Mechanisms of salt-sensitive hypertension: role of renal medullary inducible nitric oxide synthase

The goal of this study was to determine the role of renal medullary inducible nitric oxide synthase (iNOS) in the arterial pressure, renal hemodynamic, and renal excretory changes that occur in Dahl/Rapp salt-resistant (R) and salt-sensitive (S) rats during high Na intake. Forty R and S rats, equipped with indwelling arterial, venous, and renal medullary catheters, were subjected to high (8%) Na intake, and selective iNOS inhibition was achieved with continuous intravenous or renal medullary interstitial infusion of aminoguanidine (AG; 3.075 mg. kg(-1). h(-1)). After 5 days of AG, mean arterial pressure increased to 132 +/- 2% control in the S rats with high Na intake and intramedullary AG compared with 121 +/- 4% control (P < 0.05) in the S rats with high Na intake alone and 121 +/- 2% control (P < 0.05) in the S rats with high Na intake and intravenous AG. AG did not change arterial pressure in R rats. AG also caused little change in renal hemodynamics, urinary Na, or H(2)O excretion or ACh-induced aortic vasorelaxation in R or S rats. The data suggest that during high Na intake, nitric oxide produced by renal medullary iNOS helps to prevent excessive increases in arterial pressure in the Dahl S rat but not the R rat.     

Interactions between oxidative stress and inflammation in salt-sensitive hypertension

The goal of this study was to test the hypothesis that increases in oxidative stress in Dahl S rats on a high-salt diet help to stimulate renal nuclear factor-kappaB (NF-kappaB), renal proinflammatory cytokines, and chemokines, thus contributing to hypertension, renal damage, and dysfunction. We specifically studied whether antioxidant treatment of Dahl S rats on high Na intake would decrease renal inflammation and thus attenuate the hypertensive and adverse renal responses. Sixty-four 7- to 8-wk-old Dahl S or R/Rapp strain rats were maintained for 5 wk on high Na (8%) or high Na + vitamins C (1 g/l in drinking water) and E (5,000 IU/kg in food). Arterial and venous catheters were implanted at day 21. By day 35 in the high-Na S rats, antioxidant treatment significantly increased the renal reduced-to-oxidized glutathione ratio and decreased renal cortical H(2)O(2) and O(2)(*-) release and renal NF-kappaB. Antioxidant treatment with vitamins C and E in high-Na S rats also decreased renal monocytes/macrophages in the glomeruli, cortex, and medulla, decreased tumor necrosis factor-alpha by 39%, and decreased monocyte chemoattractant protein-1 by 38%. Vitamin-treated, high-Na S rats also experienced decreases in arterial pressure, urinary protein excretion, renal tubulointerstitial damage, and glomerular necrosis and increases in glomerular filtration rate and renal plasma flow. In conclusion, antioxidant treatment of high-Na Dahl S rats decreased renal inflammatory cytokines and chemokines, renal immune cells, NF-kappaB, and arterial pressure and improved renal function and damage.     

Mechanisms of salt-sensitive hypertension: role of inducible nitric oxide synthase

The goal of this study was to determine the role of inducible nitric oxide synthase (iNOS) in the arterial pressure, renal hemodynamic, renal excretory, and hormonal changes that occur in Dahl/Rapp salt-resistant (R) and salt-sensitive (S) rats during changes in Na intake. Thirty-two R and S rats, equipped with indwelling arterial and venous catheters, were subjected to low (0.87 mmol/day) or high (20.6 mmol/day) Na intake, and selective iNOS inhibition was achieved with intravenous aminoguanidine (AG, 12.3 mg. kg(-1). h(-1)). After 5 days of AG, mean arterial pressure increased to 121 +/- 3% control in the R-high Na AG rats compared with 98 +/- 1% control (P < 0.05) in the R-high Na alone rats, and S-high Na rats increased their arterial pressure to 123 +/- 3% control compared with 110 +/- 2% control (P < 0.05) in S-high Na alone rats. AG caused no significant changes in renal hemodynamics, urinary Na or H(2)O excretion, plasma renin activity, or cerebellar Ca-dependent NOS activity. The data suggest that nitric oxide produced by iNOS normally helps to prevent salt-sensitive hypertension in the Dahl R rat and decreases salt sensitivity in the Dahl S rat.     

