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Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses a-, b-, and g-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1b expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1b expression in the macula densa affects sodium excretion and saltsensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1b mRNA and protein were 30-and five-fold higher, respectively, than those of NOS1a in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1a-knockout (NOS1aKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (P,0.01) but not NOS1flox/flox mice fed a high-salt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0mmHg in WT mice (P,0.01) fed a high-salt diet. These results indicate that NOS1b is a primary NOS1 isoformexpressed in themacula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP.

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 (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P0.05). 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.

The main goal of this study was to determine whether kidney-specific induction of heme oxygenase-1 (HO-1) can prevent the development of angiotensin (Ang) II–dependent hypertension. To test this hypothesis, intrarenal medullary interstitial catheters were implanted into the left kidney of uninephrectomized mice. Infusion of cobalt protoporphyrin (CoPP; 250 μg/mL; at 50 μL/h for 48 hours) resulted in significant induction of HO-1 in the renal medulla when examined 2 weeks after the infusion with no induction observed in other organs, such as the heart or liver. Next, we examined the effect of renal-specific induction of HO-1 on the development of Ang II–dependent hypertension. CoPP or vehicle (0.1 mol/L NaOH [pH 8.3]) was infused as indicated above 2 days before implantation of an osmotic minipump, which delivered Ang II or saline vehicle at a rate of 1 μg/kg per minute. Mean arterial pressure was measured in conscious, unrestrained mice for 3 consecutive days starting on day 7 after implantation of the minipumps. Mean arterial pressure averaged 114±5, 122±4, 162±2, and 125±6 mm Hg in vehicle-, intrarenal medullary interstitial CoPP–, Ang II-, and Ang II + intrarenal medullary interstitial CoPP–treated mice, respectively (n=6 or 7). These results demonstrate that kidney-specific induction of HO-1 prevents the development of Ang II–dependent hypertension and that induction of HO-1 in the kidney may be the mechanism by which systemic delivery of CoPP lowers blood pressure in Ang II–dependent hypertension.

The purpose of this study was to test whether the melanocortin-4 receptor (MC4R) is critical in the development of hypertension associated with obesity and its metabolic disorders. MC4R-deficient homozygous (−/−) and heterozygous (+/−) and wild-type (WT) C57BL/6J mice 17 to 19 weeks old (n=5 to 7 per group) were implanted with telemetry devices for monitoring 24-hour mean arterial pressure (MAP) and heart rate (HR). After 3-day stable control measurements on normal-salt diet (NSD; 0.4% NaCl), mice received a high-salt diet (HSD; 4% NaCl) for 7 days, followed by 3-day recovery on NSD. MC4R (−/−) mice were severely obese compared with MC4R (+/−) and WT mice (body weight 48±1.5 versus 31±0.6 and 30±0.5 g respectively). On NSD, MAP was similar in all groups of mice (MC4R (−/−) 110±3 mm Hg; MC4R (+/−) 109±2 mm Hg; WT 114±2 mm Hg), and HR in MC4R (−/−) was lower than in WT (604±5 versus 645±9 bpm; P

Previous studies have indicated that the production of 20-hydroxyecisatatraenoic acid (20-HETE) is similar in the liver of C57/B6 mice and rats, but the renal production of 20-HETE is very low in this strain of mice. The present study examined the effects of induction of the renal production of 20-HETE with fenofibrate (FF) on the development of angiotensin II (Ang II)–dependent hypertension in C57BL/6J mice. The mice were divided into 4 groups and treated with vehicle (control), FF (90 mg/kg per day, IP), Ang II (1000 ng/kg per minute, SC), and Ang II plus FF. Mean arterial blood pressure (MAP) averaged 109±4 and 106±2 mm Hg in control and FF-treated mice (n=7). MAP was significantly increased in the Ang II-treated mice to 144±4 mm Hg (n=7). However, FF treatment prevented the development of Ang II–dependent hypertension, with MAP averaging 115±5 mm Hg in mice treated with both Ang II plus FF (n=7). Renal production of 20-HETE was very low in control (n=7) and Ang II-treated (n=7) mice and was increased by >2-fold in FF-treated (n=7) and Ang II plus FF-treated (n=7) mice. The levels of Cyp4A proteins were markedly increased in the kidneys of mice treated with FF and Ang II plus FF but not in the renal vasculature. These results suggest that upregulation of the production of 20-HETE in renal tubules may contribute to the blood pressure-lowering effects of FF treatment in Ang II–dependent hypertension in C57BL/6J mice.

