Masayuki Kanazawa

Endothelin-1 augments pressor response to angiotensin II infusion in rats.

To assess possible roles of endothelin in the regulation of blood pressure, we studied effects of a subpressor dose of endothelin-1 (3 micrograms/kg/day) on chronic blood pressure responses to infusion of angiotensin II and norepinephrine in rats. Rats were infused with angiotensin II at a subpressor dose (400 micrograms/kg/day i.p.) or with norepinephrine at a subpressor dose (360 micrograms/kg/day i.p.) for 6 days. Systolic blood pressure was significantly elevated during combined infusion of endothelin-1 and angiotensin II, whereas endothelin-1 alone or angiotensin II alone failed to induce any significant changes in systolic blood pressure compared with vehicle alone. This effect was sustained for the whole experimental period and was not associated with any significant changes in body weight, fluid intake, urine volume, or urinary electrolyte excretion. In contrast, combined infusion of endothelin-1 and norepinephrine failed to elevate systolic blood pressure, and no significant difference in systolic blood pressure was observed for the whole experimental period among the four groups of rats with endothelin-1 in combination with norepinephrine, endothelin-1 alone, norepinephrine alone, and vehicle alone. The present results indicate that angiotensin II and endothelin-1, but not norepinephrine and endothelin-1, work synergistically to raise the blood pressure and also suggest the possibility that endothelin-1 may modulate blood pressure control.

Antihypertensive and renal-protective effects of losartan in streptozotocin diabetic rats.

Objective To assess the renal benefits of a specific angiotensin II receptor antagonist, losartan, in diabetic rats with renal impairment. Design and methods Uninephrectomized streptozotocin diabetic spontaneously hypertensive rats (SHR) were randomly assigned to receive vehicle, or to receive losartan or captopril, or both, intraperitoneally via osmotic minipumps for 8 weeks. Results Blood pressure and urinary protein excretion in the diabetic SHR increased progressively during the experimental period. Both captopril treatment and losartan treatment completely blocked the development of hypertension in diabetic SHR. Simultaneous administration of captopril and losartan did not enhance the antihypertensive effects of losartan treatment or captopril treatment. Furthermore, losartan treatment, captopril treatment and losartan + captopril treatment all significantly decreased urinary protein excretion, urinary albumin excretion and serum creatinine to the same extent. These effects were sustained for the entire experimental period and were not associated with any significant changes in body weight, urine volume, urine sugar and urinary electrolytes excretion. These results were confirmed by morphological analysis of kidneys in each group of rats. Losartan treatment, captopril treatment and losartan + captopril treatment all significantly and effectively protected against an increase in the percentage of focal glomerular sclerosis. Losartan treatment and captopril treatment both significantly attenuated the increase in heart weight: body weight ratio. The heart weight: body weight ratio in the losartan-treated group was significantly lower than in the captopril-treated group. Conclusions These results indicate that hypertension could accelerate diabetic renal impairment and that losartan has antihypertensive and renoprotective effects in this rat model. They also suggest that the antihypertensive and renoprotective effects of captopril treatment in this rat model are caused mainly by inhibition of angiotensin II production rather than stimulation of the kallikrein-kinin system or of vasodilator prostaglandins. The difference in potency between losartan treatment and captopril treatment to attenuate the increase in heart weight: body weight ratio might partly explain the existence in the heart of angiotensin-forming pathways, which are not dependent on angiotensin converting enzyme.

Renal protective effects of chronic exercise and antihypertensive therapy in hypertensive rats with chronic renal failure

Objectives Patients with chronic renal failure are restricted to mild physical activity and tend to a lack of exercise. However, there have been few reports regarding the influence of chronic exercise on the progression of renal disease. Similarly, there are few animal models concerned with the effect of exercise training on improving renal function. Therefore, we assessed the renal effects of moderate chronic treadmill exercise in a remnant kidney model of spontaneously hypertensive rats (SHR) with chronic renal failure. We also assessed the effects of exercise and antihypertensive therapy on renal function. Design and methods Eight-week-old SHR were subjected to 5/6 nephrectomy by removal of the left kidney and excision of two-thirds of the right kidney. The rats were divided into four groups: (i) no exercise (Non-EX); (ii) moderate exercise with treadmill running (20 m/min, 0 grade incline for 60 min) (EX); (iii) EX with an angiotensin converting enzyme (ACE) inhibitor, enalapril (2 mg/kg per day, i.p.); and (iv) EX with an angiotensin receptor antagonist, losartan (5 mg/kg per day, i.p.), for 4 weeks. Results Chronic EX significantly attenuated the increase in proteinuria (P < 0.01) and significantly protected against increases in the index of glomerular sclerosis (IGS). Both enalapril and losartan with EX significantly decreased blood pressure (P < 0.001), and further decreased the IGS. In the stepwise multiple regression analysis, only antihypertensive drug remained in the model as a significant predictor of IGS (P < 0.0001). In contrast, exercise, antihypertensive drug and mean systolic blood pressure (weeks 1–4) remained in the model as a significant predictors of mean proteinuria (weeks 1–4) (all P < 0.0001). Conclusions These results suggest that exercise does not worsen renal function and has renal-protective effects in this model of rats. Moreover, the antihypertensive therapy has additional renal-protective effects in this model of rats.

