Aging and Severity of Hypertension Attenuate Endothelium-Dependent Renal Vascular Relaxation in Humans
Abstract Endothelial dysfunction may be related to cardiovascular risk factors, such as aging, hypertension, and atherosclerosis. We investigated whether aging and hypertension independently alter endothelial function in the renal circulation in humans in the absence of abnormalities in lipid and glucose metabolism. l-Arginine (500 mg/kg over 30 minutes) was intravenously administered to 33 patients with essential hypertension and 35 normotensive subjects. The l-arginine–induced increases in renal plasma flow (10.1±0.8% versus 15.8±0.9%, P<.05) and plasma cGMP (53±4% versus 82±5%, P<.05) were significantly smaller in patients with essential hypertension than in the normotensive subjects. Multivariate stepwise regression analysis showed that age (P<.0002) and the mean blood pressure (P<.0001) were independently and negatively correlated with the renal plasma flow response to l-arginine. Age (P<.002), mean blood pressure (P<.0001), and male sex (P<.05) were independently correlated with the l-arginine–induced increase in plasma cGMP. The peak change in plasma cGMP was significantly correlated with the l-arginine–induced increase in renal plasma flow (r=.63, P<.001). These findings suggest that aging and hypertension may independently impair endothelium-dependent renovascular dilation and that this effect may be caused at least in part by a decrease in nitric oxide production.
The endothelium is involved in vascular physiology via the synthesis and release of various vasorelaxant and vasoconstrictive factors.1 2 3 Stimulation of endothelial cells by acetylcholine, other agonists, and physical stimuli induces the release of EDRFs.4 5 NO, derived from l-arginine, is responsible for the biological activity of EDRF.6 7 Aging, hypertension, and atherosclerosis may alter the structure and function of vascular components (such as the endothelium, intimas, and smooth muscle cells), increasing the risk of cardiovascular and cerebrovascular diseases and renal dysfunction.8 9 10 Studies suggest that endothelial dysfunction is involved in the development of atherosclerosis. A disturbance of endothelium-dependent vasodilation in the aortic,11 forearm,12 13 coronary,14 carotid,15 and mesenteric16 arteries has been observed in elderly and hypertensive individuals and in animal models. However, because the effects of aging, hypertension, and atherosclerosis are interdependent, their individual influence on endothelial function has not been clarified. The mechanisms responsible for disturbances in endothelium-dependent vasodilation in the presence of such disorders remain unclear.
The kidney is both a target of and a contributor to hypertension. The renal vascular bed is more sensitive to vasoactive agents than are the coronary and forearm vascular beds. EDRF/NO may participate in the regulation of renal hemodynamics, specifically via its effects on the renal vasculature. Baylis et al17 demonstrated that EDRF controls renal hemodynamics in normal rats. However, the in vivo effects of aging and hypertension on endothelium-dependent renal vascular relaxation in humans have not been extensively investigated.
We investigated the effects of aging and hypertension on renal hemodynamics in normotensive control subjects and in patients with essential hypertension with normal renal function and without vascular risk factors, such as elevated levels of cholesterol and glucose, who received intravenous infusions of l-arginine.
We studied 33 Japanese patients with essential hypertension (19 men and 14 women; mean age, 48±2 years; age range, 23 to 71 years) and 35 normotensive Japanese subjects (22 men and 13 women; mean age, 46±3 years; age range, 22 to 76 years). Hypertension was defined as a systolic pressure >160 mm Hg or a diastolic pressure >95 mm Hg on at least three different occasions. Blood pressure was measured with the subject in the sitting position at an outpatient clinic at Hiroshima University School of Medicine. We excluded from study patients with secondary forms of hypertension and those subjects with a serum concentration of creatinine >2.0 mg/dL, a GFR <50 mL · min–1 · 1.48 m–2, and a RPF <300 mL · min–1 · 1.48 m–2. Individuals with severe hypertension who had a systolic blood pressure >180 mm Hg or a diastolic pressure >120 mm Hg with objective signs of hypertensive end-organ disease were also excluded. None of the patients had a history of cardiovascular or cerebrovascular disease, diabetes mellitus, hypercholesterolemia, liver disease, or renal disease, and none had taken antihypertensive drugs for at least 4 weeks before the study. Normotension was defined as a systolic pressure <140 mm Hg and a diastolic pressure <80 mm Hg. The normotensive control subjects had no history of serious disease and had taken no medications for at least 4 weeks before the study. Informed consent was obtained from all subjects. The study protocol was approved by the ethics committee of the First Department of Internal Medicine.
