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Istituto di Patologia Medica, Parma, Italy.
Istituto di Semeiotica Medica, University of Parma, Parma,
Italy.
Correspondence to Dr. Alberto Montanari, Istituto di Patologia Medica, Via Gramsci 14, I-43100 Parma, Italy. Phone: +39-0-521-991395; Fax: +39-0-521-985468; E-mail: montalbr{at}unipr.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This issue also may be of importance for human hypertension, mainly in its salt-sensitive form. Essential hypertension was associated with abnormalities in systemic and renal L-Arginine—NO pathway (13,14,15,16,17,18,19,20), similar to those of animal models of salt-sensitive, NO-deficient hypertension (21,22,23), and with a postulated stimulation of intrarenal RAS (24,25). PG inhibition as a result of clinical use of nonsteroidal anti-inflammatory drugs (NSAID) also is believed to contribute to hypertension (26). Thus, simultaneous renal NO and PG deficiency in the presence of stimulated or even normal RAS might be reasonably considered among the potential mechanisms underlying salt-sensitive hypertension in humans.
The present study therefore was designed to ascertain whether PG inhibition accentuates renal vasoconstriction and Na retention as a result of acute NO deficiency. Furthermore, we investigated whether RAS blockade at the level of AT1 receptors prevents renal actions of L-NAME when potentiated by PG inhibition. For these purposes, systemic and renal changes during L-NAME infusion were studied in Na-repleted healthy human volunteers who were pretreated with placebo (PL), losartan (LOS), indomethacin (IND), or both LOS and IND.
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Materials and Methods |
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Experimental Procedure
Participants underwent in a randomized order four L-NAME infusion studies,
each preceded by 5 d of a controlled diet that provided 250 mM Na, 80 mM K,
and 1800 Kcal. The washout period between infusions was approximately 2 wk for
men and 4 wk for women, who were studied during the follicular phase of the
menstrual cycle. Infusion studies were preceded by 3 d of PL, LOS, IND, or
both LOS and IND, respectively. LOS was given at a 50-mg single dose for 3 d
before the study at 10:00 p.m. Each participant therefore received the last
dose of LOS 10 h before the study to avoid systemic and renal hemodynamic
changes as a result of acute drug administration. Such doses of LOS counteract
effectively in humans pressor effects of L-NAME
(8) and systemic and renal
vasoconstriction as a result of exogenous AngII even after 10 h from
administration (27). IND was
administered three times a day (at 6:00 a.m., 2:00 p.m., and 10:00 p.m.) for 2
d at a dose of 75 to 125 mg/d according to the body weight (1.5 to 1.6 mg/kg
per d-1), and an additional 25-mg dose was given at 6:00 a.m. on
the infusion day, 2 h before study. Thus, IND was given with an 8-h lag after
the corresponding dose of LOS. Such doses of IND were shown previously to
produce an effective PG inhibition in Na-repleted humans with a 40 to 70% drop
in urinary excretion of both prostaglandin E2 and
6-keto-prostaglandin F1
(28,29)
and a slight positivity of Na balance in the first 2 d with UNaV matching Na
intake on the third day
(29)
After an overnight fast, experiments were initiated at 8:00 a.m. with the participant in a sitting position. A plastic indwelling catheter was placed into a cubital vein, a priming dose of 3000 mg/1.73m2 body surface area of inulin (Inutest; Laevosan Gesellschaft, Linz, Austria) and 600 mg/1.73m2 body surface area of para-aminohippuric acid (PAH) was injected, and an infusion of PAH and inulin was initiated and continued throughout the entire study using a 50-ml syringe precision pump (Perfusion Secura, Braun Melsungen, Germany) to obtain plasma levels of approximately 1.5 mg/dl for PAH and 20 mg/dl for inulin. A second indwelling catheter for blood sampling was placed immediately at the contralateral arm and kept patent by pump infusion of saline solution 1.0 ml/h. After 60 min of equilibration (-45 min time), participants emptied their bladder, then a 45-min baseline clearance period was initiated. At 45 min, after voiding, a pump infusion of 3.0 µg/kg.min-1 L-NAME in saline solution was initiated. Two additional 45-min clearance periods were performed (0 to 45 min and 45 to 90 min, respectively), then the experiment was stopped. A 300-ml tap water load was administered hourly throughout the study to ensure an appropriate urine flow. BP and pulse rate were measured every 5 min using an automatic oscillometric monitoring device (TM 2421; A and D Co. Ltd, Tokyo, Japan). Samples from urine of each clearance period were taken for excretion rates of Na and NO byproducts NO2 + NO3 (UNaV and UNOxV, respectively). Samples were drawn for plasma PAH and inulin every 15 min during the entire study and for plasma Na at -45, 0, +45, and +90 min.
