Hepatoprotective and cardioprotective effects of empagliflozin in spontaneously hypertensive rats fed a high-fat diet.

A combination of liver and heart dysfunction worsens the prognosis of human survival. The aim of this study was to investigate whether empagliflozin (a sodium-glucose transporter-2 inhibitor) has beneficial effects not only on cardiac and renal function but also on hepatic function. Adult (6-month-old) male spontaneously hypertensive rats (SHR) were fed a high-fat diet (60% fat) for four months to induce hepatic steatosis and mild heart failure. For the last two months, the rats were treated with empagliflozin (empa, 10 mg.kg-1.day-1 in the drinking water). Renal function and oral glucose tolerance test were analyzed in control (n=8), high-fat diet (SHR+HF, n=10), and empagliflozin-treated (SHR+HF+empa, n=9) SHR throughout the study. Metabolic parameters and echocardiography were evaluated at the end of the experiment. High-fat diet feeding increased body weight and visceral adiposity, liver triglyceride and cholesterol concentrations, and worsened glucose tolerance. Although the high-fat diet did not affect renal function, it significantly worsened cardiac function in a subset of SHR rats. Empagliflozin reduced body weight gain but not visceral fat deposition. It also improved glucose sensitivity and several metabolic parameters (plasma insulin, uric acid, and HDL cholesterol). In the liver, empagliflozin reduced ectopic lipid accumulation, lipoperoxidation, inflammation and pro-inflammatory HETEs, while increasing anti-inflammatory EETs. In addition, empagliflozin improved cardiac function (systolic, diastolic and pumping) independent of blood pressure. The results of our study suggest that hepatoprotection plays a decisive role in the beneficial effects of empagliflozin in preventing the progression of cardiac dysfunction induced by high-fat diet feeding.

Changes in cardiovascular autonomic control induced by chronic inhibition of acetylcholinesterase during pyridostigmine or donepezil treatment of spontaneously hypertensive rats.

Chronic treatment with acetylcholinesterase inhibitors may be a promising therapeutic strategy for treatment of cardiovascular diseases. The aim of our study was to analyze the changes in blood pressure (BP) and heart rate (HR) during 14 days of treatment with two different acetylcholinesterase inhibitors - pyridostigmine (PYR) having only peripheral effects or donepezil (DON) with both peripheral and central effects. In addition, we studied their effects on the cardiovascular response to restraint stress and on sympathovagal control of HR in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). SHR were characterized by elevated BP and increased low-frequency component of systolic BP variability (LF-SBPV), but their cardiac vagal tone and HR variability (HRV) were reduced compared with WKY. Chronic treatment with either acetylcholinesterase inhibitor decreased HR and increased HRV in both strains. PYR treatment slightly decreased BP and LF-SBPV in the dark phase of the day. Neither drug significantly altered BP response to stress, but PYR attenuated HR increase during restraint stress. Regarding sympathovagal balance, acute methylatropine administration caused a greater increase of HR in WKY than in SHR. Chronic PYR or DON treatment enhanced HRV and HR response to methylatropine (vagal tone) in WKY, whereas PYR but not DON treatment potentiated HRV and vagal tone in SHR. In conclusion, vagal tone was lower in SHR compared with WKY, but was enhanced by chronic PYR treatment in both strains. Thus, chronic peripheral, but not central, acetylcholinesterase inhibition has major effects on HR and its variability in both normotensive and hypertensive rats.

Impaired vascular ß-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries.

It was suggested that impaired ß-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased ß-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of ß-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (ß2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (ß1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated ß-adrenergic vasodilatation of conduit arteries of SHR but similar ß-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to ß-adrenergic agonists do not support major role of altered ß-adrenergic vasodilatation for high BP in genetic hypertension.

Endothelin type A receptor blockade increases renoprotection in congestive heart failure combined with chronic kidney disease: Studies in 5/6 nephrectomized rats with aorto-caval fistula.

