Eng J Med 329:1456-1462. Nevertheless, this
treatment is not justified in all newly diagnosed diabetic patients
because only approximately 30-35% of these develop progressive
kidney disease, and the long-term side effects of these drugs are
uncertain. See, e.g., Parving and Hommel, 1989, Brit Med J
299:230-233. In addition, ACE inhibitors are also presently used to
treat patients with hypertensive renal failure, including that
resulting from non-diabetic nephropathies. However, the mechanism
of renal protection, and, as noted above, the long-term side
effects of this treatment are not fully understood. Furthermore,
ACE inhibitors have been shown to negatively interact with
nonsteroidal anti-inflammatory drugs. See, e.g., Whelton, 1999, Am
J Med 106(SB):13S-24S.
[0023] In a current method of diagnosis, diabetic patients are
monitored for microalbuminuria. Persistent microalbuminuria is a
marker of widespread vascular damage and indicates the presence of
early nephropathy in type 1 and type 2 diabetes. See, e.g.,
Stehouwer et al., 1992, Lancet 340:319-323; Bojestig et al., 1996,
Diabetes Care 19:313-317; Mogensen et al., 1995, Lancet
346:1080-1084. However, the actual level of microalbuminuria may
not necessarily predict the development of overt nephropathy,
particularly among patients with a long duration of diabetes.
Bojestig et al., supra. In addition, since by the time
microalbuminuria is detected, structural renal lesions are already
present, the effectiveness of treatment to slow progression may be
substantially reduced. Bangstad et al., 1993, Diabetologia
36:523-529; Ruggenenti et al., 1998, J Am Soc Nephrol 9:2157-2169;
Fioretto et al., 1995, Kidney Int 48:1929-1935. There is a great
need to be able to predict which patients with type 1 diabetes will
develop nephropathy, and to, in general, develop a method that will
detect renal alterations that may precede the onset of significant
disease.
[0024] In summary, there is a need in the art for effective methods
for diagnosing, treating, and preventing fibrosis associated with
impairment and degradation of kidney function in a variety of
diseases and disorders, most particularly, in diabetes and
hypertension. No current research has focused on the modulation of
CTGF expression or activity as a means of preventing or treating
kidney fibrosis.
SUMMARY OF THE INVENTION
[0025] The present invention satisfies the need in the art by
providing methods for detecting, treating, and preventing renal
disorders and diseases associated with fibrosis. In particular, the
present invention provides methods for detecting, preventing, and
treating pathologies and complications associated with renal
disorders and conditions which are characterized by an
overproduction or increased deposition of extracellular matrix.
[0026] Methods Of Treatment and Prevention. The present invention
provides various approaches directed to modulation of the
overproduction of the extracellular matrix resulting in fibrosis.
Specifically, the present invention provides methods of regulating
increased accumulation of the extracellular matrix associated with
kidney fibrosis as found in various renal diseases and disorders.
These renal diseases and disorders include, but are not limited to,
all kinds of nephropathy, including glomerulonephritis,
glomerulosclerosis, and conditions resulting from glomerular
injury; diabetic nephropathy and other complications; nephritis;
interstitial disease; acute and chronic transplant rejection; renal
hypertension, including that associated with diabetes; and other
underlying causes of fibrosis. More specifically, the present
invention provides methods for preventing and treating
complications associated with the above-named renal disease and
disorders by regulating, modulating, and/or inhibiting the
expression and activity of CTGF. In particular embodiments, the
present methods are directed to the diagnosis, prevention, and
treatment of renal diseases and disorders associated with diabetes
or with hypertension.
[0027] The methods of the present invention provide for the
administration of a therapeutically effective amount of an agent
that regulates, modulates, and/or inhibits the ECM-producing
activity of CTGF. In particular, the methods of this invention are
useful for the treatment and prevention of renal disorders in
mammals, most preferably, in humans.
[0028] In one aspect, the invention provides a method of treating
complications associated with diabetes characterized by the
overproduction or overaccumulation of the extracellular matrix by
administering a therapeutically effective amount of an antibody
reactive with a CTGF polypeptide or fragments thereof, or an
antigen-binding fragment of an antibody reactive with the CTGF
polypeptide or fragments thereof.
[0029] In another aspect, the present invention provides a method
for treating and preventing complications associated with renal
disorders, particularly, diabetes and hypertension, wherein
antisense oligonucleotides which specifically bind to CTGF mRNA are
used to interrupt expression of the protein product. The antisense
oligonucleotides have a sequence capable of binding specifically
with any polynucleotide sequences encoding CTGF or fragments
thereof.
[0030] In yet a further embodiment of the present invention, a
method is provided in which small molecules are used to inhibit the
activity of CTGF or its active fragments by blocking the binding of
CTGF to its receptor, inhibiting CTGF activity and thus thereby
reducing the overproduction of the extracellular matrix associated
with the onset and/or progression of renal disorders, including
diabetes and hypertension.
[0031] The present invention further provides a method of treating
and preventing renal disorders by administering a compound that
blocks the binding interactions of or the enzymes involved in the
signal transduction pathway of CTGF.
[0032] The present invention also provides a method for treating
and preventing diabetes by administering insulin and an agent that
modulates and/or inhibits the activity of CTGF. More specifically,
the present invention discloses a method for treating and
preventing diabetes by administering insulin and an agent that
modulates, regulates, and/or inhibits the activity of CTGF
according to the methods of the present invention.
[0033] Methods for evaluating the effectiveness of anti-fibrotic
therapy, including the use of ACE inhibitors, by measuring the
levels of CTGF in a sample from a subject undergoing a course of
treatment for diseases and disorders associated with fibrosis, are
also provided.
[0034] Diagnostic Methods. The present invention is also directed
to methods for predicting which patients with diseases and
disorders associated with renal disorders will subsequently develop
progressive kidney disease. In one embodiment, the invention
provides a method for detecting and/or staging (classifying the
level, site, and spread of disease) kidney involvement in a
particular disease or disorder. In one embodiment, a method of
predicting whether a patient with diabetes will go on to develop
progressive kidney disease is provided, along with a method of
detecting the current level of kidney involvement, for example, in
a subject with diabetes as opposed to a subject without
diabetes.
[0035] The present invention is also directed to methods of
detecting the presence of pathology of a tissue characterized by an
excessive accumulation of extracellular matrix components. In one
embodiment, the method involves determining the levels of CTGF, for
example, through tissue biopsy or through non-intrusive methods,
such as, for example, collection of a urine sample. In a particular
embodiment, the method comprises determining the levels of CTGF in
a sample comprising, for example, urine or other bodily fluids from
a subject with a diabetic nephropathy, such as, for example,
diabetic glomerulosclerosis. The method can also comprise
determining the levels of CTGF in persons with progressive
sclerosis, both with and without diabetes, by determining the
levels of CTGF in urine or in other bodily fluids.
[0036] More specifically, the present invention comprises a means
of diagnosing the presence of or a predisposition to kidney
diseases and disorders, including a means for detecting and
monitoring the pathogenesis of these diseases and disorders, or for
detecting and monitoring the presence of markers for the
pathogenesis of these diseases and disorders. More specifically,
the present invention provides for diagnosing renal disorders by
measuring the levels of CTGF in a patient sample, preferably, in a
urine sample from a patient.
[0037] In one embodiment of the present invention, a method is
provided for the measurement of CTGF levels in a sample from a
patient with no known or with a suspected renal disorder.
Comparison of CTGF levels in urine samples from a patient known to
have a kidney disease or disorder, or from a patient known not to
have any kidney disease or disorder, with CTGF levels in urine
samples from a patient with no known or with a suspected kidney
disorder can be indicative of the presence of a kidney disease or
disorder. In particular, the method provides that higher levels of
CTGF are present in samples from patients having renal disorders
than in samples from patients without any renal disorder. Higher
levels of CTGF are thus indicative of the presence of a disease or
disorder associated with kidney fibrosis.
[0038] In another embodiment, the levels of CTGF can be measured by
detecting CTGF mRNA or protein in a sample. In a further
embodiment, the sample is a tissue sample and the presence of CTGF
is detected by staining of the protein in the tissue or by
determination of CTGF mRNA levels.
[0039] A preferred method of the present invention utilizes an
antibody, preferably, a monocolonal antibody, capable of
specifically binding to CTGF or active fragments thereof. The
method of utilizing an antibody to measure the levels of CTGF
allows for non-invasive diagnosis of the pathological states of
kidney diseases. In a preferred embodiment of the present
invention, the antibody is human or is humanized. The preferred
antibodies may be used, for example, in standard radioimmunoassays
or enzyme-linked immunosorbent assays or other assays which utilize
antibodies for measurement of levels of CTGF in sample. In a
particular embodiment, the antibodies of the present invention are
used to detect and to measure the levels of CTGF present in a urine
sample.
[0040] Diagnostic Kit. The present invention is further directed to
diagnostic kits for detecting and measuring the levels of CTGF in a
sample in order to detect a renal disorder or a predisposition to a
renal disorder in a subject. In one embodiment, the kit contains
antibodies specific for CTGF and reagents for detecting and
measuring CTGF in a sample. The sample can be a bodily fluid, such
as urine, or, for example, a tissue sample. In one embodiment of
the present invention, the kit comprises an immobilized antibody
which specifically recognizes CTGF and an antibody specific for
CTGF and capable of binding to an antigen component different from
the immobilized antibody. The CTGF antibody can be enzyme-labeled,
radio-labeled, or fluoroscein-labeled. The kit can also comprises
reagents necessary for detection of the antibody, and can further
comprise other reagents as desired, such as, for example,
dissolving agents, cleaning agents, and reaction terminators.
[0041] In a preferred embodiment of the invention, the kit is
packaged, for example, in a box or a container which includes the
necessary elements of the kit, and also includes instructions
relating to the use of the kit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1A and FIG. 1B depict the effects of exogenous CTGF on
mesangial cell secretion of the extracellular matrix. FIG. 1A shows
the quantities of fibronectin contained in the media at the end of
incubation determined by ELISA. FIG. 1B shows the quantities of
collagen type I contained in the media at the end of incubation
determined by ELISA.
[0043] FIG. 2 depicts CTGF gene expression in rat tissues and
cultured kidney cells. Total RNA was extracted from whole organs of
rat and from cultured rat mesangial cells and kidney fibroblasts
for Northern analysis. MC represents mesangial cells; BT represents
brain tissue; HT represents heart tissue; KT represents kidney
tissue; and KFC represents kidney fibroblast cells.
[0044] FIG. 3A, FIG. 3B, and FIG. 3C depict regulation of CTGF and
TGF-.beta. mRNA levels by exogenous TGF-.beta. and CTGF. FIG. 3A
shows results from a representative experiment. FIG. 3B shows
quantitation of mRNA bands for CTGF. FIG. 3C shows quantitation of
mRNA bands for TGF-.beta..
[0045] FIG. 4A and FIG. 4B depict expression of CTGF protein by
culture mesangial cells in the presence of exogenous TGF-.beta..
FIG. 4A shows immunoblotting using an antibody raised against full
length CTGF. FIG. 4B shows immunoblotting using an antibody raised
against a 15 amino acid sequence specific to CTGF.
[0046] FIG. 5A and FIG. 5B depict secretion of CTGF protein into
the medium of mesangial cell cultures, and the effect of heparin.
FIG. 5A shows data relating to media pooled and heparin-sepharose
treated for immunoblotting. FIG. 5B shows data relating to media
tested for CTGF content individually by ELISA prior to pooling.
[0047] FIG. 6A and FIG. 6B depict CTGF protein induction by
mesangial cells. FIG. 6A shows data relating to media pooled and
heparin-sepharose treated for immunoblotting. FIG. 6A shows data
relating to media tested for CTGF content individually by ELISA
prior to pooling.
[0048] FIG. 7 depicts the effect of high glucose concentration on
mesangial cells expression of CTGF mRNA. FIG. 7 shows samples of
pooled RNA from 6 different 100 mm culture dishes in a
representative experiment.
[0049] FIG. 8 depicts TGF-.beta. blockade of high glucose-induced
CTGF production using anti-TGF-.beta. antibody.
[0050] FIG. 9 depicts CTGF concentrations in mesangial cells
overexpressing different levels of GLUT1. Supernatants from
duplicate culture cells transduced with the GLUT1 gene denoted
MCGT1 or a transfection control LaZ gene (MCLaZ).
[0051] FIG. 10 depicts the effect of cyclic stretching on mesangial
cell expression of CTGF transcripts. At the indicated periods, RNA
was extracted and probed for CTGF message. Each lane represents the
results of the samples pooled from 24 different culture wells.
[0052] FIG. 11 depicts blockade of stimulated collagen type I
production by and anti-CTGF antibody.
[0053] FIG. 12 depicts blockade of stimulated mesangial cell
proliferation by anti-CTGF antibody.
[0054] FIG. 13A and FIG. 13B depict glomerular disease associated
with diabetes in db/db mice. FIG. 13A shows renal cortical section
from control db/m mice at 5 months of age. FIG. 13B shows renal
cortical section from diabetic db/db mice at 5 months of age. The
sections from FIG. 13A and FIG. 13B were stained with PAS for light
microscopy examination, and are examples of glomeruli demonstrating
the most severe mesangial expansion observed in the diabetic
group.
[0055] FIG. 14A, FIG. 14B, and FIG. 14C depict induction of CTGF
and fibronectin transcripts in whole kidney of diabetic db/db mice
at 5 months of age. FIGS. 14A and 14B show total RNA extracted from
whole kidneys and probed by northern analysis for CTGF mRNA and
fibronectin mRNA, respectively. The letter "C" represents
nondiabetic mice, while the letter "D" represents diabetic mice.
FIG. 4C shows quantification by denositometric analysis of the
results of the Northern analyses.
