Alternative splicing of VEGF-A and Bcl-X in diabetic nephropathy: potential for novel therapies and prognostic tools

Several alternative splicing (AS) events are associated with the development and progression of diabetic nephropathy (DN). This project looks at two of such genes. Vascular endothelial growth factor A (VEGF-A) and the apoptotic regulation gene Bcl-x. The alternatively spliced variant, VEGF-A165b is anti-angiogenic and anti-permeability but has reno-protective properties and has been shown to rescue kidney function in mouse models of DN. The first part of the study aimed to investigate novel compounds that regulate VEGF-A AS in podocytes exposed to a diabetic environment and the mechanism of action. The second half of the project involves an AS event in the Bcl-x gene which produces two splice isoforms of antagonistic effect. IL-6 is known to drive the progression of DN and is also implicated in the alternative splicing of Bcl-x. This part of the study aimed to investigate the regulation of this AS event in kidney cells grown in a diabetic environment as well as the correlation of the Bcl-xS/Bcl-xL splicing ratio with various degrees of severity in DN. Trovafloxacin (10 M), 10058-F4 (10 M), and delphinidin (10 g/ml) significantly increased the anti-angiogenic VEGF-A165b relative to pro-angiogenic VEGF-A165 in podocytes under normal glucose conditions. Furthermore, 10058-F4 and delphinidin were also found to increase the VEGF-A165b/VEGF-A165 ratio in podocytes exposed to a diabetic environment. Our data suggest that these compounds increase CLK-1 expression, a kinase known to regulate VEGF-A splice site selection. IL-6 treatment caused an increase in the Bcl-xS/Bcl-xL ratio, suggesting an influence of IL-6 on Bcl-x splicing. Bcl-xS/Bcl-xL ratio is upregulated when HEK293 cells are treated under HG, OGS+. Data from a small cohort of patients with varying stages of nephropathy indicated that an increase in the pro-apoptotic Bcl-xS in RNA extracted from urinary and leucocyte cells correlated with a decline in GFR and an increase in uACR respectively. Understanding how they are regulated and how they contribute to disease pathogenesis may open new therapeutic and prognostic avenues.<p></p>