Diabetic nephropathy (DN) is a leading cause of chronic kidney disease which is an independent risk factor for cardiovascular morbidity and mortality. Recent evidence indicates the role of inflammation in the development and progression of DN. Mesangial cells (MC) is a major kidney cell type involved in DN. Previously we demonstrated that Orai1 mediated store-operated calcium entry (SOCE) in MCs has a protective role against some detrimental intracellular pathways. Our pilot study in cultured human MCs showed that high glucose (HG)-induced secretion of inflammatory cytokines MCP1 and TNF alpha is attenuated with thapsigargin, an activator of SOCE. Therefore, we hypothesize that activation of SOCE decreases the production of inflammatory cytokines by MCs and alleviates renal inflammation and injury in DN. For aim 1, in cultured human MCs treated with normal glucose (5.6 mM) or HG (25 mM), we will manipulate SOCE function pharmacologically and biologically. The expression of selected inflammatory cytokines, transcription factor NFkB will be assessed using ELISA, qRTPCR, western blot and changes in SOCE by fura2 ratiometry. For aim2, in nondiabetic C57BLKS/J male/female mice, we will use a novel nanoparticle (NP) siRNA delivery system to achieve in vivo MC-specific Orai1 knockdown and study if the mice develop pathological changes similar to DN. Similarly, for this aim we will also use eNOS-/- db/db diabetic mice and knockdown Orai1 at 16 weeks using NP-siRNA delivery system. Addition of this group will ensure if Orai1 knockdown causes early occurrence or worsening of the renal infiltration and damage. Expression of cytokines and infiltration of inflammatory cells as macrophages, neutrophils and T lymphocytes in glomerulus in kidneys will be studied using western blot, flow cytometry, ELISA and immunohistochemistry. Renal function (urinary albumin excretion, serum and urinary creatinine level, blood urea nitrogen using ELISA kits and urine output using metabolic cages) will be evaluated. Mesangial matrix area, glomerular fibrosis and expression of matrix proteins will be assessed in kidney sections and by western blot. This study together with the use of nanoparticles will aid in the development of novel therapy for DN and reduce the subsequent cardiovascular complications.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|