[0056] FIG. 15A and FIG. 15B depict competitive RT-PCR for GAPDH
and CTGF mRNA in a single sample from diabetic mouse glomeruli.
Ethidium bromide-stained gel after PCR amplification. The lanes of
FIGS. 11A and 11B contain a constant amount of test cDNA and 2-fold
decreasing concentrations of a known amount of the specific mimic.
FIG. 15A shows competitive reverse transcriptase PCR (RT-PCR) for
GAPDH. FIG. 15B shows competitive RT-PCR for CTGF.
[0057] FIG. 16 depicts the effects of diabetes on the glomerular
expression of CTGF and fibronectin transcript levels in db/db mice,
detected by competitive RT-PCR.
[0058] FIG. 17 depicts CTGF and its recovery in normal urine. Four
aliquots were spiked with a different amount of CTGF, and a fifth
served as a control. Immunoblotting was performed using a CTGF
specific antibody.
[0059] FIG. 18 depicts the analysis of CTGF protein in urine of
diseased patients or healthy volunteers. Urine samples from 8
patients with kidney disease or 3 normal volunteers were assayed
for CTGF by immunoblotting.
DETAILED DESCRIPTION OF THE INVENTION
[0060] It is understood that the present invention is not limited
to the particular methodologies, protocols, cell lines, and
reagents, etc., described herein, as these may vary. It is also to
be understood that the terminology used herein is used for the
purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention. It must be
noted that as used herein and in the claims, the singular forms
"a," "an," and "the" include plural references unless the context
clearly dictates otherwise. Thus, for example, a reference to "an
antibody" is a reference to one or more antibodies and any
equivalents thereof known to those skilled in the art.
[0061] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Preferred methods, devices, and materials are described, although
any similar or equivalent methods can be used in the practice or
testing of the present invention. All patents, publications, and
other references cited herein are incorporated by reference herein
in their entirety.
[0062] Definitions
[0063] As used herein, the term "extracellular matrix" refers
broadly to non-cellular matrix, typically composed of proteins,
glycoproteins, complex carbohydrates, and other macromolecules.
Extracellular matrix components include, for example, collagen
types I and IV, fibronectin, laminin, and thrombospondin.
[0064] The term "fibrosis" refers to abnormal processing of fibrous
tissue, or fibroid or fibrous degeneration. Fibrosis can result
from various injuries or diseases, and can often result from
chronic transplant rejection relating to the transplantation of
various organs. Fibrosis typically involves the abnormal
production, accumulation, or deposition of extracellular matrix
components, including overproduction and increased deposition of,
for example, collagen and fibronectin.
[0065] As used herein, the terms "kidney fibrosis" or "renal
fibrosis" or "fibrosis of the kidney" refer to diseases or
disorders associated with the overproduction or abnormal deposition
of extracellular matrix components, particularly collagen, leading
to the degradation or impairment of kidney function. The terms
"disorders" and "diseases" are used inclusively and refer to any
condition deviating from normal. "Diseases" and "disorders"
include, but are not limited to, allograft and transplant
rejection, acute and chronic, and any transplant nephropathy; acute
and chronic kidney failure; autoimmune nephropathy; diabetic
nephropathy; glomerulonephritis, glomerulosclerosis, and other
forms of glomerular abnormality or injury; hypertension;
hypertrophy; interstitial disease; nephritis; sclerosis, an
induration or hardening of tissues and/or vessels resulting from
causes that include, for example, inflammation due to disease or
injury; renal-associated proliferative disorders; and other primary
or secondary nephrogenic conditions. Fibrosis associated with
dialysis following kidney failure and catheter placement, e.g.,
peritoneal and vascular access fibrosis, is also included.
[0066] It is understood that, while kidney fibrosis is the model
for discussion of the present invention, the mechanism of fibrosis
is universal. Therefore, the presently described methods, kits, and
other aspects of the present invention could also be directed to
the diagnosis, prevention, and treatment of other forms of fibrosis
and diseases and disorders associated with fibrosis and
proliferation, including, but not limited to: cardiac fibrosis,
pulmonary fibrosis, diabetic retinopathy, skin fibrosis,
scleroderma, atherosclerosis, arteriosclerosis, hypertropic
scarring, keloid formation, arthritis, liver fibrosis,
inflammation, tumor growth metastasis, other conditions related to
cell proliferation and migration, including those associated with
vascular endothelial cells, for example, angiogenesis and
neovascularization, etc.
[0067] The term "sample" is used herein in its broadest sense.
Samples may be derived from any source, for example, from bodily
fluids, secretions, or tissues including, but not limited to,
saliva, blood, urine, and organ tissue (e.g., biopsied tissue);
from chromosomes, organelles, or other membranes isolated from a
cell; from genomic DNA, cDNA, RNA, mRNA, etc.; and from cleared
cells or tissues, or blots or imprints from such cells or tissues.
A sample can be in solution or can be, for example, fixed or bound
to a substrate. A sample can refer to any material suitable for
testing for the presence of CTGF or suitable for screening for
molecules that bind to CTGF or fragments thereof. Methods for
obtaining such samples are within the level of skill in the
art.
[0068] An "antisense sequence" is any sequence capable of
specifically hybridizing to a target sequence. The antisense
sequence can be DNA, RNA, or any nucleic acid mimic or analog. The
term "antisense technology" refers to any technology which relies
on the specific hybridization of an antisense sequence to a target
sequence.
[0069] The terms "modulation" and "regulation" as used with respect
to CTGF expression or activity refer to any direction of or effect
on CTGF expression or activity as compared to normal or to
unaltered CTGF expression or activity.
[0070] Invention
[0071] A. CTGF and its Role in Fibrosis and Renal Disorders
[0072] The present invention is based on the discovery that CTGF is
an important mediator of extracellular accumulation in fibrotic
conditions, and, in particular, in fibrotic conditions associated
with renal disorders, such as diabetes and glomerular hypertension.
More specifically, the present invention is based on the discovery
that the production of CTGF by glomerular cells (in particular,
mesangial cells) is a potentially important factor in the
pathogenesis of diseases and disorders of the kidney. It was
discovered that increased levels of CTGF induced increased
production and deposition of ECM in mesangial cells. It was further
found in an analysis of urine samples that healthy subjects had no
or very minimal levels of CTGF in their urine, while the urine of
diabetic patients or patients with other renal disorders showed
increased levels of CTGF.
[0073] To demonstrate that CTGF is a critical determinant of
extracellular matrix deposition in the kidney, CTGF expression in
mesangial cells, glomeruli, and whole kidney was examined under
diabetic and non-diabetic conditions. Mesangial cells cultured in
media containing normal levels of glucose expressed low levels of
CTGF mRNA and secreted barely detectable amounts of the full length
CTGF protein. However, in a hyperglycemic environment in which
mesangial cells were exposed to elevated glucose levels,
upregulation of CTGF expression and increased protein production
were detected. Moreover, mechanical strain of mesangial cells,
exhibitive of, for example, glomerulosclerosis, glomerular
hypertension, and glomerular hypertrophy, demonstrated an
upregulated expression and protein production of CTGF in mesangial
cells. Thus, the present invention demonstrates that exposure to
conditions such as increased glucose concentrations, mechanical
force, or TGF-.beta. led to upregulated expression and protein
production of CTGF, establishing a nexus between the presence of
CTGF and renal disorders, in particular, diabetes and hypertension.
Even in the absence of hypertension, conditions that produce, for
example, glomerular hypertrophy, often a result of renal injury,
can result in induce increased capillary vessel diameter. According
to Laplace's law, vessel wall tension is correspondingly increased,
and increased mesangial cell stretching forces are likely produced.
See Cortes et al., 1997, supra.
[0074] B. Methods for Modulating and Inhibiting Activity of
CTGF
[0075] Connective Tissue Growth Factor (CTGF) is a critical
determinant of extracellular matrix deposition in kidney fibrotic
conditions. The present invention provides methods for the
diagnosis, prevention, and treatment of complications associated
with kidney fibrosis, preferably, by regulating, modulating, and/or
inhibiting the expression or activity of CTGF. More specifically,
methods of the present invention provide for the administration of
a therapeutically effective amount of an agent that regulates,
modulates, and/or inhibits the extracellular matrix producing
activity of CTGF.
[0076] Antibodies. In one embodiment of the present invention,
methods for diagnosis, prevention, and treatment of renal disorders
and diseases involve the administration of a therapeutically
effective amount of an antibody which specifically reacts with a
CTGF polypeptide or fragments thereof.
[0077] CTGF antibodies may be generated using methods well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, single chain antibodies, as well
as Fab fragments, including F(ab').sub.2 and F.sub.v fragments.
Fragments can be produced, for example, by a Fab expression
library. Neutralizing antibodies, i.e., those which inhibit dimer
formation, are especially preferred for therapeutic use.
[0078] A target polypeptide, such as CTGF or an agent that
modulates the activity and or expression of CTGF, can be evaluated
to determine regions of high immunogenicity. Methods of analysis
and epitope selection are well known in the art. See, e.g., Ausubel
et al., eds., 1988, Current Protocols in Molecular Biology, John
Wiley & Sons, Inc., New York N.Y. Analysis and selection can
also be accomplished, for example, by various software packages,
such as LASERGENE NAVIGATOR software. (DNASTAR; Madison Wis.) The
peptides or fragments used to induce antibodies should be
antigenic, but are not necessarily biologically active. Preferably,
an antigenic fragment or peptide is at least 5 amino acids in
length, more preferably, at least 10 amino acids in length, and
most preferably, at least 15 amino acids in length. It is
preferable that the antibody-inducing fragment or peptide is
identical to at least a portion of the amino acid sequence of the
target polypeptide, e.g., CTGF. A peptide or fragment that mimics
at least a portion of the sequence of the naturally occurring
target polypeptide can also be fused with another protein, e.g.,
keyhole limpet hemocyanin (KLH), and antibodies can be produced
against the chimeric molecule.
[0079] Methods for the production of antibodies are well known in
the art. For example, various hosts, including goats, rabbits,
rats, mice, humans, and others, may be immunized by injection with
the target polypeptide or any immunogenic fragment or peptide
thereof. Depending on the host species, various adjuvants may be
used to increase immunological response. Such adjuvants include,
but are not limited to, Freund's adjuvant, mineral gels such as
aluminum hydroxide, and surface-active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0080] Monoclonal and polycolonal antibodies may be prepared using
any technique which provides for the production of antibody
molecules by continuous cell lines in culture. Techniques for in
vivo and in vitro production are well known in the art. See, e.g.,
Pound, 1998, Immunochemical Protocols, Humana Press, Totowa N.J.;
Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, New York N.Y. The production of chimeric
antibodies is also well known, as is the production of single-chain
antibodies. See, e.g., Morrison et al., 1984, Proc Natl Acad Sci
81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454. Antibodies with related specificity,
but of distinct idiotypic composition, may be generated, for
example, by chain shuffling from random combinatorial immunoglobin
libraries. See, e.g., Burton, 1991, Proc Natl Acad Sci
88:11120-11123.
[0081] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents. See, e.g., Orlandi et al., 1989, Proc Natl Acad Sci
86:3833-3837; Winter and Milstein, 1991, Nature 349:293-299).
Antibody fragments which contain specific binding sites for the
target polypeptide may also be generated. Such antibody fragments
include, but are not limited to, F(ab').sub.2 fragments, which can
be produced by pepsin digestion of the antibody molecule, and Fab
fragments, which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed to allow rapid and easy identification
of monoclonal Fab fragments with the desired specificity. See,
e.g., Huse et al., 1989, Science 254:1275-1281.
[0082] Antibodies can be tested for anti-target polypeptide
activity using a variety of methods well known in the art. Various
techniques may be used for screening to identify antibodies having
the desired specificity, including various immunoassays, such as
enzyme-linked immunosorbent assays (ELISAs), including direct and
ligand-capture ELISAs, radioimmunoassays (RIAs), immunoblotting,
and fluorescent activated cell sorting (FACS). Numerous protocols
for competitive binding or immunoradiometric assays, using either
polyclonal or monoclonal antibodies with established specificities,
are well known in the art. See, e.g., Harlow and Lane. Such
immunoassays typically involve the measurement of complex formation
between the target polypeptide and a specific antibody. A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on the target polypeptide
is preferred, but other assays, such as a competitive binding
assay, may also be employed. See, e.g., Maddox et al, 1983, J Exp
Med 158:1211.
[0083] Antibodies as described above could also be used to identify
CTGF or fragments thereof in tissue, e.g., from a kidney biopsy.
The amount of CTGF present could be determined, for example, by
quantitative image analysis. CTGF mRNA levels could also be
determined, such as by reverse transcriptase polymerase chain
reaction (PCR) using portions of the biopsied tissue, e.g.,
glomeruli. In particular, in this method, mRNA from a tissue
sample, in total, or that specific for CTGF or fragments thereof,
could be transcribed to DNA and then amplified through PCR using
CTGF-specific primer sequences. Quantitation of mRNA for CTGF or
fragments thereof could be determined, for example, by a
competition reaction using equal volumes of the patient sample run
against a series of decreasing known concentrations, e.g., of a
mimic or mutant cDNA fragment.
[0084] The present invention contemplates the use of antibodies
specifically reactive with a CTGF polypeptide or fragments thereof
which neutralize the biological activity of CTGF. The antibody
administered in the method can be the intact antibody or antigen
binding fragments thereof, such as Fab, F(ab').sub.2, and F.sub.v
fragments, which are capable of binding the epitopic determinant.
The antibodies used in the method can be polyclonal or, more
preferably, monoclonal antibodies. Monoclonal antibodies with
different epitopic specificities are made from antigen containing
fragments of the protein by methods well known in the art. See,
e.g., Kohler et al., 1975, Nature 256:495-497; Ausubel, et al.,
supra.