Renal medullary endothelin-1 is decreased in Dahl salt-sensitive rats

Although it is well established that the renal endothelin (ET-1) system plays an important role in regulating sodium excretion and blood pressure through activation of renal medullary ET(B) receptors, the role of this system in Dahl salt-sensitive (DS) hypertension is unclear. The purpose of this study was to determine whether the DS rat has abnormalities in the renal medullary endothelin system when maintained on a high sodium intake. The data indicate that Dahl salt-resistant rats (DR) on a high-salt diet had a six-fold higher urinary endothelin excretion than in the DR rats with low Na(+) intake (17.8 ± 4 pg/day vs. 112 ± 44 pg/day). In sharp contrast, urinary endothelin levels increased only twofold in DS rats in response to a high Na(+) intake (13 ± 2 pg/day vs. 29.8 ± 5.5 pg/day). Medullary endothelin concentration in DS rats on a high-Na(+) diet was also significantly lower than DR rats on a high-Na(+) diet (31 ± 2.8 pg/mg vs. 70.9 ± 5 pg/mg). Furthermore, DS rats had a significant reduction in medullary ET(B) receptor expression compared with DR rats while on a high-Na(+) diet. Finally, chronic infusion of ET-1 directly into the renal medulla blunted Dahl salt-sensitive hypertension. These data indicate that a decrease in medullary production of ET-1 in the DS rat could play an important role in the development of salt-sensitive hypertension observed in the DS rat.     

Enhanced sympathoexcitatory and pressor responses to central Na+ in Dahl salt-sensitive vs. -resistant rats

An enhanced responsiveness to increases in cerebrospinal fluid (CSF) Na+ by high salt intake may contribute to salt-sensitive hypertension in Dahl salt-sensitive (S) rats. To test this hypothesis, sympathetic and pressor responses to acute and chronic increases in CSF Na+ were evaluated. In conscious young (5-6 wk old) and adult (10-11 wk old) Dahl S and salt-resistant (R) rats as well as weight-matched Wistar rats, hemodynamic [blood pressure (BP) and heart rate (HR)] and sympathetic [renal sympathetic nerve activity (RSNA)] responses to 10-min intracerebroventricular infusions of artificial CSF (aCSF) and Na+-rich aCSF (containing 0.2-0.45 M Na+) were evaluated. Intracerebroventricular Na+-rich aCSF increased BP, RSNA, and HR in a dose-related manner. The extent of these increases was significantly larger in Dahl S versus Dahl R or Wistar rats and young versus adult Dahl S rats. In a second set of experiments, young Dahl S and R rats received a chronic intracerebroventricular infusion of aCSF or Na+-rich (0.8 M) aCSF (5 microl/h) for 14 days, with the use of osmotic minipumps. On day 14 in conscious rats, CSF was sampled and BP, HR, and RSNA were recorded at rest and in response to air stress, intracerebroventricular alpha2-adrenoceptor agonist guanabenz, intracerebroventricular ouabain, and intravenous phenylephrine and nitroprusside to estimate baroreflex function. The infusion of Na+-rich aCSF versus aCSF increased CSF Na+ concentration to the same extent but caused severe versus mild hypertension in Dahl S and Dahl R rats, respectively. After central Na+ loading, hypothalamus "ouabain" significantly increased in Dahl S and only tended to increase in Dahl R rats. Moreover, sympathoexcitatory and pressor responses to intracerebroventricular exogenous ouabain were attenuated by Na+-rich aCSF to a greater extent in Dahl S versus Dahl R rats. Responses to air-jet stress or intracerebroventricular guanabenz were enhanced by Na+-rich aCSF in both strains, but the extent of enhancement was significantly larger in Dahl S versus Dahl R. Na+-rich aCSF impaired arterial baroreflex control of RSNA more markedly in Dahl S versus R rats. These findings indicate that genetic control of mechanisms linking CSF Na+ with brain "ouabain" is altered in Dahl S rats toward sympathetic hyperactivity and hypertension.     

