Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (13963 versus 15362 mmHg in the transgenic and control mice, respectively; P,0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments,. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption. Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme to biliverdin, during which both carbon monoxide (CO) gas and free iron are released. Biliverdin is then reduced to bilirubin by the ubiquitous enzyme biliverdin reductase. There are two major isoforms of HO that are responsible for the breakdown of heme, HO-1 and HO-2. HO-2 is the constitutively expressed isoform, whereas HO-1 is inducible by a wide variety of stimuli, including hypoxia, metals, ischemia, and oxidative stress. In the kidney, both isoforms of HO are present, with the highest level of expression of both isoforms in the renal medulla under basal conditions. 1,2 HO enzymes are not only expressed in the renal medulla, but their metabolites, CO and bilirubin, play an important role in the regulation of both renal vascular and tubular function. Renal medullary infusion of the general HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol, results in a significant decrease in renal medullary blood flow. 1 In the renal tubules, increases in perfusion pressure increase CO levels, and inhibition of HO activity results in significant attenuation of pressure natriuresis and development of salt-sensitive hypertension. 3

Our lab has previously demonstrated that moderate (~ 2 fold) increases in plasma unconjugated bilirubin levels are able to attenuate the development of Angiotensin II (Ang II) dependent hypertension. In order to determine the specific role of decreases in superoxide production to the blood pressure lowering affects of moderate hyperbilirubinemia (MHyB), we performed the current study in which the NADPH oxidase inhibitor, apocynin, was given to Ang II infused mice in the presence and absence of moderate hyperbilirubinemia. Apocynin (14 mM) was administered in the drinking water prior to treatment with UGT1A1 antisense morpholino (ASM; 16 μg/kg) which was administered via intravenous injection every third day. Treatments were started before the implantation of Ang II containing minipumps (1μg/kg/min) and continued throughout the 7 day protocol. Ang II infusion increased blood pressure to 145 ± 2 mmHg. Apocynin treatment alone reduced blood pressure to 135 ± 5 while MHyB alone decreased blood pressure to 118 ± 5 in Ang II infused mice. Prior inhibition of NADPH oxidase with apocynin did not result in a further decrease in blood pressure in MHyB mice which averaged 117 ± 3 (n=6/group). In aortic preparations, apocynin treatment decreased Ang II mediated superoxide production from 2433 ± 120 to 1851 ± 126 RLU/min/mg (n=4/group) which was similar to levels observed in MHyB mice alone 1473 ± 132 or in combination with apocynin 1503 ± 115. Our results indicate that MHyB lowers blood pressure via a mechanism that is independent of the inhibition of superoxide production; however, we cannot exclude the possibility that other antioxidant properties of bilirubin may be important to the antihypertensive actions in Ang II dependent hypertension. This work was supported by grants from the National Heart, Lung and Blood Institute, HL088421 and PO1HL‐51971.