[0085] In the present invention, therapeutic applications include
those using "human" or "humanized" antibodies directed to CTGF or
fragments thereof. Humanized antibodies are antibodies, or antibody
fragments, that have the same binding specificity as a parent
antibody, (i.e., typically of mouse origin) and increased human
characteristics. Humanized antibodies may be obtained, for example,
by chain shuffling or by using phage display technology. For
example, a polypeptide comprising a heavy or light chain variable
domain of a non-human antibody specific for a CTGF is combined with
a repertoire of human complementary (light or heavy) chain variable
domains. Hybrid pairings specific for the antigen of interest are
selected. Human chains from the selected pairings may then be
combined with a repertoire of human complementary variable domains
(heavy or light) and humanized antibody polypeptide dimers can be
selected for binding specificity for an antigen. Techniques
described for generation of humanized antibodies that can be used
in the method of the present invention are disclosed in, for
example, U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761; and
5,693,762. Furthermore, techniques described for the production of
human antibodies in transgenic mice are described in, for example,
U.S. Pat. Nos. 5,545,806 and 5,569,825.
[0086] In another embodiment of the present invention, a method
involves the administration of a therapeutically effective amount
of an antibody reactive to a CTGF responsive receptor, and, more
specifically, an antibody which blocks the binding of CTGF to its
cellular receptors. The method of the present invention provides
that the antibody reactive with CTGF modulates and/or inhibits the
biological activity of CTGF through the manipulation and control of
the interaction between CTGF and its receptor by inactivation of
the receptor independently of CTGF.
[0087] Antisense Oligonucleotides. The present invention provides
for a therapeutic approach which directly interferes with CTGF
expression. Specifically, a therapeutic approach which directly
interrupts the translation of CTGF mRNA into protein could be used
to bind to CTGF mRNA or to otherwise interfere with CTGF
expression. Antisense technology relies on the modulation of
expression of a target protein through the specific binding of an
antisense sequence to a target sequence encoding the target protein
or directing its expression. See, e.g., Agrawal, ed., 1996,
Antisense Therapeutics, Humana Press, Inc., Totowa N.J.; Alama et
al., 1997, Pharmacol Res 36(3):171-178; Crooke, 1997, Adv Pharmacol
40:1-449; and Lavrosky et al.,1997, Biochem Mol Med 62(1):11-22.
Antisense sequences are nucleic acid sequences capable of
specifically hybridizing to at least a portion of a target
sequence. Antisense sequences can bind to cellular mRNA or genomic
DNA, blocking translation or transcription and thus interfering
with expression of a targeted protein product. Antisense sequences
can be any nucleic acid material, including DNA, RNA, or any
nucleic acid mimics or analogs. See, e.g., Rossi et al., 1991
Antisense Res Dev 1(3):285-288; Pardridge et al., 1995, Proc Natl
Acad Sci 92(12):5592-5596; Nielsen and Haaima, 1997, Chem Soc Rev
96:73-78; and Lee et al., 1998, Biochemistry 37(3):900-1010.
Delivery of antisense sequences can be accomplished in a variety of
ways, such as through intracellular delivery using an expression
vector. See discussion, infra. Site-specific delivery of exogenous
genes is also contemplated, such as techniques in which cells are
first transfected in culture and stable transfectants are
subsequently delivered to the target site. See, e.g., Kitamura et
al., 1994, Kidney Int 43:S55-S58.
[0088] Antisense oligonucleotides of about 15 to 25 nucleic acid
bases are typically preferred as such are easily synthesized and
are capable of producing the desired inhibitory effect. Molecular
analogs of antisense oligonucleotide may also be used for this
purpose and can have added advantages such as stability,
distribution, or limited toxicity advantageous in a pharmaceutical
product. In addition, chemically reactive groups, such as
iron-linked ethylenediamine-tetraacetic acid (EDTA-Fe), can be
attached to antisense oligonucleotides, causing cleavage of the RNA
at the site of hybridization. These and other uses of antisense
methods to inhibit the in vitro translation of genes are well known
in the art. See, e.g., Marcus-Sakura, 1988, Anal Biochem
172:289.
[0089] Delivery of antisense therapies and the like can be achieved
intracellularly through using a recombinant expression vector such
as a chimeric virus or a colloidal dispersion system which, upon
transcription, produces a sequence complementary to at least a
portion of the cellular sequence encoding the target protein. See,
e.g., Slater et al., 1998, J Allergy Clin Immunol 102(3):469-475.
Delivery of antisense sequences can also be achieved through
various viral vectors, including retrovirus and adeno-associated
virus vectors. See, e.g., Miller, 1990, Blood 76:271; and Uckert
and Walther,1994, Pharacol Ther 63(3):323-347. Vectors which can be
utilized for antisense gene therapy as taught herein include, but
are not limited to, adenoviruses, herpes viruses, vaccinia, or,
preferably, RNA viruses such as retroviruses.
[0090] Retroviral vectors are preferably derivatives of murine or
avian retrovirus. Retroviral vectors can be made target-specific by
inserting, for example, a polynucleotide encoding a protein or
proteins such that the desired ligand is expressed on the surface
of the viral vector. Such ligand may be a glycolipid carbohydrate
or protein in nature. Preferred targeting may also be accomplished
by using an antibody to target the retroviral vector. Those of
skill in the art will know of, or can readily ascertain without
undue experimentation, specific polynucleotide sequences which can
be inserted into the retroviral genome to allow target specific
delivery of the retroviral vector containing the antisense
polynucleotide.
[0091] Recombinant retroviruses are typically replication
defective, and can require assistance in order to produce
infectious vector particles. This assistance can be provided by,
for example, using helper cell lines that contain plasmids encoding
all-of the structural genes of the retrovirus under the control of
regulatory sequences within the LTR. These plasmids are missing a
nucleotide sequence which enables the packaging mechanism to
recognize an RNA transcript for encapsidation. Helper cell lines
which have deletions of the packaging signal may be used. These
cell lines produce empty virions, since no genome is packaged. If a
retroviral vector is introduced into such cells in which the
packaging signal is intact, but the structural genes are replaced
by other genes of interest, the vector can be packaged and vector
virion produced.
[0092] Other gene delivery mechanisms that can be used for delivery
of antisense sequences to target cells include colloidal dispersion
and liposome-derived systems, artificial viral envelopes, and other
systems available to one of skill in the art. See, e.g., Rossi,
1995, Br Med Bull 51(1):217-225; Morris et al., 1997, Nucleic Acids
Res 25(14):2730-2736; and Boado et al., 1998, J Pharm Sci
87(11):1308-1315. For example, delivery systems can make use of
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes.
[0093] In one embodiment, a method of the present invention
administers a therapeutically effective amount of an antisense
oligonucleotide having a sequence capable of binding specifically
with any sequences of an mRNA molecule which encodes CTGF, so as to
prevent translation of CTGF mRNA.
[0094] In another embodiment of the present invention, a method is
provided in which a therapeutically effective amount of an
antisense oligonucleotide having a sequence capable of binding
specifically with any sequences of CTGF mRNA so as to prevent
translation of the mRNA.
[0095] Small Molecule Inhibitors. The present invention further
provides a method in which small molecules are used to inhibit the
activity of CTGF by blocking the binding of responsive cytokines to
the CTGF responsive receptor. For example, the present invention
provides methods of treating and preventing kidney fibrosis
utilizing small molecules that modulate, regulate and inhibit CTGF
activity.
[0096] In order to identify small molecules and other agents useful
in the present methods for treating or preventing a renal disorder
by modulating the activity and expression of CTGF, CTGF and
biologically active fragments thereof can be used for screening
therapeutic compounds in any of a variety of screening techniques.
Fragments employed in such screening tests may be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. The blocking or reduction of biological activity
or the formation of binding complexes between CTGF and the agent
being tested can be measured by methods available in the art.
[0097] Other techniques for drug screening which provide for a high
throughput screening of compounds having suitable binding affinity
to CTGF, or to another target polypeptide useful in modulating,
regulating, or inhibiting the expression and/or activity of CTGF,
are known in the art. For example, microarrays carrying test
compounds can be prepared, used, and analyzed using methods
available in the art. See, e.g., Shalon, D. et al., 1995,
International Publication No. WO95/35505, Baldeschweiler et al.,
1995, International Publication No. WO95/251116; Brennan et al.,
1995, U.S. Pat. No. 5,474,796; Heller et al., 1997, U.S. Pat. No.
5,605,662.
[0098] Identifying small molecules that modulate CTGF activity can
also be conducted by various other screening techniques, which can
also serve to identify antibodies and other compounds that interact
with CTGF and can be used as drugs and therapeutics in the present
methods. See, e.g., Enna et al., eds., 1998, Current Protocols in
Pharmacology, John Wiley & Sons, Inc., New York N.Y. Assays
will typically provide for detectable signals associated with the
binding of the compound to a protein or cellular target. Binding
can be detected by, for example, fluorophores, enzyme conjugates,
and other detectable labels well known in the art. See, e.g., Enna
et al., supra. The results may be qualitative or quantitative.
[0099] For screening the compounds for specific binding, various
immunoassays may be employed for detecting, for example, human or
primate antibodies bound to the cells. Thus, one may use labeled
anti-hIg, e.g., anti-hIgM, hIgG or combinations thereof to detect
specifically bound human antibody of the galactosyl epitope.
Various labels can be used such as radioisotopes, enzymes,
fluorescers, chemiluminescers, particles, etc. There are numerous
commercially available kits providing labeled anti-hIg, which may
be employed in accordance with the manufacturer's protocol.
[0100] For screening the compounds for cytotoxic effects, a wide
variety of protocols may be employed to ensure that one has the
desired activity. One will normally use cells, which may be
naturally occurring or modified, cell lines, or the like. The cells
may be prokaryotic or eukaryotic. For example, if one is interested
in a pathogen, where it does not matter to which epitope the
compound conjugate binds, one can combine the pathogenic cells with
each of the compounds in the presence of an antibody dependent
cytotoxic system to determine the cytotoxic effect. One may perform
this assay either prior to or subsequent to determining the effect
of the various candidate compounds on cells of the host to whom the
compound would be administered. In this way, one would obtain a
differential analysis between the affinity for the pathogenic
target and the affinity for host cells which might be encountered,
based on the mode of administration.
[0101] In some situations, one would be interested in a particular
cellular status, such as an activated state, as may be present with
T cells in autoimmune diseases, transplantation, and the like. In
this situation one would first screen the compounds to determine
those which bind to the quiescent cell, and as to those compounds
which are not binding to the quiescent cells, and screen the
remaining candidate compounds for cytotoxicity to the activated
cells. One may then screen for other cells present in the host
which might be encountered by the compounds to determine their
cytotoxic effect. Alternatively, one might employ cancer cells and
normal cells to determine whether any of the compounds have higher
affinity for the cancer cells, as compared to the normal cells.
Again, one could screen the library of compounds for binding to
normal cells and determine the effect. Those compounds which are
not cytotoxic to normal cells could then be screened for their
cytotoxic effect to cancer cells. Even where some cytotoxicity
exists for normal cells, in the case of cancer cells, where there
is a sufficient differentiation in cytotoxic activity, one might be
willing to tolerate the lower cytotoxicity for normal cells, where
the compound is otherwise shown to be effective with cancer
cells.
[0102] Instead of using cells which are obtained naturally, one may
use cells which have been modified by recombinant techniques. Thus,
one may employ cells which can be grown in culture, which can be
modified by upregulating or downregulating a particular gene. In
this way, one would have cells that differ as to a single protein
on the surface. One could then differentially assay the library as
to the effect of members of the library on cells for which the
particular protein is present or absent. In this way, one could
determine whether the compound has specific affinity for a
particular surface membrane protein as distinct from any of the
proteins present on the surface membrane.
[0103] One may differentiate between cells by using antibodies
binding to a particular surface membrane protein, where the
antibodies do not initiate the complement dependent cytotoxic
effect, for example, using different species, isotypes, or
combinations thereof. By adding the antibodies, blocking antisera
or monoclonal antibodies, to one portion of the cells, those cells
will not have the target protein available for binding to the
library member. In this way one creates comparative cells which
differ in their response based on the unavailability in one group
of a single protein. While antibodies will usually be the most
convenient reagent to use, other specific binding entities may be
employed which provide the same function.
[0104] For use in the assay to determine binding, one may use an
antibody-dependent cytotoxic system. One could use synthetic
mixtures of the ingredients, where only those components necessary
for the cytotoxic effect are present. This may be desirable where
components of blood or plasma may adversely affect the results of
the assay.
[0105] Also, while a cellular lawn is an extremely convenient way
to screen large numbers of candidates, other techniques may also
find use. These techniques include the use of multiwell plates, and
the various devices used for the preparation of the combinatorial
library, such as pins, tea bags, etc. One may grow the cells
separately in relation to the nature of the various devices, where
the device may then be contacted with the cells or have the cells
grown on the device. The device may be immersed in an appropriate
culture, seeded with the cells, or otherwise provided for contact
between the cells and the candidate compound. After adding the
cytotoxic agent, one may then analyze for lysis in a variety of
ways. For example, FACS may be used for distinguishing between live
and dead cells, [.sup.51Cr] release may be employed, or detection
of an intracellular compound in the supernatant, may serve to
detect active compounds.