Immune suppression prevents renal damage and dysfunction and reduces arterial pressure in salt-sensitive hypertension

The goal of this study was to test the hypothesis that renal infiltration of immune cells in Dahl S rats on increased dietary sodium intake contributes to the progression of renal damage, decreases in renal hemodynamics, and development of hypertension. We specifically studied whether anti-immune therapy, using mycophenolate mofetil (MMF), could help prevent increases in renal NF-kappaB activation, renal infiltration of monocytes/macrophages, renal damage, decreases in glomerular filtration rate (GFR) and renal plasma flow, and increases in arterial pressure. Seventy-four 7-to 8-wk-old Dahl S, Rapp strain rats were maintained on an 8% Na, 8% Na + MMF (20 mg.kg(-1).day(-1)), 0.3% Na, or 0.3% Na + MMF diet for 5 wk. Arterial and venous catheters were implanted at day 21. By day 35, renal NF-kappaB in 8% Na rats was 47% higher than in 0.3% Na rats and renal NF-kappaB was 41% lower in 8% Na + MMF rats compared with the 8% Na group. MMF treatment significantly decreased renal monocyte/macrophage infiltration and renal damage and increased GFR and renal plasma flow. In high-NA Dahl S rats mean arterial pressure increased to 182 +/- 5 mmHg, and MMF reduced this arterial pressure to 124 +/- 3 mmHg. In summary, in Dahl S rats on high sodium intake, treatment with MMF decreases renal NF-kappaB and renal monocyte/macrophage infiltration and improves renal function, lessens renal injury, and decreases arterial pressure. This suggests that renal infiltration of immune cells is associated with increased arterial pressure and renal damage and decreasing GFR and renal plasma flow in Dahl salt-sensitive hypertension.     

Effects of high salt intake on brain AT1 receptor densities in Dahl rats

To assess effects of dietary salt on brain AT1 receptor densities, 4-wk-old Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats were fed a regular (101 mumol Na/g) or high (1,370 mumol Na/g)-salt diet for 1, 2, or 4 wk. AT1 receptors were assessed by quantitative in vitro autoradiography. AT1 receptor densities did not differ significantly between strains on the regular salt diet. The high-salt diet for 1 or 2 wk increased AT1 receptor binding by 21-64% in the Dahl S rats in the subfornical organ, median preoptic nucleus, paraventricular nucleus, and suprachiasmatic nucleus. No changes were noted in the Dahl R rats. After 4 wk on a high-salt diet, increases in AT1 receptor binding persisted in Dahl S rats but were now also noted in the paraventricular nucleus, median preoptic nucleus, and suprachiasmatic nucleus of Dahl R rats. At 4 wk on the diet, intracerebroventricular captopril caused clear decreases in blood pressure only in the Dahl S on the high-salt diet but caused largely similar relative increases in brain AT1 receptor densities in Dahl S and R on the high-salt diet versus regular salt diet. These data demonstrate that high salt intake rapidly (within 1 wk) increases AT1 receptor densities in specific brain nuclei in Dahl S and later (by 4 wk) also in Dahl R rats. Because the brain renin-angiotensin system only contributes to salt-induced hypertension in Dahl S rats, further studies are needed to determine which of the salt-induced increases in brain AT1 receptor densities contribute to the hypertension and which to other aspects of body homeostasis.     

