SWI/SNF chromatin remodeling enzymes are associated with cardiac hypertrophy in a genetic rat model of hypertension.

Pathological cardiac hypertrophy is characterized by a sustained increase in cardiomyocyte size and re-activation of the fetal cardiac gene program. Previous studies implicated SWI/SNF chromatin remodeling enzymes as regulators of the fetal cardiac gene program in surgical models of cardiac hypertrophy. Although hypertension is a common risk factor for developing cardiac hypertrophy, there has not yet been any investigation into the role of SWI/SNF enzymes in cardiac hypertrophy using genetic models of hypertension. In this study, we tested the hypothesis that components of the SWI/SNF complex are activated and recruited to promoters that regulate the fetal cardiac gene program in hearts that become hypertrophic as a result of salt induced hypertension. Utilizing the Dahl salt-sensitive (S) rat model, we found that the protein levels of several SWI/SNF subunits required for heart development, Brg1, Baf180, and Baf60c, are elevated in hypertrophic hearts from S rats fed a high salt diet compared with normotensive hearts from Dahl salt-resistant (R) rats fed the same diet. Furthermore, we detected significantly higher levels of SWI/SNF subunit enrichment as well as evidence of more accessible chromatin structure on two fetal cardiac gene promoters in hearts from S rats compared with R rats. Our data implicate SWI/SNF chromatin remodeling enzymes as regulators of gene expression in cardiac hypertrophy resulting from salt induced hypertension. Thus we provide novel insights into the epigenetic mechanisms by which salt induced hypertension leads to cardiac hypertrophy.

Genetic variants in Arhgef11 are associated with kidney injury in the Dahl salt-sensitive rat.

A previous genetic analysis comparing the Dahl salt-sensitive (S) rat with the spontaneously hypertensive rat identified a major locus on chromosome 2 that influences proteinuria in the S rat. In the present study, blood pressure, proteinuria, and renal hemodynamics were evaluated in congenic strains with small segments of the protective spontaneously hypertensive rat genome on the S background. Proteinuria and renal function were significantly improved in the congenic strains compared with the S. The causative locus interval was narrowed to <375 kb on the basis of congenic strains, haplotype data, comparative mapping, and concordance with human genetic studies. Sequencing of the coding region of genes in this region identified 36 single nucleotide polymorphisms (13 nonsynonymous and 23 synonymous). Gene expression profiling indicated that only a few genes exhibited differential expression. Arhgef11, Pear1, and Sh2d2 were identified as important candidate genes that may be linked to kidney injury in the S rat. In particular, Arhgef11 plays an important role in the activation of the Rho-ROCK signaling pathway. Inhibition of this pathway using fasudil resulted in a significant reduction of proteinuria in treated S rats (compared with untreated S). However, no difference was observed between treated or untreated spontaneously hypertensive rat or congenic strains. The homologous region in humans was found to be associated with estimated glomerular filtration rate in the Candidate Gene Association Resource population. In summary, these findings demonstrate that allelic variants in Arhgef11, acting through the Rho-ROCK pathway, could influence kidney injury in the S as well as provide insight into human kidney disease.

Angiotensin II type 2 receptor-mediated inhibition of NaCl absorption is blunted in thick ascending limbs from Dahl salt-sensitive rats.

