Ang II-infused Rats Research Articles

Inhibition of reactive oxygen species in hypothalamic paraventricular nucleus attenuates the renin–angiotensin system and proinflammatory cytokines in hypertension

AimsTo explore whether reactive oxygen species (ROS) scavenger (tempol) in the hypothalamic paraventricular nucleus (PVN) attenuates renin–angiotensin system (RAS) and proinflammatory cytokines (PICs), and decreases the blood pressure and sympathetic activity in angiotensin II (ANG II)-induced hypertension. Methods and resultsMale Sprague–Dawley rats were infused intravenously with ANG II (10ng/kg per min) or normal saline (NS) for 4weeks. These rats were treated with bilateral PVN infusion of oxygen free radical scavenger tempol (TEMP, 20μg/h) or vehicle (artificial cerebrospinal fluid, aCSF) for 4weeks. ANG II infusion resulted in increased mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). These ANG II-infused rats also had higher levels of gp91phox (a subunit of NAD(P)H oxidase), angiotensin-converting enzyme (ACE), and interleukin-1beta (IL-1β) in the PVN than the control animals. Treatment with PVN infusion of TEMP attenuated the overexpression of gp91phox, ACE and IL-1β within the PVN, and decreased sympathetic activity and MAP in ANG II-infused rats. ConclusionThese findings suggest that ANG II infusion induces elevated PICs and oxidative stress in the PVN, which contribute to the sympathoexcitation in hypertension. Inhibition of reactive oxygen species in hypothalamic paraventricular nucleus attenuates the renin–angiotensin system, proinflammatory cytokines and oxidative stress in ANG II-induced hypertension.

Chronic infusion of enalaprilat into hypothalamic paraventricular nucleus attenuates angiotensin II-induced hypertension and cardiac hypertrophy by restoring neurotransmitters and cytokines

The renin–angiotensin system (RAS) in the brain is involved in the pathogenesis of hypertension. We hypothesized that inhibition of angiotensin-converting enzyme (ACE) in the hypothalamic paraventricular nucleus (PVN) attenuates angiotensin II (ANG II)-induced hypertension via restoring neurotransmitters and cytokines. Rats underwent subcutaneous infusions of ANG II or saline and bilateral PVN infusions of ACE inhibitor enalaprilat (ENL, 2.5μg/h) or vehicle for 4weeks. ANG II infusion resulted in higher mean arterial pressure and cardiac hypertrophy as indicated by increased whole heart weight/body weight ratio, whole heart weight/tibia length ratio, left ventricular weight/tibia length ratio, and mRNA expressions of cardiac atrial natriuretic peptide and beta-myosin heavy chain. These ANG II-infused rats had higher PVN levels of glutamate, norepinephrine, tyrosine hydroxylase, pro-inflammatory cytokines (PICs) and the chemokine monocyte chemoattractant protein-1, and lower PVN levels of gamma-aminobutyric acid, interleukin (IL)-10 and the 67-kDa isoform of glutamate decarboxylase (GAD67), and higher plasma levels of PICs, norepinephrine and aldosterone, and lower plasma IL-10, and higher renal sympathetic nerve activity. However, PVN treatment with ENL attenuated these changes. PVN microinjection of ANG II induced increases in IL-1β and IL-6, and a decrease in IL-10 in the PVN, and pretreatment with angiotensin II type 1 receptor (AT1-R) antagonist losartan attenuated these changes. These findings suggest that ANG II infusion induces an imbalance between excitatory and inhibitory neurotransmitters and an imbalance between pro- and anti-inflammatory cytokines in the PVN, and PVN inhibition of the RAS restores neurotransmitters and cytokines in the PVN, thereby attenuating ANG II-induced hypertension and cardiac hypertrophy.

