POS-352 MiR-26a-5p PROTECTS PODOCYTE FROM APOPTOSIS VIA DOWNREGULATION OF TRPC6 IN TYPE 2 DIABETIC NEPHROPATHY

Abstract

Podocyte injury is a major etiological factor in diabetic nephropathy (DN), while transient receptor potential cationic channel 6 protein (TRPC6) channels play a key role in podocyte injury. Our study is designed to investigate the regulatory effect of microRNA-26a-5p (miR-26a-5p) on DN podocyte injury induced by TRPC6 and the underlying mechanisms. 20 SPF, 8-week male Sprague-Dawley (SD) rats were used for establishing the type 2 diabetic mellitus (T2DM) group by feeding them with high fat and high sugar food for 8 weeks and intraperitoneal injection of low-dose streptozotocin (STZ, 30mg/kg). Another 10 normal male SD rats were selected as normal control group (NC). The 24h urinary albumin excretion rate (UAE), urinary creatinine (Ucr), creatinine clearance rate (CCr) and other major biochemical parameters including the fasting insulin level (FINS), serum creatinine (Scr), fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) were measured in both groups after establishing the DM models. Light and electron microscope were used to observe the pathology of kidneys and podocyte ultrastructures, respectively. Immunohistochemical method was used to show the distribution of nephrin and TRPC6 protein. To determine the expression of nephrin, TRPC6, apoptotic-associated proteins (cleaved-caspase-3 and bax/bcl-2), western blot was applied. qRT-PCR was used to detect miR-26a-5p and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2. Podocyte apoptotic rate was measured using TUNEL method. Cultured immortalized mouse podocytes (MPC5) were divided into three groups: normal glucose group (NG), hypertonic group (HM), high glucose group (HG). Before and after miR-26a-5p mimics and inhibitor transfecting, the expression and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2 were detected again using the same methods. Flow cytometry (FCM) was uesd to detect podocyte apoptosis. 1. Compared with NC group, rats in DM group exhibited disrupted biochemical conditions, for uAER, CCr, FINS, FBG, TC, TG and LDL were all significantly increased (all P <0.05) while HDL were decreased (P <0.05), along with significant insulin resistance. 2. Increased glomeruli volume, mesangial cell proliferation, thickened basement membrane and foot process fusion could be observed by light and electron microscope. 3. Immunochemistry results showed a increased expression of TRPC6 and a decreased of nephrin in DM rats than those in NC group (both P <0.05). 4. Both qRT-PCR and Western blot results in vivo and in vitro showed a notable increase in the expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 and decrease in the expression of nephrin and miR-26a-5p (P <0.05). TUNEL and flow cytometry indicated higher podocyte apoptosis in DM rats and HG group. 5. The expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 were down-regulated and nephrin expression was restored after miR-26a-5p mimics transfection (all P <0.05), while miR-26a-5p inhibitor intervention could aggravate all above changes as the effects of HG on podocytes (all P <0.05). High-glucose condition significantly decreased miR-26a-5p expression and increased TRPC6 expression and podocyte apoptosis. In DN, miR-26a-5p can protect podocyte from apoptosis via down-regulating the expression of TRPC6.