2K1C Group Research Articles

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.

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.

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.

Daily consumption of monosodium glutamate pronounced hypertension and altered renal excretory function in normotensive and hypertensive rats

This study aimed to investigate the effects of monosodium glutamate (MSG) on the levels of arterial blood pressure (ABP) and renal excretory function. Male Wistar rats were divided into 2 groups (n = 24 each) namely sham operation (SO) and 2-kidneys-1-clip (2K1C) to develop the normotensive and hypertensive model, respectively. Four weeks after the operation, each group of rats were further divided into 4 subgroups (n = 6 each) which were orally administered of either distilled water or MSG at the doses of 80, 160, or 320 mg/kg BW/day once a day for 8 weeks. The body weight, the 24-hour water intake, and the 24-hour urine output were recorded weekly. Then, each rat was anesthetized and the ABP was measured via carotid artery. The renal excretory function was examined by using the clearance technique to measure the levels of the glomerular filtration rate and the renal blood flow. The levels of serum malondialdehyde (MDA) as a marker of oxidative stress were analyzed. The expression of tumor necrosis factor alpha (TNF-α) in the kidneys was also investigated using immunohistochemistry. It was found that all doses of MSG significantly increased the ABP in both SO and 2K1C groups. All doses of MSG significantly increased the serum MDA levels and the expression of TNF-α in the kidneys of the SO groups. Long-term intake of 320 mg/kg BW MSG significantly decreased the renal excretion of salt and water in both SO and 2K1C groups. As a whole, this study demonstrated that MSG consumption contributed to an increase in oxidative stress which could lead to alterations in the cardiovascular and renal function.

DNA damage and repair on hematopoietic stem cells: impact of oxidative stress in renovascular hypertension

ABSTRACT Background This study investigated oxidative damage to bone marrow cells in the pathogenesis of renovascular hypertension (RH). Methods Male C57BL/6 J mice (10-week-old and ~23 g) were divided into two groups: Sham-operated and 2K1C, which has a stainless-steel clip placed around the left renal artery. After twenty-eight days, the animals were anesthetized for hemodynamic measurements and bone marrow cells isolation. The intracellular production of ROS, DNA damage, and DNA repair kinetics were evaluated. Results Our results show that RH increases HSCs ROS production and that the 2K1C group showed a significant reduction of HSCs in the G0/G1 phase, increased p53 expression, DNA fragmentation, low DNA repair capacity, and a higher percentage of apoptotic cells when compared with the Sham group. Conclusions Our data imply that RH can compromise the hematopoiesis by increased oxidative stress leading to impaired DNA repair activity. Furthermore, this study provides new insights into the influence of hypertension on bone marrow homeostasis. This study showed for the first time that RH leads to oxidative damage, including genotoxic, to bone marrow cells. Thus, these findings provide new insights into the consequences of RH on bone marrow cells.

A New Approach for the Development of Multiple Cardiovascular Risk Factors in Two Rat Models of Hypertension.

Cardiovascular disease (CVD) is the leading cause of death among non-communicable diseases. There is a lack of valid animal models that mimic associations among multiple cardiovascular risk factors in humans. The present study developed an animal model that uses multiple cardiovascular risk factors—namely, hypertension, hypothyroidism, and a high-fat diet (HFD). Two models of hypertension were used: renovascular hypertension (two-kidney, one clip [2K1C]) and spontaneously hypertensive rats (SHRs). The naive group was composed of normotensive rats. Twelve weeks after surgery to induce renovascular hypertension, rats in the 2K1C and SHR groups underwent thyroidectomy. The HFD was then implemented for 6 weeks. Renal function, serum redox status, biochemical CVD markers, electrocardiographic profile, blood pressure, mesenteric vascular bed reactivity, histopathology, and morphometry were investigated. Both experimental models induced dyslipidemia, renal function impairment, and hepatic steatosis, accompanied by higher levels of different inflammatory markers and serum oxidative stress. These alterations contributed to end-organ damage in all hypertensive rats. Our findings corroborate a viable alternative model that involves multiple cardiovascular risk factors and resembles conditions that are seen in humans. Both models mimicked CVD, but our data show that SHRs exhibit more significant pathophysiological changes.