Effects of exercise and β-blocker on blood pressure and baroreflexes in spontaneously hypertensive rats

Abstract Background Exercise training or β-blocker decreases high blood pressure (BP) and improves abnormal baroreflex function associated with hypertension. This study was undertaken to examine whether the effects of exercise training are additive to β-blocker in spontaneously hypertensive rats (SHR). Methods At 5 weeks of age, SHR were allocated to four groups: sedentary control, exercise training, treatment with moderate dose of bisoprolol, and their combination. Systolic BP was monitored by the tail-cuff method under restrained conditions. Sigmoidal mean arterial pressure (MAP)–heart rate (HR) reflex curves were obtained in rats at 17 weeks of age under quiet conditions before and after atenolol to ensure sympathetic blockade and to determine the vagal component of gain. After studying baroreflex function, intrinsic HR was obtained by additional administration of atropine. Results Before atenolol, both exercise training alone and bisoprolol alone lowered resting MAP and HR, and decreased upper plateau (maximal tachycardia) and lower plateau (maximal bradycardia), resulting in decreased sympathetic component of HR range (upper plateau − intrinsic HR) and increased vagal component of HR range (intrinsic HR − lower plateau). After atenolol, both exercise training alone and bisoprolol alone increased the gain of vagal component. Exercise training had no additive effect on any parameters to bisoprolol except for systolic BP and HR measured by the tail-cuff method. Conclusions Exercise training and bisoprolol have similar effects concerning resting hemodynamics and baroreflex function in SHR. Although additive effects of exercise training to bisoprolol are not evident under quiet, nonstressful conditions, some additive effects may be obtained under stress such as restrain.

Expression of cytochrome P-450 4 enzymes in the kidney and liver: regulation by PPAR and species-difference between rat and human.

Members of the cytochrome P-450 4 (CYP4) family catalyze the ω-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARα was expressed in the liver, proximal tubule and TAL. PPARγ was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARα agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARγ agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.

Kinin and angiotensin II receptor antagonists in rats with chronic renal failure : chronic effects on cardio- and renoprotection of angiotensin converting enzyme inhibitors

Objective To assess the potential of the kallikrein-kinin and renin-angiotensin systems in mediating the cardio- and renoprotective effects of angiotensin converting enzyme (ACE) inhibitors in rats with chronic renal failure. Materials and methods Spontaneously hypertensive rats (SHR) and normotensive control Wistar-Kyoto (WKY) rats subjected to five-sixths nephrectomy were randomly assigned to treatment with vehicle, a kinin antagonist (Hoe 140) or an ACE inhibitor (cilazapril) or both drugs, intraperitoneally via osmotic minipumps for 4 weeks. In addition, the effects of a chronic infusion of a specific angiotensin receptor antagonist (losartan) alone or in combination with an ACE inhibitor (enalapril) were also investigated in nephrectomized SHR for 2 weeks. Results In nephrectomized SHR and WKY rats, cilazapril alone significantly reduced systolic blood pressure, urinary protein excretion, heart weight and serum creatinine. In nephrectomized SHR, Hoe 140 alone or cilazapril in combination with Hoe 140 (7 or 70 μg/kg per day) induced no changes in these parameters, other than those associated with the effects of cilazapril alone. In nephrectomized WKY rats, cilazapril in combination with Hoe 140 (70 μg/kg per day) slightly, but not significantly, attenuated the antihypertensive effect of cilazapril but did not affect the other parameters. These results were confirmed by morphological analysis of kidneys. All the drug regimens provided effective protection against an increase in focal glomerular sclerosis. Enalapril did not modify the antihypertensive and renoprotective effects of losartan in nephrectomized SHR. Conclusions The present results indicate that the kallikrein-kinin system might not be a major factor in the cardio- and renoprotective effects of ACE inhibitors in rats with chronic renal failure.

Increased expression of urotensin II-related peptide and its receptor in kidney with hypertension or renal failure.

