Renal function and disease in zebrafish

Abstract

The kidney maintains homeostasis of our internal environment. In this thesis, the zebrafish was exploited as a model system to study human renal function and disease. The zebrafish is a useful vertebrate model for human kidney disease with an array of powerful genetic tools. The zebrafish pronephros is a simple and easily accessible system, yet with a high degree of conservation with the mammalian metanephric nephrons. Research presented here consists mainly of two parts; the glomerulus and the distal tubule. The glomerulus is the filtration apparatus of the nephron. Defective filtration leads to development of glomerular disease as represented by nephrotic syndrome. In this part of thesis (chapter 3), in order to identify novel genes that are relevant to nephrotic syndrome, the zebrafish was first used as a screening system to isolate candidate genes from human datasets, including microarray data on kidney biopsies from human nephrotic patients. Rho-GTPase activating protein, IQGAP2, was identified as a candidate gene that is enriched in the glomeruli of the human kidney, downregulated in nephrotic patients. Subsequent functional analysis of IQGAP2 in the zebrafish revealed that disruption of IQGAP2 leads to a filtration defect of the glomerulus with effacement of podocyte foot processes, a characteristic pathological feature of nephrotic syndrome. These results suggest that downregulation of IQGAP2 may be potentially relevant to pathogenesis of nephrotic syndrome in humans. In addition, this study demonstrates the strength of the zebrafish model to achieve rapid assessments of gene function in vivo. In the second part of thesis (chapter 4 & 5), we established the zebrafish as a system to investigate mechanisms underlying NaCl handling by the distal nephron. We particularly focused on sodium chloride cotransporter (NCC) expressed in the distal convoluted tubule in mammalian kidneys. NCC plays a critical role in NaCl reabsorption as evidenced by the monogenic disorders with pronounced alterations in blood pressure resulting from dysregulated NCC. The zebrafish possess the orthologue of NCC that is expressed in the pronephric distal late segment, corresponding to the mammalian distal convoluted tubule. We developed antibodies against total and phosphorylated forms of zebrafish NCC. These antibodies recognized presence of NCC in the pronephros and more importantly, phospho-antibodies could also detect changes in phosphorylation status of NCC after high salinity treatments of zebrafish. This study shows that the zebrafish pronephros can be used to analyze activity of ion transporters and fundamental function of NCC in the distal tubule is likely conserved from teleosts to mammals. Furthermore, we generated transgenic zebrafish with mCherry expression in the distal segment using the promoter from slc12a3 that encodes for NCC. This transgenic line should serve as a valuable tool for detailed analyses of function of the distal segment as well as for monitoring development of the distal nephron.