Oral Administration of Garcinia dulcis Flower Extract Lowers Arterial Blood Pressure of 2-kidneys-1-clip Renovascular Hypertensive Rat

The hexane insoluble fraction of the Garcinia dulcis (GD) flower extract comprises mainly camboginol and morelloflavone which possess potent in vivo and in vitro antioxidant properties. This study aimed to evaluate the effects of 4-week oral administration of GD flower extract on the arterial blood pressure (ABP) and the excretory function of the kidney in the 2-kidneys-1-clip (2K1C) renovascular hypertensive rats (total=12) compared to sham operated (SO) normotensive Wistar rats (total=12). Four weeks after hypertensive-induced surgery, either 50 mg/kg BW GD flower extract or vehicle was orally administered to the 2K1C or SO groups (n=6/group) daily for four weeks. ABP and the renal excretory function were studied in anesthetized rats, and expression of endothelial nitric oxide synthase (eNOS) mRNA in the isolated thoracic aorta were measured. In the 2K1C rats, GD flower extract significantly decreased ABP while increased significantly eNOS mRNA levels. GD flower extract did not exert a diuretic effect in either SO and 2K1C rats since there was no change in observed urine excretion, but it did tend to attenuated the renal tubular damage caused by renovascular hypertension. GD flower extract was anti-hypertensive in this model of renovascular hypertension and problably acts via the endothelial nitric oxide signaling pathway.

Anti-hypertensive effect of Morus alba L extract in rats


 
 
 
 Purpose: To investigate the probable antihypertensive effects of Morus alba L. extract (MALE) in renovascular hypertensive rats, and the mechanism involved in the reduction of blood pressure.
 Methods: The two-kidney one-clip (2K1C) Goldblatt model of renovascular hypertension was used in Wistar rats. The 2K1C group rats were treated with captopril (30 mg/kg), and Morus alba extract (75 and 150 mg/kg) daily for 6 weeks by intragastric administration. Systolic (SBP) and diastolic blood pressure (DBP) were measured by the tail-cuff method. Urine creatinine and urea of the rats were measured by enzyme-linked immunosorbent assay (ELISA). Superoxide dismutase (SOD) and malondialdehyde (MDA) were also evaluated using standard techniques.
 Results: In the captopril- and extract-treated groups, blood pressure decreased progressively over the course of the 6-week treatment period compared with that of the control rats (p < 0.01). The extract at 150 mg/kg also significantly (p < 0.05) increased plasma SOD activity but decreased plasma MDA concentration. Renal function significantly (p < 0.01) improved following captopril and extract (150 mg/kg) treatment.
 Conclusion: The results suggest that Morus alba extract exerts its antihypertensive effect in rats by inhibiting endothelin (ET)-converting enzyme and via its antioxidant activity. Thus, the plant extract has some potentials for development into a new drug in for use in humans.
 
 
 

Involvement of Neuropeptide Y within Paraventricular Nucleus in Electroacupuncture Inhibiting Sympathetic Activities in Hypertensive Rats.

Although electroacupuncture (EA) has been used to decrease the blood pressure (BP) clinically, the underlying mechanisms are not clearly clarified. This study aimed to assess the hypothesis that EA treatment exerts a hypotensive action via suppressing sympathetic activities and modulating neuropeptide Y (NPY) function within the paraventricular nucleus (PVN) of hypertensive rats. Male Sprague-Dawley rats were selected for the experiment, and the hypertensive models were established by the two-kidney, one-clip (2K1C) method. Then, the rats were randomly assigned to the sham group, 2K1C group, 2K1C plus EA group, and 2K1C plus sham EA group. EA treatment at the acupoints ST36 and ST40 overlying the peroneal nerves was given once a day for 30 days. The radiotelemetry system was applied to collect the arterial BP recordings. Power spectral analyses of BP variability, BP responses to ganglionic blockade, and plasma levels of norepinephrine and epinephrine were performed to assess the changes in sympathetic nerve activity. Real-time PCR and Western blots were carried out to examine the expression of NPY system in the PVN. The responses of PVN microinjection with NPY Y1R antagonist BIBO3304 were detected to check the endogenous NPY tone. The results showed that the enhanced arterial BP and sympathetic activities were effectively reduced by 30 days of EA treatment, and baroreflex sensitivity was improved in 2K1C hypertensive rats. The level of NPY mRNA and protein expression in the PVN was markedly upregulated by EA treatment in 2K1C rats. In addition, the pressor responses of PVN microinjection with NPY Y1R antagonist BIBO3304 in 2K1C models were remarkably augmented by the EA stimulation. Our results indicate that the increased NPY expression and function in the PVN induced by EA treatment contribute to antihypertensive and sympathetic suppression on hypertensive rats. The findings may elucidate the underlying mechanisms of the acupuncture to be a potential therapeutic strategy against hypertension.

