2K1C Rats Research Articles

Chronic cannabidiol treatment induces cardiovascular improvement in renovascular hypertensive rats.

Cannabidiol (CBD) is increasingly studied for its therapeutic potential in neurodegenerative diseases. Previous research on acute CBD administration has demonstrated cardiovascular benefits in hypertensive rats, including reduced mean blood pressure and oxidative stress. To investigate the long-term cardiovascular effects of chronic CBD treatment in renovascular hypertension induced by the 2-kidney-1-clip (2K1C) model. Male Wistar rats (180-200 g, 8 weeks old) underwent 2K1C or SHAM surgery. Six weeks later, rats received chronic CBD treatment (20 mg/kg, twice daily for 14 days). A combination of ex vivo, in vitro, and in vivo methods was used to assess CBD's cardiovascular effects in 2K1C hypertensive rats. Chronic CBD treatment significantly reduced blood pressure and the depressor response to hexamethonium (a ganglionic blocker). It also normalized variability in low-frequency (LF) power and LF/high-frequency (HF) ratio. CBD enhanced vasodilation and reduced vasoconstriction in the mesenteric artery of 2K1C rats, accompanied by decreased expression of aortic reactive oxygen species (ROS). Our findings suggest that chronic CBD treatment exerts antihypertensive effects by improving baroreflex sensitivity and vascular function while decreasing arterial ROS levels and sympathetic nerve activity. These results underscore CBD's potential therapeutic role in managing cardiovascular complications associated with renovascular hypertension.

OXGR1-Dependent (Pro)Renin Receptor Upregulation in Collecting Ducts of the Clipped Kidney Contributes to Na+ Balance in Goldblatt Hypertensive Mice.

The two-kidney, one-clip (2K1C) Goldblatt rodent model elicits a reduction in renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio-ability causes the accumulation of the tricarboxylic cycle intermediary, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, OXGR1 is abundantly expressed in intercalated cells (ICs) of the collecting duct (CD), thus contributing to sodium transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin system (RAS), is a key regulator of sodium reabsorption and blood pressure (BP) that is expressed in ICs. The PRR is upregulated in 2K1C rats. Here, we tested the hypothesis that chronic reduction in RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alters sodium balance and BP in 2K1C mice. To determine the role of OXGR1 in regulating the PRR in the CDs during renovascular hypertension, we performed 2K1C Goldblatt surgery (clip = 0.13 mm internal gap, 14 days) in two groups of male mice: (1) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day); (2) OXGR1-/- knockout mice. Wild-type and sham-operated mice were used as controls. After 14 days, 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p < 0.01) and a lower natriuretic response after the saline challenge test. The CK group showed upregulation of erythropoietin, augmented α-ketoglutarate, and increased PRR expression in the renal medulla. The CK of OXGR1 knockout mice and mice subjected to the OXGR1 antagonist elicited impaired PRR upregulation, attenuated SBP, and better natriuretic responses. In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.

Sarcoplasmic reticulum calcium ATPase (SERCA) proteolysis by matrix metalloproteinase-2 contributes to vascular dysfunction in early hypertension

AimsHypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction. Main methodsMale Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain. Key findingsSBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it. Significancedoxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction.

P049 RENOVASCULAR HYPERTENSIVE RATS (2K1C) HAVE REDUCED INTRACRANIAL COMPLIANCE INDUCED BY RAISED VENTRICULAR VOLUME COMPARED TO NORMOTENSIVE RATS

