2K1C Model Research Articles

Intrarenal and sympathetic premotor neuron changes in response to denervation of ischemic kidney in renovascular hypertension

AT1R upregulation in the rostral ventrolateral medulla (RVLM) contributes to sympathoexcitation and arterial hypertension in the 2‐kidney,1‐clip (2K1C) model. Our aim was to investigate whether afferent and efferent renal sympathetic nerves play a role in the modulation of Ang II receptors expression in the central nuclei (RVLM and PVN) and renal lesion markers in renovascular hypertension. Renal denervation of clipped kidneys (DnX: visible bundles were dissected + 10% phenol) was performed in Wistar male rats (n=5/group) 4.5 weeks after clipping. Ten days after DnX the mean arterial pressure was significantly (p<.05) reduced (202 ± 6 vs 178 ± 9 mmHg) in the 2K1C rats. The AT1R was upregulated in the RVLM (73%) and PVN (43%) in the 2K1C group, DnX normalized these expressions in both nuclei. The AT2R expression was increased in the RVLM (76%) and PVN (76%) of 2K1C group and DnX reduced AT2R in both regions. Renal lesion was characterized by cortical interstitium α‐smooth muscle actin (α‐SMA) immunohistochemical expression. The α‐SMA expression was augmented in the clipped (67 fold) and nonclipped (127 fold) kidneys and DnX significantly reduced α‐SMA in both kidneys (9 and 25 fold, respectively) of 2K1C rats. DnX produced no changes in control rats. Thus, our data show that renal nerves may modulate Ang II receptors in the RVLM and PVN and contribute to renal lesion in the renovascular hypertension.Supported by FAPESP and CNPQ

Early damage in 2K1C hypertension is driven by cytokine generation of parenchymal cells

The objective of this study was to define a role for chemokine generation by parenchymal cells versus circulating inflammatory cells in the initiation of renal damage in the 2K1C model of renovascular hypertension. 129 SV mice were subjected to renal artery stenosis (RAS) by placement of a polytetraethylene cuff on the right renal artery. Systolic blood pressure increased within 2 days of surgery. The cuffed kidney developed tubular atrophy, whereas the contralateral kidney developed compensatory enlargement. Immunohistochemical staining of the cuffed kidney revealed less than 2 CD3+ T cells per 200x field, and rare F4/80+ macrophages or CD20+ B cells in the first 2 weeks following RAS, with progressive increases in macrophages and T cells throughout the 6‐week observation period. Despite the relative paucity of infiltrating inflammatory cells, there was a dramatic increase in chemokine generation within the first week following RAS, as assessed by qPCR analysis of renal cortical homogenates, with 25‐fold induction of MCP‐1, 47‐fold induction of RANTES, and 15‐fold induction of MIP‐2. Chemokine generation continued to increase throughout the 6 weeks, during which circulating inflammatory cells infiltrated the cuffed kidney. Increased expression of MCP‐1 was detected in tubular epithelial cells within 1 week of RAS. We conclude that chemokine generation precedes inflammatory cell influx in RAS.

Irbesartan improves autonomic tone in a mouse model of renovascular arterial hypertension

ObjectiveActivation of renin‐angiotensin‐aldsterone and sympathetic nervous system are consequences of renovascular arterial hypertension. Here, in a model of renovascular hypertension, the role of Angiotensin‐II blockade on autonomic tone was assessed.MethodsC57bl6n mice with implanted iliac‐artery catheters received a renal‐artery clip (2‐kidneys‐1 clip, 2K1C). Sham‐surgery mice served as normal controls. Blood pressure of unrestrained, conscious animals was recorded using telemetry. Animals were randomized to Irbesartan (10 mg/kg/d per orally) or vehicle for 7 days. Cumulative intensity of the low‐frequency (LF) band of systolic blood pressure variability (SBPV) (0.15 – 0.6 Hz) was considered as sympathetic tone, the high‐frequency band (2.5–5 Hz) of heart rate variability as parasympathetic tone. Spontaneous blood‐pressure changes were used for baroreflex‐sensitivity calculation using Hemolab software. One‐way ANOVA, Newman‐ Keul test were used to assess differences. P<0.05 was deemed significant.ResultsTherapeutic‐dose Irbesartan did not lower blood pressure in the 2K1C model of arterial hypertension (Figure). However, both baroreflex and parasympathetic tone were significantly augmented by Irbesartan. Lastly, the difference of sympathetic tone between 2K1C and normal group partially vanished. Selective blockade of sympathetic tone by Metoprolol and, likewise, parasympathetic tone by Atropine confirmed the changes induced by Irbesartan on either component of autonomic nervous system (data not shown). As a mechanism, baroreflex control of heart rate was shown to be improved by Irbesartan.ConclusionAngiotensin‐receptor blocker partially normalizes both baroreflex function and parasympathetic tone in a renovascular model of arterial hypertension.

