2K1C Hypertension Research Articles

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<0.05, 2K1C:170.5±6.542; 2K1C+Nitrite15:162.4±6.712 mmHg), and improved renal function previously impaired by hypertension (urea, p<0.05; 2K1C:76.37±24.53; 2K1C+Nitrite15:47.82±13.86; creatinine, p<0.05; 2K1C:0.540±0.105; 2K1C+Nitrite15:0.3550±0.117). SOD activity decreased in the kidneys of hypertensive animals (p<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<0.05, Control:0.651±0.255; 2K1C:0.2772±0.1837; 2K1C+Nitrite15: 0.6654±0.1440 and GSH: p<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<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<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

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<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<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.

P178 CENTRAL OR PERIPHERAL INJECTION OF A FORMULATION CONTAINING THE ANGIOTENSINERGIC DIPEPTIDE ASP-ARG REDUCES ARTERIAL PRESSURE AND CARDIAC INOTROPY IN HYPERTENSIVE RATS

Background and objective: Acute injection of Angiotensin 1-2 [Ang-(1-2)] reduced arterial pressure and heart rate, and improved coronary perfusion in normotensive and hypertensive rats. Formulations that confer stability and protection against proteolysis are requisites to the feasibility peptides. Next, we evaluated in rats the cardiovascular effects of peripheral or central injections of a complex containing Ang-(1-2) surrounded by a cyclodextrin. Methods: Ethics committee approval by CEUA-FOAr/UNESP 15/2022. We used male Holtzman rats that underwent 2 kidneys, 1 clip (2K1C) hypertension or sham surgery. Six weeks later, rats were separated in different experimental sets (n=4-6 per group) and were instrumented for intracerebroventricular (i.c.v.) and intravenous (i.v.) injections while recording arterial and cardiac left ventricular pressures. The dipeptide was injected i.c.v. (1, 25, 50 and 250pmol/2 μL) or i.v. (25mmol/0.1 mL). Results: dipeptide i.c.v. (25, 50 or 250pmol) reduced arterial pressure of normotensive rats (Δ=-20±3, -20±1 and -18±7mmHg respectively, P<0.05 vs. baseline). In 2K1C, the antihypertensive effect was observed at all doses (Δ=-19±4, -26±5, -19±3 and -17±1mmHg respectively, P<0.05 vs. baseline). I.v. injection of the dipeptide evoked slight increase in arterial pressure (Δ=7±3mmHg; P<0.05 vs. baseline) that was accompanied by a positive inotropic effect (Δ=524±159mmHg/s; P<0.05 vs. baseline). In 2K1C, this same injection reduced arterial and ventricular pressures (Δ=-14±4 and -20±1mmHg respectively, P<0.05 vs. baseline), and caused negative inotropy (ΔLVdP/dtmax=-1140±170mmHg/s; P<0.05 vs. baseline). I.c.v. injection of dipeptide in anesthetized normotensive rats discretely increased arterial and ventricular pressures, while in 2K1C group it reduced arterial and ventricular pressures (Δ=-17±3 and -18±4mmHg respectively, P<0.05 vs. baseline). The ratio between the variations in contractility and mean arterial pressure revealed afterload-independent inotropic effects, i.e, the dipeptide directly affects heart performance of 2K1C. Conclusions: The formulation containing Asp1-Arg2 reduces arterial pressure and modulates cardiac function during hypertension. Whether Ang-(1-2) acts through some antagonism on Angiotensin II receptors or other paths remain to be investigated. Support: CONFAP-FAPESP/FAPEG 2019/24154-3, PROPe-UNESP 13/2022, CNPq-404079/2021-0 and FAPEG-202310267000206.

Cardiovascular and Renal Effects Induced by Alpha-Lipoic Acid Treatment in Two-Kidney-One-Clip Hypertensive Rats.

