Secreted Frizzled-related protein 1 (Sfrp1) regulates the progression of renal fibrosis in a mouse model of obstructive nephropathy - PubMed (original) (raw)

Makoto Matsuyama et al. J Biol Chem. 2014.

Abstract

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1(-/-) obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1(-/-) mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.

Keywords: Beta-catenin (B-catenin); Cell Signaling; Gene Knockout; Kidney; Pathology; Wnt Signaling.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Sfrp1 increases after unilateral ureteral obstruction (UUO). A, the Sfrp1 antibody reacted specifically with a band corresponding to Sfrp1 in lysates of Sfrp1-FLAG-expressed 293T cells, but not 293T (mock) cells by Western blotting. B, characterization of an antibody specifically recognized Sfrp1, Sfrp2, and Sfrp5. Immunoreactivity was impaired specifically with Sfrp1, but not with other Sfrp protein families (Sfrp2–5). The Sfrp2 and Sfrp5 antibodies were also immunoreacted specifically with Sfrp2 and Sfrp5 proteins, respectively. C, the levels of the Sfrp1, Sfrp2, and Sfrp5 proteins were detected by Western blot analysis in the Sham and UUO kidneys in wild-type mice (Sfrp1+/+) or Sfrp1 knock-out mice (_Sfrp1_−/−) at different time points after UUO. Actin was evaluated as an internal control. His-Sfrp2 and His-Sfrp5 were used as a positive control to detect Sfrp2 and Sfrp5 proteins, respectively.

FIGURE 2.

FIGURE 2.

Loss of Sfrp1 exacerbates the progression of renal fibrosis after UUO. A and B, representative microscopic images of the sham and UUO kidneys in wild-type (Sfrp1+/+) and Sfrp1-deficient mice (_Sfrp1_−/−). These tissue sections were prepared and stained with hematoxylin and eosin. B, higher magnification is shown in A. C, αSMA immunostaining in the obstructed kidneys. D, graph shows analysis of the percentage of αSMA-positive area in the UUO kidneys. Scale bars, 500 μm (A), 50 μm (B), 100 μm (C). *, p < 0.05.

FIGURE 3.

FIGURE 3.

Sfrp1-deficient mice are enhanced epithelial-to-mesenchymal transition after UUO. A, C, and E, immunohistochemical analyses of the mouse kidney sections with antibodies against vimentin (A), E-cadherin (C), and actinin 4 (E) in the sham and UUO kidneys after UUO. B, graph shows analysis of the percentage of vimentin-positive area in the UUO kidneys. D, the E-cadherin expression levels are given as a percentage of the UUO to sham-operated kidneys. F, renal glomerulus is unchanged in the obstructed _Sfrp1_−/− kidneys by counting actinin 4-positive glomerulus. Scale bars, 100 μm. *, p < 0.05.

FIGURE 4.

FIGURE 4.

Wnt/β-catenin pathway in the Sfrp deficient obstructed kidneys. A, detection of active- and total β-catenin, c-Myc, and cyclin D1 by Western blotting of the UUO kidneys. Actin was evaluated as an internal control. B, immunohistochemical analyses of mouse kidney sections with antibodies against active and total β-catenin in the sham and UUO kidneys at 7 days after the surgery. C and D, number of phospho-HistonH3 (Ser-10) cells in the obstructed kidneys at 7 days after UUO. C, section of the Sham and UUO kidneys in Sfrp+/+ and _Sfrp_−/− mice were subjected to immunostaining with an anti-phospho-histone H3 (Ser-10) antibody. D, graph shows analysis of the percentage of phospho-histone H3 (Ser-10)-positive cells in the UUO kidneys. Scale bars, 100 μm.

FIGURE 5.

FIGURE 5.

Non-canonical Wnt pathway in the Sfrp-deficient obstructed kidneys. A, detection of phospho- and total c-Jun, phospho- and total JNK, phospho- and total p38, and phospho- and total Ca2+/calmodulin-dependent kinase II (CaMKII) by Western blotting of the UUO kidneys. Actin was evaluated as an internal control. B, immunohistochemical analyses of mouse kidney sections with antibodies against phospho- and total c-Jun in the sham and UUO kidneys at 7 days after the surgery. C and D, number of TUNEL-positive cells in the obstructed kidneys at 7 days after UUO. C, section of the sham and UUO kidneys in Sfrp+/+ and _Sfrp_−/− mice were subjected to TUNEL staining. D, graph shows analysis of the percentage of TUNEL-positive cells in the UUO kidneys. Scale bars, 100 μm. *, p < 0.05.

FIGURE 6.

FIGURE 6.

TGF-β signaling is not altered in the obstructed _Sfrp_−/− kidneys. Detection of phospho- and total Smad3 by Western blotting of the UUO kidneys. Actin was evaluated as an internal control.

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