Bortezomib, a Proteasome Inhibitor, Attenuates Angiotensin II-Induced Hypertension and Aortic Remodeling in Rats

BackgroundHypertension is a highly prevalent disorder and a major risk factor for cardiovascular diseases. Hypertensive vascular remodeling is the pathological mal-adaption of blood vessels to the hypertensive condition that contributes to further development of high blood pressure and end-organ damage. Hypertensive remodeling involves, at least in part, changes in protein turnover. The ubiquitin proteasome system (UPS) is a major protein quality and quantity control system. This study tested the hypothesis that the proteasome inhibitor, bortezomib, would attenuate AngII-induced hypertension and its sequelae such as aortic remodeling in rats.Methodology/Principal FindingsMale Sprague Dawley rats were subjected to AngII infusion for two weeks in the absence or presence of bortezomib. Mean arterial pressure was measured in conscious rats. Aortic tissue was collected for estimation of wall area, collagen deposition and expression of tissue inhibitors of matrix metalloproteases (TIMP), Ki67 (a marker of proliferation), reactive oxygen species (ROS) and VCAM-1 (a marker of inflammation). AngII infusion increased arterial pressure significantly (160±4 mmHg vs. vehicle treatment 133±2 mmHg). This hypertensive response was attenuated by bortezomib (138±5 mmHg). AngII hypertension was associated with significant increases in aortic wall to lumen ratio (∼29%), collagen deposition (∼14%) and expression of TIMP1 and TIMP2. AngII also increased MMP2 activity, proteasomal chymotrypsin-like activity, Ki67 staining, ROS generation and VCAM-1 immunoreactivity. Co-treatment of AngII-infused rats with bortezomib attenuated these AngII-induced responses.ConclusionsCollectively, these data support the idea that proteasome activity contributes to AngII-induced hypertension and hypertensive aortic vascular remodeling at least in part by modulating TIMP1/2 and MMP2 function. Preliminary observations are consistent with a role for ROS, inflammatory and proliferative mechanisms in this effect. Further understanding of the mechanisms by which the proteasome is involved in hypertension and vascular structural remodeling may reveal novel targets for pharmacological treatment of hypertension, hypertensive remodeling or both.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Melatonin ameliorates Angiotensin II‐induced vascular endothelial damage via its antioxidative properties

Melatonin is well known to have a beneficial effect on the cardiovascular system, but it remains to be elucidated whether melatonin has a therapeutic effect on the vascular damage induced by the potential vasoactive substance angiotensin II (Ang II). In this study, the effects of melatonin on Ang II-induced vascular endothelial damage were investigated. In cultured vascular endothelial cells, Ang II stimulation increased ROS generation and inhibited eNOS phosphorylation (Ser1177), both of which were clearly restored by pretreatment with melatonin. The translocation of p47(phox) subunit of NADPH oxidase from the cytosol to plasma membrane was promoted in Ang II-treated vascular endothelial cells, which was canceled by melatonin pretreatment. In Ang II-infused rats, increased ROS generation in the aortic wall and impaired endothelial function of the aortic ring were observed, which were rescued by coadministration of melatonin. In vasculature, melatonin receptor agonist ramelteon had the antioxidative effect in the same manner as melatonin by itself. These findings suggest that melatonin directly ameliorates Ang II-induced vascular endothelial damage partly via its antioxidative properties, providing with us the potential rationale for clinical application of melatonin to the prevention from cardiovascular diseases.

Angiotensin II stimulates sympathetic neurotransmission to adipose tissue

Angiotensin II (AngII) facilitates sympathetic neurotransmission by regulating norepinephrine (NE) synthesis, release, and uptake. These effects of AngII contribute to cardiovascular control. Previous studies in our laboratory demonstrated that chronic AngII infusion decreased body weight of rats. We hypothesized that AngII facilitates sympathetic neurotransmission to adipose tissue and may thereby decrease body weight. The effect of chronic AngII infusion on the NE uptake transporter and NE turnover was examined in metabolic (interscapular brown adipose tissue, ISBAT; epididymal fat, EF) and cardiovascular tissues (left ventricle, LV; kidney) of rats. To examine the uptake transporter saturation isotherms were performed using [3H]nisoxetine (NIS). At doses that lowered body weight, AngII significantly increased ISBAT [3H]NIS binding density. To quantify NE turnover, alpha-methyl-para-tyrosine (AMPT) was injected in saline-infused, AngII-infused, or saline-infused rats that were pair-fed to food intake of AngII-infused rats. AngII significantly increased the rate of NE decline in all tissues compared to saline. The rate of NE decline in EF was increased to a similar extent by AngII and by pair feeding. In rats administered AngII and propranolol, reductions in food and water intake and body weight were eliminated. These data support the hypothesis that AngII facilitates sympathetic neurotransmission to adipose tissue. Increased sympathetic neurotransmission to adipose tissue following AngII exposure is suggested to contribute to reductions in body weight.