α-Lipoic acid (LA) is an antioxidant of endogenous production, also obtained exogenously. Oxidative stress is closely associated with hypertension, which causes kidney injury and endothelial dysfunction. Here, we evaluated the cardiovascular and renal effects of LA in the two-kidney-one-clip (2K1C) hypertension model. The rats were divided into four groups: Sham surgery (Sham), the two-kidneys-one-clip (2K1C) group, and groups treated with LA for 14 days (Sham-LA and 2K1C-LA). No changes were observed in the pattern of food, water intake, and urinary volume. The left/right kidney weight LKw/RKw ratio was significantly higher in 2K1C animals. LA treatment did not reverse the increase in cardiac mass. In relation to vascular reactivity, there was an increase in the potency of phenylephrine (PHE) curve in the hypertensive animals treated with LA compared to the 2K1C group and also compared to the Sham group. Vasorelaxation induced by acetylcholine (Ach) and sodium nitroprusside (SNP) were not improved by treatment with LA. Urea and creatinine levels were not altered by the LA treatment. In conclusion, the morphological changes in the aorta and heart were not reversed; however, the treatment with LA mitigated the contraction increase induced by the 2K1C 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.

Fenofibrate reduces cardiac remodeling by mitochondrial dynamics preservation in a renovascular model of cardiac hypertrophy

Fenofibrate, a PPAR-α agonist clinically used to lower serum lipid levels, reduces cardiac remodeling and improves cardiac function. However, its mechanism of action is not completely elucidated. In this study we examined the effect of fenofibrate on mitochondria in a rat model of renovascular hypertension, focusing on mediators controlling mitochondrial dynamics and autophagy.Rats with two-kidney one-clip (2K1C) hypertension were treated with fenofibrate 150 mg/kg/day (2K1C-FFB) or vehicle (2K1C-VEH) for 8 weeks. Systolic blood pressure and cardiac functional were in-vivo assessed, while cardiomyocyte size and protein expression of mediators of cardiac hypertrophy and mitochondrial dynamics were ex-vivo examined by histological and Western blot analyses.Fenofibrate treatment counteracted the development of hypertension and the increase of left ventricular mass, relative wall thickness and cross-sectional area of cardiomyocytes. Furthermore, fenofibrate re-balanced the expression Mfn2, Drp1 and Parkin, regulators of fusion, fission, mitophagy respectively. Regarding autophagy, the LC3-II/LC3-I ratio was increased in 2K1C-VEH and 2K1C-FFB, whereas the autophagy was increased only in 2K1C-FFB.In cultured H9C2 cardiomyoblasts, fenofibrate reversed the Ang II-induced mRNA up-regulation of hypertrophy markers Nppa and Myh7, accumulation of reactive oxygen species and depolarization of the mitochondrial membrane exerting protection mediated by up-regulation of the Uncoupling protein 2.Our results indicate that fenofibrate acts directly on cardiomyocytes and counteracts the pressure overload-induced cardiac maladaptive remodeling. This study reveals a so far hidden mechanism involving mitochondrial dynamics in the beneficial effects of fenofibrate, support its repurposing for the treatment of cardiac hypertrophy and provide new potential targets for its pharmacological function.

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.

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).

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.

Frequency-coded patterns of sympathetic vasomotor activity are differentially evoked by the paraventricular nucleus of the hypothalamus in the Goldblatt hypertension model.

