2K1C Group Research Articles

Acute superoxide scavenging restores depressed baroreflex sensitivity in renovascular hypertensive rats

In some pathological conditions such as hypertension, there is an impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In the present study we tested the hypothesis that acute superoxide scavenging would restore the reduced baroreflex sensitivity in renovascular hypertension. Male Wistar rats underwent 2-Kidney-1-Clip (2K1C) or sham surgery and were maintained untouched for six weeks to develop hypertension. After six weeks, animals from the 2K1C group were hypertensive when compared to the sham group (165 ± 9 vs. 108 ± 7 mmHg, P < 0.05). As a proof of principle for the hypertension model adopted, animals from the 2K1C group presented increased non-clipped kidney and cardiac mass index and reduced clipped kidney mass index. Regarding baroreflex, 2K1C rats presented diminished baroreflex sensitivity when compared to the sham group (2K1C + saline: −1.61 ± 0.15 vs. sham + saline: −2.79 ± 0.24 bpm mm Hg −1, p < 0.05). Moreover, acute administration of Vitamin C (150 mg/Kg, i.v.) restored baroreflex sensitivity in 2K1C rats (2K1C + Vit C: −3.08 ± 0.37 vs. 2K1C + saline: −1.61 ± 0.15 bpm mm Hg −1, p < 0.05). Furthermore, administration of apocynin (30 μg/Kg, i.v.), a NADPH oxidase inhibitor, also improved baroreflex sensitivity in the 2K1C group (2K1C + apocynin: −2.81 ± 0.24 vs. 2K1C + saline: −1.61 ± 0.15 bpm mm Hg −1, p < 0.05). In addition, autonomic blockade with either methylatropine or propranolol reduced the changes in heart rate to the same extent in all groups suggesting that improved baroreflex sensitivity by antioxidants were mediated by improvement in autonomic function. Taken together, these data suggest that NADPH oxidase-derived reactive oxygen species are involved in the blunted baroreflex sensitivity in renovascular hypertension and that acute scavenging of superoxide restores baroreflex sensitivity.

Neuronal nitric oxide synthase within paraventricular nucleus: blood pressure and baroreflex in two‐kidney, one‐clip hypertensive rats

The renin-angiotensin system is activated in the early phase of two-kidney, one-clip (2K-1C) hypertension. The paraventricular nucleus (PVN) integrates inputs regulating sympathetic outflow. The PVN receives inputs from plasma angiotensin II via projections from circumventricular organs and from renal afferent nerves transmitted via the nucleus tractus solitarii. Nitric oxide within the PVN may exert a sympathoinhibitory effect. These studies tested whether decreasing endogenous nitric oxide by introducing dominant negative (DN) constructs for neuronal nitric oxide synthase (nNOS) into PVN chronically augments hypertension and/or modulates baroreflex function. Male 6-week-old Sprague-Dawley rats underwent sham surgery or right renal artery clipping and placement of radiotelemetry transmitters. One week later, the PVN was injected bilaterally with 250 nl artificial cerebrospinal fluid containing 250 ng microl(-1) of RSV beta-galactosidase (beta-Gal), cytomegalovirus (CMV) wild-type (WT nNOS), or respiratory syncytial virus (RSV) haeme domain or RSV haemeRedF (DN nNOS). Haemodynamics were monitored for 5 weeks. Then left renal nerve electrodes were placed, and 2 days later the rats underwent baroreflex testing in the conscious state. The rise in mean arterial pressure (MAP) was significantly potentiated in the DN nNOS 2K-1C group beyond 15 days after PVN injection. By day 35, MAP in the 2K-1C groups was 152 +/- 6.3 (beta-Gal), 155.1 +/- 6.6 (WT nNOS) and 179 +/- 5.4 mmHg (DN nNOS; P < 0.01 versus all other groups). Sham-clipped rats remained normotensive. All groups displayed progressive bradycardia over time that was attenuated in the DN nNOS 2K-1C group. Baroreflex curves shifted to higher pressures, and baroreflex sensitivity of heart rate was diminished to a similar extent in all groups of 2K-1C rats. The baroreflex response of renal sympathetic nerve activity was preserved. The PVN tissue from DN nNOS rats had decreased dimerization of nNOS and generation of total nitric oxide. These findings indicate that chronic interference of nNOS dimerization required for generation of nitric oxide within the PVN potentiates the increase of blood pressure by modulating the sympathoexcitation that accompanies renovascular hypertension.

