Renal Nitric Oxide Synthase Research Articles

Acute depletion of heme by succinylacetone alters vascular responses but does not induce hypertension

Heme plays a critical role in blood pressure regulation because it is required by a number of enzymes that synthesize vascular modulators, including nitric oxide (NO), carbon monoxide (CO), guanosine 3',5'-cyclic monophosphate (cGMP), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin. The goal of this study was to examine the vascular effects of a short-term depletion of heme achieved through administration of the heme-synthesis inhibitor succinylacetone (SA), an irreversible inhibitor of aminolevulinic acid dehydratase (ALAD). Rats were depleted of heme by using a 4-day treatment with SA. This treatment impacted hemoenzyme function, decreasing renal nitric oxide synthase (NOS) activity (as indicated by decreased in vitro NOS activity), and increasing kidney microsomal heme oxygenase (HO) activity by 27%. SA treatment also resulted in enhanced reduction in blood pressure after infusions of exogenous NO donor MAHMA NONOate (at high dose) and acetylcholine (at low doses). Nevertheless, this SA treatment was insufficient to produce an overt elevation of basal arterial pressure. This latter lack of effect may be the result of multiple compensatory mechanisms for the regulation of blood pressure.

Ozone Oxidative Preconditioning Protects the Rat Kidney from Reperfusion Injury: The Role of Nitric Oxide

Ischemia/reperfusion (I/R) injury, which is commonly seen in the field of renal surgery or transplantation, is a major cause of acute renal failure. Previous studies have shown that ozone oxidative preconditioning (OzoneOP) attenuated renal I/R injury. The objective of this study was to examine the hypothesis that protective effects of OzoneOP in renal I/R injury were associated with endogenous NO. In a right-nephrectomized rat mode, anesthetized rats underwent 45 min of renal ischemia. OzoneOP (1 mg/kg) was administered before I/R injury. Rats were killed at 24, 48, and 72 h after I/R injury and blood samples and renal tissues were obtained. OzoneOP prevented the renal dysfunction induced by I/R and increased nitric oxide (NO) release and renal NO synthase (endothelial, eNOS, and inducible, iNOS) expression. In contrast, enhancement of endothelin-1 in the kidney after the reperfusion was markedly suppressed by OzoneOP. Our findings indicated that the protective effect of OzoneOP was closely related to the NO production following the increase in eNOS and iNOS expression. Ozone treatment may have important clinical implications, particularly in view of the minimizing renal damage before transplantation.

SEX DIFFERENCES IN RESPONSE TO CYCLOSPORINE IMMUNOSUPPRESSION IN EXPERIMENTAL KIDNEY TRANSPLANTATION

1. Female donors and recipients have increased risk of acute rejection and subsequent chronic allograft nephropathy (CAN), especially when cyclosporine A (CsA) is used. Decreased renal nitric oxide (NO) production is associated with chronic kidney disease. In the present study, we investigated the impact of gender, CsA dose and renal NO synthase (NOS) on CAN. 2. Kidneys from male and female F344 rats were transplanted into same-sex Lewis allograft or F344 isograft recipients and recipient rats were treated with 1.5 or 3 mg/kg per day CsA for 10 days. Grafts were removed at 22 weeks post-transplantation. Normal two-kidney F344 rats were investigated as age-matched controls. 3. Low-dose CsA was associated with accelerated CAN in female rats compared with male rats; however, with high-dose CsA, allograft females had similar pathology/function to allograft males. Isograft females (similar to isograft males) had no graft failure and only slightly, albeit significantly, greater injury than age-matched controls. Isograft females had higher renal cortical neuronal (n) NOS but lower medullary endothelial (e) NOS than isograft males. There was no difference in renal eNOS and nNOS between allograft groups. 4. In conclusion, 1.5 mg/kg per day CsA is not sufficient to prevent early graft loss in females. When the dose of CsA is doubled, allograft females and males have similar post-transplant survival. Renal NOS expression was unremarkable in any transplant group.