The paraventricular nucleus of the hypothalamus (PVN) contains premotor neurons involved in the control of sympathetic vasomotor activity. It is known that the stimulation of specific areas of the PVN can lead to distinct response patterns at different target territories. The underlying mechanisms, however, are still unclear. Recent evidence from sympathetic nerve recording suggests that relevant information is coded in the power distribution of the signal along the frequency range. In the present study, we addressed the hypothesis that the PVN is capable of organizing specific spectral patterns of sympathetic vasomotor activation to distinct territories in both normal and hypertensive animals. To test it, we investigated the territorially differential changes in the frequency parameters of the renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively), before and after disinhibition of the PVN by bicuculline microinjection. Subjects were control and Goldblatt rats, a sympathetic overactivity-characterized model of neurogenic hypertension (2K1C). Additionally, considering the importance of angiotensin II type 1 receptors (AT1) in the sympathetic responses triggered by bicuculline in the PVN, we also investigated the impact of angiotensin AT1 receptors blockade in the spectral features of the rSNA and sSNA activity. The results revealed that each nerve activity (renal and splanchnic) presents its own electrophysiological pattern of frequency-coded rhythm in each group (control, 2K1C, and 2K1C treated with AT1 antagonist losartan) in basal condition and after bicuculline microinjection, but with no significant differences regarding total power comparison among groups. Additionally, the losartan 2K1C treated group showed no decrease in the hypertensive response triggered by bicuculline when compared to the non-treated 2K1C group. However, their spectral patterns of sympathetic nerve activity were different from the other two groups (control and 2K1C), suggesting that the blockade of AT1 receptors does not totally recover the basal levels of neither the autonomic responses nor the electrophysiological patterns in Goldblatt rats, but act on their spectral frequency distribution. The results suggest that the differential responses evoked by the PVN were preferentially coded in frequency, but not in the global power of the vasomotor sympathetic responses, indicating that the PVN is able to independently control the frequency and the power of sympathetic discharges to different territories.

Deletion of the Transient Receptor Potential Vanilloid 1 Channel Attenuates Sympathoexcitation and Hypertension and Improves Glomerular Filtration Rate in 2-Kidney-1-Clip Rats.

Renal denervation lowers arterial blood pressure in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves. The TRPV1 (transient receptor potential vanilloid 1) channel is highly expressed in renal sensory nerves and detects changes in noxious and mechanosensitive stimuli, pH, and chemokines. However, the extent to which TRPV1 channels contribute to 2-kidney-1-clip (2K1C) renovascular hypertension has not been tested. We generated a novel Trpv1-/- (TRPV1 knockout) rat using CRISPR/Cas9 and 26-bp deletion in exon 3 and induced 2K1C hypertension. The majority (85%) of rat renal sensory neurons retrogradely labeled from the kidney were TRPV1-positive. Trpv1-/- rats lacked TRPV1 immunofluorescence in the dorsal root ganglia, had a delayed tail-flick response to hot but not cold water, and lacked an afferent renal nerve activity response to intrarenal infusion of the TRPV1 agonist capsaicin. Interestingly, 2K1C hypertension was significantly attenuated in male Trpv1-/- versus wild-type rats. 2K1C hypertension significantly increased the depressor response to ganglionic blockade, total renal nerve activity (efferent and afferent), and afferent renal nerve activity in wild-type rats, but these responses were attenuated in male Trpv1-/- rats. 2K1C hypertension was attenuated in female rats with no differences between female strains. Finally, glomerular filtration rate was reduced by 2K1C in wild-type rats but improved in Trpv1-/- rats. These findings suggest that renovascular hypertension requires activation of the TRPV1 channel to elevate renal afferent and sympathetic nerve activity, reduce glomerular filtration rate, and increase arterial blood pressure.

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.

Acute inhibition of nicotinamide adenine dinucleotide phosphate oxidase in the commissural nucleus of the solitary tract reduces arterial pressure and renal sympathetic nerve activity in renovascular hypertension.

A growing body of evidence suggests that oxidative stress plays a role in the pathophysiology of hypertension. However, the involvement of the reactive oxygen species (ROS) in the commissural nucleus of the solitary tract (commNTS) in development the of hypertension remains unclear. We evaluated the hemodynamic and sympathetic responses to acute inhibition of NADPH oxidase in the commNTS in renovascular hypertensive rats. Under anesthesia, male Holtzman rats were implanted with a silver clip around the left renal artery to induce 2-kidney 1-clip (2K1C) hypertension. After six weeks, these rats were anesthetized and instrumented for recording mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), and renal sympathetic nerve activity (RSNA) during baseline and after injection of apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), NSC 23766 (RAC inhibitor) or saline into the commNTS. Apocynin into the commNTS decreased MAP, RSNA, and RVR in 2K1C rats. NSC 23766 into the commNTS decreased MAP and RSNA, without changing RVR in 2K1C rats. These results demonstrate that the formation of ROS in the commNTS is important to maintain sympathoexcitation and hypertension in 2K1C rats and suggest that NADPH oxidase in the commNTS could be a potential target for therapeutics in renovascular hypertension.

