The macula densa prorenin receptor is essential in renin release and blood pressure control

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Abstract P200: Role of Macula Densa Neuronal Nitric Oxide Synthase in Control of Renin Release and Blood Pressure Recovery Following Hemorrhagic Shock

Renin is a rate limiting factor for generation of angiotensin II, which is essential for blood pressure regulation. The role of macula densa nitric oxide (NO) in renin release is not conclusive. The goal of this study was to elucidate the role of macula densa neuronal NO synthase (NOS1) in control of renin release in response to sodium challenges and hemorrhagic shock, as well as in blood pressure recovery after hemorrhagic shock. C57BL/6L mice and macula densa specific NOS1 knockout (MD-NOS1KO) mice were given 10 days of low (0.1% NaCl), normal (0.4% NaCl) and high (1.4% NaCl) sodium diet. Hemorrhagic shock was induced by withdrawing 0.4 ml whole blood from the right retro-orbital sinus. Mean arterial pressure (MAP) in conscious mice was monitored by radio-telemetry system. Plasma renin concentration (PRC) was determined by radioimmunoassay. Low sodium diet stimulated PCR by 29% (from 685 ± 32 to 883 ± 112 ng/ml/hr) in WT mice and by 16% (from 652 ± 24 to 756 ± 124 ng/ml/hr) in the MD-NOS1KO mice (n=5/group, p<0.01 vs WT). PCR was not significantly different between the WT and MD-NOS1KO mice fed a normal or high salt diet. As shown in Fig1A, following removal of 0.4ml of blood, MAP dropped to about 40mmHg in the WT mice and 35mmHg in the MD-NOS1KO mice. MAP recovered faster in WT mice than the MD-NOS1KO mice. In Fig1B, PRC increased over 200% of the basal value in WT mice, but only increased about 26% in the MD-NOS1KO mice (n=4/group, p<0.01 vs WT). We conclude that NOS1 in the macula densa facilitates renin release. Lack of macula densa NO generation impairs blood pressure recovery, which may be mediated by limiting renin release during hemorrhagic shock.

Abstract 123: A Fructose-enriched Diet Stimulates Superoxide Production in Juxtaglomerular Cells and Prevents High-salt Induced Inhibition of Plasma Renin Activity (PRA)

A fructose-enriched diet has been associated with hypertension. Western diets are rich in fructose and salt. We found that a fructose enriched diet plus high-salt induced salt sensitive hypertension. Plasma renin activity (PRA) is essential for blood pressure (BP) control. A high-salt diet decreases PRA by inhibiting renin release from juxtaglomerular (JG) cells. However it is not known if dietary fructose might impair the inhibition of renin release by high salt to promote salt sensitivity. Salt sensitive rats have enhanced levels of superoxide in the renal cortex, and we found that superoxide stimulates renin release from JG cells. Thus, we hypothesized that a fructose-enriched diet (20%) promotes salt sensitive hypertension in part by preventing high salt-induced inhibition of renin release from JG cells by enhancing superoxide production. To test this, Sprague Dawley rats were given 20% fructose in their drinking water, with normal or high salt diet (4% NaCl) for up to 4 weeks. Feeding normal rats a fructose+High-salt diet increased systolic BP by 30 mmHg whereas fructose or high-salt alone did not change BP (High-salt = 125±4, Fruct = 131±4, Fruct+High-salt = 147±7; n=6, p <0.05). A high-salt diet alone for 4 weeks decreased PRA by 85%. However, in rats fed fructose+High-salt did not decrease PRA (in ng AII/ml/hr: Ctrl = 2.51±0.72, High-salt = 0.43±0.07, Fruct = 2.71±0.9, Fruct+High-salt = 1.89±0.43; n=10, p <0.05). We next examined the role of the fructose or fructose+High-salt diet on NADPH oxidase expression in isolated JG cells. NOX4 expression was enhanced in JG cells from rats fed fructose+High-salt diet (n=4; p <0.05). Next, we measured superoxide production with Dihydroethidium and found it was higher in JG cells from rats fed fructose+high-salt diet compared to high-salt alone (% of Ctrl: High-salt = 90.7±37; fruct+High-salt = 289±85; n=4; p <0.05). We conclude that a 20% fructose-diet promotes salt sensitivity of BP. The mechanism may involve enhanced NOX4 expression and elevated superoxide levels within JG cells stimulating renin release. 15 million Americans consume 20% of their calories from fructose, and most, 4-8 times the recommended salt intake. Decreasing fructose intake could have a beneficial BP effect in hypertensive patients.

Abstract P084: Oxidative Stress Preconditioning Is Required for Leptin to Stimulate Renin Release From Mouse Juxtaglomerular Cells

Leptin is enhanced in animal models of obesity. The role of leptin in hypertension is unclear with some studies showing antihypertensive actions and others showing a pro-hypertensive role. In normotensive, non-obese, animals, infusion of leptin induces natriuresis. However, in animal models of enhanced oxidative stress, obesity, or activated renin-angiotensin system, infusion of leptin tends to increase blood pressure. Renin is a critical mediator of angiotensin-II formation, and thus blood pressure control. However, the direct effect of leptin on the release of renin from juxtaglomerular (JG) cells has not been studied. We hypothesize that under normal conditions leptin does not affect renin release whereas during high oxidative stress leptin stimulates renin release. We isolated primary cultures of mouse juxtaglomerular (JG) cells. After treatment with different agonists, renin released to the supernatant was measured by radioimmunoassay. We first tested the direct effect of murine leptin on renin release. We found that 1 hr treatment with leptin (0.1-1 uM) decreased basal renin by 20 ± 5% (p<0.05; n=8). Treatment of JG cells for longer periods (4 and 24 hrs) did not affect renin release (n=6) or total renin expression (n=4). We then tested whether oxidative stress modified the effect of leptin on renin release. For this we pre-incubated JG cells with 10 uM hydrogen peroxide (H2O2) for 1 hr. After this, the medium was removed and H2O2 was completely washed out from JG cells followed by treatment with vehicle (cont) or 10 uM leptin. We found that, in cells pre-treated with H2O2, leptin increased renin release by 49.2 ±16 %(P<0.05 vs vehicle). By Western blot, we detected the expression of the leptin receptor in lysates from JG cells. We concluded that under normal conditions leptin inhibits renin release from JG cells. However, after exposure to H2O2, leptin stimulates renin release. Our data suggest the hypothesis that oxidative stress reverses the inhibitory effect of leptin on renin release and supports a pro-hypertensive role for leptin during chronic inflammatory conditions that induces oxidative stress.