BACKGROUND: Association of congestive heart failure (CHF) and chronic kidney disease (CKD) worsens the patient's prognosis and results in poor survival rate. The aim of this study was to examine if addition of endothelin type A (ETA) receptor antagonist to the angiotensin-converting enzyme inhibitor (ACEi) will bring additional beneficial effects in experimental rats. METHODS: CKD was induced by 5/6 renal mass reduction (5/6 NX) and CHF was elicited by volume overload achieved by creation of aorto-caval fistula (ACF). The follow-up was 24 weeks after the first intervention (5/6 NX). The treatment regimens were initiated 6 weeks after 5/6 NX and 2 weeks after ACF creation. RESULTS: The final survival in untreated group was 15%. The treatment with ETA receptor antagonist alone or ACEi alone and the combined treatment improved the survival rate to 64%, 71% and 75%, respectively, however, the difference between the combination and either single treatment regimen was not significant. The combined treatment exerted best renoprotection, causing additional reduction in albuminuria and reducing renal glomerular and tubulointerstitial injury as compared with ACE inhibition alone. CONCLUSIONS: Our results show that treatment with ETA receptor antagonist attenuates the CKD- and CHF-related mortality, and addition of ETA receptor antagonist to the standard blockade of RAS by ACEi exhibits additional renoprotective actions.

Endothelin type A receptor blockade attenuates aorto-caval fistula-induced heart failure in rats with angiotensin II-dependent hypertension.

OBJECTIVE: Evaluation of the effect of endothelin type A (ET A ) receptor blockade on the course of volume-overload heart failure in rats with angiotensin II-dependent hypertension. METHODS: Ren-2 renin transgenic rats (TGR) were used as a model of hypertension. Heart failure was induced by creating an aorto-caval fistula (ACF). Selective ET A receptor blockade was achieved by atrasentan. For comparison, other rat groups received trandolapril, an angiotensin-converting enzyme inhibitor (ACEi). Animals first underwent ACF creation and 2 weeks later the treatment with atrasentan or trandolapril, alone or combined, was applied; the follow-up period was 20 weeks. RESULTS: Eighteen days after creating ACF, untreated TGR began to die, and none was alive by day 79. Both atrasentan and trandolapril treatment improved the survival rate, ultimately to 56% (18 of 31 animals) and 69% (22 of 32 animals), respectively. Combined ACEi and ET A receptor blockade improved the final survival rate to 52% (17 of 33 animals). The effects of the three treatment regimens on the survival rate did not significantly differ. All three treatment regimens suppressed the development of cardiac hypertrophy and lung congestion, decreased left ventricle (LV) end-diastolic volume and LV end-diastolic pressure, and improved LV systolic contractility in ACF TGR as compared with their untreated counterparts. CONCLUSION: The treatment with ET A receptor antagonist delays the onset of decompensation of volume-overload heart failure and improves the survival rate in hypertensive TGR with ACF-induced heart failure. However, the addition of ET A receptor blockade did not enhance the beneficial effects beyond those obtained with standard treatment with ACEi alone.

Empagliflozin Is Not Renoprotective in Non-Diabetic Rat Models of Chronic Kidney Disease.

Gliflozins (sodium-glucose transporter-2 inhibitors) exhibited renoprotective effects not only in diabetic but also in non-diabetic patients with chronic kidney disease (CKD). Controversial results were reported in experimental non-diabetic models of CKD. Therefore, we examined empagliflozin effects in three CKD models, namely, in fawn-hooded hypertensive (FHH) rats, uninephrectomized salt-loaded (UNX + HS) rats, and in rats with Goldblatt hypertension (two-kidney, one-clip 2K1C) that were either untreated or treated with empagliflozin (10 mg/kg/day) for eight weeks. Plethysmography blood pressure (BP) was recorded weekly, and renal parameters (proteinuria, plasma urea, creatinine clearance, and sodium excretion) were analyzed three times during the experiment. At the end of the study, blood pressure was also measured directly. Markers of oxidative stress (TBARS) and inflammation (MCP-1) were analyzed in kidney and plasma, respectively. Body weight and visceral adiposity were reduced by empagliflozin in FHH rats, without a significant effect on BP. Experimentally induced CKD (UNX + HS and 2K1C) was associated with a substantial increase in BP and relative heart and kidney weights. Empagliflozin influenced neither visceral adiposity nor BP in these two models. Although empagliflozin increased sodium excretion, suggesting effective SGLT-2 inhibition, it did not affect diuresis in any experimental model. Unexpectedly, empagliflozin did not provide renoprotection because proteinuria, plasma urea, and plasma creatinine were not lowered by empagliflozin treatment in all three CKD models. In line with these results, empagliflozin treatment did not decrease TBARS or MCP-1 levels in either model. In conclusion, empagliflozin did not provide the expected beneficial effects on kidney function in experimental models of CKD.