Calculations
A satisfactory steady state of plasma concentration of PAH and inulin was
obtained with our infusion technique
(8). Because variability in
plasma PAH and inulin measured throughout infusion was comparable to that
found in a duplicate analysis of single plasma samples (2.4% for PAH and 3.6%
for inulin), ERPF and GFR were estimated without measuring urinary PAH and
inulin using a constant-infusion technique
(30). Such a procedure was
preferred rather than standard urinary clearance because an unethical bladder
catheterization should be necessary to avoid errors in urine collection,
potentially of the same order of magnitude as that of measured changes in
renal hemodynamics
(8,31,32).
Constant infusion technique already has been shown to detect in humans rapid
changes in renal hemodynamics as a result of administration of different
substances such as nifedipine
(32), L-NAME
(8,31),
and AngII (33). PAH and inulin
concentrations were measured in the infusate, then multiplied for the volume
of infused solution per minute. The resulting infusion rate of PAH or inulin
was divided for each measured plasma concentration, thus obtaining four
clearance values in the baseline period and three in each drug period for both
ERPF and GFR
(8,32).
The mean values were used in the expression of data for each period.
Filtration fraction (FF) was calculated from GFR and ERPF, renal blood flow
was calculated from ERPF, and hematocrit and renal vascular resistances (RVR)
were calculated from MAP and renal blood flow. Clearance of sodium was
calculated with standard formula, and percent fractional excretion of Na
(%FENa) was calculated from clearance of sodium and GFR.
Study Drugs
PAH (20% solution) was purchased from J. Monico (Venice, Italy).
Commercially available LOS (LORTAAN; MSD, Rome, Italy; 50-mg tablets) and IND
(INDOXEN; Sigma-tau, Rome, Italy; 25-mg capsules) were used, and
pharmaceutical-grade L-NAME.HCL was obtained from Clinalfa (Laufelfingen,
Switzerland).
Analytical Methods
Na was measured by flame photometry. Plasma and infusate PAH and inulin and
urinary NOx were measured as described previously
(8).
Statistical Analyses
Data are expressed as mean ± SEM. Time-dependent effects of each
L-NAME infusion were analyzed by one-way ANOVA. Differences between various
infusions were analyzed by two-way ANOVA followed by post hoc
multiple comparisons with the Student-New-man-Keuls test. Differences at the
5% level or less were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Low-Rate L-NAME Infusion Produces Renal Vasoconstriction and Na
Retention before Any Change in MAP
L-NAME infusion after PL produced a modest but significant increase in MAP
(P < 0.001 versus baseline) only in the 45- to 90-min
period of infusion (P2), whereas renal hemodynamics was markedly altered by
L-NAME even at 0 to 45 min (P1) when MAP remained unchanged. In P1, GFR
declined by 4.7% (P < 0.05) and ERPF by 11% (P <
0.001). FF did not change, and RVR rose by 12.3% (P < 0.005).
These variations were more pronounced in the subsequent P2 period (GFR,
<0.02; ERPF, <0.05; RVR, <0.005 versus P1). FF did increase
significantly in P2 (P < 0.05 versus baseline). Na
excretion fell progressively during L-NAME infusion with both UNaV and FENa in
P2 even significantly lower than in P1 (P < 0.05).
UNOxV also declined progressively (-41% in P1, -59% in P2;
P < 0.001 for both).
AngII Blockade with LOS Prevents Systemic but Not Renal Effects of
L-NAME
At baseline, pretreatment with LOS did not affect renal hemodynamics,
whereas MAP was slightly lower than with either PL or IND (P <
0.05). LOS prevented almost completely any change in MAP during L-NAME
(P < 0.001), but it did not affect those in renal hemodynamics and
Na and NOx excretion, which essentially were the same as those with
PL.