Sex Differences in Renal Nitric Oxide Synthase, NAD(P)H Oxidase, and Blood Pressure in Obese Zucker Rats

By increasing renal oxidative stress, obesity may alter the protective effect of female sex on blood pressure (BP). The aim of this study was to determine whether female rats had altered expression and activity of renal nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase and nitric oxide synthase (NOS), enzymes important in superoxide and nitric oxide generation, respectively, and whether this relationship was altered in obesity. Male and female, lean and obese Zucker rats were fed progressively higher levels of NaCl over 54 days while BP was measured by radiotelemetry. Kidneys were harvested after euthanization. A total of 32 (n=8/body type/sex) Zucker rats were examined. On a high-salt diet (4% NaC1), male and obese rats had significantly higher mean arterial blood pressure relative to female and lean rats (mm Hg: lean male=108, lean female=99, obese male=129, and obese female=123) and reduced renal cortical NOS activity (determined by 2-way analysis of variance; P<0.05 for sex and body type). Immunoblotting revealed that cortical endothelial NOS protein abundance was reduced in obese but not in male rats. Surprisingly, lean female rats had the highest outer medullary protein levels of several NADPH oxidase subunits, including gp91phox, p47phox, and p67phox (% of lean male: 207, 196, and 151, respectively; P<0.01 for all). However, renal NADPH activity was not increased in lean females, but was significantly increased in obese rats of both sexes (P<0.05). High-NaCl diet increased BP modestly in obese females, but not at all in lean females, suggesting some loss of protection with obesity in female rats. Reduced cortical NOS activity (both in male and obese rats) and/or increased NADPH oxidase activity (obese rats) may have contributed to increased salt sensitivity of BP.

Poster 25: Effects of Exercise Training on Blood Pressure and Renal Nitric Oxide Synthases in Spontaneously Hypertensive Rats

Poster 25: Effects of Exercise Training on Blood Pressure and Renal Nitric Oxide Synthases in Spontaneously Hypertensive Rats

Gender and the Renal Nitric Oxide Synthase System in Healthy Humans

It is widely known that men with kidney disease progress to ESRD at a much greater rate than do women. The mechanism for these gender differences is not clear, but reduced availability of nitric oxide is thought to contribute to the age-related decline in renal plasma flow observed in both healthy men and women. Animal models suggest that the renal vasculature of men may be significantly more dependent on nitric oxide than that of women. Renal plasma flow response to the nonspecific nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME) was measured by para-aminohippurate clearance technique in 21 healthy, normotensive (8 male, 13 female) individuals in balance on a high-salt diet. There were striking differences between the genders in the renal hemodynamic response to L-NAME according to age, a difference that remained even after adjustment for other significant covariates. In men, the fall in renal plasma flow induced by L-NAME increased remarkably with increasing age. In women, there was no influence of age on the renovascular response to L-NAME. Neither age nor gender predicted the mean arterial pressure response to L-NAME. The renal vasculature of men becomes more dependent on nitric oxide with age compared with that of women, suggesting that any renal disease that interferes with nitric oxide production may, over time, cause existent kidney damage to progress more quickly in men relative to women.

Attenuation of reperfusion injury by renal ischemic postconditioning: The role of NO

Ischemic postconditioning (Postcond) is defined as rapid intermittent interruptions of blood flow in the early phase of reperfusion and mechanically alters the hydrodynamics of reperfusion. Although Postcond has been demonstrated to attenuate ischemia/reperfusion (I/R) injury in the heart and brain, its roles to renal I/R injury remain to be defined. In the present study, we examined the role of Postcond in I/R injury in a right-nephrectomized rat model. Postcond prevents the renal dysfunction and cell apoptosis induced by I/R and increases nitric oxide (NO) release and renal NO synthase (endothelial, eNOS and inducible, iNOS) expression. In contrast, enhancement of endothelin-1 (ET-1) in the kidney after the reperfusion was markedly suppressed by Postcond. These findings indicate that Postcond can inhibit renal I/R injury. The protective effect of Postcond is closely related to the NO production following the increase in eNOS and iNOS expression and the suppressive effect of ET-1 overproduction.