Beneficial Effects of Empagliflozin Are Mediated by Reduced Renal Inflammation and Oxidative Stress in Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein.

Gliflozins (inhibitors of sodium-glucose cotransporter 2) show many beneficial actions beyond their antidiabetic effects. The underlying mechanisms of these additional protective effects are still not well understood, especially under non-diabetic conditions. Therefore, we analyzed the effects of empagliflozin in young (3-month-old) and adult (12-month-old) male spontaneously hypertensive rats (SHR) expressing human C-reactive protein (CRP) in the liver. SHR-CRP rats are a non-diabetic model of metabolic syndrome, inflammation, and organ damage. Empagliflozin was given in a daily dose of 10 mg/kg body weight for 8 weeks. Both age groups of SHR-CRP rats treated with empagliflozin had lower body weight, decreased weight of fat depots, reduced ectopic fat accumulation in the liver and kidneys, and decreased levels of plasma insulin and ß-hydroxybutyrate. Empagliflozin effectively reduced ectopic renal fat accumulation, and was associated with decreased inflammation. Exclusively in young rats, decreased microalbuminuria after empagliflozin treatment was accompanied by attenuated oxidative stress. In adult animals, empagliflozin also improved left ventricle function. In conclusion, in young animals, the beneficial renoprotective effects of empagliflozin could be ascribed to reduced lipid deposition in the kidney and the attenuation of oxidative stress and inflammation. In contrast, hepatic lipid metabolism was ameliorated in adult rats.

Both central sympathoexcitation and peripheral angiotensin II-dependent vasoconstriction contribute to hypertension development in immature heterozygous Ren-2 transgenic rats.

Recently, we demonstrated that chronic blockade of the renin-angiotensin system (RAS) lowered the blood pressure (BP) of adult Ren-2 transgenic rats (TGR) mainly through the attenuation of central sympathoexcitation. However, the participation of central and peripheral mechanisms in the development of high BP in immature TGR remains unclear. In the present study, 6-week-old heterozygous TGR males were chronically treated with intracerebroventricular (ICV) or intraperitoneal (IP) infusions of the AT1 receptor inhibitor losartan (1 or 2 mg/kg/day) for 4 weeks. The influence of these treatments on sympathetic- and angiotensin II-dependent BP components (BP response to pentolinium or captopril, respectively) as well as on BP response to exogenous angiotensin II were determined to evaluate the participation of central and peripheral RAS in hypertension development. Chronic IP losartan administration (1 or 2 mg/kg/day) lowered the BP of immature TGR by reducing both sympathetic and angiotensin II-dependent BP components. The central action of IP-administered losartan was indicated by a reduced BP response to acute ICV angiotensin II injection. Chronic ICV administration of a lower losartan dose (1 mg/kg/day) reduced only the sympathetic BP component, whereas a higher ICV administered dose (2 mg/kg/day) was required to influence the angiotensin II-dependent BP component. Accordingly, chronic ICV losartan administration of 2 mg/kg/day (but not 1 mg/kg/day) attenuated the BP response to acute intravenous angiotensin II application. In conclusion, central sympathoexcitation seems to play an important role in hypertension development in immature TGR. Central sympathoexcitation is highly susceptible to inhibition by low doses of RAS-blocking agents, whereas higher doses also affect peripheral angiotensin II-dependent vasoconstriction.

In a Prediabetic Model, Empagliflozin Improves Hepatic Lipid Metabolism Independently of Obesity and before Onset of Hyperglycemia.