Dissociation of vascular and natriuretic Ang1–7 actions in AngII hypertensive and normotensive rats

Studies in models of Angiotensin II‐dependent hypertension have shown that kidney medullary ACE2 activity and Ang1–7 levels are reduced, suggesting there are reduced compensatory vasodilator and natriuretic Ang1–7 effects available to counterbalance the hypertension and reduced sodium excretion. To assess the possible contribution of the reduced Ang1–7 levels, Ang1–7 was administered i.v. to anaesthetized normal and chronic AngII‐infused rats (80ng/min) at doses estimated to re‐establish Ang1–7 levels. Ang1–7(110fmol/min)led to small but significant increases in arterial pressure in both groups(114±3 to 123±5 and 140±5 to 149±5 mmHg), and to decreases in total renal blood flow (7.5±0.9 to 6.4±1 and 9.5 ±1.3 to 8.4±1 ml/min, p&lt;0.05), medullary blood flow decreased only in control rats (124±22 to 113±19 PU, p&lt;0.05). Acute Ang1–7 infusions also caused significant increases in urine flow (5.1±1.1 to 10±1.6 and 5.2±0.9 to 15.6±9 μl/min) and sodium excretion (0.05±0.03 to 0.96±0.35 and 0.04±0.02 to 0.42±0.14 μmol/min) in both groups indicating a dissociation between the hemodynamic and excretory effects and supporting a direct action of Ang1–7 to inhibit tubular sodium reabsorption to the same extent in the normotensive and hypertensive rats.Supported by Polish Ministry of Science and Higher Education and the Tulane COBRE P20RR17659

Detraining Differentially Preserved Beneficial Effects of Exercise on Hypertension: Effects on Blood Pressure, Cardiac Function, Brain Inflammatory Cytokines and Oxidative Stress

AimsThis study sought to investigate the effects of physical detraining on blood pressure (BP) and cardiac morphology and function in hypertension, and on pro- and anti-inflammatory cytokines (PICs and AIC) and oxidative stress within the brain of hypertensive rats.Methods and ResultsHypertension was induced in male Sprague-Dawley rats by delivering AngiotensinII for 42 days using implanted osmotic minipumps. Rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise (ExT) for 42 days, whereas, detrained groups underwent 28 days of exercise followed by 14 days of detraining. BP and cardiac function were evaluated by radio-telemetry and echocardiography, respectively. At the end, the paraventricular nucleus (PVN) was analyzed by Real-time RT-PCR and Western blot. ExT in AngII-infused rats caused delayed progression of hypertension, reduced cardiac hypertrophy, and improved diastolic function. These results were associated with significantly reduced PICs, increased AIC (interleukin (IL)-10), and attenuated oxidative stress in the PVN. Detraining did not abolish the exercise-induced attenuation in MAP in hypertensive rats; however, detraining failed to completely preserve exercise-mediated improvement in cardiac hypertrophy and function. Additionally, detraining did not reverse exercise-induced improvement in PICs in the PVN of hypertensive rats; however, the improvements in IL-10 were abolished.ConclusionThese results indicate that although 2 weeks of detraining is not long enough to completely abolish the beneficial effects of regular exercise, continuing cessation of exercise may lead to detrimental effects.

Abstract 493: Impact of Renal Vasoconstriction on the Relationship Between Blood Pressure and Renal Injury in Angiotensin II-induced Hypertensive Rats