A Moderately High Fat Diet Promotes Salt-Sensitive Hypertension in Obese Zucker Rats by Impairing Nitric Oxide Production

The objective of this research was to examine the contribution of a moderately high fat (MHF) diet to the development of salt-sensitive hypertension in obese Zucker rats. Lean and obese Zucker rats were fed either a MHF diet or a diet of standard rat chow (control diet) for 10 weeks. From week 4 through week 10, the drinking water was supplemented with 1% NaCl. Blood pressure was measured weekly, and urinary excretion of nitric oxide metabolites (NOx) was determined at weeks 4 and 10. At week 10, renal nitric oxide synthase (NOS) activity was assessed in kidney homogenates. Blood pressures of obese, but not lean, rats on the MHF fat diet were significantly increased by salt-supplementation, whereas blood pressures of rats on the control diet were not appreciably affected. NOx excretion was increased in response to salt-supplementation in rats on the control diet, with the effect being particularly dramatic in obese rats. After salt-supplementation, NOx excretion by rats on the MHF diet was lower than rats on the control diet. In obese rats on the MHF diet, this decrease in NO production was accompanied by a reduction in renal NOS activity. These results indicate that obese rats are more inclined than lean rats to develop diet-induced hypertension in response to a moderately high fat, salt-supplemented diet. Furthermore, they suggest that MHF diet-induced defects in NO production may promote the salt-sensitivity of blood pressure in obese Zucker rats, which appear to require more NO to maintain blood pressure during a salt challenge.

Alkaline phosphatase treatment improves renal function in patients with severe sepsis or septic shock

We previously demonstrated that upregulation of renal inducible nitric oxide synthase (iNOS) during systemic inflammation is associated with proximal tubule injury. In several in vitro and animal studies alkaline phosphatase (AP) was found to be effective in attenuating the inflammatory response by dephosphorylating LPS and may prevent organ damage. The objective of this study was to investigate the effect of AP on renal iNOS expression and kidney damage in patients with severe sepsis or septic shock. Fifteen patients (nine male/six female, age 55 ± 5 years) with Gram-negative bacterial infection, two out of four SIRS criteria (<24 hours) and acute onset of end-organ dysfunction (<12 hours) were included in a randomized, double-blind, placebo-controlled phase IIa study (2:1 ratio). An intravenous bolus injection of 67.5 U/kg bovine intestinal AP was followed by a maintenance dose of 177.5 U/kg for 24 hours. Arterial blood and urine were collected at different time points and analyzed for stable metabolites of NO. iNOS mRNA was determined by quantitative real-time RT-PCR using RNA isolated from renal cells in urine. The urinary excretion of the cytosolic glutathione S-transferase-A1 (GSTA1-1), a marker for proximal tubule damage, was measured using an ELISA. Data are depicted as the median (25–75% range). NO metabolites in blood were not significantly different between AP-treated (n = 10) and placebo-treated (n = 5) patients. However, the urinary excretion of NO metabolites decreased by 80% (75–85) from 227 (166–531) at baseline to 41 (28–84) μmol/10 mmol creatinine (P < 0.05) after 24 hours of AP administration. After placebo treatment, the amount of urinary NO metabolites increased by 70% (45–570) (from 81 (64–419) to 628 (65–1,479) μmol/10 mmol creatinine, P < 0.05). Baseline expression levels of iNOS in renal cells were 42-fold induced at baseline (vs healthy subjects), and AP administration reduced this induction by 80 ± 5% (Figure ​(Figure1).1). Creatinine clearance improved by 45% (30–180) in patients treated with AP and declined by 25% (15–35) in placebo-treated patients. During the first 24 hours the amount of GSTA1-1 in urine of AP-treated patients decreased by 70% (50–80), compared with an increase of 200% (45–525) in placebo-treated patients, which correlated with urinary NO metabolites, indicating NO-induced proximal tubular damage. Figure 1 In conclusion, in septic patients, infusion of AP results in an attenuated upregulation of iNOS and, subsequent, reduced NO production in the kidney, associated with an improvement in renal function.

Discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mRen2.Lewis rats

Estrogen depletion markedly exacerbates hypertension in female congenic mRen2. Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2. Lewis rats. OVX-mRen2. Lewis exhibited an increase in systolic blood pressure (SBP) of 171 +/- 5 vs. 141 +/- 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2. Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 +/- 3 vs. 151 +/- 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 +/- 4 vs. 151 +/- 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2. Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS.

Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth rat

Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) rat. We previously showed that the Wistar-Furth (WF) rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD rats, whereas low-dose NOSI caused progression of CKD in WF rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N(G)-nitro-l-arginine methyl ester (l-NAME) (150 and 300 mg/l for 6-10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic l-NAME. SD given 150 mg/l l-NAME for 6 wk developed proteinuria and renal injury, whereas WF rats receiving 150 mg/l l-NAME for 6-10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD rat. There was little impact on renal nitric oxide synthase expression with l-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF rats, whereas WF rats showed a much smaller increment in glomerular blood pressure compared with SD rats. In conclusion, WF rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD rat. This protection may be associated with protection from glomerular hypertension.

Chronic administration of adrenomedullin attenuates the hypertension and increases renal nitric oxide synthase in Dahl salt-sensitive rats

Adrenomedullin reduces systemic blood pressure and increases urinary sodium excretion partly through the release of nitric oxide. We hypothesized that chronic adrenomedullin infusion ameliorates salt-sensitive hypertension and increases the expression of renal nitric oxide synthase (NOS) in Dahl salt-sensitive (DS) rats, because the reduced renal NOS expression promotes salt sensitivity. DS rats and Dahl salt-resistant (DR) rats were fed a high sodium diet (8.0% NaCl) for 3 weeks. The high sodium diet resulted in an increase in blood pressure and a reduction of urinary sodium excretion in association with increased renal adrenomedullin concentrations and decreased expression of renal neuronal NOS (nNOS) and renal medullary endothelial NOS (eNOS) in DS rats compared with DR rats. Chronic adrenomedullin infusion partly inhibited the increase of blood pressure and proteinuria in association with a restoration of renal nNOS and medullary eNOS expression in DS rats under the high sodium diet. The immunohistochemical analysis revealed that the restored renal nNOS expression induced by chronic adrenomedullin infusion may reflect the restoration of nNOS expression in the macula densa and inner medullary collecting duct. These results suggest that adrenomedullin infusion has beneficial effects on this hypertension probably in part through restored renal NOS expression in DS rats.

The expression and activity of renal nitric oxide synthase and circulating nitric oxide in polycystic kidney disease rats