Recent studies suggest that treatment with SGLT-2 inhibitors can reduce hepatic lipid storage and ameliorate non-alcoholic fatty liver disease (NAFLD) development beyond their glycemic benefits. However, the exact mechanism involved is still unclear. We investigated the hepatic metabolic effect of empagliflozin (10 mg/kg/day for eight weeks) on the development of NAFLD and its complications using HHTg rats as a non-obese prediabetic rat model. Empagliflozin treatment reduced neutral triacylglycerols and lipotoxic diacylglycerols in the liver and was accompanied by significant changes in relative mRNA expression of lipogenic enzymes (Scd-1, Fas) and transcription factors (Srebp1, Pparγ). In addition, alterations in the gene expression of cytochrome P450 proteins, particularly Cyp2e1 and Cyp4a, together with increased Nrf2, contributed to the improvement of hepatic lipid metabolism after empagliflozin administration. Decreased circulating levels of fetuin-A improved lipid metabolism and attenuated insulin resistance in the liver and in peripheral tissues. Our results highlight the beneficial effect of empagliflozin on hepatic lipid metabolism and lipid accumulation independent of obesity, with the mechanisms understood to involve decreased lipogenesis, alterations in cytochrome P450 proteins, and decreased fetuin-A. These changes help to alleviate NAFLD symptoms in the early phase of the disease and before the onset of diabetes.

Antihypertensive and metabolic effects of empagliflozin in Ren-2 transgenic rats, an experimental non-diabetic model of hypertension.

The new antidiabetic drugs, gliflozins, inhibit sodium-glucose transporter-2 in renal proximal tubules promoting glucose and sodium excretion. This leads not only to a significant improvement of glucose control but also to the reduction of blood pressure and body weight in both diabetic patients and experimental models. We examined whether these beneficial effects can also be achieved in a non-diabetic hypertensive model, namely in Ren-2 transgenic rats (TGR). Adult 6-month-old hypertensive TGR and their normotensive controls (Hannover Sprague-Dawley rats), were either untreated or treated with empagliflozin (10 mg/kg/day) for two months. Telemetric blood pressure monitoring, renal parameters as well as cardiac function via echocardiography were analyzed during the experiment. At the end of the study, the contribution of major vasoactive systems to blood pressure maintenance was studied. Metabolic parameters and markers of oxidative stress and inflammation were also analyzed. Empagliflozin had no effect on plasma glucose level but partially reduced blood pressure in TGR. Although food consumption was substantially higher in empagliflozin-treated TGR compared to the untreated animals, their body weight and the amount of epididymal and perirenal fat was decreased. Empagliflozin had no effect on proteinuria, but it decreased plasma urea, attenuated renal oxidative stress and temporarily increased urinary urea excretion. Several metabolic (hepatic triglycerides, non-esterified fatty acids, insulin) and inflammatory (TNF-α, leptin) parameters were also improved by empagliflozin treatment. By contrast, echocardiography did not reveal any effect of empagliflozin on cardiac function. In conclusion, empagliflozin exerted beneficial antihypertensive, anti-inflammatory and metabolic effects also in a non-diabetic hypertensive model.

Effects of systemic and renal intramedullary endothelin-1 receptor blockade on tissue NO and intrarenal hemodynamics in normotensive and hypertensive rats.

Endothelin 1 (ET-1) seems essential in salt-dependent hypertension, and activation of ETA receptors causes renal vasoconstriction. However, the response in the renal medulla and the role of tissue NO availability has never been adequately explored in vivo. We examined effects of ETA and ETB receptor blockade (atrasentan and BQ788) on blood pressure (MAP), medullary blood flow (MBF) and medullary tissue NO. Effects of systemic and intramedullary blocker application were compared in anesthetized normotensive ET-1-pretreated Sprague-Dawley rats (S-D), in salt-dependent hypertension (HS/UNX) and in spontaneously hypertensive rats (SHR). Total renal blood flow (RBF) was measured using a Transonic renal artery probe, MBF as laser-Doppler flux, and tissue NO signal using selective electrodes. In normotensive rats ET-1 significantly increased MAP, decreased RBF (-20%) and renal medullary NO. In HS/UNX rats atrasentan decreased MAP and increased medullary NO, earlier and more profoundly with intravenous infusion. In SHR atrasentan decreased MAP, more effectively with intravenous infusion; the increase in tissue NO (∼10%) was similar with both routes; however, only intramedullary atrasentan increased MBF. No consistent responses to BQ788 were seen. We confirmed dominant role of ETA receptors in regulation of blood pressure and renal hemodynamics in normotensive and hypertensive rats and provided novel evidence for the role of ETA in control of intrarenal NO bioavailability in salt-dependent and spontaneous hypertension. Under conditions of activation of the endothelin system ETB stimulation preserved medullary perfusion.