[0106] In addition, one may wish to know whether the compound has
agonist or antagonist activity. The subject assay techniques
provide for a rapid way for determining those compounds present in
the library which bind to the target protein. Once, one has
substantially narrowed the number of candidate compounds, one can
use more sophisticated assays for detecting the activity of the
compound itself. In this way, one can perform a rapid screen to
determine binding affinity and specificity, followed by a more
intensive screen to determine activity. Various techniques exist
for determining activity, where the cells may be modified, so that
a marker gene will be activated which will provide for a detectable
signal. Conveniently, the signal may be associated with production
of a dye, the production of a surface membrane protein which can be
detected with labeled antibodies, or the secretion of a protein
which can be detected in the supernatant by any of a variety of
techniques. For example, the gene that is expressed may be
luciferase modified to have a leader sequence so as to be secreted,
whereby the supernatant can then be screened for light generation
formation by using an appropriate substrate.
[0107] Various protocols may be employed for screening the library.
To some degree, this will depend upon the nature of the preparation
of the compounds. For example, the compounds may be bound to
individual particles, pins, membranes, or the like, where each of
the compounds is segregatable. In addition, the amount of compound
available will vary, depending upon the method employed for
creating the library. Furthermore, depending upon the nature of the
attachment of the compound to the support, one may be able to
release aliquots of a compound, so as to carry out a series of
assays. In addition, the manner in which the compounds are assayed
will be affected by the ability to identify the compound which is
shown to have activity.
[0108] Where the compounds are individually on a surface in a grid,
so that at each site of the grid one knows what the composition is,
one can provide a cellular lawn which is similarly organized as a
grid and may be placed in registry with the compounds bound to the
solid surface. Once the lawn and solid substrate are in registry,
one may release the compounds from the surface in accordance with
the manner in which the compounds are attached. After sufficient
time for the compounds to bind to the proteins on the cellular
surface, one may wash the cellular lawn to remove non-specifically
bound compounds. One or more washings may be involved, where the
washings may provide for varying degrees of stringency, depending
upon the desired degree of affinity. After the washings have been
completed, mammalian blood or plasma may then be added and
incubated for sufficient time for cytotoxicity. The plasma or blood
may then be removed and plaques observed, where the nature of the
compound can be determined by virtue of the position in the grid.
The plasma or blood can be free of any components that would
naturally kill the cells of the lawn.
[0109] Since the preparative process may be repeated, one could
prepare a plurality of solid substrates, where the same compounds
are prepared at the comparable sites, so that the screening could
be repeated with the same or different cells to determine the
activity of the individual compounds. In some instances, the
identity of the compound can be determined by a nucleic acid tag,
using the polymerase chain reaction for amplification of the tag.
See, e.g., International Publication No. WO93/20242. In this
instance, the compounds that are active may be determined by taking
the lysate and introducing the lysate into a polymerase chain
reaction medium comprising primers specific for the nucleic acid
tag. Upon expansion, one can sequence the nucleic acid tag or
determine its sequence by other means, which will direct the
selection of the procedure that is used to prepare the
compound.
[0110] Alternatively, one may have tagged particles where the tags
are releasable from the particle and provide a binary code that
describes the synthetic procedure for the compounds bound to the
particle. See, e.g., Ohlmeyer et al., 1993, Proc Natl Acad Sci USA
90:10922. These tags can conveniently be a homologous series of
alkylene compounds, which can be detected by gas
chromatography-electron capture. Depending upon the nature of the
linking group, one may provide for partial release from the
particles, so that the particles may be used 2 or 3 times before
identifying the particular compound.
[0111] While for the most part libraries have been discussed, any
large group of compounds can be screened analogously, so long as
the CTGF epitope can be joined to each of the compounds. Thus,
compounds from different sources, both natural and synthetic,
including macrolides, oligopeptides, ribonucleic acids, dendrimers,
etc., may also be screened in an analogous manner.
[0112] Formation of a plaque in the assay demonstrates that binding
of the member of the library to the cell, usually a surface
protein, does not interfere with the CTGF epitope binding to an
antibody, that the immune complex is sufficiently stable to
initiate the complement cascade, and that the member has a high
affinity for the target.
[0113] The subject methodology can be used in any situation where
one has a cellular target to be killed, particularly those cellular
targets having low or no CTGF epitope. Thus, the cellular target
may be a prokaryote, which is pathogenic. Various organisms
include, for example, microbacterium, Yersinia, Pseudomonas,
Bordetella pertussis, Treponema pallidum, Neisseria gonorrhoea,
Streptococcus, Hemophilus influenza, etc. Other pathogens include
eukaryotes, particularly fungi, such as Candida, Histoplasma, etc.,
and protozoa, e.g., Giardia. In addition, viruses which provide for
surface membrane proteins in infected cells, can also be the target
of the subject compounds, where the cells that are screened have
been vitally infected.
[0114] Host cells may also serve as targets, where the cells are
either abnormal or act in an adverse way to the host or treatments
of the host. For example, cancerous tissues which can be
distinguished from normal tissue can serve as a target for the
subject compounds. T or B cells associated with autoimmune diseases
or associated with GVHD or transplant rejection may also serve as
targets. Aberrant cells, regardless of their nature, so long as
they can be distinguished from normal cells, may also serve as
targets. Thus, psoriatic lesions, lymphoma cells, bacterial,
fungal, parasitic, virus infected cells, may be targets of the
subject products. Also, where one wishes to ablate a portion of
cells, without removal of all of the cells, such as cells
expressing a differentiation marker such as T cell subsets,
activated platelets, endothelial cells, hormone or cytokine
receptor expressing cells, the subject compounds may find
application.
[0115] Other screening methods for obtaining small molecules that
modulate the activity of CTGF can be found, for example,
International Publication No. WO 98/13353.
[0116] Compounds/Molecules. The present invention provides methods
for treating and preventing disorders associated with kidney
fibrosis by modulating, regulating, or inhibiting the activity of
CTGF. These methods can comprise the administration of a
therapeutically effective amount of a compound that blocks the
binding interactions or blocks enzymes involved in the signal
transduction pathway of CTGF. More specifically, the present
invention provides a method for inhibiting the activity of CTGF by
administering compounds that block the induction of CTGF.
[0117] Compounds that modulate CTGF gene expression and/or CTGF
activity in the method of the invention include agents which cause
an elevation in cyclic nucleotides in the cell. Other compounds
that may block the induction of CTGF according to the methods of
the present invention may be identified using the screening methods
described above.
[0118] In yet a further embodiment of the present invention, the
method provides for the administration of molecules that interrupt
the post-translational modification of full length CTGF or block
the activation of an inactive precursor of CTGF. As discussed
herein, exposure of mesangial cells to TGF-.beta. resulted in the
marked appearance of additional bands at 28-30 kDa which correspond
in size to the carboxy- and amino-terminal halves of the full
length CTGF molecule. As disclosed above, TGF-.beta. treatment may
result in the production of proteases or other factors capable of
cleaving the full-length molecule. Molecules that inhibit CTGF
activity may be identified using the screening methods provided
herein.
[0119] The methods of the present invention may further be used to
prevent or treat fibrosis in the kidney associated with allograft
rejection comprising administering a therapeutically effective
amount of any one of the agents described above.
[0120] The invention further provides a method for treating or
preventing diabetes by administering an effective amount of insulin
and an effective amount of an agent that regulates, modulates, or
inhibits CTGF activity as described above.
[0121] C. Pharmaceutical Formulations and Routes of
Administration
[0122] Routes of Administration. The antibodies, small molecules,
and other compounds described herein can be administered to a human
patient per se, or in pharmaceutical compositions comprising, where
appropriate, suitable carriers or excipients. The present invention
contemplates methods of treatment in which agents that modulate or
regulate the expression or activity of CTGF or fragments thereof
are administered to a patient in need, in amounts suitable to treat
or prevent the overproduction of ECM associated with CTGF. The
present methods of treatment and prevention can comprise
administration of an effective amount of the agent to a subject
which is preferably a mammalian subject, and most preferably a
human subject. In a preferred embodiment, the subject mammal and
the agent administered are of homologous origin. Most preferably,
the subject and the agent administered are human in origin.
[0123] An effective amount can readily be determined by routine
experiment, as can the most effective and convenient route of
administration and the most appropriate formulation. Various
formulations and drug delivery systems are available in the art.
See, e.g., Gennaro, ed., 1990, Remington 's Pharmaceutical
Sciences, 18.sup.th ed., Mack Publishing Co., Easton Pa. Suitable
routes of administration may, for example, include oral, rectal,
transmucosal, or intestinal administration and parenteral delivery,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, or intraocular injections. The
composition may be administered in a local rather than a systemic
manner. For example, a composition comprising an agent which
modulates, regulates, or inhibits the activity of CTGF can be
delivered via injection or in a targeted drug delivery system into
an area in which there is excess circulating CTGF or ECM
overproduction or into an area in which inhibition of CTGF activity
is desired, often in a depot or sustained release formulation.
[0124] The pharmaceutical compositions of the present invention may
be manufactured by any of the methods well-known in the art, such
as by conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. As noted above, the compositions of the present
invention can include one or more physiologically acceptable
carriers such as excipients and auxiliaries which facilitate
processing of active molecules into preparations for pharmaceutical
use. Proper formulation is dependent upon the route of
administration chosen.
[0125] For injection, for example, the composition may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable
carriers well known in the art. Such carriers enable the compounds
of the invention to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a subject. The compounds may also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides.
[0126] Pharmaceutical preparations for oral use can be obtained as
solid excipients, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinyl pyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0127] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0128] Pharmaceutical preparations for oral administration include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0129] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide, or any other suitable
gas. In the case of a pressurized aerosol, the appropriate dosage
unit may be determined by providing a valve to deliver a metered
amount. Capsules and cartridges of, for example, gelatin, for use
in an inhaler or insufflator may be formulated. These typically
contain a powder mix of the compound and a suitable powder base
such as lactose or starch.
[0130] Compositions formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion can be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Formulations for
parenteral administration include aqueous solutions of agents that
affect the activity of CTGF or fragments thereof, in water-soluble
form.
[0131] Suspensions of the active compounds may be prepared as
appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil and
synthetic fatty acid esters, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. Alternatively, the
active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0132] The compositions of the present invention may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example, subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0133] Pharmaceutical carriers for the hydrophobic molecules of the
invention could include co-solvent systems comprising, for example,
benzyl alcohol, a nonpolar surfactant, a water-miscible organic
polymer, and an aqueous phase. The co-solvent system may be the VPD
co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8%
w/v of the nonpolar surfactant polysorbate 80, and 65% w/v
polyethylene glycol 300, made up to volume in absolute ethanol. The
VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a
5% dextrose in water solution. This co-solvent system is effective
in dissolving hydrophobic compounds and produces low toxicity upon
systemic administration. Naturally, the proportions of a co-solvent
system may be varied considerably without destroying its solubility
and toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied. For example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80, the fraction size of polyethylene glycol may be
varied, other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone, and other sugars or
polysaccharides may substitute for dextrose.
[0134] Alternatively, other delivery systems for hydrophobic
molecules may be employed. Liposomes and emulsions are well known
examples of delivery vehicles or carriers for hydrophobic drugs.
Certain organic solvents such as dimethylsulfoxide also may be
employed, although usually at the cost of greater toxicity.
Additionally, the compounds may be delivered using
sustained-release systems, such as semi-permeable matrices of solid
hydrophobic polymers containing the effective amount of the
composition to be administered. Various sustained-release materials
are established and available to those of skill in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein
stabilization may be employed.
[0135] Effective Dosage. For any composition used in the present
methods of treatment, a therapeutically effective dose can be
estimated initially using a variety of techniques well known in the
art. For example, in a cell culture assay, a dose can be formulated
in animal models to achieve a circulating concentration range that
includes the IC.sub.50 as determined in cell culture. Where
inhibition of CTGF activity is desired, for example, the
concentration of the test compound which achieves a half-maximal
inhibition of CTGF activity can be determined. Dosage ranges
appropriate for human subjects can be determined using data
obtained from cell culture assays and other animal studies.
[0136] A therapeutically effective dose refers to that amount of
the molecule that results in amelioration of symptoms or a
prolongation of survival in a subject. Toxicity and therapeutic
efficacy of such molecules can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., by determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio of toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
ratio LD.sub.50/ED.sub.50. Molecules which exhibit high therapeutic
indices are preferred.
[0137] Dosages preferably fall within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. Dosages may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration, and dosage will be
chosen in view of the specifics of a subject's condition.
[0138] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
modulate or regulate CTGF activity as desired, i.e. minimal
effective concentration (MEC). The MEC will vary for each compound
but can be estimated from, for example, in vitro data, such as the
concentration necessary to achieve 50-90% activity of CTGF to
induce bone growth using the assays described herein.
[0139] Dosages necessary to achieve the MEC will depend on
individual characteristics and route of administration.
Compositions should be administered using a regimen which maintains
plasma levels above the MEC for about 10-90% of the duration of
treatment, preferably about 30-90% of the duration of treatment,
and most preferably between 50-90%. In cases of local
administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0140] The amount of composition administered will, of course, be
dependent on a number of factors, including, but not limited to,
the particular subject's weight, the severity of the affliction,
the manner of administration, and the judgment of the prescribing
physician.
[0141] Packaging. The compositions may, if desired, be presented in
a pack or dispenser device which may contain one or more unit
dosage forms containing the active ingredient. The pack may, for
example, comprise metal or plastic foil, such as a blister pack.
The pack or dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition. Suitable conditions indicated
on the label may include treatment of disorders or diseases in
which cartilage or bone induction, wound healing, neuroprotection
or the like is desired.
[0142] Receptor-Ligand Complexes. As a consequence of the above
described screening techniques, as well as other known screening
techniques which may be applied in the context of the present
invention, CTGF-ligand complexes can be formed. These complexes may
include complexes wherein the ligand is a CTGF antagonist, a CTGF
agonist, or any another compound capable of modulating the
expression and activity of CTGF. Partial agonists or antagonists of
the CTGF receptor may be useful for therapeutic or diagnostic
purposes. CTGF-agent complexes may be useful as therapeutic
entities in their own right or in methods of detecting and
quantifying CTGF levels in a sample. The measurement and
quantification of CTGF, for example, through the detection of
CTGF-ligand complexes, can be accomplished by methods available in
the art. See, e.g., Enna et al., supra.