JUXTAMEDULLARY CORTEX HAS A HIGHER DEGREE OF RENAL DAMAGE THAN THE OUTER CORTEX IN THE NON-CLIPPED KIDNEY IN TWO KIDNEY ONE CLIP RATS AFTER LONG EXPOSURE TO HYPERTENSION: PP.29.136

Objective: The present study was designed to investigate the progression of hypertensive renal damage in the non-clipped kidney of the two kidney one clip model (2K1C), 24 weeks after clipping. Design: Animals underwent clipping of the left renal artery at the age of 6 weeks (approximately 180 g) and were studied 24 weeks after clipping. One group of 12 animals was assigned to the 2K1C group, while another group of 8 animals underwent sham operation. The blood pressure was measured during the experiment in both groups. Methods: The amount of renal damage was determined semi-quantitatively using a grading system and tissue from the outer (OC) and juxtamedullary cortex (JMC) of the non-clipped kidney was analysed for mRNA and protein expression using quantitative rt- PCR and western blotting. Results: The mean arterial pressure increased after 2–4 weeks, and was 166 ± 9 mmHg in the 2K1C group and 116 ± 7 mmHg in the control group after 24 weeks. The amount of tubulointerstitial damage was higher in JMC of 2K1C group compared to OC and the controls. The mRNA expression of col1a1, n-peptidase and TIMP-1 in the 2K1C was higher in the JMC than in the OC, and than the control group. We found no difference in the expression of c-peptidase. The amount of MMP-9 protein was lower in OC of 2K1C compared to the same compartment in sham animals. No difference was found for the protein expression of TIMP-2. Conclusions: Progression of renal damage from JMC towards OC cortex has been shown earlier in genetic, low renin model of hypertension. A similar pattern of response seems to occur in 2K1C, which is a high renin model. The increased expression of n-peptidase and up-regulation of TIMP-1 may be an important mechanism in the increased collagen. The present findings indicate that blood pressure per se is the most active factor in the development of renal fibrosis.

Antihypertensive effects of Ocimum basilicum L. (OBL) on blood pressure in renovascular hypertensive rats

Ocimum basilicum L. (OBL), sweet basil, is a medicinal herb used in traditional Chinese medicine to treat cardiovascular diseases including hypertension. The objective of the study was to investigate the possible antihypertensive effects of OBL extract in renovascular hypertensive rats. The two-kidney one-clip (2K1C) Goldblatt model of renovascular hypertension was used in Wistar rats. Rats were randomized into sham, untreated 2K1C, captopril- (30 mg kg(-1) per day orally) and OBL- (100, 200, 400 mg kg(-1) per day orally) (low (L)-, medium (M)-, high (H)-OBL) treated 2K1C groups (n=10-12 per group), followed up for 4 weeks. Blood pressure, heart weight/body weight, plasma angiotensin-II and endothelin (ET)-1 were studied. OBL reduced systolic and diastolic blood pressure by about 20 and 15 mm Hg, respectively, compared with 35 and 22 mm Hg for captopril, from the lowest dose tested with no dose dependency. Cardiac hypertrophy was reduced from 3.6+/-0.7 mg g(-1) for untreated 2K1C to 3.0+/-0.6, 2.9+/-0.6 and 2.4+/-0.4 mg g(-1) for L-, M- and H-OBL, respectively, compared with 2.6+/-0.5 for sham and 3.1+/-0.4 mg g(-1) for captopril (P<0.05). Renal function was improved with captopril. Angiotensin was reduced to a lesser extent than with captopril. ET was reduced to lower concentrations (78+/-15, 80+/-22, 82+/-15 pg ml(-1) for L-, M-, H-OBL, respectively) than in sham (116+/-31 pg ml(-1)), untreated 2K1C (174+/-72 pg ml(-1)) or captopril (117+/-72 pg ml(-1)) groups. The effects of OBL on blood pressure, cardiac hypertrophy and ET, are consistent with an effect on ET-converting enzyme, and warrant further exploration.

