MXPA06009175A - Ctgf as target for the therapy of diabetic nephropathy - Google Patents

Abstract

The present invention relates to methods and compounds for treating specific early stage aspects and late stage aspects of diabetic nephropathy. Methods and compounds for treating various physiological features associated with early stage and with late stage diabetic nephropathy are also provided.

Description

CTGF AS AN OBJECTIVE FOR DIABETIC NEPHROPATHY THERAPY FIELD OF THE INVENTION The present invention relates to methods and compounds for treating specific aspects of early stage and late stage aspects of diabetic nephropathy. Methods and compounds are also provided to treat various physiological characteristics associated with the early stage and with the late stage of diabetic nephropathy.

BACKGROUND OF THE INVENTION A kidney disorder is any alteration in the normal physiology and function of the kidney. Kidney disorders can result from a wide range of acute and chronic conditions and events, including physical, chemical or biological damage, injury or trauma, disease, such as, for example, hypertension, diabetes, congestive heart failure, lupus, calciform cell anemia , and various inflammatory and autoimmune diseases, nephropathies associated with HIV, etc. Renal disorders can lead to reduced renal function, hypertension and renal failure, seriously compromising the quality of life, sometimes requiring dialysis and in certain circumstances, kidney transplantation. Diabetic nephropathy is a major long-term complication of diabetes mellitus, and that indication is primarily for kidney dialysis and kidney transplantation in the United States. (Marks and Raskin, 1998, Med Clin Norht Am., 82: 877-907). The development of diabetic nephropathy is observed in 26 to 50% of diabetic individuals Type 1 and Type 2. Consequently, diabetic nephropathy is the most common cause of end-stage renal disease, and renal failure in the Western world . Contributing risk factors associated with the development of diabetic nephropathy (and other renal disorders) in subjects with type 1 or type 2 diabetes, include hyperglycemia, hypertension, altered glomerular hemodynamics, and increased or aberrant expression of various growth factors, including transforming growth factor beta (TGFβ), insulin-like growth factor (IGF) -I, vascular endothelial growth factor (VEGF-A), and connective tissue growth factor (CTGF). English) . (See, for example, Flyvbjerg (2000) Diabetology 43: 1205-23; Brosius (2003) Exp Diab Res 4: 225-233; Gilbert et al. (2003) Diabetes Care 26: 2632-2636; and International Publication No. WO00 / 13706). Current treatment strategies aimed at slowing the progression of diabetic nephropathy using various procedures, including optimized glycemic control (through modification of diet and / or insulin therapy) and control of hypertension, have shown varying degrees of success. For example, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) administered to reduce hypertension have been shown to delay the progression or development of nephropathy and acroalbuminemia. Several clinical trials have established the benefits of ACE inhibitors and ARB in patients with diabetes. However, although ACE inhibitors have been shown to delay renal decline in patients with Type 1 diabetes, the renoprotective effect of these agents in patients with Type 2 diabetes is less clear. (Raij (2003) Am J Hypertens 16: 46S-49S). Furthermore, while glycemic control and blood pressure therapies significantly reduce the morbidity and mortality associated with diabetic nephropathy, by delaying the progression of associated pathologies, such conventional therapies do not adequately prevent the progression of the disease and thus fail. in providing a complete therapeutic effect. In addition, the administration of inhibitors of ACE or ARBs, to the current standard of health care, are not universally effective and only minimally delay, but do not eliminate, the need for kidney transplantation. Other treatment strategies have focused on one or more growth factors as therapeutic targets. Therapies directed at the inhibition of VEGF or TGFβ, either alone or in combination with ACE inhibitors or ARB, have been examined. (See, for example, De Vriese et al. (2001) J Am Soc Nephrol 12: 993-1000; Flyvbjerg et al. (2002) Diabetes 51: 3090-3094; Ziyadeh et al, (2000) Proc Nati Acad Sci 97: 8015-8020; Chen et al. (2003) Biochem Biophys Res Commun 300: 16-22; and Benigni et al. (2003) J Am Soc Nephrol 14: 1816-1824). Such therapeutic procedures, however, have not provided improvement of all aspects of renal pathology (e.g., impaired and impaired renal function and structure) associated with diabetic nephropathy. For example, the inhibition of TGFβ as a therapeutic target for diabetic nephropathy was not effective in attenuating albuminuria in db / db mice, despite the beneficial effects that such treatment had on glomerular matrix expansion. (See Ziyadeh et al, supra). In addition, while the administration of anti-VEGF antibodies to diabetic db / db mice provided benefit to the increased permeability associated with diabetes in the kidney, only minimal beneficial effects on mesangial expansion were observed. (See Flyvbjerg et al (2002), supra). Therefore, although such therapies offer promise, alone or in combination, none has resulted in the improvement of early pathological features (eg, glomerular hyperfiltration, increased glomerular filtration rate, icroalbuminuria, etc.) and late (by example, decreased glomerular filtration rate, macroalbuminuria, excessive mesangial matrix expansion, etc.) associated with chronic kidney disease, for example, diabetic nephropathy. Thus, there is a need in the art for a complete therapy, for the treatment of diabetic nephropathy that improves the symptoms of the early and late stages, and the pathologies associated with the development and progression of the disease. In addition to the above deficiencies, current therapies for diabetic nephropathy have limited applicability / efficacy due to lack of specificity. In particular, therapies directed to VEGF or TGFβ may compromise the beneficial activities of these growth factors, such as angiogenesis, tumor suppression, and proper development of the immune system. For example, while 'TGFβ has been associated with the development of fibrosis, it is also an important mediator of immune development and tumor suppression, suggesting that inhibition of TGFβ can have potentially adverse and undesirable side effects. Therefore, there is a need in the art for a more selective therapeutic procedure for diabetic nephropathy. In sum, there is a need in the art for a therapeutic procedure to treat kidney disease, in particular diabetic nephropathy, which is effective at various stages (e.g., early stage and late stage diabetic nephropathy) in the development and progression of the disease. disease. In particular, there is a need for a complete treatment for diabetic nephropathy, which is effective in treating the characteristics of the early stage and the characteristics of the late stage of diabetic nephropathy such as, for example, hyperfiltration (early stage), increased glomerular permeability (early stage), increased glomerular filtration rate (early stage), microalbuminuria (early stage), macroalbuminuria (late stage), and decreased glomerular filtration rate (late stage). There is a need for a therapeutic procedure that addresses more completely to diverse and distinct processes associated with the development and progression of diabetic nephropathy and other renal diseases. In particular, there is a need for therapies that target non-fibrotic (eg, hyperfiltration) and fibrotic (eg, mesangial matrix expansion) processes associated with diabetic nephropathy. In addition, there is a need for a therapeutic procedure to treat kidney disease in general, and diabetic nephropathy in particular, which provides structural and functional benefits. The present invention addresses these needs by identifying the role of CTGF in various processes associated with the development and progression of kidney disorders such as, for example, diabetic nephropathy, and by providing methods to inhibit and prevent these processes. The invention also addresses existing needs by providing methods and agents that can be applied to the treatment and prevention of kidney diseases, particularly, kidney disease associated with diabetes, and more particularly, diabetic nephropathy.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that the administration of the anti-CTGF antibody reduced the increase in the weight of the kidney in diabetic db / db mice. Figure 2 shows that administration of the anti-CTGF antibody reduced the clearance of creatinine in diabetic db / db mice. Figure 3 shows that administration of the anti-CTGF antibody reduced the expression of urinary albumin in diabetic db / db mice. Figure 4 shows the correlation between the levels of CTGF and VEGF in the vitreous humor.

Figure 5 shows that administration of anti-CTGF antibody reduced urinary volume in diabetic db / db mice. Figure 6 shows that anti-CTGF antibody administration reduced thickening of the basement membrane in kidneys of diabetic db / db mice. Figure 7 shows that administration of anti-CTGF antibody reduced proteinuria in a model of rat diabetic nephropathy. Figure 8 shows that administration of anti-CTGF antibody reduced BUN levels in a model of rat diabetic nephropathy. Figure 9 shows that the administration of the anti-CTGF antibody improved the glomerular filtration rate in a rat diabetic nephropathy model.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to methods and compounds for the treatment or prevention of specific aspects of early stage and late stage aspects of diabetic nephropathy, and for the treatment or prevention of various physiological characteristics associated with the Diabetic nephropathy of late stage and early stage. It is specifically contemplated that, in preferred embodiments of each of the methods described below, the preferred subject is a human subject. In one embodiment, the present invention provides a method for reducing the clearance of creatinine in a subject having or at risk of having diabetic or early-stage diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent which inhibits CTGF, which reduces the clearance of creatinine in the subject. Normal clearance levels of creatinine in humans are typically from about 97 to 137 ml / min (male adults) and 88 to 128 ml / min (in female adults). Therefore, methods for reducing creatinine clearance levels at or near these levels are specifically contemplated. Also provided herein are methods for reducing glomerular hyperfiltration in a subject having or at risk of having diabetic or early stage diabetic nephropathy, methods comprising administering to the subject a therapeutically effective amount of a agent that inhibits CTGF, as are methods to reduce glomerular hyperperfusion. In yet another aspect, the invention encompasses a method for reducing or preventing weight gain of the kidney in a subject having or at risk of having diabetes or diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent which inhibits CTGF. The invention further provides methods for normalizing glomerular filtration rate in a subject having or at risk of having diabetes or diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. Diabetic nephropathy can be, for example, early-stage diabetic nephropathy, late, incipient or evident stage. In the case in which diabetic nephropathy is of early or incipient stage, the normalization will probably be a decrease in the glomerular filtration rate, while in the case in which the diabetic nephropathy is of late or evident stage, the normalization will probably be a increase. Normal GFR in a human adult subject is approximately 120 ml / min. In the case where the subject has a high GFR above normal levels, and a decrease in GFR could be desired, methods to decrease GFR to levels below about 150 ml / min, below about 140 ml / min, below about 130 ml / min, and up to about 120 ml / min, are specifically contemplated. In the case where the subject has GFR impaired or decreased below normal, methods to increase GFR above about 15 ml / min, above about 30 ml / min, above about 60 ml / min , above about 90 ml / min, up to about 120 ml / min. In still another embodiment, the invention provides a method for reducing glomerular hypertrophy in a subject having or at risk of having diabetic or diabetic nephropathy, including early-stage diabetic nephropathy, late stage, incipient or evident stage, it comprises administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. Also provided herein are methods for reducing proteinuria in a subject having or at risk of having diabetes or diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. The invention further encompasses methods for reducing albuminuria in a subject having or at risk of having diabetes or diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. A method for reducing microalbuminuria is also described in a subject having or at risk of having diabetes or diabetic nephropathy, wherein diabetic nephropathy is early or incipient diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF, as well as a method for reducing macroalbuminuria is contemplated in a subject having or at risk of having diabetes or diabetic nephropathy, where diabetic nephropathy is late or evident stage, the method comprises administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. Normal excretion levels of urinary albumin in human adults are typically from about 15 to 30 mg per day. Macroalbuminuria is typically diagnosed when a subject has an urinary albumin excretion of approximately 30 to 300 mg / day. Macroalbuminuria is typically characterized by excretion of urinary albumin greater than about 300 mg / day. The present invention thus specifically provides methods for decreasing the excretion of urinary albumin in a subject, the method comprising administering to the subject an effective amount of an agent that inhibits CTGF, which has high excretion of urinary albumin, for example excretion levels. of urinary albumin elevated above normal. The modalities in which the excretion of urinary albumin is reduced below about 300 mg / day, below about 200 mg / day, below about 100 mg / day, below about 50 mg / day, and most preferably below about 30 mg / day, are specifically contemplated herein. In certain aspects, the invention provides a method for reducing BUN levels in a subject having or at risk of having diabetes or diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. Normal levels of BUN for human adults are in the range of 7 to 20 mg / dl. Therefore, r methods to reduce BUN levels below 20 mg / dl. The invention further provides a method for reducing the clearance of inulin in a subject who has or is at risk of having diabetes or diabetic nephropathy, the method comprises administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In specific aspects, diabetic nephropathy is late stage diabetic nephropathy or evident diabetic nephropathy. In still another embodiment, the invention provides a method for preventing, reducing the risk of, or delaying the onset of diabetic complications in a subject at risk of developing such complications, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In various modalities, diabetic complications include at least one complication selected from the group consisting of increased clearance of creatinine, increased or decreased glomerular filtration rate, thickening of the glomerular basement membrane, glomerular hyperfiltration, glomerular hyperperfusion, glomerular hypertrophy, increased excretion of urinary albumin, macroalbuminuria, macroalbuminuria, increased levels of BUN, increased clearance of inulin, kidney weight gain, and impaired renal function. The invention also encompasses a method for treating incipient diabetic nephropathy in a subject having or at risk of having incipient diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF, and a method of treating Early stage diabetic nephropathy in a subject having or at risk of having early stage diabetic nephropathy, the method comprises administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. Also contemplated herein is a method for treating obvious diabetic nephropathy in a subject having or at risk of having obvious diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