Ang II is thought to play a prominent role in the development of hypertension-induced renal disease via BP dependent and independent pathways; however the quantitative relationships between BP and renal injury have not been rigorously examined in Ang II-induced hypertension. The major goals of the present study were to assess: 1) the relationship between BP and renal injury in rats with hypertension induced by Ang II vs. renal mass reduction (RMR) and 2) the pressure-flow relationships in conscious Ang II-infused rats. One group of male Sprague-Dawley rats (Charles River) were implanted with a BP radiotransmitter and 10 days later administered Ang II (n=12; 500 ng/kg/min via osmotic minipump) or subjected to 3/4 RMR via right uninephrectomy + infarction of ∼ 1/2 of the left kidney (RKI, n=5). BP was measured continuously and kidneys were perfused fixed at 6 weeks for the assessment of renal injury. In a separate experiment, MAP and RBF (Transonic) were measured in conscious chronically instrumented rats. After recovery from surgery (∼7 days), baseline MAP and RBF were assessed (∼4 hours @ 200 Hz) on 2 consecutive days. Subsequently, rats were administered Ang II (n=6; 500 ng/kg/min) or saline (n=7; sham) via osmotic minipump and MAP and RBF were again assessed every 2-3 days for 10 days. Despite a higher average systolic BP over 6 weeks in Ang II (174±3 mmHg) vs. RKI (165±6 mmHg) rats, glomerulosclerosis (GS) was higher (p&lt;0.05) in RKI (15±7% out of 100 glomeruli) vs. Ang II (6±1% out of 100 glomeruli) rats. Moreover, the slope of the relationship between BP and %GS (Δ%GS/ΔmmHg) was greater in RKI vs. Ang II rats. Both MAP (98±2 vs. 99±3 mmHg) and RBF (8.1±1vs. 8.2±1 ml/min) were similar at baseline in Ang II and sham rats, respectively. MAP was elevated by day 3 (123±6 mmHg) and further increased to 157±5 mmHg by day 10 in Ang II rats. Conversely, RBF was decreased at day 3 (6.6±0.6 ml/min) and the vasoconstriction persisted over the experimental protocol as RBF further decreased to 5.6±0.7 ml/min at day 10 in Ang II rats. In conclusion, Ang II-induced hypertension is associated with a diminished susceptibility to renal injury as compared to rats with RMR likely due, in part, to the AngII-induced vasoconstriction, which reduces BP transmission to the renal microvasculature.

Role of H2O2 in hypertension, renin‐angiotensin system activation and renal medullary disfunction caused by angiotensin II

Activation of the intrarenal renin-angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H(2) O(2) ) contribute to hypertension. We examined whether H(2) O(2) mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II). Ang II (200 ng·kg(-1) ·min(-1) ) or saline were infused in Sprague Dawley rats from day 0 to day 14. Polyethylene glycol (PEG)-catalase (10 000 U·kg(-1) ·day(-1) ) was given to Ang II-treated rats, from day 7 to day 14. Systolic blood pressure was measured throughout the study. H(2) O(2) , angiotensin AT(1) receptor and Nox4 expression and nuclear factor-κB (NF-κB) activation were evaluated in the kidney. Plasma and urinary H(2) O(2) and angiotensinogen were also measured. Ang II increased H(2) O(2) , AT(1) receptor and Nox4 expression and NF-κB activation in the renal medulla, but not in the cortex. Ang II raised plasma and urinary H(2) O(2) levels, increased urinary angiotensinogen but reduced plasma angiotensinogen. PEG-catalase had a short-term antihypertensive effect and transiently suppressed urinary angiotensinogen. PEG-catalase decreased renal medullary expression of AT(1) receptors and Nox4 in Ang II-infused rats. Renal medullary NF-κB activation was correlated with local H(2) O(2) levels and urinary angiotensinogen excretion. Loss of antihypertensive efficacy was associated with an eightfold increase of plasma angiotensinogen. The renal medulla is a major target for Ang II-induced redox dysfunction. H(2) O(2) appears to be the key mediator enhancing intrarenal RAS activation and decreasing systemic RAS activity. The specific control of renal medullary H(2) O(2) levels may provide future grounds for the treatment of hypertension.

Sexual Dimorphism in Urinary Angiotensinogen Excretion During Chronic Angiotensin II−Salt Hypertension

