Protective Effect Of FK506on Glomerular Podocyte In Diabetic Rats

Background:Diabetic nephropathy (DN) is the most common microvascular complications of diabetes, the incidence rate increases year by year, it has a growing proportion of end-stage renal disease (ESRD). DN is a gradual process of development, Its early clinical features are increasement of glomerular filtration rate and urinary albumin excretion rate (microalbuminuria), followed by clinical proteinuria which is the major clinical manifestation in DN, proteinuria is also an independent risk factor to cause kidney damage. So it is particularly important to actively explore the mechanism of proteinuria. In recent years, the studys about mechanisms of proteinuria have achieved gratifying results that the podocyte lesions play an important role in the development of proteinuria. That the podocytes slit diaphragm proteins such as Nephrin and Podocin express and distribute abnormally is an important mechanism of proteinuria in DN. Nephrin protein was found in studying congenital nephrotic syndrome of the Finnish type by Swedish professor Karl Tryggvason in1998, the study showed that nephrin specifically expressed in the slit diaphragm,extended relative to each other between the hole area of two adjacent foot processes, and collected together to form a zipper-like porous filtration structure. Nephrin gene mutation led to protein loss and filtration barrier damage and affected the cell frame-associated protein interaction that can cause the actin cytoskeleton rearrangement and foot processes disappearment, it brought about massive proteinuria at last.FK506is a novel immunosuppressant isolated from the soil microbial culture in1984, its structure is different from cyclosporin A (CsA), but they are similar in wide immunosuppressive effects and the effect strength of FK506is10.100times as much as CsA. FK506has been widely used for the prevention and treatment for liver, kidney, small intestine, bone marrow and other organ transplantation immunosuppression reaction. Now,FK506is used more and more widely, It has been reported that FK506is effective in the treatment of Heymann nephritis, nephrotoxic serum nephritis, lupus nephritis and mesangial proliferative glomerulonephritis model, it can significantly delay glomerulosclerosis and tubular interstitial injury. Some studies have shown that FK506can obviously inhibit early renal hypertrophy and decrease urinary albumin excretion rate in diabetic rats, which provides a theoretical basis for FK506used in diabetic nephropathy, but whether its protective effect is related to the glomerular podocytes, there is no report about this issue.So this study takes Nephrin as the breakthrough point to investigate the effect of FK506on proteinuria and podocyte injury and its mechanism in diabetic rats.Objective:To investigate the protective mechanism of FK506on glomerular podocyte injury in diabetic rats.Methods:thirty-eight normal male SD rats were randomly divided into the normal control group(N,n=8), the diabetic nephropathy group(DN,n=10),the FK506treatment group(F,n=10), and the Lotensin treatment group(L,n=10).The Diabetes mellitus models were established by intra-peritoneal injection of streptozotocin at a dosage of60mg/kg in group DN、 F and L,while group F were intra-gastriced with FK506(lmg/kg/d) after4weeks, group L were intra-gastriced with Lotensin (10mg/kg/d) after4weeks,and the other two groups(group DN and group N) were treated with distilled water at the same dosage after4weeks. Blood glucose (BS) body weight(BW) and24-hour urine protein((24h Upr) were detected every4weeks, serum creatinine(SCr)、 blood urea nitrogen(BUN) and kidney weight/body weight(KW/BW) were detected at the end of the8weeks of treatment,pathological changes of nephridial tissue were observed under light microscope and electron microscope. The expression of MCP-1protein was determined by immunohistochemistry and nephrin which is podocyte-specific marker was detected by Western blotting. Results:(1)compared with group N,SCr、 BUN、 KW/BW and24h Upr in group DN、 F and group L were significantly higher(P<0.05), the above indicators except for BS were significantly lower in group F、 L than group DN(P<0.05), and no significant difference between the two group (P>0.05).(2)Under light microscope, Pathological changes of nephridial tissue in group N showed no obvious abnormality,group DN showed significantly that glomerular volume increased,mesangial cells proliferated, mesangial area broaden, basement membrane thicken.The change of the above histopathology was slighter in group F、 L than group DN (P<0.05), there was no significant difference between group F and L (P>0.05).(3)Under electron microscope, group DN showed significantly that glomerular basement membrane widened,podocyticprocess disordered,wided and fused. Glomerular basement membrane and foot processes lesions was slighter in group F、L than group DN,and no significant difference between group F and L (P>0.05).(4)Western blotting results showed that the expression of nephrin decreased by60.1%in group DN compared with group N,nephrin expression can be obviously recovered in group F ang L (P<0.05).Conclusion:FK506may reduce urinary protein,offer renal protection against DN partly by alleviating structural and functional podocyte damage through restoring nephrin expression in the podocytes.