Preeclampsia is one of the most common complications of pregnancy in humans, and is a leading cause of premature birth and fetal perinatal morbidity and mortality. The hallmark of the disorder is the manifestation of new-onset hypertension-typically after the 20th week of gestation. While the underlying cause of the disorder remains obscure, it is widely held that failure of the maternal vasculature to adequately remodel and supply the developing placenta with adequate blood flow leads to placental insufficiency and resulting ischemia. In response placental derived factors, namely the anti-angiogenic protein sFlt-1 and reactive oxygen species- are released into the maternal circulation resulting in the symptomatic phase of the. We have recently demonstrated that induction of heme oxygenase in a rodent model of placental ischemia attenuates the associated hypertension by blocking both of these pathways, presumably through byproducts carbon monoxide and bilirubin. Here, we have investigated the in vivo effects of exogenous bilirubin administration on a rodent model of placental ischemia-induced hypertension, the reduced uterine perfusion pressure (RUPP) model. RUPP animals exhibited significant increases in MAP when compared to sham animals (109 ± 3 vs 120 ± 4 mmHg, p

To evaluate the role of the renin gene in the development of hypertension in Dahl salt-sensitive rats (SS/Jr/Hsd), we derived a congenic strain of rats homozygous for the salt-resistant renin allele (S/ren rr ) and compared them with a control strain homozygous for the salt-sensitive renin allele (S/ren ss ). Mean arterial pressure was significantly higher in 12-week-old S/ren rr rats fed a high salt (8.0%) diet for 3 weeks than in S/ren ss rats or in SS/Jr/Hsd rats rederived from the foundation colony we used to generate the congenic strain (195±3 [n=49] versus 168±3 [n=17] or 161±3 [n=16] mm Hg). Mean arterial pressure was also higher in S/ren rr rats than in S/ren ss rats raised from birth on either a very low salt (0.1%) diet (119±9 [n=6] versus 100±7 [n=4] mm Hg) or a low salt (0.4%) diet (143±1 [n=22] versus 117±3 [n=10] mm Hg). Plasma renin activity of S/ren rr rats was significantly higher than that of S/ren ss rats fed a very low salt diet (5.7±2.0 versus 1.8±0.3 ng angiotensin I/mL per hour), a low salt diet (4.4±1.0 versus 1.1±0.3), or a high salt diet (1.5±0.2 versus 0.9±0.1). Urinary protein excretion was greater in S/ren rr rats than in S/ren ss rats fed a high salt diet (244.2±48.5 versus 43.6±19.5 mg/24 h), and this was associated with significant reductions in renal blood flow (3.3±0.6 versus 4.6±0.5 mL/min per gram kidney weight) and glomerular filtration rate (0.49±0.11 versus 0.82±0.08 mL/min per gram kidney weight). Captopril (20 mg/kg IV) had no effect on blood pressure in S/ren ss rats fed a low salt diet, but it lowered blood pressure by 20 mm Hg in S/ren rr rats to the same level seen in untreated S/ren ss rats. Chronic administration of captopril (5 mg/100 mL drinking water) reduced blood pressure in S/ren rr rats fed a high salt diet (170±5 mm Hg) to the same level seen in untreated S/ren ss rats, whereas it had no significant effect on blood pressure in S/ren ss rats. These results indicate that transfer of a salt-resistant renin allele to SS/Jr/Hsd rats raises plasma renin activity and augments the severity of hypertension and renal disease.

Recent studies have indicated that a deficiency in the production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the outer medulla of the kidney may contribute to the abnormalities in the renal handling of sodium and the development of hypertension in Dahl salt-sensitive rats. To determine whether a reduction in 20-HETE production in the outer medulla is sufficient to induce hypertension, an inhibitor of the renal metabolism of arachidonic acid by P450 enzymes, 17-octadecenoic acid (17-ODYA), was chronically infused directly into the outer medulla of the left kidney of uninephrectomized Lewis rats fed a high salt diet. Renal medullary interstitial infusion of 17-ODYA (400 pmol/min) reduced the formation of 20-HETE in the outer medulla of the infused kidney by 70% compared with values seen in the right kidney collected when the rat was uninephrectomized, but it had no effect on the production of 20-HETE in the renal cortex. After 5 days, mean arterial pressure rose from 115 +/- 2 to 142 +/- 2 mm Hg (n = 6) in the rats infused with 17-ODYA, while mean arterial pressure was not significantly altered in the rats infused with vehicle alone (116 +/- 1 versus 117 +/- 2 mm Hg, n = 6). These results suggest that inhibition of the renal metabolism of arachidonic acid by P450 enzymes in the outer medulla of the kidney is sufficient to induce the development of hypertension in Lewis rats fed a high salt diet and support the view that P450 metabolites of arachidonic acid play an important role in the regulation of renal function and the long-term control of arterial pressure.

