A KDM6A-KLF10 reinforcing feedback mechanism aggravates diabetic podocyte dysfunction (2019)
Figure 4

Figure 4. Podocyte-specific knockout of KDM6A in mice protects against diabetes-induced kidney injury.

A Quantitative RT-PCR evaluation of KDM6A mRNA expression in kidney glomeruli isolated from wild-type and KDM6A-knockout (KO) mice. *P < 0.05, significant difference versus wild-type controls (Wilcoxon two-sample test; n = 8).

B Western blot analysis of KDM6A expression in glomeruli and podocytes isolated from wild-type and KDM6A-KO mice. Presented experiments were performed at least three times independently.

C Changes in body weights and levels of blood glucose in wild-type or KDM6A-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between wild-type and KDM6A-KO mice with diabetes were observed during the 8-week experimental period (Wilcoxon two-sample test; n = 8).

D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type and KDM6A-KO mice with or without STZ treatment (at 8 weeks after the onset of diabetes). The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).

E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).

F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).

G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the above treated mice. Presented experiments were performed at least three times independently. Scale bars, 20 μm.

H Western blot analysis of nephrin, KDM6A and WT-1 expressed in primary podocytes isolated from the above treated mice. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).

I Effect of high glucose on the expression of podocyte-related markers in primary cultured podocytes isolated from wild-type or KDM6A-KO mice. The primary podocytes isolated from wild-type or KDM6A-KO mice were cultured in normal or high glucose (30 mM) for 48 hours. Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies. *P < 0.05 versus wild-type podocytes in normal glucose, #P < 0.05 versus wild-type podocytes in high glucose (Parametric ANOVA and a Bonferroni post hoc test; n = 3).

Data information: Data are expressed as mean ± SEM. See the exact P values for comparison tests in Appendix Table S4.