2K1C Model 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.

Abstract P201: Macula Densa (Pro)Renin Receptor Contributes to 2-Kidney, 1-Clip-Induced Renovascular Hypertension but not Ischemic Nephropathy in Mice

Our previous presentation at this meeting showed that collecting duct (CD)-specific deletion of (pro)renin receptor (PRR) or renin attenuated hypertension and kidney damage induced by the 2-kidney, 1 clip (2K1C) procedure, highlighting an essential role of the intrarenal renin-angiotensin system (RAS) in the pathogenesis of renovascular hypertension and ischemic nephropathy. Besides the CD, PRR is also expressed in the macula densa (MD), mediating renin release from the juxtaglomerular (JG) cells. Here we examined the phenotype of NKCC2-Cre-mediated MD/TAL-specific deletion of PRR (MD/TAL PRR KO) following the 2K1C procedure. Clipping-induced hypertension was partially attenuated in MD/TAL PRR KO mice compared to floxed controls (MAP on day 24: Floxed/2K1C 148.3±1.8 mm Hg vs. MD/TAL PRR KO/2K1C 138.8±2.9 mm Hg, n=6, p < 0.05), to a similar degree in mice lacking PRR or renin in the CD. However, MD/TAL PRR KO mice did not exhibit protective effects against ischemic nephropathy compared to floxed controls following the 2K1C procedure (urinary albumin/creatinine: Floxed/2K1C 65.9 ± 4.6 mg/g vs. MD/TAL PRR KO/2K1C 59.5 ± 2.7 mg/g, n=6). Furthermore, there were no significant differences in clipping-induced protein abundance of renal fibronectin, α-SMA, or proinflammatory markers such as IL-1β, TNF-α, MCP-1. The protective phenotype against hypertension observed in KO mice was paralleled with suppressed plasma renin activity, active renin content, and total prorenin/renin levels. Additionally, while clipping upregulated renin mRNA expression in both renal cortex and medulla of floxed mice, only cortical renin expression was reduced by 39% in MD/TAL PRR KO mice. The 2K1C-induced increase in circulating sPRR was attenuated by 31% in MD/TAL PRR KO mice. Overall, our results suggest that renovascular hypertension in the 2K1C model is jointly determined by the systemic and intrarenal RAS whereas ischemic nephropathy in this model solely depends on the intrarenal RAS.

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.

Idebenone protects the kidneys of rats with renovascular arterial hypertension by inhibiting oxidative stress and apoptosis.

Here, the protective effect of antioxidant Idebenone (IDB) on renovascular hypertension was studied. The two-kidney one-clip (2K-1C) model of renal hypertension was established. The rats were divided into 3 groups: sham-operation group, 2K-1C renal hypertensive rats' model group and model treated with IDB group. The mean arterial blood pressure (MBP) of rats was measured and pathological condition of kidney was observed by H&E staining. The change of renal damage biomarkers (Cre, BUN, urine proteins), inflammatory factors (IL-6, IL-1β and TNF-α), oxidative stress ratio and key factors (MDA, SOD and CAT) were assessed by kits. The apoptosis key proteins (BAD, BAX, Caspase9, GSK-3β) were detected via Western blot. The 2K-1C model of renal hypertension was established. IDB reduced the MBP, Cre, BUN, urine proteins and improved the pathological condition of 2K-1C kidney. IDB restrained the inflammation factors (IL-6, IL-1β and TNF-α) and oxidative stress in kidney of renal hypertensive rats' model. Besides, IDB suppressed the expression of apoptosis key factors (BAD, BAX, Caspase9, GSK-3β) in kidney of renal hypertensive rats' model. IDB protects the kidneys of rats with renovascular arterial hypertension by inhibiting inflammation, oxidative stress, and apoptosis. These findings might provide medication guidance for IDB in renovascular arterial 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.