The intrarenal renin-angiotensin system contributes to hypertension by regulating sodium and water reabsorption throughout the nephron. Sex differences in the intrarenal components of the renin-angiotensin system have been involved in the greater incidence of high blood pressure and progression to kidney damage in males than females. This study investigated whether there is a sex difference in the intrarenal gene expression and urinary excretion of angiotensinogen (AGT) during angiotensin II (Ang II)-dependent hypertension and high-salt (HS) diet. Male and female Sprague-Dawley rats were divided into 5 groups for each sex: Normal-salt control, HS diet (8% NaCl), Ang II-infused (80 ng/min), Ang II-infused plus HS diet, and Ang II-infused plus HS diet and treatment with the Ang II receptor blocker, candesartan (25 mg/L in the drinking water). Rats were evaluated for systolic blood pressure (SBP), kidney AGT mRNA expression, urinary AGT excretion, and proteinuria at different time points during a 14-day protocol. Both male and female rats exhibited similar increases in urinary AGT, with increases in SBP during chronic Ang II infusion. HS diet greatly exacerbated the urinary AGT excretion in Ang II-infused rats; males had a 9-fold increase over Ang II alone and females had a 2.5-fold increase. Male rats displayed salt-sensitive SBP increases during Ang II infusion and HS diet, and female rats did not. In the kidney cortex, males displayed greater AGT gene expression than females during all treatments. During Ang II infusion, both sexes exhibited increases in AGT gene message compared with same-sex controls. In addition, HS diet combined with Ang II infusion exacerbated the proteinuria in both sexes. Concomitant Ang II receptor blocker treatment during Ang II infusion and HS diet decreased SBP and urinary AGT similarly in both sexes; however, the decrease in proteinuria was greater in the females. During Ang II-dependent hypertension and HS diet, higher intrarenal renin-angiotensin system activation in males, as reflected by higher AGT gene expression and urinary excretion, indicates a mechanism for greater progression of high blood pressure and might explain the sex disparity in development of salt-sensitive hypertension.

Upregulation of the Na+-K+-2Cl− cotransporter 1 via histone modification in the aortas of angiotensin II-induced hypertensive rats

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in diverse models of hypertension. We hypothesized that NKCC1 is upregulated via histone modification in the aortas of angiotensin II (Ang II)-induced hypertensive rats. An osmotic mini-pump containing Ang II was implanted in the subcutaneous tissues of the backs of Sprague-Dawley (SD) rats for 7 days. The systolic blood pressure was recorded every day by the tail-cuff method. On days 3 and 7, the mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The levels of Nkcc1 mRNA and protein in the aortas were measured using real-time PCR and Western blotting, respectively. The histone modifications and recruited proteins at the Nkcc1 promoter were determined by chromatin immunoprecipitation. The inhibition of concentration-response curves to phenylephrine by bumetanide, an inhibitor of NKCCs, was greater in Ang II-infused rats than in sham-operated (sham) rats . The levels of Nkcc1 mRNA and protein in the aortas increased gradually as Ang II was infused into the rats. Acetylated histone H3 (H3Ac), an activating histone code, was increased but trimethylated histone H3 at lysine 27 (H3K27me3), a repressive histone code, was greatly decreased in Ang II-infused rats compared with sham. RNA polymerase II was recruited to the Nkcc1 promoter with increased KDM6b. We conclude that the NKCC1 is upregulated via histone modification in the aortas of Ang II-induced hypertensive rats. Thus, we suggest that this ion transporter is epigenetically upregulated by histone modification or DNA demethylation upon the development of hypertension.

Metformin treatment of angiotensin II‐hypertensive rat decreases phenylephrine‐mediated increased contraction in pudendal arteries

Hypertension is a major risk factor for erectile dysfunction (ED). Increased circulating angiotensin II (AngII) levels are associated with ED. Data from our laboratory showed that AngII infusion resulted in increased corpus cavernosum (CC) contractility, resulting in ED. Metformin treatment restored normal CC tone and erectile function in AngII‐infused rats. Pudendal artery (PA), the major artery providing blood flow to the penis, is important for normal erectile function. This study tested the hypothesis that AngII infusion will result in increased PA contractility, thus limiting blood flow to penis, and that metformin treatment will restore normal PA contractility. Male Sprague‐Dawley rats were implanted with mini‐osmotic pumps containing saline or AngII (70 ng/min, 28 days). Animals were then treated with metformin (500mg/Kg/day) or vehicle during the last week of AngII infusion. AngII infused rats displayed ED which was associated with increased phenylephrine‐mediated contraction in PA (% of the U‐46619 contraction) (Emax= 400.7% ± 14.47 in AngII vs Emax= 208.6% ± 7.79 in sham). Metformin treatment restored erectile function in AngII infused animals, and reversed the AngII‐mediated increase in PA contractility (Emax= 277.2% ± 14.55 in AngII+metformin vs Emax= 400.7% ± 14.47 in AngII). We conclude that metfromin treatment may be beneficial to restore normal erectile function.

