Effects Of Fluvastatin And High Glucose On Cell Proliferation, Apoptosis And Klotho Protein Of Human Renal Tubular Epithelial Cell

The 21st century has the most diabetogenic environment in human history. In 2007, there were 246 million people with diabetes in the world, but by 2025, that number is estimated to reach 380 million. The increase in prevalence of diabetes will be greater in the developing countries, and the problems of diabetes are now seen as a major global public health concern. Therefore,diabetes is now the major cause of end-stage kidney failure throughout the world in both developed and emerging nations,and it is the primary diagnosis causing kidney disease in 20~40% of people starting treatment for end stage renal disease worldwide.Data from the National Health and Nutrition Examination Surveys showed that approximately 10% of all adults and nearly one-quarter of adults over 60 reporting statin use. Statins modify circulating lipid levels by inhibiting the conversion of HMG-CoA to mevalonate. Thus,statins have a well-established role in reducing levels of total cholesterol,low-density lipoprotein cholesterol,triglycerides and increasing high-density lipoprotein cholesterol. The clinical benefits of statins were generally assumed to result from their ability to lower serum total- and LDL -cholesterol levels; however, recent observations suggest that some of the clinical benefits associated with statin therapy may be independent of their cholesterol -inhibiting action. Statins not only inhibit cholesterol synthesis, but also affect production of many other compounds with mevalonic acid pathway, such as ubiquinone, dolochol, farnesyl pyrophosphate (FPP) and geranylgeranyl pyropho -sphate (GGPP). The last two compounds are involved in a number of cellular processes including cell signalling, differentiation and proliferation, and that might be the reason of protective effects against kidney disease.Objective: The purpose of the study was to investigate the effects of high glucose and fluvastatin on cell proliferation, apoptosis and klotho protein of human renal tubular epithelial cell(HK-2), and explored the mechanism.Methods: HK-2 cells were incubated with normal glucose (5.5mmol/L), mannitol, high glucose (25.0mmol/L), fluvastatin in different concentrations(0.01μmol/L~1mmol/L)for different time(0h,12h,24h,48h,72h)with or without MVA, GGPP or FPP. The proliferation of HK-2 was determined by MTT colorimetry. The apoptotic morphology of HK-2 was detected by Hoechst 33258 staining. The protein expression of caspase-3, caspase-8, caspase-9, klotho and RhoA were detected by western blot.Results: High glucose induced HK-2 cell proliferation at an early phase of (24~48h), decreased HK-2 cell proliferation later at 72h. Incubation with normal glucose, fluvastatin in different concentrations(0.01μmol/L~1mmol/L)decreased HK-2 cell proliferation. The inhibitory of(0.1μmol/L~10μmol/L)fluvastatin on HK-2 was prominent in high glucose status. The percentage of antiproliferation of fluvastatin in the high glucose status was in a time-dose dependent effect, especially fluvastatin at the concentration of 10μmol/L for 72h.In the normal glucose status, 10μmol/L fluvastatin induced HK-2 apoptosis in a time-dependent manner. High glucose induced apoptosis; fluvastatin in low concentrations (0.1, 1μmol/L) decreased apoptosis in the high glucose status, while apoptotic ratio of 10μmol/L fluvastatin was the similar to HG. HG stimulated the activities of caspase-3, caspase-8 and caspase-9 in a time-dependent manner. In the high and normal glucose status, fluvastatin induced the activities of caspase-3, caspase-8 and caspase-9 in a time-dependent manner. (0.1, 1μmol/L) fluvastatin inhibited the activities of caspase-3, caspase-8 and caspase-9 in the high glucose status, while 10μmol/L fluvastatin reduced caspase-3, caspase-9 protein at 48h in the high glucose status, caspase-8 protein expression was the similar to high glucose without fluvastain.High glucose decreased klotho protein expression P<0.05, fluvastatin induced significantly over-expression of klotho protein P<0.05. The effect of fluvastatin on klotho protein was abrogated by MVA and GGPP, but not by FPP, meanwhile fluvastatin inhibited high glucose-induced RhoA expression P<0.05.Conclusion: High glucose induced a dual effect involving an early phase of increasing cell proliferation (24~48h), and a late phase of decreasing cell prolifera -tion (72h). Incubation with normal and high glucose, fluvastatin inhibited HK-2 cell proliferation. High glucose induced HK-2 apoptosis, and 10μmol/L fluvastatin induced HK-2 apoptosis in a time-dependant manner both in the normal and high glucose status. (0.1, 1μmol/L) fluvastatin decreased high glucose-induced apoptosis, while HG induced proapoptotic protein (caspase-3, caspase-8 and caspase-9) above and beyond 10μmol/L fluvastatin. HG reduced anti-apoptotic klotho protein expression, Fluvastatin inhibited RhoA/Rho kinase pathway activated by high glucose, resulting in up-regulation of klotho expression in HK-2. It was concluded that low-level fluvastatin could inhibit HK-2 apoptosis induced by high glucose.