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Long term in vivo two-photon imaging of podocyte injury. The pictures of panel a show that a subset of podocytes (arrowheads) in Chet larvae at 4 dpf only express eGFP and not NTR-mCherry and therefore are not vulnerable to MTZ treatment (representative image of n = 3 experiments, scale bar represents 25 μ m). Panel b shows the morphological changes in the glomerulus during treatment with 5 mM MTZ as seen in the 3D reconstructions of long term 2-PM of Chet larvae beginning at 4 dpf. After approximately 5 hours, dilation of Bowman's space occurred which decreased in the following time as shown by the double arrows at t = 5:40, 8:00, 12:20 hours (scale bar represents 25 μ m). Panel c shows single frames of the detachment of two adjacent podocytes (arrowheads) between t = 9 and 10 hours (asterisks indicate a capillary loop which is covered by podocytes, scale bar represents 10 μ m). The time series of single frames over 8 hours in panel d shows retraction of major processes of a single podocyte during treatment with 5 mM MTZ (scale bar represents 10 μ m).
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Podocytes have a unique 3D structure of major and interdigitating foot processes which is the
prerequisite for renal blood filtration. Loss of podocytes leads to chronic kidney disease ending in end
stage renal disease. Until now, the question if podocytes can be replaced by immigration of cells along
the glomerular basement membrane (GBM) is under...
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... exclude that podocytes were still attached to the GBM but did not express mCherry due to the downregulation of podocin, we performed PAS and methylene blue staining of cross sections and transmission electron microscopy (TEM). The histological cross sections in Supplementary Figure 3a and b revealed areas along the capillaries which were not covered by podocytes in con- trast to control larvae. Figure 2b and Supplementary Figure 3c show broad areas of denuded GBM (arrowheads Control glomeruli show pronounced staining for nephrin and podocin as well as a normal glomerular appearance, whereas glomeruli after MTZ treatment show decreased mCherry (podocytes) fluorescence as well as weak staining for nephrin and podocin (representative images of n = 3 individual experiments, scale bar represents 10 μ m). ...
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... histological cross sections in Supplementary Figure 3a and b revealed areas along the capillaries which were not covered by podocytes in con- trast to control larvae. Figure 2b and Supplementary Figure 3c show broad areas of denuded GBM (arrowheads Control glomeruli show pronounced staining for nephrin and podocin as well as a normal glomerular appearance, whereas glomeruli after MTZ treatment show decreased mCherry (podocytes) fluorescence as well as weak staining for nephrin and podocin (representative images of n = 3 individual experiments, scale bar represents 10 μ m). Using TUNEL assay, we investigated the appearance of cell-death in MTZ treated (f) and control larvae (g). ...
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... strain ET (Tg(wt1a:eGFP); mitfa w2/w2 ; roy a9/a9 ) 1 . This new strain, named Chet (Cherry and ET; Tg(nphs2:Eco.NfsB-mCherry); Tg(wt1a:eGFP); mitfa w2/w2 ; roy a9/a9 ; Supplementary Figure 1), expresses eGFP in podocytes and parietal epithelial cells. Interestingly, not all podocytes expressed mCherry-NTR together with eGFP at 4 dpf (yellow, Fig. 3a). We found some podocytes which expressed eGFP but not mCherry (green, Fig. 3a, arrowhead) which were not affected by the MTZ treatment ...
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... Chet (Cherry and ET; Tg(nphs2:Eco.NfsB-mCherry); Tg(wt1a:eGFP); mitfa w2/w2 ; roy a9/a9 ; Supplementary Figure 1), expresses eGFP in podocytes and parietal epithelial cells. Interestingly, not all podocytes expressed mCherry-NTR together with eGFP at 4 dpf (yellow, Fig. 3a). We found some podocytes which expressed eGFP but not mCherry (green, Fig. 3a, arrowhead) which were not affected by the MTZ treatment ...
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... Movie 2 shows a Chet glomerulus (4 dpf) treated with MTZ over a time period of 24 hours. As shown in Fig. 3b, we found that the fluorescence intensity and the number of MTZ-sensitive podocytes decreased during observation. Furthermore, a dilation of Bowman's space was observed in 83.6% (SD = 25.7%, Supplementary Figure 5) of Chet larvae compared to 6.1% (SD = 3.9%) of control larvae, which started to resolve approximately at t = 12 hours ...
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... in Fig. 3b, we found that the fluorescence intensity and the number of MTZ-sensitive podocytes decreased during observation. Furthermore, a dilation of Bowman's space was observed in 83.6% (SD = 25.7%, Supplementary Figure 5) of Chet larvae compared to 6.1% (SD = 3.9%) of control larvae, which started to resolve approximately at t = 12 hours (Fig. 3b, ...