Maintaining Normal Blood Pressure in a Renovascular Stenosis Model of Hypertension in Adult Lewis Rats: Putative Physiological Modulation of the Renin‐Angiotensin System

Prolonged unilateral renal artery stenosis using a Goldblatt Two-Kidney-One Clip (2K1C) technique is a validated scientific approach to inducing experimental hypertension in laboratory animals. This patho-physiological modulation is associated with the activation of the renin-angiotensin system with increased renin and angiotensin II release to initiate hypertension. This mode of experimental hypertension has been demonstrated for many rat strains including Sprague Dawley, Brown Norway and Wistar Kyoto but has not been fully characterized in syngeneic Lewis rats. The objective of this study was to develop and characterize a unilateral renal artery stenosis model of hypertension in adult male Lewis rats using a 2K1C method for hypertension studies in our laboratory. Thirty animals were randomly assigned to two groups to undergo sham or the 2K1C surgical procedure under isoflurane-induced anesthesia; approval was granted by our institutional animal ethics committee (A2404, n =15 per group). Hemodynamic parameters including heart rate; systolic and diastolic blood pressure were monitored weekly for 6 weeks, using a volume-pressure tail cuff and a CODA pressure computer. Plasma concentrations of critical biomarkers of the renin-angiotensin system were quantified using standard ELISA assays and renal histopathological changes were assessed microscopically. Unilateral renal arterial stenosis caused marked renal atrophy, glomerulosclerosis and interstitial fibrosis, but a compensatory renal hypertrophy was observed in contralateral kidneys. Interestingly, unilateral renal arterial stenosis failed to induce hypertension over the six week period. The resistance to develop hypertension in the 2K1C group was associated with a significant decrease in total plasma renin (P = 0.023), an increase in the ratio of plasma angiotensin (1-7) to total renin (P = 0.034) and a decrease in the ratio of angiotensin II to total renin (P = 0.013). There were no changes in plasma electrolytes, glucose, creatinine or urea. These data demonstrate that adult male Lewis rats may have physiologically modulated the activation of the renin-angiotensin system to maintain a homeostatic balance and resist hypertension following unilateral renal artery stenosis.

Regulation of connexins genes expression contributes to reestablishes tissue homeostasis in a renovascular hypertension model

Connexins (Cx) are essential for cardiovascular regulation and maintenance of cardio-renal response involving the natriuretic peptide family. Changes in the expression of connexins promote intercellular communication dysfunction and may induce hypertension, atherosclerosis, and several other vascular diseases. This study analyzed the expression of the genes involved in the renin-angiotensin system (RAS) and the relation of the connexins gene expression with the renovascular hypertension 2K1C in different tissues. The insertion of a silver clip induced renovascular hypertension 2K1C into the left renal artery. Biochemical measurements were made using commercial kits. Gene expression was evaluated in the liver, heart, and kidneys by RT-PCR. The genes investigated were LDLr, Hmgcr, Agt, Ren, Ace, Agtr1a, Anp, Bnp, Npr1, Cx26, Cx32, Cx37, Cx40 and Cx43. All genes involved in the RAS presented increased transcriptional levels in the 2K1C group, except hepatic Agt. The natriuretic peptides (Anp; Bnp) and the receptor genes (Npr1) appeared to increase in the heart, however, Npr1 decreased in the kidneys. In hepatic tissue, hypertension promoted increased expression of Cx32, Cx37, and Cx40 genes however, Cx26 and Cx43 genes were not influenced. Expression was upregulated for Cx37 and Cx43 in cardiac tissue in the 2K1C group, but Cx40 did not demonstrate any difference between groups. The stenotic kidney showed an upregulated expression for Cx37 vs Sham and contralateral kidney, although Cx40 and Cx43 were downregulated. Hypertension did not modify the transcriptional expression of Cx26 and Cx32. Therefore, this study indicated that RAS and cardiac response were regulated transcriptionally by renovascular hypertension 2K1C. Moreover, the results of connexin gene expression demonstrated differential transcriptional regulation in different tissues studied and suggest a relationship between cardiac and renal physiological changes as an adaptive mechanism to the hypertensive state.