The present invention contemplates the use of the present methods in combination with other therapies. In one embodiment, the method is used in combination with another therapy, for example, to further increase the therapeutic effect on certain pathological events, etc. The two treatments can be administered at the same time or consecutively, for example, during a course of treatment time or after the progression and remission of the disease. In another embodiment, the method is used in combination with another therapeutic method that has a similar or different mode of action, for example, "inhibitors of ACE, ARB, statin, inhibitor of S final product of advanced glycation (AGE), etc. Thus, in a particular embodiment, the present invention provides a method for treating diabetic nephropathy in a subject having or at risk of having diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF, in combination with an inhibitory amount of an angiotensin-converting enzyme inhibitor.The present invention further provides a method for treating diabetic nephropathy in a subject having or at risk of having diabetic nephropathy, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF, in combination with a inhibitory nature of an angiotensin receptor blocker. Methods for treating progressive renal failure in a subject are provided in one embodiment, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In still another embodiment, the invention provides a method for reducing the risk or delaying the onset of the development of macroalbuminuria in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In a further embodiment, there is also provided a method for reducing the risk or delaying the onset of the development of macroalbuminuria in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In a particular aspect, the invention relates to the present discovery that CTGF is identified herein as a critical factor in early stage progressive diseases, including diabetic renal complications and vitreorretine disorders. Therefore, in one aspect, the invention relates to a method for treating or preventing the early stage aspects of a progressive disease in a subject having or at risk of having such a disease, the method comprising administering to the subject a Therapeutically effective amount of an agent that inhibits CTGF. In a further aspect, the progressive disease is associated with a growth factor different from CTGF, and, in a specific aspect, the other growth factor is VEGF. In one aspect, the progressive disease is a kidney disease, and, in a particular aspect, the progressive disease is associated with diabetes or with diabetic complications, or is diabetic nephropathy. The invention further encompasses a method for improving renal function in a subject having or at risk of impaired renal function, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. As summarized in the foregoing description, the invention relates to the discovery that anti-CTGF therapy is effective in the treatment or prevention of various physiological characteristics of early-stage and late-stage diabetic nephropathy. Accordingly, it is contemplated that the present invention provides methods for treating or preventing a renal disorder associated with at least one of the selected characteristics of the following: increased creatinine clearance; increased glomerular filtration or glomerular hyperfiltration, proteinuria; increased excretion of urinary albumin; increased glomerular volume; glomerular hypertrophy; increased renal weight; thickening of the glomerular basement membrane; reduced glomerular filtration rate, - increased levels of BUN; and increased clearance of inulin. In each case, the methods comprise administering to a subject in need of such treatment an effective amount of an agent that inhibits CTGF. These methods specifically cover the administration to a subject of the agent that inhibits CTGF for the express purpose of preventing progression to or development of any of the complications described above. The invention further encompasses the use of an agent that inhibits CTGF in the manufacture of a medicament to reduce the clearance of creatinine; reduce glomerular hyperfiltration; reduce glomerular hyperperfusion; reduce glomerular hypertrophy; reduce the thickness of the glomerular basement membrane; reduce the excretion of urinary albumin; reduce proteinuria; reduce albuminuria; reduce macroalbuminuria; reduce macroalbuminuria; reduce BUN levels; normalize glomerular filtration rate; reduce the clearance of inulin; reduce or prevent the weight gain of the kidney; prevent, reduce the risk of, or delay the onset of diabetic complications; for the treatment or prevention of the progression of incipient diabetic nephropathy; the treatment or prevention of the progression of early stage diabetic nephropathy; the treatment or prevention of the progression of obvious diabetic nephropathy; treat or prevent progressive renal failure; treat or prevent the development of early stage aspects of progressive disease in a subject; and improving renal function in a subject who has or is at risk of having diabetes or diabetic nephropathy. In various embodiments, the agent is selected from the group consisting of an antibody, a small molecule inhibitor, an antisense nucleic acid, and a siRNA. In preferred embodiments of the methods described above, the subject is a human subject. In any of the methods described above, it is particularly contemplated that the agent that inhibits CTGF may be a polypeptide, polynucleotide, or small molecule; for example, an antibody that binds to CTGF, an antisense molecule, siRNAs, small molecule chemical compounds, etc. In particular, the present invention contemplates that inhibition of CTGF can be achieved by any of the methods well known in the art for modulating the expression and activity of CTGF. The use of the anti-CTGF agent, for example, the human monoclonal antibody directed against CTGF, is preferred, although any method of expressing the inhibition of the gene encoding CTGF, the inhibition of CTGF production, or the inhibition of activity of CTGF, is contemplated by the present invention. For example, small molecule compounds can be used to inhibit CTGF expression, production or activity thereof. Since the expression of CTGF is inhibited by the cyclic nucleotide, such a combination may include, for example, a cyclic nucleotide analogue or a phospodiesterase (PDE) inhibitor. (See, for example, Duncan et al. (1999) FASEB J 13: 1774-1786). In addition, polynucleotides that include small interfering ribonucleic acids (siRNAs), microRNAs (miRNAs), ribozymes, and anti-sense sequences, can be used in the present methods to inhibit the expression and / or production of CTGF. (See, for example, Kondo et al. (2000) Biochem Biophys Res Commun 278: 119-124). Such techniques are well known to those skilled in the relevant art. Exemplary embodiments for use in the methods of the present invention are described, for example, in International Publication No. W02004 / 108764, which is incorporated by reference herein in its entirety.

DETAILED DESCRIPTION OF THE INVENTION It should be understood that the invention is not limited to particular methodologies, protocols, cell lines, assays and reagents described herein, since these may vary. It should also be understood that the terminology used herein is intended to describe the particular embodiments of the present invention, and is not intended in any way to limit the scope of the present invention as described in the appended claims. It should be noted that as used herein and in the appended claims, the singular forms, "a, an," and "the," include plural references, unless the context clearly dictates otherwise. Thus, for example, a reference to "a fragment" includes a plurality of such fragments, a reference to an "antibody" is a reference to one or more antibodies already equivalent thereto, known to those skilled in the art, and so on. Unless defined otherwise, all technical and scientific terms used herein have the same meanings that are commonly understood by a person of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and detailing the methodologies, reagents, and tools reported in publications that may be used in connection with the invention. Nothing in the present should be considered as an admission that the invention is not authorized to antedate such description by virtue of the prior invention. The practice of the present invention will employ, unless otherwise stated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill in the art. Such techniques are fully explained in the literature. See, for example, Gennaro, A.R. , ed. (1990) Remington's Pharmaceutical Sciences, 18th ed. , Mack Publishing Co.; Colowick, S. et al. , eds., Methods In Enzymology, Academic Press, Inc .; Handbook of Experimental Immunology, Vols. 1-4 (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., Eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press, -. Ausubel, F.M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., Eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press), - PCR (Introduction to Biotechniques Series), 2nd ed. (Newton &Graham eds., 1997, Springer Verlag). The invention relates in part to the discovery that the connective tissue growth factor (CTGF) plays a key role in the early stage specific aspects of kidney disease including, for example, glomerular hyperfiltration, increased glomerular permeability, the increased glomerular filtration rate, macroalbuminuria, etc. CTGF had previously been associated with specific aspects of late stage renal disease, for example, glomerulosclerosis and tubulointerstitial fibrosis, but it had not been identified as a critical objective • to affect various characteristics of early-stage renal disease.