Chronic central infusion of aldosterone leads to sympathetic hyperreactivity and hypertension in Dahl S but not Dahl R rats

Six-week-old Dahl salt-sensitive (S) and -resistant (R) rats received for 2 wk an intracerebroventricular infusion of aldosterone (Aldo) (22.5 ng/h) or vehicle containing artificial cerebrospinal fluid (aCSF) with 0.15 M Na+. At 8 wk, mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in conscious rats at rest, in response to air stress, and to an intracerebroventricular injection of the alpha2-adrenoceptor agonists guanabenz or ouabain. Baroreflex control of RSNA and HR was estimated by using intravenous phenylephrine and nitroprusside. In Dahl S but not Dahl R rats, Aldo raised resting MAP by 20-25 mmHg, doubled sympathoexcitatory and pressor responses to air stress and sympathoinhibitory and depressor responses to guanabenz, and impaired baroreflex function. In Dahl S but not Dahl R rats, Aldo significantly increased content of ouabain-like compounds (OLC) in the hypothalamus and attenuated excitatory responses to ouabain. Aldo did not affect water intake, plasma electrolytes, or OLC in plasma and adrenal glands. In another set of three groups of Dahl S rats, Aldo dissolved in aCSF containing 0.16, 0.15, or 0.14 M Na+ was infused intracerebroventricularly for 2 wk. CSF Na+ concentration ([Na+]) showed only a nonsignificant increase, but resting MAP increased from 111 +/- 3 mmHg in rats with Aldo in 0.14 M Na+ to 131 +/- 3 and 147 +/- 3 mmHg with Aldo in 0.15 and 0.16 M Na+, respectively (P < 0.05 for both). These findings indicate that in Dahl S rats, intracerebroventricular infusion of Aldo causes similar central responses as high salt intake, i.e., increases in brain OLC content, sympathetic hyperreactivity, and hypertension. The extent of the increase in blood pressure (BP) by intracerebroventricular Aldo depends on the [Na+] in the vehicle. In Dahl R rats, intracerebroventricular Aldo did not increase brain OLC, sympathetic reactivity, and BP, suggesting that in this rat strain, a decrease in central responsiveness to mineralocorticoids may contribute to its salt-resistant nature.     

Infiltrating T lymphocytes in the kidney increase oxidative stress and participate in the development of hypertension and renal disease

The present studies examined the role and mechanism of action of infiltrating T lymphocytes in the kidney during salt-sensitive hypertension. Infiltrating T lymphocytes in the Dahl salt-sensitive (SS) kidney significantly increased from 7.2 ± 1.8 × 10(5) cells/2 kidneys to 18.2 ± 3.9 × 10(5) cells/2 kidneys (n = 6/group) when dietary NaCl was increased from 0.4 to 4.0%. Furthermore, the expression of immunoreactive p67(phox), gp91(phox), and p47(phox) subunits of NADPH oxidase was increased in T cells isolated from the kidneys of rats fed 4.0% NaCl. The urinary excretion of thiobarbituric acid-reactive substances (TBARS; an index of oxidative stress) also increased from 367 ± 49 to 688 ± 92 nmol/day (n = 8/group) when NaCl intake was increased in Dahl SS rats. Studies were then performed on rats treated with a daily injection of vehicle (5% dextrose) or tacrolimus (0.25 mg·kg(-1)·day(-1) ip), a calcineurin inhibitor that suppresses immune function, during the period of high-NaCl intake (n = 5/group). In contrast to the immune cell infiltration, increased NADPH oxidase expression, and elevated urine TBARS excretion in vehicle-treated Dahl SS fed high salt, these parameters were unaltered as NaCl intake was increased in Dahl SS rats administered tacrolimus. Moreover, tacrolimus treatment blunted high-salt mean arterial blood pressure and albumin excretion rate (152 ± 3 mmHg and 20 ± 9 mg/day, respectively) compared with values in dextrose-treated Dahl SS rats (171 ± 8 mmHg and 74 ± 28 mg/day). These experiments indicate that blockade of infiltrating immune cells is associated with decreased oxidative stress, an attenuation of hypertension, and a reduction of renal damage in Dahl SS rats fed high salt.     

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.     