NO reduces NaCl absorption by thick ascending limbs (TALs) by inhibiting the Na/K/2Cl cotransporter (NKCC2). We have shown that NO-induced inhibition of Na transport is reduced in Dahl salt-sensitive rat (SS) TALs. Angiotensin II increases NO production in TALs via angiotensin II type 2 receptor (AT(2)R). It is unknown whether AT(2)Rs regulate TAL NaCl absorption and whether this effect is reduced in SS rats. We hypothesized that AT(2)R activation decreases TAL Na transport via NO, and this effect is blunted in SS rats. In the presence of angiotensin II type 1 receptor antagonist losartan, AT(2)R activation with angiotensin II inhibited NKCC2 activity by 32±7% (P<0.03). AT(2)R antagonist PD-123319 abolished the effect of angiotensin II. Activation with the AT(2)R-selective agonist CGP42112A (10 nmol/L) decreased NKCC2 activity by 29±6% (P<0.03). The effect of CGP42112A on NKCC2 activity was blocked by PD-123319 and by NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. In Dahl salt-resistant rat TALs, 1 nmol/L of CGP42112A decreased NKCC2 activity by 23±4% (P<0.01). In SS TALs, it had no effect. TAL AT(2)R mRNA did not differ in SS versus salt-resistant rats. We conclude the following: (1) TAL AT(2)R activation decreases Na absorption; (2) this effect is mediated by AT(2)R-induced stimulation of NO; (3) AT(2)R-induced reduction of NKCC2 activity is blunted in SS rats; and (4) defects in AT(2)R/NO signaling rather than decreased AT(2)R expression likely account for the blunted effect in SS TALs. Impaired AT(2)R-mediated signaling in TALs could contribute to the Na retention associated with salt-sensitive hypertension.

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat.

The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.

Possible role of brain salt-inducible kinase 1 in responses to central sodium in Dahl rats.

In Dahl salt-sensitive (S) rats, Na(+) entry into the cerebrospinal fluid (CSF) and sympathoexcitatory and pressor responses to CSF Na(+) are enhanced. Salt-inducible kinase 1 (SIK1) increases Na(+)/K(+)-ATPase activity in kidney cells. We tested the possible role of SIK1 in regulation of CSF [Na(+)] and responses to Na(+) in the brain. SIK1 protein and activity were lower in hypothalamic tissue of Dahl S (SS/Mcw) compared with salt-resistant SS.BN13 rats. Intracerebroventricular infusion of the protein kinase inhibitor staurosporine at 25 ng/day, to inhibit SIK1 further increased mean arterial pressure (MAP) and HR but did not affect the increase in CSF [Na(+)] or hypothalamic aldosterone in Dahl S on a high-salt diet. Intracerebroventricular infusion of Na(+)-rich artificial CSF caused significantly larger increases in renal sympathetic nerve activity, MAP, and HR in Dahl S vs. SS.BN13 or Wistar rats on a normal-salt diet. Intracerebroventricular injection of 5 ng staurosporine enhanced these responses, but the enhancement in Dahl S rats was only one-third that in SS.BN13 and Wistar rats. Staurosporine had no effect on MAP and HR responses to intracerebroventricular ANG II or carbachol, whereas the specific protein kinase C inhibitor GF109203X inhibited pressor responses to intracerebroventricular Na(+)-rich artificial CSF or ANG II. These results suggest that the SIK1-Na(+)/K(+)-ATPase network in neurons acts to attenuate sympathoexcitatory and pressor responses to increases in brain [Na(+)]. The lower hypothalamic SIK1 activity and smaller effect of staurosporine in Dahl S rats suggest that impaired activation of neuronal SIK1 by Na(+) may contribute to their enhanced central responses to sodium.

Increased resistance to LPS-induced myocardial dysfunction in the Brown Norway rats versus Dahl S rats: roles of inflammatory cytokines and nuclear factor kappaB pathway.

We previously demonstrated that hearts from Brown Norway (BN) rats were more resistant to ischemic injury than hearts from Dahl S (SS) rats. Here we determined the susceptibility to LPS-induced cardiomyopathy in these rats and examined the involvement of inflammatory signaling. Both strains were treated with LPS (20 mg/kg) via i.p. injection for 6 h. Myocardial function was assessed by the Langendorff system, and proinflammatory cytokines were measured by the enzyme-linked immunosorbent assay. LPS significantly reduced left ventricular developed pressure in both strains. Interestingly, the decrease of left ventricular developed pressure in BN rat hearts was approximately 25% less than that in SS rat hearts. Furthermore, LPS significantly reduced the peak rate of contraction and the peak rate of relaxation in SS hearts but not in BN hearts. No differences in LPS-induced decreases in coronary flow rate were observed between BN and SS rats. In addition, LPS-induced increases in proinflammatory cytokines, TNF-alpha, IL-1beta, and IL-6, were significantly lower in both plasma and hearts of BN rats compared with production in SS rats. LPS notably up-regulated the expression of proinflammatory enzymes, iNOS and cyclooxygenase 2, in SS hearts but not in BN hearts. Interestingly, LPS did not stimulate Toll-like receptor 4 or its adaptor myeloid differentiation factor 88 expression in the hearts of either strain but did increase IkappaB and P65 phosphorylation, less prominently in BN hearts than in SS hearts. These data indicate that reduced production of proinflammatory cytokines and diminished nuclear factor kappaB activation are major mechanisms by which BN hearts are more resistant to LPS-induced myocardial dysfunction than SS hearts.