Nitric oxide (NO) influences tubular fluid and electrolyte transport, and hence possibly also fluid accumulation in renal cysts. The expression and activity of intrarenal constitutive NO synthase (cNOS) [neuronal NOS, nNOS and endothelial NOS, eNOS] and inducible NOS (iNOS) and plasma nitrite/nitrate (PNOx) concentration were assessed in homozygous Han:SPRD polycystic kidney disease (PKD) rats (cy/cy), heterozygous Han:SPRD PKD rats (cy/+), homozygous normal Han:SPRD littermates (+/+) and Sprague Dawley rats (sd). The results showed: 1) nNOS expression was decreased in proximal tubules and thick ascending limbs of the loop of Henle in cy/cy and cy/+ rats compared to +/+ and sd rats (p<0.05). nNOS was weakly expressed in the epithelium of small cysts and unexpressed in epithelium of large cysts. 2) iNOS expression was increased in proximal tubular epithelial cells in cy/+ rats compared to +/+ rats and sd rats (p<0.01). iNOS expression in cyst epithelium was decreased in cy/+ rats (p<0.05) and absent in cy/cy rats. 3) eNOS expression was similar in the endothelium of intrarenal arteries in all groups. 4) The activity of renal cNOS was decreased in cy/cy and cy/+ rats; the activity of iNOS was decreased only in cy/cy rats, with no significant difference among the other three groups. 5) PNOx concentration was higher in cy/cy rats than in the other three groups, and correlated positively with plasma creatinine and urea. In conclusion, NOS expression and activity decreased as cysts developed, suggesting that NO downregulation is involved in the pathogenesis of PKD.

Protection against puromycin aminonucleoside-induced chronic renal disease in the Wistar-Furth rat

The Wistar-Furth (WF) rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) rat, and protection was associated with preserved renal nitric oxide (NO) production. This study examined CRD induced with repeated administration of puromycin aminonucleoside (PAN). SD PAN developed nephrotic range proteinuria (>1 g/24 h), and at 15 wk severe renal injury developed and the glomerular filtration rate (GFR) was reduced to approximately 10% of sham. Total NO production, renal NO synthase (NOS) activity, and renal neuronal (n) and medullary endothelial (e)NOS abundance were reduced in the SD PAN. WF PAN exhibited less severe initial proteinuria (>400 mg/24 h), which abated within weeks, whereas GFR was normal and injury was minimal at 15 wk. Total NO production and renal NOS activity and abundance were significantly elevated compared with SD PAN. NOS mRNA (nNOS, eNOS, and inducible NOS) was not altered in WF, whereas SD showed significant increases in NOS gene expression with PAN. In conclusion, WF showed resistance to a second model of CRD with maintained renal NOS activity compared with SD.

Renal neuronal nitric oxide synthase protein expression as a marker of renal injury

Animal studies suggest that nitric oxide deficiency occurs in the remnant after 5/6 removal of renal mass. The present studies investigated the time course in relation to progression of renal disease, as well as the impact on individual renal nitric oxide synthase (NOS) isoforms. Rats were studied from 2 to 11 weeks after 5/6 ablation/infarction (A/I) of renal mass, with acceleration of progression by high protein and salt intake, in some groups. Measurements were made before sacrifice of 24-hour protein and creatinine excretion, blood was taken for creatinine and blood urea nitrogen (BUN) determination and the kidneys were investigated histologically for structural damage, abundance of endothelial NOS (eNOS) and neuronal NOS (nNOS), and in some groups for in vitro NOS activity. A time-dependent fall in glomerular filtration rate (GFR) and rise in proteinuria and glomerular sclerosis developed after 5/6 A/I. The nNOS abundance in cortex and medulla was decreased relative to shams, in all but the mildest injury and there was a strong, steep correlation between level of glomerular sclerosis and the degree of reduction in renal nNOS. Where measured, cortical NOS activity correlated with the nNOS abundance. In contrast, the eNOS abundance was either increased or unchanged in rats post A/I. Renal nNOS abundance was reduced in the 5/6 A/I model of renal disease when plasma creatinine> approximately 1 mg/dL and when> approximately 20% of remaining glomeruli were sclerosed.

Protection of wistar furth rats from chronic renal disease is associated with maintained renal nitric oxide synthase.