Background and Objective: Intracerebroventricular (ICV) infusions of increased volumes of saline can be used to study intracranial compliance (ICC). Since the two-kidneys,1-clip (2K1C) rat model of renovascular hypertension has an enhanced baseline level of intracranial pressure (ICP), the present study aimed to verify the effect of ICV infusions of 0.15 M NaCl on ICP and ICC in 2K1C rats. Methods: Male Holtzman rats (180-210 g) were randomly separated to be normotensive (NT; sham surgery, n = 5) or with 2K1C (n = 5), done by a partial left renal artery occlusion. Six weeks after, rats were anaesthetized with urethane (1.2 g/kg body weight, i.v.), tracheostomized, placed under artificial ventilation, and had an ICP sensor and a needle inserted into the lateral ventricle for, respectively, ICP recording and ICV 0.15 M infusions (30, 60 and 90 μL, at 100 μL/min). There was a 20-minute interval between each infusion. ICC was calculated by the ratio of volume change to ICP change [ICC = ΔV (μL)/Δ ICP (mmHg)]. Results: Baseline MAP, ICP, and P2/P1 ratio of 2K1C rats (166 ± 6 mmHg; 15.7 ± 1.7 mmHg; 1.6 ± 0.1, respectively) were higher than the ones in NT rats (94 ± 5 mmHg; 10.1 ±1.4 mmHg; 0.7 ± 0.1 respectively; p&lt;0.05 vs 2K-1C). ICV infusions induced increased ICP in 2K1C rats (19 ±0.5; 42 ±1.2 and 55 ±5.5 mmHg, vs NT: 8 ±0.6; 25 ±3.2 and 36 ± 4.9 mmHg, respectively at 30, 60 and 90 μL infusion; p&lt;0.05). In addition, 2K1C rats presented a reduced ICC only at 30 μl infusion (5.6 ±1.4, vs NT: 11.4 ±0.9; p&lt;0.05). Conclusions: These findings suggest that renovascular hypertension impairs ICC to the well-documented increase in ICP. Support: FAPESP, CNPq.

Abstract 54: The kidney-OVLT axis in renovascular hypertension: Individual and combined effects of afferent renal denervation and OVLT lesion in 2K1C rats.

We have recently reported that afferent renal denervation (ARDN) markedly attenuates the pathogenesis of hypertension in the 2 kidney 1 clip (2K1C) model in the rat. This was associated with a reduction in neurogenic pressor activity (NPA). We have also recently shown that lesion of the organum vasculosum of the lamina terminalis (OVLT), a forebrain circumventricular organ, had nearly identical effects in 2K1C rats as ARDN. Together these findings suggest that a neural pathway from the kidney to the OVLT plays an important role in 2K1C hypertension. Based on these findings we hypothesized that the combined effect of ARDN and lesion of the OVLT (OVLTx) on arterial pressure in 2K1C rats would not be greater than the individual treatments. Male Sprague-Dawley rats were randomly selected to receive either OVLT lesion (x) or sham-OVLTx. One week later, rats were instrumented with telemeters to measure mean arterial pressure (MAP). One week later, renal artery stenosis was created by placing a clip on the left renal artery and then, rats were subjected to ARDN by periaxonal capsaicin or sham treatment of the clipped kidney creating 4 experimental groups: sham+sham (n=6), sham+ARDN (n=6), OVLTx+sham (n=5), OVLTx+ARDN (n=5). MAP was measured daily for 28 days, and at the end of the protocol, NPA was assessed by measuring the peak MAP response to ganglionic blockade. MAP was similar in all 4 groups at the beginning of the study averaging 108±3 mmHg. MAP increased to 180±10 mmHg in the sham+sham group compared to 130±13mmHg in sham+ARDN, 142±18mmHg in OVLTx+sham rats and 158±14mmHg in OVLTx+ARDN rats (p&lt;0.001). NPA in Sham+sham rats was significantly higher (-44 ± 8 mmHg) compared to sham+ARDN (-18 ± 3 mmHg), OVLTx+sham (-21 ± 2 mmHg) and OVLTx+ARDN (-19 ± 4 mmHg) rats (p&lt;0.05). Notably, the combined effects of OVLTx+ARDN on MAP and NPA were not significantly different from either OVLTx (p=1.00 and p=0.819) or ARDN (p=0.113 and p= 0.878) alone. In summary, ARDN and OVLTx attenuated hypertension and NPA similarly in 2K1C-HTN rats but the combination of these treatments did not produce any additional effect on MAP or NPA. These findings are consistent with the hypothesis that afferent renal nerves and the OVLT are linked in a common neural pathway required for the development of hypertension following renal artery stenosis.

O65 SARCOPLASMIC CALCIUM ATPASE (SERCA) PROTEOLYSIS BY MATRIX METALLOPROTEINASE (MMP)-2 CONTRIBUTES TO VASCULAR DYSFUNCTION IN EARLY HYPERTENSION

