An intronic locus determines SHROOM3-expression and potentiates renal allograft fibrosis

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 DESCRIPTION (provided by applicant): Tubulo-interstitial fibrosis (TIF) contributes to the progression of chronic kidney disease (CKD) from varied etiologies, including chronic allograft nephropathy (CAN), to end stage renal failure. However, sensitive predictor of kidney fibrosis and effective antifibrotic therapy for CKD do not exist. Therefore, it is critical for us to identiy markers of and drug targets for TIF. Preliminary data demonstrates that SHROOM3 expression in 3 months renal allografts biopsies was associated with indices of poor allograft function (higher CADI score and decline in eGFR) at 12 months post transplantation, but not at 3 months. Furthermore, theSHROOM3rs17319721 SNP (i.e. A/G or A/A) is associated with a higher SHROOM3 expression at 3 months and a greater propensity to developaΔ CADI (chronic allograft index score) ≥2at 12 months, compared to the G/G allele. In the presence of the risk allele regulation of expression appears to be mediated via a Wnt/βcatenin/ TCF7L2 dependent pathway. Furthermore, expression of SHROOM3 enhanced the profibrotic effects of TGFβ. Based on these preliminary data, the central hypothesize of this proposal is that the SHROOM3 snprs17319721 functions as a cisacting expression quantitative trait locus of SHROOM3 to facilitate TGFβ1 signaling contributes to renal fibrosis leading to progression of CAN and CKD. To test this hypothesis, the following specific aims are proposed: Specific aim 1: To validate that SHROOM3 genotype correlates with expression in a second transplant cohort (1a); To determine the correlation of SHROOM3 genotype and expression with long-term graft outcomes (1b); and To determine SHROOM3 protein expression and localization in the allograft (1c).Specific aim 2: (2a)To determine the transcriptional regulation of SHROOM3 expression by the rs1731972 SNP; (2b) To map the molecular interaction between the TCF7L2/βcatenin complex and the consensus binding sequence encompassing rs17319721; (2c) To investigate the mechanism of SHROOM3facilitated TGFβ signaling in kidney fibrosis. Specific aim 3: To determine the role of SHROOM3 on kidney fibrosis in CAN and CKD murine models in vivo. An inducible and tissues pecific SHROOM3 knockdown model (3a) and an overexpression transgenic model (3b) will be used to examine SHROOM3's function in these models of renal fibrosis.Innovation:SHROOM3is a novel candidate gene for which both a mutant allele and expression predict the decline of renal function and the progression of TIF in renal allografts. This is the first description of a CKD associated SNP that functions as an eQTL. Furthermore, a mechanism for transcriptional regulation via the in tronic SNP is proposed. The current research plan will build upon these initial findings to further delineate transcriptional regulation of SHROOM3, the mechanism by which it contributes the development of fibrosis in vitro and its significance in vivo. The proposed research is of Significance because it will determine the role ofShroom3 in the development and progression of CAN and CKD and its potential as a therapeutic target for the prevention of fibrosis.