Division of Nephrology, Departments of 1 Medicine and 2 Physiology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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Nitric oxide (NO) regulates
renal O2 consumption, but the source of NO mediating this
effect is unclear. We explored the effects of renal NO production on
O2 consumption using renal cortex from mice deficient
(/
) in endothelial (e) nitric oxide synthase (NOS). O2
consumption was determined polarographically in slices of cortex from
control and eNOS(
/
) mice. NO production was stimulated by
bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an
NOS inhibitor. Basal O2 consumption was higher in
eNOS(
/
) mice than in heterozygous controls (919 ± 46 vs.
1,211 ± 133 nmol O2 · min
1 · g
1;
P < 0.05). BK and Ram decreased O2
consumption significantly less in eNOS(
/
) mice [eNOS(
/
): BK
19.0 ± 2.8%, Ram
20.5 ± 3.3% at 10
4 M;
control: BK
29.5 ± 2.5%, Ram
34 ± 1.6% at
10
4 M]. The NO synthesis inhibitor
nitro-L-arginine methyl ester (L-NAME)
attenuated this decrease in control but not eNOS(
/
) mice. An NO
donor inhibited O2 consumption similarly in both groups independent of the presence of L-NAME. These results
demonstrate that NO production by eNOS is responsible for regulation of
renal O2 consumption in mouse kidney.
bradykinin; renal physiology; ramiprilat; nitric oxide
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INTRODUCTION |
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REGULATION OF O2 consumption by nitric oxide (NO) has been demonstrated in skeletal and cardiac muscle and the kidney (19, 25, 31). The effects of NO on renal O2 consumption have been shown at the level of the intact kidney, cortical and medullary tissue, and isolated renal tubules (13, 16). The effect of NO appears to occur at the level of the mitochondria through direct inhibition of elements of the electron transport chain (6, 7). Thus, in addition to regulating renal blood flow through its effect on vascular tone, NO could affect O2 levels, particularly in the normally hypoxic renal medulla, playing a role in modulating renal tubular injury.
NO is produced by a family of NO synthases (NOS) consisting of three major isoforms, neuronal (nNOS), inducible (iNOS), and endothelial (eNOS; see Refs. 14 and 15). All three of these isoforms have been detected in the kidney, with nNOS predominantly present in the macula densa, iNOS in the medullary thick ascending limb and collecting ducts, and eNOS in glomerular capillaries, intrarenal arteries and arterioles, vasa recta, proximal tubules, and thick ascending limbs (2, 4, 14, 15, 29, 30). NOS activity in microdissected segments of the rat kidney was found to be greatest in the inner medullary collecting duct, moderate in glomeruli and vasa rectae, and significantly less in other structures (30). This agrees with previous studies showing higher NOS activity in the medulla than in the cortex (15, 21).
NO production in the kidney regulates renal hemodynamics, tubuloglomerular feedback, and sodium excretion (14, 15). It also appears to play a role in injury during glomerular and tubulointerstitial inflammation and ischemic acute renal failure (14). More recently, a role in regulation of renal O2 consumption has been described (13, 16). However, the endogenous source of NO influencing renal O2 consumption is unknown. Therefore, we elected to explore the effects of stimulation of endogenous NO production on renal O2 consumption in vitro using tissue from mice deficient in the eNOS isoform.
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METHODS |
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Reagents. S-nitroso-N-acetylpenicillamine (SNAP), bradykinin, nitro-L-arginine methyl ester (L-NAME), succinate, and sodium cyanide were purchased from Sigma. Ramiprilat was a gift from Hoechst Marion Roussel (New Brunswick, NJ).
Animals studied.
Homozygous mutant eNOS(/
) mice and heterozygous eNOS(+/
) mice
were generated through interbreeding of heterozygous eNOS(+/
) mice in
our laboratory. These mice were originally developed by Shesely et al.
(26). The eNOS mice were genotyped by Southern analysis of
DNA as described previously (26). All protocols were
approved by the Institutional Animal Care and Use Committee of New York
Medical College and conformed to the current National Institutes of
Health and American Physiological Society guidelines for the care and
use of laboratory animals.
Preparation of kidney tissue slices and measurement of
O2 consumption.
Heterozygous and homozygous mice were of similar weights [eNOS(+/):
26.9 ± 1.1 g; eNOS(
/
): 24.5 ± 0.6;
P > 0.05]. Heterozygous mice were used rather than
wild-type mice because of the difficulty in obtaining wild-type mice
from mating of the heterozygotes and because heterozygotes and
wild-type mice had shown similar responses in the past (19,
26). Mice were anesthetized with pentobarbital sodium (65 mg/kg
ip). The left kidneys were immediately removed, decapsulated, and
weighed. Thin slices of cortex (~1 mm, weight 15-25 mg) were
prepared. Tissues were incubated in Krebs bicarbonate solution
[containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 glucose, pH 7.4] bubbled with 21%
O2-5% CO2-74% N2 at 37°C for
2 h.
Effect of bradykinin and ramiprilat on O2
consumption.