Methods for treating or preventing renal disorders include, for example, diabetic nephropathy, and methods for treating or preventing associated pathologies are specifically contemplated. The present invention provides the methods and compositions for reducing or improving in a subject, the complications associated with different, multiple pathological processes, associated with kidney disorders, for example, diabetic nephropathy, by inhibiting CTGF. In some embodiments, the subject is an animal, more preferably a mammal, and most preferably a human. The present invention also provides the compositions for use in the methods described herein. Such compositions may include small molecule compounds; peptides and proteins that include antibodies or functionally active fragments thereof; and polynucleotides that include small interfering ribonucleic acids (siRNAs), microRNAs (miRNAs), ribozymes, and anti-sense sequences. (See, for example, Zeng (2003) Proc Nati Acad Sci USA 100: 9779-9784; and Kurreck (2003) Eur J Biochem 270; 1628-1644). The present invention is based in part on the discovery of the unexpected benefits of inhibition of CTGF in the treatment of multiple and specific aspects of renal disorders, for example, diabetic nephropathy. The present invention provides the data demonstrating that inhibition of CTGF reduced various pathological aspects of kidney disease, not previously associated with CTGF. In certain aspects, the present invention provides evidence that inhibition of CTGF provides a therapeutic method to treat or prevent specific physiological aspects of diabetic nephropathy, previously associated with biological and pathological VEGF activities, such as, for example, glomerular hyperfiltration and hyperperfusion.

Diabetic Ephropathy Diabetes is a leading cause of morbidity and mortality worldwide, with approximately 40% of all individuals with diabetes developing diabetic nephropathy, requiring either renal dialysis or transplantation. Diabetes is the leading cause of end-stage kidney disease, and therefore, any individual diagnosed with diabetes is at risk of developing diabetic nephropathy. The progression of diabetic nephropathy is characterized by a clearly predictable pattern of events. In general, the time course of development of diabetic nephropathy is as follows. Glomerular hyperfiltration and renal hypertrophy occur in the first few years after onset of diabetes and are reflected by an increased glomerular filtration rate (eg, from a normal glomerular filtration rate of approximately 120 ml / min to approximately 150 ml / min in humans). During the first 5 years of diabetes, pathological changes, such as glomerular hypertrophy, thickening of the glomerular basement membrane, and expansion of the glomerular mesangial volume, are observed. The glomerular filtration rate returns gradually to normal. After 5 to 10 years of diabetes, individuals begin to excrete small amounts of albumin in the urine (macroalbuminuria). Macroalbuminuria (diabetic individuals with macroalbuminuria are referred to as having incipient diabetic nephropathy) is an important predictor of progression to evident diabetic nephropathy (characterized, in part, by macroalbuminuria or overt proteinuria). The thickening of the basement membrane and the expansion of the glomerular volume observed in the early stages of the disease can accumulate in late-stage diabetic nephropathy, leading to obliteration of the capillary lumen, and sooner or later, to glomerulosclerosis. Once obvious diabetic nephropathy is present, a gradual decline in glomerular filtration rate occurs, and approximately half of the individuals reach end-stage renal disease in 7 to 10 years. Clinically, the stages of development and progression of diabetic nephropathy in humans have been well described. Stage I diabetic nephropathy is associated with increased (eg, glomerular) renal filtration (eg, hyperfiltration, resulting from increased blood flow through the kidneys and glomeruli), increased glomerular filtration rate, glomerular hypertrophy, and kidneys enlarged Stage II diabetic nephropathy is a clinically silent phase associated with continuous hyperfiltration and renal hypertrophy. Thickening of the glomerular basement membrane and mesangial expansion also occurs. Stage III diabetic nephropathy (also known as incipient diabetic nephropathy) is associated with macroalbuminuria and microproteinuria. Macroalbuminuria is defined as 30 to 300 mg / day of urinary albumin in a 24-hour collection, 20 to 200 μg / min of urinary albumin, or 30 to 300 μg / mg of creatinine in a point collection. The kidneys progressively lose the ability to filter waste, and blood levels of creatinine and urea-nitrogen increase. Thickened glomerular basement membrane and mesangial expansion continue to occur, with increasing severity. Stage IV diabetic nephropathy (also known as obvious diabetic nephropathy) is associated with macroalbuminuria (eg, clinical albuminuria) and levels of creatinine and blood urea-nitrogen (BUN) in the blood continue to rise. Macroalbuminuria is defined as more than 300 mg / day of urinary albumin in a 24-hour collection, more than 200 μg / min of urinary albumin, or more than 300 μg / mg of creatinine in. the collection by points. Once obvious diabetic nephropathy occurs, the glomerular filtration rate gradually falls over a period of several years. Stage V diabetic nephropathy occurs with end-stage renal disease and renal failure.

Hyperfiltration and Hyperperfusion Early-stage diabetic nephropathy is associated with impaired renal function, characterized in part by glomerular hyperfiltration and hyperperfusion. Glomerular hyperfiltration is a glomerular adaptation to the loss of nephroids, associated with hyperglycemia and diabetes. With the loss of the functional mass of nephroids, the remaining functional nephroids become hypertrophied and adopt an increased workload, which attempts to minimize the complete loss of renal function. As a result, glomerular hyperfiltration and hyperperfusion occur. Glomerular hyperfiltration and hyperperfusion are reflected as the increased glomerular filtration rate. The glomerular filtration rate is a measurement of the volume of filtering performed by the kidneys per minute. The measurement of glomerular filtration rate in human subjects has been accepted as the best total index of renal function in health and disease. (Smith, Diseases of the kidney and urinary tract, In: Structure and Function in Health and Disease, New York, Oxford Univ. Press, 1951: 836-887). The glomerular filtration rate can be determined by various methods, such as by measuring the urinary clearance of a filtration marker, such as inulin, iotalamate or iohexol. More commonly, the glomerular filtration rate is estimated by determining the clearance of creatinine, a protein produced by the muscle and released into the blood. The clearance of creatinine (often expressed - as ml / min) can be determined by comparing the level of creatinine collected in urine in a given period of time, for example 12 hours or 24 hours, with the level of creatinine in blood. A typical creatinine clearance rate is approximately 97 to 137 ml / min in male adults, and approximately 88 to 128 ml / min in female adults. In clinical practice, the clearance of creatinine is more frequently estimated from the -concentration of serum creatinine. The clearance of creatinine is directly related to the excretion of urinary creatinine and inversely to the concentration of serum creatinine. Various formulas that provide estimates of creatinine clearance, and therefore estimates of glomerular filtration rate, using parameters such as serum creatinine concentration, age, sex, and body size, have been developed and they are standards in the matter. (See, for example, Cockcroft and Gault (1976) Nephron 16: 31-41; Levey et al (1999) Annals or Internal Medicine 130: 462-470; Rule et al (2004) Ann Intern Med 141: 929-937 ). The methods and compounds of the present invention reduced the clearance of creatinine in an animal model of diabetes. (See, for example, Example 1). Therefore, the present invention provides methods and compounds for reducing clearance of creatinine in a subject with increased or elevated creatinine clearance or in which the clearance of creatinine is elevated above normal levels. The present invention demonstrates that the inhibition of CTGF (for example, by the administration of an antibody to CTGF) reduces the clearance of creatinine associated with nephropathy, and in particular, diabetic nephropathy. Increased clearance of creatinine is associated with glomerular hyperfiltration, hyperperfusion, hypertrophy, and increased glomerular filtration rate, and is indicative of impaired or impaired renal function in the early stages of the development of nephropathy, for example, nephropathy diabetic In one aspect, the present invention provides the methods and compounds for reducing the clearance of creatinine by inhibiting CTGF. In another aspect, the present invention provides methods and compounds for reducing permeability of glomerular creatinine and restoring glomerular selectivity and glomerular function by inhibition of CTGF. In another aspect more, methods and compounds are provided to treat or prevent glomerular hypertrophy, hyperfiltration and hyperperfusion associated with hyperglycemia or diabetes, by inhibiting CTGF. In yet another aspect, methods and compounds for treating or preventing glomerular hypertrophy, hyperfiltration, and hyperperfusion associated with kidney diseases, and, in particular, diabetic nephropathy, by inhibiting CTGF are provided. In one aspect, kidney disease is early-stage diabetic nephropathy. In other aspects, the present invention provides the methods and compounds - for reducing the rate of glomerular filtration in a subject, with an increased glomerular filtration rate, by inhibiting CTGF. In one aspect, the present invention provides methods and compounds for reducing glomerular filtration rate, by administering to a subject having or at risk of having an impaired or increased glomerular filtration rate, of an agent that inhibits CTGF. In one aspect, impaired glomerular filtration and increased glomerular filtration rate are associated with early-stage kidney disease. In certain embodiments, the present invention provides methods and compounds for treating a kidney disorder associated with or characterized by increased clearance of creatinine, by administering to a subject having or at risk of having the disorder, an agent that inhibits CTGF. , treating or preventing in this way the disorder. In other embodiments, the present invention provides methods and compounds for the treatment of a renal disorder associated with or characterized by increased glomerular filtration and glomerular hyperfiltration by administration to a subject having or at risk of having the disorder, of an agent which inhibits CTGF, treating or preventing the disorder in this way. It was found that the methods and compounds of the present invention increase the glomerular filtration rate in an animal model of late stage diabetic nephropathy. (See E pg 3). Therefore, the present invention provides the methods and compounds for increasing or normalizing the glomerular filtration rate in a subject, with an impaired or reduced glomerular filtration rate, or in which the glomerular filtration rate is below normal. , by the inhibition of CTGF. In one aspect, the present invention provides the methods and compounds for. increase or normalize the glomerular filtration rate by administering to a subject having or at risk of having an impaired or reduced glomerular filtration rate of an agent that inhibits CTGF. In yet another aspect, the impaired glomerular filtration rate and reduced glomerular filtration rate are associated with diabetic nephropathy of late or overt stage renal disease. In one aspect, the present invention provides methods and compounds for treating or preventing a renal disorder associated with impaired glomerular filtration rate and decreased glomerular filtration rate, when administered to a subject having or at risk of having the disorder, an agent that inhibits CTGF, treating or preventing the disorder in this way. In yet another aspect, the impaired glomerular filtration rate and the reduced glomerular filtration rate are associated with late stage kidney disease. It is contemplated that the present methods can be applied to improve renal function, to normalize the rate of glomerular filtration, to reduce hyperfiltration and glomerular hyperperfusion, or to reduce "creatinine clearance in a subject, with any clinically accepted standard of measurement. indicative of kidney disease or kidney disease, or a subject at risk of developing such renal disease., the subject has diabetic kidney disease. In various modalities, the subject has stage I kidney disease, stage II kidney disease, stage III kidney disease, stage IV kidney disease, or stage V kidney disease. The present methods are applied to prevent, reduce or delay the initiation of renal complications associated with early-stage kidney disease in a subject at risk of developing such complications, or the manufacture of a medicament for a subject, preferably a human subject, having any of the disorders and characteristics associated with the kidney disease discussed in the present. In one aspect, the subject has diabetes. Diabetes "can be determined by any measurement accepted and used by those skilled in the art." A human subject could be diagnosed with diabetes with a blood glucose level greater than about 20 mg / dL (as determined by a glucose test. fasting blood, an oral glucose tolerance test or a random blood glucose test.) Therefore, in certain aspects, it is contemplated that a human subject having a blood glucose level above about 200 mg / dl is a suitable subject for treatment with the methods or use of the medicaments provided by the present invention Other suitable subjects contemplated for treatment with the present methods have impaired glomerular filtration rate In one embodiment, the human subject has a filtration rate glomerular or above the normal glomerular filtration rate, for example, above approx. 120 ml / min. Therefore, it is contemplated that a subject, having a glomerular filtration rate above about 120 ml / min, above about 130 ml / min, above about 140 ml / min, or above about 150 ml / min is a suitable subject for treatment with the methods or use of the medicaments provided by the present invention. It is further contemplated, in various embodiments, that methods for reducing the glomerular filtration rate in a subject with increased glomerular filtration rate (eg, in a subject with hyperfiltration and glomerular hyperperfusion) can be applied to reduce the glomerular filtration rate in a human subject, at a level below 150 ml / min, in or about 140 ml / min, below about 130 ml / min, or at a level of about 120 ml / min. The methods and compounds of the present invention reduced the increase in kidney weight associated with diabetes and early stage diabetic nephropathy in an animal model of diabetes. (See Example 1). Therefore, a method of treating or preventing a kidney disorder associated with increased kidney weight, the method comprises administering to a subject having or at risk of having a disorder, an agent that inhibits CTGF, treating or preventing this mode the disorder, is contemplated by the present invention. The invention further contemplates a method for treating or preventing a renal disorder associated with increased glomerular volume, the method comprising administering to a subject having or being suspected of having the disorder, an agent that inhibits CTGF, thereby treating or preventing the disorder. The present methods and compounds are also applied to prevent, reduce or delay the onset of renal complications associated with late stage kidney disease in a subject at risk of developing such complications, or the manufacture of a medicament for a subject, preferably a human subject, who has any disorder or conditions associated with the early stage kidney disease discussed herein. In one aspect, the human subject has a glomerular filtration rate below a normal glomerular filtration rate, for example, below about 120 ml / min. Therefore, it is contemplated that a human subject having a glomerular filtration rate below about 120 ml / min, below about 90 ml / min, below about 60 ml / min, below about 30 ml / min, or below approximately15 ml / min is a suitable subject for treatment with the methods or use of the medicaments provided by the present invention. It is further contemplated, in various embodiments, that methods for increasing the glomerular filtration rate in a human subject with reduced or impaired glomerular filtration rate (for example in a subject with obvious diabetic nephropathy) can be applied to increase the filtration rate glomerular at a level above about 15 ml / min, above about 30 ml / min, above about 60 ml / min, above about 90 l / min, and up to a level of about 120 ml / min . In certain modalities, the kidney disorder is associated with type 1 or type 2 diabetes. In other modalities, the kidney disorder is diabetic nephropathy.

