Mechanistic analysis of compounds that modulate VEGF-A splicing in podocytes with therapeutic potential for diabetic nephropathy

Abstract

Vascular endothelial growth factor A (VEGF-A) has an alternatively spliced variant, VEGF-A165b, formed when a distal 3 splice site in exon 8 is selected. The anti-angiogenic and anti-permeability VEGF-A165b has reno-protective properties and has been shown to rescue kidney function in diabetic nephropathy (DN) mouse models (PMID 25542969). We previously identified three new compounds that can increase the expression of the VEGF-A165b isoform. Two of these compounds, named ESSO1 and ESSO3, were discovered through a screen of synthetic compounds that alter VEGF-A splicing (PMID 33941763). A third compound, delphinidin, was found to be the key regulator of VEGF-A splicing in a natural blueberry and sea-buckthorn extract (DIAVIT) (PMID 30865689). This study aimed to investigate the signalling mechanism through which these three compounds regulate VEGF-A splicing and increase the protective VEGF-A165b isoform in renal podocytes. Human podocytes were exposed to a diabetic environment (glucose soup [GS]: 25 mM glucose, 1 ng/ml TNF-, 1 ng/ml IL-6, and 100 nM insulin), in comparison to a normal glucose (5.5 mM glucose) and an osmotic control (5.5 mM glucose + 19.5 mM mannitol) control, for 48 hours. RNA and protein were extracted for RT-PCR and Western blotting analysis of splice isoforms. Immunofluorescence and cell viability assays were also used. Immunofluorescence showed no significant difference in nephrin expression for all treatments compared to control, suggesting that normal podocyte function is not affected by these compounds. Trypan blue exclusion assay, used to assess the percentage cell viability of podocyte cells, showed that they do not affect podocyte cell viability. By using various inhibitor treatments, we have found several molecules involved in signalling, like JNK and splicing kinases SRPK1 and CLK1 for ESSO1, splicing factor hnRNPH and kinase CLK1 for ESSO3 and AMPK, splicing factor SRSF6 and kinase CLK1 for delphinidin. Other molecules, like ATM, E2F1, and hnRNPA1, were ruled out. Interestingly, all three compounds significantly increased expression of the splicing kinase CLK1, which has been involved in VEGF-A splicing regulation, suggesting that all three compounds may be increasing the VEGF-A165b/panVEGF-A165 ratio through signalling through this kinase. Studies of human biopsies showed that CLK1 is downregulated in DN patients compared to healthy volunteers. The development of a CLK1 activator as a DN therapy seems, therefore, a plausible opportunity.