Cooperation of augmented calcium sensitization and increased calcium entry contributes to high blood pressure in salt-sensitive Dahl rats.

Salt hypertensive Dahl rats are characterized by sympathoexcitation and relative NO deficiency. We tested the hypothesis that the increased blood pressure (BP) response to fasudil in salt hypertensive Dahl rats is due to augmented calcium sensitization in the salt-sensitive strain and/or due to their decreased baroreflex efficiency. BP reduction after acute administration of nifedipine (an L-type voltage-dependent calcium channel blocker) or fasudil (a Rho kinase inhibitor) was studied in conscious intact rats and in rats subjected to acute NO synthase inhibition or combined blockade of the renin-angiotensin system (captopril), sympathetic nervous system (pentolinium), and NO synthase (L-NAME). Intact salt-sensitive (SS) Dahl rats fed a low-salt diet had greater BP responses to nifedipine (-31 ± 6 mmHg) or fasudil (-34 ± 7 mmHg) than salt-resistant (SR) Dahl rats (-16 ± 4 and -17 ± 2 mmHg, respectively), and a high-salt intake augmented the BP response only in SS rats. These BP responses were doubled after acute NO synthase inhibition, indicating that endogenous NO attenuates both calcium entry and calcium sensitization. Additional pentolinium administration, which minimized sympathetic compensation for the drug-induced BP reduction, magnified the BP responses to nifedipine or fasudil in all groups except for salt hypertensive SS rats due to their lower baroreflex efficiency. The BP response to the calcium channel blocker nifedipine can distinguish SS and SR rats even after calcium sensitization inhibition by fasudil, which was not seen when fasudil was administered to nifedipine-pretreated rats. Thus, enhanced calcium entry (potentiated by sympathoexcitation) in salt hypertensive Dahl rats is the abnormality that is essential for their BP increase, which was further augmented by increased calcium sensitization in salt-sensitive Dahl rats.

Addition of Endothelin A-Receptor Blockade Spoils the Beneficial Effect of Combined Renin-Angiotensin and Soluble Epoxide Hydrolase Inhibition: Studies on the Course of Chronic Kidney Disease in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats.

INTRODUCTION: Previous studies in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX) have shown that besides pharmacological blockade of the renin-angiotensin system (RAS) also increasing kidney tissue epoxyeicosatrienoic acids (EET) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for degradation of EETs, and endothelin type A (ETA) receptor blockade retards chronic kidney disease (CKD) progression. This prompted us to evaluate if this progression will be alleviated by the addition of sEH inhibitor and ETA receptor antagonist to the standard complex blockade of RAS (angiotensin-converting enzyme inhibitor plus angiotensin II type 1 receptor blocker) in rats with established CKD. METHODS: The treatment regimens were initiated 6 weeks after 5/6 NX in TGR, and the follow-up period was 60 weeks. RESULTS: The addition of sEH inhibition to RAS blockade improved survival rate, further reduced albuminuria and renal glomerular and kidney tubulointerstitial injury, and attenuated the decline in creatinine clearance - all this as compared with 5/6 NX TGR treated with RAS blockade alone. Addition of ETA receptor antagonist to the combined RAS and sEH blockade not only offered no additional renoprotection but, surprisingly, also abolished the beneficial effects of adding sEH inhibitor to the RAS blockade. CONCLUSION: These data indicate that pharmacological strategies that combine the blockade of RAS and sEH could be a novel tool to combat the progression of CKD. Any attempts to further extend this therapeutic regimen should be made with extreme caution.

Renoprotection Provided by Additional Diuretic Treatment in Partially Nephrectomized Ren-2 Transgenic Rats Subjected to the Combined RAS and ETA Blockade.

