RARRES1 of Renal Tubular Epithelial Cells Aggravates Renal Fibrosis through Interaction with KHDRBS1/Src to Enhance Phosphorylation of STAT3

Background: The underlying mechanisms of Renal fibrosis are uncertain. RARRES1, a retinoic acid receptor protein, is normally found in podocytes but upregulates in renal tubules during disease. This study aims to investigate the role and mechanism of RARRES1 in renal fibrosis. Methods: 1. NEPTUNE and C-PROBE cohorts studied the link between RARRES1 expression in the renal tubular interstitium and CKD outcomes. 2. RNAscope confirmed the RARRES1 gene expression in renal tubular cells in CKD patients. Using Nephroseq data, we correlated RARRES1 expression with CKD fibrosis, with validation by Immunofluorescence staining. 3. Tubular cell-specific RARRES1 knockout mice were subjected to UUO-D10 and FA-D14 models to assess renal function and evaluate fibrosis. 4. Mass Spectrometry, CO-IP, and molecular cloning identified RARRES1 interactions and specific binding domains. 5. Protein interaction analysis and genetic manipulation were used to investigate the downstream mechanism. Results: 1. RARRES1 was upregulated in the renal tubular interstitium of CKD patients. Higher RARRES1 expression in renal tubules correlated with lower GFR and worse outcomes in CKD patients. 2. RARRES1 gene expression positively correlated with fibrosis, as validated by immunofluorescence staining of α-SMA. 3. RARRES1 knockout in tubular cells alleviated renal injury and fibrosis in UUO-D10 and FAN-D14 mouse models. 4. KHDRBS1 was identified as an interacting protein of RARRES1 by CO-IP, with amino acids 263-269 of RARRES1 being crucial for this interaction. 5. Overexpression of RARRES1 and knockdown of KHDRBS1 in HK-2 cells led to a significant decrease in fibrosis markers within renal tubular epithelial cells. 6. KHDRBS1 interacts with STAT3 and Src, and overexpression of RARRES1 in HK-2 cells lead to a notable increase in phosphorylated STAT3 (p-STAT3). Additionally, RARRES1 knockdown in primary human renal tubular epithelial cells significantly reduced fibronectin expression upon TGF-β stimulation and the nuclear translocation of p-STAT3. Conclusion: Elevated RARRES1 in CKD renal tubular cells exacerbates fibrosis via interaction with KHDRBS1, Src and STAT3, which promotes STAT3 phosphorylation and nuclear translocation. Targeting this RARRES1-KHDRBS1-Src axis may offer a therapeutic approach for renal fibrosis.