Overexpression of HIF-1α transgene in the renal medulla attenuated salt sensitive hypertension in Dahl S rats

Hypoxia inducible factor (HIF)-1α-mediated gene activation in the renal medulla in response to high salt intake plays an important role in the control of salt sensitivity of blood pressure. High salt-induced activation of HIF-1α in the renal medulla is blunted in Dahl S rats. The present study determined whether the impairment of the renal medullary HIF-1α pathway was responsible for salt sensitive hypertension in Dahl S rats. Renal medullary HIF-1α levels were induced by either transfection of HIF-1α expression plasmid or chronic infusion of CoCl? into the renal medulla, which was accompanied by increased expressions of anti-hypertensive genes, cyclooxygenase-2 and heme oxygenase-1. Overexpression of HIF-1α transgenes in the renal medulla enhanced the pressure natriuresis, promoted the sodium excretion and reduced sodium retention after salt overload. As a result, hypertension induced by 2-week high salt was significantly attenuated in rats treated with HIF-1α plasmid or CoCl?. These results suggest that an abnormal HIF-1α in the renal medulla may represent a novel mechanism mediating salt-sensitive hypertension in Dahl S rats and that induction of HIF-1α levels in the renal medulla could be a therapeutic approach for the treatment of salt-sensitive hypertension.     

Effects of thiol antioxidant on reduced nicotinamide adenine dinucleotide phosphate oxidase in hypertensive Dahl salt-sensitive rats

Recent studies implicate of reactive oxygen species (ROS) in hypertension; however, whether reactive oxygen species promote hypertensive derangements is not fully clear. We thus investigated the effects of an antioxidant, N-acetyl-L-cysteine, on hypertensive Dahl salt-sensitive rats. High-salt intake for 4 weeks markedly elevated systolic arterial pressure, urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the enzyme activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with the elevated expression of its subunits gp91phox and p47phox at the levels of mRNA and protein. Supplement with N-acetyl-L-cysteine reduced the increase in systolic arterial pressure and counteracted the elevation of urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the increases in NADPH oxidase activity/expression in high-salt-loaded Dahl salt-sensitive rats. N-acetyl-L-cysteine supplement ameliorated plasma and urinary levels of thromboxane B(2) (an end metabolite of thromboxane A(2)), associated with improvement of both the abnormal contraction and the impaired nitric oxide-dependent relaxation in renal arteries. These results revealed that oxidative stress mediates hypertensive changes in Dahl salt-sensitive rats, because thiol antioxidant N-acetyl-L-cysteine attenuated the augmentation of local ROS production by diminishing the elevation of NADPH oxidase expression and ameliorated renal/vascular hypertensive changes.     

NADPH oxidase contributes to renal damage and dysfunction in Dahl salt-sensitive hypertension

The goal of this study was to test the hypothesis that NADPH oxidase contributes importantly to renal cortical oxidative stress and inflammation, as well as renal damage and dysfunction, and increases in arterial pressure. Fifty-four 7- to 8-wk-old Dahl salt-sensitive (S) or R/Rapp strain rats were maintained for 5 wk on a high sodium (8%) or high sodium + apocynin (1.5 mmol/l in drinking water). Arterial and venous catheters were implanted on day 21. By day 35 in the high-Na S rats, mRNA expression of renal cortical gp91phox, p22phox, p47phox, and p67phox NADPH subunits in S rats increased markedly, and treatment of high-Na S rats with the NADPH oxidase inhibitor apocynin resulted in significant decreases in mRNA expression of these NADPH oxidase subunits. At the same time, in apocynin-treated S rats 1) renal cortical GSH/GSSG ratio increased, 2) renal cortical O2(.-) release and NADPH oxidase activity decreased, and 3) renal glomerular and interstitial damage markedly fell. Apocynin also decreased renal cortical monocyte/macrophage infiltration, and apocynin, but not the xanthine oxidase inhibitor allopurinol, attenuated decreases in renal hemodynamics and lowered arterial pressure. These data suggest that NADPH oxidase plays an important role in causing renal cortical oxidative stress and inflammation, which lead to decreases in renal hemodynamics, renal cortical damage, and increases in arterial pressure.     