Wistar Furth (WF) rats do not develop renal injury after severe reduction of renal mass. Because clinical and animal studies suggested that nitric oxide (NO) deficiency occurs and may contribute to chronic renal disease (CRD), the status of the NO system in WF versus Sprague Dawley (SD) rats was examined with the 5/6 renal ablation/infarction (A/I) model of CRD. Eleven weeks after A/I, SD rats developed proteinuria, severe kidney damage, decreased renal function, and marked decreases in total and renal NO synthase (NOS), specifically neuronal NOS. In contrast, WF rats exhibited elevated baseline and maintained post-A/I total NO production, with no decrease in renal cortex NOS activity despite a decrease in remnant neuronal NOS abundance. When low-dose chronic Nomega-nitro-L-arginine methyl ester treatment was added for WF A/I-treated rats, rapid progression of CRD was observed. In conclusion, elevated NO production in WF rats was associated with protection from the progression of CRD after renal mass reduction. The protection might be attributable to greater total and renal NO-generating capacity and increased nephron number, compared with SD rats. NOS inhibition rendered WF rats susceptible to progression, suggesting a possible critical threshold for NO production, below which renal injury occurs.

Sexual dimorphism in the aging kidney: Effects on injury and nitric oxide system

Male gender confers enhanced susceptibility to development of age-dependent kidney damage. In other models of progressive renal disease, development of injury is linked to declines in renal nitric oxide synthase (NOS) capacity. We investigated the in vitro characteristics of the renal NOS system in young (3 to 5 months), middle-aged (11 to 13 months) and old (18 to 22 months) male and female Sprague-Dawley rats. NOS activity (pmol [3H]-arginine converted to [3H]-citrulline/mg protein/minute) is reduced in the soluble fraction of renal cortex from old versus young males but not females. In contrast, NOS activity in the soluble fraction of cerebellum is not altered by age or gender. The abundance of endothelial NOS (eNOS) and neuronal (nNOS) is reduced in renal cortex of old versus young males but is unchanged in female cortex. In renal medulla, eNOS protein is reduced with age in both males and females. We found no difference in abundance of either eNOS or nNOS protein in the cortex of young male and female rats. The incidence and severity of glomerular damage increases markedly with age in the male and only slightly in the female. These findings indicate that a relative reduction occurs in renal NOS in the male kidney with advancing age, whereas NOS protein and activity is maintained during aging in females. This, together with the marked age-dependent kidney damage seen in the male, suggests that the renal NO deficiency in the aging male rat may contribute to the age-dependent kidney damage.

A high-fat, refined-carbohydrate diet affects renal NO synthase protein expression and salt sensitivity.

Chronic consumption of a high-fat, refined-carbohydrate (HFS) diet causes hypertension. In an earlier study, we found increased nitric oxide (NO) inactivation by reactive oxygen species (ROS) and functional NO deficiency in this model. Given the critical role of NO in renal sodium handling, we hypothesized that diet-induced hypertension may be associated with salt sensitivity. Female Fischer rats were fed an HFS or a standard low-fat, complex-carbohydrate (LFCC) rat chow diet starting at 2 mo of age for 2 yr. Arterial blood pressure, renal neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), and inducible NO synthase (iNOS) protein and nitrotyrosine abundance (a marker of NO inactivation by ROS), and urinary NO metabolite excretion were measured. To assess salt sensitivity, the blood pressure response to a high-salt (4%) diet for 1 wk was determined. After 2 yr, renal nNOS and urinary NO metabolite excretion were significantly depressed, whereas arterial pressure, eNOS, iNOS, and nitrotyrosine were elevated in the HFS group but remained virtually unchanged in the LFCC group. Consumption of the high-salt diet resulted in a significant rise in arterial pressure in the HFS, but not in the LFCC, group. Thus chronic consumption of an HFS diet results in hypertension and salt sensitivity, which may be in part due to a combination of ROS-mediated NO inactivation and depressed renal nNOS protein expression.

Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene.