PG Inhibition Potentiates Both Systemic and Renal Effects of
L-NAME
Baseline MAP, renal hemodynamics, and Na excretion were not affected by IND
pretreatment. However, when L-NAME was infused, MAP rose significantly even in
P1 (P < 0.001 versus both baseline and LOS alone). The
rise in MAP was markedly accentuated in P2 (P < 0.001
versus both PL and LOS). GFR fell more with IND than with PL or LOS
(P < 0.01 for both) in P1 and in P2 (P < 0.02 and
0.005, respectively). The same potentiation of L-NAME effects by IND was
observed for ERPF and for GFR (P < 0.02 in P1, P <
0.01 in P2 versus both PL and LOS) and RVR (P < 0.001
versus both PL and LOS in P1 and P2). Both UNaV and %FENa were
significantly more reduced in comparison with PL, LOS, or IND+LOS (P
< 0.005 in P1, P < 0.001 in P2). Changes in FF were the same as
those with both PL and LOS. Neither baseline UNOxV nor its decrease
along L-NAME infusion was modified by IND pretreatment.
Potentiation by PG Inhibition of L-NAME Effects Is Prevented by AngII
Blockade
When L-NAME was infused after pretreatment with LOS+IND, the rise in MAP
seen in P1 with IND alone was prevented (P < 0.001), with values
not different from baseline, PL, and LOS alone. Similar results were obtained
for GFR (P < 0.02 versus IND), ERPF (P <
0.02), RVR (P < 0.005), UNaV (P < 0.005), or %FENa
(P < 0.005). In P2 MAP was significantly lower than after both PL
(P < 0.001) and IND alone (P < 0.001) without
significant differences from baseline and LOS alone. Changes in GFR, ERPF,
RVR, UNaV, or %FENa were significant smaller than those observed with IND
alone (P < 0.02, <0.005, <0.001, <0.001, <0.001
respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both baseline renal hemodynamics and Na excretion on the third day of IND were the same as those after PL, in agreement with previous studies (28,29). When L-NAME was infused after IND, MAP rose significantly (+5%) even in the early infusion period; its later increase reached +10%, and both renal vasoconstriction and decline in GFR were enhanced, with FF increasing, however, to the same extent as after PL (+8%). This suggests that the obvious accentuation by IND of changes in glomerular hemodynamics took place mainly at the afferent arteriolar side. The finding of no further rise in FF also indicates that simultaneous greater increase in renal perfusion pressure did not contribute to potentiated vasoconstriction, provided that such an increase did not exceed 10% of baseline values. Actually, with much greater variations in MAP as those obtained by others in humans receiving high doses of L-NAME (35,36), a disproportionate efferent vasoconstriction could be expected with consequent additive effects on FF as shown in animal studies (1,5,6,9).
Because Na excretion was impaired further by IND in both absolute and fractional terms, potentiation of L-NAME antinatriuresis resulted from an increase in Na reabsorption rather than from the drop in GFR. Urinary excretion of NOx was not affected by IND at baseline or during L-NAME, suggesting that potentiated effects of L-NAME were not due to a different degree of NO deficiency. Our data demonstrate, therefore, that endogenous PG substantially modulate L-NAME actions in humans, including vasoconstriction in both systemic and renal vasculature and Na retention.
Baseline renal hemodynamics and Na excretion were not changed by LOS, as previously reported in Na-repleted healthy humans (37). However, LOS abolished the slight but definite rise in MAP observed from 45 to 90 min, thus confirming that systemic vasoconstriction as a result of NOS inhibition is at least attenuated by RAS blocking agents (8,21). At the renal level, however, changes in hemodynamics and Na handling were not prevented by LOS. This indicates that the unopposed action of endogenous AngII accounted for vasoconstriction in systemic circulation but not for vasoconstriction and Na retention in the kidney (8). AT1 receptor blockade also prevented the potentiation by IND of pressor action of L-NAME. This demonstrates that endogenous AngII mediates potently effects of acute NO deficiency in systemic circulation, vasodilator PG modulating only partially AngII vasoconstriction.
As the main finding of the present study, AT1 receptor blockade failed to affect renal actions of L-NAME alone, but it prevented their potentiation by PG inhibition. Because AT1 blockade was effective only when PG synthesis was inhibited, our data indicate that vasodilating PG counteract renal vaso-constricting and antinatriuretic action of endogenous AngII even when NO synthesis is reduced substantially.