ROLE OF SPINAL AT1, IONOTROPIC GLUTAMATERGIC AND GABAERGIC RECEPTORS ON CONTROL OF RENAL SYMPATHETIC NERVE ACTIVITY IN 2K‐1C RATS

Previous studies performed in our laboratory showed a higher glutamate and angiotensin receptors activation, as well as an alteration in GABAergic pathways in central nuclei involved in the cardiovascular control that may lead to sympathoexcitation in renovascular hypertensive rats (2K‐1C). However, the role of the spinal cord neurons on the renal sympathoexcitation remains unclear in this model. Thus, we aimed to assess the influence of spinal glutamatergic and GABAergic (ionotropic receptors), as well as angiotensin II type 1 receptors (AT1) receptors on the renal sympathetic nerve activity (rSNA) in the 2K‐1C rats. Male Wistar rats (250–300 gr) were used to induce renovascular hypertension by clipping the renal artery with a silver clip (0.2 mm). After six weeks, a polyethylene catheter (PE‐10) was inserted into the subarachnoid space and advanced to the T10–11 vertebrae level in urethane‐anaesthetized rats (1.2 g/Kg, iv). All experiments were approved by the Institutional Ethical Committee of UNIFESP. The effects of intrathecal (i.t.) injection of kynurenic acid (KYN) (160 nmol in 2 μl), losartan (Los) (2nmol in 2 μl) or bicuculline (Bic) (8 mM in 2 μl) on blood pressure (BP) and rSNA were investigated. KYN i.t. injection induced a larger and significant fall on rSNA in the 2K‐1C compared to control rats. Intrathecally Los evoked a larger and significant fall in rSNA in the 2K‐1C compared to control group. KYN decreased BP similarly in both, control and 2K‐1C groups, however, Los significantly decreased BP in the 2K‐1C group only. Intrathecally Bic triggered a more intense BP and rSNA reponses in 2K‐1C compared to control rats. Gene expression analysis by qRT‐PCR showed a spinal (T11–12) AT1 mRNA upregulation in the 2K‐1C rats (1.71‐fold change), while no changes were observed in AT2, ionotropic glutamate and GABA receptors subunits. Therefore, our data show that ionotropic spinal cord excitatory amino acid, GABAergic and AT1 receptors play a significant role in the control of rSNA in the 2K‐1C model that may contribute to the maintenance of hypertension. The participation of spinal AT1 receptors seems to be more important in the establishment of sympathoexcitation in this model. The origin of those projections remains unclear.Support or Funding InformationCNPq, FAPESP (2018/01898‐4) and CAPES

Renal blood flow and oxygen metabolism in renovascular hypertension

Renovascular hypertension affects ~1–4% of the hypertensive population and represents the second‐leading cause of secondary hypertension. There is an urgent clinical need to gain insight into the aetiology of renovascular hypertension to form the basis of new treatment targets. We hypothesize that high blood pressure in these conditions aims to sustain perfusion thus optimising organ blood flow and tissue oxygen homeostasis.Male Wistar Rats with (2K1C, n=5) or without (control, n=3) renal artery clip to generate renovascular hypertension were used. Six weeks after renal artery clipping, renal hemodynamics and oxygen metabolism were evaluated under isoflurane anaesthesia. Renal blood flow was measured in the clipped kidney using an ultrasound probe, glomerular filtration rate was measured using FITC‐inulin and the arterio‐venous difference in oxygen content was used to determine the clipped kidney oxygen consumption. Despite mean arterial pressure being higher in the 2K1C group when compared with controls (134±9 vs 97±10 mmHg, p=0.02), blood flow (7±1 vs 7±1 ml/min, p=0.823), glomerular filtration rate (0.42±0.1 vs 0.53±0.1 ml/min, p=0.45) and oxygen consumption (1.0±0.12 vs 0.6±0.1 mlO2/min, p=0.06) were not significantly different between 2K1C and control animalsThis preliminary data suggests that high blood pressure in 2K1C animals is sustaining normal renal perfusion, oxygen homeostasis and renal hemodynamics despite the reduction of arterial diameter.Support or Funding InformationBritish Heart Foundation funded research.

