pdx1 Knockout Leads to a Diabetic Nephropathy– Like Phenotype in Zebrafish and Identifies Phosphatidylethanolamine as Metabolite Promoting Early Diabetic Kidney Damage

Abstract

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. Here, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison to the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing (RNA-seq) of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine (PtdE) in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD showing signs of the early disease progression already in larval stage and several selective features of later DKD in adult mutants.

Thiosulfate sulfurtransferase prevents hyperglycemic damage to the zebrafish pronephros in an experimental model for diabetes

“…: I-19/01) and carried out in accordance with the approved guidelines. Embryos of the Tg(wt1b:EGFP) 17 line were raised and staged as described according to hours post fertilization (hpf) 36 . Embryos were kept in egg water at 28.5 °C with 0.003% 1-phenyl-2-thiourea (Sigma) to suppress pigmentation.…”

“…hTST was diluted to 13.5 µg/µL and 1 µg/µL in 0.1% BSA/PBS. One nanoliter of morpholino or hTST [1 µg/µl] was injected into the yolk sac of one cell or two cell stage embryos 16 , 17 , 37 .…”

“…Images were taken using a Leica DFC420 C camera, attached to a Leica MZ10 F modular stereo microscope. Alterations of pronephros were quantified by measuring the glomerular length, width and neck size using the Leica LAS V4.8 software 17 .…”

“…The ability to introduce targeted mutations using sequence specific transcription activators like effector nucleases (TALENs) or the clustered regularly interspaced short palindromic repeats (CRISPR) system has made the zebrafish a highly attractive model for studying the consequences of loss of function alleles 11 – 13 . The implications of the null mutation of pdx1 (pancreatic and duodenal homeobox 1) in zebrafish were investigated after the successful formation of the Zebrafish Mutation Project 14 – 17 . Homozygous pdx1 mutations lead to disruption of pancreatic islet development and glucose homeostasis 15 .…”

Zebrafish: A Model to Study and Understand the Diabetic Nephropathy and Other Microvascular Complications of Type 2 Diabetes Mellitus

“…In zebrafish, Pdx1 regulates embryonic glucose metabolism by activating insulin gene expression in developing pancreatic progenitor cells; it was also demonstrated, in a recent study conducted by Wiggenhauser et al, that pdx1 Knockout could lead to a phenotype similar to that of DN in Zebrafish, and they also identified phosphatidylethanolamine as a metabolite that could potentially be promoting early diabetic kidney damage [ 55 ]. Adult pdx1 mutant zebrafish have smaller bodies, lower survival rates, and lower amounts of beta cells and insulin.…”