The Pathogenesis And Therapeutic Approach Of Diabetic Nephropathy In Zucker Diabetic Fatty Rats

At the present time, more and more life-style related ailments are threatening our health. A worldwide epidemic of Diabetes Mellitus, a metabolic syndrome, is characterized by defects in insulin secretion, insulin resistance, or both, contributing to variety of metabolic disorders, such as cardiovascular diseases, hepatic steatosis, inflammations, oculopathy and renal diseases. It is estimated that globally 150 million people got diabetes and this number is increasing by millions every year. In China, 40 million people have been diagnosed with diabetes, where type II diabetes (insulin resistance) takes a proportion of 90%.Diabetic Nephropathy is a leading cause of end-stage renal diseases and an independent risk factor for all-cause and cardiovascular mortalities in diabetic patients. Diabetic nephropathy accounts for 30–40% of patients requiring renal replacement therapy. It is structurally characterized by an early thickening of tubular and glomerular basement membranes due to an excessive accumulation of the extracellular matrix (ECM). Excessive deposition of ECM in the glomerular mesangium and tubulointerstitium is closely associated with a progressive decline in renal functions in diabetes. In human diabetic nephropathy, the extent of tubulointerstitial fibrosis is well correlated with renal dysfunction. During diabetes, the collagen fibers undergo various physicochemical changes including loss of flexibility, greater resistance to enzymatic digestion, alterations in viscosity, changes in ligand binding activity, and other suprastructural modifications. Although normal collagen cross-linking is necessary to stabilize fibrils and to provide distensibility, elasticity and mechanical strength to tissues, excessive collagen cross-linking during diabetes may significantly damage the kidneys and interfere with normal renal functions. So far, the mechanism of Diabetic Nephropathy has not been well understood, and there is still no effective therapeutic treatment in the management and reversal of renal fibrosis neither.Zucker diabetic fatty (ZDF) rat is a well-recognized genetic model of non–insulin-dependent (type 2) diabetes (T2D) and obesity. ZDF rats have a defective leptin receptor; they display hyperphagic, obese, insulin resistance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, hepatosteatosis, abnormal cardiac lipid metabolism and cardiac fibrosis. Besides, ZDF rats get glomerular and tubulointerstitial lesion, enhanced BUN level, decreased creatinine clearance and proteinuria. In contrast to ZDF rat, Zucker Lean (ZL) rat, as healthy animal, is often applied as the control model. We examined renal fibrosis and renal functions in both ZL and ZDF rats with biochemical assays and tissue staining, by which four important clinically diagnostic index of renal functions have been assessed. With the increased glomerular permeability and decreased tubular re-absorbance, Serum albumin, Serum creatinine, BUN and UA levels are abnormal in non-fasted ZDF rats, while biopsy result exhibits a mass of renal fibrosis in diabetic animals as well, suggesting our model animals have got severe renal dysfunction that was generated from structural abnormality.In order to acknowledge the concentration and composition of ECM in renal fibrosis, a new colorimetric method was adopted for collagen assay, in which collagen was categorized into soluble and insoluble fractions. The soluble collagen is most recent one, correlating to collagen biosynthesis; On the other hand, the klunky insoluble fraction level depends on the collagen bio-degradation. With aging or under pathological change, soluble collagen undergoes structural changes and transforms to cross-linking insoluble one. The latter one is responsible for the disorganization and malfunction of kidney tissue, due to its rigidness, which could be seen in ZDF rats obviously.After a series of evaluation, we confirmed that ZDF rats were benign animal model for research on the genetic regulatory pathway involved in diabetic nephropathy . Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitors superfamily and is the primary physiologic inhibitor of urokinase-type plasminogen activator (uPA). As the main inhibitor of plasminogen activation, PAI-1 has been involved in a variety of activities in the pathological processes such as inhibition of fibrinolysis, ECM turnover, activation of several proenzymes and latent growth factors, all promoting tissue fibrosis and sclerosis. In this study, PAI-1 has been identified as a critical mediator of glomerulosclerosis and interstitial fibrosis. Our results clearly demonstrated that upregulation of renal PAI-1 gene expression markedly inhibited collagen bio-degradation, which was consistent with an increase in renal collagen accumulation, insoluble and soluble collagen contents in ZDF rats. We also identified that Transforming growth factor-β1 (TGF-β1) and Angiotensin II type 1 receptor (AT1), two potent regulators of the plasmin system, acted on excess collagen deposition by stimulating PAI-1 gene expression to inhibit collagen bio-degradation, and by inducing collagen biosynthesis at the same time. PAI-1/TGF-β1/AT1 constitutes an relatively intact gene pathway to regulate the ECM protein level, and their overexpression contributes the renal fibrosis in ZDF rats. Numerous reports indicate that lasting hyperglycemia and hyperleptinemia play an crucial role in the upregulation of TGF-β1 and AT1 transcription, which, taken together with our findings of PAI-1/TGF-β1/AT1 regulatory pathway, helps in building the exact relationship between diabetes and diabetic nephropathy.Salacia oblonga has been used as anti-diabetic medicine for several thousand years in Ayurvedic medical literature. Increasing evidences suggest the regulatory effect of this herb on exogenous nutrients, including inhibition of carbohydrates absorption (α-glucosidase inhibitor activity) and lipids transportation (pancreatic lipase inhibitor activity) in small intestine. It has been reported to improve postprandial hyperglycemia, hyperlipidemia, cardiac fibrosis, hepatic steatosis and fatty liver etc, indicating that Salacia oblonga is a potential therapeutic approach to the treatment of diabetic nephropathy. We investigated the effect of mangiferin-quantified water extract of Salacia oblonga root on renal fibrosis in ZDF rats. Salacia oblonga extract (SOE) normalized Serum albumin, Serum creatinine, BUN and UA levels, reduced both soluble and insoluble collagen content in kidney tissue, and suppressed the overexpression of PAI-1/TGF-β1/AT1 gene pathway in ZDF rats.SOE containing defined content of mangiferin exhibits the effectiveness in diminishing renal fibrosis. However, as the extract of herbal medicine, its complex ingredients limit us to ascertain the exact therapeutic target in gene pathway. Therefore, in further study, the investigation on the major active component of SOE, mangiferin, with gene-knockout experimental model could provide us a deeper insight into the pathologic and pharmacological mechanism of diabetic nephropathy.