Background and objective: Hypertension-induced increased matrix metalloproteinase (MMP)-2 activity proteolyzes extra- and intracellular proteins in aortas and contributes to vascular dysfunction. MMP-2 proteolyzes sarcoplasmic reticulum calcium ATPase (SERCA) in the heart during ischemia and reperfusion injury, thus impairing cardiac function. SERCA activity is also impaired in the arteries of hypertensive animals. We investigated whether hypertension-induced increased MMP-2 activity in aorta contributes to SERCA proteolysis, vascular remodeling and dysfunction. Methods: Aortas from male Sprague-Dawley (SD) rats, incubated or not with angiotensin II (AngII) and MMP inhibitors, ONO-4817 or doxycycline (Doxy), were used for zymography and co-immunoprecipitation of SERCA with MMP-2. Male SD rats were also submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with Doxy from third to seventh day post-surgery. Systolic blood pressure (SBP) was assessed basally and daily by tail-cuff plethysmography. After 7 days, aortas were collected for gel and in situ zymography, western blot to SERCA, ATPase activity assay, vascular reactivity to phenylephrine and acetylcholine, Ki-67 immunofluorescence and hematoxylin/eosin stain. A7r5 cells were incubated or not with AngII, in the presence or absence of SERCA inhibitor, tapsigargin, to assess intracellular calcium concentrations. Results: AngII increased MMP-2 activity in aortas and MMP-2 co-immunoprecipitated with SERCA. SBP was increased in 2K-1C rats and Doxy did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Intracellular calcium concentrations were increased in A7r5 cells incubated with AngII and/or SERCA inhibitor, tapsigargin. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and Doxy decreased Ki-67 cell proliferation. In 2K-1C rats, arterial contraction to phenylephrine was accentuated but Doxy ameliorated only the impaired relaxation to acetylcholine. Conclusion: Doxy reduced MMP-2 activity in the aortas of 2K-1C rats and prevented the ATPase activity impairment and MMP-2-induced SERCA proteolysis and vascular dysfunction.

P224 EFFECT OF SODIUM NITRITE ON RENAL DAMAGE FROM 2-KIDNEY, 1-CLIP (2K-1C) HYPERTENSION IN RATS

Renovascular hypertension is characterized by oxidative stress and decreased availability of nitric oxide (NO). Nitrite is a physiologically recycled metabolite that generates NO. Treatment with sodium nitrite has antihypertensive effects due to its antioxidant capacity. However, the renal effects of this treatment are not known in 2K1C hypertension. This study aims to evaluate the effect of treatment with sodium nitrite on kidney damage caused by 2K1C hypertension. Hypertension was induced by cleavage of the left renal artery in Wistar rats. Two weeks after surgery, the animals were treated with two doses (1 mg/kg/day or 15 mg/kg/day) of sodium nitrite or vehicle for 4 weeks, by gavage. Blood pressure (BP) was checked weekly by tail plethysmography. To assess renal function, plasma urea and creatinine were determined. Kidney oxidative stress was evaluated through the activity of catalase (CAT), superoxide dismutase (SOD), levels of reduced glutathione (GSH), lipid peroxidation, and superoxide (O2-) levels. Treatment with 15 mg/kg/day of sodium nitrite reversed the increase in BP in 2K1C rats (p&lt;0.05, 2K1C:170.5±6.542; 2K1C+Nitrite15:162.4±6.712 mmHg), and improved renal function previously impaired by hypertension (urea, p&lt;0.05; 2K1C:76.37±24.53; 2K1C+Nitrite15:47.82±13.86; creatinine, p&lt;0.05; 2K1C:0.540±0.105; 2K1C+Nitrite15:0.3550±0.117). SOD activity decreased in the kidneys of hypertensive animals (p&lt;0.05, Control:0.7176±0.06426; 2K1C:0.636±0.0888; 2K1C+Nitrite1:0.703±0.0866; 2K1C+Nitrite15:0.648±0.173). As for CAT and GSH, there was a decrease in activity in hypertensive animals, with improvement in the group treated with nitrite 15 mg/kg/day (CAT p&lt;0.05, Control:0.651±0.255; 2K1C:0.2772±0.1837; 2K1C+Nitrite15: 0.6654±0.1440 and GSH: p&lt;0.05, Control:44.80±10.59; 2K1C: 29,62±7,253 2K1C+Nitrite15:44.25±10.07). Lipid peroxidation increased by hypertension was reduced with treatment at the highest dosage (p&lt;0.05, Control:0.314±0.158; 2K1C:0.497±0.113; 2K1C+Nitrite15:0.278±0.084). O2- levels were decreased in all groups treated with nitrite (p&lt;0.05, 2K1C:3.844±1.598; 2K1C+Nitrite1:0.9643±0.2063; 2K1C+Nitrite15:0.4431±0.1838). Our results suggest that sodium nitrite treatment improves 2K1C renal damage through its antioxidant activity. Acknowledgments: FAPEMIG# APQ-00946-23, CNPq# 302076/2022-0

Ile-Pro-Pro attenuates sympathetic activity and hypertension.

