VEGF reduces cytotoxicity and promotes survival in hCIPs, but the signalling pathway through which VEGF acts is unknown (Foster et al. 2003). In endothelial cells VEGF promotes survival by stimulating a pathway, which results in the phosphorylation of AKT (Nishino et al. 2002). Tyrosine phosphorylation of nephrin, a component of the slit diaphragm, can induce the serine/threonine phosphorylation of AKT (Huber et al. 2003). We investigated whether VEGF could interact with nephrin to induce the phosphorylation of AKT and hence induce a survival pathway in podocytes.

hCIPs were serum starved overnight, then treated with 1 nM VEGF for 20 mins. Protein was extracted and quantified. 60 µg of the protein was boiled and then run on an SDS PAGE gel, transferred to a PVDF membrane and probed with 5 µg/ml anti-phospho-AKT, stripped and reprobed with 5 µg/ml anti-AKT. Bands were then visualised using chemiluminescence and analysed using densitometry. 100 µl of the same protein samples were incubated overnight using 1 µg/ml phosphotyrosine antibody, then immunoprecipitated using protein A/G agarose beads. The immunoprecipitate and supernatant were then analysed by Western blotting as above. The membrane was then probed using a nephrin antibody (1:500). HCIPs and nephrin mutated hCIPs (NMhCIPs) were also serum starved for 16hrs and left in a cell suspension for 4hrs. Apoptosis was assayed using Annexin V/propidium iodide staining and quantified using flow cytometry.

1 nM VEGF significantly reduced phosphorylation of total AKT by 32.7 ± (S.E.M.);14.8 % compared to serum starvation, which induced phosphorylation of total AKT by 78.5 ± 23.4 % in hCIPS (paired Ttest, p≤0.05, n = 3). A 180 kDa band corresponding to nephrin was seen in protein samples that had been treated with VEGF and immunoprecipitated with anti-phospho-tyrosine, whereas in untreated samples the band was found in the supernatant. Serum starvation and cell suspension significantly induced apoptosis in NMhCIPs, 25.3 ± 2.0 % compared to hCIPs, 2.5 ± 0.6 % (p≤0.0001, unpaired Ttest, n = 4), however it reduced necrosis in NMhCIPs, 4.4 ± 0.5 % compared to ± 2.3 % (p≤0.005, unpaired Ttest, n = 4).

These results show that VEGF can induce the phosphorylation of nephrin, which may significantly influence podocyte apoptosis. However, this does not appear to be through phosphorylation of AKT.

This work was supported by BHF BB2000003 and Wellcome Trust 69029.