Role of plasmacytoid dendritic cells in vascular dysfunction in mice with renovascular hypertension

Endothelial dysfunction and inflammation are clinically significant risk factors for cardiovascular diseases in hypertension. Although immune cells play a role in hypertension, the impact of plasmacytoid dendritic cells in established renovascular hypertension-induced cardiovascular complications is not fully understood. We investigated plasmacytoid dendritic cells' contribution to arterial endothelial dysfunction and inflammation in renovascular hypertension.A two-kidney one-clip (2K1C) model for four weeks in both male and female mice was used to induce renovascular hypertension. We treated mice with or without anti-PDCA-1 antibodies for one week to deplete the plasmacytoid dendritic cells. Renovascular hypertension causes cardiac hypertrophy, lung edema, and microvascular endothelial dysfunction associated with inflammation induction in mice. Moreover, renovascular hypertension affects the profile of immune cells, including dendritic cells and macrophages, with variations between male and female mice. Interestingly, the depletion of plasmacytoid dendritic cells significantly reduces blood pressure, cardiac hypertrophy, lung edema, inflammation, and oxidative stress and improves microvascular endothelial function via the endoplasmic reticulum (ER) stress, autophagy, and mTOR-dependent mechanisms.Plasmacytoid dendritic cells significantly contribute to the development of cardiovascular complications in renovascular hypertension by modulating immune cells, inflammation, oxidative stress, and ER stress.

Apelin-13: a novel approach to suppressing renin production in RVHT.

Renovascular hypertension (RVHT) is characterized by renal artery stenosis and overactivated renin-angiotensin system (RAS). Apelin, known for its negative modulation of RAS, has protective effects against cardiovascular diseases. The role and mechanisms of the primary active form of apelin, apelin-13, in RVHT are unclear. In this study, male Sprague-Dawley rats were divided into control, two-kidney one-clip (2K1C) model, and 2K1C with apelin-13 treatment groups. Renin expression was analyzed using immunohistochemistry and molecular techniques. Full-length (pro)renin receptor (fPRR) and soluble PRR (sPRR) levels were assessed via Western blotting, and cAMP levels were measured using ELISA. Plasma renin content, plasma renin activity (PRA), angiotensin II (ANG II), and sPRR levels were determined by ELISA. Human Calu-6 and mouse As4.1 cells were used to investigate renin production mechanisms. The 2K1C model exhibited increased systolic blood pressure, plasma renin content, PRA, sPRR, and ANG II levels, while apelin-13 treatment reduced these elevations. Apelin-13 inhibited cAMP production, renin mRNA expression, protein synthesis, and PRR/sPRR protein expression in renal tissue. In Calu-6 cells, cAMP-induced fPRR and site-1 protease (S1P)-derived sPRR expression, which was blocked by cAMP-responsive element-binding protein (CREB) inhibition. Apelin-13 suppressed cAMP elevation, CREB phosphorylation, fPRR/sPRR protein expression, and renin production. Recombinant sPRR (sPRR-His) stimulated renin production, which was inhibited by the PRR decoy peptide PRO20 and S1P inhibitor PF429242. These findings suggest that apelin-13 inhibits plasma renin expression through the cAMP/PKA/sPRR pathway, providing a potential therapeutic approach for RVHT. Understanding the regulation of renin production is crucial for developing effective treatments.NEW & NOTEWORTHY Our research elucidated that apelin-13 inhibits renin production through the cAMP/PKA/soluble (pro)renin receptor pathway, presenting a promising therapeutic approach for renovascular hypertension (RVHT) by targeting renin expression mechanisms. These findings underscore the potential of apelin-13 as a novel strategy to address RVHT.

Abstract P221: Soluble (pro)renin Receptor Contributes To 2-kidney, 1-clip-induced Renovascular Hypertension And Ischemic Nephropathy In Mice