Isoleucine-proline-proline (Ile-Pro-Pro, IPP) is a natural food source tripeptide that inhibits angiotensin-converting enzyme (ACE) activity. The aim of this study was to determine the central and peripheral roles of IPP in attenuating sympathetic activity, oxidative stress and hypertension. Male Sprague-Dawley rats were subjected to sham-operated surgery (Sham) or two-kidney one-clip (2K1C) surgery to induce renovascular hypertension. Renal sympathetic nerve activity and blood pressure were recorded. Bilateral microinjections of IPP to hypothalamic paraventricular nucleus (PVN) attenuated sympathetic activity (-16.1 ± 2.5%, P < 0.001) and hypertension (-8.7 ± 1.5 mmHg, P < 0.01) in 2K1C rats by inhibiting ACE activity and subsequent angiotensin II and superoxide production in the PVN. Intravenous injections of IPP also attenuated sympathetic activity (-15.1 ± 2.1%, P < 0.001) and hypertension (-16.8 ± 2.3 mmHg, P < 0.001) via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. The duration of the effects of the intravenous IPP was longer than those of the PVN microinjection, but the sympatho-inhibitory effect of intravenous injections occurred later than that of the PVN microinjection. Intraperitoneal injection of IPP (400 pmol/day for 20 days) attenuated hypertension and vascular remodeling via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. These results indicate that IPP attenuates hypertension and sympathetic activity by inhibiting ACE activity and oxidative stress. The sympathoinhibitory effect of peripheral IPP is mainly caused by the ACE inhibition in PVN, and the antihypertensive effect is related to the sympathoinhibition and the arterial ACE inhibition. Long-term intraperitoneal IPP therapy attenuates hypertension, oxidative stress and vascular remodeling.

Activation AMPK in Hypothalamic Paraventricular Nucleus Improves Renovascular Hypertension Through ERK1/2-NF-κB Pathway.

Hypertension is a globally prevalent disease, but the pathogenesis remains largely unclear. AMP-activated protein kinase (AMPK) is a nutrition-sensitive signal of cellular energy metabolism, which has a certain influence on the development of hypertension. Previously, we found a down-regulation of the phosphorylated (p-) form of AMPK, and the up-regulation of the angiotensin II type 1 receptor (AT1-R) and that of p-ERK1/2 in the hypothalamic paraventricular nucleus (PVN) of hypertensive rats. However, the exact mechanism underlying the relationship between AMPK and AT1-R in the PVN during hypertension remains unclear. Thus, we hypothesized that AMPK modulates AT1-R through the ERK1/2-NF-κB pathway in the PVN, thereby inhibiting sympathetic nerve activity and improving hypertension. To examine this hypothesis, we employed a renovascular hypertensive animal model developed via two-kidney, one-clip (2K1C) and sham-operated (SHAM). Artificial cerebrospinal fluid (aCSF), used as vehicle, or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, an AMPK activator, 60μg/day) was microinjected bilaterally in the PVN of these rats for 4weeks. In 2K1C rats, there an increase in systolic blood pressure (SBP) and circulating norepinephrine (NE). Also, the hypertensive rats had lowered expression of p-AMPK and p-AMPK/AMPK, elevated expression of p-ERK1/2, p-ERK1/2/ERK1/2 and AT1-R, increased NF-κB p65 activity in the PVN compared with the levels of these biomarkers in SHAM rats. Four weeks of bilateral PVN injection of AMPK activator AICAR, attenuated the NE level and SBP, increased the expression of p-AMPK and p-AMPK/AMPK, lessened the NF-κB p65 activity, decreased the expression of p-ERK1/2, p-ERK1/2/ERK1/2 and AT1-R in the PVN of 2K1C rats. Data from this study imply that the activation of AMPK within the PVN suppressed AT1-R expression through inhibiting the ERK1/2-NF-κB pathway, decreased the activity of the sympathetic nervous system, improved hypertension.