Macroalbuminuria The evidence of early clinical nephropathy, including diabetic nephropathy, is the appearance of low but abnormal levels of albumin in the urine, a condition termed as macroalbuminuria. Individuals with macroalbuminuria are referred to as having incipient nephropathy or, if associated with diabetes, incipient diabetic nephropathy. Diabetic individuals with macroalbuminuria have an increased risk in a 42% progression to evident diabetic nephropathy, in comparison to those with normoalbuminuria (Bruno et al., 2003, Diabetes Care 26: 2150-2155). Therefore, the onset and development of macroalbuminuria in individuals with diabetes is associated with the increased risk of progression to overt diabetic nephropathy (eg, macroalbuminuria) and eventual end-stage renal disease and renal failure. (See, for example, Mogensen and Christensen (1984) N Engl J Med 311: "89-93; Mogensen et al. (1983) Diabetes 32 [Suppl 2]: 64-78; Viberti et al (1982) Lancet 1: 1430-1432). Macroalbuminuria can be determined by several methods, including: (1) the measurement of the albumin to creatinine ratio in a random spot urine collection; (2) 24-hour urine collection with creatinine, allowing simultaneous measurement of the clearance of creatinine, and (3) synchronized collection (eg, every 4 hours or overnight) Normal excretion of urinary albumin in humans is less than 30 μg / mg creatinine (point collection), less than 30 mg / 24 hours (24-hour collection), or less than 20 μg / min (synchronized collection) Macroalbuminuria in humans who have urinary albumin excretion from 30 to 299 μg / mg of creatinine (point collection, 30 to 299 mg / 24 hours (24-hour collection) or 20 to 199 μg / min (synchronized collection). Macroalbuminuria (for example, clinical albuminuria) in humans is the position of urinary albumin excretion greater than or equal to 300 μg / mg creatinine (point collection), more than or equal to 300 mg / 24 hours (24-hour collection) ) or more than or equal to 200 μg / min (synchronized collection). The present invention demonstrates for the first time that the inhibition of CTGF (for example, by the administration of an antibody to CTGF) reduces the excretion of urinary albumin associated with nephropathy, and in particular, diabetic nephropathy. (See, for example, Example 1). Increased excretion of urinary albumin is associated with changes in the permeability and selectivity of glomerular albumin, and is indicative of impaired or impaired renal function in early stages of the development of nephropathy. In one aspect, the present invention provides methods for reducing the excretion of urinary albumin by inhibiting CTGF. In another aspect, the present invention provides methods for reducing permeability to glomerular albumin and for restoring glomerular selectivity by CTGF inhibition. In still another aspect, the present invention provides methods for reducing macroalbuminuria by inhibiting CTGF. Through the reduction of macroalbuminuria and the excretion of urinary albumin, the present methods, therefore, provide a means to treat early stage kidney disease and incipient nephropathy. As described above, in early-stage kidney disease, the onset and development of macroalbuminuria (eg, incipient nephropathy) is associated with an increased risk of macroalbuminuria development, evident nephropathy, and early-stage kidney disease, and kidney failure in individuals with diabetes. The methods and compositions of the present invention, therefore, are also applied to the prevention, reduction or delay of the onset of or reduction in the risk of developing renal complications associated with late stage kidney disease, including macroalbuminuria, evident nephropathy, and end-stage renal disease, and renal failure, in a subject at risk of developing such complications. The present invention demonstrates that inhibition of CTGF (e.g., by administration of an antibody to CTGF) reduces proteinuria, BUN levels, and creatinine clearance associated with nephropathy. Increased proteinuria, BUN levels, and creatinine clearance are indicators of impaired or impaired renal function and the development of nephropathy. In one aspect, the present invention provides methods and compounds for reducing proteinuria by inhibiting CTGF. In still another aspect, the present invention provides methods and compounds for reducing BUN levels by inhibiting CTGF. In yet another aspect, the methods and compounds are provided to reduce the clearance of creatinine by inhibition of CTGF. The present invention demonstrates that inhibition of CTGF (e.g., by administration of an antibody to CTGF) improves renal function. Since diabetic nephropathy progresses to late-stage kidney disease, the decline in glomerular filtration rate, as measured, for example, by decreased clearance of inulin, is an indicator of impaired or impaired renal function. The present invention further demonstrates that inhibition of CTGF (e.g., by administration of an antibody to CTGF) improved the rate of impaired or reduced glomerular filtration associated with late stage kidney disease. (See Example 3). In one aspect, the present invention provides methods and compounds for increasing the rate of glomerular filtration by CTGF inhibition. In still another aspect, the present invention provides the methods and compounds for decreasing the clearance of inulin by CTGF inhibition. In yet another aspect, methods and compounds are provided for treating or preventing impaired renal function, in particular impaired renal function associated with nephropathy, such as diabetic nephropathy by inhibition of CTGF. In other aspects, nephropathy is associated with decreased glomerular filtration rate, macroalbuminuria, or evident nephropathy. Late-stage diabetic nephropathy is associated with various pathological and morphological changes in the kidney. Such changes include mesangial expansion, associated with increased matrix production and accumulation of the mesangial extracellular matrix; expansion of mesangial cells; thickening of the glomerular basement membrane, which in late-stage diabetic nephropathy is associated with glomerulosclerosis; and development of tubulointerstitial fibrosis. (Gilbert et al (1999) Kidney Int 56: 1627-1673). Glomerulosclerosis and tubulointerstitial fibrosis are the key structural markers of late-stage kidney disease, of advanced diabetic nephropathy with renal failure, resulting in a reduction in glomerular filtration rate and, possibly, end-stage renal disease. and kidney failure. Prior to the present invention, CTGF had been associated with characteristics of late stages of renal pathology, specifically, excess extracellular matrix production, excess mesangial matrix expansion, and development of glomerulosclerosis and tubulointerstitial fibrosis. (See, for example, International Publication No. WO00 / 13706). It was thought that other factors, for example, VEGF, were responsible for the processes, pathologies and various characteristics associated with early stage kidney disease, for example, hyperfiltration and increased glomerular patency. In contrast, the present invention provides the data demonstrating that it is CTGF that plays a key role in the development and progression of the early stage, as well as the late stage aspects of nephropathy, and thus represents an ideal target for a complete and effective procedure to diabetic nephropathy. The present invention provides methods to treat and prevent various early stage clinical and pathological aspects, as well as late stage diabetic nephropathy. Specifically, the methods and compositions of the present invention are useful for treating or preventing glomerular hyperfiltration and expansion of the mesangial matrix. Therefore, the present invention contemplates methods for treating various aspects of kidney disease, including features of early diabetic kidney disease, such as, for example, hypertrophy and renal and glomerular hyperfiltration (measures such as increased clearance of creatinine, increased excretion of urinary albumin, increased speed of glomerular filtration, etc.), and characteristics of late-stage diabetic kidney disease (decreased glomerular filtration rate, expansion of the mesangial matrix, thickening of the basement membrane, etc.). The present invention provides the methods, and compositions for use herein, for treating a disorder or condition wherein the connective tissue growth factor (CTGF) is a mediating factor. Various disorders associated with CTGF have been described in the literature; however, until the present invention, CTGF was mainly associated with fibroproliferative conditions, particularly those associated with TGFβ. Although numerous disorders involve fibroproliferative processes, and the treatment of these disorders using therapies directed to provide or prevent the activity of CTGF have been suggested, the present invention extends to the understanding and, thus, to the use of therapies directed to CTGF for the treatment of various conditions and complications, not fibroproliferative associated with diabetic nephropathy and renal disorders. The methods of the present invention, for example, the inhibition of CTGF, effectively reduce hyperfiltration by the kidney and normalize or restore renal function as measured, for example, by glomerular filtration rate, urinary albumin excretion, albuminuria and / or proteinuria. Thus, the methods and compositions of the present invention can be used to "treat patients at risk for diabetic nephropathy, including, for example, early-stage diabetic nephropathy and incipient diabetic nephropathy." Such subjects include individuals diagnosed with hyperglycemia, hypertension and / or diabetes. Additionally, the methods of the present invention can be used to treat patients diagnosed with a renal disorder such as glomerulosclerosis, glomerulonephritis or diabetic nephropathy. The methods of the present invention, for example, the inhibition of CTGF, reduce the expansion of the mesangial matrix and the thickening of the glomerular basement membrane. Thus, the methods of the present invention can be used to treat patients at risk of diabetic nephropathy to prevent albuminuria, reduced glomerular filtration rate, and the like. Such objects include individuals diagnosed with hyperglycemia, hypertension and / or diabetes. In addition, the methods of the present invention can be used to treat patients who have obvious diabetic nephropathy or other renal disorder such as glomerulosclerosis, glomerulonephritis, etc. Therefore, in one aspect, the present invention contemplates methods for treating or preventing the processes associated with early stage kidney disease or late stage kidney disease by the inhibition of CTGF. These pathological functions include, for example, hyperfiltration, albuminuria, proteinuria, glomerular hypertrophy and expansion of the mesangial volume. The use of the present methods to treat or prevent the aspects of the early stage and late stage of kidney disease, previously associated with VEGF and TGFβ, is specifically contemplated. As stated above, the methods can be used to treat patients at risk of diabetic nephropathy or an associated pathology, and to treat patients who have a renal disorder such as glomerulosclerosis, glomerulonephritis, diabetic nephropathy, etc. The present invention contemplates the use of the present methods in combination with other therapies. In one embodiment, the method is used in combination with another therapy, for example, to further increase the therapeutic effect on certain pathological events, etc. The two treatments can be administered at the same time or consecutively, for example, during a course of treatment time or after the progression and remission of the disease. In yet another embodiment, the method is used in combination with another therapeutic method having a similar or different mode of action, for example, an inhibitor of ACE, ARB, statin, inhibitor of the final product of advanced glycation (AGE), etc. . Current therapeutic methods for treating diabetic nephropathy are known to one of skill in the art, and include, for example, ACE inhibitors, angiotensin receptor blockers, statins, end-product inhibitors of advanced glycation, gene therapy with the growth factor of hepatocytes, pyridoxa ina, Enapril, PPAR antagonists, sulphonylurea antagonists, matrix metalloproteinase inhibitors, COX-2 inhibitors, pirfenidone, sulodexide, high-dose thiamine and Benfotiamine, blockers of calcium channels, etc. The use of any of these therapeutic agents in combination with the use of the methods of the present invention is specifically contemplated. The present invention represents the first time that the therapeutic efficacy of two distinct pathological aspects associated with kidney disease has been demonstrated (eg, early stage characteristics and late stage characteristics of diabetic nephropathy). Although anti-CTGF therapy is exemplified herein using a human monoclonal antibody directed against CTGF, any method of inhibiting the expression of the gene encoding CTGF, inhibiting the production of CTGF, or inhibiting the activity of CTGF it is contemplated by the present invention. For example, small molecule compounds can be used to inhibit CTGF expression, production or activity thereof. Since the expression of CTGF is inhibited by the cyclic nucleotide, such a compound may include, for example, a cyclic nucleotide analog or a phosphodiesterase (PDE) inhibitor. (See, for example, Duncan et al. (1999) FASEB J 13: 1774-1786). In addition, polynucleotides that include small interfering ribonucleic acids (siANRs), microRNAs (miRNAs), ribozymes, and anti-sense sequences can be used in the present methods to inhibit the expression and / or production of CTGF. (See, for example, Kondo et al. (2000) Biochem Biophys Res Commun 278: 119-124; and SIMO et al., supra). Such techniques are well known to those skilled in the relevant art. The present invention provides exemplary evidence that the methods described herein, using an anti-CTGF monoclonal antibody in animal models of diabetes, provide improvement in creatinine clearance and glomerular hypertrophy, and a decrease in kidney weight, an indicator of glomerular fibrosis and mesangial expansion induced by TGFβ and mediated by CTGF. In this way, the methods of the present invention improve the two pathologies that contribute to diabetic nephropathy, for example, mesangial expansion and glomerular filtration. In certain aspects, the present invention provides methods and compositions for treating a disorder associated with TGFβ, by inhibiting CTGF. In other aspects, the present invention provides methods and compositions for treating a disorder associated with VEGF by inhibiting CTGF. In still other aspects, the present invention provides methods and compositions for treating a disorder associated with TGFβ and VEGF by inhibiting CTGF. The methods and compositions for treating a disorder associated with other growth factors, eg, IGF-1, endothelin, etc., are also contemplated in the present invention. In one aspect, the present invention provides a method for treating or preventing a renal disorder associated with increased clearance of creatinine, the method comprising administering to a subject having or at risk of having the disorder, an agent that inhibits CTGF. (for example, it inhibits or reduces the expression of CTGF or the activity of CTGF), treating or preventing in this way the renal disorder. In yet another aspect, the present invention provides methods for treating or preventing a renal disorder associated with increased gomerular filtration and hyperfiltration by administration to a subject having or at risk of having the disorder, of an agent that inhibits CGTF. , treating or preventing the disorder in this way. In yet another aspect, the present invention provides methods for treating or preventing a renal disorder associated with thickening of the basement membrane, by administering to a subject having or at risk of having the disorder, a queyLhhibe CTGF agent, thereby treating or preventing the disorder. In yet another aspect, the present invention provides methods for treating or preventing a renal disorder associated with increased urine volume, by administering to a subject having or at risk of having the disorder, an agent that inhibits CTGF, treating or thus preventing the disorder. A method is also provided for the treatment of a renal disorder associated with increased urinary albumin excretion by administering to a subject having the disorder or at risk of having the disorder, of an agent that inhibits CTGF, by treating or preventing this way the kidney disorder. In one aspect, the present invention provides methods for reducing the clearance of creatinine in a subject in need of such treatment, the method comprising administering to the subject an agent that inhibits CTGF. Methods are also provided to reduce the excretion of urinary albumin, to reduce glomerular filtration and glomerular hyperfiltration, to reduce glomerular volume expansion, or to reduce the increase in kidney weight in a subject in need of such treatment, the methods comprise administering to the subject an agent that inhibits CTGF. In one embodiment, the present invention provides a method for treating or preventing proteinuria associated with kidney disease, the method comprising administering to a subject having or at risk of having kidney disease, an agent that inhibits CTGF. In a further embodiment, proteinuria is albuminuria. In respective modalities, albuminuria is microalbuminuria or macroalbuminuria. In yet another embodiment, the present invention provides a method for treating or preventing thickening of the basement membrane in the kidney, the method comprising administering to a subject having or at risk of having the thickening of the basement membrane in the kidney. , an agent that inhibits CTGF. In yet another embodiment, the present invention provides a method for reducing or preventing increased urinary volume, by administering to a subject having or at risk of having increased urinary volume, an agent that inhibits CTGF. The methods of the present invention include administering to a subject in need of a therapeutically effective amount of an agent that inhibits CTGF (e.g., that reduces the expression or activity of CTGF). In certain embodiments, the agent is an antibody to CTGF.