The Goldblatt two-kidney, one-clip (2K1C) model features renovascular hypertension and ischemic nephropathy due to overactivated renin-angiotensin system (RAS). Soluble (pro)renin receptor (sPRR), the extracellular domain of (pro)renin receptor (PRR), is primarily generated by site-1 protease (S1P) and implicated as a potential regulator of the RAS and renal function. Here, we examined the function and underlying mechanism of sPRR in 2K1C model by pharmacological inhibition of S1P and genetic mutagenesis of S1P cleavage site. Male 2-mo-old C57BL/6 mice were subjected to the 2K1C procedure or sham operation for 1 month, followed by a 14-day infusion of vehicle or a specific S1P inhibitor PF429242 (PF) (30 mg/kg/day via minipump). The 2K1C procedure induced a 2-fold increase in plasma sPRR, which was reduced by 50% following PF treatment. This was paralleled by improvement of renovascular hypertension (MAP:142.1±1.3 vs. 129.9±2.8mmHg, n=7, p < 0.05) and cardiac hypertrophy (HW/BW:6.3±0.3 vs. 5.0 ± 0.3mg/g, n=7, p < 0.01) accompanied with suppressed cortical and medullary renin activity, active renin content, prorenin content and total renin content in clipped kidney. Meanwhile, cortical and medullary renin mRNA level in clipped kidney was increased 3.8-fold and 5.3-fold, which was reduced by 41% and 55% respectively, following PF treatment. The level of urinary albumin, KIM-1 and NGAL was elevated 5.1-fold, 4.0-fold and 5.7-fold after 2K1C surgery, which was blunted 35%, 49% and 59%, respectively, following PF treatment. Secondly, we employed a novel mouse model with mutagenesis of the cleavage site in PRR (termed as Mutant mice) to verify the role of endogenous sPRR in 2K1C model. Similarly, the hypertensive response and ischemic nephropathy of Mutant mice to 2K1C procedure was blunted (24-day MAP: 2K1C/WT 142.5±2.1 vs. 2K1C/Mutant 128.2±3.2mmHg; 24-day urinary albumin/creatinine: 2K1C/WT 64.9±7.2 vs. 2K1C/Mutant 33.0±1.9mg/g, n=6, p < 0.05), in parallel with attenuated response of systemic and intrarenal RAS. Together, consistent results with the different approaches have established an important role of S1P-derived sPRR in regulation of the RAS and thus the pathogenesis of 2K1C-induced renovascular hypertension and ischemic nephropathy.

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.

Hibiscus attenuates renovascular hypertension-induced aortic remodeling dose dependently: the oxidative stress role and Ang II/cyclophilin A/ERK1/2 signaling.

Introduction: The high levels of angiotensin II (Ang II) can modify the vascular tone, enhance vascular smooth muscle cells (VSMCs) proliferation and hypertrophy and increase the inflammatory cellular infiltration into the vessel wall. The old herbal nonpharmacological agent, Hibiscus (HS) sabdariffa L has multiple cardioprotective impacts; thus, we investigated the role of HS extract in amelioration of renovascular hypertension (RVH)-induced aortic remodeling. Materials and methods: Thirty-five rats (7/group) were randomly allocated into 5 groups; group: I: Control-sham group, and RVH groups; II, III, IV, and V. The rats in RVH groups were subjected to the modified Goldblatt two-kidneys, one clip (2K1C) for induction of hypertension. In group: II, the rats were left untreated whereas in group III, IV, and V: RVH-rats were treated for 6weeks with low dose hibiscus (LDH), medium dose hibiscus (MDH), and high dose hibiscus (HDH) respectively. Results: We found that the augmented pro-contractile response of the aortic rings was ameliorated secondary to the in-vivo treatment with HS dose dependently. The cyclophilin A (CyPA) protein levels positively correlated with the vascular adhesion molecule-1 (VCAM-1) and ERK1/2, which, in turn, contribute to the reactive oxygen species (ROS) production. Daily HS intake modified aortic renovation by enhancing the antioxidant capacity, restraining hypertrophy and fibrosis, downregulation of the metastasis associated lung adenocarcinoma transcript (MALAT1), and cyclophilin A (CyPA)/ERK1/2 levels. Discussion: Adding to the multiple beneficial effects, HS aqueous extract was able to inhibit vascular smooth muscle cell proliferation induced by 2K1C model. Thus, adding more privilege for the utilization of the traditional herbal extracts to attenuate RVH-induced aortopathy.