Under combined AT1 blockade and PG inhibition, L-NAME affected renal hemodynamics and Na excretion to the same extent as that after PL or LOS alone. Thus, there was a substantial component of renal response to L-NAME independent of both AngII and PG and possibly related to either abrogation of vasodilatory-natriuretic action of baseline NO or unopposed effect of endogenous vasoconstrictors other than AngII, such as endothelin (32).
The present human data agree with those that show that intrarenal PG inhibition in anesthetized dogs accentuated renal vasoconstriction, elevation in MAP, and impaired volume-dependent natriuresis as a result of intrarenal NOS inhibition (10,11,12). All of these alterations were corrected substantially by captopril (12) but not by verapamil (38), suggesting that such a potentiation was AngII dependent (12). In that animal model, however, marked stimulation of sympathetic nervous system, endothelin, and RAS itself was to be expected as a result of anesthesia (5,11,39), and the dependence on AngII of effects of PG inhibition could well reflect an activation of RAS rather than interactions among NO, PG, and AngII at their baseline levels of activity. Actually, RAS activity is inhibited or at least unchanged in both short-term PG inhibition and early phases of acute NO deficiency in humans (28,29,36,40,41,42). In addition, renal vasoconstriction as a result of NG-monomethyl-L-arginine infusion may be reduced in comparison with Na-repleted state just when endogenous RAS is activated by Na restriction (42). Because any contribution of a stimulated RAS was ruled out in our human model, our findings indicate that endogenous PG and NO interact at baseline with intrarenal AngII in maintaining renal function. Their reduced availability might lead to an AngII-dependent impairment of the kidney in vasodilating and excreting Na appropriately even when intrarenal AngII is not stimulated or suppressed by high Na intake.
Our findings also may be relevant for our understanding of interactions among NO, PG, and AngII in hypertensive conditions with potentially low NO availability. Human essential hypertension was shown to be associated with low circulating levels of NOx (13), slow whole-body NO turnover (13), and impairment in NO-dependent vasodilation in both peripheral vasculature (14,15,16) and kidney (17,18,19). These alterations were much more evident in salt-sensitive elderly individuals (15,19), who also displayed abnormal responses of plasma NOx to manipulation of Na intake (20). Such abnormalities parallel closely those of some animal models of salt-sensitive hypertension typically related to NO deficiency (21,22,23). A reduced production of NO in the kidney relative to an enhanced activity of RAS was proposed as an important step in the development of salt sensitivity in hypertension (24). In addition, a selective abnormal activation of intrarenal RAS, as determined by a blunted renal sensitivity to exogenous AngII and an exaggerated renal vasodilation to captopril, was suggested to play a role in a subset of hypertensive patients (nonmodulators) (25). Our findings show, however, that NO deficiency may lead to marked AngII-dependent changes in renal function in humans under conditions of maximal suppression of RAS as a result of Na repletion, provided that PG production was impaired.
A state of reduced PG production is common in hypertensive humans (26,43), as a result of the widespread clinical use of NSAID. Such drugs were shown to increase MAP (44), principally in salt-sensitive individuals (45), to increase the probability to initiate an antihypertensive therapy (46), and to blunt the actions of some antihypertensive drugs (43,44,47). In this view, our data support the concept that impaired renal NO synthesis and depletion of PG contribute to salt-sensitive hypertension through an AT1 receptor—dependent mechanism, irrespective of the degree of intrarenal RAS activation.
Caution should be taken in extending our results obtained in acute studies of NOS inhibition in young healthy individuals to a chronic condition that generally develops later in life, such as salt-sensitive, essential hypertension. It is worth noting, however, that NO-dependent vasodilation declines with age in both systemic vasculature (15) and kidney (18), and elderly people are known to be more sensitive to the hypertensive action of NSAID (43,44,45,46,47). Taken together, such aspects suggest that interactions among NO, PG, and AngII shown in this study could become even more important under chronic conditions in elderly hypertensive individuals.
In conclusion, the present human study indicates that endogenous vasodilator PG in the presence of impaired NO synthesis plays a role in modulating the effects of AngII in both kidney and systemic circulation. At the kidney level, a preserved PG production seems to be fully protective toward both vasoconstriction and Na retention resulting from the unopposed action of endogenous AngII. The clinical relevance of these observations pertains to human hypertensive conditions associated with impaired NO availability. In such a setting, PG inhibition may act as a further, harmful hypertensive mechanism, independent of any abnormality of RAS regulation.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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