In a preferred embodiment, the antibody is a monoclonal antibody to CTGF. In another preferred embodiment, the antibody is a human or humanized antibody to CTGF. In another, more mode, the agent is a small molecule. In yet another embodiment, the agent is an antisense oligonucleotide. Several agents have been identified that inhibit CTGF. Antibodies that bind to CTGF are described in U.S. Patent No. 5,408,040; International Publication No. WO99 / 07407; Publication International No W099 / 33878, and International Publication Do not . WO00 / 35936. An exemplary antibody for use in the methods of the present invention has been described in International Publication No. WO2004 / 108764, incorporated by reference herein in its entirety. Such antibodies or fragments thereof can be administered by various means known to those skilled in the art. For example, antibodies are frequently injected intravenously, intraperitoneally or subcutaneously. Small molecule inhibitors of CTGF expression and / or activity thereof have also been described; for example, International Publication No. W096 / 38172 identifies cAMP modulators such as cholera toxin and 8Br-cAMP as inhibitors of CTGF expression.

Therefore, compounds identified as, for example, prostaglandin and / or prostacyclin analogues such as Iloprost (see, for example, International Publication No. WO00 / 02450; Ricupero et al. (1999) Am J Physiol 277: L1164- 1171; also see Ertl et al. (1992) Am Rev Respir Dis 145: A19), and potentially the phosphodiesterase IV inhibitors (see, for example, Kohyama et al. (2002) Am J Respir Cell Mol Biol 26: 694 -701), can be used to modulate the expression of CTGF. Also, inhibitors of protein kinases activated by the serine / threonine mitogen, particularly p38, the cyclin-dependent kinase, for example CDK2, and the glycogen synthase kinase (GSK) -3 have also been implicated in the expression decreased CTGF.

(See, for example, Matsuoka et al. (2002) Am J Physiol Lung Cell Mol Physiol 283: L103-L112; Yosi ichi et al. (2001) Eur J Biochem 268: 6058-6065; International Publication No. WO01 / 38532 and International Publication No. O03 / 092584). Such agents can be used to reduce the expression of CTGF and thereby improve or prevent pathological processes induced by CTGF in joint disorders. Such compounds can be formulated and administered according to established procedures within the art. Antisense technologies, including small interference ribonucleic acids (siRNA), micro-RNA (miRNA), ribozymes, and anti-sense sequences directed at the expression of CTGF, can also be used to treat joint disorders. (See, for example, Zeng (2003) Proc Nati Acad Sci USA 100: 9779-9784; and Kurreck (2003) Eur J Biochem 270: 1628-1644). Antisense constructs that target the expression of CTGF have been described and used to reduce the expression of CTGF in various cell types. (See for example, Publication International? O. W096 / 38172; International Publication No. WO00 / 27868; International Publication No. WO00 / 35936; International Publication? O. WO03 / 053340; Kothapalli et al. (1997) Cell Growth Differ 8 (1): 61-68; Shimo et al. (1998) J Biochem (Tokyo) 124 (1): 130-140; and Uchio et al. (2004) Wound Repair Regen 12: 60-66). Such antisense constructs can be used to reduce the expression of CTGF and thereby improve or prevent pathological processes induced by CTGF in disorders of the joints. Such constructs can be designed using appropriate vectors and expression regulators for specific expression of cells or tissue and for constitutive or inducible expression. Such genetic constructions can be formulated and administered according to established procedures within the art.