[0143] D. Diagnostics
[0144] The present invention is further directed to a method of
detecting or diagnosing the presence of pathology of a tissue
characterized by an excessive accumulation of the extracellular
matrix components, in particular, those associated with renal
disorders. One method involves the detection or diagnosis of
diabetes, including diabetic nephropathy and diabetic
glomerulosclerosis. In a preferred method, the detection or
diagnosis is accomplished by measuring CTGF levels in a urine
sample from a patient. In one embodiment, the method includes
determining the level of CTGF in a first urine sample and comparing
this level to the level of CTGF present in a normal urine sample,
i.e., a sample from a subject without a renal disorder. An elevated
level of CTGF in the first sample is indicative of the pathological
condition in question, for example, diabetes or hypertension. In
particular, individuals without any renal disorders, normal levels
of CTGF may be at or close to zero. In diabetic patients or in
patients experiencing infection or other trauma, levels of CTGF may
be significantly increased. Thus, the presence of kidney fibrosis
could be identified by detecting increases in levels of CTGF in a
sample. In a preferred method, the sample is a non-intrusive sample
such as a urine sample. Assessment of CTGF levels in a urine sample
can be accomplished, for example, by ELISA using a CTGF-specific
antibody. Detection of CTGF levels could be indicative of the
advancement or worsening of diabetic hypertension or other renal
disorders prior to the onset of renal complications, providing for
a method of early-stage detection and diagnosis. Furthermore, CTGF
levels can serve as a predictor of, for example, which diabetic
patients have a predisposition to develop kidney diseases and
disorders.
[0145] More generally, detection of CTGF levels, including levels
of unique forms or fragments of CTGF, may be obtained through
immunoassay methods, for example, ELISAs, RIAs, or any other assays
which utilize an antibody to detect the presence of a protein
marker. The ELISA and RIA methods are preferred and may be used,
for example, with the monoclonal antibodies of the present
invention to detect levels of CTGF. In a preferred method of the
invention, urine samples are obtained first from patients suspected
or known to have a renal disease or disorder. Levels of CTGF in
this first sample are measured, for example, through immunoassay,
and are compared with the CTGF levels in a second sample, the
second sample being obtained from a patient known to have a renal
disorder or from a patient known not to have any renal disorder, to
determine the presence or progression of a kidney disease. The same
methods may be used to monitor the progression of a kidney
disease.
[0146] More generally, antibodies specific for a target
polypeptide, such as antibodies specific for CTGF, are useful in
the present invention for diagnosis of renal disorders and diseases
associated with aberrant expression of CTGF. Diagnostic assays for
CTGF can include methods utilizing the antibody and a label to
detect CTGF in a sample from a patient suspected of having a renal
disorder or disease. The sample could comprise, for example, body
fluids, cells, tissues, or extracts of such tissues, including, for
example, glomeruli microdissected from biopsy material. Protocols
employed to screen for and identify antibodies having the desired
specificity can also be used for the detection of CTGF or the
target polypeptide in the sample.
[0147] Preferably, in the diagnostic methods of the present
invention, normal or standard values for CTGF expression are
established in order to provide a basis for the diagnosis of the
existence of a renal disease or disorder or a predisposition to a
renal disease or disorder. In one of the methods of the present
invention, this is accomplished by combining body fluids or cell
extracts taken from normal subjects with antibody to CTGF under
conditions suitable for complex formation. Such conditions are well
known in the art. The amount of standard complex formation may be
quantified by comparing levels of antibody-target complex in the
normal sample with a dilution series of positive controls, in which
a known amount of antibody is combined with known concentrations of
purified CTGF. Standard values obtained from normal samples may be
compared, for example, in a specific embodiment, with values
obtained from samples from subjects suspected of having a kidney
disease or disorder, or having a predisposition to a kidney disease
or disorder, associated with kidney fibrosis. Deviation between
standard and subject values establishes the presence of or
predisposition to the disease state. The diagnostic methods of the
present invention may also be directed to the detection of a
predisposition or susceptibility to a renal disorder. This can be
accomplished, for example, by detecting a marker indicative of a
predisposition or susceptibility to develop a particular disorder,
for example, diabetes. The marker can comprise, for example, a
genetic polymorphism.
[0148] Monoclonal antibodies can be detected by methods discussed,
for example, supra. Monoclonal antibodies against CTGF can be
conjugated to an appropriate enzyme such as horseradish peroxidase,
protein ferritin, enzyme alkaline phosphatase,
.beta.-D-galactosidase etc. These enzyme-linked antibody
preparations can be mixed with, for example, urine samples that
contain unknown amounts of CTGF in an indirect ELISA. Direct or
sandwich ELISAs could also be performed using the same
antibodies.
[0149] RIA techniques may also be used to measure levels of CTGF
in, for example, urine. For example, CTGF may be radioactively
labeled and mixed with monoclonal antibodies specific for CTGF and
a serum sample containing an unknown amount of unlabeled CTGF.
Binding competition between the labeled and unlabeled CTGF with the
monoclonal antibody occurs. By measuring the amount of
radioactivity of the reaction mixture, the amount of CTGF present
in the sample can be quantitatively determined. See, e.g., U.S.
Pat. Nos. 4,438,209 and 4,591,573. Non-competitive RIAs can also be
performed.
[0150] Polynucleotide sequences encoding CTGF can be used for the
diagnosis of conditions or diseases associated with increased
levels of CTGF expression. For example, polynucleotide sequences
encoding CTGF may be used in hybridization or PCR assays of fluids
or tissues from biopsies to detect CTGF expression. The form of
such qualitative or quantitative methods may include Southern or
northern analysis, dot blot or other membrane-based technologies;
PCR technologies; dip stick, pin, chip and ELISA technologies. All
of these techniques are well known in the art and are the basis of
many commercially available diagnostic kits.
[0151] The present invention additionally provides methods for
evaluating the effectiveness of anti-fibrotic therapy, including
the use of ACE inhibitors, by measuring the levels of CTGF in a
sample from a subject undergoing a course of treatment for diseases
and disorders associated with fibrosis. CTGF levels can be measured
in samples, for example, urine sample, taken from the subject at
various points before, during, and after a course of treatment. The
efficacy of a treatment can be evaluated with reference to the
variation in CTGF levels present in the samples taken at different
stages of a course of treatment.
[0152] Kits. The present invention provides kits for detecting CTGF
in samples, in particular, in fluid samples. In a preferred
embodiment, the diagnostic kits of the present invention contain
reagents for measuring levels of CTGF in urine samples. In a
particular embodiment, this kit comprises a monoclonal antibody
specific for CTGF bound to a support and a second monoclonal
antibody specific for a different CTGF epitope and enzyme-labeled.
The kit further comprises reagents for detecting the enzyme-labeled
monoclonal antibody. The reagent kit employs immunological methods
in measuring CTGF in the urine sample, thus allowing for the
detection and monitoring of kidney disorders and diseases. In
particular embodiments, the kit allows for the detection and
monitoring of fibrotic and sclerotic disorders resulting from, for
example, diabetes and hypertension. In another embodiment, the kit
comprises a radio-labeled or fluorescein-labeled antibody in place
of the enzyme-labeled antibody.
[0153] In one embodiment, the diagnostic kit of the present
invention comprises elements useful in the detection of CTGF in
tissue samples, using immunohistochemical techniques. The kit could
be used in conjunction with, for example, a software program which
allows for quantitative measurement of the levels of CTGF in the
tissue sample by image analysis or other comparative techniques.
See, e.g., Riser et al., 1996, supra. Another embodiment provides a
diagnostic kit for detecting and measuring levels of CTGF mRNA in
tissue samples. In one embodiment, the kit comprises reagents used
to reverse transcribe CTGF mRNA to DNA. The kit can further
comprise reagents necessary to amplify CTGF-specific DNA, including
primers complementary to polynucleotides encoding CTGF or fragments
thereof. The kit can also include a competitive mimic or mutant
cDNA for use in quantifying the level of CTGF mRNA present in the
sample.
[0154] In a preferred embodiment, the diagnostic kit of the present
invention is packaged and labeled, for example, in box or container
which includes the necessary elements of the kit, and includes
directions and instructions on the use of the diagnostic kit.
[0155] The following examples explain the invention in more detail.
The following preparations and examples are given to enable those
skilled in the art to more clearly understand and to practice the
present invention. The present invention, however, is not limited
in scope by the exemplified embodiments, which are intended as
illustrations of single aspects of the invention only, and methods
which are functionally equivalent are within the scope of the
invention. Indeed, various modifications of the invention in
addition to those described herein will become apparent to those
skilled in the art from the foregoing description and accompanying
drawings. Such modifications are intended to fall within the scope
of the appended claims.
EXAMPLES
[0156] Unless otherwise stated, the following materials and methods
were used in the examples of the present invention.
[0157] Cells And Tissue Culture. The mesangial cells were a
cloned-line derived from outgrowths of Fischer rat glomeruli, and
upon serial passage, these mesangial cells continue to express key
markers. (See, e.g., Riser et al., 1998, J Am Soc Nephrol
9:827-836.) The medium used was RPMI 1640 with penicillin and
streptomycin and, unless otherwise noted, 5 mM glucose. The growth
medium contained 20% NU-SERUM media supplement (Collaborative
Research, Bedford Mass.). Unless otherwise noted, mesangial cells
were cultured for approximately 4 days in growth medium, and when
reaching confluency, were washed twice with serum-free medium and
incubated for 2448 hours under serum-deprived of 0.5% FCS (fetal
calf serum) conditions. The cultures were then incubated for a
designated period in fresh maintenance medium (0.5% FCS), with or
without experimental treatments. At the concentration of FCS used
in these studies, no active TGF-.beta.1, TGF-.beta.2, or
TGF-.beta.3 was detectable in the fresh medium, as determined by a
highly sensitive mink lung bioassay. (See, e.g., Riser et al.,
1996, supra.) The renal fibroblasts used in Northern analysis were
mouse tubulointerstitial fibroblasts (TFB). (See, e.g., Alverez, et
al., 1998, Kidney Int 41:14-23.)
[0158] Animals And Specimen Collection. Diabetic male db/db mice
and their nondiabetic db/m littermates were obtained from Jackson
Laboratories (Bar Harbor Me.). The db/db mouse carries a defective
receptor gene for leptin, a key weight control hormone. (See, e.g.,
Hummel et al., 1966, Science 153:1127-1128.) These mice become
obese at 3 to 4 weeks of age and develop hyperglycemia. Associated
nephropathy includes proteinuria and mesangial expansion with
increased mesangial matrix that develops by 5 to 7 months. (See,
e.g., Cohen et al., 1995, J Clin Invest 95:2338-2345.) In the
present experiments, mice were sacrificed at the age of 5 months.
Blood glucose levels were determined during the study and at
sacrifice, using a calorimetric method based on the glucose
oxidase-peroxidase reaction and supplied in a kit form (Glucose
Procedure No. 510 kit, Sigma Diagnostics, St. Louis Mo.). Following
a 24 hour acclimation to metabolic cages, two consecutive 24 hour
urine samples were collected. At the end of the collection period,
the lower part of the cage including the collection funnel was
rinsed with distilled water and the final sample volume recorded.
Protein concentration in the urine was measured according to a
method for quantifying microgram quantities of protein utilizing
protein-dye binding. (See, e.g., Bradford, 1976, Anal Biochem
72:248-254.)
[0159] After anesthesia by an oxygen/ether mixture, the abdominal
cavity was opened, a 23 gauge needle was inserted into the aorta
and the kidneys were perfused with four (4) ml of ice cold
perfusion buffer of (RPMI with 4% BSA) containing 10 mM vanadyl
ribonucleoside complex (VRC), an RNase inhibitor (Gibco/BRL, Grand
Island, N.Y.). Chilled 0.9% saline was poured over the kidneys
during this perfusion. The kidneys were then removed, and the right
kidney was frozen in liquid nitrogen for subsequent RNA extraction
and Northern analysis. Fine sagittal slices of the left kidney were
rapidly obtained. One section was fixed in 3.8% paraformaldehyde,
embedded in parafilm and stained with periodic acid Schiff (PAS)
for light microscopic evaluation. The remaining slices were used
for glomerular microdissection and reverse transcription and
polymerase chain reaction (RT-PCR) of the isolated glomeruli. The
methods used were a modification of known methods for determining
glomerular mRNA levels. (See, e.g., Peten et al., 1993, Kidney Int
Suppl 39:S55-S58.) Tissue sections were placed in a buffer of HBSS
containing 10 mM VRC, and then 50 glomeruli were dissected from
each kidney in less than 50 minutes. The glomeruli were next
transferred to a PCR tube with 30 .mu.l of rinse buffer (HBSS
containing 5 mM DTT and 50 units/ml of human placental ribonuclease
inhibitor (Boeringer Mannheim, Indianapolis Ind.). Following
centrifugation, the supernatant was removed and microscopically
examined for the accidental presence of glomeruli. Seven
microliters of a lysis solution (rinse buffer containing 2% Triton
X-100) were added, and the samples were stored at -70.degree. C.
until processed. All of these procedures were carried out at
4.degree. C.
