2K1C Model Research Articles

Benefical therapeutic effect of Chinese Herbal Xinji'erkang formula on hypertension-induced renal injury in the 2-kidney-1-clip hypertensive rats.

Increase in evidence shows that the role of kidney injury in hypertension is important. Xinji'erkang (XJEK), a Chinese herbal formula, has been identified as an effective preparation in the treatment of coronary heart disease and myocarditis. We have previously demonstrated that XJEK attenuate oxidative stress and hypertension target organ damage. The aim of this study was to assess the renal protective function of XJEK. Two Kidney One Clip (2K1C) model was adopted to induce hypertension in rats. We submitted male Sprague Dawley (150-180) g rats to either renal artery clipping or sham operation. Renal hypertension was established after four weeks of surgery. Rats were randomized divided into the four groups: sham-operated group (Sh-Op) (n=10), two-kidney, one-clip hypertension group (2K1C) (n=10), Xinji'erkang treatment group (XJEK) (n=10) and Fosinopril (n=10) treatment group. Drugs were administered orally daily for four weeks. Systolic pressures were measured every week using the tail-cuff apparatus. 24h before death, urine samples were collected for detect of urinary proteins. The kidney weight (KW) index was expressed as kidney weight/body weight (KW/BW). The histological changes were investigated by hematoxylin and eosin and Van Gieson staining. Immunohistochemical assay was employed to observe the intra-renal transforming growth factor-β1 (TGF-β1) protein expression. Serum creatinine (SCR) and blood urea nitrogen (BUN) were assayed by automatic biochemical analyzer. ELISA kit was used to assay Angiotensin II (Ang II) and TGF-β1 content in serum. Administration of XJEK markedly alleviated the rise in blood pressure and declined LKW/BW ratio. Histo-pathological injuries including hypertrophic glomerular, glomerular sclerosis, glomerular and interstitial fibrosis were attenuated. XJEK also decreased SCR, BUN, urinary proteins in 24h urine, serum Ang II and TGF-β1 concentrations and the intra-renal TGF-β1 protein expression. XJEK therapy in the 2K1C hypertensive rats affects the rise in blood pressure and ameliorates the severity of kidney injury. The protective effect is most likely due to the ability of XJEK to affect the Renin-Angiotensin-Aldosterone System (RAAS) and the TGF-β systems.

Evaluation of CART-, glucagon-, and insulin-immunoreactive cells in the pancreas of an experimental rat model of unilateral renal artery stenosis.

Hypertension is one of the most frequently occurring diseases worldwide. Approximately 10% of the population with hypertension reveal the secondary type of hypertension. The aim of this study was to evaluate the cells containing CART, insulin and glucagon in the pancreas of rats with renovascular hypertension. An experimental model of hypertension in rats according to Goldblatt (2K1C model of hypertension) was used in the study. The experimental material (pancreas) was collected in the 6th week of the study. Cells containing CART, insulin and glucagon were evaluated using immunohistochemical and morphometric methods. Pancreatic islet cells were evaluated based on the number and intensity of staining. The investigation showed an increase in the number and immunoreactivity of CART containing cells, 6 weeks after partial unilateral ligation of the renal artery. There was a significant decrease in the number of glucagon-IR cells. Although intensity of staining these cells did not change. No differences were observed in the number and staining affinity of insulin-containing cells. On the basis of the study it can be stated that the endocrine system of pancreas undergoes changes in the course of renovascular hypertension. This may affect the production of hormones and contribute to the development of possible hypertension complications.

Deficient prolylcarboxypeptidase gene and protein expression in left ventricles of spontaneously hypertensive rats (SHR)

Prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine peptidase that inactivates angiotensin II and activates pre-kallikrein, is thought to have anti-hypertensive and anti-proliferative roles in cardiovascular homeostasis. We hypothesized that PRCP function may be altered in heart tissue under conditions that predispose to left ventricle hypertrophy (LVH) in rats. We therefore used real-time PCR and western-blotting to examine the mRNA and protein expression of PRCP in the hearts of spontaneously hypertensive rats (SHR) at pre-hypertensive (5-week-old) and hypertensive (16-week-old) stages compared with age-matched hypertensive (2 kidney-1 clip; 2K-1C) rats and normotensive Wistar rats. PRCP mRNA expression was significantly reduced in hearts of 5- and 16-week-old SHR compared with age-matched Wistar controls, 2K-1C hypertensive rats and sham-operated normotensive rats. There were no significant differences in the PRCP mRNA and protein expression levels in hearts from hypertensive renovascular and sham-operated normotensive rats. Prolonged treatment of SHR with the AT1 receptor antagonist losartan (40mg/kg, gavage for 8 weeks) reduced the left ventricular weight/body weight ratio (LVW/BW), as well as the mRNA expression of collagen type 1, collagen type 3 and MMP9 in left ventricular tissue, without affecting PRCP gene and protein expression. Our results suggest that diminished PRCP gene and protein expression might be constitutionally involved in the SHR phenotype. In addition, since neither the development of arterial hypertension in the 2K-1C model nor its successful treatment in SHR altered PRCP gene and protein expression in heart tissue, it appears unlikely that PRCP function is regulated by the renin–angiotensin system or by afterload conditions.

Quantitative and qualitative evaluation of CART-containing cells in adrenal glands of male rats with hypertension

Adrenal activity is stimulated and secretion of stress hormones is increased during advanced stages of renovascular hypertension. The literature suggests that the neuropeptide, cocaine and amphetamine regulated transcript (CART), might regulate adrenal secretory function and thus could influence its activity. We assessed potential quantitative and qualitative changes in the cells that contained CART in the adrenal glands of rats with renovascular hypertension. The renal arteries of ten rats were subjected to a clipping procedure, i.e., two-kidney one-clip (2K1C) model of arterial hypertension, and after 6 weeks each rat developed stable hypertension. CART was localized using immunohistochemistry. CART was detected in a large population of cells in the medulla, sparse nerve fibers in the cortex and the capsule of the adrenal gland. The population of CART-positive cells in adrenal glands of two kidney-one clip (2K1C) treated rats was greater and their immunoreactivity was increased compared to controls. Similarly, the length, width, area and diameter of CART-immunoreactive cells were significantly greater in the hypertensive rats than in controls. We demonstrated that renovascular hypertension alters the number and immunoreactivity of CART-containing cells in adrenal glands.

Comparison of AMG 416 and cinacalcet in rodent models of uremia

BackgroundAMG 416 is a novel peptide agonist of the calcium-sensing receptor (CaSR). This report describes the activity of AMG 416 in two different rodent models of uremia, compared in each case to cinacalcet, an approved therapeutic for secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease on dialysis.MethodsAMG 416 was administered as a single intravenous (IV) bolus in a severe, acute model of renal insufficiency (the “1K1C” model) and plasma parathyroid hormone (PTH) and serum calcium levels were monitored for 24 hours. In a chronic, less severe model of renal dysfunction, the 5/6 nephrectomy (5/6 Nx) model, AMG 416 was administered as a once-daily IV bolus for 28 days. Both studies included a control (vehicle) group and a comparison cinacalcet group (po dosing at 30 mg/kg and 10 mg/kg for the 1K1C and 5/6 Nx studies, respectively).ResultsAdministration of AMG 416 by IV bolus injection into rats with acute renal dysfunction (1K1C model) resulted in a sustained reduction in plasma PTH from the initial elevated values. Following a single IV bolus (0.5 mg/kg), AMG 416 caused a substantial drop in PTH levels which remained approximately 50% below their initial level at 24 hrs. In the same model, oral treatment with cinacalcet (30 mg/kg) resulted in an acute drop in PTH which almost returned to the starting level by 24 hours after dosing. In the 5/6 Nx chronic uremia model, daily IV dosing of AMG 416 over 4 weeks (1 mg/kg) resulted in a sustained reduction in PTH, with approximately 50% of the initial level observed 48 hours post treatment throughout the study. Cinacalcet treatment (10 mg/kg) in the same model resulted in acutely lowered plasma PTH levels which returned to placebo levels by 24 hours post-dose. Consistent with the reductions in plasma PTH, reductions in serum calcium were observed in both AMG 416- and cinacalcet-treated animals.ConclusionsAs a long-acting CaSR agonist suitable for administration by the IV route, AMG 416 is a potential new therapy for the treatment of CKD patients with SHPT receiving hemodialysis.

