The proposed studies in this LBRN proposal will investigate the renal functional significance of the heme-heme oxygenase (HO)-carbon monoxide (CO) system during recurrent insulin-induced hypoglycemia (RIIH). HO catalyzes the conversion of heme to iron, biliverdin and CO. Biliverdin is rapidly reduced to bilirubin, which is a major antioxidant in mammalian systems. CO inhibits vascular smooth muscle cell growth and apoptosis, relaxes vascular smooth muscle, and promotes diuresis. Two major HO isozymes have been identified: HO-1 is an inducible heat shock protein (HSP32), while HO-2 is constitutive. AngII levels have been reported to be elevated in type I diabetic patients promoting hyperfiltration and hypertension in these patients with eventual renal injury. Increased AngII levels are associated with oxidative stress, which is a well-established stimulus for HO-1 induction. Recent reports have indicated that HO-1 expression is increased during AngII induced hypertension, and potentially plays a role in renal protection. Therefore, AngII induced hypertension in type I diabetic patients may increase HO-1 levels to promote CO production, and this increase in CO may help to buffer AngII and superoxide mediated hypertension. Hyperglycemia has been reported to alter circulating AngII levels in diabetes potentially promoting renal disease. However, the source of glycemic induction of AngII remains to be elucidated. Type I or insulin- dependent diabetics are known to have fluctuating blood glucose levels in that hyperglycemia is treated with insulin promoting various bouts of hypoglycemia. Thus, determining the mechanism of AngII induction, and the underlining mediators of AngII induced hypertension could provide a novel treatment to protect against the onset of renal failure. The proposed studies will test the hypothesis that chronic insulin injections and hypoglycemia promote hypertension and renal damage via an increase in renal angiotensin II. In the proposed studies indices of renal injury (alpha-Actin expression in mesangial cells, afferent arteriolar wall thickness, cortical cell proliferation, and macrophage infiltration) will be measured along with renal (RBF, GFR, RVR, sodium excretion, potassium excretion, urine volume) and systemic (TPR, MAP) function. In addition, HO protein expression, in the kidneys, of vehicle treated hypoglycemic normotensive and hypertensive rats will be measured. A potential mechanism for HO induction by AngII will be evaluated through EPR measurements of oxidative stress, NOS isoforms, nitrite/nitrate levels and inhibiting NADPH oxidase by apocynin. A precursor of heme, delta-aminolevulinic acid (DALA) will be used to increase HO-mediated formation of CO. The HO inhibitors, zinc protoporphyrin-IX (ZnPP) in vivo and chromium mesoporphyrin (CrMP) in vitro will be used to decrease CO production. In preliminary studies Sprague Dawley rats treated with chronic low dose insulin injections sustained blood glucose levels of 30mg/dl, after 2 days of treatment. A significant increase in circulating angiotensin II, heme oxygenase levels and carboxyhemoglobin levels was measured. Mean arterial pressure was 10-15mmHg higher in insulin treated rats as compared to saline injected controls. Thus, our preliminary data suggest that chronic insulin treatment and hypoglycemia promotes an increase in angiotensin II, which activates the HO-CO system, through an elevation in blood pressure.