Project Summary/Abstract Consumption of a western diet (WD) is a major contributor to the epidemic of obesity and associated insulin resistance, hypertension and kidney disease that afflict our Veteran population. Chronic kidney disease is increasing despite widespread use of renin-angiotensin-aldosterone system (RAAS) inhibition and aggressive blood pressure control. Higher circulating aldosterone levels and excess mineralocorticoid receptor (MR) activation, prevalent in obese, insulin resistant individuals, may be driving the increased risk for progression of kidney disease, often in the face of adequate blood pressure control. Tubulointerstitial fibrosis plays a major role in the development and progression of kidney disease. What little is known about MR activation in tubulointerstitial fibrosis has been extrapolated from vascular studies where the development of endothelial cell stiffness can be directly related to reductions in bioavailable nitric oxide (NO). Lowered NO levels activate transglutaminase 2 (TG2) known to promote the extracellular matrix remodeling, synthesis of fibrotic products and macrophage infiltration that lead to fibrosis. My pilot data indicate that consumption of a WD promotes MR activation resulting in lower endothelial nitric oxide synthase (eNOS) activity, cell stiffness, macrophage infiltration, a shift to an inflammatory M1/M2 polarization and vascular fibrosis. Preliminary data link activation of the endothelial cell MR and TG2 activation but the role of endothelial MR activation and its regulation of eNOS and TG2 in tubulointerstitial fibrosis remain to be determined. In this context, the Objectives have been designed to investigate the mechanisms by which over-nutrition promotes tubulointerstitial fibrosis through the endothelial aldosterone/MR system. We posit that the endothelial MR regulates bioavailable NO and TG2, leading to stiffness and immune system activation. We will use a high fat/sugar/salt diet (WD) known to activate the aldosterone/MR system and an endothelial-cell specific MR knockout mouse (ECMR-/-) model to more specifically implicate the endothelial MR in tubulointerstitial fibrosis. Fibrosis will be determined by light and electron microscopy, expression of fibrotic markers and magnetization transfer MRI. To address Objective 1, I will measure endothelial cell stiffness using atomic force microscopy, vascular reactivity using intravital microscopy, and resistance using ultrasound imaging. To address Objective 2, I will focus on monocyte chemotaxis and Mφ polarization through FACS and flow cytometry analysis of immune markers and M2 polarization along with other measures of inflammation, such as cytokine expression. Objective 3 has been designed to investigate the importance of MR-, NO-, and TG2-dependent actions on kidney fibrosis. I will employ a NO-donor and/or a TG2 inhibitor and measure oxidative, inflammatory, extracellular cross-linking enzymes and fibrotic pathways to gauge molecular ...