[OP.4D.01] FUNCTIONAL ANALYSIS OF TMEM63C IN ZEBRAFISH REVEALS ITS ROLE AS A NOVEL CANDIDATE FOR ALBUMINURIA AND CHRONIC KIDNEY DISEASE

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Abstract

Objective:

The Munich Wistar Frömter (MWF) rat represents a rat model for chronic kidney disease (CKD) with the development of albuminuria, hypertension and structural kidney damage. By genomic analysis in MWF we have recently identified a novel candidate gene for albuminuria, i.e. transmembrane protein 63c (tmem63c). We set out to test the relevance of tmem63c by analysing its role for the functional integrity of the glomerular filtration barrier (GFB) in zebrafish.

Design and method:

The transgenic zebrafish line Tg(fabp10a:gc-EGFP) expressing the vitamin D binding protein tagged with enhanced green fluorescent protein (EGFP) was used for analysis of GFB function. Upon damage of GFB gc:EGFP leaks through the glomerulus and disappears from the vasculature mimicking an albuminuria phenotype. We used morpholino knockdown as well as CRISPR/Cas9-mediated somatic mutants of tmem63c to reduce its levels in developing zebrafish embryos. We quantified gc:EGFP in the vasculature by fluorescence microscopy and analysed gc:EGFP in embryo 3 (E3) water at 120 hours post fertilization (hpf) by dot blot technique. For evaluation of structural kidney development we compared the embryos with reduced tmem63c with controls of Tg(wt1b:EGFP) in which EGFP is expressed in pronephric tissue under Wilms’ tumor suppressor (wt1b) promoter.

Results:

Knock-down of tmem63c in Tg(fabp10a:gc-EGFP) resulted in brain and pericardial edema and caused a marked decline in gc:EGFP fluorescence in the vasculature (p < 0.0001 vs. control). In addition, gc:EGFP could be detected in the E3 water of tmem63c deficient embryos, but not in controls by dot blot analysis. Analysis of Tg(wt1b:EGFP) at 48 hpf demonstrated enlargement of glomeruli and convoluted proximal tubuli in embryos with reduced tmem63c as compared to controls.

Conclusions:

We confirmed a functional role of tmem63c for albuminuria development by demonstrating a GFB defect in tmem63c deficient zebrafish embryos. In addition, tmem63c deficiency associated with development of edema and structural kidney changes that supports its role for kidney function. The role of tmem63c is currently largely unknown, but our findings provide the basis for further translational research of this novel candidate for albuminuria and CKD.

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