Proteomic comparison of kidneys from wild-type (WT; n = 8) and mutant (MU; n = 8) zebrafish. Gene ontology (GO) enrichment analysis and KEGG analysis. A Hierarchical clustering for differentially expressed proteins between WT and MT. Downregulated proteins in MU compared to WT in B GO Biological Process analysis; C GO Cellular component analysis; D GO Molecular function analysis. Upregulated proteins in MU compared with WT in E GO Biological Process analysis; F GO Cellular component analysis; G GO Molecular function analysis. H KEGG pathways related to downregulated proteins in MU compared to WT. I KEGG terms associated with upregulated proteins in MU vs. WT. Enrichments are reported as false discovery rates (FDR) ≤ 0.05. The twenty most enriched pathways are represented

Analysis of morphological parameters in wild-type and GLA-mutant mitochondria in cells from proximal and distal renal tubules. A Electron microscopy showing representative micrographs of mitochondria (red arrows) in cells from wild-type and mutant proximal and distal tubules; B Mitochondrial form factor and aspect ratio plotted against each other in proximal tubule; C Statistical comparison using mean with 95% confidence intervals (CI) for form factor and aspect ratios in mitochondria from proximal tubule cells; D Mitochondrial form factor and aspect ratio plotted against each other in distal tubule; E Statistical comparison using mean and 95% confidence intervals (CI) for form factor and aspect ratios in mitochondria from distal tubule cells; F, G Mitochondrial morphological parameters comparison in proximal tubules and distal tubules; H, I Mitochondrial volume and density determination in proximal and distal tubules. Values are represented as mean with upper 95% CI. Data were analyzed using Mann–Whitney test. *p-value ≤ 0.05; ****p-value ≤ 0.00001; ns = non-significant

Mitochondrial cristae morphology and area determination in proximal and distal tubules performed using TEM images and total antioxidant determination (TAC) in wild-type and GLA-mutant Zebrafish. A, B Individual score grades across the whole mitochondrial population analyzed in cells from renal proximal and distal tubules; C, D Total cristae area in cells from proximal and distal tubules; E, F Cristae volume in proximal and distal renal tubules; G, H Cumulative cristae mean scores for proximal and distal renal tubules, I TAC levels in adult (9-months-old) zebrafish kidney tissue lysates. For mitochondria cristae morphology quantification data represented as mean with upper 95% confidence interval. For TAC values are represented as violin plot representing the median and IQ ranges. Data were analyzed using Mann–Whitney test; ****p-value ≤ 0.00001; **p-value ≤ 0.001

Immunohistochemical analysis of the expression of selected proteins in wild-type and mutant zebrafish renal tissues. A Representative image of IHC analysis targeting mitochondrial marker Sod2 and lysosomal marker Cd63 in kidneys from wild-type and mutant ZF renal tissues; B Quantification of immunohistochemical staining of sections from wild-type and mutant ZF kidneys. Signal intensity is significantly higher in wild-type than in mutant specimens for CD63 and Sod2; C Heatmap showing protein expression of CD63) and Sod2 in mutant and wild-type kidney tissues, normalized as percent of positive pixels. Data analysis using Mann–Whitney test; **p-value ≤ 0.001