Pharmaceutical Formulations and Administration Routes The compositions of the present invention may be distributed directly or in pharmaceutical compositions containing excipients, as is well known in the art. The present methods of treatment may comprise administering an effective amount of a compound of the present invention to a subject having or at risk of diabetic nephropathy; particularly a disorder associated with, for example, glomerular hyperfiltration and glomerular hyperperfusion, macroalbuminuria, incipient diabetic nephropathy, macroalbuminuria, evident nephropathy, etc. In a preferred embodiment, the subject is a mammalian subject, and in a more preferred embodiment, the subject is a human subject. An effective amount, for example, a dose of the compound or drug can be easily determined by routine experimentation, as can also be an effective and convenient route of administration and an appropriate formulation. Various formulations and drug delivery systems are available in the art.

(See, for example, Gennaro, ed. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Li bird and Gilman, eds. (2001) The Pharmacological Basis of Therapeutics, supra). Suitable routes of administration may, for example, include oral, rectal, topical, nasal, pulmonary, ocular, intestinal and parenteral administration. Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration. Secondary routes of administration include intraperitoneal, intra-arterial, intra-articular, intracardiac, intrascisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration. The indication to be treated, together with the physical, chemical and biological properties of the drug dictate the type of formulation and the route of administration to be used, as well as whether local or systemic distribution could be preferred. The pharmaceutical dosage forms of a compound of the invention can be provided in an instant release, controlled release, sustained release or target drug delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols , and lyophilized formulations. Depending on the administration route used, special devices may be required for the application or administration of the drug, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks. The pharmaceutical dosage forms are frequently composed of the drug, one or more excipients and a container / closure system. One or multiple excipients, also referred to as inactive ingredients, may be added to a compound of the invention to improve or facilitate the manufacture, stability, administration, and safety of the drug, and may provide a means to achieve a release profile desired of the drug. Therefore, the type of excipient (s) to be added to the drug may depend on various factors, such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing process. Pharmaceutically acceptable excipients are available in the art, and include those listed in the various pharmacopoeias. (See, for example, USP, JP, EP and BP, FDA website page (www.fda.gov), Inactive Ingredient Guide 1996, and Handbook of Pharmaceutical Additives, ed. Ash; Synapse Information Resources, Inc. 2002). The pharmaceutical dosage forms of a compound of the present invention can be manufactured by any of the methods well known in the art., such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tablet-making, suspension, extrusion, spray drying, levigation, emulsification, (nano / micro) encapsulation, entrapment or process lyophilization. As noted above, the compositions of the present invention may include one or more physiologically acceptable inactive ingredients, which facilitate the processing of the active molecules in preparations for pharmaceutical use. The proper formulation is dependent on the desired route of administration. For intravenous injection, for example, the composition can be formulated in. aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidine or citrate for pH adjustment of the formulation, and a tonicity agent, such as, for example, sodium chloride or dextrose. For transmucosal or nasal administration, semisolids, liquid formulations or patches, which possibly contain penetration enhancers, may be preferred. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated in liquid or solid dosage forms and as instant or controlled / sustained release formulations. Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft gelatin capsules, liquids, gels, syrups, suspensions, and emulsions. The compounds may also be formulated in rectal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. Oral solid dosage forms can be obtained using excipients, which may include fillers, disintegrators, binders (dry and wet), dissolution retarders, lubricants, glidants, anti-stick agents, cation exchange resins, wetting agents, antioxidants, preservatives, coloring and flavoring agents. These excipients may be of synthetic or natural source. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrolidone, silicates, silicon dioxide, sodium benzoate, sorbitol, starches, stearic acid or a salt thereof, sugars (for example dextrose, sucrose, lactose, etc.), talcum, tragacanthus oleagin, vegetable oils (hydrogenated) and waxes. Ethanol and water can serve as granulation aids. In certain cases, the coating of the tablets with, for example, a taste masking film, a stomach acid resistant film, or a release retarding film, is desirable. Natural and synthetic polymers, in combination with dyes, sugars and organic solvents or water, are frequently used to coat the tablets, resulting in the dragees. When a capsule is preferred over a tablet, the drug powder, suspension or solution thereof can be distributed in a compatible soft or hard gelatin capsule. In one embodiment, the compounds of the present invention can be administered topically, such as through a skin patch, a semi-solid or a liquid formulation, for example a gel, a (micro-) emulsion, an ointment, a solution, a (nano / micro) -suspension, or foam. Penetration of the drug into the skin and underlying tissues can be regulated, for example, using penetration enhancers; the choice and appropriate combination of lipophilic, hydrophilic and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsifiers; by adjusting the pH; and the use of complexing agents. Other techniques, such as iontophoresis, can be used to regulate the penetration of the skin of a compound of the invention. Transdermal or topical administration could be preferred, for example, in situations in which local administration with minimal systemic exposure is desired. For administration by inhalation, or administration to the nose, the compounds for use according to the present invention are conveniently distributed in the form of a solution, suspension, emulsion or semi-solid aerosol from pressurized containers, or a nebulizer, usually with the use of a propellant, for example, halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas. For topical aerosols, hydrocarbons such as butane, isobutene and pentane are useful. In the case of a pressurized aerosol, the appropriate dose unit can be determined by the provision of a valve to distribute a measured quantity. Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator, can be formulated. These typically contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Compositions formulated for parenteral administration by injection are usually sterile and can be presented in unit dosage forms, for example, in ampules, syringes, injection pens, or in multiple dose containers, the latter usually containing a preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as buffers, tonicity agents, viscosity increasing agents, surfactants, suspending and dispersing agents, antioxidants, polymers. biocompatibles, agents uelantes and preservatives. Depending on the injection site, the vehicle may contain water, a synthetic or vegetable oil, and / or organic cosolvents. In certain cases, such as with a lyophilized product or a concentrate, the parenteral formulation could be reconstituted or diluted prior to administration. Depot formulations, which provide controlled or sustained release of a compound of the invention, can include injectable suspensions of nano / microparticles or nano / micro or non-micronized crystals. Polymers such as poly (lactic acid), poly (glycolic acid), or copolymers thereof, can serve as controlled / sustained release matrices, in addition to others well known in the field. Other deposit distribution systems can be presented in the form of implants and pumps that require incision. Suitable carriers for intravenous injection for the molecules of the invention are well known in the art and include water-based solutions containing a base, such as, for example, sodium hydroxide, to form an ionized compound, sucrose or sodium chloride. sodium as a tonicity agent, for example, the buffer contains phosphate or histidine. Co-solvents such as, for example, polyethylene glycols can be added. These water-based systems are effective in dissolving the compounds of the invention and produce low toxicity after "systemic administration." The proportions of the components of a system in solution can be varied considerably, without destroying the characteristics of solubility and toxicity. , the identity of the components can be varied, for example, low toxicity surfactants, such as polysorbates or poloxamers, can be used, such as polyethylene glycol and other co-solvents, biocompatible polymers such as polyvinylpyrrolidone can be Aggregates, and other sugars and polyols can substitute dextrose For the composition useful for the present methods of treatment, a therapeutically effective dose can be estimated initially using a variety of techniques well known in the art. can be based on concentrates effective conditions established in cell culture assays. Appropriate dose ranges for human subjects can be determined, for example, using data obtained from animal studies, and cell culture assays. A therapeutically effective dose or amount of a compound, agent or drug of the present invention refers to an amount or dose of the compound, agent or drug that results in an improvement of the symptoms or a prolongation of survival in a subject. The toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determination of LD50 (the lethal dose at 50% of the population) and ED50 (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 LD50 / ED50 ratio. Agents that show high therapeutic indices are preferred. The effective amount or the therapeutically effective amount is the amount of the compound or pharmaceutical composition that will promote the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, physician or other clinician, for example, reducing the clearance of creatinine, glomerular hyperfiltration and hyperperfusion, the excretion of albumin in urine, or microalbuminuria, or the treatment of early or late diabetic nephropathy, etc. Doses fall preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The doses may vary within this range, depending on the dosage form employed and / or the route of administration used. The exact formulation, the route of administration, the dose and the dose range should be chosen according to the methods known in the art, in view of the specifications of a condition of the subject. The amount and range of doses can be adjusted individually to provide the plasma levels of the active portion, which are sufficient to achieve the desired effects, for example, the regulation of glucose metabolism, the decrease in blood glucose levels, etc., for example, the minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated from, for example, in vi tro data and animal experiments. The doses needed to achieve MEC will depend on the individual characteristics and the route of administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of the agent or composition administered may be dependent on a variety of factors, including the sex, age and weight of the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. The present compositions may, if desired, be presented in a dispensing container or device containing one or more dosage unit forms containing the active ingredient. Such a container or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in jars. The container or dispensing device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for the treatment of an indicated condition. These and other embodiments of the present invention will readily occur to those of ordinary skill in the art, in view of the description herein.

EXAMPLES The invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the invention. These examples are provided solely to illustrate the claimed invention. The present invention is not limited in scope by the exemplified embodiments, which are intended to be illustrations of the simple aspects of the invention, only. Any methods that are functionally equivalent are within the scope of the invention. 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 from the appended figures. It is intended that such modifications fall within the scope of the appended claims.