No benefit of HIF prolyl hydroxylase inhibition for hypertensive renal damage in renovascular hypertensive rats.

Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified "preconditioning" approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

Heart rate fragmentation in renovascular hypertensive rats

Although rapid oscillations of the heart rate (HR) are attributed to vagal modulation of the heart, a pattern of ultra-rapid HR variation has been named heart rate fragmentation (HRF). It is a recently proposed approach to evaluate sino-atrial instability characterized by the presence of numerous inflections points in a series of successive values of cardiac intervals. HRF is increased in aging and coronary diseases. Hypertension markedly alters HRV indices and might affect HRF as well. This study aimed to investigate the HRF in renovascular hypertension in rats. Wistar rats were anesthetized, and hypertension was surgically induced by partial constriction of the left renal artery with a silver clip with a 0.2 mm gap. In one kidney one clip (1K1C) hypertension model, the contralateral kidney was removed. In two kidneys one clip (2K1C) model, the contralateral kidney was kept intact. Sham-operated control rats underwent to same surgical procedures without receiving the clip around the renal artery. After 45 days (2K1C) or 60 days (1K1C), animals were instrumented with a catheter into the femoral artery, and on the following day, their arterial pressure (AP) was directly recorded without the effect of anesthesia. For HRF analysis, pulse interval (PI) series were generated and transformed into a sequence of symbols “-1,” “0,” or “1” when the difference between successive PI values (transitions) was negative, zero, or positive, respectively. Following, sequences of 4 consecutive symbols were classified according to the number of transitions. The total percentage of inflection points (PIP) and the percentage of sequences with zero (W0), one (W1), two (W2), or three (W3) inflection points were quantified. As expected, the arterial pressure increased in both groups of hypertension rats compared to control counterparts. All HRF indices were found similar in 1K1C hypertensive rats compared to their control counterparts. Nevertheless, in 2K1C hypertensive rats HRF was found to be lower than normotensive controls (PIP: 64±7 vs. 79±1% and W2: 30±1 vs. 39±1% in 2K1C and normotensive controls, respectively). In conclusion, high blood pressure itself does not affect HRF, although, depending on the model of hypertension used, HRF can be markedly impaired. The interpretation of these findings is challenging, and the high levels of angiotensin in the 2K1C model may affect cardiac pacemaker cells, making their action potentials more stable than unstable. However, the specific mechanisms by which this could happen still need to be studied. Funding Sources: FAPESP, CAPES and CNPq This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dracocephalum moldavica L. Extract ameliorates intestinal inflammation by regulating gut microbiota and repairing the intestinal barrier in 2k1c rats

When used in ethnomedicine, Dracocephalum moldavica L. (DML) and its bioactive extract protect against several diseases caused by inflammation and oxidative stress. However, the effect of DML on intestinal inflammation in hypertension has not been thoroughly investigated. This study focused on the effects of DML extract on intestinal inflammation in two-kidney, one clip (2K1C) rats. Male Wistar rats were divided into three groups with daily intragastric administration of either saline (control group and model group) or DML extract (treatment group, 50 mg/kg per day) for 4 weeks. In the model and treatment groups, 2K1C hypertension was induced by clipping the left renal artery. The profiles of stool microbiota, the levels of short-chain fatty acids (SCFAs), the integrity of the gut barrier, oxidative stress biomarkers and inflammatory factors were analyzed in rats supplemented with or without DML extract. This study revealed the alleviation of high blood pressure in 2K1C rats treated with DML extract. The DML extract attenuated gut microbiota dysbiosis in the 2K1C rats by increasing the abundance of the genus Lactobacillus and by decreasing the abundance of Papillibacter, Desulfovibrio and Anaerobiospirillum genera. Treatment with the DML extract increased the levels of SCFAs and repaired the intestinal barrier, it reduced the release of oxidative stress biomarkers and inflammatory factors and suppressed the expression of TLR4 and NF-?B mRNAs. We conclude that the DML extract may alleviate intestinal inflammation by adjusting the profile of gut microbiota and enhancing the intestinal barrier.