The normotensive and hypertensive subjects received a diet containing 170 mmol of sodium chloride per day for 1 week before the study. Throughout the study, caloric intake (40 cal · kg–1 · d–1) and the potassium (100 mmol/d) and calcium (40 mmol/d) content of the diet were kept constant. All subjects ate meals that were prepared in the Hiroshima University Hospital kitchen. Dietary compliance was assessed by measuring the 24-hour urinary excretion of sodium, chloride, and potassium throughout the study. l-Arginine was infused beginning at 8:30 am. Subjects fasted overnight for at least 12 hours and were kept in a supine position in a quiet, dark, and air-conditioned room maintained at a constant temperature (22°C to 25°C) throughout the study. A 19-gauge polyethylene catheter (Terumo Co) was inserted into the right antecubital vein for infusion of PAH, inulin, and l-arginine. Another catheter was inserted into the left antecubital vein to obtain blood samples. After a 30-minute rest period, subjects received an initial bolus dose of 8.0 mg/kg of PAH and 16 mg/kg of inulin. PAH and inulin were subsequently infused at constant rates of 12 and 20 mg/min, respectively, with a syringe pump (Terfusion; Terumo Co) throughout the study.18 l-Arginine (500 mg/kg) was administered over 30 minutes with an infusion pump (PEI-1000; Pal Medical Co) beginning 60 minutes after initiation of the PAH and inulin infusions. A 30-minute recovery period was allowed after the end of the l-arginine infusion. Blood pressure and heart rate were measured at 1-minute intervals with a TM2420 monitor (AND Co) attached to the upper left arm. The mean blood pressure was defined as the diastolic pressure plus one third of the pulse pressure. Blood samples were obtained for determinations of the serum concentrations of PAH and inulin, and the plasma concentrations of cGMP and h-ANP at 0, 15, 30, and 60 minutes after the start of the l-arginine infusion. Baseline fasting serum concentrations of total cholesterol, creatinine, glucose, and electrolytes were obtained at 0 minutes.
In the preliminary study, to examine whether the effects of l-arginine on renal hemodynamics are due to its contribution to the release of NO, we administered d-arginine, the enantiomer of l-arginine, as a control for l-arginine. The effects of l- and d-arginine on renal hemodynamics and plasma cGMP in 7 normotensive male subjects (mean age, 26±2 years; age range, 23 to 35 years) were measured. These studies were done in a double-blind, randomized fashion on a separate day. d-Arginine infusion was performed in a protocol identical to that described for l-arginine. d-Arginine caused a significant increase in RPF and a significant decrease in RVR. The responses of RPF and RVR to l-arginine were significantly greater than those to d-arginine (RPF 20.4±2.7% versus 7.0±1.6%, P<.05, and RVR −28.1±2.9% versus −10.2±1.9%, P<.05), whereas the response of blood pressure was similar in both infusions (l-arginine: −8.1±1.3% versus d-arginine: −7.3±1.4%). The infusion of d-arginine also did not significantly alter the GFR. The percent increase in plasma cGMP was significantly higher during l-arginine infusion than during d-arginine infusion (129±19% versus 18±3%, P<.05). Changes in h-ANP were similar in both infusions (22±4% versus 20±3%). Renal vasorelaxation and plasma cGMP responses were much greater during l-arginine as compared with d-arginine. The small increase in cGMP in response to d-arginine may be caused by an increase in h-ANP. It has been reported that other amino acids, which are not substrate for NO, also produce renal vasodilation.19 Although the precise mechanism is unclear, only one third of the effects of l-arginine on RPF can be explained by these effects of amino acid. These findings suggest that exogenous l-arginine infusion may cause a renal vasorelaxation mostly through the release of NO rather than a nonspecific effect of amino acid.
The l-arginine used for intravenous administration was l-arginine hydrochloride (Morishita Ruseru Pharmaceutical Co). d-Arginine was d-arginine hydrochloride (Sigma Chemical Co). The administered inulin was Inutest (Laevosan-Gesellschaft Co), and the PAH was p-aminohippurate (Daiichi Pharmaceutical Co).