Dracocephalum moldavica L. Extract Ameliorates Hypertension Through Modulating the Interaction Between miRNAs and Gut Microbiota in 2K1C Rats

Aim: To investigate the therapeutic effects of Dracocephalum moldavica L. extract (DME) against hypertension and elucidate the underlying molecular mechanism. Method: A two-kidney one-clip (2K1C) model was established in male Wistar rats, and DME or saline was administered via oral gavage for 4 weeks. Subsequently, the blood pressure was monitored. Fecal microRNAs (miRNAs), gut microbes, and host metabolites were evaluated through miRNA sequencing, 16S rRNA gene sequencing, and serum metabolomics analysis, respectively. Results: DME administration attenuated 2K1C-induced hypertension in rats. Additionally, DME alleviated the dysregulation of miRNA expression and gut microbiota dysbiosis in 2K1C rats. The correlation analysis confirmed the association of the differentially expressed miRNAs with gut microbes. Furthermore, non-targeted metabolomics analysis revealed different metabolic profiles in the serum of 2K1C rats compared to control rats. Interestingly, the changes in those serum metabolites were partially attenuated by DME. The metabolite enrichment analysis demonstrated that the differentially expressed metabolites were mainly involved in three metabolic pathways (Arachidonic acid metabolism, TCA cycle, and Alanine, aspartate and glutamate metabolism). Conclusion: DME can be suggested as an effective intervention in the prevention of hypertension through modulating the miRNAs, gut microbiota, and host metabolites. Our findings may give new insights regarding the treatment of hypertension.

The Total Denervation of the Ischemic Kidney Induces Differential Responses in Sodium Transporters' Expression in the Contralateral Kidney in Goldblatt Rats.

The Goldblatt model of hypertension (2K-1C) in rats is characterized by renal sympathetic nerve activity (rSNA). We investigated the effects of unilateral renal denervation of the clipped kidney (DNX) on sodium transporters of the unclipped kidneys and the cardiovascular, autonomic, and renal functions in 2K-1C and control (CTR) rats. The mean arterial pressure (MAP) and rSNA were evaluated in experimental groups. Kidney function and NHE3, NCC, ENaCβ, and ENaCγ protein expressions were assessed. The glomerular filtration rate (GRF) and renal plasma flow were not changed by DNX, but the urinary (CTR: 0.0042 ± 0.001; 2K-1C: 0.014 ± 0.003; DNX: 0.005 ± 0.0013 mL/min/g renal tissue) and filtration fractions (CTR: 0.29 ± 0.02; 2K-1C: 0.51 ± 0.06; DNX: 0.28 ± 0.04 mL/min/g renal tissue) were normalized. The Na+/H+ exchanger (NHE3) was reduced in 2K-1C, and DNX normalized NHE3 (CTR: 100 ± 6; 2K-1C: 44 ± 14, DNX: 84 ± 13%). Conversely, the Na+/Cl- cotransporter (NCC) was increased in 2K-1C and was reduced by DNX (CTR: 94 ± 6; 2K-1C: 144 ± 8; DNX: 60 ± 15%). In conclusion, DNX in Goldblatt rats reduced blood pressure and proteinuria independently of GRF with a distinct regulation of NHE3 and NCC in unclipped kidneys.

The hydrogen sulfide donor 4-carboxyphenyl-isothiocyanate decreases blood pressure and promotes cardioprotective effect through reduction of oxidative stress and nuclear factor kappa B/matrix metalloproteinase (MMP)-2 axis in hypertension

AimsOur aim was to evaluate whether the hydrogen sulfide (H2S) donor, 4-carboxyphenyl-isothiocyanate (4-CPI), exerts cardioprotective effect in the two kidney- one clip (2K-1C) rats through oxidative stress and MMP-2 activity attenuation and compare it with the classical H2S donor, Sodium Hydrosulfide (NaHS). Materials and methodsRenovascular hypertension (two kidneys-one clip; 2K-1C) was surgically induced in male Wistar rats. After two weeks, normotensive (2K) and hypertensive rats were intraperitoneally treated with vehicle (0.6 % dimethyl sulfoxide), NaHS (0.24 mg/Kg/day) or with 4-CPI (0.24 mg/Kg/day), for more 4 weeks. Systolic blood pressure (SBP) was evaluated weekly by tail-cuff plethysmography. Heart function was assessed by using the Millar catheter. Cardiac hypertrophy and fibrosis were evaluated by hematoxylin and eosin, and Picrosirius Red staining, respectively. The H2S was analyzed using WSP-1 fluorimetry and the cardiac oxidative stress was measured by lucigenin chemiluminescence and Amplex Red. MMP-2 activity was measured by in-gel gelatin or in situ zymography assays. Nox1, gp91phox, MMP-2 and the phospho-p65 subunit (Serine 279) nuclear factor kappa B (NF-κB) levels were evaluated by Western blotting. Key findings4-CPI reduced blood pressure in hypertensive rats, decreased cardiac remodeling and promoted cardioprotection through the enhancement of cardiac H2S levels. An attenuation of oxidative stress, with inactivation of the p65-NF-κB/MMP-2 axis was similarly observed after NaHS or 4-CPI treatment in 2K-1C hypertension. SignificanceH2S is a mediator that promotes cardioprotective effects and decreases blood pressure, and 4-CPI seems to be a good candidate to reverse the maladaptive remodeling and cardiac dysfunction in renovascular hypertension.