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension.

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-β signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-β/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-β/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.

Nitric oxide at the CVLM is involved in the attenuation of the reflex bradycardia in renovascular hypertensive rats

Hypertension is associated to an increase in central oxidative stress and an attenuation of the baroreflex control of arterial pressure. The present study evaluated the effect of alterations in the levels of nitric oxide (NO) and superoxide anion in the caudal ventrolateral medulla (CVLM), a key area of the brainstem for the baroreflex control of arterial pressure, in renovascular hypertensive rats (2K1C). Baseline mean arterial pressure (MAP), heart rate (HR), and reflex bradycardia were evaluated 30days after renal artery occlusion in anesthetized (urethane, 1.2g/kg, i.p.) 2K1C or normotensive (SHAM) rats. The MAP, HR, and baroreflex control of HR were evaluated before and after CVLM microinjections of the non-selective NOS inhibitor l-NAME (10nmol), the NO precursor l-ARG (50nmol), or the antioxidant ascorbic acid, Vit C (10nmol). In both 2K1C and SHAM animals, CVLM microinjection of l-NAME produced a decrease in MAP, whereas l-ARG induced a significant increase in MAP. However, microinjection of Vit C into the CVLM produced a decrease in MAP and HR only in 2K1C and not in SHAM rats. Cardiovascular effects produced by microinjection of l-ARG into the CVLM were abolished by prior microinjection of l-NAME in the CVLM of 2K1C and SHAM rats. Microinjection of l-NAME into the CVLM increased the sensitivity of reflex bradycardia in 2K1C animals. In contrast, the CVLM microinjection of l-ARG reduced reflex bradycardia only in SHAM rats. Vit C in the CVLM did not change reflex bradycardia in either 2K1C or in SHAM rats. These results suggest that increased oxidative stress in the CVLM during hypertension contributes to the reduced baroreflex sensitivity and to maintain hypertension in the 2K1C model.

Antioxidant Protective Effects of the Resveratrol on the Cardiac and Vascular Tissues from Renal Hypertensive Rats

Background: Accumulating reactive oxygen species (ROS) is involved in cellular signaling and function disturbances due to the oxidative stress, which contributes to several diseases. The consequences of ROS activity represent an im-portant mechanism on the pathogenesis of vascular diseases, such as hypertension. Increased blood pressure observed in renal hypertension of the 2 kidneys-1 clip (2K-1C) model involves increased ROS levels in the cardiovascular system. Resveratrol, a polyphenolic compound primarily found in red wine, has many biological and pharmacological proper-ties. Considering the antioxidant properties of resveratrol, the present study was aimed to investigate the effects of the chronic treatment with resveratrol on cardiovascular system from renal hypertensive rats. Results: 2K-1C hypertension presented increased blood pressure, which was reduced at the end of the fifth week of resveratrol treatment. The cardiac hypertrophy index and the basal levels of ROS in rat aortic rings were also reduced by resveratrol treatment. Conclusions: The present findings clearly show the protective effects from resveratrol on the blood pressure, the car-diac growth and the vascular ROS generation in renal hypertension.

A novel vascular clip design for the reliable induction of 2-kidney, 1-clip hypertension in the rat.

