Development of an Angiotensin II Induced Kidney Injury Model in Zebrafish

Introduction: Chronic kidney disease (CKD) is a complex disease with multiple etiologies (obesity, diabetes, hypertension, etc.) and several mechanisms involved (inflammation, fibrosis, metabolism dysregulation, oxidative stress, etc.). Zebrafish share 70% of human disease-causing genes, including many that are required for kidney development and disease progression. The kidney’s blood filtration unit (glomerulus) and the structure of the nephron segments are also highly conserved between human and zebrafish, both morphologically and molecularly. However, there are key differences. For example, in zebrafish, kidney development stops at the mesonephros stage, whereas in mammals, it progresses to the metanephric stage. A major advantage to using zebrafish is that female zebrafish can lay hundreds of eggs, facilitating high throughput screening of new therapeutic targets. We previously developed a proteinuria reporter zebrafish line (NLD3). The aim of this study was to develop and characterize a model of kidney disease on top of the NLD3 zebrafish model using the vasoactive peptide angiotensin II (AngII) that activates the Renin Angiotensin System (RAS). Method: On day 0, NLD3 zebrafish were bred and eggs collected. Embryos were treated with different doses of AngII (0.5, 5, 20, 50µM) for 2 or 3 days post fertilisation (dpf). At 4 dpf, a Nanoluciferase assay was performed on the fish medium to determine the level of proteinuria. Once the optimal dose/duration of incubation was found, treatment with various drugs targeting the RAS, Angiotensin receptor blocker (ARB) and Angiotensin Converting Enzyme inhibitors (ACEi) were tested to mitigate the effects of AngII. Bulk RNAseq on whole embryos was performed at 4 dpf. Results: AngII at 20 and 50µM doses induced kidney dysfunction when treatment commenced from 1 dpf or 2 dpf. No side effects on cardiac function were observed. Further experiments were conducted with a 2-day incubation period (2 dpf to 4 dpf) with 20µM of AngII. Incubation with the ACEi ramipril and captopril, commenced one day prior to AngII treatment, did not improve the proteinuria. Similar results were observed with Olmesartan and Losartan (angiotensin receptor blockers). However, Losartan Carboxylic Acid (LCA – the active metabolite of losartan) improved kidney function without any detrimental effects on zebrafish development. The bulk RNAseq analysis performed on all embryos showed the broad effect of AngII. Indeed, a gene ontology (GO) analysis highlighted an increased expression of genes involved in the NAFLD signaling pathway after treatment with AngII. The addition of LCA induced the overexpression of genes from the cAMP mediated signaling pathway. Conclusion: AngII can induce significant and reproducible proteinuria in zebrafish larvae that can be reduced by losartan active metabolite. More targeted tissue-specific RNAseq experiments are currently being performed to further characterize the changes in gene expression on the glomerulus and tubulus and estimate the relevance to human data sets. Once relevance of zebrafish AngII-driven kidney disease is established to humans, this model could be used for validating selected targets in a translatable model to human kidney disease. This work has been funded by Novo Nordisk. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.