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Modeling Renal Fibrosis in Zebrafish Using Aristolochic Acid I

Chronic kidney disease (CKD) is a major global health problem that is characterized by a slow and progressive loss of kidney function. The endpoint of virtually all progressive renal diseases is fibrosis, characterized histologically by an excessive accumulation and deposition of extracellular matrix (ECM) disrupting the normal histo-architecture of the organ.

The number of renal fibrosis patients doubled from 1990 to 2016 making it increasingly urgent to generate and validate models for a better understanding of the disease and to find potential novel therapies. Although they offer a high throughput screening capacity, in vitro models have limitedly been utilized due to the fact that they cannot recapitulate the complex interaction among various cell types. In contrast, rodents have been used extensively to model the disease and to elucidate the pathogenesis and underlying mechanisms of renal fibrosis. However, none of these in vivo models are suitable for screening of large compounds libraries owing to their high costs and labor-intensive procedures. Therefore, it would be very useful to be able to develop an animal model for therapy testing which is cost-efficient and mirrors well the characteristics of human renal fibrosis.

Zebrafish (Danio rerio) are small vertebrates with highly conserved physiology to humans and with a high degree of conservation to the human genome also with respect to pharmaceutical drug targets. By combining these features with easy handling and speed, they have emerged as a cost-efficient and valid alternative for disease modeling and large-scale drug screening over the last decade. Recent studies also show that the zebrafish is a suitable and cost-effective model to study fibrosis.

Aristolochic acid (AA)-induced nephropathy in humans is characterized by progressive renal interstitial fibrosis and urothelial malignancy and has been observed after unintentional oral intake of Aristolochia species. Of interest, similar results as found in patients were observed in rats after chronic AA treatment. For instance, when rats were s.c. injected daily with 10 mg/kg AA, tubular necrosis associated with lymphocytic infiltrates and tubular atrophy surrounded by interstitial fibrosis was present at day 10 and day 35, respectively. Moreover, C57Bl/6J male mice subjected to daily i.p. administration of AA (3.5 mg/kg) already developed clear renal fibrosis from 5 days onwards.

With the aim to explore the possibility to generate a zebrafish model of renal fibrosis, in this study the fibrogenic renal effect of aristolochic acid I (AAІ) after immersion was assessed. Our results reveal that larval zebrafish at 15 dpf (days post-fertilization) exposed for 8 days to 0.5 µM AAI showed clear signs of AKI (acute kidney injury). The damage resulted in the relative loss of the functional glomerular filtration barrier. Conversely, we did not observe any deposition of collagen, nor could we immunodetect α-SMA, a hallmark of myofibroblasts, in the tubules. In addition, no increase in gene expression of fibrogenesis biomarkers after whole animal RNA extraction was found. As zebrafish have a high capability for tissue regeneration possibly impeding fibrogenic processes, we also used a tert/− zebrafish line exhibiting telomerase deficiency and impaired tissue homeostasis. AAI-treated tert/− larvae displayed an increased sensitivity towards 0.5 µM AAI. Importantly, after AAI treatment a mild collagen deposition could be found in the tubules. The outcome implies that sustained AKI induced by nephrotoxic compounds combined with defective tert/− stem cells can produce a fibrotic response. However, we further show that the limited time slot and overall induced toxicity dramatically limits the feasibility to deploy AAI as a chemical to set-up a renal fibrosis model in zebrafish.

Nothwitstanding the current outcome, it is anticipated that finding the right conditions to model renal fibrosis in zebrafish is a matter of time. In that respect, we have suggested a few alternative approaches, like the use of other fibrogenic compounds, and the overexpression of TGF-b1a by a mifepristone-inducible LexPR system. Possibly, also the use of a genetic ablation approach expressing nitroreductase in the nephron, in combination with chemical renal stress, could be advantageous.


Date:13 Oct 2015 →  20 Aug 2020
Keywords:Renal fibrosis, Zebrafish
Disciplines:Biomarker discovery and evaluation, Drug discovery and development, Medicinal products, Pharmaceutics, Pharmacognosy and phytochemistry, Pharmacology, Pharmacotherapy, Toxicology and toxinology, Other pharmaceutical sciences
Project type:PhD project