Roles For VEGF-C/NRP2Axis In Regulating Apoptosis And Autophagy Of Renal Tubular Epithelial Cells During Serum Deprivation

Objective To investigate the changes of vascular endothelial growth factor C(VEGF-C) expression during renal ischemia/reperfusion injury and the roles of VEGF-C and its receptor NRP-2in regulating apoptosis and autophagy of NRK52E cells under serum deprivation condition.Methods (1) To investigate the relationship between renal ischemia-reperfusion injury and VEGF-C in vivo:Establish mice renal IRI model,collect blood specimens and detect the levels of Scr and BUN.Periodic acid-Schiff stain (PAS) was used to evaluate the severity of acute kidney injury. Use immunohistochemistry and Western blot to examine the expression of VEGF-C and its distribution.(2) To observe how VEGF-C and its receptor NRP-2affect the apoptosis of NRK52E when deprived of serum:Serum deprivation at various time points stimulates NRK52E cell,Western blot was used to detect the expression of active Caspase3and total caspase-3; Use siRNA to knockdown the expression of VEGF-C and NRP-2respectly,RT-PCR and Western blot was used to examine the expression of VEGF-C and NRP-2in negative siRNA transfected cells and specific siRNA transfected cells.24h after VEGF-C or NRP-2specific siRNA transfected,cells were serum deprivation for another24h,and then cell flow cytometry was performed to analyze the cell apoptosis rates in each group. Western Blot was used to detect changes of anti-apoptosis protein Bcl-2expression. After4h of VEGF-C incubation, Cells were serum starved with recombinant wild-type VEGF-C at different concentration for another24h,CCK8was used to evaluate cell viability.(3) To observe the effection of VEGF-C and its receptor NRP-2on the autophagy of NRK52E cells induced by Serum deprivation:Serum deprivation at different time point stimulates NRK52E cells respectively, use Western Blot to detect LC3protein expression;Cell immunofluorescence was performed to investigate the location and expression of LC3in NRK52E cells after serum deprivation for24h; The transmission electron microscope was used to detect the ultrastructure changes of NRK52E cells. The expression of VEGF-C,NRP-2was knockdowned by specific siRNA transfection,and then the changes of LC3induced by Serum deprivation was examined by Western Blot.Cells were incubated with Bafilomycin Al in serum deprivation condition,and then CCK8was used to evaluate cell viability. Cells were treated with Bafilomycin Al in serum deprivation medium, The fold-change in LC3-II levels in VEGF-C siRNA transfected group in the presence or absence of Bafilomycin A1was calculated.Results (1) The levels of serum creatinine and blood urea nitrogen were significantly increased in a time-dependent manner after reperfusion in mice. Notably, the renal function was significantly aggravated at24h and partially restored at48h after reperfusion. Compared with the sham group, expansion of renal tubulars, typical protein cast, renal tubular epithelial cell swelling and necrosis,loss of brush border, and inflammatory cells invasion were observed in kidney tissues after renal ischemia/reperfusion injury, but no obvious pathological changes of glomeruli were detected. The expression of VEGF-C in IR24h group was significantly higher than in the sham group,especially at the junction of renal cortex and medulla. The expression of VEGF-C was increased in a time-dependent manner confirmed by Western blotting.(2) As the extension of serum deprivation, the expression of cell activated caspase3was increased, while the expression of total caspase3was reduced. The level of the apoptosis induced by serum deprivation was significantly increased after VEGF-C and NRP-2gene konckdowned respectively; Compared with serum deprivation stimulation, expression of the Bcl-2protein decreased more obviously after the application of VEGF-C siRNA. VEGF-C significantly preserved cell viability in cells subjected to serum deprivation.(3) Serum deprivation increased the expression of LC3II. Mitochondria swelling, increased cavitation, and the degradation autophagy vesicles could be observed after serum deprivation by transmission electron microscope.The expression of LC3II protein, induced by serum deprivation, was further increased after koncking down the expression of VEGF-C or NRP-2respectively. Cell viabilty was markedly decreased after Bafilomycin A1incubation.The fold-change of LC3-â…¡ in VEGF-C siRNA transfected NRK52E cells after Bafilomycin A1treatment were reduced compared to control cells.Conclusions VEGF-C was up-regulated in renal ischemia reperfusion injury.VEGF-C and its receptor NRP-2were essential for maintain the survival of NRK52E cells under serum deprivation condition. VEGF-C protects NRK52E cells against serum starvation. VEGF-C/NRP-2axis plays an important role in regulating autophagy of NRK52E cells induced by serum deprivation.