Sprr2f protects against renal injury by decreasing the level of reactive oxygen species in female mice

Abstract

Renal injury leads to chronic kidney disease, with which women are not only more likely to be diagnosed than men but have poorer outcomes as well. We have previously shown that expression of small proline-rich region 2f (Sprr2f), a member of the small proline-rich region (Sprr) gene family, is increased several hundredfold after renal injury using a unilateral ureteral obstruction (UUO) mouse model. To better understand the role of Sprr2f in renal injury, we generated a Sprr2f knockout (Sprr2f-KO) mouse model using CRISPR-Cas9 technology. Sprr2f-KO female mice showed greater renal damage after UUO compared with wild-type (Sprr2f-WT) animals, as evidenced by higher hydroxyproline levels and denser collagen staining, indicating a protective role of Sprr2f during renal injury. Gene expression profiling by RNA sequencing identified 162 genes whose expression levels were significantly different between day 0 and day 5 after UUO in Sprr2f-KO mice. Of the 162 genes, 121 genes were upregulated after UUO and enriched with those involved in oxidation-reduction, a phenomenon not observed in Sprr2f-WT animals, suggesting a protective role of Sprr2f in UUO through defense against oxidative damage. Consistently, bilateral ischemia-reperfusion injury resulted in higher serum blood urea nitrogen levels and higher tissue reactive oxygen species in Sprr2f-KO compared with Sprr2f-WT female mice. Moreover, cultured renal epithelial cells from Sprr2f-KO female mice showed lower viability after oxidative damage induced by menadione compared with Sprr2f-WT cells that could be rescued by supplementation with reduced glutathione, suggesting that Sprr2f induction after renal damage acts as a defense against reactive oxygen species.

Keywords: acute renal injury; chronic renal injury; ischemia-reperfusion injury; reactive oxygen species; small proline-rich region 2f.

Graphical abstract

Fig. 1.

Small proline-rich region 2f (Sprr2f) expression in genitourinary tissues. Representative photomicrographs (magnification: ×20) of hematoxylin and eosin-stained sections (top) and Zs-Green fluorescent reporter show Sprr2f expression from the same section (bottom). Green fluorescent protein driven by the Sprr2f promoter was observed in all epithelial cells of the kidney (A), ureter (B), and bladder (C) in both male and female mice. It was also seen in the endometrium (D) of female mice and the epididymal epithelium of male mice (E). Scale bar = 50 μm.

Fig. 2.

CRISPR/Cas9 strategy and genotyping for small proline-rich region 2f (Sprr2f) knockout (KO) mice. A: Sprr2f exon 2 was targeted for deletion (coding exons shown as solid black bars; noncoding exons shown as blue lines). Forward primer (Fw) labeled by green arrows and reverse (Rv) primer labeled by red arrows were used for validation and genotyping. B: PCR genotyping of wild-type (WT) Sprr2f^+/+, heterozygous Sprr2f^+/−, and homozygous KO Sprr2f^−/− mice. C: Sprr2f mRNA expression in wild-type Sprr2f^+/+ (n = 3) and homozygous KO Sprr2f^−/− kidneys (n = 3) were validated by quantitative RT-PCR.

Fig. 3.

Small proline-rich region 2f knockout (Sprr2f-KO) mice display greater acute kidney injury after ischemia-reperfusion injury (IRI). Blood urea nitrogen (BUN) levels were measured 24 h after sham operation (n = 3) or IRI (n = 10) in wild-type (Sprr2f-WT) and Sprr2f-KO mice. BUN levels were dramatically increased after IRI in both Sprr2f-WT and Sprr2f-KO mice. Mean BUN levels in Sprr2f-KO mice were significantly higher than in Sprr2f-WT mice.

Fig. 4.

Unilateral ureteral obstruction (UUO) induces greater renal fibrosis in small proline-rich region 2f knockout (Sprr2f-KO) mice compared with wild-type (Sprr2f-WT) mice. A: no significant difference in hydroxyproline levels in kidney tissues harvested from Sprr2f-WT (n = 2) and Sprr2f-KO (n = 2) mice at day 0 (D0), whereas a significant increase in hydroxyproline content in Sprr2f-KO mice (n = 11) compared with Sprr2f-WT mice (n = 12, *P = 0.022) was observed at day 14 (D14) after UUO. B: representative images of picrosirius red staining of kidney sections of Sprr2f-WT and Sprr2f-KO mice at D0 and D14 (n = 2 in each group) after UUO (magnification: ×20). Arrows indicate collagen fibers stained in dark red. Scale bar = 50 μm.

Fig. 5.

Gene expression profiling of small proline-rich region 2f knockout (Sprr2f-KO) mice after unilateral ureteral obstruction (UUO). A: volcano plot of gene expression profiles of Sprr2f-KO mice at day 0 (n = 4) and day 5 (n = 3) after UUO. Genes significantly upregulated (blue dots) or downregulated (red dots) were identified by the Bonferroni-Holm method (family-wise error rate < 0.05). The y-axis is −log₁₀; P value, representing the negative log of unadjusted P value at base 10; the x-axis is log₁₀ fold, representing the negative log of fold change at base 10. The cutoff line on P value on the y-axis is P < 0.05. B: heatmap of genes differential expressed at day 0 and day 5 after UUO. C: pathways enriched in differentially expressed genes after UUO identified using PANTHER classification system were ranked by adjusted P value.

Fig. 6.

Small proline-rich region 2f (Sprr2f) protects against reactive oxygen species in vitro and in vivo. A: renal epithelial cells from wild-type Sprr2f^+/+ mice (n = 6) and knockout (KO) Sprr2f^−/− mice (n = 7) were treated for 24 h with 20 µM menadione. The percentage of live cells was determined by a Cyquant cell proliferation assay. Cells from KO Sprr2f^−/− mice showed significantly lower survival compared with cells from wild-type Sprr2f^+/+ mice. B: pretreatment of cells with GSH rescued renal epithelial cells from menadione-induced cell death in both wild-type Sprr2f^+/+ and KO Sprr2f^−/− mice. No significant difference was observed between pretreated Sprr2f^+/+ and KO Sprr2f^−/− mice in response to menadione. n.s., Not significant. C: representative dihydroethidium (DHE) staining in kidneys from wild-type Sprr2f^+/+ (n = 2) and KO Sprr2f^−/− (n = 2) 24 h after ischemia-reperfusion injury (IRI). Scale bar = 75 μm. D: quantification of the DHE signal normalized against the DAPI signal showed significantly higher DHE levels in Sprr2f^−/− mice (53 image fields) compared with Sprr2f^+/+ mice (44 image fields).