Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis
Madhav C. Menon, … , John Cijiang He, Barbara Murphy
Madhav C. Menon, … , John Cijiang He, Barbara Murphy
Published December 1, 2014
Citation Information: J Clin Invest. 2015;125(1):208-221. https://doi.org/10.1172/JCI76902.
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Research Article Nephrology

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis

Abstract

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7–like 2–dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2–mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell–specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

Authors

Madhav C. Menon, Peter Y. Chuang, Zhengzhe Li, Chengguo Wei, Weijia Zhang, Yi Luan, Zhengzi Yi, Huabao Xiong, Christopher Woytovich, Ilana Greene, Jessica Overbey, Ivy Rosales, Emilia Bagiella, Rong Chen, Meng Ma, Li Li, Wei Ding, Arjang Djamali, Millagros Saminego, Philip J. O’Connell, Lorenzo Gallon, Robert Colvin, Bernd Schroppel, John Cijiang He, Barbara Murphy

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Figure 6

Shroom3 knockdown in vivo inhibits canonical TGF-β1/SMAD3 signaling and profibrotic gene expression and abrogates renal fibrosis in a murine model.

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Shroom3 knockdown in vivo inhibits canonical TGF-β1/SMAD3 signaling and ...
Male Rosa-RTTA/COLTGM mice were fed Dox for 3 weeks to obtain Shroom3 knockdown in all cell types; non-Dox-fed littermates of corresponding genotypes were used as controls (n = 5 pairs; Supplemental Figure 6, A–C). Left UUO was surgically created, and mice were perfused 10 days later. (A) Relative Shroom3 and Col1a1 transcript levels, by RT-PCR (normalized to Gapdh), from whole cortices of control and UUO kidneys of non-Dox-fed and Dox-fed animals (n = 5). (B) WBs of phospho-SMAD3, SMAD3, SHROOM3, total GFP, and β-actin from mouse kidney cortex lysates from UUO kidneys of non-Dox-fed and Dox-fed mice. (C) Representative images from non-Dox-fed control kidneys, non-Dox-fed UUO kidneys, and Dox-fed UUO kidneys. Shown are periodic acid–Schiff stain (PAS; original magnification, ×10), picrosirius red stain (original magnification, ×40), and COL1A1 IF (TRITC–Alexa Fluor) performed on snap-frozen kidney cortex sections (original magnification, ×40). (D and E) Morphometric quantification of threshholded ×40 images (n = 5 animals; 10 random hpfs/animal), showing area of (D) picrosirius red staining and (E) COL1A1 IF as a percentage of total hpf area. (A, D, and E) Values are mean ± SEM. *P ≤ 0.05, **P < 0.001, ***P < 0.0001 between means, ANOVA with post-test Bonferroni comparison.