[0160] Experimental samples from control and diabetic mice were
thawed on ice and then subjected to 2 additional freeze/thaw cycles
to lyse the glomeruli. The RT reaction was then carried out using a
cDNA synthesis kit (Boehringer Mannheim), with oligo(dT) as a
primer. Reactions containing glomeruli, but without added reverse
transcriptase, or without glomeruli, but with reverse
transcriptase, served as negative controls. The reaction mixture
was incubated for 60 minutes at 42.degree. C., and then chilled to
4.degree. C. for 10 minutes. Samples were then diluted at a ratio
of 1:10 in distilled water and frozen at -70.degree. C. until PCR
was completed.
[0161] Evaluation Of Renal Tissue By Light Microscopy. Five to 6
nonconsecutive 6 .mu.m sections per kidney were PAS stained and
examined. Mesangial sclerosis was scored on a scale of zero to four
(0-4), wherein zero (0) represents no lesion; one (1) represents
minimal mesangial expansion; two (2) represents mesangial expansion
and/or basement membrane thickening; three (3) represents marked
mesangial thickening, some collapsed lumina, and occasional lobule
with full sclerosis; and four (4) represents a diffuse collapse of
capillary lumina, and sclerosis involving 75% or more of the tuft.
A total of 100-150 glomeruli per kidney were scored by an observer
blinded as to the origin of the specimens. Only glomerular profiles
showing a mesangial region that could be unequivocally evaluated
were scored.
[0162] Competitive PCR and Northern blotting. All PCR were
performed using the GENEAMP DNA amplification kit (Perkin-Elmer
Cetus, Norwalk Conn.) and a 9600 thermal cycler (Perkin Elmer). For
quantitation, a competitive PCR reaction was run using a cDNA
mimic. Thirty-eight cycles of replication were used. Five PCR tubes
were set up for each sample. Each tube in a series contained a
fixed amount of the wild-type cDNA along with decreasing
concentrations of the mimic cDNA. The products were separated by
agarose gel electrophoresis and visualized by ethidium bromide
staining. Bands were digitized by scanning densitometry (SCANMASTER
3+ densitometer; Howtek, Hudson N.H.) and quantified with image
analysis (NIH Image, v. 1.59 from Twilight Clone BBS, Silver
Springs Md.). A plot of the ratio of wild type/mimic vs. the
reciprocal of the input mutant concentration was constructed and
the amount of glomerular cDNA determined from the resulting linear
regression. Northern analysis was carried out as previously
described, following pulverization of samples in a liquid-nitrogen
cooled stainless-steel mortar and homogenization in 1.0 ml of RNA
STAT-60 reagent (Tel-Test Inc., Friendswood Tex.). Probes for
individual mRNAs and the corresponding cDNAs, were labeled with
.sup.32P by random hexamer priming using the PRIME-1 kit (Sigma).
Autoradiograms were digitized by scanning densitometry and
quantified as described above.
[0163] Primers, probes, and cDNA mimics. Primers for CTGF were
designed and synthesized based on conserved sequences between the
human and mouse CTGF (fisp 12) gene. The primers, Primer R and
Primer F, were as follows: Primer F: 5'-GAG TGG GTG TGT GAC GAG CCC
AA G G-3' and Primer R: 5' ATG TCT CCG TAC ATC TTC CTG TAG T-3'.
The amplification product was 558 bp in size. The sequence was
confirmed by cloning into a PCR script (Invitrogen Corp., Carlsbad
Calif.). Two clones were sequenced and were identical. A
competitive cDNA mimic was produced using a PCR mimic construction
kit (K 1700-1, Clontech Laboratories, Palo Alto Calif.). For each
mimic, two composite primers (3' and 5') were first made containing
the CTGF target gene sequence, plus a 20-nucleotide stretch
designed to hybridize to opposite strands of a heterologous DNA
fragment provided in the kit. The desired primer sequences were
then incorporated into this fragment during PCR amplification. A
dilution of the first PCR reaction was then amplified using only
the gene-specific primers. This ensured that all mimic molecules
had complete gene-specific sequences. The mimic was then purified
by passage through CHROMA SPIN TE-100 columns (Clontech). By this
method, the size (200-650 bp) could be adjusted by choosing the
appropriate sequences of the generic DNA fragment for the composite
primers. The resulting cDNA competes on an equal basis for the same
primers in the same reaction. An amplimer of the CTGF mimic was 496
bp in size.
[0164] Primers (Primer F and Primer R) for rat fibronectin cDNA
were Primer F: 5' TGC CAC TGT TCT CCT ACG TG 3' and Primer R:
5'-ATG CTT TGA CCC TTA CAC GG 3'. A competitive mimic for
fibronectin was constructed as described above. Products of
amplification were approximately 312 bps (sample) and 474 bps
(mimic). Primers for GAPDH (Clontech) produced an amplification
fragment of 985 bp. A GAPDH competitive mimic was constructed as
described above, and produced a fragment of 604 bp. The cDNA probe
for Northern analysis was from a sequence of human CTGF shared by
rat and mouse.
[0165] Production of recombinant CTGF and anti-CTGF antibodies.
Recombinant human CTGF protein (rhCTGF) was generated using a
baculoviral expression system. A human CTGF open reading frame of
1047 bp was amplified using primers engineered with BamHI sites
immediately flanking the ATG start codon and TGA stop codon
(forward primer 5'-GCT CCG CCC GCA GTG GGA TCC ATG ACC GCC GCC-3';
reverse primer 5'-GGA TCC GGA TCC TCA TGC CAT GTC TCC GTA-3'). A
clone designated as Clone DB60R32 was used as a template, which
contains the entire 2075 bp CTGF cDNA. The amplified product was
subcloned into the BamHI site of PFASTBAC1 vector (Gibco/BRL),
analyzed for insert orientation, and verified by sequencing of both
DNA strands. Generation of recombinant baculovirus containing the
CTGF cDNA was performed as outlined by Gibco/BRL (pFastBac
expression system). Recombinant baculovirus stocks were isolated,
expanded to high virus titer, and used to infect High Five insect
cells for expression of CTGF. The recombinant CTGF was purified by
heparin sepharose affinity chromatography as described previously.
Peak fractions containing rhCTGF were determined by immunoblotting
and Coomassie staining of sodium dodecyl sulfate
(SDS)-polyacrylamide gels.
[0166] Two anti-CTGF antibodies were used. The first, anti-CTGF
polyclonal designated, pAb839, was prepared by immunizing rabbits
with a keyhole limpet hemocyanin-coupled synthetic peptide
corresponding to amino acids 329-343 (CPG DND IFE SLY YRK) that is
unique to the carboxy terminus of CTGF. The production of antibody
was monitored by ELISA with the peptide conjugated to BSA and
absorbed to plastic. The anti-CTGF antibodies were
affinity-purified by passage through a CPG DND IFE SLY
YRK-Sepharose peptide column using standard protocols (See, e.g.,
Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor N.Y.). Peptide blocking
studies confirmed the monospecificity of pAb839 for CTGF in western
immunoblotting assays. Western immunoblot analysis also revealed
that pAb839 recognized CTGF only in a reduced conformation. The
second antibody, pIgY3 polyclonal, was raised in chickens by
immunizing with purified baculovirus-derived full-length rhCTGF
protein, and was subsequently affinity purified through a
rhCTGF-Sepharose column. (See, e.g., Kothapalli et al., 1997, Cell
Growth Differ 8:61-68).
[0167] ELISA. The amount of specific extracellular matrix
components secreted into the culture medium was quantified by
ELISA, using procedures described in the art. (See, e.g., Riser et
al., 1992, J Clin Invest 90:1932-1943.) It was previously
determined in mesangial cell cultures, that media containing 0.5-1%
FCS was optimal for the recovery of fibronectin and collagen. (See,
e.g., Riser et al., 1992, supra.) Experimental samples of culture
medium were tested in triplicate. Purified matrix components,
diluted in the same medium, were run (0.5-500 ng/well) as
standards. All antisera were tested for specificity before their
use by immunoblotting, with and without blocking, using the
extracellular matrix standards. Color intensity was measured with a
TITERTEK MULTISCAN MCC/340 plate reader (Flow Laboratories, McLean
Va., and the results analyzed using a curve-fitter computer program
(Interactive Microware Inc., State College Pa.).
[0168] An indirect ELISA was used to quantitate CTGF levels in the
conditioned media. Microtiter wells were coated with media samples
or the rhCTGF standard (50 .mu.l/well) for 2 hours at room
temperature in a 96-well plate. The wells were washed 4 times with
Dulbecco's phosphate buffered saline (D-PBS) and then incubated
with pIgY3 antibody at 1.25 .mu.g/ml (50 .mu.l/well) in a blocking
buffer of 1% BSA, 0.05% Tween 20 in D-PBS for 60 minutes. After
thorough washing with D-PBS, an HRP-conjugated rabbit anti-chicken
IgG (Zymed Laboratories Inc., South San Francisco Calif.) was added
to all wells at a 1:6400 dilution in blocking buffer for 30
minutes. The substrate, TMB-ELISA (Gibco/BRL) was added at room
temperature for 15 minutes. The reaction was stopped with 1 M
sulfuric acid and the color developed measured at 450 nm in an
ELISA multiscan spectrophotometer (Molecular Devices, Sunnyvale
Calif.). The amount of CTGF protein present in samples was
determined by using a logarithmic standard curve using serial
dilutions of 3 pg to 3 ng/well of rhCTGF standard antigen.
[0169] Heparin sepharose precipitation and immunoblotting. To
analyze for CTGF protein expression, conditioned media were
collected and the heparin-binding proteins precipitated by
end-over-end mixing for 4 hours at 4.degree. C. with Heparin
Sepharose CL-6B beads (Pharmacia, Pisctaway N.J.). The beads were
washed three times with an ice cold RIPA lysis buffer comprised of
150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1%
deoxycholate, 0.1% SDS and 2 mM EDTA. The bound proteins were then
eluted by boiling in a SDS sample buffer comprised of 62 mM
Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol and bromophenol blue for 5
minutes under either non-reducing, or reducing conditions
containing 5% mercaptoethanol. The eluted heparin-binding proteins
were resolved in 4-20% SDS-polyacrylamide gels and
electrophoretically transferred to nitrocellulose filters
(Schleicher and Schuell, Keene N.H.) for 2 hours at 140 mA. The
filters were blocked with blocking buffer comprised of TTBS; 150 mM
NaCl, 50 mM Tris, 0.2% Tween-20, 5% BSA, pH 7.4 for 2 hours at room
temperature, and then probed for CTGF by incubation for 40 minutes
with an anti-CTGF antibody at 0.5 .mu.g/ml in the blocking buffer.
After extensive washing at 37.degree. C., the filters were
incubated with either a HRP-conjugated donkey anti-rabbit IgG
(Amersham, Arlington Heights Ill.), or a HRP-conjugated rabbit
anti-chicken IgG (Zymed) at a 1:12,000 dilution in the blocking
buffer. Immunoreactivity was detected by using a SUPERSIGNAL
chemiluminescent substrate (Pierce, Rockford Ill.).
[0170] Additional reagents. Purified extracellular components used
as standards were rat collagen I (Upstate Biotechnology Inc., Lake
Placid N.Y.) and rat fibronectin (Chemicon International Inc.,
Temecula Calif.). The corresponding antibodies, polyclonal anti-rat
collagen I and anti-rat fibronectin were used in ELISA. In
preliminary experiments, the polyclonal anti-rat collagen I
antibody did not cross-react with fibronectin or laminin, whereas
the anti-rat fibronectin antibody did not cross-react with collagen
I or laminin. The TGF-.beta. used for stimulation experiments was
human TGF-.beta.2 (Celtrix Corporation, Santa Clara Calif.). This
recombinant cytokine was produced in Chinese hamster ovary (CHO)
cells and then purified by previously reported techniques (See,
e.g., 1991, Ogawa et al., 1991, Meth Enzymol 198:317-327). A
monoclonal antibody designated as I.D 11.1, neutralizes
TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 (Genzyme Corporation,
Cambridge Mass.).
[0171] Statistical analysis. Data was expressed as means.+-.SEM
(Standard Error Mean). For tissue culture data, unless otherwise
noted, differences between two groups were evaluated using a paired
Student's t-test. A paired test was utilized because of the cloned
nature of the mesangial cells studied. In the case where results
were normalized to corresponding control values, the data was
analyzed by a one-sample t test with a hypothesized mean of 100% to
compare the test group with the control. A paired two-sample t test
was used to examine differences between 3 test groups. In both
cases, a Holm's test was then applied post hoc to adjust for
multiple comparisons. (See, e.g., Holm, 1979, Scan J Statist
6:65-70). For histological data, the mean sclerosis score was
calculated in the glomeruli of each kidney and the statistical
difference between the diabetic and control groups determined in a
non-paired t-test.
[0172] A. CTGF is an Important Factor in the Pathogenesis of Renal
Diseases, Including Diabetes
[0173] The examples provided in the present invention, provides the
first evidence demonstrating the production of CTGF protein by
glomerular cells and its role as a potentially important factor in
the pathogenesis of diabetic glomerulosclerosis. Hyperglycemia and
glomerular hypertension are two major, well-known, casual factors
of diabetic glomerulosclerosis. Prior to the present invention, the
role of CTGF in the development of glomerulosclerosis has not been
studied. The following examples, demonstrate that CTGF stimulates
cultured mesangial cells to produce, deposit, and accumulate
extracellular matrix components. The examples also demonstrate that
the induction of endogenous CTGF is triggered by increased glucose
concentrations, exogenous TGF-.beta., and mechanical strain.