OBJECTIVE: Our previous study in heterozygous Ren-2 transgenic rats (TGR) demonstrated that long-term treatment with endothelin receptor A (ETA) blocker atrasentan added to the renin-angiotensin system (RAS) blockade had renoprotective effects in a model of chronic kidney disease (CKD) induced by partial nephrectomy. Since ETA blockade is known to cause edema, we were interested whether diuretic treatment added to this therapy would be beneficial. DESIGN AND METHODS: Partial nephrectomy (NX) was performed at the age of 3 months in TGR rats which were subjected to: (i) RAS blockade alone (angiotensin receptor blocker losartan and angiotensin converting enzyme inhibitor trandolapril), (ii) combined RAS (losartan and trandolapril) and ETA receptor blockade (atrasentan), or (iii) diuretic (hydrochlorothiazide) added to the combined RAS + ETA blockade for 50 weeks following NX. RESULTS: At the end of the study systolic blood pressure and cardiac hypertrophy were similarly decreased in all treated groups. Survival was significantly improved by ETA receptor blockade added to RAS blockade with no further effects of diuretic treatment. However, additional diuretic treatment combined with RAS + ETA blockade decreased body weight and had beneficial renoprotective effects - reductions of both kidney weight and kidney damage markers. Proteinuria gradually increased in rats treated with RAS blockade alone, while it was substantially lowered by additional ETA blockade. In rats treated with additional diuretic, proteinuria was progressively reduced throughout the experiment. CONCLUSION: A diuretic added to the combined RAS and ETA blockade has late renoprotective effects in CKD induced by partial nephrectomy in Ren-2 transgenic rats. The diuretic improved: renal function (evaluated as proteinuria and creatinine clearance), renal morphology (kidney mass, glomerular volume), and histological markers of kidney damage (glomerulosclerosis index, tubulointerstitial injury).

Sympathectomy-induced blood pressure reduction in adult normotensive and hypertensive rats is counteracted by enhanced cardiovascular sensitivity to vasoconstrictors.

The effect of chemical sympathectomy on cardiovascular parameters and the compensatory role of adrenal hormones, the renin-angiotensin system, and cardiovascular sensitivity to vasoconstrictors were studied in spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. Sympathectomy was induced in 20-week-old rats by daily intraperitoneal guanethidine administration (30 mg/kg b.w.) for 2 weeks. Basal blood pressure (BP), heart rate (HR), and restraint stress-induced cardiovascular changes were measured by radiotelemetry. The BP response to catecholamines was determined in rats with implanted catheters. Sympathectomy decreased BP only transiently, and after 14-day guanethidine treatment, BP returned to basal values in both strains. Sympathectomy permanently lowered HR, improved baroreflex sensitivity, and decreased the low-frequency domain of systolic blood pressure variability (a marker of vascular sympathetic activity). Guanethidine also attenuated the BP and HR responses to restraint stress. On the other hand, the BP response to catecholamines was augmented in sympathectomized rats, and this was not due to the de novo synthesis of vascular adrenergic receptors. Sympathectomy caused adrenal enlargement, enhanced the expression of adrenal catecholamine biosynthetic enzymes, and elevated plasma adrenaline levels in both strains, especially in WKY rats. Guanethidine also increased the plasma levels of aldosterone and corticosterone in WKY rats only. In conclusion, sympathectomy produced a transient decrease in BP, a chronic decrease in HR and improvement in baroreflex sensitivity. The effect of sympathectomy on BP was counteracted by increased vascular sensitivity to catecholamines in WKY rats and SHRs and/or by the enhanced secretion of adrenal hormones, which was more pronounced in WKY rats.

Role of angiotensin II in chronic blood pressure control of heterozygous Ren-2 transgenic rats: Peripheral vasoconstriction versus central sympathoexcitation.