Urotensin II-related peptide (URP) is a novel vasoactive peptide that shares urotensin II receptor (UT) with urotensin II. In order to clarify possible changes of URP expression in hypertension and chronic renal failure (CRF), the expressions of URP and UT were studied by quantitative RT-PCR and immunohistochemistry in kidneys obtained from spontaneous hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and WKY with CRF due to 5/6 nephrectomy. Expression levels of URP mRNA and UT mRNA were significantly higher in the kidneys obtained from SHR compared with age-matched WKY (at 5-16 and 16 weeks old, respectively). A dissection study of the kidney into three portions (inner medulla, outer medulla and cortex) showed that the expression levels of URP mRNA and UT mRNA were highest in the inner medulla and the outer medulla, respectively, in both SHR and WKY. The expression levels of URP and UT mRNAs were greatly elevated in the remnant kidneys of CRF rats at day 56 after nephrectomy, compared with sham-operated rats (about 6.5- and 11.9-fold, respectively). Immunohistochemistry showed that URP immunostaining was found mainly in the renal tubules, vascular smooth muscle cells and vascular endothelial cells. UT immunoreactivity was localized in the renal tubules and vascular endothelial cells. These findings suggest that the expressions of URP and UT mRNAs in the kidney are enhanced in hypertension and CRF, and that URP and its receptor have important pathophysiological roles in these diseases.

Endothelin receptors in ischemic rat brain and Alzheimer brain.

Summary: Endothelin (ET) binding sites in male Wistar rat brains subjected to a 20-min four-vessel occlusion (transient forebrain ischemia model) which induces hip-pocampal neuron death, and in human brains with Alzheimer disease, were mapped by quantitative in vitro autoradiography employing [125I]ET-1 as a radioligand. Rats were decapitated 4 or 7 days after ischemia. In the rat brain, the [125I]ET-1 binding sites were remarkably increased in the hippocampal CA1 and dentate gyrus, ventral thalamic nucleus, and cortical vessels 4 and 7 days after ischemia, when many reactive astroglia were observed. The [125I]ET-1 binding sites decreased in the cerebral cortex affected by Alzheimer disease. The binding was abolished by 1 μM unlabeled ET-1, ET-3, sarafotoxin S6b, and BQ788 (an ETB antagonist) but not by BQ123 (an ETA antagonist), suggesting that the [125I]ET-1 binding sites are as ETB receptors. The present findings raise the possibility that a glial ET system could be responsible for the occurrence of ischemic neuron cell death.

Atorvastatin upregulates nitric oxide synthases with Rho-kinase inhibition and Akt activation in the kidney of spontaneously hypertensive rats

Objective 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins reduce blood pressure and have beneficial effects in cardiovascular and kidney diseases. The present study examined the effect of chronic treatment with atorvastatin (ATV) on the expression of nitric oxide synthase (NOS) and the activity of Rho-kinase and Akt in the kidney of spontaneously hypertensive rats (SHRs). Methods SHRs were treated with ATV for 8 weeks and the SBP was measured. The expressions of endothelial, neuronal and inducible NOS (eNOS, nNOS and iNOS, respectively) proteins in the kidney were examined by immunoblot analysis. The activity of eNOS, Rho-kinase and Akt in the kidney was examined by assessing the phosphorylation of eNOS, ezrin/radixin/moesin (ERM) and Akt, respectively. Results ATV reduced the SBP without changing the plasma cholesterol levels. ATV increased eNOS expression in the cortex and medulla and nNOS expression in the medulla, whereas it did not affect iNOS expression. Although it upregulated eNOS expression in the kidney, ATV decreased the levels of phosphorylated eNOS in the cortex and did not affect the ratio of phosphorylated eNOS to total eNOS in the medulla. ATV also inhibited Rho-kinase activity and enhanced Akt activity in the kidney of SHRs. Conclusion ATV upregulates eNOS and nNOS expressions with Rho-kinase inhibition and Akt activation in the kidney of SHRs. The renal nitric oxide system, Rho-kinase and Akt may contribute to the antihypertensive and renoprotective effects of statins.

Effects of benidipine, a calcium antagonist, on urinary kallikrein excretion and renal impairment in experimental diabetes

Objective To assess the long-term effects of different antihypertensive agents on urinary protein excretion and kallikrein excretion in diabetic rats with renal impairment. Methods Uninephrectomized streptozotocin diabetic Wistar-Kyoto rats were randomly assigned to receive vehicle, a calcium antagonist (benidipine) or an angiotensin converting enzyme inhibitor (captopril) for up to 12 weeks. Active kallikrein was determined by its kininogenase activity, and generated kinins were measured by radioimmunoassay. Total kallikrein was also determined by measuring kininogenase activity after inactive kallikrein had been activated with trypsin. Results Urinary protein excretion increased significantly in diabetic rats compared with non-diabetic rats. Urinary active kallikrein excretion was significantly reduced in diabetic rats, whereas urinary total kallikrein excretion was unchanged, resulting in a reduced percentage of active to total kallikrein compared with that in non-diabetic rats. Benidipine and captopril reduced blood pressure and attenuated the development of diabetic renal impairment in a similar manner. However, only benidipine attenuated the decreases in urinary active kallikrein excretion and the ratio of active to total kallikrein in diabetic rats. Conclusions Although pathophysiological relevance of impaired urinary kallikrein activation to the development of diabetic renal impairment remains to be determined, our result might suggest a new mechanism by which calcium antagonists protect the kidney from diabetic renal impairment.