PS-BPB08-1: ANGIOTENSIN II AT1A RECEPTORS IN THE PROXIMAL TUBULES PLAY A CRITICAL ROLE IN THE DEVELOPMENT OF TWO-KIDNEY, ONE-CLIP GOLDBLATT HYPERTENSION

Objective: It is well recognized that the activation of the renin-angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two-kidney, one-clip Goldblatt hypertension (2K1C) in animal models, but the roles of intratubular angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target, Na+/H+ exchanger 3 (NHE3) in the proximal tubules have not been determined previously. In the present study, we tested the hypothesis that genetic deletion of AT1a receptors selectively in the proximal tubules of the kidney attenuates the development of 2K1C Goldblatt hypertension via AT1a receptor-mediated, Na+/H+ exchanger 3 (NHE3)-dependent mechanisms. Design and Methods: To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.12 mm internal diameter, on the left renal artery for 4 weeks in adult male and female wild-type (WT), global AT1areceptor knockout (Agtr1a-/- ), proximal tubule (PT)-specific Agtr1a-/- (PT-Agtr1a-/- ), or PT-Nhe3-/- mice, respectively. Results: Systolic blood pressure increased in a time-dependent manner reaching a maximal response by Week 2–3 in wild-type mice (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n = 12, P < 0.01). 2K1C Goldblatt hypertension in WT mice was associated with significant increases in renin mRNA expression in the clipped kidney (Control: 2066 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P < 0.01), and decreases in renin mRNA expression in the nonclipped right kidney (1738 ± 341 copies/ng RNA, P < 0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C Goldblatt hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P < 0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney (P < 0.01). In contrast to WT mice, the development of 2K1C hypertension was completely attenuated in global Agtr1a-/- (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n = 9, n.s.), PT-Agtr1a-/- mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 4 mmHg, n = 12, n.s.), or PT-Nhe3-/- mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n.s.). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a-/- mice, but it was decreased in the nonclipped kidney of PT-Agtr1a-/- mice (P < 0.05). Conclusions: Taken together, our results suggest that the activation of intratubular RAS in the proximal tubules and increase of NHE3-mediated proximal tubular Na+ reabsorption play a key role in the development of 2K1C Goldblatt hypertension in mice.

S-51-6: RENOVASCULAR HYPERTENSION AND BRAIN: ROLE OF THE RENIN-ANGIOTENSIN SYSTEM AND SODIUM

Renovascular hypertension is a type of secondary hypertension. Renovascular hypertensive 2-kidney, 1-clip (2K1C) rats have an increased activity of the renin-angiotensin system, and it is an experimental model to study renovascular hypertension. Renovascular hypertensive 2K1C rats have an imbalance of RAS components in the brain, an impairment of the baroreflex and they present an increase in daily intake of hypertonic NaCl solution. Therefore, we focused on studying: 1) the effects of overexpression of RAS components in the in the nucleus of the solitary tract (NTS), a brainstem area involved with cardiovascular control, 2) the brain mechanisms involved with the sodium appetite (operationally defined as an ingestion of hypertonic NaCl solution) in 2K1C rats. In adult Holtzman rats, the left renal artery was partially occluded by a silver clip with an opening of 0.2 mm to induce 2K1C. The experiments started after 6 weeks (hypertension plateau). We observed that increasing angiotensin type 2 receptor (AT2R) expression in the NTS attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex observed in 2K1C rats. Furthermore, an observed decrease in mRNA levels of angiotensin-converting enzyme 2 in the NTS of 2K1C rats was restored to control levels after viral-mediated increases in AT2R at this site. Virally mediated blockade of an intracellular pathway activated by angiotensin II (ANG II) acting in type 1 receptor (AT1R) in the NTS attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex. Daily 0.3 M NaCl intake transiently increased from the 2nd to the 5th week after 2K1C surgery. On the 6th week, in spite of comparable daily 0.3 M NaCl intake between 2K1C and sham rats, water deprivation (used to induce sodium appetite) produced a greater increase 0.3 M NaCl intake in 2K1C rats. Moreover, this intake is totally blocked by central AT1R and mineralocorticoid receptor antagonism. These data demonstrated that ANG II acting in the AT1R in the NTS is involved with the development of hypertension and baroreflex impairment in 2K1C and that AT2 overexpression at the same site can counteract these effects. In addition, the results demonstrated the participation of ANG II and aldosterone acting centrally in the enhanced sodium intake in water deprived 2K1C rats.