Elevated sympathetic activity precedes the arterial hypertension in the Goldblatt model

Despite the extensive use of the Goldblatt model of hypertension (two kidney one clip, 2K1C) sympathetic mechanisms responsible for both the development and maintenance of the elevated blood pressure remain unclear. We tested the hypothesis that sympathetic nerve activity (SNA) is elevated by the third week after clipping and before development of hypertension. Using the in situ working heart brainstem preparation, male Wistar rats were divided into three groups: Sham (n=7), and 2K1C (3 weeks) of which some were hypertensive (2K1C‐H, n=6) or and others normotensive (2K1C‐N, n=9) as determined a priori. At comparable perfusion rates, pressures were higher in both 2K1C groups (2K1C‐H: 76 ± 1; 2K1C‐N: 74 ± 3 vs Sham: 60 ± 2 mmHg, p&lt; 0.05). The respiratory modulation of SNA was significantly elevated in both 2K‐1C groups (e.g. 2K1C‐H: 47.7 ± 6.1; 2K1C‐N: 32.8 ± 2.8 vs Sham: 20.5 ± 2.5 μV, p&lt; 0.05). Pre‐collicular transection reduced SNA in all rat groups (2K1C‐H: 32.5 ± 7.5, 2K1C‐NH: 48 ± 6.9 vs Sham: 13.2 ± 1%, p&lt; 0.05). Subsequent transection at the medullary‐spinal cord abolished SNA in Sham and 2K1C‐N but decreased it by 57± 5.5% in 2K1C‐H. We conclude, SNA can be raised prior to onset of hypertension by the 3rd week after clipping and this, in part, originates from enhanced respiratory modulation. Supported by CAPES and British Heart Foundation.

Upregulation of AT1R and iNOS in the Rostral Ventrolateral Medulla (RVLM) Is Essential for the Sympathetic Hyperactivity and Hypertension in the 2K-1C Wistar Rat Model

We hypothesized that upregulation of angiotensin type 1 receptor (AT(1)R) and inducible nitric oxide (NO) synthase (iNOS) within the rostral ventrolateral medulla (RVLM) could contribute to two-kidney, one-clip (2K-1C) hypertension. The experiments were performed in male Wistar rats, 6 weeks after the renal surgery. The animals were divided into control (SHAM, n = 18) and hypertensive groups (2K-1C, n = 18). Bilateral tissue punches were taken from sections containing the RVLM to perform iNOS gene expression analyses by the real-time PCR technique, and AT(1)R and iNOS protein expression analyses by western blotting. In addition, we injected losartan (1 nmol), an AT(1)R antagonist, and aminoguanidine (250 pmol), an iNOS inhibitor, bilaterally into the RVLM to analyze the mean arterial pressure (MAP) and renal sympathetic nerve activity (rSNA). iNOS mRNA expression levels were greater (P < 0.05) in the 2K-1C group compared to the SHAM group. Furthermore, the AT(1)R and iNOS protein expression were significantly increased in the RVLM of 2K-1C rats compared to SHAM rats. Injection of losartan into the RVLM reduced the MAP (11%) and rSNA (18%) only in the 2K-1C rats, whereas injection of aminoguanidine in the same region decreased the MAP (31%) and rSNA (34%) in hypertensive rats. The present study suggests that upregulation of AT(1)R and iNOS in the RVLM is important in the maintenance of high blood pressure and renal sympathetic activation in 2K-1C hypertension.