Nucleic acid binding of annexin A2 is regulated through angiotensin II/AT1 signaling in kidneys of hypertensive rats (1088.2)

The aim was to identify new targets that regulate gene expression at the post‐transcriptional level in hypertension. Heparin affinity chromatography was applied to enrich putative nucleic acid‐binding proteins from kidneys of 2K1C hypertensive rats. A 0.2mm silver clip was used to induce hypertension (sham: 109±8 mmHg, 2K1C: 146±6 mmHg, P<0.05). We found a 36 kDa protein that showed an increased affinity to the nucleic acid mimetic in the 2K1C model. This factor was identified by LCMSMS and western blotting as Annexin A2 (ANXA2), a RNA‐binding protein known to influence mRNA stability and translation. Immunofluorescence staining revealed widespread distribution of ANXA2 throughout the kidney with high levels in endothelial cells, the cells of the Bowman capsule and in the medullary collecting ducts. Infusion of angiotensin II (AngII) reproduced our findings in the 2K1C animals and the AT1a blocker Losartan abolished the effect on Annexin A2. Strikingly, we observed no change in total Annexin A2 protein levels or localization, suggesting that post‐translational modification modulates Annexin A2 function via ANGII/AT1a signalling. In conclusion, AngII regulates the nucleic acid binding activity of Annexin A2 in the kidneys of rats with hypertension and, thus, may be an important regulator of gene expression in this setting.Grant Funding Source: Supported by Lars Hierta Foundation, Magnus Bergvall Foundation, Swedish Society for Medical Researc

Evaluation of polyherbal formulation (SJT-HT-03) for antihypertensive activity in albino rats.

Background:Hypertension is an incurable pathological condition and lifelong therapy is required. Long term use of conventional synthetic anti-hypertensive drugs is associated with a spectrum of toxic effects. However, therapeutic interventions using herbal drugs for hypertension have gained considerable attention worldwide.Aim:To evaluate the anti-hypertensive activity of polyherbal formulation (SJT-HT-03).Materials and Methods:The polyherbal formulation (SJT-HT-03) comprises of leaves of Aegle marmelos L., fruits of Benincasa hispida Thunb., Garcinia indica Thouars, and flowers of Musa paradiasica L., Rosa indica L., Hibiscus rosa sinensis L. Selected plants as mentioned above were collected, dried and extracted with different solvents. Formulation SJT-HT-03 (250 mg/kg, p.o.), was evaluated using two kidney one clip (2K1C) model and deoxycorticosterone acetate (DOCA)-salt-induced hypertension model using the enalapril (10 mg/kg, p.o.) and hydrochlorothiazide (5 mg/kg, p.o.) as a reference standard drug in respective models.Results:SJT-HT-03 significantly reduced (P < 0.001, one-way analysis of variance followed by Turkey's multiple comparison tests) systolic as well as diastolic blood pressure (BP) in 2K1C and DOCA-salt model. Further, SJT-HT-03 has shown a significant reduction (P < 0.01) in angiotensin converting enzyme (ACE) activity in serum, clipped kidney as well as in lungs in 2K1C model, whereas significant reduction (P < 0.05) in serum Na+ and increase in serum K+ level in DOCA model.Conclusion:Polyherbal formulation SJT-HT-03 possess significant anti-hypertensive activity by producing direct depressant effect on heart, inhibition of ACE, aldosterone antagonistic as well as diuretic effect and thereby act on multiple targets to achieve optimal effect.

Sympathoactivation and rho-kinase-dependent baroreflex function in experimental renovascular hypertension with reduced kidney mass.

