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 4

TGF-β1 increases SHROOM3 in a β-catenin/TCF7L2–dependent manner.

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TGF-β1 increases SHROOM3 in a β-catenin/TCF7L2–dependent manner. (A) SHR...
(A) SHROOM3 mRNA transcript levels (normalized to GAPDH) in PRCECs treated for 24 hours with TGF-β1 (2.5 and 5 ng/ml) relative to cells without TGF-β1 treatment. Values are mean ± SEM (n = 3). *P < 0.05, ANOVA with post-test Bonferroni comparison. (B and C) Representative WBs of (B) SHROOM3 and β-actin in PRCECs treated with TGF-β1 (5 ng/ml) for 30 and 48 hours and (C) SHROOM3, cyclin D1, and β-actin in HK2 cells treated with AG-L-67051 (0.3 or 0.6 μM) for 48 hours (n = 3 sets each). (D) HK2 tubular cells were treated with and without TGF-β1 (5 ng/ml) in the presence or absence of the β-catenin inhibitor quercetin (10 μM). Shown are representative WBs of SHROOM3 and β-actin at 30 hours (n = 3 independent sets). (E) HK2 cells (cultured in 6-cm dishes), infected with lentiviral constructs containing either scrambled or TCF7L2 shRNA, were treated with TGF-β1 (5 ng/ml) for 48 hours. Shown are representative WBs of SHROOM3, TCF7L2, and β-actin from duplicate experiments (n = 3 sets).