| Over the years, the number of end stage renal diseases (ESRD) has been going up steadily due to the increased incidences of chronic kidney disease (CKD). Multiple causes can induce CKD but it is renal fibrosis that is commonly recognized as the single factor that leads to ESRD. Although there have been encouraging progress in studies regarding the mechanisms of renal fibrosis and numerous therapeutic approaches have been proposed in an attempt to control factors such as hypertension, diabetes, and proteinuria that may quicken the progression of renal disease, there seem to be no effective measures as yet in terms of preventing the development of CKD. This shows that most of the theoretical researches done so far cannot yet be applied to clinical use which really means that the prevention of CKD is still far from satisfying our expectations.Objective:1. To investigate the effects of Ffluvastatin on proliferation and fibronectin (FN) biosynthesis of human renal tubular epithelial cell induced by Angiotensin II.2. To observe Fluvastatin's influence onto expression of IL-6 in kidney,renal interstitial fibrosis,potocyte, renal function, proteinuria and to discuss statins'protective effects on renal tissues damaged by diabetes mellitus (DM).3. To explore the effect of Rho-kinase signal pathway in renal interstitial fibrosis of diabetic nephropathy(DN) and the renopective mechanism of fluvastatin.4. To investigate the effects and mechanism of fluvastatin on high glucose induced klotho expression, proliferation and opoptosis of human renal tubular epithelial cell(HK-2) Method:1①Cultured human renal tubular epithelial cells were treated with AngII at different concentrations (10-9～10-5 mol/L respectively).②Cells were treated with 10-6 mol/L AngII and different concentrations of fluvastatin(10-9～10-5 mol/L respectively).③The proliferation of renal tubular epithelial cells were determined by MTT colorimetry at different time(24h,.48h,72h).④Cells were treated with AngII (10-6 mol/L )for different time (6h, 12h, 24h, 48h, 72h, and 96h), added different concentrations of fluvastatin(10-9--10-5mol/L respectively)for 48h. FN expression by renal tubular cell was detected by both western blot and immunofluorescence.2 Streptozocin(STZ)-induced diabetic model of CD-1 mice were studied. Diabitic mice were divided into three groups depend on different doses of Flufastatin treatment (5mg/kg/d,25mg/kg/d,125mg/kg/d)for 4 weeks and in dose 25mg/kg/d for 8 and 12weeks. All groups have the normal control and diabetic control. The mice were sacrificed at the end of the first, second and third month. Mouse body weight ,weight of one kidney, serum glucose(Glu), cholesterol(ch), triglyceride(TG), urea nitrogen(BUN), creatinine(Cr), and ratio of urinary albumin /creatinine (Alb/cr)were measured before and were killed. IL-6, fibronecin (FN) ,α-Smooth muscle actin (α-SMA) and WT-1 in renal tissue were detected by ELISA or Western blotting and immunohistochemistry. PAS stain was used to observe pathomorphological changes in the renal tissues.3 Human renal proximal tubular epithelial cells(HK-2 cells) were cultured in vitro . Rho-kinase activity was expressed as phosphorylation of myosin-phosphatase target 1 (p-MYPT1). The level of p-MYPT1 and fibronectin (FN) stimulated by high glucose was determined by Western blot at the time of 6h, 12h, 24h. Same marks were detected when high glucose cultured HK-2 cells treated with different concentrations (10-7, 10-6and 10-5 mol/L) of fluvastatin for 12h,and when co-treated by10-5 mol/L of fluvastatin and lysophosphatidic acid (LPA), MVA, GGPP, FPP respectively for 12h .4 HK-2 cells were incubated with normal glucose,high glucose,fluvastatin in different concentrations for different time. HK-2 cells proliferation and apoptosis were determined by MTT and Hoechst33258 respectively. The protein expression of caspase-3, klotho and RhoA were detected by western blotting. Furthermore, the effects of fluvastatin combined with or without mevalonate (MVA), GGPP and FPP (essential molecules for isoprenylation of the small GTPase Rho) on klotho were observed.Result:1.①AngII can induce proliferation of human renal tubular epithelial cell(P<0.05), it was the most significantly increased for 48h, especially at the concentration of 10-5 mol/L, this effect is reduced along with the lowering concentration.②Fluvastatin can attenuate the proliferation of human renal tubular epithelial cell induced by AngII (P<0.05), that was significantly at the concentration of 10-5 mol/L for 48h.③Fibronectin synthesis by renal tubular epithelial cells cultured in AngII medium increased. The expression of FN started increasing after 12h, and achieved peak after 72h.