The 2-kidney, 1-clip (2K1C) model has provided many insights into the pathogenesis of renovascular hypertension. However, studies using the 2K1C model often report low success rates of hypertension, with typical success rates of just 40-60%. We hypothesized that these low success rates are due to fundamental design flaws in the clips traditionally used in 2K1C models. Specifically, the gap widths of traditional silver clips may not be maintained during investigator handling and these clips may also be easily dislodged from the renal artery following placement. Therefore, we designed and tested a novel vascular clip possessing design features to maintain both gap width and position around the renal artery. In this initial study, application of these new clips to the left renal artery produced reliable and consistent levels of hypertension in rats. Nine-day application of clips with gap widths of 0.27, 0.25, and 0.23 mm elicited higher mean arterial blood pressures of 112 ± 4, 121 ± 6, and 135 ± 7 mmHg, respectively (n = 8 for each group), than those of sham-operated controls (95 ± 2 mmHg, n = 8). Moreover, 8 out of 8 rats in each of the 0.23 and 0.25 mm 2K1C groups were hypertensive, whereas 7 out of 8 rats in the 0.27 mm 2K1C group were hypertensive. Plasma renin concentrations were also increased in all 2K1C groups compared with sham-operated controls. In summary, this novel clip design may help eliminate the large degree of unreliability commonly encountered with the 2K1C model.

Role of oxidative stress reduction on AT 1 receptor‐dependent cardiovascular alterations in renovascular hypertension

Renovascular hypertension is associated with increased renal sympathetic nerve activity (rSNA), baroreflex dysfunction and oxidative stress. We tested whether AT1 receptor blockade would produce systemic antioxidant effects and, comparatively, whether oxidative stress reduction with an antioxidant would produce cardiovascular alterations. Losartan (Lo, 30mg/kg/day) or vitC (vC,150mg/kg/day) were orally administered for 1 week by the 6th week after left renal artery clipping (2K1C). Systemic oxidative stress was evaluated by the balance of blood concentrations of TBARS and glutathione (GSH). Both Lo and vC significantly (p<0.05) normalized TBARS (from 1.6±0.1 to 1.0±0.1 and 1.1±0.1nmol/ml, respectively) and GSH (from 3.4±0.4 to 5.2±0.3 and 5.2±0.2μmol/g Hb) in the 2K1C group. Hypertension (172±3mmHg) and rSNA (162±4spikes/s) were significantly reduced after Lo (115±9 and 96±8) and vC (133±6 and 118±12). Both Los and vC increased renal baroreflex sensitivity for pressor (67% and 105%,) and depressor responses (140% and 120%,) in the 2K-1C rats. Thus, our data suggest that AT1-dependent cardiovascular dysfunctions in the 2K1C model are largely mediated by systemic oxidative stress. Supported by FAPESP

Role of cytochromeP-450 metabolites in the regulation of renal function and blood pressure in 2-kidney 1-clip hypertensive rats

Alterations in renal function contribute to Goldblatt two-kidney, one-clip (2K1C) hypertension. A previous study indicated that bioavailability of cytochrome P-450 metabolites epoxyeicosatrienoic acids (EETs) is decreased while that of 20-hydroxyeicosatetraenoic acids (20-HETE) is increased in this model. We utilized the inhibitor of soluble epoxide hydrolase cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB) and HET-0016, the inhibitor of 20-HETE production, to study the role of EETs and 20-HETE in the regulation of renal function. Chronic c-AUCB treatment significantly decreased systolic blood pressure (SBP) (133 ± 1 vs. 163 ± 3 mmHg) and increased sodium excretion (1.23 ± 0.10 vs. 0.59 ± 0.03 mmol/day) in 2K1C rats. HET-0016 did not affect SBP and sodium excretion. In acute experiments, renal blood flow (RBF) was decreased in 2K1C rats (5.0 ± 0.2 vs. 6.9 ± 0.2 ml·min(-1)·g(-1)). c-AUCB normalized RBF in 2K1C rats (6.5 ± 0.6 ml·min(-1)·g(-1)). HET-0016 also increased RBF in 2K1C rats (5.8 ± 0.2 ml·min(-1)·g(-1)). Although RBF and glomerular filtration rate (GFR) remained stable in normotensive rats during renal arterial pressure (RAP) reductions, both were significantly reduced at 100 mmHg RAP in 2K1C rats. c-AUCB did not improve autoregulation but increased RBF at all RAPs and shifted the pressure-natriuresis curve to the left. HET-0016-treated 2K1C rats exhibited impaired autoregulation of RBF and GFR. Our data indicate that c-AUCB displays antihypertensive properties in 2K1C hypertension that are mediated by an improvement of RBF and pressure natriuresis. While HET-0016 enhanced RBF, its anti-natriuretic effect likely prevented it from producing a blood pressure-lowering effect in the 2K1C model.

