Fig 2: Generation of c-Maf conditional knockout mice. (A) Schematic targeting strategy for the generation of floxed alleles in the c-Maf locus and the detected genotyping bands for each allele. KI, knock-in; L-HA, left homology arm; R-HA, right homology arm. (B) Genotyping PCR screening of floxed and deleted alleles, including floxed c-Maf (493 bp), Ayu1-Cre (269 bp) and c-Maf deletion (993 bp). (C) HE staining of lens sections from control (c-Maffl/fl) and c-Maffl/fl; Ayu1-Cre mice at E14.5. The control mice were c-Maffl/fl. (D) Immunostaining for c-MAF in lens fiber cells of control (c-Maf+/+) and c-Maffl/fl; Ayu1-Cre mice at E14.5. (E) Genotyping PCR signals for deletion confirmed that c-Maf knockout was successfully induced by tamoxifen injection. All scale bars=100 µm.

Fig 3: IHC revealed the expression of c-MAF and E-cadherin in the lenses of control and c-Maf?TAM mice. (A) Bright-field and IHC staining images of the lens. IHC staining for (B) germinative zone, (C) posterior and (D) anterior pole of the lens in the c-Maf?TAM and control mice. IHC staining images are merged: E-Cadherin in green, c-MAF in red and DAPI in blue. n=4 each (1 male, 3 females). Scale bars=100 µm.

Fig 4: c-Maf?TAM mice develop severe renal glycosuria, basic aminoaciduria, and albuminuria.Changes in (A) urine glucose, (B) water intake, and (C) urine volume in c-Maf?TAM and c-Maffl/fl groups (n = 7 per group). c-Maf?TAM group showed higher urinary glucose level, water intake, and urine volume. Loss of c-Maf did not alter glucose homeostasis, as determined by (D) 4-hour fasting blood glucose level, (E) glucose tolerance test after 10 hours of fasting, (F) serum insulin level, and (G) serum glucagon level. Glucose tolerance was examined before and up to 120 minutes after glucose solution injection (n = 11 in c-Maffl/fl and n = 15 in c-Maf?TAM groups). (H) Urinary basic amino acid levels increased in the c-Maf?TAM mice compared with the c-Maffl/fl mice. (I) c-Maf?TAM group showed higher urinary albumin levels than the c-Maffl/fl group (n = 6 per group). (J) IHC staining for albumin expression in the c-Maf?TAM group showed decreased granular signals in the proximal tubules compared with the c-Maffl/fl group (n = 3 per group). A analyses were performed on TAM(10d). Scale bars: 100 µm. A–I were presented as the mean and the standard error of the mean (SEM). To assess whether differences between c-Maf?TAM and c-Maffl/fl mice were statistically significant, a minimum of 3 biological replicates were analyzed using Welch’s t test, and a P value < 0.05 was considered significant. *P < 0.05, and ***P < 0.001. White circles: c-Maffl/fl groups, and black circles: c-Maf?TAM groups.

Fig 5: RNA-Seq analysis of the relationship between c-Maf and renal features.(A) Volcano plot of overall gene expression: 261 genes were upregulated (red) and 113 genes were downregulated (blue) in c-Maf?TAM mice. (B) Top 10 pathways of the DEGs in c-Maf?TAM mice. (C) Heatmap of the expression of 123 genes related to SLC-mediated transmembrane transport between c-Maf?TAM and c-Maffl/fl mice (n = 4 per group). (D) Megalin and (E) cubilin gene expression levels were lower in c-Maf?TAM mice than in c-Maffl/fl mice (n = 10 per group). (F) A tendency for decreased megalin protein expression levels in c-Maf?TAM mice than in c-Maffl/fl mice (n = 4 per group). Scale bars: 100 µm. For RNA-Seq transcriptome analysis, gene expression values were calculated using the quantile method. Gene expression was compared between c-Maffl/fl control mice and c-Maf?TAM mice using the Empirical Analysis DGE tool (edgeR test) in the CLC Genomics Workbench (Version 10.1.1; QIAGEN). False discovery rate (FDR) adjustments (81) were applied to correct for multiple testing, and DEGs were identified using an FDR cutoff of 0.05. D and E were presented as the mean and the standard error of the mean (SEM). To assess whether differences between c-Maf?TAM and c-Maffl/fl mice were statistically significant, a minimum of 3 biological replicates were analyzed using Welch’s t test, and a P value < 0.05 was considered significant. *P < 0.05, and ***P < 0.001. White circles: c-Maffl/fl groups, and black circles: c-Maf?TAM groups.