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... a few hours, we observed the detachment of podocytes. As shown in Supplementary Movie 2 most podo- cytes detached in clusters and in a synchronized fashion in each half of the pronephric glomerulus. In Fig. 3c, two neighboring podocytes detached from their capillary (Fig. 3c, asterisk) between t = 9 and 10 hours (Fig. 3c, arrowheads). In 77.1% (SD = 13.6%) of the larvae at least one of the remaining, non-detached podocytes retracted its major processes during the treatment with MTZ as shown in Fig. 3d and in Supplementary Movie ...
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... a few hours, we observed the detachment of podocytes. As shown in Supplementary Movie 2 most podo- cytes detached in clusters and in a synchronized fashion in each half of the pronephric glomerulus. In Fig. 3c, two neighboring podocytes detached from their capillary (Fig. 3c, asterisk) between t = 9 and 10 hours (Fig. 3c, arrowheads). In 77.1% (SD = 13.6%) of the larvae at least one of the remaining, non-detached podocytes retracted its major processes during the treatment with MTZ as shown in Fig. 3d and in Supplementary Movie ...
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... a few hours, we observed the detachment of podocytes. As shown in Supplementary Movie 2 most podo- cytes detached in clusters and in a synchronized fashion in each half of the pronephric glomerulus. In Fig. 3c, two neighboring podocytes detached from their capillary (Fig. 3c, asterisk) between t = 9 and 10 hours (Fig. 3c, arrowheads). In 77.1% (SD = 13.6%) of the larvae at least one of the remaining, non-detached podocytes retracted its major processes during the treatment with MTZ as shown in Fig. 3d and in Supplementary Movie ...
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... in each half of the pronephric glomerulus. In Fig. 3c, two neighboring podocytes detached from their capillary (Fig. 3c, asterisk) between t = 9 and 10 hours (Fig. 3c, arrowheads). In 77.1% (SD = 13.6%) of the larvae at least one of the remaining, non-detached podocytes retracted its major processes during the treatment with MTZ as shown in Fig. 3d and in Supplementary Movie ...
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The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix c...
Citations
... Tubular reabsorption was assessed after injection of 10 kDa Alexa-647 dextran (Sigma), as described 18 . 16 h after dextran injection, larvae were prepared for cryosectioning with the LeicaSM 200R cryomicrotome (6 µm, Leica, Germany), as described before 48 . Finally, slides were stained with Hoechst 33342 and 2 µg/ml CF770-conjugated wheat-germ agglutinin (biotium) for 10 min at RT. Proteinuria of sham and CTNS-mCherry mRNA injected ctns −/− [Tg(l-fabp:DBP:eGFP)] larvae was assessed by DBP-GFP fluorescence. ...
Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns−/− zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS−/− kidney cells and injection into ctns−/− zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns−/− zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns−/− larvae, and restoration of the zebrafish pronephros function.
... There are several transgenic zebrafish lines that make these experiments achievable. There are wt1a and wt1b reporter lines that have been utilized in multiple studies [123,[157][158][159][160][161]. There are also lines such as nphs1::GFP and nphs2::GFP that demarcate the slit diaphragm. ...
... These podocytes also express bacterial nitroreductase (NTR), which reduces the normally benign chemical metronidazole (MTZ), producing a cytotoxic effect [162,163]. This inducible podocyte-damage line has also been used to follow podocyte movements after administered injury, where researchers found that damaged cells are largely static in a 24-h post-injury period [160]. ...
Podocytes are exquisitely fashioned kidney cells that serve an essential role in the process of blood filtration. Congenital malformation or damage to podocytes has dire consequences and initiates a cascade of pathological changes leading to renal disease states known as podocytopathies. In addition, animal models have been integral to discovering the molecular pathways that direct the development of podocytes. In this review, we explore how researchers have used the zebrafish to illuminate new insights about the processes of podocyte ontogeny, model podocytopathies, and create opportunities to discover future therapies.
... 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 treatment with podocyte-directed drugs [10][11][12] . Exemplary for morpholino-guided gene knockdowns, we targeted two disease-causing genes for which zebrafish models have been established. ...
... 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 h 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 . ...
... Initially this model has been established to deplete pancreatic beta-cells and has later been modified to a dose-dependent depletion of podocytes 10,33 . We have shown that upon treatment with high dose MTZ (5 mM) zebrafish rapidly develop proteinuria and classic morphologic changes known from human nephrotic syndrome such as severe podocyte foot process effacement and later podocyte detachment 11,34 . Lately, we could show that when dose-dependently only a smaller subset of podocytes is depleted, a more chronic course of disease is initiated. ...