Sexual dimorphism in oxidant status in spontaneously hypertensive rats

Male spontaneously hypertensive rats (SHR) have a blunted pressure-natriuresis relationship and enhanced oxidative stress compared with female SHR. Furthermore, oxidative stress contributes to abnormal renal Na+ handling and renal damage in hypertension. The aim of this study was to determine whether a sex difference exists in renal inner medullary hydrogen peroxide (H2O2) levels and/or antioxidant systems in SHR and the influence of sex steroids on these systems. Thirteen-week-old intact and gonadectomized male and female SHR were placed in metabolic cages for 24-h urine collection. Renal inner medullas were isolated for antioxidant activity assays and Western blot analysis or for measurements of H2O2 using Amplex Red. Studies verified that male SHR had greater Na+ reabsorption compared with female SHR. Male SHR had enhanced urinary excretion of H2O2 compared with female SHR. Gonadectomy decreased H2O2 excretion in males and increased H2O2 excretion in females, suggesting that testosterone stimulates total body oxidative stress and estrogen suppresses levels of total body oxidative stress. There was not a sex difference in inner medullary H2O2 levels. Male SHR had a testosterone-dependent increase in inner medullary SOD activity, and both intact and gonadectomized males had high levels of inner medullary catalase activity compared with females. The results of this study showed that there was a sexual dimorphism in Na+ handling and oxidant status. We hypothesize that there is a testosterone-sensitive increase in whole body reactive oxygen species production that results in a compensatory increase in the inner medullary antioxidant capability possibly to normalize Na+ handling.     

Increases in CSF [Na+] precede the increases in blood pressure in Dahl S rats and SHR on a high-salt diet

In Dahl salt-sensitive (S) and salt-resistant (R) rats, and spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, at 5-6 wk of age, a cannula was placed in the cisterna magna, and cerebrospinal fluid (CSF) was withdrawn continuously at 75 microl/12 h. CSF was collected as day- and nighttime samples from rats on a regular salt intake (0.6% Na+; R-Na) and then on a high salt intake (8% Na+; H-Na). In separate groups of rats, the abdominal aorta was cannulated and blood pressure (BP) and heart rate (HR) measured at 10 AM and 10 PM, with rats first on R-Na and then on H-Na. On H-Na, CSF [Na+] started to increase in the daytime of day 2 in Dahl S rats and of day 3 in SHR. BP and HR did not rise until day 3 in Dahl S rats and day 4 in SHR. In Dahl R and WKY rats, high salt did not change CSF [Na+], BP, or HR. In a third set of Dahl S rats, sampling of both CSF and BP was performed in each individual rat. Again, significant increases in CSF [Na+] were observed 1-2 days earlier than the increases in BP and HR. In a fourth set of Dahl S rats, BP and HR were recorded continuously by means of radiotelemetry for 5 days on R-Na and 8 days on H-Na. On H-Na, BP (but not HR) increased first in the nighttime of day 2. In another set of Dahl S rats, intracerebroventricular infusion of antibody Fab fragments binding ouabain-like compounds (OLC) with high affinity prevented the increase in BP and HR by H-Na but further increased CSF [Na+]. Finally, in Wistar rats on H-Na, intracerebroventricular infusion of ouabain increased BP and HR but decreased CSF [Na+]. Thus, in both Dahl S and SHR on H-Na, increases in CSF [Na+] preceded the increases in BP and HR, consistent with a primary role of increased CSF [Na+] in the salt-induced hypertension. An increase in brain OLC in response to the initial increase in CSF [Na+] appears to attenuate further increases in CSF [Na+] but at the "expense" of sympathoexcitation and hypertension.     