Effects of chronic antioxidant treatment on AT‐1 receptor, NAD(P)H oxidase subunits and main antioxidant enzymes gene expression in the kidneys of 2K‐1C rats

Previous studies showed that chronic treatment with an antioxidant: vitamin C (vC) improves hypertension and sympathetic vasomotor hyperactivity in an Ang II‐dependent model of renovascular arterial hypertension. To test the hypothesis that oxidative stress is associated with NAD(P)H oxidase activation by Ang II, gene expressions of AT‐1 receptor (AT1R), NAD(P)H oxidase subunits (p47phox and gp91phox) and main antioxidant enzymes were evaluated in the renal cortex of 2 kidney‐1 clip model (2K1C, 6 weeks after clipping) rats before and after vC (150mg/kg/day). Gene expression of AT1R was elevated (51%) in the clipped kidney (CK) and reduced (47%) in the nonclipped kidney (NK) of 2K1C group. vC reduced AT1R expression only in the CK (31%). Elevated gene expression of p47phox and gp91phox was observed only in the CK of 2K‐1C group (184% and 132%, respectively) and vC produced no changes in these expressions. Gene expression of catalase was reduced in the CK (70%) and NK (83%) of 2K1C rats. vC increased gene expression of glutathione peroxidase (GPx) in the CK (185%) and NK (212%) of 2K1C. The present study suggests that renal oxidative stress may be related to elevated AT1R and NAD(P)H oxidase subunits gene expression in the CK of 2K1C rats and vitC treatment may produce beneficial effects by AT1R downregulation in the CK and increase in GPx gene expression in the kidneys of 2K1C rats.Supported by FAPESP (07/56925‐1)

Chronic Antioxidant Treatment Improves Arterial Renovascular Hypertension and Oxidative Stress Markers in the Kidney in Wistar Rats

Sympathetic vasomotor hyperactivity and baroreflex dysfunction are involved in the development and maintenance of renovascular arterial hypertension. We hypothesized that angiotensin (Ang) II-dependent oxidative stress contributes to the pathophysiology of the two-kidney, one-clip (2K-1C) model. The mean arterial pressure (MAP), baroreflex, and renal sympathetic nerve activity (rSNA) were evaluated after chronic administration of an antioxidant, vitamin C (vitC 150 mg/kg/day) in male Wistar 2K-1C rats. Additionally, the mRNA levels of Ang II subtype 1 receptor (AT(1)R), NAD(P)H oxidase subunits (p47phox and gp91phox), and major antioxidant enzymes were evaluated in the renal cortex. After vitC treatment, the MAP (170 +/- 4 vs. 133 +/- 6 mm Hg; P < 0.05) and rSNA (161 +/- 5 vs. 118 +/- 12 spikes/s; P < 0.05) were significantly reduced only in the 2K-1C group. VitC improved the baroreflex control of heart rate (HR) and rSNA. The expression of AT(1)R, p47phox, and gp91phox was elevated (51, 184, and 132%, respectively) in the clipped kidney of 2K-1C group. VitC downregulated AT(1)R in the clipped kidney (31%). Catalase (CAT) expression was reduced in clipped (70%) and nonclipped (83%) kidneys of 2K-1C rats. VitC treatment augmented the expression of glutathione peroxidase (GPx) in both clipped (185%) and nonclipped (212%) kidneys of the 2K-1C group. The present study suggests a role for oxidative stress in the cardiovascular and sympathetic alterations in renovascular hypertension, associated with changes in the expression of AT(1)R, NAD(P)H oxidase subunits, and antioxidant enzymes in the kidney.