Our previous studies demonstrated that chronic systemic blockade of renin-angiotensin system (RAS) lowered blood pressure (BP) of Ren-2 transgenic rats (TGR) by the attenuation of both angiotensin II-dependent and sympathetic vasoconstriction. Since systemic RAS blockade also inhibits brain RAS, we were interested which effects on these two types of vasoconstriction will have the central RAS blockade in hypertensive TGR rats. Adult male heterozygous TGR rats and their Hannover Sprague Dawley (HanSD) controls were subjected to chronic systemic or intracerebroventricular administration of either angiotensin type 1 receptor blocker losartan or direct renin inhibitor aliskiren for 4 weeks. Additional groups of TGR and HanSD rats were used for the evaluation of acute peripheral and brain effects of angiotensin II. Both chronic systemic and intracerebroventricular administrations of losartan or aliskiren normalized BP of TGR animals. BP effect of brain RAS blockade was based solely on the reduced sympathetic vasoconstriction, while systemic RAS blockade attenuated both angiotensin II-dependent and sympathetic vasoconstriction. Surprisingly, neither peripheral nor central pressor effects of acute angiotensin II administration were enhanced in TGR compared to HanSD rats. In conclusion, sympathoinhibition represents the main mechanism of BP reduction in heterozygous TGR rats subjected to chronic brain or systemic RAS blockade, while peripheral attenuation of angiotensin II-dependent vasoconstriction during systemic RAS blockade is less important. Our data suggest that the participation of angiotensin II in BP control of adult heterozygous TGR rats is shifted from peripheral vasoconstriction to central sympathoexcitation. Similar mechanisms cannot be excluded in human essential hypertension.

Which sympathoadrenal abnormalities of adult spontaneously hypertensive rats can be traced to a prehypertensive stage?

Alterations of sympathoadrenal and sympathoneural systems have been suggested to be involved in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). To evaluate the ontogenetic changes of these systems, mRNA and protein expressions of catecholaminergic system genes were measured in adrenal glands and sympathetic ganglia, and the catecholamine levels were determined in adrenal glands, sympathetic ganglia and plasma of prehypertensive (4-week-old) and hypertensive (24-week-old) SHR. Vascular sympathetic innervation was visualized in the femoral artery by glyoxylic acid. In the adrenal glands of prehypertensive SHR, the expression of catecholamine biosynthetic enzymes Ddc, Dbh and Pnmt was lower than in aged-matched Wistar-Kyoto rats. In contrast, the adrenal content of dopamine, noradrenaline and adrenaline was higher in prehypertensive SHR (141%, 123% and 120% of Wistar-Kyoto rats, respectively, p < 0.01). In the adrenal glands of adult SHR, the expression of catecholamine biosynthetic enzymes Th, Ddc, Dbh and Pnmt was decreased along the amounts of dopamine and noradrenaline (50% and 38%, respectively, p < 0.001). The expression levels of Ddc and Dbh enzymes were also downregulated in the sympathetic ganglia of both prehypertensive and adult SHR. At both ages, the density of sympathetic innervation was twofold higher in SHR compared to Wistar-Kyoto rats (p < 0.001). In conclusion, adrenal catecholamine content was increased in prehypertensive SHR, whereas it was reduced in SHR with established hypertension. Surprisingly, downregulation of catecholamine biosynthetic enzymes was observed in both the adrenal medulla and sympathetic ganglia of SHR at both ages. Thus, this downregulation might be a compensatory mechanism that counteracts the vascular sympathetic hyperinnervation seen in SHR of both ages.

Exaggerated blood pressure response to fasudil or nifedipine in hypertensive Ren-2 transgenic rats: role of altered baroreflex.

Basal calcium sensitization is decreased in spontaneously hypertensive rats, although their blood pressure (BP) response to acute Rho-kinase inhibition is enhanced. Using fasudil (Rho-kinase inhibitor) or nifedipine (L-VDCC blocker), we evaluated the contribution of calcium sensitization and calcium entry to BP maintenance in hypertensive transgenic Ren-2 rats (TGR) focusing on the influence of major vasoactive systems and/or baroreflex efficiency on BP responses to these two drugs. Homozygous TGR and normotensive Hannover Sprague-Dawley (HanSD) control rats aged 5, 11, or 22 weeks were used. The acute BP-lowering effects of fasudil or nifedipine were studied in intact rats, nitric oxide-deficient L-NAME-pretreated rats and rats subjected to combined blockade of the renin-angiotensin system (RAS), sympathetic nervous system (SNS) and nitric oxide synthase (NOS). Fasudil- or nifedipine-induced BP reduction increased during hypertension development in TGR. By contrast, the nifedipine-induced BP response decreased, whereas the fasudil-induced BP response increased with age in HanSD controls. Our data indicated a major contribution of nifedipine-sensitive calcium entry and relative attenuation of calcium sensitization in hypertensive rats compared with normotensive controls. The BP responses to fasudil or nifedipine were enhanced by NOS inhibition and combined blockade in normotensive HanSD rats but not in hypertensive TGR. In conclusion, calcium  sensitization is attenuated by endogenous nitric oxide in normotensive HanSD rats but not in hypertensive TGR. Moreover, BP reduction elicited by acute Rho-kinase inhibition is partially compensated by enhanced sympathetic vasoconstriction. The decreased compensation in hypertensive rats with impaired baroreflex efficiency explains their greater BP response to fasudil than in normotensive animals.