Effects of Endothelin Receptor Antagonists on Renal Hemodynamics in Angiotensin II-Infused Rats on High NaCl Intake

Aim: The aim was to investigate effects of selective endothelin (ET) receptor antagonists on renal hemodynamics and dynamic renal blood flow autoregulation (RBFA) in angiotensin II (Ang II)-infused rats on a high NaCl intake. Methods: Sprague-Dawley rats received Ang II (250 ng/kg/min, s.c.) and an 8 % NaCl diet for 14 days after which renal clearance experiments were performed. After baseline measurements animals were administered either: (a) saline vehicle; (b) ET_A receptor antagonist BQ-123 (30 nmol/kg/min); (c) ET_B receptor antagonist BQ-788 (30 nmol/kg/min); or (d) BQ-123 + BQ-788, for six consecutive 20-minute clearance periods. Results: BQ-123 reduced arterial pressure (AP) and selectively increased outer medullary perfusion versus vehicle (p<0.05). These effects were attenuated or abolished by combined BQ-123 and BQ-788. BQ-788 reduced renal blood flow and increased renovascular resistance (p<0.05). Ang II-infused rats on high NaCl intake showed abnormalities in dynamic RBFA characterized by an impaired myogenic response that were not significantly affected by ET receptor antagonists. Conclusion: In hypertensive Ang II-infused rats on a high-NaCl intake selective ET_A antagonism with BQ-123 reduced AP and specifically increased OM perfusion and these effects were dependent on intact ET_B receptor stimulation. Furthermore, ET receptor antagonists did not attenuate abnormalities in dynamic RBFA.

HSP27/HSPB1 as an adaptive podocyte antiapoptotic protein activated by high glucose and angiotensin II

Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFβ1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN.

Abstract P264: Interaction of Collecting Duct-Derived Prorenin and Soluble Prorenin Receptor Increases Intraluminal Renin Activity and Augments Intratubular Angiotensin II Formation in Ang II-Dependent Hypertensive Rats

Upregulation of collecting duct (CD)-derived renin (CD renin) in angiotensin II (Ang II)-dependent hypertension may provide a pathway for intratubular Ang II formation by acting on angiotensinogen (AGT) delivered from proximal tubule segments. Recently, a prorenin/renin receptor (PRR) has been cloned and shown to enhance renin and prorenin activation. The soluble form of the PRR (sPRR) is augmented in the renal inner medulla of chronic Ang II-infused rats. The present study was performed to determine if renin is secreted into the lumen by the CD cells in chronic Ang II-infused rats and to establish the functional contribution of sPRR to the enhanced renin activity in distal nephron segments. Accordingly, urinary levels of renin ( uRen ) and Ang II ( uAngII ) were measured by RIA in chronic Ang II-infused male Sprague-Dawley rats [80 ng/min, SC minipumps for 14 d, n=10] and sham-operated rats [n=10]. Systolic blood pressure increased in the Ang II rats by Day 5 and continued to increase throughout the study (Day 13; Ang II: 175±10 vs. sham: 116±2 mmHg; p &lt;0.05). Although plasma renin activity (PRA) was suppressed in the Ang II-infused rats, renal medullary renin content was significantly augmented (12,605±1,343 vs. 7,956±765 ng Ang I/h/mg; p &lt;0.05). The excretion of uAngII was also increased (3,813±431 vs. 2,080±361 fmol/day; p &lt;0.05). In addition, renin and prorenin excretion rates increased progressively and were markedly augmented by Day 13 of Ang II infusion [renin (8.6±1.5 vs. 2.8±0.5x10 -6 Enzyme Units Excreted (EUE) /day; prorenin: 15.8 ± 2.8 vs. 2.6 ± 0.7x10 -3 EUE /day, p &lt;0.05). Renin and prorenin protein levels examined by Western Blot in the urine were similarly increased. Importantly, the incubation of urine samples of Ang II-infused rats with recombinant human prorenin showed increased Ang I formation compared to sham-operated rats. In conclusion, in chronic Ang II-infused rats, the presence of sPRR in the urine reflects augmented enzymatic activity of prorenin secreted by the principal cells of the CD, which increase intratubular Ang II de novo formation in the distal nephron segments thus contributing to enhanced sodium reabsorption during Ang II-dependent hypertension.