CYP450 4A inhibition attenuates O 2 induced arteriolar constriction in chronic but not acute Goldblatt hypertension

We explored the role of 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) in oxygen-induced vasoconstriction in a normal renin form of hypertension [the 1 kidney-1 clip Goldblatt hypertensive rat (1K1C)] and a high renin form of hypertension [the 2 kidney-1 clip Goldblatt hypertensive rat (2K1C)]. A silver clip was placed around the left renal artery of adult Sprague–Dawley males. The right kidney was removed in the 1K1C group and left intact in the 2K1C group. Arteriolar responses to elevation of O 2 concentration in the superfusion solution from 0% O 2 to 21% O 2 were determined in the in situ cremaster muscle before and after inhibition of cytochrome P450 4A ω-hydroxylase (CYP450 4A) with N-methyl-sulfonyl-12, 12-dibromododec-11-enamide (DDMS). Arteriolar constriction to elevated PO 2 was enhanced in the chronic 1K1C but not the acute 1K1C or 2K1C. DDMS eliminated O 2-induced arteriolar constriction in the 9-week 1K1C, but had no effect in the 2-week 1K1C, and only partially inhibited O 2-induced constriction of arterioles in the 4-week 2K1C rat. These findings indicate that although the CYP4A/20-HETE system contributes to arteriolar constriction in response to elevated PO 2 in the established stage of 1K1C renovascular hypertension, physiological alterations in other mechanisms are the primary determinants of O 2-induced constriction of arterioles in the early and developing stages of 1K1C and 2K1C hypertension.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan [10 mg x kg(-1) x day(-1); N = 5] or the superoxide scavenger tempol [0.2 mmol x kg(-1) x day(-1); N = 5], which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Heart rate and arterial pressure variability in the experimental renovascular hypertension model in rats

This study was conducted in one kidney, one clip (1K1C) Goldblatt hypertensive rats to evaluate vascular and cardiac autonomic control using different approaches: 1) evaluation of the autonomic modulation of heart rate (HR) and systolic arterial pressure (SAP) by means of autoregressive power spectral analysis 2) assessment of the cardiac baroreflex sensitivity; and 3) double blockade with methylatropine and propranolol.The 1K1C group developed hypertension and tachycardia. The 1K1C group also presented reduction in variance as well as in LF (0.23±0.1 vs. 1.32±0.2 ms2) and HF (6.6±0.49 vs. 15.1±0.61 ms2) oscillations of pulse interval. Autoregressive spectral analysis of SAP showed that 1K1C rats had an increase in variance and LF band (13.3±2.7 vs. 7.4±1.01 mmHg2) in comparison with the sham group. The baroreflex gain was attenuated in the hypertensive 1K1C (−1.83±0.05 bpm/mmHg) rats in comparison with normotensive sham (−3.23±0.06 bpm/mmHg) rats. The autonomic blockade caused an increase in the intrinsic HR and sympathetic predominance on the basal HR of 1K1C rats. Overall, these data indicate that the tachycardia observed in the 1K1C group may be attributed to intrinsic cardiac mechanisms (increased intrinsic heart rate) and to a shift in the sympathovagal balance towards cardiac sympathetic over-activity and vagal suppression associated to depressed baroreflex sensitivity. Finally, the increase in the LF components of SAP also suggests an increase in sympathetic activity to peripheral vessels.

Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 ± 8 mmHg versus 167 ± 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P < 0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P < 0.05). A 21-fold increase ( P < 0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C + doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline.

Cardiac and aortic structural alterations due to surgically‐induced menopause associated with renovascular hypertension in rats