Baroreflex Sensitivity, Cardiac and Kidney Remodeling and Deterioration in Vasoactive Substances Content in Blood in Experimental Model of Renovascular Hypertension. Action of Natural Flavone, Luteolin

This study aimed to analyze pharmacological actions of phenolic compound luteolin on the renal and cardiac hypertrophy, blood pressure (BP), baroreflex sensitivity (BRS), levels of epoxyeicosatrienoic acids (EETs), prostaglandin E-2 (PGE-2) and endothelin-1 (E1) in plasma in the 2 kidney - 1 clip (2K-1C) model of renovascular hypertension (RVH). All animals, were randomized into 2 groups: control (normal) I - sham-operated, II- RVH male Wistar rats, which after 4 weeks of surgical intervention secondly randomized to control II group, treated 0.1% dimethyl sulphoxide (DMSO) and main group - with luteolin in 15 DMSO, 3 mg/kg body weight, intraperitonially, during 2 weeks. ET-1, EETs and PGE2 levels investigated in carotid artery blood plasma and analyzed using ELISA kits. All data statistically analyzed using the SPSS-10.0 program. In RVH rats BP increased by 32%, cardiac and right kidney hypertrophy and reduction in parasympathetic component of BRS by 40% and sympathetic by 39%. The plasma level of total trans-EETs and PGE2 in RVH rats decreased by 44% and 50% respectively, while the level of ET-1 increased by 67%. Two weeks treatment with luteolin lowered BP, improved parasympathetic, without marked changes in sympathetic component of BRS. Deremodeling of cardiac and renal hypertrophy under prolonged treatment with luteolin accompanied with increasing in the level of EETs by 44%, PGE-2 by 50% and markedly reducing of plasma content of ET-1 (by 60%). Inhibition of EET hydrolase using low doses of luteolin provides beneficial cardio and renoprotective action in experimental model of RVH.

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.

S-07-2: DISCOVERY OF NEUROENDOTHELIAL CELLS AND THEIR ROLE IN HYPERTENSION AND AGING

Objective: Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow and in the development of renal and cardiovascular diseases. Tissue-specific EC dysfunction can contribute to several different diseases including hypertension. Recent transcriptomic studies identified many EC subtypes in multiple organs including the brain and the kidneys, however, their functions are incompletely understood. The present study aimed to explore physiological functional significance and cardiovascular disease and hypertension relevance of a newly discovered minority subtype of scattered ECs expressing neuronal nitric oxide synthase (Nos1). Design and method: A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, mTORgof/lof), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, two-kidney one-clip (2K1C) model of renovascular (Goldblatt) hypertension (RVHT), whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. Results: Our studies identified and characterized, for the first time, a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, therefore we named them neuroendothelial cells (NECs). NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. NECs are found only in the three organs that exhibit the best blood flow autoregulation capacity, with the highest density in the brain&gt;kidney&gt;heart (NEC/EC (%) 8.42+/-0.79, 3.21+/−0.33, 1.73+/−0.07, respectively). NEC density is reduced with aging in the brain and the kidney (NEC/EC (%) 4.675, and 1.850, respectively, p &lt; 0.01, 2.5 years old compared to 2-month-old). The number of NECs increased significantly in the hypo-perfused, hypoxic clipped (CK) kidney, but reduced in the hyperperfused non-clipped (NCK) kidney in RVHT. Intravital multiphoton microscopy (MPM) of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations. Newly established NEC gain-of-function mouse models exhibited increased endothelium-dependent vasodilation and diminished agonist-induced vascular contractility in brain and kidney resistance arterioles compared to controls. In addition, preliminary single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Conclusions: These new vascular anatomy and hemodynamic findings strongly suggest sensory, blood flow and/or baroreceptor functions of NECs as well as a role in the autoregulation of organ blood flow and hypertension pathogenesis.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Effects of asperglaucide on endothelium function in 2K1C hypertensive rats

Background and Aims : This study aimed to investigate the effects of Asperglaucide (ASP) on endothelial nitric oxide synthase (eNOS) expression, vascular fluidity and vascular endothelial function in the two-kidney one-clip (2K-1C) model of renovascular arterial hypertension.

Improved method for surgical induction of chronic hypertension in mice.

