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.
View: Text | PDF
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

×

Figure 7

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

Options: View larger image (or click on image) Download as PowerPoint
Selective Shroom3 knockdown in tubular cells inhibits canonical TGF-β1/S...
To achieve Shroom3 knockdown exclusively in tubular cells, PAX8-RTTA/COLTGM mice were fed Dox for 3 weeks; non-Dox-fed littermates of the corresponding genotype were used as controls. Dox feeding of PAX8-RTTA/COLTGM mice induced Shroom3 knockdown and GFP production in tubular cells (Supplemental Figure 7). Left UUO was surgically created, and mice were perfused 10 days later. (A) Relative Col1a1 mRNA, by RT-PCR (normalized to Gapdh). (B) Representative WBs for phospho-SMAD3, SMAD3, Shroom3, total GFP, and β-actin between Dox-fed and non-Dox-fed PAX8-RTTA mice. (C–E) Representative microphotographs (C; original magnification, ×40) from kidney sections of picrosirius red staining and COL1A1 IF, and corresponding morphometric quantifications for (D) Sirius red and (E) COL1A1 (n = 5 animals; 10 random hpfs/animal). Data are mean ± SEM. *P ≤ 0.05, **P < 0.001 between means, ANOVA with post-test Bonferroni comparison.