Renal phosphodiesterase 4B is activated in the Dahl salt-sensitive rat.

Reduced beta-adrenoreceptor signaling is associated with increased sympathoadrenal activity in hypertensive patients and animal models of hypertension. However, the mechanism that accounts for this characteristic decline in beta-adrenergic signaling is unclear. In the present study, we investigated renal phosphodiesterase 4B, which metabolizes cAMP. Immunoblot analysis detected only the phosphodiesterase 4B4 isoform present in kidney tissue from spontaneously hypertensive rats, hypertensive Dahl salt-sensitive (SS) rats, and Dahl salt-resistant rats. The phosphorylated (activated) form of the protein was present at 2-fold greater levels in Dahl SS rats than in spontaneously hypertensive rats and Dahl salt-resistant rats, whereas the unphosphorylated form of the protein was reduced by approximately one half in SS animals. In accord with immunoblot data, rolipram-inhibitable cAMP hydrolyzing activity, a measure of PDE4 activity, was approximately 3-fold greater in kidney cytosolic extracts from SS rats than in extracts from spontaneously hypertensive rats and salt-resistant rats. Phosphodiesterase 4B expression was detected by immunohistochemistry in the renal vasculature, proximal tubules, and distal tubules. These results raise the possibility that increased PDE4 activity, specifically phosphodiesterase 4B4 activity, reduces beta-adrenergic signaling in the kidney and contributes to salt-sensitive hypertension in the Dahl SS rat.

Effects of aldosterone and angiotensin II receptor blockade on cardiac angiotensinogen and angiotensin-converting enzyme 2 expression in Dahl salt-sensitive hypertensive rats.

BACKGROUND: We previously reported that a high-sodium diet activates the local renin-angiotensin-aldosterone system (RAAS) in cardiovascular tissues of Dahl salt-sensitive hypertensive (DS) rats. Angiotensin-converting enzyme 2 (ACE2) is a novel regulator of blood pressure (BP) and cardiac function. The effect of blockade of aldosterone or angiotensin II (Ang II) on cardiac angiotensinogen and ACE2 in DS rats is unknown. METHODS: The BP, plasma renin activity (PRA), plasma aldosterone concentration (PAC), heart weight, endothelium-dependent relaxation (EDR), and messenger RNA (mRNA) levels of collagen III, angiotensinogen, ACE, and ACE2 in the heart were measured in DS rats and in Dahl salt-resistant (DR) rats fed high or low salt diets. The rats were treated orally with or without eplerenone (100 mg/kg/d), candesartan (10 mg/kg/d), or both drugs combined for 8 weeks. RESULTS: A high salt diet increased BP (140%), heart/body weight (132%), and collagen III mRNA levels (146%) and decreased PRA and PAC concomitant with increased expression of cardiac angiotensinogen mRNA and decreased mRNA levels of ACE2 in DS rats. Eplerenone or candesartan significantly decreased the systolic BP from 240 +/- 5 mm Hg to 164 +/- 4 mm Hg or to 172 +/- 10 mm Hg, respectively (P < .05). Eplerenone or candesartan partially improved heart/body weight and cardiac fibrosis, improved EDR and decreased cardiac ACE and angiotensinogen mRNA levels in DS rats. Candesartan increased ACE2 mRNA levels in the heart. Combination therapy normalized BP and further improved cardiac hypertrophy, fibrosis, and EDR. CONCLUSIONS: In DS rats, blockade of aldosterone or Ang II protects cardiac hypertrophy and fibrosis by inactivation of the local RAAS in the heart.

Hyperhomocysteinemia associated with decreased renal transsulfuration activity in Dahl S rats.