Alpha1 Na,K-ATPase and Na,K,2Cl-cotransporte/D3mit3 loci interact to increase susceptibility to salt-sensitive hypertension in Dahl S(HSD) rats

BACKGROUND: Essential (multigenic) hypertension is a complex multifactorial disease whose genetic etiology has not been unraveled on a major locus-effect investigative paradigm. As with other complex genetic diseases, applying an interacting loci paradigm could be critical in the elucidation of genetic determinants. Having defined the alpha1 Na,K-ATPase (alpha1NK) as a hypertension susceptibility gene in Dahl salt-sensitive (Dahl S) rats, we determined whether alphaINK interacts with another renal epithelial Na transporter to increase susceptibility to salt-sensitive hypertension. We focused on alpha1NK and Na,K,2Cl-cotransporter (NKC) as an a priori candidate interacting gene pair because they comprise a functionally linked Na transport system in renal thick ascending limb of Henle (TALH) epithelial cells and exhibit altered function in prehypertensive Dahl S rats in contrast to Dahl salt-resistant normotensive (Dahl R) rats. MATERIAL AND METHOD: Cosegregation analysis of alphaNK and NKC loci was done in a (Dahl S x Dahl R) F2 cohort characterized for blood pressure by radiotelemetry using the D2mghII microsatellite marker in the alpha1NK gene and the D3mit3 microsatellite marker close to the NKC gene (NKC/D3mit3 locus). Single locus and digenic analyses were performed to establish the individual and interactive genetic contribution to salt-sensitive hypertension. Molecular analysis was then done to support the NKC gene as the likely candidate gene interacting with alpha1NK in Dahl salt-sensitive hypertension pathogenesis. RESULTS: Compared with respective single locus analysis, digenic analysis of 96 F2 (Dahl S x Dahl R) hybrid male rats revealed cosegregation of alpha1NK and NKC/D3mit3 loci as interacting pair with salt-sensitive hypertension with markedly increased significance for systolic (one-way ANOVA p = 10(-6)), diastolic (p = 10(-5)), and mean arterial (p = 10(-6)) blood pressures. Concordantly, two-way ANOVA detected interaction between alpha1NK and NKC loci in determining the levels of systolic (p = 0.004), diastolic (p = 0.008), and mean arterial (p = 0.006) pressures. To unravel potential NKC molecular dysfunction(s) involved in hypertension pathogenesis, we investigated putative differences between Dahl S and Dahl R rats in nucleotide sequence and isoform gene expression of the renal-specific Na,K,2Cl-cotransporter. Molecular analysis revealed an inversion of alternatively spliced NKC-isoform ratios (4B:4A:4F) between Dahl S and Dahl R prehypertensive kidneys supported by four mutations in intron-3 immediately upstream to alternatively spliced exons 4B, 4A, and 4F. No nucleotide changes were detected within the aminoacid encoding exons of NKC. CONCLUSIONS: Altogether, these current data and previous characterization of the role of the Q276L alpha1NK molecular variant in Dahl S hypertension provide cumulative compelling evidence that alpha1NK and NKC/D3mit3 loci interact to increase susceptibility to hypertension in Dahl S rats and that NKC is the likely candidate gene that interacts with alpha 1NK. More importantly, the data substantiate gene interaction as an operative mechanism in multigenic hypertension.     

Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension

We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial (22)Na(+) transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat.     

Altered peripheral noradrenergic activity in intact and sinoaortic denervated Dahl rats

Development of salt-induced hypertension in Dahl salt-sensitive (S) rats is dependent on sympathetic overactivity which may be partially related to arterial baroreflex dysfunction and, therefore, is regionally selective. Our first experiment was designed to determine which regions have elevated sympathetic activity in Dahl S compared with Dahl salt-resistant (R) rats. Weanling (4-week-old) female Dahl R and S rats were fed low or high salt diets (0.13% and 8% NaCl) until 10 weeks of age. Norepinephrine (NE) synthesis was blocked with alpha-methyl-p-tyrosine, and the fractional decline of NE concentration was measured in various tissues. Dahl S rats with increases in both arterial pressure and left ventricular weight demonstrated increased NE turnover in the sinoatrial node, the atrial appendages, the cardiac ventricles, and the renal cortex. In all of these tissues except the cardiac ventricle, increases were associated with high salt intake. Our second experiment was designed to test if arterial baroreflex dysfunction could account for regional increases in sympathetic activity. Separate groups of Dahl R and S rats fed high salt were subjected to either sham surgery or sinoaortic baroreceptor denervation 1 week prior to turnover determinations. Sinoaortic baroreceptor denervation abolished differences in NE turnover between salt-fed Dahl R and S rats in the cardiac sinoatrial node and the atrial appendages, but not in the cardiac ventricles and the renal cortex. Sinoaortic baroreceptor denervation also abolished differences between salt-fed Dahl S and R rats in the spleen but not the duodenum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antihypertensive effects of chronic anti-TGF-beta antibody therapy in Dahl S rats