Example 1: Treatment of Early Stage Characteristics of Diabetic Nephropathy The methods of the invention were used to demonstrate the broad spectrum efficacy in an animal model for certain aspects of early stage diabetic nephropathy, as follows. Eight-week-old mice that had a loss-of-function mutation in the leptin receptor (Ob-R; encoded by the db gene) were obtained from Harlan, Indianapolis, IN. These db / db mice serve as an animal model of type 2 diabetes in obese, and, in particular, a model of type 2 diabetic nephropathy in obese, characterized by early aspects of diabetic nephropathy including, for example, renal hyperfiltration and proteinuria with minimal development of interstitial fibrosis. This is an animal model of early stage diabetic nephropathy, rather than late-stage diabetic nephropathy, as evidenced by the minimal development of interstitial fibrosis. The homozygous db / db mice (diabetics) are hyperglycemic at 8 weeks of age. The homozygous db / db (diabetic) and db / + heterozygous (non-diabetic) animals were treated (intraperitoneal injection) either with the anti-CTGF monoclonal antibody (aCTGF) (prepared as described in International Publication No. WO2004 / 108764 or by the cell line identified by the ATCC Accession No. PTA-6006, deposited on May 20, 2004) or the human control IgG (clgG). In all animals, an initial injection of 300 μg of the antibody was followed by doses of 100 μg administered 3 times a week for 60 days. Blood samples were collected and body weights were measured at the beginning of and periodically throughout the treatment period. The consumption of food was also recorded. Table 1 below shows the average body weight (BW), blood glucose level (BG), and food intake (FC) on day 0 and day 60 in db / + mice treated with clgG, db / + mice treated with aCTGF, diabetic db / db mice treated with clgG, and db / db mice treated with ocCTGF. All data are expressed as the Media + SEM. The number of mice per group (n) was in the range of 9 to 15. Non-diabetic animals (db / +) that had polycystic kidneys were excluded from the analysis. As shown in Table 1, there was a clear distinction between diabetic animals (db / db) and non-diabetic animals (db / +) with respect to body weight, blood glucose levels and feed intake. Treatment with either anti-CTGF or clgG antibody did not significantly affect total weight gain, blood glucose levels, or food intake in diabetic (db / db) or non-diabetic (db / +) animals.

TABLE 1 The data is expressed as the mean + SEM. * P < 0.01 versus db / + mice. Inhibition of Progression of Early Stage Characteristics of Diabetic Nephropathy After the period of treatment with anti-CTGF antibody described above, various measurements of renal function and nephropathy were obtained, including kidney weight, clearance of the creatinine, the excretion of urinary albumin, and the volume of urine. Table 2 below shows the average weight of the kidneys (), the clearance of creatinine (CrCl), and the excretion of 24-hour urinary albumin (UAE) at day 60 in db / + mice treated with clgG, db mice / + treated with aCTGF, db / db mice treated with clgG, and db / db mice treated with aCTGF. All data are expressed as the Mean ± SEM. The number of mice per group (n) was in the group of 9 to 15. As previously established, non-diabetic animals (db / +) that had polycystic kidneys were excluded from the analysis. TABLE 2 The data are expressed as the mean ± SEM. ** P < 0.01 versus db / + mice. * P < 0.01 versus db / + mice and P < 0.05 db / db mice treated with clgG. ? P < 0.01 verus db / db mice treated with clgG. DP < 0.01 versus db / + mice and db / db mice treated with clgG. As shown in Table 2, the db / db mice showed hyperfunctional kidneys as indicated by renal enlargement (e.g., increased kidney weight) (Figure 1), increased clearance of creatinine (Figure 2), and excretion Increased urinary albumin (Figure 3, * P < 0.01 versus db / + treated with anti-CTGF). The diabetic animals treated with the anti-CTGF antibody showed reduced gain in kidney weight compared to the diabetic animals treated with clgG. The clearance of creatinine in db / db animals treated with clgG was approximately twice the value observed in db / +, indicating impaired renal function, hypertrophy and hyperfiltration in diabetic animals. The excretion of urinary albumin was also increased in diabetic db / db animals compared to the urinary albumin excretion observed in non-diabetic db / + animals. The db / db animals treated with the anti-CTGF antibody "had creatinine clearance and levels of urinary albumin excretion markedly lower than those observed in the db / db animals treated with clgG." Specifically, the diabetic mice treated with the anti-CTGF had creatinine clearance levels 82% lower than those observed in diabetic mice treated with clgG.Diabetic mice treated with anti-CTGF had 69% lower urinary albumin excretion levels than those observed in the diabetic mice treated with clgG. provide evidence of a dramatic improvement in renal function in mice treated with the anti-CTGF antibody.The treatment of non-diabetic animals using the methods of the invention showed no adverse effects on the weight or renal function. of anti-CTGF antibody to diabetic animals resulted in reduced p that of the kidney, clearance of creatinine and excretion of urinary albumin. In addition, diabetic mice (db / db) showed an increased urinary volume compared to non-diabetic mice (db / +). Administration of the anti-CTGF antibody as described above, reduced urine volume in diabetic db / db mice. (See Figure 5, * P < 0.01 versus db / + treated with anti-CTGF). These data indicated that administration of anti-CTGF antibody to diabetic animals reduced urinary volume. These results also indicated that inhibition of CTGF provides a method to reduce the increased urinary volume, associated with diabetic nephropathy, and therefore provides a method to improve renal function. He . Analysis of changes in glomerular volume (for example, reduction in glomerular volume expansion) and thickening of the basement membrane further demonstrated the efficacy of CTGF inhibition in the treatment and prevention of the development and progression of diabetic nephropathy. As shown in Figure 6 (* not different from db / + treated with anti-CTGF), treatment of diabetic animals (db / db) with the anti-CTGF antibody reduced the thickening of the basement membrane. Taken together, these data showed that treatment of diabetic animals (db / db) with anti-CTGF antibody reduced kidney hypertrophy (as evidenced by lower kidney weight in diabetic animals treated with anti-CTGF) and restored renal function (as evidenced by a reduced increase in creatinine clearance and the rate of urinary excretion in diabetic animals treated with anti-CTGF). These results also indicated that inhibition of CTGF provided a method to reduce glomerular permeability and hyperfiltration, as well as the reduction of mesangial expansion and thickening of the basement membrane. Therefore, inhibition of CTGF provides a therapeutic method to treat the early stage characteristics of diabetic nephropathy.

Example 2: CTGF Involved in Early Stage Characteristics of Progressive Vitreoretinal Disorders The association between CTGF and ocular disease, including retinal disorders, has previously been established (See for example, International Publication No. WO 03/049773). Here, the relationships between the CTGF and VEGF ocular concentrations and the degree of neovascularization and fibrosis were examined to determine the correlation, if any, between the expression of CTGF and VEGF in the vitreous humor. A correlation could be suggestive of the involvement of CTFG in the early stage and late-stage aspects of ocular disorders. Vitreous undiluted samples (0.5 to 1 ml) were obtained at the start of a flat-area vitrectomy in patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), macular roughness, or macular orifice. Samples of vitreous fluids were collected in sterile tubes, immediately frozen in dry ice and stored at -80 = C until they were evaluated for CTGF and VEGF. Neovascularization associated with all retinal disorders was scored as follows: grade 0, without neovascularization; grade 1, immobile neovascularization, only with non-perfused glial vessels present; and grade 2, active neovascularization, with prefused prerretinal capillaries. (See Aiello et al. (1994) N Engl J Med 331: 1480-1487). The levels of CTGF and VEGF in vitreous samples were measured by ELISA. In summary, vitreous samples were centrifuged at 14,000 rpm for 15 minutes at 2C and the supernatant was collected. CTGF levels were measured by the sandwich ELISA assay using two monoclonal antibodies to human CTGF, each of which specifically recognizes a region other than the N-terminal region of CTGF as follows. Microtiter plates were coated overnight at 4aC with the anti-CTGF capture monoclonal antibody (10 μg / ml) in coating buffer (50 mM sodium borate, pH 9.6). The plates were blocked with 100 μl of 1% BSA in phosphate buffered saline for 2 hours at room temperature, and then washed with the wash buffer (phosphate buffered saline containing 0.05% Tween 20). The vitreous samples were diluted 5 times in assay buffer (50 mM TRIS, pH 7.7, 0.1% BSA, 4 mM magnesium chloride, 400 mM zinc chloride, 0.05% sodium azide, 50 mg / 1 heparin sodium and 0.1% of Triton X-100). 50 μl of the diluted vitreous sample was added to each well, together with 50 μl of the biotinylated monoclonal antibody for human anti-CTGF detection (diluted in the test buffer). The plates were incubated for 2 hours at 37 aC, washed with wash buffer, and incubated with 100 μl / well of streptavidin-conjugated alkaline phosphatase (1 μg / ml diluted in the assay buffer) (Jackson Immunoresearch Laboratories) for 1 hour at room temperature. After this incubation, the plates were washed with wash buffer and 100 μl of substrate solution (1 mg / ml, p-nitrophenyl phosphate, Sigma Chemical Co.) in diethanolamine buffer (1 M diethanolamine, 0.5 mg magnesium chloride). mM, 0.02% sodium azide, pH 9.8) was added to each well. The absorbance was read at 405 nm on a Bio-Rad microplate reader. Recombinant human CTGF was used, purified as a standard. Vitreous levels of CTGF were determined by a sandwich ELISA assay, commercially available according to the manufacturer's instructions (R &D Systems). A significant positive correlation was observed between CTGF levels and the degree of neovascularization (p = 0.0153). As shown in Table 3 below, patients with the highest degree (grade 2) of neovascularization had significantly higher CTGF levels in patients with a lower degree (grade 0 or grade 1) of neovascularization. Figure 4 shows intravitreal levels of CTGF or VEGF that were positively correlated (r = 0.544, p = 0.001). Significantly, these results demonstrate for the first time a direct correlation between the levels of CTGF and VEGF measured from the same sample, at the same stage of the disease.

TABLE 3 The results of the experiments described above, showed that CTGF is present in the human vitreous humor and its concentration correlated significantly and strongly with the presence and degree of neovascularization, and that the vitreous humor levels of CTGF correlated strongly with the levels of the vitreous humor of CTGF. While it has been established that CTGF is associated with the development and progression of ocular fibrosis and other aspects of late stage retinal disease, the present results taken in conjunction with the results shown in example 1 and example 3 herein, involve to CTGF as well as to VEGF co or a critical factor in the development of the early stage of progressive disease, including diabetic nephropathy and various vitreorretinal disorders. Therefore, the present invention provides methods for treating the early retinopathy (eg, neovascularization) and late retinopathy (eg, fibrosis) stages such as PVR, PDR, etc.