The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.
... 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] . ...
... 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 . ...
... The copyright holder for this preprint (which this version posted April 10, 2021. ; https://doi.org/10.1101/2021.04.09.439067 doi: bioRxiv preprint changes known from human nephrotic syndrome such as severe podocyte foot process effacement and later podocyte detachment 11,33 . Lately, we could show that when dose-dependently only a smaller subset of podocytes is depleted, a more chronic course of disease is initiated. ...
The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo . Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments.
Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences.
All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.
... Cryosections (6 μm) were cut on a Microm HM 560 microtome (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and immunofluorescence staining was performed as previously described [21]. Nephrin staining was carried out overnight at 4˚C with 1:2000 rabbit anti-zebrafish nephrin (gift of Dr. A. Majumdar, Uppsala, Sweden) and Alexa-Fluor-647 antirabbit antibody (Invitrogen, Carlsbad, California, USA). ...
... PCRs were performed as previously described [21]. In brief, DreamTaq DNA Polymerase (Thermo Fisher Scientific) was used for RT-PCR and RT-qPCR was carried out with iQ SYBR green Supermix (BioRad, Hercules, California, USA) on an iCycler Thermal Cycler (BioRad). ...
... An excellent method to specifically induce damage to podocytes in zebrafish larvae has been carried out with the nitroreductase-metronidazole system [10,26]. Podocyte apoptosis, formation of pseudocysts, podocyte detachment and proteinuria can be induced by adding metronidazole to the medium without injection [21,27]. Although this model is very robust and provides easily reproducible results, it is limited to one zebrafish strain. ...
Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.
... This leads to rapid onset of proteinuria and edema resembling human nephrotic syndrome. 5,6,14 The aim of this study was to examine the glomerular response upon partial podocyte depletion and to investigate the applicability of this model for human FSGS. ...
... 27 To date the only inducible model of podocyte depletion that combines the ease of adding a reagent to the medium with the advantage of cell-specific ablation is the NTR/MTZ model. 5,14 In this study, we chose a short treatment with lowdose MTZ to deplete a subset of podocytes so that larvae survived long enough to investigate glomerular response and adaption to injury. ...
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.
... E-mail: wilhelm.kriz@urz.uni-heidelberg.de studies in zebrafish, it was found that podocytes are stationary cells and do not move on the glomerular basement membrane (GBM), even if challenged to close areas of bare GBM after podocyte detachment (Endlich et al., 2014;Endlich et al., 2017;Siegerist et al., 2017). Moreover, there is yet no solution to the puzzle of why viable podocytes that are lost by detachment from the GBM need to be replaced by immigrating cells: why do viable detaching podocytes not start a de novo reintegration onto the GBM? ...
This study presents a theoretical analysis of the problems related to the inability of podocytes to proliferate. The basis of these problems is the very high rate of glomerular filtration. Podocytes do not in general die by apoptosis or necrosis but are lost by detachment from the glomerular basement membrane (GBM) as viable cells. Podocytes situated on the outside of the filtration barrier and attached to the GBM only by their foot processes are permanently exposed to the flow dynamic forces of the high filtration rate tending to detach them from the GBM. The major challenge seems to consist of the high shear stresses on the foot processes within the filtration slits due to filtrate flow. Healthy podocytes are able to resist this challenge, injured podocytes are not, and may undergo foot process detachment, leading to a gap in the podocyte cover of the GBM. This represents a mortal event. Like a dam break, such a leak cannot be repaired. The ongoing exposure to filtrate flow prevents any attempt to close the gap, thus preventing any regeneration including cell proliferation. An improvement of this precarious situation consists of healing by scarring that may involve only one lobule of the glomerulus, permitting the remaining lobules to maintain filtration. An answer to the question of which waste product requires such a high filtration rate for its excretion may be in the huge quantity of circulating peptides, a problem that dates far back in evolution.
... However, given that podocytes produce high amounts and, therefore, are responsible for maintaining the bioavailability of Vegfa for endothelial cells, even unspecific damage to the podocyte itself may reduce Vegfa signaling to endothelial cells. As an example, it has been shown that endothelial cells have significantly fewer fenestrations as a result of podocyte cell ablation induced by the use of a nitroreductase (NTR)/metronidazole (MTZ) model [18,21,22]. As proposed by Schumacher et al., reduction of VEGF binding to heparan sulfate may induce paracrine VEGF signaling in the glomerular endothelium [13], while a decrease in VEGFA may be causative for the narrowing of the fenestrae of the endothelial cells as demonstrated in a murine VEGFA conditional knockout model [20]. ...