Menopause and hypertension independently alter cardiovascular remodelling, but little is known about their effect on left ventricular and aortic wall remodelling. Eight-weeks-old Wistar rats were divided into four groups of six animals each: Sham group, OVX group (ovariectomized rats), 2K1C (two-kidneys, one-clip rats) and OVX + 2K1C group and kept until 19 weeks. Blood pressure (BP) increased 12% in OVX group, 35% in 2K1C and OVX + 2K1C groups compared with sham group. Vaginal cytology showed Sham and 2K1C rats cycling normally, whereas OVX and OVX + 2K1C rats were persistently in dioestrus or proestrus. At euthanasia, left ventricle (LV) and thoracic aorta were removed and analysed (immunohistochemistry and stereology). LV mass/tibia length ratio and cross-sectional area of cardiomyocytes increased in all groups except Sham. The intramyocardial vascularization reduced 30% in comparison with Sham group, with no difference among OVX, 2K1C and OVX + 2K1C groups. The cardiac interstitium increased more than 45% in both 2K1C and OVX + 2K1C groups compared with Sham, but there was no significant difference between Sham and OVX groups. Nuclei number of LV cardiomyocyte diminished in OVX group, followed by 2K1C group and OVX + 2K1C group, with no difference between the 2K1C and the OVX + 2K1C groups. There was positive immunostaining for angiotensin II AT1 receptor in smooth muscle cell layer of aortic tunica media in all groups. These results show that both ovariectomy and renovascular hypertension enhance BP as a single stimulus and therefore produce adverse cardiac remodelling. However, renovascular hypertension exerts a far greater influence than surgically-induced menopause in this parameter.

Beneficial effects of docosahexaenoic acid on active avoidance performance in 1K-1C hypertensive rats

The present study evaluated the role of chronic docosahexaenoic acid (DHA) supplementation on active avoidance learning task performance in experimental hypertension. Male Wistar rats were randomly divided into five experimental groups as follows: control, sham, DHA treated, 1K-1C hypertensive, and 1K-1C hypertensive + DHA treated. Hypertension was induced in 1K-1C rats via placing a silver clip (0.20-mm ID) around the left renal artery following a right uninephrectomy. DHA (36 mg/kg/day) was given to the treatment groups for 60 days by gastric gavage. Arterial blood pressure was measured by using the tail-cuff method. Active avoidance responses were determined by an automated shuttle-box. In brain (cerebrum) and hippocampus tissues, thiobarbituric acid reactive substances (TBARS) and nitrite levels were measured by fluorometric methods. DHA supplementation decreased blood pressure in hypertensive rats. Data from active avoidance training indicated that performance of active avoidance learning tasks were significantly impaired in 1K-1C hypertensive rats, but was completely restored by DHA supplementation. Increased cerebrum TBARS levels in 1K-1C rats were abolished by DHA administration. Cerebrum nitrite levels were lower in the DHA, 1K-1C and 1K-1C + DHA treated groups compared to controls. Hippocampus nitrite levels were lower in DHA treated and 1K-1C hypertensive rats compared to controls and higher in 1K-1C + DHA treated rats compared to the 1K-1C group. Our data indicates that DHA supplementation improves the performance of active avoidance learning tasks which is impaired in experimental hypertension. These affirmative changes might be due to a DHA-induced decrease in lipid peroxidation which may in turn limit the consumption of nitric oxide (NO) which promotes active avoidance learning.

The role of docosahexaenoic acid on visual evoked potentials in one kidney‐one clip hypertension

To investigate the effects of polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) on visual evoked potentials (VEPs) in a one kidney-one clip (1K-1C) hypertension model in rats. Five experimental groups were formed: a control group, a sham group, a group supplemented with DHA, a 1K-1C group, and a 1K-1C + DHA group. The DHA groups were treated for 60 days. In the 1K-1C groups, the right kidney was removed and a silver clip with a 0.2-mm gap was placed on the left renal artery. The DHA-supplemented rats had lower blood pressure than their respective controls (p < 0.01). The increased brain and retina thiobarbituric acid reactive substances (TBARS) levels in hypertensive rats were abolished by DHA administration. The brain nitrite levels were lower in the DHA, 1K-1C and 1K-1C + DHA groups compared with the control group (p < 0.01), and the retina nitrite level was higher in the 1K-1C + DHA group compared with the DHA and 1K-1C groups (p < 0.01). There was an improvement of P(2), N(2) and P(3) components following DHA supplementation in 1K-1C hypertensive rats compared with the 1K-1C group. The present study suggests that DHA supplementation has the potential to prevent VEP changes caused by an experimental model of hypertension. This state might be related to the lipid peroxidation lowering effect of DHA.

