The Research Of TRPC6 Expression And Relationship With Podocyte Injury And Repair

Objective:Slit diaphragm(SD)between podocyte food processes is a key component in glomerular filtration barrier. Mutations discovered by Winn et al. and Reiser et al[1,2] in the gene encoding TRPC6, a non-selective cation channel of the TRP family expressed in podocyte foot processes, have been shown to cause focal segmental glomerulosclerosis(FSGS). Recent researches demonstrated that TRPC6 mediated calcium signaling pathway and participated in preserving normal physiological function of kidney. In our paper, expression of transient receptor potential cation channel 6 (TRPC6) and its related function in conditional immortalized mouse podocyte clone 5 (MPC5)were investigated to provide the evidence of injury and repair of podocyte at molecule level.Method:1 Conditional immortalized mouse podocyte clone 5(MPC5)culture: according to the references [3], the resurgent MPC5 cell was cultured under condition of 33℃. More than 80% coverage could be achieved after average 3-4 days. Then the condition was changed to 37℃, and the cell would differentiate to mature podocyte cell prior to later experiment after 10-14 days'culture.2 The expression of TRPC6 in differentiated MPC5 was detected by RT-PCR and Western blotting method.3 Relationship between TRPC6 and cytoskeletal protein : differentiated MPC5 were divided into two groups: control group(group 1, without any treatment), treated by cytochalasin D(group 2, which was added 2.5 μmol/mL cytochalasin D to induce cytoskeletal depolymerization for 6 hours), and the amount of TRPC6 expression in two groups was compared through Western blotting method.4 Podocyte injury and repair : containing control group, puromycin- aminonucleoside(PAN)nephropathy podocyte model group, dexamethasone treatment group (0.01μmol/L, 7 d), captopril treatment groups(10μmol: 12 h, 24 h, 48 h, 72 h), all-trans retinoic acid(ATRA) treatment groups(0.2μmol: 24 h, 48 h, 72 h, 96 h, 120 h). TRPC6 expression in these groups was compared with each other through Western blotting method.Results:1 MPC5 cells can be cultured in two different phenotypes. MPC-5 cells grew under permissive conditions(at 37℃with 100 U/mLγ-interferon). These cells proliferate and maintain an epithelial phenotype with cobblestone-like morphology under permissive conditions . MPC-5 cells grew under nonpermissive conditions(14 days at 37℃, noγ-interferon). In contrast, culturing under nonpermissive conditions stopped proliferation and induced conversion into arborized cells.2 The specific RT- PCR band of TRPC6 was detected in differentiated MPC5 was 181 bp corresponding with our design results. The specific protein band of TRPC6 was detected in differentiated MPC5 with the size of 110.3 Compared to control group, TRPC6 protein expression amount in cytochalasin D treatment group increased significantly(P<0.01).4 The expression of TRPC6 protein in PAN group increased significantly (P<0.01)relative to control group.5 The results of three drugs treatment:(1) Compared to control group, TRPC6 protein expression in groups treated by dexamethasone increased(P<0.05), TRPC6 protein expression decreased significantly(P<0.01)compared with PAN model group;(2) Compared to control group, TRPC6 protein expression in groups treated by captopril increased ( P<0.05 ) , TRPC6 protein expression decreased (P<0.05)compared with PAN model group. Especially in 24 h treatment group, the expression of TRPC6 protein decreased significantly(P<0.01)compared with PAN model group; (3) Compared to control group, TRPC6 protein expression in groups treated by ATRA increased(P<0.05), TRPC6 protein expression decreased(P<0.05)compared with PAN model group. Especially in 72 h treatment group, the expression of TRPC6 protein decreased significantly(P<0.01)compared with PAN model group.Conclusions:1 The expression of TRPC6 is verified in differentiated MPC5.2 TRPC6 protein expression increased significantly when podocyte was injured by cytochalasin D and PAN;After dexamethasone, captopril and ATRA treatment, TRPC6 protein expression decreased in the injured podocyte;3 The expression of TRPC6 protein in groups treated by captopril and ATRA varied with different treatment time, the best treatment time were 24 h and 72 h for captopril and ATRA, respectively.