Date of Award
Doctor of Philosophy
Field of Study
Graduate School of Biomedical Sciences
J. Thomas Cunningham
Robert T. Mallet
Glomerular mesangial cells (MCs) are the major source of extracellular matrix (ECM). One of the early pathological changes in diabetic nephropathy (DN) is accumulation of ECM in glomeruli. Multifunctional store-operated Ca2+ entry (SOCE) regulates MC function. However, whether and how SOCE in MCs contributes to pathophysiology of DN remains unknown. The aim of the study was to investigate association of SOCE in MCs with ECM protein expression and the underlying mechanism using both in vitro and in vivo systems.
Study I was to determine the effect of diabetes on SOCE. In cultured human MCs, we found that prolonged high glucose (HG) treatment (7 days) significantly increased SOCE and membrane currents through store-operated channels (SOC). These responses were abolished by SOC inhibitors. Consistently, prolonged HG treatment also increased the abundance of SOC proteins STIM1 and Orai1. HG also increased STIM1, but not Orai1 mRNA expression. Furthermore, both STIM1 and Orai1 proteins were also increased in the glomeruli/renal cortices of diabetic rats.
Study II determined the influence of SOCE in MCs on ECM protein expression. We found that activation of SOC by thapsigargin reduced the abundance of fibronectin and collagen IV while inhibitors of SOC had opposite effects. Knockdown of Orai1 in human MCs increased fibronectin abundance. The HG induced increase in fibronectin was attenuated by SOCE. Using a nanoparticle siRNA delivery system, specific knockdown of Orai1 in MCs in mice increased glomerular fibronectin and collagen IV protein content and mesangial expansion.
Study III determined the mechanism for inhibition of ECM protein expression by SOCE. We found that activation of SOC attenuated TGFβ1 mediated phosphorylation and translocation of Smad3, a known fibrotic pathway in MCs. However, there was no change in the production or secretion of TGFβ1 by MCs. Orai1 knockdown in MCs in mice increased the activation of Smad3.
Taken together, our results indicate that SOCE in MCs may be increased in the late stage of diabetes, which suppresses ECM protein expression by inhibiting TGFβ1-smad3 pathway.
"Store operated calcium entry in glomerular mesangial cells and diabetic nephropathy" Fort Worth, Tx: University of North Texas Health Science Center;