[0174] As demonstrated in Example 2, CTGF mRNA is expressed in the
whole kidney of normal animals, and that its level is high in
comparison to the heart and brain, suggesting that endogenously
produced CTGF may be involved in the normal turnover of renal
extracellular matrix. However, the low levels of constitutive CTGF
mRNA expression demonstrated in cultured mesangial cells suggest
that this cell type may have a controlling mechanism for CTGF
formation different from that in the cells forming the bulk of the
renal tissue, i.e. tubular epithelial cells. The low expression of
CTGF mRNA observed in mesangial cells under unstimulated conditions
is associated with an apparent release of small quantities of CTGF
protein into the culture medium, see Example 3. The CTGF protein
was present as a 36 and 38 kD molecular species. The larger protein
is equivalent in size to the full-length CTGF molecule predicted
from gene analysis, whereas the smaller peptide may represent a
differential N-glycosylation in the CTGF N-terminal half. It was
observed that in both insect and mammalian cells, pretreatment with
tunicamycin, that inhibits the N-glycosylation of glycoproteins,
the larger CTGF band is reduced in its migration to localize with
the smaller moiety. These molecular species observed in mesangial
cells are similar in size to that secreted by vascular endothelial
and fibroblast cells (See, e.g., Bradham et al., 1991, supra;
Kothapalli et al., 1997, supra; and Steffen et al., 1998, Growth
Factors 15:199-213.) The small amounts of CTGF detected in the
conditioned medium of mesangial cultures were not the result of low
levels of its synthesis, but were rather due to the restricted
release of the protein into medium. This was indicated by the
ability of sodium-heparin to dramatically increase the levels of
CTGF protein measured in the media. (See Example 3.) These results
suggest that as much as 80% of the CTGF synthesized by mesangial
cells remains cell- or matrix-bound. In a quantitative assay it is
shown that, in the presence heparin, mesangial cells secrete
approximately 7 ng of CTGF per 10.sup.6 cells in each 24 hour
period. (See Example 3.)
[0175] Given that CTGF stimulates extracellular matrix
accumulation, it was examined whether known factors implicated in
the development of diabetic glomerulopathy alter CTGF mRNA
expression. High extracellular glucose concentrations markedly
increased the levels of CTGF mRNA as well as the production of CTGF
protein in mesangial cells. (See, Example 4.) In a similar manner,
TGF-.beta. also upregulates the expression of CTGF mRNA and
protein. With strong upregulation, as occurred in response to
TGF-.beta., there was a marked induction of a small molecular
weight CTGF species, which according to its size of .about.18 kD,
is approximately half of the full-length CTGF molecule. (See,
Example 2 and FIG. 6.) The size and properties of this small
molecular weight CTGF species recovered from a heparin-sepharose
column indicated that it contains both the thrombospondin 1 and the
C-terminal modules of CTGF. The small molecular species
demonstrated in mesangial cells following stimulation, may have
distinct biological activities as compared to the whole molecule.
Since TGF-.beta. secretion in mesangial cells is stimulated by
increased ambient glucose concentrations, the observed induction of
CTGF by high glucose may occur indirectly, mediated by the action
of TGF-.beta.. The neutralization studies described in Example 5
demonstrated a direct role for the cytokine in the process, since
incubation with TGF-.beta. antibodies resulted in a complete
blockade of CTGF stimulation. (See FIG. 8.)
[0176] Cyclic mechanical strain was also examined as a possible
regulatory element in CTGF expression. The results demonstrated
that stretching is a potent stimulus for the upregulation of CTGF
mRNA levels (See Example 7 and FIG. 10). The rapid induction of
CTGF mRNA following stretch suggests that TGF-.beta. production
and/or activity may not be required to mediate the initial effects
of mechanical strain. Cyclic strain induces TGF-.beta.1 synthesis
and activation, but this effect is only evident after 48-72 hours
of mechanical stimulation. (See, e.g., Riser et al., 1992, supra).
These studies also demonstrated that TGF-.beta. and CTGF are able
to autoinduce their own expression in mesangial cells. (See,
Example 3 and FIG. 3.) This autoinducing action of CTGF is the
first time that such action has been observed for CTGF.
Furthermore, this action appears to be selective, since exogenous
CTGF has no effect on TGF-.beta. transcript levels. (See, FIG. 3.)
These findings suggested that once stimulated by TGF-.beta., CTGF
mRNA levels in mesangial cells may remain elevated even in the
absence of additional TGF-.beta. activity resulting in a continued
enhancement of extracellular matrix synthesis and deposition, which
may explain the prevalent inability to totally block extracellular
matrix production in mesangial cells and in the mesangium by
TGF-.beta. neutralization. (See, e.g., Border et al., 1990, supra;
Sharma et al., 1996, supra; and Ziyadeh et al., 1994, supra.)
[0177] Quantitative glomerular expression of CTGF mRNA in db/db
mice (See, Example 10), demonstrated that CTGF action is a factor
in the initiation of glomerular extracellular matrix deposition in
diabetes. While CTGF mRNA is expressed in normal glomeruli, the
levels are dramatically upregulated by 28-fold, after a short
period of diabetes and before the onset of overt glomerular disease
(See Example 10). As demonstrated in these examples, CTGF mRNA
upregulation occurred at a time when glomerular fibronectin mRNA
levels were increased. However, the glomerular mesangial expansion
was minimal and proteinuria insignificant. As compared to
glomeruli, the much lower upregulation of CTGF observed in the
whole kidney as demonstrated in Example 10 shows that the CTGF is,
at least in the early phases of nephropathy, primarily involved in
the induction of the glomerular alterations. However, in the more
advanced stages of diabetic nephropathy, CTGF may be an important
inducer of tubulointerstitial disease.
[0178] In summary, the following examples essentially demonstrate
that, in addition to enhanced glomerular TGF-.beta. expression,
CTGF upregulation is an important factor in the excess deposition
of the extracellular matrix by mesangial cells. This CTGF
upregulation is driven by a combination of high glucose
concentrations and cellular mechanical stain via pathways that are
both dependent and independent of TGF-.beta. stimulation.
[0179] B. Experimental Data Demonstrating Nexus Between the
Presence of CTGF and the Onset and Progression of Renal Disorders,
Including Diabetes
Example 1
[0180] CTGF-Induced Changes In Extracellular Matrix Production Of
Mesangial Cells. To determine the effects of exogenous CTGF on
mesangial cell production of the extracellular matrix,
serum-depleted cells were exposed for 48 hours to media containing
20 ng/ml of rhCTGF. For comparison purposes, additional cultures
were incubated in media without exogenous CTGF, but containing
either 2 ng/ml of TGF-.beta., or 20 mM glucose. As anticipated,
exogenous TGF-.beta. and the high glucose concentration increased
the amount of secreted fibronectin by 23 and 30%, respectively,
over that of controls as shown in FIG. 1A. The presence of
exogenous CTGF in the media also effectively stimulated fibronectin
secretion by 45%. Like fibronectin, the quantity of secreted
collagen type I was also increased by 64% CTGF, as well as by 50%
TGF-.beta. or 22% high glucose as shown in FIG. 1B.
Example 2
[0181] Renal And Mesangial Cell CTGF Expression: Regulation By
TGF-.beta.. It was determined whether cultured rat mesangial cells
expressed CTGF mRNA, and the results were compared to those from
whole kidney. Northern analysis demonstrated a single 2.4 kb CTGF
transcript in mesangial cells and whole kidney, but in contrast no
detectable message was evident in cultured kidney fibroblasts as
demonstrated in FIG. 2. When compared to other tissues, the most
abundant expression was in the kidney, being approximately 20-fold
higher than in the brain.
[0182] To determine if TGF-.beta. was a regulatory factor in
mesangial cell expression of CTGF message, cells were
serum-depleted, exposed to 2 ng/ml of TGF-.beta. for 24 hours, the
mRNA was then probed. Changes in TGF-.beta. transcript levels were
also monitored. Exogenous TGF-.beta. exposure increased the
expression of CTGF mRNA greater than 4-fold as shown in FIG. 3A and
FIG. 3B, whereas TGF-.beta. mRNA increased 80% (see FIG. 3A and
FIG. 3C). To determine whether CTGF was capable of regulating its
own expression, or that of TGF-.beta., mesangial cells were also
exposed to 20 ng/ml of rhCTGF. As shown in FIG. 3A and FIG. 3C,
this treatment did not alter the level of TGF-.beta. mRNA, but in
contrast, strongly autoinduced CTGF message as demonstrated in FIG.
3A and FIG. 3B.
[0183] To demonstrate whether low CTGF mRNA expression in
unstimulated mesangial cells was associated with a detectable
production of the corresponding protein, and to determine the
effects of TGF-.beta., cells were serum depleted and then cultured
for an additional 24 hours in fresh maintenance medium in the
presence or absence of 2 ng/ml exogenous TGF-.beta.. The
conditioned medium was subsequently heparin-sulfate precipitated
and analyzed by immunoblotting using two different anti-CTGF
antibodies. Immunoblotting with pIgY3 antibody, raised against the
full-length rhCTGF, demonstrated that under basal, unstimulated
conditions mesangial cells secreted very small amounts of CTGF (see
FIG. 4A). However, upon exposure to TGF-.beta., the secretion of
CTGF protein was markedly stimulated. The predominant product
detected in these cultures migrated to the same position as the
recombinant standard. Immunoblotting of the same samples with the
pAb839 antibody, raised against a 15 amino acid sequence unique to
CTGF, confirmed the identity of the protein detected (see FIG.
4B).
Example 3
[0184] Detection Of CTGF In Mesangial Cells. The CTGF protein
detected In mesangial cell cultures above represents free molecules
present in the media. The existence of a heparin binding domain
within CTGF suggests that a substantial portion of the synthesized
and released protein exists bound to proteoglycans, or to
fibronectin, present on the cell surface or in the extracellular
matrix. To ascertain whether this was the case, and to determine
the time course for appearance of CTGF in the extracellular
environment under unstimulated conditions, mesangial cell cultures
were serum-deprived and then fresh maintenance media containing 50
.mu.g/ml of sodium heparin was added. Conditioned media were
collected after defined incubation periods and the majority of the
sample pooled and heparin-sulfate precipitated. Immunoblotting of
the 4 hour samples produced faint CTGF bands at approximately 36
and 39 kD (see, FIG. 5A). The intensities of these bands increased
sharply by 24 hours and remained elevated throughout the 72 hour
incubation period. At 48 and 72 hours, when the full-length CTGF
bands were intense, a faint additional band with an electrophoretic
mobility of approximately 20 kD could also be detected. As
previously demonstrated, in the absence of sodium heparin, the CTGF
present in the media was barely detectable, suggesting that the
majority of CTGF protein produced was bound to the cell and/or
substrate. Because immunoblotting is largely a qualitative assay,
individual supernatants were also evaluated by ELISA prior to their
pooling and precipitation, and the results were expressed on a per
cell basis. This highly quantitative assay demonstrated a time
dependent increase in CTGF, with approximately 7 ng/10.sup.6 cells
being secreted in a 24-hour period as shown in FIG. 5B. The amount
of CTGF secreted in the medium during the total 72-hour period was
reduced to 20% in the absence of heparin.
[0185] In a subsequent experiment, the regulation of secreted CTGF
by TGF-.beta. was reexamined, in the presence of heparin.
Accordingly, mesangial cells were serum-deprived, then incubated
for 48 hours in a maintenance media containing 50 .mu.g/ml of
sodium heparin and 2 ng/ml TGF-.beta.. Immunoblotting of pooled,
precipitated media samples indicated that TGF-.beta. markedly
increased the secretion of full-length (36-39 kDa) CTGF as
demonstrated in FIG. 6A. However, even more pronounced was the
induction of the molecule(s) appearing at 18-20 kDa. This smaller
moiety corresponds in size to half of the full-length CTGF
molecule. Quantitative analysis by ELISA of the individual samples,
prior to being pooled and precipitated for immunoblotting,
demonstrated a 2.5-fold enhancement of total secreted CTGF in
response to TGF-.beta. treatment (see FIG. 6B).
Example 4
[0186] Mesangial Cell Expression Of CTGF: Regulation By Glucose
Concentrations. To determine if CTGF expression might also be
altered by the ambient concentration of glucose, mesangial cell
cultures continuously grown in 5 mM glucose were incubated for 14
days in growth media containing 35 mM glucose. The time was chosen
because previous studies demonstrated that this period was required
for the full induction of ECM protein production. (See, e.g.,
Pugliese et al., 1997, J Am Soc Nephrol 8(3):406-414.) As shown in
FIG. 7, mesangial cells grown in medium containing 5 mM glucose
concentration demonstrated minimal levels of CTGF message. However,
following long-term exposure to an increased glucose concentration,
mesangial cell transcripts for CTGF were markedly upregulated,
reaching a 7-fold level above control, as determined by
quantitative image analysis as depicted in FIG. 7.
[0187] To examine the effects of high glucose exposure on the
secretion of CTGF protein, serum-deprived cultures that shortened
the exposure time to 48 hours, and included sodium heparin in the
medium were used. This protocol allowed a comparison to the effects
of TGF-.beta.. Immunoblotting of pooled and precipitated media
samples indicated that exposure to 20 mM of glucose increased the
amount of CTGF secreted as shown in FIG. 6A. Interestingly,
however, this stimulation appeared to be limited to the full-length
molecule only. Quantitation of secreted CTGF protein by ELISA,
prior to pooling and precipitation demonstrated a 2-fold induction
by high extracellular glucose levels as demonstrated in FIG. 6B,
which is an increase similar to that induced by TGF-.beta., under
the experimental conditions selected. To determine if the observed
increase in CTGF could be due to an osmolar effect, the experiments
were repeated using a mannitol. Under these conditions, there was
no induction of CTGF released as measured by ELISA (5 mM glucose,
2.39.+-.0.28 ng/10.sup.6 cells; 5 mM glucose plus 15 mM mannitol,
1.94.+-.0.32), and no change in the distribution of CTGF forms
secreted as determined by immunoblotting.
