Quality Control and Fold Change. Total samples (n = 16), with sex-matched groups: MtM: Mutant Male (n = 4), MtF: Mutant Female (n = 4), WtM: Wild type Male (n = 4), WtF: Wild type Female (n = 4). (A) Pearson Correlation for pairwise comparison of mRNA FPKM between samples, rounded to 2 decimals; (B) Heatmap clustering of variance stabilized counts for all genes binned into five hundred clusters, showing that both sex and condition are clustering together; (C) PCA analysis of variance stabilized counts for all genes, with batch correction for both Sex and Replicates; (D) Volcano plot of Log2 fold change (LFC) for all genes using DESeq2 differential expression analysis, with resulting −log10 adjusted p-values. (Negative LFC means LFC (Mutant Mt) < LFC (Wild type Wt). A high confidence set of genes are displayed with gene symbols (adjusted p-value < 0.000001 and absolute value of LFC > 1.7).

Gene ontology (GO) enrichment analysis of pathways upregulated and downregulated in renal tissues from the mutant, compared to wildtype ZF. Data refers to GO Biological Process (BP: (A) and (B), respectively), Cellular component (CC, (C) and (D), respectively) and Molecular function (MF: (E) and (F), respectively). In all cases, the twenty most enriched pathways are reported. FDR ≤ 0.05.

KEGG pathway enrichment analysis and heatmap of m6pr and atf6 genes in mutant compared to wildtype ZF. (A) KEGG pathways associated with the upregulated genes; (B) KEGG pathways associated with the downregulated genes; (C) heatmap (percentage column normalized, where dark blue is the maximum per column) showing the expression of the selected downregulated genes m6pr and atf6.

Immunohistochemical and Western blot detection of selected proteins in kidneys from WT and M ZF reveals protein expression disturbances in mitochondria and lysosomes. (A) Representative IHC staining specific for mitochondrial marker isocitrate dehydrogenase (NAD⁽⁺⁾)3 alpha (Idh3a) and lysosomal marker cathepsin B (Ctsb) in kidney tissue sections from WT and M ZF (upper right and upper left panels, and lower right and lower left panels, respectively; (B) Quantification of immunohistochemical staining of sections from WT and M ZF kidneys. Signal intensity is significantly higher in WT than in M for both proteins. (C) Representative immunoblots of Ctsb and Idh3a in kidney tissue section from WT and M ZF; (D) Quantification of immunoblots from WT and M ZF kidneys. (Mann–Whitney test U * p < 0.05). Wildtype (WT), mutant (M). ns: not significant. Scale bar (bottom left corner, in black) = 100 µm.