Superoxide anions in the paraventricular nucleus mediate the enhanced cardiac sympathetic afferent reflex and sympathetic activity in renovascular hypertensive rats

An enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation in renovascular hypertension. The present study was designed to determine the role of superoxide anions in the paraventricular nucleus (PVN) in mediating the enhanced CSAR and sympathetic activity in renovascular hypertension in the two-kidney, one-clip (2K1C) model. Sinoaortic denervation and vagotomy were carried out, and renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded under anesthesia. The CSAR was evaluated by the response of RSNA to the epicardial application of capsaicin. Superoxide anion levels and NAD(P)H oxidase activity in the PVN increased in 2K1C rats and were much higher in 2K1C rats than in sham-operated (sham) rats after the epicardial application of capsaicin or PVN microinjection of ANG II. In both 2K1C and sham rats, PVN microinjection of the superoxide anion scavenger tempol or the NAD(P)H oxidase inhibitor apocynin abolished the CSAR, whereas the SOD inhibitor diethyldithiocarbamic acid (DETC) potentiated the CSAR. Tempol and apocynin decreased but DETC increased baseline RSNA and MAP. ANG II in the PVN caused larger responses of the CSAR, baseline RSNA, and baseline MAP in 2K1C rats than in sham rats. The effects of ANG II were abolished by pretreatment with tempol or apocynin in both 2K1C and sham rats and augmented by DETC in the PVN in 2K1C rats. These results indicate that superoxide anions in the PVN mediate the CSAR and the effects of ANG II in the PVN. Increased superoxide anions in the PVN contribute to the enhanced CSAR and sympathetic activity in renovascular hypertension.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase‐2 activity and expression in a model of renovascular hypertension

Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg.kg(-1).day(-1)), HCTZ (20 mg.kg(-1).day(-1)) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Exercise Training in Two Kidney One Clip Rats Decreases Blood Pressure via Predominantly Nitric Oxide Mechanisms in Paraventricular Nucleus

Exercise training (ExT) decreases MAP in hypertension but the mechanism in the two kidney‐one clip (2K1C) model with Ang II and sympathetic components is not known. Plasma Ang II acts on circumventricular organs that project to paraventricular nucleus (PVN) whose efferent output influences sympathetic tone. We hypothesized that voluntary wheel ExT in 2K1C rats for 6 or 12 wk would enhance GABA and nitric oxide (NO) sympathoinhibitory mechanisms in the PVN. Bicuculline (BIC 200 pmol) or L‐NAME 200 μg was microinjected into PVN in awake sedentary (SED) or ExT sham clipped (SC) and 2K1C rats. At 6 wk ExT in SC or 2K1C did not change resting MAP or HR vs SED groups. BIC evoked greater rise in MAP and HR in SC ExT vs SED but not in 2K1C rats. After BIC, RSNA increased similarly in SED and ExT in both SC and 2K1C. At 12 wk resting MAP was 27 mmHg lower in 2K1C ExT vs SED rats (P&lt; 0.05). After L‐NAME in 2K1C, RSNA increased 24±9% in SED vs 79±16% in ExT (P&lt;0.02). After BIC, RSNA increased less in 2K1C ExT vs SED rats (P&lt;0.01). No differences in responses to L‐NAME or BIC occurred in SC SED vs ExT rats at 12 wk. In 2K1C rats when hypertension is not Ang II dependent (12 wk), ExT significantly decreases MAP. 12 wk but not 6 wk ExT in 2K1C rats exerts an inhibitory effect on RSNA mediated at least partly by PVN nitritergic mechanisms. After longer ExT inhibitory actions of NO on excitatory neurotransmitters/pathways in PVN may predominate over those for GABA. HL‐079102