Afferent innervation of the ischemic kidney contributes to renal dysfunction in renovascular hypertensive rats.

The ablation of renal nerves, by destroying both the sympathetic and afferent fibers, has been shown to be effective in lowering blood pressure in resistant hypertensive patients. However, experimental studies have reported that the removal of sympathetic fibers may lead to side effects, such as the impairment of compensatory cardiorenal responses during a hemodynamic challenge. In the present study, we evaluated the effects of the selective removal of renal afferent fibers on arterial hypertension, renal sympathetic nerve activity, and renal changes in a model of renovascular hypertension. After 4weeks of clipping the left renal artery, afferent renal denervation (ARD) was performed by exposing the left renal nerve to a 33mM capsaicin solution for 15min. After 2weeks of ARD, we found reduced MAP (~ 18%) and sympathoexcitation to both the ischemic and contralateral kidneys in the hypertensive group. Moreover, a reduction in reactive oxygen species was observed in the ischemic (76%) and contralateral (27%) kidneys in the 2K1C group. In addition, ARD normalized renal function markers and proteinuria and podocin in the contralateral kidney. Taken altogether, we show that the selective removal of afferent fibers is an effective method to reduce MAP and improve renal changes without compromising the function of renal sympathetic fibers in the 2K1C model. Renal afferent nerves may be a new target in neurogenic hypertension and renal dysfunction.

Comparative analysis of relative gene expression data reveals novel microRNAs in the artery of rats with renovascular hypertension

Hypertension is associated with impaired vascular endothelial function. However, the regulatory mechanisms of vascular dysfunction in rats with renovascular hypertension (RVH) remain poorly understood. In this study, the 2-kidney- 1-clip (2K1C) hypertensive rat model was utilized. Next-generation sequencing was then used to detect microRNA (miRNA) expression profiling in the arteries of 2K1C rats. We identified 17 miRNAs that were differentially expressed in the 2K1C group compared with the sham group, of which 9 were downregulated and 8 were upregulated. These differentially expressed miRNAs were found to be associated with immune/inflammatory and metabolic pathways, which are involved in vascular dysfunction. Treatment with losartan maintained the expression of the differentially expressed miRNAs, miR-31a-5p and miR-142-3p, and the levels of TNF-?, IL-1?, IL-6 and MCP-1, indicating that the differentially expressed miRNAs and their associated immune/inflammatory pathways play a pivotal role in the modulation of the vascular dysfunction in 2K1C rats. Our study provides valuable information about miRNA expression in the arteries of 2K1C rats, expanding our understanding of the complex molecular mechanisms underlying the vascular dysfunction in rats with RVH.

Tissue-specific transcriptional regulation of epithelial/endothelial and mesenchymal markers during renovascular hypertension.

Epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition are processes that can occur under different biological conditions, including tissue healing due to hypertension and oxidative stress. The purpose of the present study was to evaluate the differences in gene expression of epithelial/endothelial and mesenchymal markers in different tissues. A two-kidney, one-clip (2K1C) renovascular hypertension rat model was used. Hypertension was induced by the clipping of the left renal artery; the rats were randomized into sham and 2K1C groups and monitored for up to 4 weeks. The gene expressions of E-cadherin (E-cad), N-cadherin (N-cad), α-smooth muscle actin (α-SMA), collagen I (COL1A1), collagen III (COL3A1) and hepatocyte growth factor (HGF) were determined by reverse transcription-PCR. The levels of the cytokines transforming growth factor-β1, tumor necrosis factor-α, interleukin (IL)-4, IL-6 and IL-10 were evaluated using ELISAs. The levels of thiobarbituric acid reactive substances and thiol groups were measured to evaluate oxidative stress. All analyses were performed on the liver, heart and kidneys tissues of sham and model rats. The 2K1C animals exhibited a higher systolic blood pressure, as well as cardiac hypertrophy and atrophy of the left kidney. Fibrotic alterations in the heart and kidneys were observed, as was an increase in the collagen fiber areas, and higher levels of inflammatory cytokines, which are associated with the increased expression of fibroproliferative and anti-fibrotic genes. Renovascular hypertension regulated epithelial/endothelial and mesenchymal markers, including E-cad, N-cad, α-SMA and COL1A1 in the kidneys and heart. EMT in the kidneys was mediated by an increased level of inflammatory and profibrotic cytokines, as well as by oxidative stress. The data in the present study suggested that the expression of epithelial/endothelial and mesenchymal markers are differentially regulated by hypertension in the liver, heart and kidneys.

Effects of Hibiscus Sabdariffa Calyces Aqueous Extract on the Antihypertensive Potency of Captopril in the Two-Kidney-One-Clip Rat Hypertension Model.

Hibiscus sabdariffa aqueous extract (HS) is often used as complementary therapy for hypertension. However, some studies have shown that coadministration with a conventional antihypertensive drug can affect drug potency. We compared the effects of HS plus captopril (CAP) coadministration to HS and CAP administration alone on blood pressure and renin-angiotensin-aldosterone system (RAAS) biomarkers in the rat two-kidney-one-clip (2K1C) model of hypertension. Male Sprague Dawley rats were randomly divided into seven groups (n=6/group), a normal control (SHAM) group, and six 2K1C groups. In 2K1C animals, hypertension was induced using a stainless microclip (inner diameter of 0.20 mm). Four weeks after 2K1C surgery, blood pressure was significantly higher than in the SHAM group. Then, model rats were randomly divided into negative control (2K1C, no treatment), positive control (4.5 mg captopril/200 g body weight [BW] orally [p.o.]), HS alone (30 mg/200 g BW; p.o.), and 3 co-treatment groups receiving HS (15, 30, or 60 mg/200 g BW; p.o.) plus 4.5 mg/200 g BW captopril. The treatments were performed for two weeks. Blood pressure was significantly reduced by all the drug treatments to near the level of SHAM controls. Plasma renin level, serum angiotensin converting enzyme (ACE) activity, and plasma angiotensin II level were also significantly elevated in the 2K1C group compared to the SHAM group. Both serum ACE activity and plasma angiotensin II level were significantly reduced to near SHAM group levels by all the drug treatments. Hibiscus sabdariffa aqueous extract alone can reduce blood pressure. This extract appears could be used as a supplement with captopril but may not provide any additional benefit.

Alteration of the Gut Microbiota and Its Effect on AMPK/NADPH Oxidase Signaling Pathway in 2K1C Rats.

Background The purpose of this study was to evaluate the alteration of the gut microbiota and its effect on adenosine monophosphate-activated protein kinase (AMPK)/nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) signaling pathway in two-kidney one-clip (2K1C) rats. Methods The 2K1C rat models were established. The rats were randomly divided into the following 2 groups: 2K1C group and sham group. Alterations of the gut microbiota were analyzed based on the high throughput sequencing method. Plasma concentrations of short chain fatty acids (SCFAs) were measured by chromatography. The protein expression of phosphorylated AMPK and acetyl-CoA carboxylase (ACC) was determined by western blotting. NADPH oxidase activity was measured by a luminometer. Results Microbial community analyses revealed that the structure and composition of the gut microbiota were significantly disrupted in 2K1C rats when compared to sham rats. This disruption was associated with the drastic increase in relative abundance of the genera Prevotella and the decrease in SCFA-producing bacterial population. We further confirm that SCFAs produced by the gut microbiota influence NADPH oxidase activity through AMPK. Conclusions Our data implicated the important role of gut microbiota in the regulation of AMPK/NADPH oxidase signaling pathway.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-β-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.