Heme oxygenase-1 (HO-1) induction has been previously demonstrated to reduce body weight and prevent type II diabetes in obese mice. However, the role of increased bilirubin production in this response is unknown. Gilbert’s syndrome is derived from a mutation in the hepatic UGT1A1 gene which results in decreased conjugation and increased levels of plasma biliribin independent of changes in HO-1. In order to determine the role of increased plasma bilirubin on the development of type II diabetes and obesity, we placed mice whose only functioning UGT1A1 gene contained the human Gilbert’s syndrome mutation (UGT1A1*28, n=4) as well as C57BL/6J control mice (n=5) on a high fat (60%) diet for 35 weeks. Plasma bilirubin levels were significantly increased in the UGT*28 mice as compared to controls and averaged 1.65 ± 0.2 vs. 1.18 ± 0.1 mg/dL (P

Preeclampsia is characterized by abnormal placentation that is thought to arise from inadequate cytotrophoblast migration and invasion of the spiral arteries. The resulting reduced perfusion of the utero-placental unit promotes the development of hypertension in preeclampsia. Beta Epithelial Na+ Channel (βENaC) protein, a mediator of cytotrophoblast migration in vitro, is decreased in placentas of preeclamptic patients. We have recently reported that hemeoxygenase-1 (HO-1) and its by-products reduce blood pressure in placental ischemic rats. Since HO and its byproducts can regulate ENaC expression, including βENaC, are important mediators of migration, the goal of this study was to test the hypothesis that HO-1 induction 1) stimulates βENaC expression and migration in cultured cytotrophoblasts and 2) restores βENaC expression in ischemic placentas. Cobalt protoporhyrin (CoPP, 1 - 10 μM, 48 hrs) stimulated HO-1 induction in cultured cytotrophoblasts (BEWO cells), upregulated βENaC expression, and increased spontaneous migration by 39.2 ± 6.0% (n=7, p

We have recently demonstrated that chronic, low level treatment with carbon monoxide releasing molecules (CORMs) prevents the development of obesity in response to a high fat diet. The objective of this study was to test the hypothesis that chronic, low level treatment with CORM can reverse established obesity via a mechanism independent of food intake. This hypothesis was tested by treating 24 week old DIO C57BL6/J mice with a low level of the CO releasing compound, CORM-A1 (5 mg/kg body weight, i.p.; n=4) or the inactive compound iCORM-A1 (n=6) every 48-hours for 30 weeks while maintaining a high fat (60%) diet. Initial body weights (BW) were not different between the groups; however, at 30 weeks of treatment, CORM-A1 significantly reduced body weight as compared to iCORM-A1 (32.7±2.9 g (62% of initial BW) vs. 52.1±13.2 g; pLBM /min vs.70.4±16.6 mL/kg LBM /min; p=0.07). However, weight loss in CORM-A1 treated mice was independent of differences in motor activity (4665±2396 cm vs. 5339±3325 cm, p=0.81) and food intake (3.1±0.2 g/day vs. 3.5±0.1 g/day; p=0.1). These results demonstrate that chronic, low level treatment with a CO donor at levels which do not raise blood carboxyhemoglobin levels reverses established obesity in mice fed a high fat diet through a mechanism independent of food intake. Our results suggest that chronic, low level treatment with CO releasing compounds may be a novel therapy for the reversal of obesity. This work was supported by grants from the NHLBI, PO1HL-51971, HL088421 and 1T32HL105324.

Introduction: Obesity or high fat diet (HF) are risk factors for the development of hypertension. We examined the hypothesis that targeting the vascular system with human heme oxygenase -1 (HO-1) may attenuate both vascular and adipocyte dysfunction in vivo and in vitro. Methods: Lentivirus (Lenti) construct expressing human HO-1 under the control of endothelium specific promoters VE-cadherin (VECAD-HO-1) and VECAD −GFP (control) were used to treat mice, using a bolus injection into the renal artery, and kept on a high fat diet for 26 weeks. Human HO-1 gene expression was sustained for 9 months. For in vitro studies, human EC were cultured with Lenti- VECAD-HO-1 and added to Lenti-VECAD-GFP or control. The conditioned media (CM) from ECs was harvested and 10% CM was added to adipogenic media to measure paracrine effect on adipogenesis in human Mesenchymal Stem Cells (MSCs) derived adipocytes. Signaling molecules were measured by western blot. Results: Lentiviral transduction with VECAD-HO-1 construct attenuated the increase in blood pressure (from 149.9 ± 2.4 to 118 ± 2.0 mmHg, p Conclusion: Targeting HO-1 to ECs resulted in the attenuation of blood pressure and the prevention of body weight gain resulting in increased ANG-1, PDGF, and VGEF levels and the reprogramming of MSCs derived adipocytes to produce healthy and smaller adipocytes with the release of adiponectin.

