C-reactive protein promotes diabetic kidney disease : mechanism and potential application

Abstract

Diabetic kidney disease (DKD) is a predominant cause of end-stage renal disease, resulting in significant socio-economic burden. Previous studies have suggested that C-reactive protein (CRP) enhances DKD by augmenting renal inflammation and fibrosis via TGF-β/Smad3 signaling pathway. The mechanisms by which CRP enhances DKD remains largely unclear. In this thesis, the roles of two differently expressed genes NLRP3 and ACSM3 on CRP related DKD were investigated based on RNA sequencing results from kidneys of CRPwt-db/db and CRPtg-db/db mice, as well as pathological mechanisms and potential therapeutic applications.

Firstly, we investigated the role of NLRP3 in CRP related DKD. We demonstrated that CRP enhances DKD by activating NLRP3 inflammasome and the phosphorylation of Smad3. Blockade of CRP receptor abolishes CRP signaling, and suppresses the phosphorylation of Smad3 and activation of NLRP3 inflammasome in vitro. Furthermore, genetic deletion or pharmacological inhibition of Smad3 eliminates the activation of NLRP3 inflammasome in diabetic kidneys or CRP/high glucose treated cells. Notably, we discovered that p-Smad3, upregulated by CRP, binds to the NLRP3 gene promoter, thereby increasing the expression.

Secondly, we investigated the role of ACSM3 in CRP related DKD. ACSM3 ranks the top 1 down-regulated gene in RNA sequencing analysis. ACSM3 is localized on the mitochondrial outer membrane and functions as a fatty acid-catalyzing enzyme. Ferroptosis, is a newly identified form of non-apoptotic cell death in DKD characterized by iron-dependent lipid peroxidation. These findings suggest that ACSM3 may contribute to DKD through ferroptosis. Indeed, we found that overexpression of CRP in diabetic mice significantly enhances ferroptosis and declines ACSM3 in kidneys. Interestingly, deletion of Smad3 alleviates ferroptosis and reverses the loss of ACSM3 in diabetic kidneys. Consistently, the ferroptosis induced by CRP is suppressed by SIS3, or the blockade of CRP receptor which are associated with the upregulation of ACSM3 in human kidney epithelial cells (HK-2). Moreover, pharmacological inhibition or overexpression of ACSM3 supresses ferroptosis in high glucose treated HK-2 cells.

Thirdly, we screened chemical compounds from Chinese herbal medicine potentially targeting ACSM3. The high through-output virtual screening selected compounds from the databases of 112,000 compounds. Simultaneously, the analysis of prescriptions from 29 Chinese Medicine Masters in Nephrology revealed 25 Chinese herbs applied for kidney disease. 16 compounds that target ACSM3 and are commonly used for kidney diseases were then selected, and followed by the experimental verification of 8 compounds according to docking score, oral bioavailability and drug like score. The cellular thermal shift assay and surface plasmon resonance assay suggested Rhapontin, an active ingredient from Chinese herb Rumex obtusifolius L., is the top candidate. Furthermore, we demonstrated that Rhapontin attenuates ferroptosis and reverses the loss of ACSM3 in HK-2 cells treated with high glucose/CRP.

Taken together, we discovered novel mechanisms that CRP aggravates DKD through Smad3-NLRP3 mediated inflammation and Smad3/ACSM3 mediated ferroptosis. Importantly, for the first time, our study shows that targeting ACSM3 by Rhapontin attenuates ferroptosis. Thus, the development of new therapies targeting CRP, Smad3, NLRP3 and ACSM3 holds promise for improving outcomes in DKD patients.