Electroacupuncture on stomach 36 point (ST‐36) reduces hypertension through endothelial nitric oxide synthase (eNOS) activity in the stomach meridian

Acupuncture has been used in Asia to treat hypertension for a long time. We hypothesized that acupuncture on ST-36 activates eNOS signaling mechanisms to reduce hypertension. We tested the hypothesis using the hamster two-kidney, one-clip renal hypertension (2K1C) model. 30 minute daily electroacupuncture (EA) treatment for 5 days reduced mean arterial pressure from 160.0±7.6 to 129.5±8.8 mmHg (mean±SEM), compared to 115.0±7.2 mmHg in sham-operated hamsters. We used NO-sensitive microelectrodes to measure periarteriolar NO concentration. EA increased periarteriolar NO concentration from 82.3±10.2 nM to 135.1±12.6 nM in the 2K1C hamster cheek pouch microcirculation. Hypertension reduced eNOS protein relative to the sham-operated control, as measured by Western blotting. EA prevented the reduction of eNOS associated with hypertension and showed even higher eNOS expression than the sham-operated control in stomach and hamster cheek pouch tissues, which are on the stomach meridian. Analysis of liver tissue indicated that EA on ST-36 caused only a slight increase in eNOS expression in hypertensive animals compared with the non-treatment 2K1C group. The liver eNOS levels in 2K1C-treated hamsters were less than in sham-operated hamsters. We conclude 1) stimulation of eNOS is a mechanism stimulated by ST-36 elecroacupuncture to reduce blood pressure; 2) ST-36 elecroacupuncture-induced reduction of blood pressure works through the stomach meridian (supported by NIH grant 5RO1 HL 70634).

An increase in the synthesis and release of calcitonin gene-related peptide in two-kidney, one-clip hypertensive rats

Previous investigations have indicated that capsaicin-sensitive sensory nerves play an important role in modulation of the peripheral resistance of the circulation system. In the present study, we examined the role of capsaicin-sensitive sensory nerves in two-kidney, one-clip (2K1C) renovascular hypertension rats. Systolic blood pressure (BP) was monitored by the tail-cuff method throughout the experiment. Concentrations of calcitonin gene-related peptide (CGRP) in the plasma, the level of CGRP mRNA in dorsal root ganglia (DRG) and the density of CGRP immunoreactive (CGRP-ir) fibers in mesenteric artery were measured. Blood pressure was significantly elevated at day 10 postoperation (BP was 143±10 and 114±7 mm Hg for 2K1C and Sham groups, respectively, p<0.05). Treatment with capsaicin, which selectively depletes neurotransmitters in sensory nerves, enhanced hypertensive responses to clipping (BP was 168±7 and 143±10 mm Hg at day 10 postoperation for Cap1+2K1C and 2K1C groups, respectively, p<0.05), and BP in the rats treated with a second injection of capsaicin was greater than that in the rats treated with a single injection of capsaicin (At day 30 postoperation, BP was 199±7 and 166±9 mm Hg for Cap2+2K1C and 2K1C groups, respectively, p<0.01; mean arterial pressure was 185.2±6.6 and 150.5±4.1 mm Hg for Cap2+2K1C and 2K1C groups, respectively, p<0.01). The expression of α-CGRP mRNA in DRG (122.87±3.67 arbitrary units, p<0.05), the level of CGRP in the plasma (75.40±4.99 pg/ml, p<0.01) and the density of CGRP-ir fibers in mesenteric artery (525.67±31.42 intersections, p<0.05) were significantly increased in 2K1C rats. Treatment with capsaicin, a single injection or a second injection, prevented the increased in the expression of CGRP mRNA in DRG. However, the decreased level of CGRP was only observed in the rats treated with a second capsaicin. These results suggest that in 2K1C hypertensive rats, the activity of capsaicin-sensitive sensory nerves is increased, which is playing a compensatory depressor role to partially counteract the increase in blood pressure, and that the cardiovascular actions of CGRP is mediated by the α-CGRP isoform.

