Renal Ischemia-reperfusion In Podocyte Injury Mechanism Research

ObjectiveTo investigate the effects of Angâ…¡on podocyte after ischemia/ reperfusion and to discuss the possible mechanism.Methods(1)The rat models induced by ischemia and reperfusion of kidney were established.These rats were sacrified at 0.5h,1h,3h,6h,12h and 24h after reperfusion. The blood samples were obtained for the measurement of serum creatinine, the level of Angâ…¡in plasma and kidney tissue was tested by ELISA. The change of renal histology was observed by electron microscope.(2)The ischemia/reperfusion rats were treated with angiotensin receptor blockers(ARB)-Losartan.Tested the serum creatinine and the histology change of podocyte.The expression of the nephrin and podocin in renal tissue were examined by indirect immunofluorescence staining and Western-blot.(3)The conditionally immortalized human podocyte cell lines were cultured.The histology change of the cell was observed,and the expression of nephrin and podocin on the differentiated podocytes was tested by indirect immunofluorescence.Then the cells were cultured in 95%N₂ incubater for 1h,3h and 6h to estabilish model.The level of Angâ…¡in supernatant was measured by ELISA.(4)Human podocyte cell lines were stimulated with angiotensinâ…¡at different concentrations,and then ARB and PKCI were added.Western-blot was applied to assess protein expression of PKC,and the change of nephrin,podocin was analyzed by flow cytometry.Results(1)Following the I/R injury,the serum creatinine was increased,and the level of Angâ…¡in serum and kidney tissue were also increased.The podocyte injury was observed,and the expression of nephrin and podocin was decreased,as compared to the control group.(2)The ARB can prevent the change of podocyte.(3)The conditionally immortalized human podocyte cell lines proliferate at a temperature of 33℃,and transform into a quiescent,differentiated phenotype after transfer to 37℃.Podocyte markers,nephrin and podocin,were expressed when the cells were kept at 37℃for 14 days.The Angâ…¡secretion of podocytes did not significantly change after the cells were cultured in high concentration of N₂.(4)The survival rate of podocyte stimulated with Angâ…¡was in dose dependent manner.In cultured podocyte cells stimulated with angiotensinâ…¡,the activity of PKC was increased and the expression of nephrin,podocin was decreased.In cultured podocyte cells treated with ARB or PKCI,the change was reversed partly.Conclusion:1.The injury was observed in podocyte after I/R.2.Angâ…¡mediated this change,and ARB can protect podocyte from ischemia/reperfusion injury on renal histology and function,3.The effect of Angâ…¡on podocyte may be,at least in part,due to its activation of PKC signaling pathway.PKCI can reversed the effect partly.