Construction And Preliminary Study Of Zebrafish Kidney Diseases And Regeneration Models

The kidney, derived from intermediate mesoderm, is one of the most important organs in human. Two principal functions of the kidney are to remove waste from the blood and to balance ions and metabolite concentrations in the blood within physiological ranges. In vertebrates, nephron is the basic unit of the kidney, which is composed of three structures:the glomerulus, the ducts and the collecting ducts. Glomerulus has the function of waste filtration from blood, ducts can reabsorb the useful ions and metabolite, while the waste will be transported by collecting ducts. The nephrons with defective development and/or structural damage will result in the functional disorders and kidney diseases. Serious kidney diseases can not be cured because the diseases come with complex pathogenesis and many complications. The Glomurular filtration rate (GFR) and proteinuria are clinically used in monitoring the function of the kidney. In clinic, most of the kidney diseases will finally develop into end-stage kidney failure, which can only be treated by dialysis and kidney transplantation. Thus, it is crucial to develop method to get renal repair after kidney damage. It has been reported that there are some evidences of the kidney damage repair in the adult mammals, but the mechanism of the kidney damage repair has remained unknown. Therefore, it is necessary to study gene regulation of the kidney development, the pathogenesis of kidney diseases and the molecular mechanism of kidney regeneration. In my work, I focused on kidney development, kidney diseases and kidney regeneration in zebrafish.1, constructing zebrafish glomerulus filtration barrier (GFB) model; 2, ENU screen for the mutants affecting zebrafish pronephric ducts; 3, constructing zebrafish kidney regeneration model. The first part is crucial for monitoring the function of zebrafish kidney at any time. The second part, ENU screen, will provide several genetic mutants with defective pronephric ducts, and maybe it is very helpful for further mechanism study of kidney development and diseases in human. Construction of zebrafish kidney regeneration model makes contributions to exploring the mechanism of kidney regeneration.In the first part of my work, I have constructed several transgenic zebrafish lines using gfp/rfp driven by pronephric specific genes’promoter:Tg(podocin:eGFP) and Tg(podocin:dsRed)expressing in podocytes; Tg(pax2a:eGFP)expressing in the intermediate mesoderm and the pronephric ducts; Tg(cdhl7-eGFP), Tg(cdh17:dsRed) and Tg(enpep:dsRed)expressing in pronephric ducts. When these transgenic lines crossed with the zebrafish transgenic lines Tg(flkl:mcherry)a line with red fluorescence in blood vessel, We can observe zebrafish glomerulus filtration barrier. In the second part of my research, I obtained mutants from 30 F2 families after screen of 262 mutaginized genomes. These mutants have abnormal pronephric ducts at 3.5dpf. I classified mutants into three groups according to the pronephric ducts phenotype at 3.5dpf:1, the delayed and/or defective development of pronephric ducts; 2, the defective migration of pronephric ducts; 3, the overgrown pronephric ducts. One interesting mutant isV3-5. It showed greatly diluted pronephric ducts, a phenotype before cystic pronephric ducts. Constructing zebrafish kidney regeneration model is the third part of my work. In this part, I constructed transgenic fish using NTR driven by pronephric gene podocin and cdhl7’s promoters. These lines can be used for investigation of renal regeneration and a starting material for ENU screen for renal regeneration mutants.In conclusion, we have constructed several kidney specific Tg lines for study of renal development and regeneration, and obtained a number of mutants with defective pronephric ducts from genetic screen.