Fibrogenesis plays a major role in chronic kidney disease (CKD) and remains a vexing issue for clinicians. Our laboratory has sought to understand molecular mechanisms of renal fibrosis by examining cellular responses to fibrogenic stimuli such as TGF-beta. Our studies identified Smad anchor for receptor activation (SARA) as a potentially anti-fibrotic molecule. SARA was originally described as a TGF-beta receptor adaptor molecule that facilitates Smad signaling, but is now known to have a broader role as a scaffolding protein that assembles molecules in complexes to direct their function. Our published and preliminary data indicate that SARA inhibits cells from assuming a fibrogenic phenotype. Further, SARA is downregulated in cells that are subjected to fibrogenic stimuli, concomitant with the cells becoming fibrogenic. Its expression also is markedly reduced in kidneys undergoing fibrosis. These findings suggested that preventing SARA down-regulation would be antifibrotic. We now present data indicating that ectopic expression of SARA prevents both aristolochic acid-induced tubulointerstitial fibrosis in conditionally SARA-transgenic mice and nephrocyte-associated fibrosis in Drosophila. Together, our findings support the hypothesis that maintaining SARA expression could delay or prevent kidney fibrosis. The overall goal of this proposal is to determine the actions and regulation of SARA in CKD. To address our hypothesis we propose three specific aims. First, we will characterize the antifibrotic actions of SARA. We have developed a transgenic mouse that conditionally expresses SARA under the control of Chicken-beta-actin-Cre. Our preliminary data indicate that inducing expression with the PDGFRbeta promoter is protective against fibrosis. We will induce tissue-specific expression of SARA in these mice to determine the cellular locus of SARA’s antifibrotic actions. Further, we will determine whether SARA downregulation is necessary and sufficient to cause fibrosis by using Crisper technology to develop a conditional SARA-knockout mouse, and we will evaluate the role of putative splice variants of SARA by expressing different domains of the SARA protein in cultured proximal tubular cells or renal pericytes. In the second aim, we will characterize the regulation of SARA expression by examining the role of a TGF-beta-inducible transcriptional repressor of SARA that we have identified, BHLHE40. We also will determine the mechanism of action of another antifibrotic molecule, soluble Klotho, which we have observed to stimulate and act through SARA expression. Finally, we will determine effector pathways that mediate SARA’s antifibrotic actions. We will elucidate a novel mechanism by which SARA inhibits the fibrogenic actions of beta-Catenin, and we will identify new effector mechanisms of SARA through RNA-Seq of TGF-beta-treated mouse cells and drosophila undergoing pericardial nephrocyte-associated fibrosis. Together these studies will define a novel antifibrotic molecule and its potential role in regulating the progression of CKD.
|Effective start/end date||8/24/17 → 6/1/19|
- National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK105055-02 REVISED)