Hydrogen Sulfide Inhibits Plasma Renin Activity

The development of renovascular hypertension depends on the release of renin from the juxtaglomerular (JG) cells, a process regulated by intracellular cAMP. Hydrogen sulfide (H2S) downregulates cAMP production in some cell types by inhibiting adenylyl cyclase, suggesting the possibility that it may modulate renin release. Here, we investigated the effect of H2S on plasma renin activity and BP in rat models of renovascular hypertension. In the two-kidney-one-clip (2K1C) model of renovascular hypertension, the H2S donor NaHS prevented and treated hypertension. Compared with vehicle, NaHS significantly attenuated the elevation in plasma renin activity and angiotensin II levels but did not affect plasma angiotensin-converting enzyme activity. Furthermore, NaHS inhibited the upregulation of renin mRNA and protein levels in the clipped kidneys of 2K1C rats. In primary cultures of renin-rich kidney cells, NaHS markedly suppressed forskolin-stimulated renin activity in the medium and the intracellular increase in cAMP. In contrast, NaHS did not affect BP or plasma renin activity in normal or one-kidney-one-clip (1K1C) rats, both of which had normal plasma renin activity. In conclusion, these results demonstrate that H2S may inhibit renin activity by decreasing the synthesis and release of renin, suggesting its potential therapeutic value for renovascular hypertension.

Characterization of cardiac remodeling in a large animal “one-kidney, one-clip” hypertensive model

Objective. The aim of this study is to characterize cardiac remodeling in a large animal model of hypertension. Methods. 23 sheep were subjected to unilateral nephrectomy followed by clamping of the remaining renal artery to 60% (“one kidney-one clip”, 1K1C) 3 weeks later. Blood pressure (BP) was monitored invasively over 73±28 days. Cardiac function was assessed with magnetic resonance imaging and compared with 12 size-matched controls. Detailed atrial histopathological analysis was performed. Results. In the 1K1C animals, BP rose from baseline to reach a plateau by 4 weeks (systolic BP: 107±12 to 169±27, diastolic BP: 71±10 to 118±29 mmHg, both p< 0.0001); cardiac hypertrophy was significant when compared with controls with increased left ventricular weight [left ventricular (LV)/body wt: 2.7±0.5 vs 2.1±0.2 g/kg, p=0.01] as well as bi-atrial enlargement (right atrial, RA: 22.9±4.9 vs 15.7±2.8g, p=0.003; left atrial, LA: 35.5±6.7 vs 20.9±4.1g, p=0.0003); cardiac magnetic imaging demonstrated significantly increased LA volumes (end-diastolic volume: 42.9±6.8 vs 28.7±6.3 ml, p< 0.0001) and reduced LA ejection fraction (24.1±3.6 vs 31.6±3.0%, p=0.001) while LV function was relatively preserved (42.3±4.7 vs 46.4±4.1%, p=0.1); degeneration and necrosis of atrial myocytes were evident with increased atrial lymphocytic infiltration and interstitial fibrosis. Conclusions. The ovine 1K1C model produces reliable and reproducible hypertension with demonstrable cardiac end-organ damage.