This study examined the role of transforming growth factor-beta (TGF-beta) in the development of hypertension and renal disease in 9-wk-old male Dahl salt-sensitive (Dahl S) rats fed an 8% NaCl diet for 3 wk. The rats received an intraperitoneal injection of a control or an anti-TGF-beta antibody (anti-TGF-beta Ab) every other day for 2 wk. Mean arterial pressure was significantly lower in Dahl S rats treated with anti-TGF-beta Ab (177 +/- 3 mmHg, n = 12) than in control rats (190 +/- 4 mmHg, n = 17). Anti-TGF-beta Ab therapy also reduced proteinuria from 226 +/- 20 to 154 +/- 16 mg/day. Renal blood flow, cortical blood flow, and creatinine clearance were not significantly different in control and treated rats; however, medullary blood flow was threefold higher in the treated rats than in the controls. Despite the reduction in proteinuria, the degree of glomerulosclerosis and renal hypertrophy was similar in control and anti-TGF-beta Ab-treated rats. Renal levels of TGF-beta1 and -beta2, alpha-actin, type III collagen, and fibronectin mRNA decreased in rats treated with anti-TGF-beta Ab. To examine whether an earlier intervention with anti-TGF-beta Ab would confer additional renoprotection, these studies were repeated in a group of 6-wk-old Dahl S rats. Anti-TGF-beta Ab therapy significantly reduced blood pressure, proteinuria, and the degree of glomerulosclerosis and renal medullary fibrosis in this group of rats. The results indicate that anti-TGF-beta Ab therapy reduces blood pressure, proteinuria, and the renal injury associated with hypertension.     

Mineralocorticoid receptor activation: a major contributor to salt-induced renal injury and hypertension in young rats

Excessive salt intake is known to preferentially increase blood pressure (BP) and promote kidney damage in young, salt-sensitive hypertensive human and animal models. We have suggested that mineralocorticoid receptor (MR) activation plays a major role in kidney injury in young rats. BP and urinary protein were compared in young (3-wk-old) and adult (10-wk-old) uninephrectomized (UNx) Sprague-Dawley rats fed a high (8.0%)-salt diet for 4 wk. The effects of the MR blocker eplerenone on BP and renal injury were examined in the high-salt diet-fed young UNx rats. Renal expression of renin-angiotensin-aldosterone (RAA) system components and of inflammatory and oxidative stress markers was also measured. The effects of the angiotensin receptor blocker olmesartan with or without low-dose aldosterone infusion, the aldosterone synthase inhibitor FAD286, and the antioxidant tempol were also studied. Excessive salt intake induced greater hypertension and proteinuria in young rats than in adult rats. The kidneys of young salt-loaded rats showed marked histological injury, overexpression of RAA system components, and an increase in inflammatory and oxidative stress markers. These changes were markedly ameliorated by eplerenone treatment. Olmesartan also ameliorated salt-induced renal injury but failed to do so when combined with low-dose aldosterone infusion. FAD286 and tempol also markedly reduced urinary protein. UNx rats exposed to excessive salt at a young age showed severe hypertension and renal injury, likely primarily due to MR activation and secondarily due to angiotensin receptor activation, which may be mediated by inflammation and oxidative stress.