Functional Study of map3k15 in Zebrafish Pronephros

Background: MAP3K15 is highly expressed in renal tissues, but its role in kidney remains unclear. We identified a MAP3K15 frameshift mutation in a family with idiopathic membranous nephropathy, and knockout mice showed spontaneous podocyte injury and proteinuria. To investigate its role, we generated map3k15 knockout zebrafish via CRISPR/Cas9 to examine effects on pronephros development and function. Methods: CRISPR/Cas9-generated map3k15 knockout lines were validated by qRT-PCR and WISH. Edema and morphological changes were examined microscopically. Podocin staining and Tg(gtshβ:GFP) assessed renal structures, while proteinuria and FITC-dextran assays evaluated glomerular function. RNA-seq and qRT-PCR identified kidney-related gene changes. Results: map3k15 is expressed early during embryonic development and localizes to the pronephric tubules. Two stable map3k15 konckout lines were generated, with significantly reduced mRNA expression. Compared to WT, mutants developed progressive edema by 2 dpf, mainly in the head, pericardium, and yolk. Immunofluorescence showed markedly decreased Podocin expression in glomeruli, along with proximal tubule abnormalities. SDS-PAGE confirmed proteinuria, and 70 kDa FITC-dextran clearance assays indicated compromised glomerular barrier. Transcriptomic profiling identified 6,498 differentially expressed genes, with significant downregulation of nephrogenic markers (pax2a, lhx1a, wt1a, nphs2), consistent with qRT-PCR. Conclusion: This study generated map3k15 knockout zebrafish via CRISPR/Cas9 and showed that map3k15 loss leads to pronephric defects, including edema, impaired filtration, and proteinuria, underscoring its essential role in pronephros development and function.