Pyridostigmine attenuates hypertension by inhibiting activation of the renin-angiotensin system in the hypothalamic paraventricular nucleus.

Activation of the renin-angiotensin system (RAS) triggers oxidative stress and an inflammatory response in the hypothalamic paraventricular nucleus (PVN), in turn increasing the sympathetic hyperactivity that is a major cause of hypertension. Pyridostigmine has cardioprotective effects by suppressing the RAS of myocardial tissue. However, whether pyridostigmine attenuates hypertension by inhibiting the RAS of the PVN remains unclear. We thus investigated the effect and mechanism of pyridostigmine on two-kidney one-clip (2K1C)-induced hypertension. 2K1C rats received pyridostigmine, or not, for 8weeks. Cardiovascular function, hemodynamic parameters, and autonomic activity were measured. The PVN levels of pro-/anti-inflammatory cytokines, oxidative stress, and RAS signaling molecules were evaluated. Our results showed that hypertension was accompanied by cardiovascular dysfunction and an autonomic imbalance characterized by enhanced sympathetic but diminished vagal activity. The PVN levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS), NOX-2, and malondialdehyde (MDA) increased; those of IL-10 and superoxide dismutase (SOD) decreased. Moreover, the RAS signaling pathway was activated, as evidenced by increased levels of the angiotensin-converting enzyme (ACE), angiotensin II (Ang II), and the Ang II type 1 receptor (AT1R) and a decreased AT2R level. Pyridostigmine lowered blood pressure and improved cardiovascular function, associated with restoration of the autonomic balance. Meanwhile, pyridostigmine decreased PVN IL-6, TNF-α, ROS, NOX-2, and MDA levels and increased IL-10 and SOD levels. Additionally, pyridostigmine suppressed PVN ACE, Ang II, and AT1R levels and increased AT2R expression. Pyridostigmine attenuated hypertension by inhibiting PVN oxidative stress and inflammation induced by the RAS.

Comparative assessment of CacyBP/SIP, β-catenin and cannabinoid receptors in the adrenals of hypertensive rats.

Taking into account homeostatic disorders resulting from arterial hypertension and the key importance of CacyBP/SIP, β-catenin and endocannabinoids in the functioning of many organs, it was decided to assess the presence and distribution of CacyBP/SIP, β-catenin, CB1 and CB2 in the adrenal glands of hypertensive rats of various aetiology. The study was conducted on the adrenal glands of rats with spontaneous and renovascular hypertension. The expression of CacyBP/SIP, β-catenin, CB1 and CB2 was detected by immunohistochemistry and real-time PCR method. The results of the present study revealed both lower gene expression and immunoreactivity of CacyBP/SIP in the adrenal glands of all hypertensive groups compared to the normotensive rats. This study demonstrated a reduction in the immunoreactivity and expression of the β-catenin, CB1 and CB2 genes in the adrenals of 2K1C rats. While in SHR, the reaction showing β-catenin and CB1 was very weak or negative, and the expression of CB2 in the adrenal glands of these rats increased. The results of this study show, for the first time, marked differences in the expression of CacyBP/SIP, β-catenin and CB1 and CB2 cannabinoid receptors in the adrenal glands of rats with primary (SHR) and secondary hypertension (2K1C).

Ang II-induced contraction is impaired in the aortas of renovascular hypertensive animal model.