BackgroundDysregulation of the autonomic nervous system is frequent in subjects with cardiovascular disease. The contribution of different forms of renovascular hypertension and the mechanisms contributing to autonomic dysfunction in hypertension are incompletely understood. Here, murine models of renovascular hypertension with preserved (2-kidneys-1 clip, 2K1C) and reduced (1-kidney-1 clip, 1K1C) kidney mass were studied with regard to autonomic nervous system regulation (sympathetic tone: power-spectral analysis of systolic blood pressure; parasympathetic tone: power-spectral analysis of heart rate) and baroreflex sensitivity of heart rate by spontaneous, concomitant changes of systolic blood pressure and pulse interval. Involvement of the renin-angiotensin system and the rho-kinase pathway were determined by application of inhibitors.ResultsC57BL6N mice (6 to 11) with reduced kidney mass (1K1C) or with preserved kidney mass (2K1C) developed a similar degree of hypertension. In comparison to control mice, both models presented with a significantly increased sympathetic tone and lower baroreflex sensitivity of heart rate. However, only 2K1C animals had a lower parasympathetic tone, whereas urinary norepinephrine excretion was reduced in the 1K1C model. Rho kinase inhibition given to a subset of 1K1C and 2K1C animals improved baroreflex sensitivity of heart rate selectively in the 1K1C model. Rho kinase inhibition had no additional effects on autonomic nervous system in either model of renovascular hypertension and did not change the blood pressure. Blockade of AT1 receptors (in 2K1C animals) normalized the sympathetic tone, decreased resting heart rate, improved baroreflex sensitivity of heart rate and parasympathetic tone.ConclusionsRegardless of residual renal mass, blood pressure and sympathetic tone are increased, whereas baroreflex sensitivity is depressed in murine models of renovascular hypertension. Reduced norepinephrine excretion and/or degradation might contribute to sympathoactivation in renovascular hypertension with reduced renal mass (1K1C). Overall, the study helps to direct research to optimize medical therapy of hypertension.

Mesenchymal Stem Cells (MSC) Prevented the Progression of Renovascular Hypertension, Improved Renal Function and Architecture

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×105 cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT1 were elevated, whereas AT2 levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.

Abstract 188: Loss of Renal Prolyl Carboxypeptidase in Mice With Chronic Kidney Disease

There are 26 million adults with chronic kidney disease (CKD) in the U.S. and the incidence continues to increase. It is well documented that the activation of the renin angiotensin system and the elevated formation of angiotensin (Ang) II both contribute to renal pathophysiology in CKD. Emerging evidence suggests that the Ang II degrading protease prolyl carboxypeptidase (PCP) is renoprotective. Thus, we investigated protein expression and activity of renal PCP using immunofluorescence, western blot and mass spectrometry in a mouse model of CKD. Renal injury in male C57Bl6 mice was caused by constriction of the left renal artery using silver clips (2K1C-method). Blood pressure measurements by radiotelemetry revealed a significant increase of 36.1 ± 3.9 mm Hg in 2K1C animals compared with control animals 1 week after clip placement (p&lt;0.0001). Using immunofluorescence and confocal microscopy, PCP was localized in the Bowman’s capsule of the glomerulus and in proximal and distal renal tubules. Western blot analysis showed PCP was significantly reduced in clipped 2K1C kidneys compared to unclipped kidneys of the 2K1C mice or compared to control mice (clipped 0.04 ± 0.02 vs unclipped 0.58 ± 0.16 vs control 0.65 ± 0.18, p &lt; 0.05). In addition, renal PCP enzyme activity was found to be markedly reduced in 2K1C kidneys as assessed by mass spectrometric based enzyme assays (clipped 37.1 ± 4.3 pmol Ang-(1-7)/h/μg vs unclipped 77.3 ± 12.3 pmol Ang-(1-7)/h/μg vs control 120.7 ± 14.7 pmol Ang-(1-7)/h/μg, p &lt; 0.01). In contrast, protein expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected. Notably, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion (clipped 34.6 ± 3.1 vs unclipped 52.1 ± 4.0 vs control 1.2 ± 2.1, p &lt; 0.0001) and renal fibrosis (clipped 57.5 ± 0.9 vs unclipped 33.0 ± 0.7 vs control 3.3 ± 0.2, p &lt; 0.0001). Results suggest that PCP is suppressed in chronic kidney injury and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PCP. Therefore, Ang II processing by PCP may have clinical implications in patients with renal pathologies.