Assays and Parameters of Renal Hemodynamics
Samples of venous blood were placed in tubes containing EDTA-Na (1 mg/mL) and polystyrene tubes. The tubes containing EDTA were promptly chilled in an ice bath. Plasma and serum were immediately separated by centrifugation at 3100g at 4°C for 10 minutes and at 1000g at room temperature for 10 minutes, respectively, and were stored at −80°C until assayed. Serum concentrations of total cholesterol, creatinine, glucose, and electrolytes and urinary electrolytes were determined by routine chemical methods. Plasma renin activity was measured using a radioimmunoassay (Gamma Coat PRA, Baxter Travenol Co). The plasma level of cGMP was measured by radioimmunoassay using a cGMP kit (Yamasa Shoyu Co), and the level of plasma h-ANP was also measured by radioimmunoassay (Amersham Co). The RPF was estimated from PAH clearance. The serum concentration of PAH was analyzed by spectrophotometry. The GFR was estimated from inulin clearance.20 The serum concentration of inulin was analyzed by the anthrone method.21 RVR was calculated by dividing the mean blood pressure by the renal blood flow, and the filtration fraction was calculated by dividing the GFR by the RPF. RPF, GFR, and RVR were normalized to the body surface area divided by 1.48 m2 (1.48 m2 is the average body surface area of the Japanese population).
Comparisons of baseline parameters between the two groups were made using unpaired Student’s t test. Differences were compared using one-way ANOVA for repeated measures followed by Scheffé’s F test. Relationships between variables, such as age and mean blood pressure and the renal hemodynamic responses to l-arginine, were determined by linear regression analysis. Multivariate analysis using multiple stepwise regression was performed to determine the relationships between the responses of renal hemodynamics and cGMP to l-arginine and age, gender, body mass index, mean blood pressure, serum concentrations of creatinine, cholesterol, and glucose, and the plasma renin activity. All results are presented as mean±SEM. A value of P<.05 was considered to be statistically significant. Multivariate analysis was performed with the SAS (Statistical Analysis System) program package.
The mean blood pressure and RVR were significantly higher in the hypertensive subjects than in the normotensive subjects (Table 1⇓). Other parameters were similar in both groups.
Effects of l-Arginine on Mean Blood Pressure and Heart Rate
The mean blood pressure decreased promptly after the initiation of the l-arginine infusion and immediately returned to the baseline level after the end of the infusion. The heart rate gradually increased during the l-arginine infusion and then gradually returned to the baseline level during the recovery period. The maximal percent changes in the mean blood pressure and heart rate were similar in the hypertensive patients and normotensive subjects (Fig 1⇓).
Effects of l-Arginine on Renal Hemodynamics
Fig 2⇓ shows the effects of l-arginine infusion on RPF, GFR, RVR, and filtration fraction in patients with essential hypertension and in normotensive control subjects. The maximal l-arginine–induced increase in RPF was smaller in the patients with essential hypertension (10.1±0.8%) than in the normotensive control subjects (15.8±0.9%) (P<.05). The hypertensive patients showed smaller maximal decreases in the RVR (−14.3±0.7%) and the filtration fraction (−2.1±0.7%) compared with the normotensive control subjects (RVR −20.1±0.8%; filtration fraction −8.9±0.8%) (P<.05). RPF, RVR, and filtration fraction returned to baseline levels at 30 minutes after the end of l-arginine infusion. The infusion of l-arginine did not significantly alter the GFR in either group.
Age was inversely correlated with the maximal RPF response to l-arginine (r=−.45, P<.001) in all subjects (Fig 3⇓). The maximal RPF response to l-arginine was significantly correlated with the baseline mean blood pressure (r=−.56, P<.001) (Fig 4⇓). There was no significant correlation between the renal hemodynamic responses and other variables. Multivariate stepwise regression analysis showed that age (P<.0002) and mean blood pressure (P<.0001) were independently related to the maximal RPF response to l-arginine in all subjects (Figs 3⇓ and 4⇓). There was no independent correlation between the maximal RPF response to l-arginine and gender, body mass index, creatinine level, cholesterol level, glucose level, or plasma renin activity (Table 2⇓).
Effects of l-Arginine on the Plasma Concentrations of cGMP and h-ANP
Baseline plasma concentrations of cGMP were similar in both groups (Table 1⇑). The percent increase in cGMP was significantly smaller in the hypertensive patients than in the normotensive control subjects (53±4% versus 82±5%, P<.001) (Fig 5⇓, left). The administration of l-arginine significantly increased the level of h-ANP in both groups (from 6.7±0.3 to 8.1±0.4 pmol/L in the hypertensive patients and from 6.9±0.3 to 8.5±0.5 pmol/L in the normotensive control subjects; P<.01). The percent change in h-ANP was similar in both groups (Fig 5⇓, right).
There was a significant inverse correlation between age and the l-arginine–induced increase in cGMP (r=−.36, P<.01) in all subjects (Fig 3⇑). The baseline mean blood pressure and the l-arginine–induced increase in cGMP were negatively correlated (r=−.51, P<.001) in all subjects (Fig 4⇑). There was no significant correlation between the cGMP response and the other variables. Age (P<.002), mean blood pressure (P<.0001), and male sex (P<.05) were independently associated with the cGMP response to l-arginine (Table 2⇑).