ABSTRACTChronic hypertension can be induced in mice by one-kidney one-clip (1K1C) or two-kidney one-clip surgery, transgenic overexpression of angiotensinogen and renin, administration of deoxycorticosterone acetate-salt, supplying Nitro-L-arginine methyl-ester in the drinking water and Angiotensin-II infusion. Although each model has its own pros and cons, selection of a model that mimics human hypertensive disease accurately is essential to ensure rigor and reproducibility in hypertension research. 1K1C mice represent an efficient, budget-friendly, and translationally capable model; however, their use in preclinical research has remained largely hindered due to concerns about potential technical complexity and lack of reported information regarding procedure-related mortality rates. Here, we describe in detail an improved version of the 1K1C surgery in mice that has zero intraoperative mortality and excellent survival rates in a long-term setting and permits the development of stable chronic hypertension and its target organ complications. Key to this outcome is unilateral nephrectomy 1 week after renal artery clipping to decelerate the blood pressure (BP) increase, which allows the organism to adapt better to the BP rise. The technical and animal welfare improvements presented here may promote the acceptance of the 1K1C model.

Anti-hypertensive effects of Callisia fragrans extract on Reno-vascular hypertensive rats

ABSTRACT Objectives This study aims to investigate the anti-hypertensive effects of aqueous extract of Callisia fragrans and their underlying mechanism using a two-kidney one-clip (2K1C) model of reno-vascular hypertension in rats. Methods The reno-vascular hypertensive rats were treated with C. fragrans leaf extract (100 and 500 mg/kg; p.o.) and a reference drug, captopril (20 mg/kg; p.o.), for 4 weeks. The blood pressure and heart rate were recorded using a tail-cuff. The heart weight, left ventricular wall thickness, and serum creatinine and urea levels were measured. A spectrophotometric assay was used to analyze the angiotensin-converting enzyme (ACE) inhibition activity of the extract and the reference drug. The total volume and the concentration of sodium, potassium, and chloride in urine samples were evaluated. Results C. fragrans extract significantly reduced both systolic and diastolic blood pressures in the reno-vascular hypertensive rats. No significant difference in the heart rate was observed between each animal group. C. fragrans extract reduced the 2K1C-induced increase in the heart and body weight ratio and the left ventricular wall thickness. Moreover, the extract also attenuated the increase in serum urea induced by the 2K1C treatment. C. fragrans extract inhibited ACE activity in vitro with an IC50 of 20.97 ± 3.94 µg/ml. The urine output and urinary electrolyte excretion significantly increased in C. fragrans extract-treated rats. Conclusions These findings demonstrated that C. fragrans extract can mitigate hypertension and alleviate ventricular hypertrophy and renal dysfunction in reno-vascular hypertensive rats, at least in part via ACE activity inhibition and diuretic property.

Estradiol Supplement or Induced Hypertension May Attenuate the Angiotensin II Type 1 Receptor Antagonist-Promoted Renal Blood Flow Response to Graded Angiotensin II Administration in Ovariectomized Rats.

Backgrounds Estrogen replacement therapy (ERT) and hypertension may influence females' renin-angiotensin system (RAS) and its components. The angiotensin II (Ang II) type 1 receptor (AT1R) antagonist (losartan) may promote renal blood flow (RBF), and it is widely used in the clinic to control hypertension. The main objective of this study was the effects of estradiol or induced hypertension on RBF response to Ang II in losartan-treated ovariectomized (OVX) rats. Methods Two groups of OVX rats were treated with placebo (group 1) and estradiol (group 2) for period of four weeks, and another group of OVX rats was subjected to induce hypertension by two-kidney one clip (2K1C) model (group 3). All the groups were subjected to the surgical procedure under anesthesia, and AT1R was blocked by losartan. RBF and renal vascular resistance (RVR) responses to Ang II administration were determined and compared. Results Mean arterial (MAP) and renal perfusion (RPP) pressures in group 3 and uterus weight (UT) in group 2 were significantly more than other groups (P < 0.05). Ang II infusion resulted in dose-related percentage change increase in RBF and decrease in RVR. However, these responses in the OVX-estradiol and OVX-hypertensive rats were significantly lower than in the OVX-control group (P < 0.05). For instance, at the dose of 1000 ng/kg/min of Ang II administration, the percentage change of RBF was 45.1 ± 10.4%, 17.9 ± 2.3%, and 16.7 ± 4.7% in the groups of 1 to 3, respectively. Conclusion Losartan prescription in some conditions such as hypertension or ERT could worsen RBF and RVR responses to Ang II.