assessment-of-kidney-function-in-mouse-models-of-glomerular-disease

Assessment of Kidney Function in Mouse Models of 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 ...

techniques-to-assess-kidney-function-mouse-models-glomerular

Techniques to Assess Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. This protocol describes a full kidney work-up that should be ...

University of Bristol

University of Exeter

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.

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-A b only. VEGF-A b 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-A b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A b has opposite effects to VEGF-A on the expression of genes involved in endothelial cell migration and proliferation.

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

The cell surface protein CD44 is involved in diverse physiological processes, and its aberrant function is linked to various pathologies such as cancer, immune dysregulation, and fibrosis. The diversity of CD44 biological activity is partly conferred by the generation of distinct CD44 isoforms through alternative splicing. We identified an unexpected function for the ubiquitous hyaluronan-degrading enzyme, hyaluronidase 2 (HYAL2), as a regulator of  splicing. Standard CD44 is associated with fibrotic disease, and its production is promoted through serine-arginine-rich (SR) protein-mediated exon exclusion. HYAL2 nuclear translocation was stimulated by bone morphogenetic protein 7, which inhibits the myofibroblast phenotype. Nuclear HYAL2 displaced SR proteins from the spliceosome, thus enabling HYAL2, spliceosome components (U1 and U2 small nuclear ribonucleoproteins), and  pre-mRNA to form a complex. This prevented double-exon splicing and facilitated the inclusion of CD44 exons 11 and 12, which promoted the accumulation of the antifibrotic CD44 isoform CD44v7/8 at the cell surface. These data demonstrate previously undescribed mechanisms regulating  alternative splicing events that are relevant to the regulation of cellular phenotypes in progressive fibrosis.

Nuclear hyaluronidase 2 drives alternative splicing of  pre-mRNA to determine profibrotic or antifibrotic cell phenotype.

Diabetic Nephropathy: Novel Molecular Mechanisms and Therapeutic Avenues.

Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b).

Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury.

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-Ab, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-A/VEGF-Ab 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-Ab 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.

ArticleTotal : 2	Year
Publication title Cited by 12	2018
Publication title

Article	Year
The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer. Oncotarget\| PubMed ID: 26452038	2015
VEGF-A b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney. The Journal of physiology\| PubMed ID: 28574576	2017
Nuclear hyaluronidase 2 drives alternative splicing of pre-mRNA to determine profibrotic or antifibrotic cell phenotype. Science signaling\| PubMed ID: 29162741	2017
Diabetic Nephropathy: Novel Molecular Mechanisms and Therapeutic Avenues. BioMed research international\| PubMed ID: 29359147	2017
Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury. Nephron\| PubMed ID: 29393270	2018
Modulation of VEGF-A Alternative Splicing as a Novel Treatment in Chronic Kidney Disease. Genes\| PubMed ID: 29462869	2018

Sebastian Oltean

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