Figure 6. CRISPR/Cas9 mediated point mutation in the Zfp24 binding site in the Sox9 promoter abrogates AKI-induced Sox9 upregulation.

A transgenic knock-in mouse was generated using CRISPR/Cas9 and a point mutation was introduced in the Zfp24 binding site within the murine Sox9 promoter. (a) Schematic representation of the single point mutation in the Zfp24 binding site within the murine Sox9 promoter. Heterozygous mutant mice were crossed to generate littermate control (WT) and promoter mutant (Prom^mut) mice. Representative DNA sequencing data confirming the mutation is shown here. These mice were then challenged with bilateral renal ischemia (30 min), cisplatin (30 mg/kg, single intraperitoneal injection) nephrotoxicity, or glycerol-induced rhabdomyolysis (7.5 ml/kg 50% glycerol in the hind-leg muscles) followed by examination of the severity of AKI using biochemical and histological analysis. (b-f) Blood urea nitrogen, serum creatinine, renal Ngal gene expression (qPCR), and histological analysis (H&E) showed that mutations in the Zfp24 binding site within the Sox9 promoter results in significantly worsened renal impairment in the IRI, cisplatin, and rhabdomyolysis-associated mouse models of AKI. The asterisk in the histological images depicts damaged tubules. Histopathologic analysis and tubular damage were scored by calculation of the percentage of tubules that showed dilation, epithelium flattening, cast formation, loss of brush border and nuclei, and denudation of the basement membrane. In all the bar graphs (n = 8 biologically independent samples from3–4 independent experiments), experimental values are presented as mean ± S.D. The height of error bar = 1 S.D., and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey’s multiple-comparison test was carried out, and statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 100 μm.