Angiotensin-(1–7) attenuates angiotensin II-induced cardiac remodeling associated with upregulation of dual-specificity phosphatase 1

Chronic hypertension induces cardiac remodeling, including left ventricular hypertrophy and fibrosis, through a combination of both hemodynamic and humoral factors. In previous studies, we showed that the heptapeptide ANG-(1-7) prevented mitogen-stimulated growth of cardiac myocytes in vitro, through a reduction in the activity of the MAPKs ERK1 and ERK2. In this study, saline- or ANG II-infused rats were treated with ANG-(1-7) to determine whether the heptapeptide reduces myocyte hypertrophy in vivo and to identify the signaling pathways involved in the process. ANG II infusion into normotensive rats elevated systolic blood pressure >50 mmHg, in association with increased myocyte cross-sectional area, ventricular atrial natriuretic peptide mRNA, and ventricular brain natriuretric peptide mRNA. Although infusion with ANG-(1-7) had no effect on the ANG II-stimulated elevation in blood pressure, the heptapeptide hormone significantly reduced the ANG II-mediated increase in myocyte cross-sectional area, interstitial fibrosis, and natriuretic peptide mRNAs. ANG II increased phospho-ERK1 and phospho-ERK2, whereas cotreatment with ANG-(1-7) reduced the phosphorylation of both MAPKs. Neither ANG II nor ANG-(1-7) altered the ERK1/2 MAPK kinase MEK1/2. However, ANG-(1-7) infusion, with or without ANG II, increased the MAPK phosphatase dual-specificity phosphatase (DUSP)-1; in contrast, treatment with ANG II had no effect on DUSP-1, suggesting that ANG-(1-7) upregulates DUSP-1 to reduce ANG II-stimulated ERK activation. These results indicate that ANG-(1-7) attenuates cardiac remodeling associated with a chronic elevation in blood pressure and upregulation of a MAPK phosphatase and may be cardioprotective in patients with hypertension.

Comparison of ROCK and EGFR Activation Pathways in the Progression of Glomerular Injuries in AngII-Infused Rats

Aim: The roles of rho-kinase (ROCK) and epidermal growth factor receptor (EGFR) were studied using an angiotensin II (AngII)-dependent hypertension rat model. Method: Male Wistar rats were infused with AngII at a rate of 400 ng/kg body weight (BW)/min for 14 days. Effects of ROCK inhibitor, fasudil (20 mg/kg BW), and EGFR inhibitor, gefitinib (3 mg/kg BW), were studied. Results: AngII infusion increased blood pressure (BP; 220 ± 19 mmHg) as well as the number of proliferating cells in glomeruli judged by Ki67 and proliferating cell nuclear antigen immunostaining and urinary protein excretion (118 ± 19 mg/day). AngII also decreased p27 expression and increased cyclin D1 expression in glomeruli, as well as induced dissociation of the nephrin- and podocin-immunostaining patterns in podocytes. Treatment with fasudil or gefitinib completely inhibited glomerular cell proliferation without changing the BP. Although the decreased p27 expression was reversed by both treatments, cyclin D1 induction was abolished only by gefitinib. Fasudil significantly reduced proteinuria (57.2 ± 17.5 mg/day), but not gefitinib (133.3 ± 30.9 mg/day). The dissociation of podocin and nephrin was ameliorated by fasudil, but not by gefitinib. Conclusion: ROCK and EGFR have distinct roles in proteinuria and glomerular cell proliferation in this model.

Augmented intratubular renin and prorenin expression in the medullary collecting ducts of the kidney as a novel mechanism of angiotensin II-induced hypertension

Augmented intratubular renin and prorenin expression in the medullary collecting ducts of the kidney as a novel mechanism of angiotensin II-induced hypertension

PPARγ activation suppresses angiotensin II-induced production of KLF5 in rat vascular smooth muscle cells

ObjectiveThe mechanisms underlying the inhibitory effects of PPARγ agonists on Ang II-induced VSMC proliferation and the Ang II/KLF5-dependent signalling pathway remain unclear.MethodsSprague–Dawley rats received Ang II (150 ng/kg/min) with or...