Abstract 71: Novel Bach1 Modulators Increase HMOX1 and Suppress Hypertension in the Goldblatt Model of Renovascular Hypertension

We report for the first time a novel class of compounds that specifically modulate the Bach1 transcriptional repressor pathway. In cellula, the compounds selectively inhibit the activity of the transcriptional repressor Bach1 resulting in transcription of a network of antioxidant and cytoprotective genes including HMOX1. HPP-1971, a member of this class, is a potent and selective Bach1 inhibitor that induces HMOX1 &gt; 40-fold with a potency of 408 nM in human lung fibroblasts. To assess activity in vivo, we tested HPP-1971 in the Goldblatt model of renovascular hypertension. Sprague Dawley rats were implanted with in dwelling pressure telemeter probes and subsequently underwent sham surgery or placement of a 0.25 mm silver clip around the left renal artery. HPP-1971 treatment, dosed orally at 1,3,10 and 30 mpk, commenced three days following clip surgery, and continued for 18 days. At study completion, blood pressure, clipped kidney weight, renal HMOX1 enzyme activity and plasma aldosterone levels were measured (see Table). HPP-1971 attenuated both kidney atrophy and the increase in blood pressure in a dose dependent manner with significant differences seen at 3, 10 and 30 mpk (p&lt;.0001). HMOX1 enzyme activity in the clipped kidney increased with treatment, reaching a maximum of 5.7 ± 0.9 nM/hr/mg at 30 mpk relative to sham operated animals (p&lt;.0001). Plasma aldosterone levels increased in 2K1C animals compared to sham controls but were reduced by HPP-1971 treatment. These findings define a novel role for Bach1 suppressors in counteracting the influence of the RAAS system on hypertension and kidney atrophy in the 2K1C model of renovascular hypertension.

Abstract 55: PPAR-δ Binding to Heme Oxygenase 1 Promoter Prevents Angiotensin II Induced Vascular and Adipocyte Dysfunction in a Model of Renovascular Hypertension

Introduction: Renin-Angiotensin System (RAS) induces oxidative stress and contributes to various pathological conditions including insulin resistance and the metabolic syndrome. Recent studies have shown the role of PPARδ-agonist in attenuation of Angiotensin II induced oxidative stress.The aim of this study was to explore the effectiveness of a PPARδ-agonist in the prevention of Ang II-induced vascular and adipocyte dysfunction and the possible interaction between PPARδ and HO-1 system in a model of enhanced oxidative stress, the Goldblatt 2K1C model. Methods: We first established a direct stimulatory effect of the PPARδ-agonist (GW 501516) on the HO-1 gene by demonstrating increased luciferase activity in COS-7 cells transfected with a luciferase-HO-1 promoter construct. SD rats were divided in 4 groups: sham operated animals, 2K1C rats and 2K1C rats treated with GW 501516, in the absence or presence of the HO activity inhibitor, stannous mesoporphyrin (SnMP). Results: 2K1C animals had increased blood pressure, visceral adiposity, adipocyte hypertrophy, increased inflamatory cytokines, increased circulatory and adipose tisssue levels of renin and Ang II along with increased adipose tissue gp91 phox expression (p&lt;0.05) when compared to sham operated animals. Treatment with GW 501516 increased adipose tissue HO-1 and adiponectin levels (p&lt;0.01) along with enhancement of Wnt10b and β-catenin expression. HO-1 induction was accompanied by the decreased expression of Wnt 5b, Mest and C/EBPα levels and an increased number of small adipocytes (p&lt;0.05). These effects of GW501516 were reversed in 2K1C animals exposed to SnMP (p&lt;0.05). Conclusion: This novel study corroborates the existence of a PPARδ-HO-1 axis whose direct stimulation via the administration of an exogenous PPARδ-agonist induces HO-1 expression, abates RAS associated adipocyte and vascular dysfunction and induces adipocyte remodeling with smaller adipocytes while decreasing inflammation and increasing adiponectin secretion.