Hemodynamic Response to Gabapentin in Conscious Spontaneously Hypertensive Rats.

Ligands of auxiliary α2δ subunit of voltage-dependent calcium channels (VDCCs) decrease elevated L-type VDCCs surface expression in arterial myocytes and arterial constriction in spontaneously hypertensive rats (SHR). However, their effect on blood pressure (BP) is unclear. In this study, we investigated the hemodynamic response to acute and chronic administration of gabapentin, a ligand of auxiliary α2δ subunit of VDCCs, in adult SHR with established neurogenic hypertension. The acute gabapentin administration lowered BP and heart rate more in conscious SHR than Wistar-Kyoto rats. Both nifedipine (L-type VDCCs blocker) and ω-conotoxin GVIA (N-type VDCCs blocker) also decreased BP more in SHR, but only gabapentin and ω-conotoxin GVIA abolished the nitroprusside-induced reflex tachycardia of baroreceptor-heart rate control. Hypotensive effect of gabapentin was accompanied by a reduction of (1) plasma norepinephrine level, (2) depressor response to ganglionic blocker pentolinium, (3) power of low frequency component of systolic BP variability, and (4) pressor response of mesenteric vascular bed to periarterial nerve stimulation, suggesting the decrease of peripheral sympathetic nerve transmission. Moreover, gabapentin effects on BP and baroreflex were absent in sympathectomized rats. In conclusion, the acute (but not chronic) administration of gabapentin lowered BP more in SHR than in Wistar-Kyoto rats. Besides the known L-type VDCCs involvement in the vascular effect of gabapentin, our data revealed the important role of N-type VDCCs in acute gabapentin effect on sympathetic control of BP. Gabapentin-induced changes of sympathetic nerve transmission indicated major hemodynamic mechanism of the acute response to this drug.

Renin-angiotensin system blockade alone or combined with ETA receptor blockade: effects on the course of chronic kidney disease in 5/6 nephrectomized Ren-2 transgenic hypertensive rats.

BACKGROUND: Early addition of endothelin (ET) type A (ETA) receptor blockade to complex renin-angiotensin system (RAS) blockade has previously been shown to provide better renoprotection against progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). In this study, we examined if additional protection is provided when ETA blockade is applied in rats with already developed CKD. METHODS: For complex RAS inhibition, an angiotensin-converting enzyme inhibitor along with angiotensin II type 1 receptor blocker was used. Alternatively, ETA receptor blocker was added to the RAS blockade. The treatments were initiated 6 weeks after 5/6 NX and the follow-up period was 50 weeks. RESULTS: When applied in established CKD, addition of ETA receptor blockade to the complex RAS blockade brought no further improvement of the survival rate (30% in both groups); surprisingly, aggravated albuminuria (588 ± 47 vs. 245 ± 38 mg/24 h, p < 0.05) did not reduce renal glomerular injury index (1.25 ± 0.29 vs. 1.44 ± 0.26), did not prevent the decrease in creatinine clearance (203 ± 21 vs. 253 ± 17 µl/min/100 g body weight), and did not attenuate cardiac hypertrophy to a greater extent than observed in 5/6 NX TGR treated with complex RAS blockade alone. CONCLUSIONS: When applied in the advanced phase of CKD, addition of ETA receptor blockade to the complex RAS blockade brings no further beneficial renoprotective effects on the CKD progression in 5/6 NX TGR, in addition to those seen with RAS blockade alone.