Up‐regulation of the human‐specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Figures

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  FIGURE 1. Overexpression of CHRFAM7A reduces renal injury in mice with unilateral ureteral obstruction (UUO). (A) The CHRFAM7A gene was expressed only in mice of the CHRFAM7A KI group, but not in mice of the WT group. CHRFAM7A gene was confirmed to be expressed in kidneys of CHRFAM7A KI mice, but not in the kidneys of WT mice. (B) Detection of serum creatinine (Scr) and blood urea nitrogen (BUN) levels in mice (n = 5). (C) H&E staining in the kidneys from the four groups of mice (×200). Arrows indicate inflammatory cells, circles indicate renal tubular dilation and triangles indicate edema of renal tubular epithelial cells. Scale bar = 100 μm. (D, E) Detection of KIM‐1 mRNA expression by q‐PCR and protein expression by Western‐blot in kidneys from the indicated groups of mice (n = 5). **p < 0.01 vs. WT group; ## p < 0.01 vs. WT‐UUO group. CHRFAM7A KI, CHRFAM7A knock in; WT, wild type.

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  FIGURE 2. Overexpression of CHRFAM7A reduces unilateral ureteral obstruction (UUO)‐induced inflammatory cytokines and grow factors. (A, B) IL‐6 levels in serum and kidney were measured by ELISA (n = 5). All values are expressed as mean ± SD. (C–H) The expression of inflammatory factors IL‐1β, IL‐6, TNF‐α, CCL2, CD206 and FIZZ1in mouse kidney was detected by q‐PCR. Up‐regulation of CHRFAM7A reduced mRNA expression of IL‐1β, IL‐6, TNF‐α and CCL2 in response to UUO mice but increased the expression of CD206 and FIZZ1 compared with WT‐UUO mice (n = 5). **p < 0.01 vs. WT group; # p < 0.05, ## p < 0.01 vs. WT‐UUO group.

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  FIGURE 3. Overexpression of CHRFAM7A delays the progression of renal fibrosis in mice after unilateral ureteral obstruction (UUO). (A]) Representative images and quantitation of Masson's trichrome staining in kidneys from the indicated groups of mice (×200). Scale bar = 100 μm. (B, C) The expression of α‐SMA and fibronectin (FN‐1) in kidneys was detected by q‐PCR. The overexpression of CHRFAM7A reduced the mRNA expression of these fibrotic factors in the kidneys of UUO mice (n = 5). WT‐UUO group. (D) Western blot was used to detect the protein expression of α‐SMA in kidney tissues from indicated groups of mice (n = 4). Semi‐quantitative analysis of α‐SMA for the Western blot data is shown. **p < 0.01 vs. WT group; ## p < 0.01 vs. WT‐UUO group.

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  FIGURE 4. Overexpression of the CHRFAM7A gene inhibits UUO‐induced TGF‐β1/Smad2/3 signalling pathway activation. (A, B) mRNA expression of TGF‐β1 and Smad3 in mouse kidney tissues was detected by q‐PCR. The high expression of CHRFAM7A reduced gene expression of those factors in the kidneys of UUO‐injured mice (n = 5). (C, D) Western‐blot was used to detect the protein expression of TGF‐β1 and Smad2/3 in kidney tissues from above four groups of mice; densitometric analysis of the Western blot data for these factors was shown. *p < 0.05, **p < 0.01 vs. WT group; # p < 0.05, ## p < 0.01 vs. WT‐UUO group.

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  FIGURE 5. Overexpression of CHRFAM7A decreases expression of fibrotic markers stimulated by recombinant human TGF‐β1 in HK‐2 cells. (A) Fluorescence images of HK‐2 cells with transfection of pLVX‐GFP‐CHRFAM7A plasmid. Scale bar = 100 μm. (B) Human‐specific CHRFAM7A is highly expressed in HK‐2 cells transfected with the CHRFAM7A plasmid. (C, D) q‐PCR results showed that overexpression of the human‐specific CHRFAM7A gene reduced TGF‐β1‐induced increases of fibrotic markers α‐SMA and fibronectin in HK‐2 cells. Δ p < 0.05 vs. HK‐2 negative control; *p < 0.05, **p < 0.01 vs. empty vector; # p < 0.05, ## p < 0.01, vs. empty vector + TGF‐β1 group.

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  FIGURE 6. Overexpression of CHRFAM7A inhibits TGF‐β1‐induced epithelial‐mesenchymal transition of HK‐2 cells. (A) Representative brightfield images demonstrate morphological changes seen in HK‐2 cells treated with TGF‐β1 after transfection of human specific CHRFAM7A plasmid or empty vector (×100). (a) HK‐2 cells were transfected with CHRFAM7A plasmid. (b) HK‐2 cells were transfected with CHRFAM7A plasmid, followed by TGF‐β1 stimulation (20 ng/ml) for 24 h. (c) HK‐2 cells were transfected with empty plasmid, (d) HK‐2 cells were transfected with empty plasmids, followed by TGF‐β1 TGF‐β1stimulation (20 ng/ml) for 24 h. (B) Immunofluorescent staining for mesenchymal marker vimentin in HK‐2 cells. Scale bar = 100 μm. (C–E) q‐PCR results indicated that the overexpression of CHRFAM7A inhibited the TGF‐β1‐induced increase in of N‐cadherin and vimentin in HK‐2 cells. (F–H) Western blot was used to detect the protein expression of TGF‐β1, Smad2/3 and vimentin in HK‐2 cells from the indicated groups. The overexpression of CHRFAM7A inhibited TGF‐β1‐induced expression of TGF‐β1, Smad2/3 and vimentin in HK‐2 cells. ΔΔ p < 0.01 vs. HK‐2 negative control; **p < 0.01 vs. empty vector; ## p < 0.01, vs.…