Abstract Differences in the renal metabolism of arachidonic acid by cytochrome P450 have been reported in the spontaneously hypertensive rat (SHR) and Wistar-Koyto rats, but the contribution of this system to the development of hypertension is unclear. The present study compared renal P450 activity and blood pressure in SHR and Brown-Norway rats (BN) under control conditions and in response to an elevation in sodium intake; genetic linkage analysis was performed in an F 2 population (n=219) derived from these strains. Basal renal P4504A enzyme activity measured by conversion of [ 14 C]arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) was significantly greater in the kidneys of adult SHR (n=7) than of BN (n=8) (82±7 versus 60±5 pmol/min per milligram protein). Renal 20-HETE production fell 45% in SHR and 22% in BN in which salt intake was elevated by drinking of saline instead of water for 2 weeks. Mean arterial pressure averaged 157±3 mm Hg in SHR (n=9) and 100±2 mm Hg in BN fed a normal salt diet, and it rose to 170±7 mm Hg ( P P 2 population. The P4504A genotype did not cosegregate with baseline mean arterial pressure in the F 2 population; however, significant linkage was observed with the change in mean arterial pressure after sodium intake of the rats was elevated. The degree of linkage differed in male and female rats, and the highest LOD score (3.6) was observed in male F 2 rats with a BN grandfather. These findings suggest that the difference in renal P450 activity in SHR and BN does not contribute to the development of hypertension in this F 2 population, but it may play some role in determining the blood pressure response to an elevation in salt intake.

The Dahl salt-sensitive rat (SS/Jr) is a widely used animal model of salt-sensitive hypertension. SS/Jr rats are believed to be highly inbred and uniformly sensitive to the hypertensinogenic effects of sodium chloride, but we have recently observed that SS/Jr rats from Harlan Sprague Dawley, Inc, exhibit considerable variability in their blood pressure response to supplemental dietary salt. To test the possibility that commercially available SS/Jr rats are genetically contaminated and therefore no longer fully inbred, we performed molecular genetic studies and blood pressure measurements in several groups of SS/Jr rats purchased from Harlan Sprague Dawley. We found molecular evidence of heterozygosity and/or atypical allelic variants involving loci on at least five different chromosomes. Many of the rats also failed to exhibit a salt-sensitive blood pressure phenotype. We conclude that SS/Jr rats being sold by the only commercial vendor of Dahl rats in the United States are genetically contaminated and resistant to the hypertensinogenic effects of salt. These findings raise serious questions about the interpretation of research conducted with SS/Jr rats obtained from Harlan Sprague Dawley.

The ultimate goal in the treatment of myocardial infarction (MI) is the delivery of therapeutic agents in a timely fashion that would be able to protect the heart from the deleterious effects of prolonged ischemia or the effects of repeated bouts of ischemia. The ideal agent would “know” when to become active, specifically in the affected area of the heart to limit side effects, and could be turned “off” after the ischemia is resolved. This “smart” approach would allow for treatment to protect the specific region of the heart most at risk for damage during ischemia while at the same time ending the therapy when the ischemic incident is resolved. This type of approach would foster rapid treatment with minimal outside intervention during the critical early phase of MI. In this issue of Hypertension , Tang et al describe a novel gene therapy system for ischemic heart injury using an ischemia-sensing biosensor to activate the expression of a therapeutic gene (heme oxygenase-1 [HO-1]), which acts to limit the extent of ischemic injury.1 Because expression of the HO-1 gene is under the control of the ischemia biosensor, its expression is then subsequently turned off after the tissue is adequately oxygenated when proper blood flow is restored to the heart. This permits gene expression to be controlled at an unprecedented level, driven specifically by the pathological stimulus and lasting only as long as the stimulus persists. The ischemic biosensor developed by Tang et al is composed of an oxygen-sensing transactivator (OST), which is a GAL4 DNA-binding domain fused to the oxygen-dependent degradation domain (ODD) of the …