Bradykinin or ramiprilat, at concentrations of 107 to
10
4 mol/l, was added in a cumulative
concentration-dependent manner. They were used to measure the effects
of stimulation of endogenous NO production on renal cortical
O2 uptake. The response to these drugs was also examined
after preincubation with the NOS inhibitor L-NAME
(10
3 mol/l) to determine the role of NO in the regulation
of O2 uptake. Each drug, with and without
L-NAME, was used in groups of six eNOS(+/
) and
eNOS(
/
) mice.
Effect of NO donor on O2 consumption.
SNAP at concentrations of 107 to 10
4 mol/l
was added in a cumulative concentration-dependent manner to assess the
effects of exogenous NO on renal cortical O2 uptake. The
response to SNAP was also examined after preincubation with
L-NAME (10
3 mol/l). Each condition was tested
in groups of six eNOS(+/
) and eNOS(
/
) mice.
Statistical analysis. All data are expressed as means ± SE. Statistical analysis of baseline O2 consumption was performed using Student's t-test. Changes in O2 consumption caused by drug treatment were analyzed using two-way ANOVA followed by multiple comparisons using the Tukey test (Sigma-Stat; Jandel). Statistical significance was achieved at P < 0.05.
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RESULTS |
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Basal O2 consumption.
Baseline O2 consumption in renal cortical tissue from
heterozygous eNOS(+/) mice was significantly lower than in homozygous eNOS(
/
) mice [eNOS(+/
): 919 ± 46 nmol
O2 · min
1 · g
1,
n = 6; eNOS(
/
): 1,211 ± 133 nmol
O2 · min
1 · g
1,
n = 5; P < 0.05; Fig.
1]. Inhibition of NOS with
L-NAME significantly increased O2 consumption
in tissue from the heterozygous eNOS(+/
) mice (1,061 ± 62 nmol
O2 · min
1 · g
1,
n = 6; P < 0.05) but had no
significant effect in the eNOS(
/
) mice (1,185 ± 143 nmol
O2 · min
1 · g
1,
n = 5; P = not significant; Fig. 1).
|
Effect of bradykinin and angiotensin-converting enzyme inhibitor on
tissue O2 consumption.
The effect of stimulation of endogenous NO production on renal cortical
O2 consumption was tested using bradykinin
(107 to 10
4 mol/l, n = 6)
and the angiotensin-converting enzyme (ACE) inhibitor ramiprilat
(10
7 to 10
4 mol/l, n = 6).
Cumulative doses of bradykinin caused significant concentration-dependent decreases in O2 consumption in
renal cortical tissue from both groups of mice [eNOS(+/
): from
4.2 ± 1.8 to
29.5 ± 2.5%; eNOS(
/
): from
5.6 ± 2.0 to
19.0 ± 2.8%], although the decrease was less in the
deficient mice [P < 0.05 for eNOS(+/
) vs.
eNOS(
/
) at the two highest bradykinin concentrations; Fig. 2]. The response to bradykinin was
significantly attenuated by L-NAME in tissue from
heterozygous eNOS(+/
) mice (from
1.3 ± 0.6 to
20.8 ± 2.4%; P < 0.05 vs. 10
4 and
10
5 mol/l bradykinin) but not from eNOS-deficient mice
(from
3.2 ± 1.8 to
16.0 ± 2.8%).
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Effect of NO donors on renal cortical tissue O2
consumption.
Cumulative doses of the NO-releasing agent SNAP (107 to
10
4 mol/l, n = 6) caused similar
concentration-dependent decreases in O2 consumption in
tissue from both groups of mice [eNOS(+/
): from
11.4 ± 1.8 to
59.7 ± 1.1%; eNOS(
/
): from
4.6 ± 2.1 to
60.1 ± 6.1%; Fig. 4]. The
maximum level of inhibition was not different between the groups.
L-NAME had no effect on SNAP-induced decreases in
O2 consumption (Fig. 4).
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DISCUSSION |
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NO has been shown to directly regulate renal O2 consumption in intact animals, isolated kidney tissue, and isolated renal tubules (13, 16). However, the endogenous source of NO responsible for this effect has not been explored. The results presented here demonstrate for the first time that the eNOS isoform in the kidney plays a primary role in the production of NO, which regulates renal O2 consumption in intact tissue.
Mice homozygous for disruption of the eNOS gene [eNOS(/
)] have no
detectable eNOS by immunohistochemistry, have lower body weights, and
develop hypertension (26). Heterozygotes [eNOS(+/
)] have detectable, although reduced, eNOS staining and blood pressures that are minimally, but not significantly, elevated (26).
Baseline renal cortical O2 consumption in heterozygous mice
was significantly reduced compared with eNOS-deficient mice, suggesting
a basal level of NO production in the renal cortex that is exerting an effect on O2 consumption. This is supported by the ability
of L-NAME to increase O2 consumption in
heterozygotes but not homozygous deficient mice.
