| Objective:As an integral member of the filtration barrier in the kidney glomerulus, the podocyte, glomerular visceral epithelial cell, is in a unique position, synthesizes the physiological and pathologic extracellular matrix, and elaborates various cytokines and their receptors. Podocyte is exposed to chemical stimulus from the urinary space (Bowman's capsule), and subjected to the injury factors from all kind of kidney diseases. Angiotensin ? (Ang ?), a key effector of renin-angiotensin system(RAS), has been considered as an important risk factor in the progression of kidney diseases. Previous studies have reported that Ang ? can induce podocyte apoptosis both in in vivo and in vitro. However, the molecular mechanisms of Ang ?-induced podocyte apoptosis remain unknown. A critical molecule associated with apoptosis in epithelial cells and neurons is the non-receptor protein tyrosine kinase, c-Abl, which is localized in both nucleus and cytoplasm. In the present study, we evaluated c-Abl expression in podocytes, and explored the role of c-Abl in Ang ?-induced podocyte apoptosis and its molecular mechanism.Methods:Part1:24male Wistar rats (Group B, C, E and F) were assigned to receive Ang ? (400ng·kg-1min-1, Group B and E) by osmotic minipump, of which12rats (Group C and F) were assigned to receive Telmisartan (3mg·kg-1d-1).12rats received normal saline as control groups (Group A and D). Animals were sacrificed at day14(Group A, B and C), and day28(Group D, E and F) respectively. Renal pathological change and podocyte apoptosis were evaluated using transmission electron microscop and TUNEL assay. Expression of c-Abl mRNA and protein was examined by Realtime-PCR, Western blot, and immunohistochemistry staining.Part2:Conditionally immortalized mouse podocytes were used in vitro. Podocytes were cultured for2weeks after thermoswitching from33?to37?. Cells were serum-free for at least12hours prior to drug stimulation.(1) Expression of c-Abl in control cells was examined by immunofluorescence staining.(2) Cultured podocytes were treated with Ang ? from10-9M to10-6M and for various times (0h?3h?6h?12h? 24h). Expression of c-Abl mRNA and protein was examined by Realtime-PCR and Western blot.(3) Cells were treated with Ang ? (10-8M) in the presence or absence of c-Abl inhibitor, Src-I1, and podocyte apoptosis was analysed by flow cytometry and Hoechst-33342staining.(4) Cells were treated with Ang ? (10-8M) in the presence or absence of specific c-Abl siRNA, and podocyte apoptosis was analysed by flow cytometry and Hoechst-33342staining.(5) Cells transfected with c-Abl plamid or c-Abl siRNA were treated with Ang II (10"8M) and then apoptosis was analysed by flow cytometry and Hoechst-33342staining.Part3:Conditionally immortalized mouse podocytes were used in vitro. Podocytes were cultured for2weeks after thermoswitching from33?to37?. Cells were serum-free for at least12hours prior to stimulation.(1) Podocytes were treated with Ang ? (10"8M) for various times (0h?3h?6h?12h?24h) in the presence or absence of c-Abl inhibitor, Src-Il, and c-Abl phosphorylation at Y245and Y412was carried out by Western blotting.(2) Podocytes were treated with Ang ? (10-8M) for various times (0h?3h?6h?12h?24h) in the presence or absence of c-Abl inhibitor, Src-Il, and the nuclear and cytoplasmic c-Abl expression were analysed by Western blotting and immunofluorescence.(3) Podocytes were treated with Ang ? (10-8M) for6h in the presence or absence of c-Abl inhibitor, Src-Il, and the nuclear c-Abl and p53were quantified by co-immunoprecipitation and Western blotting.ResultsPart1:(1)24h urinary protein was increased in Ang ?-infused rats at day14and further increased at day28(P<0.05). Urinary protein was decreased in Group C and F (P<0.05).(2) At day14, Ang ? infusion induced podocyte apoptosis and the apoptotic podocytes were further increased at day28.(3) Ang ? infusion induced the foot process fusion.(4) c-Abl expression was mainly localized in the cytoplasm and nucleus of the podocytes and renal tubular cells. Expression of c-Abl mRNA and protein was increased in Ang ?-infused rats at day14and more obvious at day28(P<0.05). Expression of c-Abl mRNA and protein was inhibited in Group C and F (P<0.05).Part2:(1) c-Abl expression was mainly localized in the cytoplasm and nucleus of control podocytes in vitro.(2) Expression of c-Abl mRNA and protein was increased in Ang ?-treated cells (P<0.05), and the effects was Ang ? dose-and time-dependent.(3) Ang II induced podocyte apoptosis, and down-regulation of c-Abl expression by c-Abl inhibitor (Src-I1) or specific siRNA inhibited Ang ?-induced podocyte apoptosis. Conversely, podoctyes transfected with c-Abl plasmid showed enhanced apoptosis.Part3:(1) Ang ? stimulated c-Abl phosphorylation at Y245and Y412.(2) Western blot analysis of the nuclear fraction shown that the c-Ab1protein was significantly increased in the podocytes exposed to Ang ?; however, the cytoplasmic c-Abl did not show any changes. c-Abl inhibitor, Src-I1, inhibited Ang?-induced nuclear increase of c-Abl.(3) Expression of nuclear p53was increased in Ang ?-treated cells (P<0.05). c-Abl inhibitor, Src-I1, significant inhibited Ang ?-stimulated p53expression in the cell nucleus (P<0.05). c-Abl-p53complex was increased in Ang ?-treated podocytes.Conclusion:(1) c-Abl expression has been demonstrated in rat podocytes in vivo and mouse podocytes in vitro.(2) Ang? induces podocyte apoptosis, and c-Abl inhibitor or specific siRNA can inhibit Ang ?-induced popdocyte apoptosis. In contrast, the overexpression of c-Abl can increase Ang ?-induced popdocyte apoptosis.(3) Podocytes treated with Ang ? show an increase in nucleus c-Abl expression, and nucleus p53expression, these results indicated that c-Abl mediates Ang ?-induced apoptosis in podocytes by p53. |
PDF Full Text Request