A new inflammation pathway in diabetic kidney disease - Diabetic kidney disease (DKD) is the most common cause of end stage renal disease. Immunological and inflammatory mechanisms play a significant role in DKD development. Infiltration of circulating inflammatory cells into kidney is mediated by locally produced cytokines. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce inflammatory cytokines in DKD. Therefore, inhibiting MC-derived cytokine production should protect kidney from diabetic injury. Inhibitor of MyoD family A (I-mfa) is a transcription modulator regulating transcription activity of multiple genes. Recent studies revealed that I-mfa inhibits intracellular Ca2+ signaling. It is known that cytosolic Ca2+ signals can inhibit pro-inflammatory cytokine production in many cell types. Recently, we found, for the first time that I-mfa was expressed in glomerular MCs. However, its function in kidney and in MCs is not known. Our pilot studies showed that diabetes and high glucose (HG) increased I- mfa protein contents in MCs in mice and humans. Overexpressing I-mfa in human MCs increased production of inflammatory cytokines. Global deletion of I-mfa decreased macrophage infiltration into glomerulus of diabetic mice. We further found that HG stimulated production of inflammatory cytokines by MCs, which was inhibited by activation of store-operated Ca2+ channel (SOC). In vivo downregulation of SOC in MCs increased macrophage infiltration into glomerulus in mice. Furthermore, both I-mfa and HG significantly inhibited SOC- mediated Ca2+ entry (SOCE) in MCs. Single cell RNA sequencing (scRNA-seq) showed that deletion of I-mfa increased mRNA level of orai1 (the gene encoding SOC protein) in MCs. Moreover, I-mfa had physical interactions with hepatocyte nuclear factor 4α (HNF4α), a transcription factor which has multiple binding sites at the promoter region of orai1. We, thereby hypothesize that increased abundance of I-mfa contributes to inflammatory responses in DKD by stimulating cytokine production in MCs by inhibiting Orai1-mediated SOCE via HNF4α. Three specific aims will be addressed. Aim I will investigate that I-mfa stimulates inflammatory cytokine production/secretion by MCs using both in vitro and in vivo systems. We will manipulate I-mfa levels in cultured human MCs (in vitro) and in MCs of mice with and without DKD (in vivo), and evaluate the resultant effects on production of pro-inflammatory cytokines and glomerular inflammatory responses and renal injury. Aim II will determine that SOCE is an anti-inflammatory mechanism by suppressing inflammatory cytokine production/secretion by MCs using both in vitro and in vivo systems. We will manipulate SOC activity in cultured human MCs (in vitro) and in MCs of mice with and without DKD (in vivo), and assess the resultant responses on production of pro-inflammatory cytokines and renal inflammation and injury. Aim III will delineate the signaling pathway by which I-mfa suppresses SOCE via HNF4α in MCs. We will use a variety of biochemical and molecular tools to identify a novel signaling pathway of I-mfa/HNF4α/Orai1/SOCE in MCs. This study will identify a new mechanistic pathway in regulation of glomerular inflammatory responses in DKD.
