A Fluorescence-Based Assay for Proteinuria Screening in Larval Zebrafish (<i>Danio rerio</i>)

Hanke, Nils; King, Benjamin L.; Vaske, Bernhard; Haller, Hermann; Schiffer, Mario

doi:10.1089/zeb.2015.1093

Cited by 27 publications

(23 citation statements)

References 16 publications

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“…Previous studies which investigated glomerular filtration with eGFP‐labelled DBP in zebrafish focused on the measurement of fluorescence intensity in the eye (Hanke et al . ) or in the vasculature (Kotb et al . ).…”

Section: Discussionmentioning

confidence: 99%

4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

et al. 2016

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Section: Discussionmentioning

confidence: 99%

4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

et al. 2016

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“…To investigate whether the edema phenotype was caused by proteinuria we used our established zebrafish proteinuria model in a transgenic Tg(l-fabp:DBP:EGFP) zebrafish that expresses a 78 kDa green fluorescent plasma protein that can be monitored in the retinal plexus of the fish [22,23]. In miR-143 injected zebrafish, plasma protein fluorescence was significantly lower compared to control indicating loss of this protein from the circulation (Fig.…”

Section: Mir-143 Overexpression In Zebrafish Larvae Causes Edema and mentioning

confidence: 99%

Overexpression of TGF-β Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans

Müller-Deile

Gellrich

Schenk

et al. 2016

Cell Physiol Biochem

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Background: TGF-β is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-β. Since miR-143-3p (miR-143) is described as a TGF-β inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. Methods: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-β. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. Results: We detected a time dependent, TGF-β inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. Conclusion: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

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“…Other indirect methods (retinal plexus fluorescence intensity and intravascular fluorescence) of proteinuria quantification are published on the basis of this transgenic line. 32 However, they are highly dependent on the (variable) expression of the transgene and lack a direct proof of protein excretion. 32 Here, we present a novel proteinuria assay in zebrafish by DBF staining in sodium dodecylsulfate-polyacrylamide gel electrophoresis gels, that is independent of transgenic background (Figure 1c).…”

Section: Discussionmentioning

confidence: 99%

“…32 However, they are highly dependent on the (variable) expression of the transgene and lack a direct proof of protein excretion. 32 Here, we present a novel proteinuria assay in zebrafish by DBF staining in sodium dodecylsulfate-polyacrylamide gel electrophoresis gels, that is independent of transgenic background (Figure 1c). 33 Surprisingly, the differential band at 25 kDa that distinguishes between homozygous KO and heterozygous controls represents a low-molecular-weight protein, which stands in contrast to glomerular high-molecular-weight proteinuria in humans and mice.…”

Section: Discussionmentioning

confidence: 99%

Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout

Jobst-Schwan

Hoogstraten

Kolvenbach

et al. 2019

Kidney International

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Recently, recessive mutations of MAGI2 were identified as a cause of steroid-resistant nephrotic syndrome (SRNS) in humans and mice. To further delineate the pathogenesis of MAGI2 loss of function, we generated stable knockout lines for the two zebrafish orthologues magi2a and magi2b by CRISPR/Cas9. We also developed a novel assay for the direct detection of proteinuria in zebrafish independent of transgenic background. Whereas knockout of magi2b did not yield a nephrotic syndrome phenotype, magi2a -/larvae developed ascites, periorbital edema, and proteinuria, as indicated by increased excretion of low molecular weight protein. Electron microscopy demonstrated extensive podocyte foot process effacement. As in human SRNS, we observed genotype/phenotype correlation, with edema onset occurring earlier in zebrafish with truncating alleles (5-6 days post fertilization) versus hypomorphic alleles (19-20 days post fertilization). Paradoxically, corticosteroid treatment exacerbated the phenotype, with earlier onset of edema. In contrast, treatment with cyclosporine A or tacrolimus had no significant effect. Although RhoA signaling has been implicated as a downstream mediator of MAGI2 activity, targeting of the RhoA pathway did not modify the nephrotic syndrome phenotype. In the first CRISPR/Cas9 zebrafish knockout model of SRNS, we found that corticosteroids may have a paradoxical effect in the setting of specific genetic mutations.

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A Fluorescence-Based Assay for Proteinuria Screening in Larval Zebrafish (Danio rerio)

Cited by 27 publications

References 16 publications

4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

Overexpression of TGF-β Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans

Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout

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