Renin-angiotensin system plays a critical role in blood pressure control, and the abnormal activation of the AT1 receptor contributes to the development of renovascular hypertension. This study aimed to evaluate the underlying cellular signaling for AT1 receptor activation by Ang II and to compare this mechanism between aortas from 2K-1C and 2K rats. Effects of antagonists and inhibitors were investigated on Ang II-induced contractions in denuded or intact-endothelium aortas. The AT1 receptor antagonist abolished Ang II-induced contraction in 2K-1C and 2K rat aortas, while AT2 and Mas receptors antagonists had no effect. Endothelial nitric oxide synthase inhibition increased the maximal effect (Emax) of Ang II in 2K, which was not changed in 2K-1C aortas. It was associated with lower eNOS mRNA levels in 2K-1C. Endothelium removal increased the Emax of Ang II in 2K-1C and mainly in 2K rat aortas. Nox and COX inhibition did not alter Ang II-induced contraction in 2K and 2K-1C rat aortas. However, AT1 expression was higher in 2K-1C compared to 2K rat aortic rings, whereas expression of phosphorylated (active) IP3 receptors was lower in 2K-1C than in 2K rats. These results demonstrate that endothelium removal impairs Ang II-stimulated contraction in the aorta of 2K-1C rats, which is associated with the reduction of IP3 receptor phosphorylation and activation. In addition, eNOS plays a critical role in Ang II-induced contraction in 2K rat aortas. It is possible that the high Ang II plasma levels could desensitize AT1 receptor in 2K-1C rats, leading to impaired IP3 receptors activation.

Garcinia dulcis Extract Alleviates Inflammation in Kidney and Liver of the 2-Kidney-1-Clip Hypertensive rat.

Garcinia dulcis (GD) extract possesses anti-hypertensive property that are poorly characterized. This study aimed to investigate an anti-inflammatory effect of GD flower extract in the 2-kidney-1-clip (2K1C) hypertensive compared to sham operative (SO) rat. Male Wistar rats were divided into 2 groups; the 2K1C group in which a silver clip was placed around renal artery to induce hypertension, and the SO normotensive group. Four weeks later, each group of rats were further divided into 2 subgroups, each subgroup was orally gavaged of either corn oil (vehicle) or 50 mg/kg BW GD extract daily for 4 weeks. The malondialdehyde (MDA) levels in serum, liver, and kidney were determined. Hematoxylin and eosin staining was carried out for histological examination, Periodic acid - Schiff staining for glomerular injury, Masson's trichrome staining for renal fibrosis, and immunohistochemistry for either tumor necrosis factor alpha (TNF-α) or endothelial nitric oxide synthase (eNOS) investigation. Taken together, our results demonstrated that GD flower extract decreased the MDA level in both serum and liver and kidney tissue and suppressed the expression of TNF-α in both liver and kidney of 2K1C hypertensive rats. Mesangial cell proliferation, expansion of mesangial matrix, widening Bowman's capsule space, congestion of glomerular capillary and vessel, cloudy swelling of renal tubular epithelial cell, and renal fibrosis were observed in the kidneys of 2K1C rats. Therefore, we concluded that GD flower extract can alleviate liver and kidney inflammation in which partially attenuates the glomerular injury in the 2K1C rat.

Chronic red laser treatment induces hypotensive effect in two-kidney one-clip model of renovascular hypertension in rat.

To evaluate whether the chronic effect of photobiomodulation therapy (PBM) onsystolic arterial pressure (SAP)from two kidneys one clip (2K-1C) hypertension animal models can cause a hypotensive effect. Serum levels of nitric oxide were also analyzed and the assessment of lipid peroxidation of the thoracic aorta artery. Male Wistar rats were used. Hypertensive animals (2K-1C) with Systolic arterial pressure (SAP) greater than or equal to 160mmHg were used. Systolic arterial pressure (SAP) was determined by the tail plethysmography technique. Normotensive (2K) and hypertensive (2K-1C) rats were treated to PBM for 4weeks using a laser whose irradiation parameters were: red wavelength (λ) = 660nm: operating continuously; 56s per point (3 points) spot size = 0.0295 cm2; average optical power of 100 mW; energy of 5.6J per point; irradiance of 3.40 W/cm2; fluency of 190J/cm2 per point. The application was on the animals tails, at 3 different points simultaneously, in contact with the skin. To assess serum nitrite and nitrate (NOx) levels, blood collection was performed after chronic PBM treatment, 24h after the last laser application. The evaluation of the lipid peroxidation of the thoracic aorta artery was performed by measuring the concentration of hydroperoxide by the FOX method. Chronic photobiomodulation therapy (PBM) by red laser (660nm) can induce a hypotensive effect in 64% of 2K-1C hypertensive animals, which we say responsive animals. There was no difference in serum NO levels 24h after the last red laser application, between treated and non-treated groups. Aortic rings from 2K-1C hypertensive animals present a higher lipid peroxidation. The chronic PBM treatment by red laser decreased aortic rings lipid peroxidation in hypertensive responsive groups, compared to control. our results indicate that chronic PBM made by red laser has an important hypotensive effect in renovascular hypertensivemodels, by a mechanism that involves decrease in oxidative stress from vascular beds.

