Store-Operated Calcium Entry in Mesangial Cells And Glomerular Inflammatory Responses: Role of Interleukin-6

Abstract

Purpose: Emerging evidence indicates that immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). The early features of DN include accumulation of extracellular matrix (ECM) in the glomerular mesangium. Inflammatory cell infiltration mediated by locally produced cytokines/chemokines contributes to the histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. Interleukin-6 (IL-6) has dual roles acting as an inflammatory or anti-inflammatory cytokine in a cell context manner. We have previously demonstrated that the Orai-1 mediated store-operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine if and how SOCE in MCs regulated IL-6 by MCs and macrophage infiltration into glomerulus. Methods: In cultured human MCs, levels of IL-6 were examined using ELISA in the presence of normal glucose (5 mM D-glucose) with/without an activator (thapsigargin at 1 µM) of SOCE. Immunoblot analysis was used to study the expression of various proteins in the whole cell lysates of MCs. In the human MCs, IL-6 was overexpressed using IL-6 plasmid while Orai 1 was knockdown using siRNA against human Orai1 using transfection reagents. In wild type C57BL6 mice, Orai-1 channel protein in MCs was knocked down using the targeted nanoparticle-siRNA delivery system at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine macrophages infiltration in glomeruli using F4/80 as a marker. Results: In cultured human MCs, activation of SOCE by thapsigargin significantly increased IL-6 expression level which was attenuated by inhibitor of SOCE, GSK7975A and knockdown of Orai 1. IL-6 overexpression reduced the expression of ECM proteins in MCs. In vivo knockdown of Orai1 in MCs induced infiltration of F4/80 stained macrophages into the glomeruli in the mice treated with nanoparticle/Orai1 siRNA for 5 days compared to the control mice. Conclusion: SOCE positively regulates IL-6 expression in MCs which in turn suppresses the ECM proteins. Orai-1 mediated SOCE inhibits the glomerular macrophage infiltration in mice. Thus SOCE in MCs has protective responses against glomerular inflammation and fibrosis.