Elevated plasma homocysteine (Hcys) has been reported to participate in the development of arterial and glomerular sclerosis in Dahl salt-sensitive hypertensive (SS) rats. The mechanism resulting in hyperhomocysteinemia in these animals remains unknown. Disposal of Hcys in the kidneys plays an important role in regulating the plasma Hcys level. We, therefore, examined the activities and expressions of the enzymes involved in the metabolism of Hcys in the kidneys of SS rats, compared with that of Brown Norway rats and SSBN13 rats, a consomic subcolony of SS rats that carries a substituted chromosome 13 from Brown Norway rats. High-performance liquid chromatography analysis demonstrated that plasma Hcys levels were significantly higher in SS rats. The conversion of S-adenosylhomocysteine into Hcys via S-adenosylhomocysteine hydrolase by renal tissue was not different among these 3 rat strains. However, the metabolic rate of Hcys into cysteine was markedly reduced in the SS rat kidneys. The mRNA and protein levels of cystathionine beta-synthase (CBS), one of the key enzymes in the transsulfuration pathway in the kidneys, were significantly lower in SS rats. In microdissected nephron segments, CBS mRNA was shown to be mainly present in renal proximal tubules (PTs). The mRNA levels of CBS in the PTs were also significantly decreased in SS rats, accompanied by a reduced CBS activity in PTs. We conclude that hyperhomocysteinemia is associated with a decreased activity and expression of CBS in renal PTs because of the defect of chromosome 13 in SS rats.

Renal medullary nitric oxide deficit of Dahl S rats enhances hypertensive actions of angiotensin II.

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng. kg(-1). min(-1)) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 +/- 8 to 117 +/- 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng. kg(-1). min(-1) did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 +/- 2 to 138 +/- 2 mmHg (P < 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of L-arginine (300 microg. kg(-1). min(-1)) into the renal medulla of Dahl S rats. Intravenous infusion of L-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.

The dual AngII/AVP receptor gene N119S/C163R variant exhibits sodium-induced dysfunction and cosegregates with salt-sensitive hypertension in the Dahl salt-sensitive hypertensive rat model.

BACKGROUND: Essential hypertension is a prevalent complex polygenic disease and a major risk factor for cardiovascular disease, the leading cause of death in developed countries. Because of its complex and multifactorial nature, its genetic determinants still remain largely unknown. The Dahl salt-sensitive hypertensive rat model exhibits impaired sodium handling, which is hypothesized to play a key role in the pathophysiology of polygenic hypertension. Thus, genes associated with renal regulation of salt and water balance are a priori likely candidates for a causative role in hypertension pathogenesis. The functional properties and renal-specific expression of the recently characterized AngII/AVP receptor suggest a putative modulator role in tubular sodium and fluid reabsorption. Based on these observations, we investigated the potential involvement of the AngII/AVP receptor in salt-sensitive hypertension. MATERIALS AND METHODS: We performed cosegregation analysis of the AngII/AVP receptor locus with salt-sensitive hypertension in an F2 (Dahl S X Dahl salt-resistant [R]) hybrid male cohort characterized for blood pressure by radiotelemetry after 8 weeks of high salt challenge. Further molecular analysis was done to identify putative AngII/AVP receptor molecular variants that could account for the AngII/ AVP receptor involvement in salt-sensitive hypertension pathogenesis. RESULTS: The AngII/AVP receptor was mapped to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker by radiation hybrid mapping analysis. Quantitative trait locus (QTL) analysis detected a highly significant linkage of the AngII/AVP receptor locus with high blood pressure (LRS = 13.8, p= 0.0002). Molecular characterization of the Dahl S and Dahl R AngII/AVP receptor cDNAs revealed two amino acid substitutions in the Dahl S AngII/AVP receptor (N119S, C163R) when compared to the Dahl R AngII/AVP receptor. These mutations are associated with an increased receptor affinity for both ligands (AVP and AngII) and an enhanced G(s)-coupling by the receptor resulting in increased activation of adenylate cyclase with concomitant increase in cAMP production. CONCLUSIONS: The observed molecular dysfunction in the Dahl S AngII/AVP receptor is consistent with increased tubular sodium and fluid reabsorption observed in Dahl S rats. Interestingly, the AngII/AVPr locus is within the narrowed chromosome 1 QTL region for blood pressure detected in different rat intercross linkage analyses. Altogether, the data strongly suggest that the AngII/AVP receptor is a hypertension susceptibility gene in the Dahl S rat model, as well as raises the hypothesis that it too underlies the chromosome 1 blood pressure QTL identified in other hypertension rat models.

