Apoptosis plays a vital role in cell homeostasis during development and disease. Bcl-x, a member of the Bcl-2 family of proteins, is a mitochondrial transmembrane protein that functions to regulate the intrinsic apoptosis pathway. An alternative splicing (AS) event in exon 2 of Bcl-x results in two isoforms of Bcl-x with antagonistic effects on cell survival: Bcl-xL (long isoform), which is anti-apoptotic, and Bcl-xS (short isoform), which is pro-apoptotic. Bcl-xL is the most abundant Bcl-x protein and functions to inhibit apoptosis by a number of different mechanisms including inhibition of Bax. In contrast, Bcl-xS can directly bind to and inhibit the anti-apoptotic Bcl-xL and Bcl-2 proteins, resulting in the release of the pro-apoptotic Bak. There are multiple splice factors and signaling pathways that influence the Bcl-xL/Bcl-xS splicing ratio, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), transcription factors, and cytokines. Dysregulation of the AS of Bcl-x has been implicated in cancer and diabetes. In cancer, the upregulation of Bcl-xL expression in tumor cells can result in resistance to chemotherapeutic agents. On the other hand, dysregulation of Bcl-x AS to promote Bcl-xS expression has been shown to be detrimental to pancreatic β-cells in diabetes, resulting in β-cell apoptosis. Therefore, manipulation of the splice factor, transcription factor, and signaling pathways that modulate this splicing event is fast emerging as a therapeutic avenue in the treatment of cancer and diabetes.

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-A_xxxa), as well as a family of isoforms with anti-angiogenic properties (VEGF-A_xxxb). 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-A_xxxb, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-A_xxx/VEGF-A_xxxb 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.

Key points: Progressive depletion of all vascular endothelial growth factor A (VEGF‐A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over‐expression of VEGF‐A₁₆₅b only. VEGF‐A₁₆₅b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub‐podocyte space coverage, produced by VEGF‐A depletion. VEGF‐A₁₆₅b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF‐A₁₆₅b has opposite effects to VEGF‐A₁₆₅ on the expression of genes involved in endothelial cell migration and proliferation. Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF‐A). However, little is known about the contribution of VEGF‐A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF‐A₁₆₅b (resulting from alternative usage of a 3′ splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad‐transgenic model, that over‐expression of VEGF‐A₁₆₅b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF‐A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub‐podocyte space coverage is reduced when VEGF‐A is depleted, all of which are rescued in VEGF‐A₁₆₅b over‐expressors. VEGF‐A₁₆₅b restores the expression of platelet endothelial cell adhesion molecule‐1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down‐regulates genes involved in migration and proliferation of endothelial cells, otherwise up‐regulated by the canonical isoform VEGF‐A₁₆₅. The results of the present study indicate that manipulation of VEGF‐A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

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.

There is evidence to suggest that abnormal angiogenesis, inflammation, and fibrosis drive diabetic nephropathy (DN). However, there is no specific treatment to counteract these processes. We aimed to determine whether DIAVIT, a natural Vaccinium myrtillus (blueberry) and Hippophae Rhamnoides (sea buckthorn) extract, is protective in a model of type II DN. Diabetic db/db mice were administered DIAVIT in their drinking water for 14 weeks. We assessed the functional, structural, and ultra-structural phenotype of three experimental groups (lean+vehicle, db/db+vehicle, db/db+DIAVIT). We also investigated the angiogenic and fibrotic pathways involved in the mechanism of action of DIAVIT. Diabetic db/db mice developed hyperglycaemia, albuminuria, and an increased glomerular water permeability; the latter two were prevented by DIAVIT. db/db mice developed fibrotic glomeruli, endothelial insult, and glomerular ultra-structural changes, which were not present in DIAVIT-treated mice. Vascular endothelial growth factor A (VEGF-A) splicing was altered in the db/db kidney cortex, increasing the pro-angiogenic VEGF-A₁₆₅ relative to the anti-angiogenic VEGF-A₁₆₅b. This was partially prevented with DIAVIT treatment. Delphinidin, an anthocyanin abundant in DIAVIT, increased the VEGF-A₁₆₅b expression relative to total VEGF-A₁₆₅ in cultured podocytes through phosphorylation of the splice factor SRSF6. DIAVIT, in particular delphinidin, alters VEGF-A splicing in type II DN, rescuing the DN phenotype. This study highlights the therapeutic potential of natural drugs in DN through the manipulation of gene splicing and expression.