The Study Of The Protective Mechanisms Of Rosiglitazone On Kidneys Of Diabetic Rats

Background and Objective: Diabetic nephropathy, both a microvascular complication of diabetes and one of the major causes of mortality in diabetic patients, was characterized by glomerular hyperfiltration, hypertrophy, mesangial dilation and basement membrane thickening in the early stage and glomerulosclerosis in the end stage. Theses typical pathophysiological changes were associated with a number of factors and mechanisms, such as dynamic action of blood, nonenzymatic glycation end products, vasoactive substances, cytokines, growth factors, etc. Recent findings had proved that peroxisome proliferator activated receptor Y (PPAR y ) was a nuclear receptor as well as a ligand-regulated transcription factor that controled gene expression by binding to specific response elements(PPRE), thus playing an important part in the development of diabetic nephropathy. Thiazolidinediones (TZDs) were recognized as highly selective synthetic ligands for PPAR Y and commonly used for NIDDM. They could lower blood glucose level by improving insulin sensitivity of peripheral tissues. Rosiglitazone, the most powerful TZD, showed renal protective effects since it could markedly reduce proteinuria and improve renal pathological changes in Zucker fatty rats. But the mechanisms were not clear. So we used streptozotocin(STZ)-induced diabetic rat models to clarify the possible mechanisms of renal protective effects of rosiglitazone by treating with rosiglitazone and a series of measurements such as biochemical, immunohistochemical and molecularbiological methods, ect.Methods: Male Wistar rats were randomly divided into three groups—Group R, DM and C. After 10 hours fast, the rats in Group R and Group DM were performed peritoneal injection of 60 mg/Kg STZ and the rats in Group C were performed injection of sham citrate buffer with the equal volume. 72 hours later, the rats in Group R and Group DM whose blood glucose was above 16.7mmol/l and urineglucose was positive(+ + H------h + + +) were identified as diabetic rats, whereasthose in Group R and Group DM whose blood glucose was less than 16.7mmol/l and urine glucose was negative were excluded. ?Group R: n=6, diabetic rats, were treated with 5mg/Kg rosiglitazone by daily gavage; ?Group DM, n=6, diabetic rats, received equal volume normal saline by daily gavage; ?Group C, n=6, normal rats, received equal volume normal saline by daily gavage. During the period of the experiment, free food and water without insulin were given to these rats. At the end of the eighth week of the experiment, the quantity of 24hr urinary protein, body weight, blood glucose and creatinine clearance rate(CCr) were measured, and then animals were sacrificed and the kidneys were removed, weighted and used for further experiments. PPAR Y expression of renal tissue was determined by immunohistochemistry, RT-PCR semiquantitive method and Western blotting. TGF-3 i expression was compared by using immunohistochemistry staining and RT-PCR method. Immunohistochemistry method was used to measure Proliferating Cell Nuclear Antigen (PCNA).Results:1. Physical and biochemical indexes: Blood glucose and urinary protein of the rats in Group DM and Group R were much higher than that in Group C(P<0.01). CCr of each group was normal. The quantity of 24hr urinary protein of the rats in Group R was much lower than that in Group C(P<0.05). There was no statistical difference of blood glucose between the rats of Group R and of Group DM, which indicated that rosiglitazone did not influence glucose matabolism on these diabetic rats.2. Renal pathological examination: Increased volume of glomeruli of the rats inGroup DM and Group R was seen under microscope. There were no renal pathological changes in Group C rats.3. Immunohistochemistry: ?PPARY expression in the renal tissue of Group DM and Group R rats was higher than that of Group C rats(P<0.01), while PPAR Y expression of Group R was lower than that of Group DM rats(P<0.05). ?TGF- P i expression in the renal tissue of Group DM and Group R rats was higher than that of Group C rats(P<0.01), while TGF- P i expression of Group R is lower than that of Group DM rats(P<0.05). ?There were more PCNA positive cells in the renal tissue of Group DM and Group R rats than that of Group C rats(P<0.05), while there were fewer PCNA positive cells in the renal tissue of Group R rats than that of Group DM rats(P<0.05).4. RT-PCR: PPAR Y mRNA and TGF- P i mRNA expression in the renal tissue of Group DM and Group R rats were higher than that of Group C rats(P<0.01), while PPAR Y mRNA and TGF- P , mRNA expression of Group R rats is lower than that of Group DM rats(P<0.05).5. Western blot: There was the same PPAR Y expression pattern at protein level as at mRNA level.Conclusion: Rosiglitazone could significantly reduce urinary protein of STZ-induced experimental diabetic rats and exhibit direct renal protective effect possibly by activating PPAR Y , downregulating TGF- P i, inhibiting cell proliferation and extracellular matrix synthesis and improving interstitial fibrosis without decreasing blood glucose level.