Stimulators of NO production, bradykinin and ramiprilat, decreased O2 consumption significantly more in heterozygous mice possessing eNOS activity compared with homozygous deficient mice. This decrease was attenuated by L-NAME in heterozygotes, demonstrating the involvement of NOS activation, whereas in eNOS-deficient mice, L-NAME had no effect. In both heterozygous and homozygous mice, an NO donor produced similar decreases in O2 consumption, suggesting that other mechanisms of regulation of O2 consumption by NO in these animals are intact.
The results presented here differ in some respects from previous observations of regulation of O2 consumption by NO in cardiac tissue from eNOS-deficient mice (19). Bradykinin decreases cardiac tissue O2 consumption by ~20% in wild-type and heterozygous deficient mice, and L-NAME completely reverses the effect. eNOS-deficient mice have no decrease in cardiac tissue O2 consumption in response to bradykinin, suggesting that eNOS is the predominant source of NO in cardiac tissue (19).
In the kidney, bradykinin decreases O2 consumption in both heterozygous (29.5% decrease) and homozygous (19% decrease) deficient mice. The ability of bradykinin to decrease O2 consumption in the eNOS-deficient mouse kidney may be related to the expression of both nNOS and iNOS in the kidney, whereas cardiac tissue has predominantly eNOS. However, L-NAME was not able to fully inhibit the decrease in O2 consumption induced by bradykinin. This may have been due to other effects of bradykinin or ramiprilat or to a small effect mediated by another NOS isoform (i.e., iNOS or nNOS). In the latter case, the inability to detect an effect of L-NAME on the bradykinin-induced decrease in O2 consumption in eNOS-deficient mice could reflect lower sensitivity of the other isoforms to inhibition or too few data points to detect a small difference.
Modulation of O2 consumption by NO may play a role in the normal kidney and in various pathophysiological processes. NO produced by eNOS has been postulated to play a role in regulation of the normal extracellular fluid volume through vasodilatation and regulation of tubuloglomerular feedback (5). NO produced in glomeruli and other cortical microvessels or proximal tubules might be expected to alter sodium reabsorption through a direct effect on the Na+-K+-ATPase or through diminished O2 consumption, leading to less availability of ATP (13, 17). During DOCA salt-induced hypertension in rats, tubular eNOS expression is increased (3). It has been suggested that tubular NO production in these animals is adaptive, leading to inhibition of salt reabsorption (3). The mechanisms outlined above may underlie this effect through inhibition of O2 consumption by increased NO production.
Previous work from our laboratory has demonstrated the importance of eNOS-derived NO in the regulation of tissue O2 consumption in vitro (16, 19, 25, 31). Production of both NO and eNOS expression is decreased in the failing heart, concomitant with increases in O2 consumption (23, 27). Drugs that improve survival in heart failure, such as ACE inhibitors, amlodipine, and nitrates, also decrease O2 consumption in the failing human heart via stimulation of NO production, suggesting that lowering of myocardial O2 consumption may be another cardioprotective role of NO (18). In the kidney during heart failure, renal blood flow typically decreases in association with increased renal vascular resistance (8). Levels of eNOS are increased in both the cortex and medulla in rats with a model of decompensated congestive heart failure and may serve to protect the renal circulation, especially the medulla, in the face of increased endothelin (1). Increased production of NO in response to ACE inhibitors in this situation might protect the kidney both through renal vasodilatation and decreased O2 consumption. The medulla in particular, because of its lower O2 tension, seems to be more sensitive to the effects of NO on lowering O2 consumption (13).
ACE inhibitors also play an important role in renoprotection in a variety of proteinuric renal diseases (12, 20). Several mechanisms for this protective effect have been postulated, including decreased intraglomerular pressure, decreased production, and/or increased proteolysis of extracellular matrix and alterations in growth factor production (12, 20). Production of high levels of NO in glomeruli, primarily by iNOS in infiltrating mononuclear cells, has been implicated in immunological glomerular injury in animal models, although results with nonspecific inhibitors of NOS have been contradictory (10, 22). Production of low levels of NO by constitutively expressed eNOS may exert protective effects through maintaining blood flow, decreasing matrix production, and inhibiting mesangial cell proliferation (24, 28), although NO may also decrease cytokine-stimulated protease activity (9). eNOS expression is upregulated in glomeruli in human glomerulonephritis, including lesions of minimal to mild IgA nephropathy, minimal to mild lupus nephritis, and minimal change disease but tends to drop in moderate to severe proliferative lesions (11). Stimulation of production of NO by eNOS in response to ACE inhibitors, in addition to preserving glomerular blood flow, might also exert a protective effect by decreasing O2 consumption. This raises the possibility of other NO donors, or drugs that stimulate NO production, as potential therapeutic agents in these diseases as well.
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ACKNOWLEDGEMENTS |
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Ramiprilat was the kind gift of Hoescht Marion Roussel.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants PO-1 HL-43023 and HL-50142 (T. H. Hintze) and by the Westchester Artificial Kidney Center (S. Adler).
Address for reprint requests and other correspondence: S. Adler, Nephrology, 19 Bradhurst Ave., Atrium North, Suite 0100, Hawthorne, NY 10532 (E-mail: stephen{at}nymc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2000; accepted in final form 13 December 2000.
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