Genetic Deletion of AT1a Receptor or Na+/H+ Exchanger 3 Selectively in the Proximal Tubules of the Kidney Attenuates Two-Kidney, One-Clip Goldblatt Hypertension in Mice.

The roles of angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of two-kidney, 1-clip (2K1C) Goldblatt hypertension have not been investigated previously. The present study tested the hypothesis that deletion of the AT1a receptor or NHE3 selectively in the proximal tubules of the kidney attenuates the development of 2K1C hypertension using novel mouse models with proximal tubule-specific deletion of AT1a receptors or NHE3. 2K1C Goldblatt hypertension was induced by placing a silver clip (0.12 mm) on the left renal artery for 4 weeks in adult male wild-type (WT), global Agtr1a−/−, proximal tubule (PT)-specific PT-Agtr1a−/− or PT-Nhe3−/− mice, respectively. As expected, telemetry blood pressure increased in a time-dependent manner in WT mice, reaching a maximal response by Week 3 (p < 0.01). 2K1C hypertension in WT mice was associated with increases in renin expression in the clipped kidney and decreases in the nonclipped kidney (p < 0.05). Plasma and kidney Ang II were significantly increased in WT mice with 2K1C hypertension (p < 0.05). Tubulointerstitial fibrotic responses were significantly increased in the clipped kidney (p < 0.01). Whole-body deletion of AT1a receptors completely blocked the development of 2K1C hypertension in Agtr1a−/− mice (p < 0.01 vs. WT). Likewise, proximal tubule-specific deletion of Agtr1a in PT-Agtr1a−/− mice or NHE3 in PT-Nhe3−/− mice also blocked the development of 2K1C hypertension (p < 0.01 vs. WT). Taken together, the present study provides new evidence for a critical role of proximal tubule Ang II/AT1 (AT1a)/NHE3 axis in the development of 2K1C Goldblatt hypertension.

Nobiletin resolves left ventricular and renal changes in 2K-1C hypertensive rats

This study investigated the effects of nobiletin on cardiorenal changes and the underlying mechanisms involved in two-kidney, one-clip (2K-1C) hypertension. 2K-1C rats were treated with nobiletin (15 or 30 mg/kg/day) or losartan (10 mg/kg/day) for 4 weeks (n = 8/group). Nobiletin (30 mg/kg) reduced high levels of blood pressure and circulating angiotensin II and angiotensin-converting enzyme activity in 2K-1C rats. Left ventricular (LV) dysfunction and remodelling in 2K-1C rats were alleviated in the nobiletin-treated group (P < 0.05). Nobiletin reduced the upregulation of Ang II type I receptor (AT1R)/JAK (Janus kinase)/STAT (signal transducer and activator of transcription) protein expression in cardiac tissue of 2K-1C rats (P < 0.05). The reduction in kidney function, and accumulation of renal fibrosis in 2K-1C rats were alleviated by nobiletin (P < 0.05). Overexpression of AT1R and NADPH oxidase 4 (Nox4) protein in nonclipped kidney tissue was suppressed in the nobiletin-treated group (P < 0.05). The elevations in oxidative stress parameters and the reductions in antioxidant enzymes were attenuated in 2K-1C rats treated with nobiletin (P < 0.05). In summary, nobiletin had renin-angiotensin system inhibitory and antioxidant effects and attenuated LV dysfunction and remodelling via restoration of the AT1R/JAK/STAT pathway. Nobiletin also resolved renal damage that was related to modulation of the AT1R/Nox4 cascade in 2K-1C hypertension.