Example 5
[0188] TGF-.beta. Block Of High Glucose-Induced CTGF Production. To
determine if TGF-.beta. is responsible for CTGF production by
mesangial cells in the presence of high glucose, mesangial cells
were cultured for 14 days in the presence of either 5 mM glucose or
20 mM glucose, and were seeded and grown under the same glucose
conditions for an additional 8 day period. On day 4, the cultures
were serum-deprived, and half received 20 .mu.g/ml of an antibody
that neutralizes TGF-.beta.1, 2 and 3 activity. Fresh antibody was
added daily, and the media was replaced 24 hours prior to
collection. Measurement of CTGF secretion by ELISA demonstrated a
stimulatory effect of high glucose as depicted in FIG. 8. However,
neutralization of TGF-.beta. activity in these cultures blocked the
induction of CTGF by high glucose. While the constitutive secretion
of CTGF in the presence of normal concentrations of glucose also
appeared somewhat reduced by the presence of a TGF-.beta. antibody,
this change was not statistically significant (p=0.09). Also
non-significant (p=0.075) was the difference in CTGF levels in
normal glucose- and high glucose-treated cells when TGF-.beta. was
neutralized (see FIG. 8).
Example 6
[0189] Glucose transporter expression and CTGF production.
Mesangial cells transduced with the human glucose transporter 1
(GLUT1) gene producing a line designated, MCGT1, demonstrated a
10-fold increase in GLUT1 protein, a 5-fold increase in glucose
uptake and a 2-3 fold increase in the synthesis of collagen types I
and IV, fibronectin and laminin, as compared to a control mesangial
cell line, designated MCLacZ, and transduced with the bacterial
P-galactosidase gene. These cell lines were used to demonstrate
that the increase in intracellular glucose, rather than simply the
extracellular glucose concentration per se, is the major
determinant of exaggerated extracellular matrix formation by
mesangial cells in culture. (See, e.g., Helig et al., 1995, supra.)
To determine if CTGF is also increased in this in vitro model of
diabetes, MCCT1 and MCLacZ cells were seeded and grown for 48 hours
in RPMI with 20% NuSerum, 8 mM glucose. Cells were then washed
twice in serum-free media and fresh RPMI containing 1% FCS added.
Conditioned media were collected 24 hours later and CTGF protein
levels were determined by ELISA. Approximately 80 ng of
CTGF/10.sup.6 cells was detected in the media of the control MCLacZ
cultures whereas the level nearly doubled (147 ng/10.sup.6 cells)
in MCGT1 cultures as shown in FIG. 9.
Example 7
[0190] Mesangial Cell Expression Of CTGF: Regulation By Cyclic
Mechanical Strain. To determine if cyclic mechanical strain was
also a factor capable of altering mesangial cell expression of
CTGF, cells were seeded into collagen-coated flexible-bottom
plates, then after overnight incubation, and subjected to either
stretch or maintained under static conditions. Stretching was set
at 3 cycles per minute and 19% maximum elongation using the
computer-controlled system previously described in Riser et al.,
1992, supra, and Riser et al., 1996, supra. This degree of
stretching was chosen to approximate the mechanical force
experienced by mesangial cells in vivo. (See, e.g., Cartes et al.,
1997, supra.)
[0191] At the designated periods, the cells were lysed and total
RNAs extracted and probed for CTGF transcripts. Cyclic stretching
induced a rapid and marked increase in CTGF message as shown in
FIG. 9. Quantitative image analysis of the Northern blot showed
that levels of CTGF mRNA increased more than 2-fold by 4 hours and
remained elevated at this level after 8 hours of stretching.
Additional experiments demonstrated that CTGF transcripts were
significantly increased even after 48 hours of stretch.
Example 8
[0192] Blockade Of Stimulated Collagen Production By Anti-CTGF
Antibody.
[0193] Mesangial cells were grown for 4 days in RPMI medium with
20% NU-SERUM media supplement (Collaborative Research), the medium
was replaced with one containing 1% FCS (serum-deprived conditions)
and 0 or 5 ng/ml of TGF-.beta.2. Half of the cultures received
anti-CTGF antibody (goat affinity purified, pGAP) and the other
half received non-immune goat IgG. Fresh antibody was added daily,
and the media was replaced 24 hours prior to collection. Media from
individual wells were tested by ELISA. As shown in FIG. 11,
treatment with anti-CTGF antibody did not alter the production of
baseline collagen, but completely blocked the increased production
due to TGF-.beta.. There was no significant difference between the
amount of collagen produced in unstimulated cultures as compared to
that of cultures stimulated by TGF-.beta., but treated by anti-CTGF
antibody.
Example 9
[0194] Blockade Of Stimulated Mesangial Cell Proliferation by
Anti-CTGF Antibody. Mesangial cells were grown for four days in
RPMI medium with 20% NU-SERUM media supplement (Collaborative
Research), the medium was replaced with one containing 1% FCS
(serum-deprived conditions) and 0 or 5 ng/ml of TGF-.beta.2. Half
of the cultures received anti-CTGF antibody (goat affinity
purified, pGAP) and the other half non-immune goat IgG. Fresh
antibody was added daily, and the media was replaced 24 hours prior
to collection. Media from individual wells were tested by ELISA. As
shown in FIG. 12, TGF-.beta. treatment significantly induced (87%)
mesangial cell proliferation. Treatment with anti-CTGF antibody
significantly reduced (approximately 50%), the induction of cell
proliferation. The same antibody treatment had no effect on basal
proliferation, i.e., under unstimulated conditions.
Example 10
[0195] CTGF Expression In Experimental Diabetic Nephropathy. To
determine if CTGF is upregulated in early diabetic nephropathy,
studies were carried out on diabetic db/db mice, and the results
compared to those from age-matched nondiabetic db/m littermates. At
5 months of age, approximately 3.5 months after the onset of
diabetes, animals were evaluated for blood glucose levels, total
weight, proteinuria, and mesangial expansion. At the time of
sacrifice, mean blood glucose levels, as well as body weights, were
significantly greater in the db/db animals as shown in the
following Table 1.
1 TABLE 1 Control db/m Diabetic db/db Blood Glucose 142 .+-. 19.0
mg/dL, n = 8 485 .+-. 58.0, n = 6 P < 0.001 Weight 29.2 .+-.
1.00 g, n = 8 43.1 .+-. 7.30, n = 6 P < 0.001 Proteinuria 2.32
.+-. 1.07 mg/24 h, n = 10 2.78 .+-. 0.93, n = 9 P = 0.330
Glomerular Sclerosis 0.101 .+-. 0.048, n = 17 0.649 .+-. 0.369, n =
6 P = 0.003
[0196] As shown in FIG. 13, inspection of the renal tissue by light
microscopy demonstrated that the diabetic animals exhibited
noticeable, but minimal, glomerular changes consistent with early
diabetic glomerulosclerosis, i.e. mild mesangial matrix expansion
without apparent tubulointerstitial disease. In addition, Table 1
shows that the level of proteinuria was not significantly greater
than in controls. Semiquantitative analysis of the glomerular
changes demonstrated that the observed mesangial expansion in the
diabetic animals was indeed consistent, but of mild intensity. A
value of zero (0) represents no lesion and a value of one (1)
represents minimal mesangial expansion in the majority of
glomeruli, without basement membrane thickening.
[0197] Northern analysis of whole kidney RNAs indicated that the
CTGF message levels were markedly increased in 4 out of 5 diabetic
mice as shown in FIG. 14A. These changes were mirrored by parallel
changes in fibronectin transcript levels. Quantitation of results
yielded a mean 103% increase in CTGF expression while fibronectin
levels were 80% greater than in the controls as shown in FIG. 14B
and FIG. 14C. Moreover, transcript levels were detected in
competitive RT-PCR for CTGF mRNA in a single sample from diabetic
mouse glomeruli as compared to the control GAPDH sample as depicted
in FIG. 15A and FIG. 15B. Analysis of microdissected glomeruli
identified multiple animals (5 diabetic and 5 control groups), that
by a competitive and quantitative RT-PCR method (described above),
identified a low, but measurable, transcript level of CTGF in the
glomeruli of control animals. (See FIG. 16.) In mice with diabetes,
the level of CTGF was dramatically increased by 27-fold. (See FIG.
16.) The upregulation of glomerular CTGF mRNA was accompanied by a
nearly 5-fold increase in the amount of fibronectin mRNA. These
large differences were not due to dissimilar glomerular size
resulting from diabetic hypertrophy, since the level of GAPDH
message was not significantly increased in diabetic animals as
compared to controls (control, 1.39.+-.0.524.times.10.sup.-1
attomoles/glomerulus; diabetic 2.59.+-.0.307; P>0.05).
Therefore, a dramatic increase in CTGF expression was documented at
a time when changes in the kidney were minimal.
[0198] C. Detection of CTGF in Samples as an Indicator of Renal
Diabetes Associated Disorders
Example 11
[0199] The Presence And Stability Of Urinary CTGF. To examine
whether CTGF protein was secreted in the urine, and to examine the
stability of the CTGF molecule after its secretion in the urine,
samples of urine were collected from a healthy donor and divided
into five, 25-ml aliquots. Various amounts of rhCTGF, ranging from
25 to 750 ng, were added to four of the five aliquots (the "spiked
samples"). The fifth aliquot served as a control, receiving no
added CTGF (the "unspiked sample). All samples were frozen, and
stored at -70.degree. C., then later thawed and clarified by
centrifugation. Following heparin sepharose quantitative extraction
of samples, immunoblotting was performed using CTGF specific
antibody as indicated in FIG. 17. The results identified the
presence of a scarcely detectable level of CTGF secretion in the
urine of the unspiked sample. Further, the stability of the CTGF
protein in the urine was demonstrated by the progressive increase
in CTGF recovered in the spiked samples. Comparison of sample lanes
with that containing freshly added rhCTGF (35 ng) indicated that
CTGF was largely, if not entirely, preserved.
Example 12
[0200] CTGF In The Urine Of Renal Patients. The quantity and/or
molecular form of CTGF present in the urine that might be altered
in patients with established nephropathy, including that associated
with diabetes, was investigated. Urine samples from 8 ambulatory
patients being treated for a variety of kidney diseases, of which 3
had a history of diabetes, were collected and frozen during routine
visits (Nephrology and Hypertension Clinic, Henry Ford Hospital).
Similarly, samples were also obtained from 3 normal healthy
volunteers with no history of kidney disease. All samples were
later batch-thawed and processed above. CTGF was detected in 1 of 3
normal volunteers, and in all patient samples as shown in FIG. 18.
Immunoreactive CTGF appeared in 3 different molecular forms. A CTGF
band (doublet) was present in 1 control sample and 4 of 8 patients
samples. Interestingly, a large molecular weight band,
approximately 200 kDA, was present in every patient sample,
appearing only as a very faint band in a single control sample.
This large band likely represents CTGF in complex with a second,
unknown urinary protein. Even more intriguing was a unique small
CTGF fragment, approximately 9-12 kDa. This smaller moiety appears
to be equivalent to the heparin binding C-terminal quarter fragment
of CTGF, and was present in the urine of all 3 diabetic patients,
but not present in nondiabetic patients or healthy controls.
Interestingly, this product may compare to the CTGF fragment
produced by mesangial cells in culture when stimulated by high
glucose concentrations of TGF-.beta..
[0201] In a separate experiment, CTGF in human urine samples was
measure by ELISA, and presented in the following Table 2.
2TABLE 2 Patient Population Patient Healthy Kidney Diabetic: No
Number Control Disease Kidney Disease 1 0.81 0.36 0.68 2 0.43 1.22
7.12 3 0.84 5.46 5.68 4 0.67 2.53 0.40 5 5.72 0.56 6 0.78 3.84 7
3.47 Mean CTGF 0.69 2.79 3.05 .+-.SE 0.10 0.83 1.12
[0202] Table 2 compares the amounts of CTGF detected in healthy
volunteers to patients with kidney disease (in some cases
associated with diabetes), or to patients with 5 to 10 years with
diabetes, but without kidney disease. Each group had 4 to 7 samples
from different individuals. Amounts of CTGF/ml were first
determined by ELISA, comparing sample values to a standard curve
using serial dilutions of a known quantity of rhCTGF. To
standardize results (i.e. to account for variation in the
production of urine), amounts of CTGF (CTGF/ml) were then divided
by the urine creatinine from the same patient, determined from the
same urine.
[0203] The results demonstrate that healthy individuals demonstrate
consistently low levels of urinary CTGF. However, among those with
kidney disease the mean level of CTGF increased 4-fold. In those
patients with diabetes, but as yet undiagnosed kidney disease,
there was a similar 4.4-fold increase. It was expected that because
only approximately 40% of those with diabetes will go on to develop
nephropathy, a similar percent of patients would exhibit increased
CTGF levels. Interestingly, of the 6 diabetic patients tested, 3,
or 50%, demonstrated clearly elevated CTGF levels. The remaining
patients appeared to have values similar to those of the healthy
volunteers.
[0204] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims. All
patents, publications, and other references cited herein are
incorporated by reference herein in their entirety.
Sequence CWU 1
1
7 1 25 DNA Homo sapiens 1 gagtgggtgt gtgacgagcc caagg 25 2 25 DNA
Homo sapiens 2 atgtctccgt acatcttcct gtagt 25 3 20 DNA Rattus
norvegicus 3 tgccactgtt ctcctacgtg 20 4 20 DNA Rattus norvegicus 4
atgctttgac ccttacacgg 20 5 33 DNA Homo sapiens 5 gctccgcccg
cagtgggatc catgaccgcc gcc 33 6 30 DNA Homo sapiens 6 ggatccggat
cctcatgcca tgtctccgta 30 7 15 PRT Homo sapiens 7 Cys Pro Gly Asp
Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys 1 5 10 15
* * * * *