Adrenomedullin (AM) is a potent depressor peptide whose vascular action is suggested to involve nitric oxide (NO) release. To explore the role of endogenous AM in vascular and renal function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists AM(22-52) and CGRP(8-37) on the renal perfusion pressure (RPP) of kidneys isolated from AM transgenic (TG)/heterozygote knockout (KO) mice and wild-type littermates (WT). Furthermore, we evaluated the renal function and histology 24 hours after bilateral renal artery clamp for 45 minutes in TG, KO, and WT mice. Baseline RPP was significantly lower in TG than in KO and WT mice (KO 93.4+/-4.6, WT 85.8+/-4.2, TG 72.4+/-2.4 mm Hg [mean+/-SE], P<0.01). ACh and AM caused a dose-related reduction in RPP, but the degree of vasodilatation was smaller in TG than that in KO and WT (%DeltaRPP 10(-7) mol/L ACh: KO -48.1+/-3.9%, WT -57.5+/-5.6%, TG -22.8+/-4.8%, P<0.01), whereas N(G)-nitro-L-arginine methyl ester (L-NAME) caused greater vasoconstriction in TG (%DeltaRPP 10(-4) mol/L: KO 33.1+/-3.3%, WT 55.5+/-7.2%, TG 152.6+/-21.2%, P<0.01). Both AM antagonists increased RPP in TG to a greater extent compared with KO and WT mice (%DeltaRPP 10(-6) mol/L CGRP(8-37): KO 12.8+/-2.6%, WT 19.4+/-3.6%, TG 41.8+/-8.7%, P<0.01). In mice with ischemic kidneys, serum levels of urea nitrogen and renal damage scores showed smaller values in TG and greater values in KO mice (urea nitrogen: KO 104+/-5>WT 98+/-15>TG 38+/-7 mg/dL, P<0.05 each). Renal NO synthase activity was also greater in TG mice. However, the differences in serum urea nitrogen and renal damage scores among the 3 groups of mice were not observed in mice pretreated with L-NAME. In conclusion, AM antagonists increased renal vascular tone in WT as well as in TG, suggesting that endogenous AM plays a role in the physiological regulation of the vascular tone. AM is likely to protect renal tissues from ischemia/reperfusion injury through its NO releasing activity.

Tumor necrosis factor–α–induced hypertension in pregnant rats results in decreased renal neuronal nitric oxide synthase expression

Background Preeclampsia is associated with increases in plasma levels of tumor necrosis factor–α (TNF-α), a cytokine known to contribute to endothelial dysfunction. We recently reported that a twofold elevation in plasma TNF-α produces significant reductions in renal function and hypertension in pregnant rats. The purpose of this study was to determine the role of the nitric oxide (NO) system in TNF-α–induced hypertension in pregnant rats. Methods Tumor necrosis factor–α (50 ng/day) was chronically infused starting at day 14 of gestation. Mean arterial pressure, 24-h urinary nitrite/nitrate excretion, and renal nitric oxide synthase (NOS) protein expression by Western blot analysis was measured at day 19 of gestation. Results A twofold increase in plasma TNF-α levels in pregnant rats resulted in a significant increase in arterial pressure (97 ± 3.6 v 116 ± 2.1 mm Hg, pregnant versus TNF-α pregnant, respectively, P < .05), but no significant change in urinary nitrite/nitrate excretion (22.0 ± 1.9 v 20.8 ± 2.5 μmol/24 h, pregnant versus TNF-α pregnant, respectively), a measure of whole body NO production. As abnormalities in renal production of NO would not be reflected in the measure of whole body NO production, changes in renal NOS protein levels were determined. The protein expression of both neuronal (nNOS) and inducible (iNOS) nitric oxide synthase were significantly decreased in the medulla of TNF-α pregnant rats (nNOS: 10.6 ± 0.7 v 8.2 ± 0.8 densitometric units, P < .05; and iNOS: 19.2 ± 0.9 v 15.4 ± 0.8 densitometric units, P < .05, pregnant versus TNF-α pregnant, respectively). Conclusion: The hypertension associated with a chronic twofold increase in TNF-α in pregnant rats is associated with significant decreases in renal nNOS and iNOS protein production.