J Physiol. 2017 Oct 1;595(19):6281-6298. doi: 10.1113/JP274481.

VEGF-A165b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms in the kidney

Megan Stevens1,2,4, Christopher R. Neal2, Andrew H. J. Salmon1,2, David O. Bates3, Stevens J. Harper1,2 and Sebastian Oltean1,2,4

1Schoool of Physiology, Pharmacology and Neurosciences, University of Bristol, UK

2Bristol Renal, School of Clinical Sciences, University of Bristol, UK

3Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, UK

4Present address: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, UK

Correspondence should be addressed to: Megan Stevens and Sebastian Oltean, Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, St Luke’s Campus, Heavitree Road, Exeter, EX1 2LU, UK. Tel: 01392727417 Email: s.oltean@exeter.ac.uk and m.stevens2@exeter.ac.uk

 

Abstract

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 the 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-expressers. VEGF-A165b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and the glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro, and down-regulates genes involved in the migration and proliferation of endothelial cells, which are otherwise up-regulated by the canonical isoform VEGF-A165a. The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

PMID: 28574576

 

Supplement

Chronic kidney disease (CKD) is a major health concern worldwide resulting in an increasing burden on health resources. CKD and proteinuria are linked to an increased risk of cardiovascular disease and mortality [1-3]. Therefore, there is a critical requirement to determine the mechanism of glomerular dysfunction in CKD, and to find new ways to manipulate it for therapeutic benefit.

Alternative splicing, where a single gene transcript can give rise to multiple proteins depending on the way the gene is spliced, it estimated to occur in >94% of human genes [4,5]. Alternative splicing is a tightly regulated process; however, changes to its regulation can result in cellular dysfunction and disease. There is an increasing number of splice isoforms that have been implicated in CKD, including vascular endothelial growth factor A (VEGF-A) [6]. The use of an alternative 3’ splice site in exon 8 of VEGF-A results in the expression of a functionally opposite family of isoforms, which includes VEGF-A165b. This anti-angiogenic, anti-permeability isoform has been shown to be protective in CKD [6].

This is the first study to examine the effects of podocyte-specific VEGF-A165b over-expression when all other VEGF-A isoforms are depleted. To do this we used a quad-transgenic mouse model; constitutive over-expression of human VEGF-A165b under a nephrin promoter mouse crossed with a podocyte-specific inducible VEGF-A-knock out (KO) mouse (VEGF-A-KO x Neph-VEGF-A165b). We assessed albuminuria and the permeability of individual glomeruli, ultra-structural changes in the glomeruli, and the mechanism of action of VEGF-A165b in vivo and how this differs from that of VEGF-A165a in conditionally immortalised glomerular endothelial cells (ciGEnCs) in vitro.

The progressive depletion of VEGF-A in the kidneys of the VEGF-A-KO mice resulted in albuminuria (a significantly elevated urinary albumin creatinine ratio [uACR] compared to WT control mice) at 10 and 14 weeks post-induction with doxycycline. However, when the VEGF-A-KO mice over-expressed the VEGF-A165b isoform, the albuminuria was significantly prevented (Figure 1).

 

 

Similarly, the glomeruli isolated from VEGF-A-KO mice displayed a significantly increased permeability to water (water permeability normalised to glomerular area: LpA/Vi) at 10 and 14 weeks post-induction of the KO compared to WT controls. The increased LpA/Vi was prevented in VEGF-A-KO x Neph-VEGF-A165b mice at 10 weeks, but although decreased at 14 weeks, it was not significant (Figure 2). The oncometric assay used to measure the LpA/Vi is a far more sensitive measure of the permeability of the glomerular filtration barrier than the uACR; therefore, we believe that the VEGF-A165b was not able to rescue this phenotype at 14 weeks due to the further depletion of VEGF-A observed.

 

 

Further to the increased permeability of the glomerular filtration barrier, VEGF-KO-A mice developed glomerular ultra-structural changes as observed with transmission electron microscopy (Figure 3). VEGF-A-KO mice developed a thickening of the glomerular basement membrane and a decrease in the number of endothelial fenestrations, indicating a loss of the normal fenestrated phenotype. The sub-podocyte space coverage was also decreased and an increase in the slit width between the podocyte foot processes was observed, indicating the beginnings of foot process effacement. As nephrin expression was not found to be altered in the VEGF-A-KO mice, the slit modifications are indicated to be due to a rearrangement of slit protein (including podocin and nephrin) bonds. However, in the VEGF-A-KO x Neph-VEGF-A165b mice, the was no basement membrane thickening, no reduction in the sub podocyte space coverage, and no increase in the slit width; therefore, VEGF-A165b alone appears to be effective at maintaining the ultra-structure of the glomerular filtration barrier.

 

 

Although ultra-structural changes were observed, we did not detect any glomerulosclerosis in the renal cortex of the VEGF-A-KO mice, which is likely to be due to the mild phenotype that develops in this model. However, we did observe a decrease in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the glomeruli of VEGF-A-KO mice, which was prevented when they over-expressed VEGF-A165b. In addition, the capillary density and circumference were also decreased in the VEGF-A-KO glomeruli, indicating collapsing of the glomerular capillaries. In the VEGF-A-KO x Neph-VEGF-A165b mice, VEGF-A165b played a protective role in preserving the capillary density and circumference, which suggests that the protective mechanism of VEGF-A165b in vivo is mediated via actions on the endothelial compartment.

Regarding the mechanism of action of VEGF-A165b in the glomeruli, we show that although it increases the expression of VEGF receptor 2 (VEGFR2) both in vivo and in ciGEnCs, it fails to induce phosphorylation of the receptor (Figure 4). However, like VEGF-A165a, VEGF-A165b did result in the phosphorylation of ERK1 and Akt, which are pro-survival factors downstream of VEGFR2. This suggests that although VEGF-A165b is inhibitory to VEGFR2, it may be signalling via other receptors in GEnCs, which are yet to be determined.

 

 

Finally, a VEGF-A PCR array revealed a decrease in the expression of some mRNAs known to be involved in the migration and proliferation pathways, including SHC1 and ACTG1, when ciGEnCs were stimulated with VEGF-A165b compared to VEGF-A165a. However, when there is a knock down of VEGF-A in the glomeruli in vivo, VEGF-A165b has the opposite effect and increased the expression of SHC1 and ACTG1. Therefore, it is apparent that a change in the balance of the two isoforms can result in the downstream signalling pathways that are activated being altered. This also gives strength to the hypothesis that VEGF-A165b is acting through other receptors in the glomerulus.

In conclusion, the present study has further demonstrated the protective effects of VEGF-A165b in a model where all other VEGF-A isoforms are depleted. In addition, it has provided some novel mechanistic evidence regarding the importance of the VEGF-A splicing ratio in kidney function. Therefore, future studies should aim to find therapeutic avenues in CKD that aim to switch splicing towards VEGF-A165b.

 

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