The stenotic kidney of Rag1 mice subjected to 2K1C hypertension is protected from atrophy (P3078)

Abstract Renal artery stenosis is a common finding in patients with cardiovascular disease. Recent studies have provided evidence that innate immunity plays a critical role in the development of chronic renal disease in renovascular hypertension. We have previously demonstrated that interstitial infiltration of lymphocytes is an early event in the development of renal atrophy in the 2K1C model of renovascular hypertension. We therefore sought to test the hypothesis that lymphocytes play a role in the development of progressive atrophy/fibrosis in this model. We subjected mice to renal artery stenosis through placement of a polytetrafluoroethylene cuff on the right renal artery. In accordance with our previous observations, wild type mice subjected to RAS developed renal atrophy within 2 weeks following placement of the cuff (70.5 +/- 11.9%). Chemokine expression in renal cortex of the stenotic kidney reflected predominantly a Th1 response (STAT1, STAT4, TBX21); CCL2, CCL17, CCL12, and TNF mRNA expression were also upregulated, but not IL-6 and IL-10. In striking contrast to wild type mice, Rag1 mice developed significantly less atrophy (23.1 +/- 10.4, p&amp;lt;0.0037 vs wild type). Unlike wild type mice, histologic evaluation of renal cortex of Rag1 mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. We propose that T cell-mediated inflammation plays a critical role in the development of renal atrophy in 2K1C mice.

Effects of Oryeongsan improved vascular relaxation in hypertension models

Body fluid volumes, which are regulated by the kidneys, are related to blood volume and arterial blood pressure. Oryeongsan (ORS), composition of five herbal medicines, has been widely used to traditional prescription for the treatment of body fluid disorders. The purpose of this study is to investigate the effect of ORS on vascular reactivity and related mechanism in the hypertension model. The experimental models were designed two types of hypertension as follows; 2 kidney 1 clip (2K1C) model and L‐NAME‐induced model. Both 2K1C‐ and L‐NAME‐ induced models were treated with two different dose of ORS (10 and 100 mg/kg/day) for 2 weeks. Blood pressure was markedly increased in both 2K1C and L‐NAME‐induced hypertension models. Interestingly, treatment with ORS significantly improved the impairments of acetylcholine (ACh)‐, sodium nitroprusside (SNP)‐, and atrial natriuretic peptide (ANP)‐induced vascular relaxation of aortic rings in both hypertension models. Treatment with ORS also significantly increased cyclic GMP (cGMP) levels of aortic rings (p&lt;0.01). In addition, both 2K1C and L‐NAME‐induced hypertension models increased ANP, aldosterone levels, and renin activity in plasma, by contrast, treatment with ORS attenuated those levels and activity. These results suggest that ORS ameliorates vascular dysfunction in 2K1C and L‐NAME‐induced hypertension models.

Losartan Reduces Oxidative Stress Within the Rostral Ventrolateral Medulla of Rats With Renovascular Hypertension

Previous studies showed that the microinjection of antioxidants or the overexpression of superoxide dismutase within the rostral ventrolateral medulla (RVLM) reduces hypertension and sympathoexcitation in the 2-kidney, 1-clip (2K-1C) model. In this study, we hypothesized that angiotensin II (ANG II) type 1 receptor (AT1R) is involved in the oxidative stress within the RVLM and contributes to cardiovascular dysfunction in renovascular hypertension. Losartan (30mg/kg/day, oral gavage) was administered for 7 consecutive days by week 5 after implantation of the clip (gap width = 0.2mm). Mean arterial pressure, baroreflex, and renal sympathetic nerve activity (rSNA) were evaluated. Superoxide production was evaluated by dihydroethidium (DHE) staining within the RVLM and within a control area. Systemic oxidative stress was characterized by measurement of thiobarbituric acid reactive substances (TBARS) and total glutathione (tGSH) in the blood. AT1R blockade significantly (P < 0.05) reduced hypertension by approximately 20% (n = 11) and sympathoexcitation to the kidneys by approximately 41% (n = 6) in the 2K-1C rats. Losartan treatment increased the baroreflex sensitivity of rSNA to pressor (67%) and depressor (140%) stimuli in the 2K-1C rats. AT1R blockade caused a significant (66%) reduction in DHE staining within the RVLM but not within the control area, reduced plasma TBARS (from 1.6±0.1 to 1.0±0.1 nmol/ml), and increased tGSH (from 3.4±0.4 to 5.2±0.3 μmol/g Hb) in the 2K-1C group only. Our findings suggest that the beneficial effects of ANG II blockade in renovascular hypertension are partly due to preferential reduction of oxidative stress in the RVLM.