Effects of angiotensin-converting enzyme inhibition and calcium channel blockade on cardiac apoptosis in rats with 2K1C (two-kidney/one-clip) renovascular hypertension

Apoptosis plays a role in the regulation of heart mass and architecture, and might contribute to the cardiac remodelling seen in renovascular hypertension. It is not known whether the beneficial effects of angiotensin-converting enzyme (ACE) inhibition or calcium channel blockade on cardiac remodelling are linked to the modulation of apoptosis. To test this hypothesis, we established four groups of rats: (i) sham-operated controls, (ii) a group that underwent the two-kidney/one-clip (2K1C) procedure, (iii) a group with 2K1C treated for 12 weeks with quinapril (6mg·day-1·kg-1), and (iv) a group with 2K1C treated for 12 weeks with diltiazem (24mg·day-1·kg-1). Treatment started 2 weeks after clipping. Systolic blood pressure was reduced to a similar extent by quinapril and diltiazem (2K1C, 223±19mmHg; 2K1C+quinapril, 149±15mmHg; 2K1C+diltiazem, 160±40mmHg; both P&amp;lt;0.01 compared with 2K1C alone). Left ventricular weight, interstitial fibrosis and perivascular fibrosis were reduced significantly by both drugs. The apoptotic index (apoptotic cells/total cell number) was increased 21.6-fold (P&amp;lt;0.01) after quinapril treatment as compared with the 2K1C group, but was not affected by calcium channel blockade. In conclusion, our study demonstrates that ACE inhibition, in contrast with calcium channel blockade, may cause regression of cardiac hypertrophy/remodelling in 2K1C renovascular hypertensive rats through enhanced apoptosis.

Hypertension Plus Diabetes Mimics the Cardiomyopathy Induced by Nitric Oxide Inhibition in Rats

We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM). Prospective trial. University laboratory. Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester [L-NAME], 75 micro mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B(2) (TXB(2), the stable metabolite of thromboxane A(2)) were also measured. In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME-treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p < 0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB(2) concentrations. These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy.

Platelet-derived growth factor receptor-β in myocyte was upregulated by angiotensin II

To observe the regulatioon of platelet-derived growth factor (PDGF) receptor-β in myocyte stimulated by angiotensin II (AngII) at both integrated and cellular levels and reveal the signal transduction mechanism in cell, two kidneys, one clip (2K1C) renal hypertension were performed by placing a sliver clip around the left renal artery. Blood pressure and the ratio of left ventricular weight to body weight were measured at 4 and 8 weeks after operation. The content of AngII in heart was detected by radioimmunology assay; the protein level of PDGF receptor-β in heart was measured by Western blot analysis. The alteration of PDGF receptor-β stimulated by AngII and several inhibitors was observed on cultured neonatal rat ventricular myocyte (NRVM). The content of AngII in heart of 2K1C renal hypertensive rat at 4 and 8 weeks after operation was increased. Compared with sham group, 4 and 8 weeks after operation, PDGF receptor-β in heart of 2K1C group was upregulated by 100.3% and 127.1% (P< 0.05), respectively. This upregulation could be inhibited by captopril. For cultured myocyte, PDGF receptor-β was increased by 47.1% after being stimulated by AngII and this upregulation could be inhibited by losartan which was an inhibitor of AT1 receptor. PLC inhibitor (U73122) and MEK inhibitor (PD98059) could partly inhibit PDGF receptor-β upregulation induced by AngII. These results suggested that AngII could upregulate PDGF receptor-β in myocyte by its AT1 receptor and this effect was at least partly dependent on PLC and extracellular signal-regulated kinase (ERK).