④Fluvastatin inhibited FN expression by Ang II , the 10-5mol/L of fluvastatin had the most significant effect, and 10-6 mol/L had less effect than the 10-5mol/L, from 10-9 to 10-7mol/L, the fluvastatin had no effects on the expression of FN.2. (1) Blood sugar level, urinary albumin/creatinine ration, urea nitrogen in blood, kidney weight/body weight ratio, IL-6 , FN,α-SMA, renal corpuscle area, ratio of mesenterium matrix versus area of renal corpuscle in mice treated with STZ were higher than those in mice without STZ (P<0.05, 0.01 respectively ), WT-1 in mice with STZ was lowered compared with those mice STZ free (P<0.05). The marks mentioned above in all groups with Fluvastatin were decreased while WT-1 was increased compared to the group without Fluvastatin 1 or 2 months later (P<0.05, 0.01 respectively) . In addition, ratio of urinary albumin /creatinine as well as the IL-6 level tapered, presenting a time-dependent nature. (2) Renal pathology by PAS-stained show that STZ mice enlarged their area of glomerulus and area of renal corpuscle, and their mesenterium as well as extracellular matrix became proliferated obviously with time elapse. The mice treated with Fluvastatin alleviated their proliferation of mesenterium and extracellular matrix compared to those with the similar model but without Fluvastatin. Immunohistochemisty show that expression of FN,α-SMA in STZ mice were increased significantly compare with normal control (P<0.01) , and in STZ mice treated with Fluvastatin were decreased significantly compare to those mice without Fluvastatin (P<0.05,0.01). (3) Serum cholesterol and triglyceride in all groups unchanged (P>0.05).3. High glucose enhance the expression of p-MYPT1 and FN at the time of 6h, 12h, 24h compared with the time of 0h in culured HK-2cells( P<0.05 respectively). The increase of FN expression stimulated by high glucose was in time-dependent fashion and reached the peak at 24h(P<0.01).The increased level of p-MYPT1 reached the peak at 12h(P<0.01). Fluvastatin decreased high glucose-mediated level of p-MYPT1 and FN in dose-dependent manner (P<0.05). The inhibitory effect of fluvastatin on up-regulation of p-MYPT1 and FN stimulated by high glucose was reversed by MVA, GGPP respectively (P<0.05) , partially neutralized by LPA(P<0.05) but was unchanged by FPP (P>0.05).4. High glucos increased HK-2 cells proliferation at 24～48h(<0.05) while decreased proliferation at 72h (<0.05). When after HK-2 incubated with high glucose 24～48h, the rate of apoptosis and cleaved caspase-3 increased, RhoA expression increased meanwhile klotho expression decreased at the time of 12h and in a time-dependent manner (<0.05 respectively). Fluvastatin blunted the high glucose -induced response and ameliorated the decrease in klotho expression towards high levels in a dose-dependent (<0.05 respectively). This regulatory effect on clotho was abolished by the addition of MVA and GGPP, but not FPP.Conclusion:1. Fluvastatin protects against AngII-induced proliferation and FN synthesis of human renal tubular epithelial cell in a dose-dependent fashion.2. Fluvastatin is able to reduce high glucose-induced proteinuria and improve renal function, alleviate proliferation of mesangial cells and extracellular matrix. The beneficial effect of Fluvastatin might be caused by inhibiting expression of IL-6, FN,α-SMA in renal tissue and preserving WT-1 expression in podocytes against high glucose-induced injury, so it can play the role of amelioration impairment of diabetic nephropathy early and prevent renal fibrosis. The renalprotective effects of Fluvastatin are independent on its lipid-lowering function.3. Rho-kinase may be one of the initiation signals of renal interstitial fibrosis of DN. The mechanism of decrease renal interstitial fibrosis in DN with Fluvastatin is associated with its effect of inhibiting Rho-kinase signaling pathway.4. High glucose can reduce HK-2 clotho expression and enhance cells proliferation and apoptosis。The mechanism of statins improving these effects by high glucose is related with statins inactivating the RhoA pathway, resulting in over-expression of klotho, which may contribute to the novel target of statins towards on treatment DN .In clousion, Challenges exist in the treatment of diabetic nephropathy due to its complicated pathologic mechanism and considerable cytokines, signal pathways and crosstalking. Statins is expected to play a potent role in the treatment of diabetic nephropathy because of its pleiotropic effects on Ras, inflammation, oxidative stress, renal fibrosis and klotho beyond lipid lowering. Key words:...
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