The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

Vascular endothelial growth factor a (VEGF-A) signaling is essential for physiological and pathological angiogenesis. Alternative splicing of the VEGF-A pre-mRNA gives rise to a pro-angiogenic family of isoforms with a differing number of amino acids (VEGF-Axxxa), as well as a family of isoforms with anti-angiogenic properties (VEGF-Axxxb). The biological functions of VEGF-A proteins are mediated by a family of cognate protein tyrosine kinase receptors, known as the VEGF receptors (VEGFRs). VEGF-A binds to both VEGFR-1, largely suggested to function as a decoy receptor, and VEGFR-2, the predominant signaling receptor. Both VEGFR-1 and VEGFR-2 can also be alternatively spliced to generate soluble isoforms (sVEGFR-1/sVEGFR-2). The disruption of the splicing of just one of these genes can result in changes to the entire VEGF-A/VEGFR signaling axis, such as the increase in VEGF-A165a relative to VEGF-A165b resulting in increased VEGFR-2 signaling and aberrant angiogenesis in cancer. Research into this signaling axis has recently focused on manipulating the splicing of these genes as a potential therapeutic avenue in disease. Therefore, further research into understanding the mechanisms by which the splicing of VEGF-A/VEGFR-1/VEGFR-2 is regulated will help in the development of drugs aimed at manipulating splicing or inhibiting specific splice isoforms in a therapeutic manner.

Vascular endothelial growth factor a (VEGF-A) is a prominent pro-angiogenic and pro-permeability factor in the kidney. Alternative splicing of the terminal exon of VEGF-A through the use of an alternative 3' splice site gives rise to a functionally different family of isoforms, termed VEGF-Axxxb, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-Axxx/VEGF-Axxxb isoform balance has recently been reported in several kidney pathologies, including diabetic nephropathy (DN) and Denys-Drash syndrome. Using mouse models of kidney disease where the VEGF-A isoform balance is disrupted, several reports have shown that VEGF-A165b treatment/over-expression in the kidney is therapeutically beneficial. Furthermore, inhibition of certain splice factor kinases involved in the regulation of VEGF-A terminal exon splicing has provided some mechanistic insight into how VEGF-A splicing could be regulated in the kidney. This review highlights the importance of further investigation into the novel area of VEGF-A splicing in chronic kidney disease pathogenesis and how future studies may allow for the development of splicing-modifying therapeutic drugs.