Differential nNOS gene expression in salt-sensitive and salt-resistant Dahl rats.

BACKGROUND: Several indications exist to suggest that an impaired production of nitric oxide might have a role in the development of salt-sensitive hypertension. OBJECTIVE: To examine whether the gene expression of the nitric oxide synthases (NOS) is altered in the salt-sensitive Dahl rat compared with that in the salt-resistant Dahl rat. DESIGN AND METHODS: The abundance of NOS mRNA was measured by RNase protection assay in different organs of salt-resistant and salt-sensitive Dahl rats. In addition, the zonal expression of NOS genes in the kidney under salt load and salt restriction was determined. RESULTS: The abundance of endothelial NOS mRNA was similar between the salt-resistant and salt-sensitive Dahl rat strains in all organs. Inducible NOS mRNA was not detectable by RNase protection assay in any organ. Neuronal NOS (nNOS) mRNA expression, however, was about 50% lower in brain and kidney of salt-sensitive Dahl rats than in salt-resistant Dahl rats. Within the kidney, nNOS mRNA levels were significantly decreased in salt-sensitive Dahl rats compared with those in salt-resistant Dahl rats, in cortex, outer and inner medulla (50, 40 and 30%, respectively) under all dietary conditions. A comparison of renal nNOS gene expression in Dahl rats with that in salt-insensitive Sprague- Dawley rats revealed that the abundance of renal nNOS was similar in salt-sensitive Dahl and Sprague-Dawley rats, but was increased in salt-resistant Dahl rats relative to that in Sprague-Dawley rats. CONCLUSION: These data suggest that nNOS gene expression is increased in salt-resistant Dahl rats compared with that in salt-sensitive Dahl rats. This increased nNOS expression of the salt-resistant Dahl strain might play a part in compensating for a defect of renal salt excretion in the Dahl strains.

A mechanism of oxygen free radical production in the Dahl hypertensive rat.

OBJECTIVE: To determine if oxygen free radicals derived from xanthine oxidase are involved in the development of salt-induced hypertension. Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction and provide a mechanism for lesion formation. METHODS: Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a low-salt, high-salt or high-salt + tungsten diet for 4 wk. In vivo production of superoxide (O2-) was detected by the reduction of a tetranitroblue tetrazolium (TNBT) dye in the rat mesentery, while plasma hydrogen peroxide (H2O2) production levels were determined using a modified electrochemical electrode technique. RESULTS: The tungsten diet lowered the blood pressure of Dahl-S rats compared to high-salt-treated Dahl-S rats, but had no effect on blood pressure in Dahl-R rats. Light absorption of formazan deposits revealed that tungsten-treated Dahl-S rats had reduced TNBT staining along the endothelium of arterioles and venules compared to hypertensive, high-salt-treated Dahl-S rats. In addition, tungsten-treated Dahl-S rats had a lower plasma H2O2 concentration compared to hypertensive, high-salt-treated Dahl-S rats. CONCLUSIONS: These findings indicate that xanthine oxidase-derived oxygen free radicals are involved in the pathogenesis of salt-induced hypertension.

Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats.

The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+ exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were cultured, grown on coverslips, and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope-based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 h. Baseline [Ca2+]i values measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]i values were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM, which resulted in movement of Na+ out of and Ca2+ into these cells (reverse-mode Na+/Ca2+ exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i (Delta[Ca2+]i) with reduction in Nae was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+]i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Delta[Ca2+]i in R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction of R but not S MCs, an increase which was abrogated by prior 24-h PMA pretreatment and corresponded to reduction in the 70-kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+/Ca2+ exchange activity in MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+ exchange regulation pathway in MCs of S rats.