The 6-O-endosulfatases (sulfs) are important enzymatic components involved in the regulation of heparan sulfate by altering the sulfatation pattern. Specifically in the kidney, sulfs have been implicated in the glomerular podocyte-endothelial cell crosstalk and in the preservation of the glomerular filtration barrier (GFB) in different mouse models. Since it has been shown that in zebrafish larvae, Sulf1, Sulf2a, and Sulf2b are expressed in the pronephric kidney we set out to establish if a reduction in sulf expression leads to GFB dysfunction. Here, we show that a reduced sulf expression following morpholino (MO) induced knockdown in zebrafish larvae promotes damage to the GFB leading to renal plasma protein loss from the circulation. Moreover, a combined knockdown of Sulf1, Sulf2a, and Sulf2b is associated with severe morphologic changes including narrowing of the fenestration between glomerular endothelial cells as well as thickening of the glomerular basement membrane and podocyte foot process effacement, suggesting that glomerular damage is an underlying cause of the circulatory protein loss observed after MO injection. Additionally, we show that a decrease in sulf expression reduces the bioavailability of VegfA in the glomerulus of the pronephros, which may contribute to the structural changes observed in the glomeruli of morphant fish. Furthermore, consistent with previous results, knockdown of the sulfs is associated with arteriovenous malformations in particular in the tail region of the larvae. Overall, taken together our results suggest that 6-O-endosulfatases are important in the preservation of GFB integrity and a reduction in their expression levels induces phenotypic changes that are indicative of renal protein loss.
... Podocyte-depletion has been shown one of the initial steps in the development of glomerulosclerosis. 1 In focal and segmental glomerulosclerosis (FSGS), podocytes are injured and parietal epithelial cells (PECs) are activated thus forming cellular lesions on the glomerular tuft. 2 Recently, Kuppe et al. showed that upon activation, PECs develop a cuboidal phenotype and are more prone to contribute to sclerotic lesions. 3 Since experimental procedures in rodents are not ideally suitable for high-throughput drug screening assays, we and others used the larval zebrafish pronephros as a vertebrate model 4 to study glomerular morphology 5 and permselectivity of the glomerular filtration barrier. 6 At 48 hours past fertilization, zebrafish develop a single glomerulus attached to a pair of tubules. ...
... 4 As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) model for podocyte depletion, since the prodrug MTZ is activated exclusively in podocytes expressing the NTR under control of the nphs2 promotor leading to rapid onset of proteinuria and edema resembling human nephrotic syndrome. 7,5,6 The aim of this study was to examine the glomerular response upon mild podocyte depletion and to investigate the applicability of this model for human FSGS. ...
... As shown before, the NTR/MTZ model is a valuable tool to investigate glomerular adaption and regeneration after podocyte loss by enabling specific podocyte-depletion. 7,5 In this study, we adapted the injury so that larvae survived long enough to investigate glomerular response. As described before, MTZtreated larvae developed periocular edema, which is a hallmark for proteinuria 7 which we verified using clearance of highmolecular-weight dextran. ...
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, podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Herein we show that podocyte-depleted zebrafish larvae develop acute proteinuria, severe foot process effacement and activate PECs which create cellular lesions and deposit extracellular matrix on the glomerular tuft. We therefore propose that this model shows features of human FSGS and show its applicability for a high-throughput drug screening assay.
... mitfaw2/w2; mpv17a9/a9) and "Cherry" (Tg(nphs2:GAL4-VP16); Tg(UAS:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the E. coli-derived enzyme nitroreductase (NTR) under the control of the podocyte-specific podocin promotor. 17 We then generated fusion pairs of "ET" and "Cherry" as previously described and initiated a metronidazole (MTZ) treatment at 96 hpf for 24 hours. As shown in Figure 2E, one parabiont showed the mcherry þ pronephros, whereas the other parabiont displayed the wt1a þ pronephros. ...
Proteinuria can be induced by impairment of any component of the glomerular filtration barrier (GFB). To determine the role of circulating permeability factors on glomerular damage, we developed a parabiosis-based zebrafish model to generate a common circulation between zebrafish larvae. A morpholino-mediated knockdown of a podocyte specific gene (nephronectin) was induced in one zebrafish larva which was then fused to an un-manipulated fish. Notably, proteinuria and glomerular damage were present in the manipulated fish and in the parabiotically-fused partner. Thus, circulating permeability factors may be induced by proteinuria even when an induced podocyte gene dysregulation is the initiating cause.