Example 3: Treatment of Late Stage Characteristics of Diabetic Nephropathy The effect of anti-CTGF therapy was examined in an animal model of late-stage diabetic nephropathy. As previously described using this animal model, rats with diabetes mellitus showed high susceptibility to unilateral renal ischemic reperfusion, resulting in rapidly progressive nephropathy and renal failure of the final stage, associated with the development of fibrosis, atrophy of the kidney. , and severely compromised glomerular filtration rate (See, for example, Melin et al (1997) Kidney Int. 52: 985-991.) In this animal model of diabetes mellitus, ischemia severely impaired renal function in diabetic rats. animal model, the renal effects on the effect of the kidney and the pathology of hyperglycemia and ischemia are similar to those observed in late-stage diabetic nephropathy in humans, end-stage renal disease (ESRD) .Diabetes mellitus was induced in male Sprague Dawley rats by a single intravenous dose of streptozotocin (STZ) (50 mg / kg). Unilateral renal (IR) was achieved in a kidney by clamping the left renal artery for 30 minutes, thereby preventing blood flow to the left kidney. Treatment with the anti-CTGF monoclonal antibody (i.p. 5 mg / kg) was started one day before reperfusion of renal ischemia (e.g., 2 weeks after the development of diabetes) and continued 3 times a week for 10 weeks. The control animals that did not receive the anti-CTGF antibody were administered with PBS (i.p. 5 ml / kg). The blood samples were obtained from the vein of the tail. Clinical blood chemistry, performed by Quality Clinical Labs, Inc. (Mountain View, CA), was analyzed at week 0, 4, 8 and 10. The total 24-hour urinary protein was determined at weeks 5 and 9. Individual rats were placed in metabolic cages and 24-hour urine specimens were collected. The volume of urine was measured and the urine protein was analyzed using a BCA protein assay kit (Pierce Chemical Co.). Glomerular filtration rate (GFR) is the most widely used measurement of renal function. Inulin clearance is a measurement of the glomerular filtration rate. In these experiments, the glomerular filtration rate (eg, renal function) was determined for individual kidneys by measuring urine volume and clearance of inulin. Urine was collected via a cannulated ureter and blood was collected from the femoral artery. The volume of urine was estimated gravimetrically. Inulin concentration was determined using the anthrone method. The clearance of the inulin, indicator of GFR, was determined using the formula: (Uconc x Uvo?) / SCOnc- At the end of the experiment the kidneys were removed for biochemical and histopathological evaluation. The data is represented as the mean +/- SEM. The data were compared within the experimental groups at each time point using the one-way analysis of variance (ANOVA) and the Student-Newman-Keuls method (SIGMASTAT). When only two groups were compared, a t- test was used (two samples assuming equal variance analysis tool, Microsoft Excel). A value of P <; 0.05 was considered significant. The animals administered with a simple dose of "STZ became diabetic, as indicated by elevated blood glucose levels, increased blood glucose levels from less than 200 mg / dL in control animals (not treated with STZ) to levels greater than 600 mg / dL in animals treated with STZ, indicating that these animals were diabetic.RNRI of non-diabetic animals (for example, not treated with STZ), did not increase blood glucose levels above that of the control animals (data not shown) Blood glucose levels remained elevated in animals treated with STZ throughout the 10 weeks after unilateral renal IR (data not shown).

Late-stage proteinuria The microalbuminuria characteristic of early-stage diabetic nephropathy progresses to macroalbuminuria and late-stage proteinuria. In animals with diabetes mellitus, significant increases in total 24-hour urinary protein were observed (eg, late-stage proteinuria) indicating increased glomerular hyperfiltration and development of renal failure. As shown in Figure 7, the total protein in urine in non-diabetic animals (negative control + PBS; IR + PBS) was approximately 100 mg / 24 hours. (In Figure 7, * greater than the non-diabetic (p <0.001), # less than DM + IR + PBS (p <0.05) to the corresponding weeks). Diabetic animals with renal IR, however, had total urinary protein levels exceeding 350 mg / 24 hours. Administration of the anti-CTGF antibody to diabetic animals with renal IR resulted in a significant reduction in total urinary protein from 24 hours to weeks 5 and 9, to approximately 250 mg / 24 hours, and 250 mg / 24 hours , respectively, compared to untreated diabetic animals. (See figure 7). These data showed that the administration of an antibody to CTGF reduced proteinuria in diabetic animals. These results indicated that inhibition of CTGF provides a therapeutic means to decrease renal hyperfiltration.

These results demonstrate for the first time that anti-CTGF therapy is useful to prevent the development and progression of late-stage proteinuria.

Neither blood urea triogen (BUN) Increased BUN levels are indicators of impaired renal function associated with late-stage diabetic nephropathy.Significant increases in BUN levels were observed in these diabetic animals. the control non-diabetic animals (negative control + PBS, IR + PBS) were below 20 mg / dl at 0, 4 and 10 weeks of the study.In diabetic animals with renal IR, the BU? levels increased from approximately 22 mg / dl at week 0, up to more than 40 mg / dl at 4 weeks (See figure 8, * greater than negative control + PBS and IR + PBS (p <0.01) # less than DM + IR + PBS (p <0.01) at 4 weeks.) Administration of anti-CTGF monoclonal antibody to diabetic animals with renal IR resulted in a reduction of BUN levels at 4 weeks (up to about 30 mg / dl) and at 10 weeks (up to approximately 35 mg / dl), compared to n to that observed in diabetic animals without the administration of the anti-CTGF antibody. (See figure 8). These results showed for the first time that anti-CTGF therapy is useful for reducing BUN levels in diabetic animals, indicating that inhibition of CTGF provides a therapeutic procedure to improve renal function.

Glomerular filtration rate The glomerular filtration rate was determined for individual kidneys for each of the various experimental conditions described above. In control animals (eg, non-diabetic, non-IR), GFR was greater than 0.3 ml / minute / kidney / 100 g. Non-diabetic animals with renal IR had a GFR of approximately 0.28 ml / minute / kidney / 100 g. The diabetic animals without renal IR had a GFR of approximately 0.17 ml / minute / kidney / 100 g. (Data not revealed) . The rate of glomerular filtration was drastically reduced in the ischemic kidney of animals with diabetes mellitus at 10 weeks, up to approximately 0.01 ml / minute / kidney / 100 g. (See figure 9). The administration of the anti-CTGF antibody significantly improved the glomerular filtration rate in individual kidneys in diabetic animals affected by renal IR, at a level of less than 0.035 ml / minute / kidney / 100 g. These data showed that the administration of an antibody to CTGF increased the glomerular filtration rate in diabetic animals in late stage kidney disease. These results demonstrated for the first time that anti-CTGF therapy is effective in the improvement of glomerular filtration in late-stage diabetic nephropathy, and therefore, provides a therapeutic procedure to improve renal function in renal disease. late stage. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the following description. Such modifications are intended to fall within the scope of the appended claims. All references cited herein are incorporated by reference herein, in their entirety.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (24)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing the clearance of creatinine in a subject who has or is at risk of having diabetes or early stage diabetic nephropathy.

2. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing glomerular hyperfiltration in a subject who has or is at risk of having diabetes or early-stage diabetic nephropathy.

3. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing glomerular hyperperfusion in a subject who has or is at risk of having diabetes or early-stage diabetic nephropathy.

4. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing the excretion of urinary albumin in a subject having or at risk of having diabetes or diabetic nephropathy.

5. The use of an agent that inhibits CTGF for the manufacture of a medicament to reduce or prevent the weight gain of the kidney, in a subject who has or is at risk of having diabetes or diabetic nephropathy.

6. The use of an agent that inhibits CTGF for the manufacture of a medicament for normalizing glomerular filtration rate in a subject who has or is at risk of having diabetes or diabetic nephropathy.

7. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing glomerular hypertrophy in a subject who has or is at risk of having diabetes or diabetic nephropathy.

8. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing proteinuria in a subject who has or is at risk of having diabetes or diabetic nephropathy.

9. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing albuminuria in a subject who has or is at risk of having diabetes or diabetic nephropathy.

10. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing microalbuminuria in a subject having or at risk of having diabetes or diabetic nephropathy.

11. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing macroalbuminuria in a subject who has or is at risk of having diabetes or diabetic nephropathy.

12. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing BUN levels in a subject who has or is at risk of having diabetes or diabetic nephropathy.

13. The use of an agent that inhibits CTGF for the manufacture of a medicament for reducing the clearance of inulin in a subject who has or is at risk of having diabetes or diabetic nephropathy.

1 . The use of an agent that inhibits CTGF for the manufacture of a medicament to prevent, reduce the risk of, or delay the onset of diabetic complications in a subject at risk of developing such complications.

15. The use of an agent that inhibits CTGF for the manufacture of a medicament for treating incipient diabetic nephropathy in a subject who has or is at risk of having incipient diabetic nephropathy.

16. The use of an agent that inhibits CTGF for the manufacture of a medicament for treating incipient diabetic nephropathy in a subject who has or is at risk of having early-stage diabetic nephropathy.

17. The use of an agent that inhibits CTGF for the manufacture of a medicament for treating diabetic nephropathy evident in a subject who has or is at risk of having evident diabetic nephropathy.

18. The use of an agent that inhibits CTGF in combination with an inhibitory amount of an angiotensin-converting enzyme inhibitor for the manufacture of a medicament for treating diabetic nephropathy in a subject having or at risk of having diabetic nephropathy.

19. The use of an agent that inhibits CTGF in combination with an inhibitory amount of an angiotensin receptor blocker for the manufacture of a medicament for treating diabetic nephropathy in a subject having or at risk of having diabetic nephropathy.

20. The use of an agent that inhibits CTGF for the manufacture of a medicament for treating or preventing aspects of the early stage of a progressive disease in a subject who has or is at risk of having such a disease.

21. The use of an agent that inhibits CTGF for the manufacture of a medicament for treating insufficient progressive kidney in a subject.

22. The use of an agent that inhibits CTGF for the manufacture of a medicament for improving renal function in a subject who has or is at risk of impaired renal function.

23. The use of an agent that inhibits CTGF in the manufacture of a drug to reduce the clearance of creatinine; reduce glomerular hyperfiltration; reduce glomerular hyperperfusion; reduce glomerular hypertrophy; reduce the thickness of the glomerular basement membrane; reduce the excretion of urinary albumin; reduce proteinuria; reduce albuminuria; reduce microalbuminuria; reduce macroalbuminuria; reduce BUN levels; normalize glomerular filtration rate; reduce the clearance of inulin; reduce or prevent the weight gain of the kidney; prevent, reduce the risk or delay the onset of diabetic complications; treat or prevent the progression of incipient diabetic nephropathy; treat or prevent the progression of early stage diabetic nephropathy; treat or prevent the progression of obvious diabetic nephropathy; treat or prevent progressive renal failure; treat or prevent the development of aspects of the early stage of progressive disease in a subject; and improving renal function in a subject who has or is at risk of having diabetes or diabetic nephropathy.

24. The use in accordance with the claim 23, wherein the agent is selected from the group consisting of an antibody, a small molecule inhibitor, an antisense nucleic acid and a siRNA.