Renin-Dependent Hypertension in Mice Requires the NLRP3- Inflammasome

Objective: Recent studies have implicated an enhanced secretion of IL1β through activation of the Nod-like receptor family, pyrin domain containing 3 (Nlrp3)-inflammasome as the pro-inflammatory signal in animal models of the gout, chronic kidney disease (CKD), atherosclerosis, metabolic syndrome, type 2 diabetes mellitus, but its contribution to hypertension is not established. We aimed to demonstrate the role of the Nlrp3-inflammasome in the two-kidneys, one clip (2K1C) Goldblatt model of hypertension in Nlrp3-/- and apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc)-/- and wild type control male mice. Study design: 2K1C hypertension was generated by narrowing left renal artery using U-shaped stainless steel clip. BP and heart rate were recorded intra-arterially with a computerized data-acquisition system in conscious mice. Plasma renin activity and concentration were measured by radioimmunoassay. Renal transcript levels of Nlrp3, Asc, Casp1, IL1A, IL1β, and IL6 were assessed by RT-Q-PCR. Results: Results show that Nlrp3-/- and Asc-/- mice are protected from developing hypertension and have lower circulating levels of plasma renin and serum amyloid A (SAA) and IL6, in comparison to wild type (WT) control mice. RNA levels of SAA, Nlrp3, and IL1β are increased in the ischemic kidney of the WT control mice. Administration of anti-IL1β antibody to the WT control mice attenuated the increase of blood pressure and renin in the 2K1C model. The results suggest that NALP3 inflammasome has an impact on blood pressure and renin secretion during renal hypoperfusion induced by renal arterial clip and contributes to renin-dependent renovascular hypertension. Conclusion: These findings show that Nlrp3-inflammasome mediated production of IL1 is linked to the development of renovascular hypertension and might be a novel target for the treatment of renovascular hypertension, CKD and other inflammatory diseases with hypertension.

277 DEVELOPING A CANINE MODEL TO TEST ELECTROACUPUNCTURE TREATMENT OF HYPERTENSION

Introduction: : Hypertension causes cardiovascular mortality and morbidity. Recent studies of electroacupuncture (EA) have demonstrated benefits in rodent models of hypertension, but EA has not been proven in larger animal models and human trials report mixed results. Methods: A canine model of hypertension was developed using the Goldblatt 1K1C method, removing one kidney and clipping the renal artery (RA) on the remaining kidney to reduce flow up to 80%. Implantable hemodynamic monitors were implanted to record acute and chronic arterial blood pressure (BP). EA was delivered at the ST-36 by an external stimulator or an implanted lead/stimulator. Continuous ambulatory BP was recorded before, during, and after EA stimulus to determine effects on BP. Results: Three of 8 dogs studied had complications from the 1K1C procedure and 1 dog removed from the study due to aggressive behaviour. Four dogs were followed for the duration of the study. Two had increases in BP in the first 2-4 weeks which returned to near baseline; it was subsequently confirmed that both clips had dislodged from the RA. The remaining 2 dogs had sustained increases in BP with the IK1C procedure. Acute and chronic EA cycling over time produced no sustained changes in BP. Conclusions: In the canine 1K1C model, significant RA occlusion is required to develop hypertension but too much risks the animal's survival. Effective EA for hypertension in dogs requires a) use of alternate acupoints b) better identification of ST-36 and/or c) more effective stimulation methods than achieved in this study.

Intravital microscopy reveals endothelial dysfunction in resistance arterioles in Angiotensin II-induced hypertension