Type I collagen proteolysis by matrix metalloproteinase-2 contributes to focal adhesion kinase activation and vascular smooth muscle cell proliferation in the aorta in early hypertension

IntroductionIncreased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. MethodsMale Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. Results2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. ConclusionsIncrease in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

Effects of aurantiamide on a rat model of renovascular arterial hypertension.

Asperglaucide (ASP) is an aurantiamide, an effective constituent of purslane (Portulaca oleracea L.), a safe to eat greenery. Effects of ASP on endothelial function, endothelial nitric oxide synthase (eNOS) expression, vascular fluidity, renal and vascular reactive oxygen, and nitrogen species (ROS/RNS) production was examined in the two-kidney one-clip (2K-1C) rat model of renovascular arterial hypertension. ASP toxicity, dose dependent eNOS gene expression and protein levels were also analyzed in human umbilical vein endothelial cells (HUVEC). The 2K-1C model of hypertension was created via surgery and mean blood pressure (MBP) was measured by tail-cuff method during four weeks of ASP treatment. Erythrocyte deformability was monitored by rotational ektacytometry, while vascular constrictor and dilator responses were determined in organ baths. eNOS gene expression and protein levels were assessed in thoracic aorta and HUVEC. MBP was significantly decreased in hypertensive rats treated with ASP. Endothelium dependent vascular dilator and constrictor responses were also considerably improved following ASP treatment. There was a notable increase in red blood cell deformability in hypertensive rats treated with ASP as compared to hypertensive rats alone. A significant increase was observed in eNOS gene expression and protein levels in both normotensive and hypertensive rats treated with ASP. Treatment of HUVEC with 3µM ASP notably increased eNOS mRNA and protein levels. In conclusion, ASP lowered blood pressure, improved endothelium-mediated relaxation, decreased renovascular ROS/RNS production in hypertensive rats. ASP also increased eNOS protein expression in aorta and HUVEC at nontoxic doses. ASP may have future potential as an anti-hypertensive agent.

Renal and hypothalamic inflammation in renovascular hypertension: role of afferent renal nerves.

Renal denervation (RDN) is a potential therapy for drug-resistant hypertension. However, whether its effects are mediated by ablation of efferent or afferent renal nerves is not clear. Previous studies have implicated that renal inflammation and the sympathetic nervous system are driven by the activation of afferent and efferent renal nerves. RDN attenuated the renal inflammation and sympathetic activity in some animal models of hypertension. In the 2 kidney,1 clip (2K1C) model of renovascular hypertension, RDN also decreased sympathetic activity; however, mechanisms underlying renal and central inflammation are still unclear. We tested the hypothesis that the mechanisms by which total RDN (TRDN; efferent + afferent) and afferent-specific RDN (ARDN) reduce arterial pressure in 2K1C rats are the same. Male Sprague-Dawley rats were instrumented with telemeters to measure mean arterial pressure (MAP), and after 7 days, a clip was placed on the left renal artery. Rats underwent TRDN, ARDN, or sham surgery of the clipped kidney and MAP was measured for 6 wk. Weekly measurements of water intake (WI), urine output (UO), and urinary copeptin were conducted, and urine was analyzed for cytokines/chemokines. Neurogenic pressor activity (NPA) was assessed at the end of the protocol calculated by the depressor response after intraperitoneal injection of hexamethonium. Rats were euthanized and the hypothalamus and kidneys removed for measurement of cytokine content. MAP, NPA, WI, and urinary copeptin were significantly increased in 2K1C-sham rats, and these responses were abolished by both TRDN and ARDN. 2K1C-sham rats presented with renal and hypothalamic inflammation and these responses were largely mitigated by TRDN and ARDN. We conclude that RDN attenuates 2K1C hypertension primarily by ablation of afferent renal nerves which disrupts bidirectional renal neural-immune pathways.NEW & NOTEWORTHY Hypertension resulting from reduced perfusion of the kidney is dependent on renal sensory nerves, which are linked to inflammation in the kidney and hypothalamus. Afferent renal nerves are required for chronic increases in both water intake and vasopressin release observed following renal artery stenosis. Findings from this study suggest an important role of renal sensory nerves that has previously been underestimated in the pathogenesis of 2K1C hypertension.