Rosiglitazone (peroxisome proliferator-activated receptor-gamma) counters hypertension and adverse cardiac and vascular remodeling in 2K1C hypertensive rats

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists have been shown controlling blood pressure (BP) in spontaneously hypertensive rats and salt-sensitive hypertensive rats. The present study aims to test the hypothesis that PPAR-gamma agonist rosiglitazone has beneficial effects on cardiac and vascular adverse remodeling in a model of renovascular hypertension (two-kidneys–one-clip, 2K1C model). Wistar rats were divided into four groups ( n=6): SHAM group, 2K1C, 2K1C+HYD (treated with hydralazine for 5 weeks) and 2K1C+ROSI (treated with rosiglitazone for 5 weeks). The left ventricle (LV), thoracic aorta (Ao) and common carotid artery (CCA) were analyzed. The BP did not show significant difference at the end of the experiment in groups 2K1C+ROSI, 2K1C+HYD and SHAM. The LV mass was smaller in 2K1C+ROSI compared with the other groups. The intima-media thickness was smaller in 2K1C+ROSI compared with untreated 2K1C ones, but not in 2K1C+HYD; 2K1C and 2K1C+HYD showed smaller Ao and CCA density of smooth muscle cell nuclei, and smaller surface density of the elastic lamellae than SHAM. The Ao and CCA circumferential wall tension and tensile stress were greater in 2K1C than in SHAM. Hypertrophied cardiomyocytes were seen in 2K1C, but not in 2K1C+ROSI and SHAM; 2K1C+ROSI had enhanced volume and length densities of intramyocardial arteries than 2K1C. The volume density of cardiac interstitium was greater in 2K1C and 2K1C+HYD than in SHAM. In conclusion, PPAR-gamma agonist rosiglitazone has beneficial effects controlling BP, reducing vascular adverse remodeling, and preserving intramyocardial vascularization in renovascular hypertensive rats (2K1C model).

Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension

Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-beta, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction.

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

EFFECTS OF CHRONIC VITAMIN C TREATMENT ON CARDIAC AND RENAL BAROREFLEXES IN RENOVASCULAR HYPERTENSION

Oxidative stress seems to be involved in the development and maintenance of renovascular hypertension. The aim of the present study was to evaluate the effects of chronic administration of an antioxidant ‐ vitamin C (VitC) ‐ on arterial blood pressure (AP), baroreflex control of heart rate (HR) and on renal sympathetic nerve activity (RSNA) in the two kidney one clip hypertension (2K‐1C) model. Control and 2K‐1C male Wistar rats were treated with two different doses of VitC (150 and 250mg/kg/day) by gavage for seven days. VitC 150mg/kg decreased basal AP in 2K‐1C (23±7mmHg, n=13) as well as in control (C: 12±3mmHg, n=12) rats, with no changes in HR. In contrast, VitC 250mg/kg did not reduce significantly AP; however, there was a tendency of reduction in both groups. The baroreflex control of HR and RSNA was evaluated by bolus injection of phenylephrine. Both doses of VitC increased baroreflex sensibilitiy for bradicardic responses (C+Vit C150: 58%, n=6; 2K‐1C+VitC150: 47%, n=9; C+VitC250: 49%, n=8; 2K‐1C+VitC250: 128%, n=7) and RSNA (Cl+VitC150: 62%, n=6 and 2K‐1C+VitC150: 153%, n=6). Taken altogether, the present study suggests a possible role of oxidative stress in the baroreflex dysfunction in renovascular hypertension. Apparently, the actions of VitC on baroreflex control and on AP responses are independent. Supported by FAPESP.

Abstract 1173: Gq Signaling Mediates High Blood Pressure and Hypertrophy in Two Divergent Models of Hypertension