It is known that hypertension is associated with endothelial dysfunction and that Angiotensin II (Ang II) is a key player in the pathogenesis of hypertension. We aimed to elucidate whether endothelial dysfunction is a specific feature of Ang II-mediated hypertension or a common finding of hypertension, independently of underlying etiology. We studied endothelial-dependent vasorelaxation in precapillary resistance arterioles and in various large-caliber conductance arteries in wild-type mice with Ang II-dependent hypertension (2-kidney 1-clip (2K1C) model) or Ang II-independent (volume overload) hypertension (1-kidney 1-clip model (1K1C)). Normotensive sham mice were used as controls. Aortic mechanical properties were also evaluated. Intravital microscopy of precapillary arterioles revealed a significantly impaired endothelium-dependent vasorelaxation in 2K1C mice compared with sham mice, as quantified by the ratio of acetylcholine (ACh)-induced over S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced vasorelaxation (2K1C: 0.49±0.12 vs. sham: 0.87±0.11, P=0.018). In contrast, the ACh/SNAP ratio in volume-overload hypertension 1K1C mice was not significantly different from sham mice, indicating no specific endothelial dysfunction (1K1C: 0.77±0.27 vs. sham: 0.87±0.11, P=0.138). Mechanical aortic wall properties and endothelium-dependent vasorelaxation, assessed ex vivo in rings of large-caliber conductance (abdominal and thoracic aorta, carotid and femoral arteries), were not different between 2K1C, 1K1C and sham mice. Endothelial dysfunction is an early feature of Ang II- but not volume-overload-mediated hypertension. This occurs exclusively at the level of precapillary arterioles and not in conduit arteries. Our findings, if confirmed in clinical studies, will provide a better understanding of the pathophysiological mechanisms of hypertension.

The role of AT1 receptor-mediated reproductive function in renovascular hypertension in male rats

There is an association between hypertension and reproductive dysfunction. Angiotensin II (Ang II) is involved in the pathogenesis of hypertension and the regulation of reproduction. The present study aimed to determine whether the angiotensinergic system mediates the effects of hypertension on reproductive function in male rats subjected to a two-kidney, one-clip (2K1C) model. Sexual behavior parameters, gametogenesis and plasma concentrations of Ang II, testosterone, prolactin and corticosterone were evaluated in male rats 28days after 2K1C or sham surgery and losartan (Los) treatment (a type 1 angiotensin II (AT1) receptor antagonist) or vehicle (V) treatment. The animals were divided into Sham+V, 2K1C+V, Sham+Los and 2K1C+Los groups. The 2K1C+V group showed a hypertensive response, inhibition of sexual behavior, spermatogenesis dysfunction, and increases in plasma Ang II and prolactin. Conversely, plasma testosterone decreased, and plasma corticosterone remained constant. Losartan treatment normalized blood pressure and prevented the changes in plasma testosterone and prolactin, sexual behavior and spermatogenesis in the 2K1C+Los group. In addition, losartan treatment caused an additional increase in circulating Ang ll in both groups (Sham+Los and 2K1C+Los). Together, these results suggest that Ang ll, acting through the AT1 receptor, modulates behavioral and endocrine parameters of reproductive function during renovascular hypertension. In addition, the effects of circulating Ang II on plasma testosterone and prolactin seem to contribute to the spermatogenic and sexual dysfunctions in hypertensive rats.

The Classic Renovascular (Goldblatt) Hypertension (RVHT) is Mediated by Succinate/GPR91 Signaling

The novel metabolic receptor GPR91 is key in the activation of the renin‐angiotensin system (RAS). The ligand of GPR91 is the TCA‐cycle intermediate succinate which quickly accumulates in hypoxic tissues. Succinate injection iv in mice causes acute elevations in blood pressure (BP) which is RAS and GPR91 dependent. In the 2‐ kidney, 1‐clip (2K1C) model of RVHT, there is reduced blood flow to the clipped kidney (CK) and hypertension is renin (RAS)‐ dependent. We tested the hypothesis that succinate/GPR91 signaling is involved in the development of RVHT using a novel approach to the 2K1C model as described by Lorenz et al. Wild type (WT) and GPR91 KO mice were either sham operated or a soft cuff was placed on the left renal artery. During 3 weeks, BP was measured daily, and urine and plasma collected weekly. At 3 weeks, systolic BP (SBP) was unchanged in both WT and KO sham groups (WT: 108±2 to 105±14; KO: 104±2 to 91±11 mmHg). SBP increased in cuffed WT from 111±0 to 131±8 mmHg (peaked at week 2 at 148±6 mmHg, P&lt;0.05), but did not change in the cuffed KO (103±13 to 106±9 mmHg). At week 3, kidneys were excised for further analysis. Renin expression increased 3.4±07‐ fold in WT CK, decreased 50% in WT non‐CK (NCK), and was unchanged in the KO CK and NCK. Renal fibrosis in the CK was also improved in GPR91 KO mice. We concluded that GPR91/succinate signaling is a major player in the control of intra‐renal renin (RAS) and BP in RVHT.