The peak increase in cGMP, which is a marker of NO production, was significantly correlated with the maximal RPF response to l-arginine (r=.63, P<.001) (Fig 6⇓).
Atherosclerosis, hypercholesterolemia, hypertension, and advancing age can impair endothelium-dependent vascular relaxation in the coronary14 22 and forearm12 13 23 arteries in humans. Although it is widely accepted that the kidney is important in the pathogenesis, maintenance, and progression of hypertension, renal endothelial function has not been extensively investigated in hypertensive patients. We recently reported that endothelium-dependent renal vasodilatation was impaired even in patients with mild essential hypertension and normal renal function and who had no objective evidence of end-organ damage as compared with normotensive control subjects.24 The present study is the first to demonstrate the effects of aging and blood pressure on renal endothelial function in humans. The present results showed that the l-arginine–induced increase in RPF was negatively correlated with age and the baseline blood pressure and that the l-arginine–induced increase in cGMP was inversely correlated with age, the baseline blood pressure, and male sex.
The present findings suggest that endothelium-dependent renovascular relaxation and the production of NO-cGMP in response to l-arginine were inhibited by aging. These findings are consistent with the results of previous studies showing that aging impairs the acetylcholine-induced endothelium-dependent vascular dilation in the coronary,14 22 25 26 forearm,12 13 and renal27 arteries in humans. Previous studies suggest that atherosclerosis begins in childhood and progresses with age.28 29 Studies in rats have shown that increased age is associated with impaired endothelium-dependent vascular relaxation in response to acetylcholine, bradykinin, and ATP in the mesenteric arteries, the cerebral artery, and the thoracic aorta.9 11 16 However, in humans or rats, age does not influence the effects of such endothelium-independent vasodilators as sodium nitroprusside and papaverine,15 16 suggesting that the endothelium-dependent vasorelaxation is selectively impaired by aging.
On the other hand, Reckelhoff and Manning30 have reported that with advancing age, the renal vasculature of rats becomes more responsive to the vasoconstrictor effects of NG-nitro-l-arginine methyl ester inhibition of NO synthase, suggesting that NO may play a progressively more important role in the control of renal function as aging progresses. Their findings are in disagreement with data obtained in previous rat studies of mesenteric arteries,9 the cerebral artery,11 and the thoracic aorta,16 in which endothelium-dependent vascular relaxation was impaired with aging. Sabbatini et al31 reported that acute ischemic renal failure is worse in old rats than in young rats even in the absence of age-related glomerulosclerosis and that the greater renal impairment after acute ischemic renal failure in old rats is likely related to reduced NO levels. Hollenberg et al27 also showed that the renal response to acetylcholine was attenuated in the aging human. This discrepancy may be caused by differences in vascular beds and races.
In the present study, the renal vasorelaxant response to the infusion of l-arginine was blunted in patients with essential hypertension as compared with normotensive control subjects. The blood pressure was inversely correlated with the renal vasorelaxant response to l-arginine, which suggests that the severity of hypertension exerts a major influence on renal endothelial function. Experimental data suggest that endothelial dysfunction develops as the blood pressure rises,3 4 suggesting that a modification of the l-arginine–NO-cGMP pathway may be a secondary, not a primary, event in the pathogenesis, maintenance, and progression of hypertension. The present findings suggest that endothelial dysfunction may be a consequence rather than a cause of hypertension. However, our previous findings24 32 33 suggest that endothelial dysfunction may be present even in the early stage of hypertension and that changes in renal endothelial function may be a cause of hypertension. Whether endothelial dysfunction is a cause or an effect of hypertension, the present findings suggest that an elevated blood pressure may be an important independent contributor to the development of endothelial dysfunction.
NO stimulates cytosolic guanylate cyclase and increases cGMP. h-ANP, another humoral compound, also increases cGMP production by stimulating cytosolic guanylate cyclase.34 In the present study, a similar l-arginine–induced increase in h-ANP was observed in hypertensive patients and normotensive control subjects. Thus, differences in the l-arginine–induced increase in cGMP between these groups may have been caused by differences in the production of NO. Hishikawa et al35 demonstrated that h-ANP was increased by both l-arginine and a saline vehicle in humans, suggesting that a volume load may elevate h-ANP.
In the present study, multivariate analysis showed that the percent increase in the plasma level of cGMP, which is an index of NO production, in response to l-arginine infusion was independently correlated with age, the baseline blood pressure, and male sex. The peak change in cGMP was significantly correlated with the l-arginine–induced increase in the RPF. Therefore, a decrease in NO production may be a mechanism for the blunted renal vasorelaxant response to l-arginine associated with aging and an increase in blood pressure. The present results also indicated that male sex contributed independently to the decrease in NO production, suggesting that it may be a cardiovascular risk factor.
