Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis

Abstract

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern frequently found in patients with nephrotic syndrome that often progress to end-stage kidney disease. The initial step in development of this histologically defined entity is injury and ultimately depletion of podocytes, highly arborized interdigitating cells on the glomerular capillaries with important function for the glomerular filtration barrier. Since there are still no causal therapeutic options, animal models are needed to develop new treatment strategies. Here, we present an FSGS-like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission electron microscopy. By intravenous injection of fluorescent high-molecular weight dextran, we confirmed leakage of the size selective filtration barrier. Additionally, we observed severe podocyte foot process effacement of remaining podocytes, activation of proximal tubule-like parietal epithelial cells identified by ultrastructural cytomorphology, and expression of proximal tubule markers. These activated cells deposited extracellular matrix on the glomerular tuft which are all hallmarks of FSGS. Our findings indicate that glomerular response to podocyte depletion in larval zebrafish resembles human FSGS in several important characteristics. Therefore, this model will help to investigate the disease development and the effects of potential drugs in a living organism.

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

“…Lately, we could show that when dose-dependently only a smaller subset of podocytes is depleted, a more chronic course of disease is initiated. In that model, zebrafish embryos resemble the phenotype known from human FSGS including activation of parietal epithelial cells that migrate to the glomerular tuft and deposit extracellular matrix 12 .…”

“…Embryos with a Tg(nphs2:GAL4), Tg(UAS:Eco.nfsB-mCherry) background obtained from double transgenic incrosses and selected for strong and homogenous mCherry fluorescence in podocytes at 72 hours past fertilization (hpf), were podocyte-depleted by treating embryos with 5 mM metronidazole (MTZ) dissolved in 0.1% DMSO in E3 medium or 0.1% DMSO in E3 as a vehicle control from 96 to 120 hpf as described before 11 . To induce focal and segmental glomerulosclerosis (FSGS)-like disease in zebrafish embryos, partial podocyte depletion was performed as described before 12 .…”

“…Endogenous normalization candidates were selected from pretrials. Herein, smallRNA sequencing was performed from isolated glomeruli of zebrafish larvae (Tg(nphs2:GAL4); Tg(UAS:Eco.nfsb-mCherry), ZFIN: ZDB-FISH-160601-2 backcrossed to mitfa w2/w2 ) treated with MTZ and DMSO as described above 12 with slight changes. Glomeruli were isolated at 6 dpf manually by micropipetting after slightly disrupting embryos in a tissue homogenizer.…”

“…As established and widely used kidney disease models, we injected 1-cell-stage embryos with antisense morpholino oligonucleotides targeting wt1a translation initiation (translation blocking MOs: TBM) and nphs1 splicing of the intron between deposition) as we have shown before 12 . As shown in Figure 2 A, the phenotype progressively developed during the disease course over 4 days after the washout of MTZ with a lower concentration with increasing lethality until 9 dpf (Fig.…”

“…Patients with nephrotic syndrome develop high-molecular weight proteinuria together with hypoproteinemic edema as typical characteristics 7 . A multitude of studies used larval zebrafish to model proteinuric glomerular diseases may it be with the help of morpholino-guided knockdown of podocyte genes 8,9 or by treatment with podocyte-directed drugs [10][11][12] .…”