Role of new podocyte-associated proteins in the renal ultrafiltration barrier

Abstract

Chronic kidney disease (CKD) is a major health problem and an economical burden affecting people worldwide. The main causes of CKD are diabetes and hypertension and patient numbers keep increasing. In many cases, CKD is progressive leading to end stage renal disease (ESRD), a condition that can be treated only through chronic dialysis or renal transplantation. One of the first clinical signs of CKD is proteinuria due to impaired function of the glomerulus and glomerular podocyte cells. Podocyte damage and eventual loss has been shown to be a common key pathogenic event in many glomerular disease processes leading to CKD and ESRD. The overall aim of this thesis was to identify and characterize podocyte-specific or enriched proteins in glomerular homeostasis and disease with the goal of discovering potential novel molecular targets for the treatment of CKD.

In study I & II we investigated the role of actin cytoskeleton-associated protein Coro2b. Using confocal and stimulated emission depletion (STED) immunofluorescence microscopy we show that Coro2b is expressed early during glomerulogenesis and only in the podocyte within the kidney cortex of man and mouse. Additionally, STED microscopy revealed localization towards the apical plasma membrane of the podocyte foot processes (FPs). Analysis of Coro2b expression in patient biopsies revealed that it is differentially expressed in diabetic nephropathy (DN) and not in IgA nephropathy (IgAN) or membranous nephropathy. Constitutive and podocyte-specific knock out (KO) of Coro2b in BL6 mice did not affect glomerular development, however targeting the podocyte FP actin cytoskeleton in a protamine sulphate injury model resulted in altered response in the podocyte-specific Coro2b KO mice when compared to their wildtype (wt) littermate controls. In zebrafish embryos, knock-down of Coro2b with morpholinos resulted in podocyte loss/de-differentiation, FP effacement and impaired pronephric filtration barrier function.

In study III we show that orphan GPCR Gprc5a is only expressed in the podocyte within the kidney cortex and downregulated in patients with DN. The constitutive KO of Gprc5a lead to elevated levels of profibrotic markers TGF-beta, EGFR and Col1a1, as well as to mesangial matrix expansion and thickening of the glomerular basement membrane in aging mice. Nephropathy, caused by STZ induced diabetes, was more severe in Gprc5a KO mice than in their littermate controls, as shown by higher albuminuria and increased mesangial matrix expansion, presence of totally sclerotic glomeruli and more podocyte FP effacement. In cell culture experiments with immortalized human podocytes, we demonstrated that the overexpression of Gprc5a inhibits the activation or expression of EGFR, TGF-beta and SMAD2/3, while the silencing of Gprc5a increases the activation and expression of the same proteins after EGF stimulation.

In study IV we demonstrated that orphan Gprc5b is highly enriched in podocytes and is localized to the apical membrane. Interestingly, Gprc5b is upregulated in DN, IgAN and lupus nephritis. Generation of a podocyte-specific KO mouse line in combination with lipopolysaccharide induced nephropathy, RNA sequencing and cell culture experiments, we showed that Gprc5b activates pro-inflammatory NF-kappaB/p65 and increases cytokine levels of ccl2 IL-6 an M-CSF1 in podocytes. Gprc5b overexpression also increases the activation/phosphorylation of EGFR and beta-catenin, while Gprc5b-KO glomeruli show decreased recruitment of CD45 positive leukocytes. In study V we demonstrated through expressional profiling that ZFYVE28 is a novel podocyte-enriched protein that localizes to the FPs. Overexpression of ZFYVE28 in cultured human podocytes leads to enhanced EGFR and SMAD2 activation and redistribution. The constitutive and podocyte-specific KO of ZFYVE28 in BL6 mice showed no developmental or phenotypical changes compared to wt littermates in both health and a model of glomerulonephritis. Altogether, this thesis provides clinical and biological insights into podocyte organization during health and glomerular disease.