Abstract
Background and Aims
Although focal and segmental glomerulosclerosis (FSGS) has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, glomerular podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Therefore, novel drug-screening assays are needed. Unfortunately, simple cellular in vitro-based screening assays are not ideal as glomerular architecture and crosstalk between glomerular cells is insufficiently modelled. Therefore, reliable animal models are still required for drug development, which unfortunately are not ideal for high-throughput applications. To date, due to its size, easy maintenance and breeding, zebrafish larvae are the simplest vertebrate model that are used in high-content screenings. Until today, it was unclear whether zebrafish can be used as a model for human FSGS. We therefore aimed to investigate whether partial podocyte-depletion in larval zebrafish leads to formation of FSGS-like disease and if the model can be used for screening purposes.
Method
We used a transgenic zebrafish model of pharmacogenetic podocyte depletion: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express the bacterial nitroreductase under control of the podocin promotor and can be dose-dependently ablated upon administration of metronidazole. Proteinuria was quantified using in vivo confocal laser scanning microscopy of intravenously administered high-molecular-weight fluorescent dextran. Plastic-embedded larvae where histologically and morphometrically assessed using HE, PAS and Jone’s silver staining after metronidazole washout. Glomerular ultrastructure was assessed using transmission electron microscopy of ultrathin sections. Immunofluorescence staining was carried out on kryosections to investigate extracellular matrix deposition (collagen-1, laminin), cellular proliferation (pcna) as well as parietal cell origin and activation (pax2a).
Results
To partially deplete podocytes, larvae where treated with 80 µM metronidazole from 4-6 days post fertilization, so that a subset of podocytes was depleted. In contrast to controls, podocyte-depleted larvae developed severe whole-body edema (Fig. A). Dynamic in vivo imaging of intravascular 500 kDa fluorescent dextran revealed massive leakage of the glomerular filtration barrier. Ultrastructural and immunofluorescent evaluation showed broad foot process effacement of remaining podocytes (Fig. D) and massive decrease of the slit diaphragm component podocin. Moreover, we found numerous sub-podocyte space pseudocysts (asterisk in Fig. D), microvillous transformation and formation of podocytic tight junctions as well as parietovisceral adhesions of the two layers of Bowman’s capsule. Parietal epithelial cells where activated, changed their phenotype towards a cuboidal shape, began to proliferate as demonstrated by pcna immunofluorescence and where recruited to cellular lesions on the glomerular tuft as demonstrated by the presence of cuboidal pax2a+ cells on the glomerular tuft (arrowheads Fig. B). Moreover, we found significant extracellular matrix deposition by the pax2a+ cells as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C).
Conclusion
Herein we show that upon podocyte-depletion, zebrafish larvae develop important functional and morphological features of human FSGS such as severe proteinuria and edema, podocyte foot process effacement, activation of parietal epithelial cells which contribute to cellular lesions and deposit extracellular matrix on the glomerular tuft. We conclude that this model resembles the human disease in important features and therefore propose its applicability for a high-throughput drug screening assay for FSGS.
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