Hypertension (HBP) is associated with increased levels of AngII and NE, which bind to Gq-coupled receptors. Many animal models and humans with HBP have increased levels of G protein receptor kinase 2 (GRK2) in vascular smooth muscle(VSM), which causes HBP. We tested whether inhibiting VSM Gq signaling by expressing of an intracellular peptide (GqI) decreased HBP and cardiac hypertrophy, in two divergent murine models of HBP: the 2 kidney, 1 clip (2K1C)model and a genetic mouse model that overexpresses G protein receptor kinase 2 (GRK2) in VSM. Methods: GqI mice and their non-transgenic littermate controls (C) were subjected to 2K1C. Conscious BP was measured via radiotelemetry and echo(m-mode) was performed before and 4 wks post 2K1C. Increasing doses of AngII were added to aortic rings. To test the efficacy of GqI in the 2K1C model, we also treated mice with Losartan, an AngII-receptor blocker. We crossbred GRK2 mice (have HBP) with GqI. We measured BP, echo, and did immunohistology on these hearts. Results: GqI mice had lower BP vs. C after 2K1C (111 ± 3 vs. 125 ± 4 mmHg, n = 8,8). Although, there was a decrease in BP in GqI mice following 2K1C, cardiac hypertrophy still occurred. There were similar increases in ventricular wall thickness (VWT) between C and GqI 4 weeks post 2K1C (C: 1.00 ±.05 vs. .79 ±.03 mm, n = 10,15; GqI: .93 ± .03 vs. .76 ± .02 mm, n = 20,19). There were also similar decreases in cardiac function after 2K1C measured by fractional shortening (FS) in C (35.7 ± 1.3 vs. 26.0 ± 1.9%, n = 10,15) and GqI (34.2 ± 0.8 vs. 25.9 ± 1.4, n = 20,19). The dose response curve to AngII in aortic rings was right-shifted 2.4 fold in GqI mice vs. C, suggesting decreased AngII sensitivity in GqI mice. Losartan normalized VWT in both C and GqI after 2K1C (0.77 ± .03 and 0.77 ± .02 mm) and FS (32.9 ± 0.7 and 32.0 ± 0.9%, n = 10,8). Hybrid GRK2/GqI mice had decreased BP vs. GRK2 (87.5 ± 6.3 vs. 111.6 ± 3.1 mmHg, n = 5,7). Hybrid GRK2/GqI mice also had less VSM hypertrophy than GRK2 (23.8 ± 1.9 vs. 32.9 ± 2.5 um, n = 6,3). Conclusions: We show that Gq signaling is important in 2 models of HBP. It appears that Gq signaling contributes to high BP through both AngII signaling and VSM hypertrophy. These data suggests that inhibition of VSM Gq signaling may provide future therapeutic strategies to treat HBP and HF.

Development of potent oral nanoparticulate formulation of coenzyme Q10 for treatment of hypertension: Can the simple nutritional supplements be used as first line therapeutic agents for prophylaxis/therapy?

Coenzyme Q10 (CoQ10) is an antioxidant with well-established pharmacological activities against several chronic diseases; however, it is marketed only as a nutritional supplement without any claims of its therapeutic activity and one of the reasons for this could be the poor oral bioavailability rendering difficulties in administering this molecule to achieve therapeutic concentrations. Therefore, the present investigation was aimed at improving the oral bioavailability of CoQ10 by delivering it as nanoparticulate formulation. Biodegradable nanoparticulate formulations based on poly(lactide-co-gylcolide) (PLGA) were prepared by emulsion technique using quaternary ammonium salt didodecyldimethylammonium bromide (DMAB) as a stabilizer. The effect of initial CoQ10 loading on entrapment efficiency and the particle size was studied using 5–75% initial load resulting in good entrapment efficiency (61–83%) without any appreciable increase in the particle size for 5–30% loading (107–110 nm). However, 50% and 75% led to increase in particle size with no appreciable changes in entrapment efficiency. The intestinal uptake of CoQ10 as a suspension in carboxymethylcellulose (CMC), a commercial formulation and the developed nanoparticulate formulation was studied in male Sprague–Dawley (SD) rats and found to be 45%, 75% and 79%, respectively, suggesting that solubility and permeability related problems of CoQ10 were overcome by nanoparticulate formulation. Furthermore, the developed nanoparticulate formulation was evaluated for its therapeutic potential in renal hypertensive animals (Goldblatt 2K1C model), demonstrating improved efficacy at a 60% lowered dose as compared to CoQ10 suspension and superior efficacy than the commercial formulation at an equal dose. Together, these results indicate the potential of nanotechnology in improving the therapeutic value of molecules like CoQ10, facilitating its usage as first line therapeutic agent thus revolutionizing its role in current medical therapy.