Rhapontin alleviates C-reactive protein induced diabetic kidney disease through Smad3-ACSM3 mediated ferroptosis

Abstract

<p><strong>Background: </strong></p><p>Accumulated evidence indicated that C-reactive protein (CRP) enhances diabetic kidney disease (DKD) via Smad3 signaling pathway. Acyl-CoA synthetase medium-chain family member 3 (ACSM3) locates on the membrane of mitochondria to catalyze fatty acids. Ferroptosis is one type of cell deaths, featured with iron-dependent phospholipid peroxidation. This study determined whether CRP promotes DKD via Smad3-ACSM3 mediated ferroptosis and explore potential therapeutics.</p><p><br></p><p><strong>Methods:</strong></p><p>CRP transgenic (Tg)×db/db, CRPtg×db/dm, Smad3 knockout (KO)×db/db and Smad3 KO×db/m mice were used in the study. Differentially expressed genes in CRPtg×db/db mice were analyzed by the RNA sequencing. To evaluate protective role of ACSM3, AAVs with ACSM3 overexpression were administered into db/db mice. In vitro, HK-2 cells were treated with CRP with/without blocking of CRP receptor by CD32b antibody or treated with Smad3 inhibitor SIS3 were employed. Ferroptosis indexes were measured by IF, WB and qPCR. High through output molecular docking and cellular thermal shift assay (CETSA) were conducted to identify candidate compounds targeting ACSM3.</p><p><br></p><p><strong>Results:</strong></p><p>Overexpression of CRP in diabetic mice significantly enhanced ferroptosis in kidneys. The RNAseq result indicated that ACSM3 level was significantly downregulated in CRPtg×db/db mice, compared with CRPtg×db/dm mice. Interestingly, deletion of Smad3 alleviated the ferroptosis and reversed ACSM3 deficiency in diabetic kidneys. Overexpression of ACSM3 in diabetic mice led to ferroptosis alleviation in kidneys. Consistently, the ferroptosis induced by CRP in vitro were reversed by SIS3, or the blockade of CRP receptor, or the overexpression of ACSM3. Furthermore, through the high through output molecular docking prediction and CETSA, we found that Rhapontin was one of potential compounds to suppress ferroptosis by targeting ACSM3.</p><p><br></p><p><strong>Conclusion:</strong></p><p>Targeting on ACSM3 and SMAD3 have therapeutic potential for CRP induced ferroptosis in DKD.</p>