The impact of obesity has been well described in the progression of chronic kidney disease (CKD) with diabetes, hypertension and even IgA nephropathy, however the basis for how obesity contributes to progressive kidney disease is unclear. Obesity is often associated with elevated insulin levels and the state of insulin resistance at the level of muscle and adipose tissue is accepted. The impact of elevated insulin on the kidney is less well established. Although insulin resistance is a key contributor to podocyte injury, the role of insulin and the insulin receptor in the proximal tubular cell has not been well studied. In our recent published study we demonstrate that lack of the insulin receptor in proximal tubular cells of the mouse kidney was renoprotective upon challenge with a high fat diet. We further demonstrated that insulin down-regulates the cystathione y lyase (CSE) in the mouse kidney with concomitant inhibition of hydrogen sulfide generation and reduction of AMPK activity. The reduced hydrogen sulfide may be a key step as addition of hydrogen sulfide restores AMPK activation despite exposure to insulin in proximal tubular cells. In this new VA Merit proposal, in aim 1 we will examine the role of AMPK as the major pathway that is downstream of insulin in tubular cells by examining mice that are deficient in the insulin receptor and AMPK a1 or AMPK a2 catalytic subunits at the tubular level. With our new method of spatial metabolomics we will determine whether key metabolites are regulated at the tubular level in the kidney specific proximal tubular insulin receptor knockout (KPTIRKO) mouse. Identification of metabolites in the kidney, blood and urine will indicate if a circulating biomarker could indicate engagement of the tubular IR and indicate the status of tubular AMPK activity. In aim 2, we will test the hypothesis that insulin rapidly stimulates proteasomal degradation of CSE via regulation of Nox4 activity.