NO provokes vasodilation, inhibits the aggregation and adhesion of platelets, and participates in the suppression of smooth muscle cell proliferation.36 Therefore, a deficiency in NO production may reduce the antiaggregatory properties of the endothelium and promote intravascular clotting, leading to the development of atherosclerosis and an increased incidence of angina pectoris, myocardial infarction, and stroke.
We evaluated the effects of intravenous infusions of l-arginine on the renal circulation in humans. This method is useful in estimating the activity of the endothelial l-arginine–NO-cGMP pathway. The effects of the intravenous infusions of l-arginine have been studied on other vascular beds, such as femoral37 and pulmonary38 circulations. The administration of specific NO synthase inhibitors, such as NG-nitro-l-arginine methyl ester and NG-monomethyl-l-arginine, and of agonists that stimulate NO release, such as acetylcholine and bradykinin, would have allowed us to draw more specific conclusions concerning the role of basal and stimulated releases of NO in the renal circulation. However, because the intravenous infusion of NO synthase inhibitors may increase the blood pressure and vascular resistance and reduce the vascular blood flow, these agents may lead to adverse effects in hypertensive individuals. We therefore decided not to use these agents, primarily because of ethical concerns.
In the present study, we demonstrated that d-arginine caused a small but significant increase in RPF. These data coupled with the previous studies that demonstrate other basic amino acid effects on RPF suggest that all of the response to l-arginine is not caused by NO. We should take into account the specificity of using l-arginine as a specific probe for NO.
Because in the present study we measured renal hemodynamics without urine collection (constant infusion technique),18 we did not perform the urinary cGMP measurements. The measurement of urinary cGMP is a more sensitive method of measuring renal NO release than plasma cGMP. This measurement would have allowed us to draw more specific conclusions concerning the NO production in the renal circulation.
Endothelial dysfunction is usually manifested by an impairment in the endothelium-dependent vasorelaxation related to the decreased production of EDRFs and/or by the increased production of endothelium-derived contracting factors. Küng and Lüscher11 have suggested that the effects of age and hypertension on endothelium-dependent vasodilation involve different mechanisms. Aging blunts the production of NO, whereas hypertension enhances the production of endothelium-derived constricting factors. However, the present results suggest that impaired endothelium-dependent renal vasodilation in hypertensive patients may result from a decrease in the production of EDRF/NO. We suggest that a decrease in NO production may be the most important mechanism underlying the age- and hypertension-induced impairment of endothelium-dependent renal vasodilation. However, we did not investigate other possible mechanisms in the present study and thus cannot exclude such possibilities as a decrease in the number and/or affinity of receptors for endothelium-dependent vasoactive agents on the endothelial membrane, the release of endothelium-derived hyperpolarizing factor, or the decreased sensitivity of smooth muscle cells to vasodilatory agents and/or the increased sensitivity of smooth muscle cells to vasoconstrictors.
The number of subjects studied is relatively small compared with large epidemiological trials. However, significant relations were observed between the renal responses to l-arginine and age or the baseline blood pressure. A more significant independent correlation between the renal response to l-arginine and age would be shown with a larger sample size.
In conclusion, the endothelium-dependent renal vasodilation observed in response to l-arginine was impaired in patients with essential hypertension as compared with normotensive control subjects because of the blunted response of NO-cGMP. In addition, increased age and an elevated blood pressure impaired the endothelium-dependent renal vasorelaxation and the production of NO. Thus, morphological alterations in the endothelium associated with aging and hypertension may lead to a decrease in the production of NO, which may impair endothelium-dependent responses. A blunting of the endothelium-dependent vasorelaxation may increase the risk of vascular complications in the elderly and in hypertensive persons.
Selected Abbreviations and Acronyms
|EDRF(s)||=||endothelium-derived relaxing factor(s)|
|GFR||=||glomerular filtration rate|
|h-ANP||=||human atrial natriuretic peptide|
|RPF||=||renal plasma flow|
|RVR||=||renal vascular resistance|
This study was supported in part by grants-in-aid for Scientific Research (07407065 and 08457639) from the Ministry of Education, Science and Culture of Japan and a Foundation for Total Health Promotion grant (1992). The authors thank Yuko Omura for secretarial assistance.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, California, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-620-I-621).
- Received August 5, 1996.
- Revision received October 10, 1996.
- Accepted January 28, 1997.
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