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Hypertension. 1996;27:1165-1172

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(Hypertension. 1996;27:1165-1172.)
© 1996 American Heart Association, Inc.

Articles

Effect of Salt Intake and Inhibitor Dose on Arterial Hypertension and Renal Injury Induced by Chronic Nitric Oxide Blockade

Sergio Seiji Yamada; Ana Lúcia Sassaki; Clarice Kazue Fujihara; Denise Maria Avancini Costa Malheiros; Gilberto De Nucci; Roberto Zatz

From the Renal Division, Department of Clinical Medicine, University of São Paulo (Brazil) School of Medicine, and Department of Pharmacology, State University of Campinas (Brazil) School of Medicine.

Correspondence to Roberto Zatz, MD, PhD, Laboratório de Fisiopatologia Renal, Av Dr Arnaldo, 455, 3-s/67, 01246-903 São Paulo SP, Brazil.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Long-term nitric oxide blockade by N{omega}-nitro-L-arginine methyl ester (L-NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure-natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake.


Key Words: nitric oxide • kidney • salt • pressure natriuresis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Chronic inhibition of NO synthesis promotes progressive arterial hypertension and renal parenchymal injury consisting primarily of glomerular ischemia, glomerulosclerosis, and interstitial expansion.1 2 The exact mechanisms underlying the functional and structural changes encountered in this model remain to be determined.

The pathogenetic role of salt retention in this model is particularly controversial. Several investigators have reported evidence that sodium retention contributes to the development of hypertension in this model,3 4 that administration of pressor4 and subpressor5 doses of NO inhibitors induces salt sensitivity, and that a deficient NO response may underlie spontaneously occurring salt sensitivity.6 Consistent with these findings, other studies have shown that acute NO inhibition shifts the pressure-natriuresis relationship to the right, thereby impairing renal sodium excretion capability.7 8 Similar results were obtained by chronic infusion of an NO inhibitor directly into the renal medulla.9 However, other studies of chronic NO inhibition failed to disclose any effect on BP of concomitant salt restriction10 or excess.11 12 In addition, sodium balance was found not to be affected in these rats,13 casting doubt as to whether salt retention occurs in this model. The reasons for these conflicting results are unclear. However, it must be noted that these studies have used widely varying doses of NO inhibitors. Conceivably, lower-grade NO inhibition might subtly impair renal sodium excretory capability and promote salt-dependent hypertension, whereas severe and generalized vasoconstriction14 might predominate with more complete NO inhibition. Indeed, those studies indicating salt sensitivity in this model tended to use relatively low or even subpressor doses of NO inhibitors.3 4 5 Additionally, in none of these studies was sodium intake varied between extremes, such as needed, for instance, to demonstrate salt sensitivity in Dahl rats.6

In the present study, we examined renal and systemic hemodynamics and renal histology at two different levels of NO inhibition combined with three widely differing levels of salt intake. In addition, we examined the effect of chronic NO inhibition on the pressure-natriuresis relationship. In this manner, we were able to explore in detail the interaction of NO inhibition and salt intake in this model.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
One hundred and ninety-six adult male Munich-Wistar rats, obtained from an established colony at the University of São Paulo and weighing 239 to 295 g, were used in this study. Rats received tap water ad libitum and commercially available rat chow (Harlan Teklad) containing 25% protein and sodium chloride at either 0.06% sodium (LS diet), 0.5% sodium (SS diet), or 3.2% sodium (HS diet). All experimental procedures were conducted in accordance with our institutional guidelines.

Experimental Groups
Rats were distributed among nine experimental groups. (1) Group LS0: Rats received an LS diet and no drug treatment. (2) Group LS3: Rats received an LS diet and the NO inhibitor L-NAME, 0.12 mmol/L, corresponding to a daily ingestion of approximately 3 mg/kg. In preliminary experiments, this dose was shown to promote little or no increase in BP even after several weeks of treatment. (3) Group LS25: Rats received an LS diet and 1 mmol/L L-NAME, corresponding to a daily ingestion of approximately 25 mg/kg. In preliminary experiments, this dose was shown to reproducibly promote progressive systemic hypertension. (4) Group SS0: Rats received an SS diet and no drug treatment. (5) Group SS3: Rats received an SS diet and 0.12 mmol/L L-NAME in the drinking water, corresponding to an average intake of approximately 3 mg/kg per day. (6) Group SS25: Rats received an SS diet and 1 mmol/L L-NAME in the drinking water, corresponding to an average intake of approximately 25 mg/kg per day. (7) Group HS0: Rats received an HS diet and no drug treatment. (8) Group HS3: Rats received an HS diet and 0.06 mmol/L L-NAME, corresponding to a daily ingestion of approximately 3 mg/kg. Since rats on an HS diet drank twice as much water as those on an LS diet, L-NAME concentrations in the drinking water were correspondingly adjusted to ensure that HS3 rats received the same dose of the inhibitor as SS3 and LS3 rats, namely, 3 mg/kg per day. (9) Group HS25: Rats received an HS diet and 0.5 mmol/L L-NAME, corresponding to a daily ingestion of approximately 25 mg/kg. As in the preceding group, L-NAME concentrations in the drinking water were adjusted to compensate for the higher water intake of rats on an HS diet. All salt regimens were started 1 week before L-NAME treatment was initiated.

Pressure-Natriuresis Curves
In 8 additional rats of group SS0, 11 of group SS3, 12 of group SS25, 15 of group HS0, 14 of group HS3, and 14 of group HS25, the pressure-natriuresis curve was determined at 2 weeks of treatment with a modified form of the method described by Roman and Cowley.15 Rats were anesthetized with 100 mg/kg thiobutabarbital IP and placed on a temperature-controlled surgical table. Rectal temperature was maintained at 37±0.5°C. The femoral artery was cannulated with PE-50 tubing for continuous monitoring of mean arterial pressure by a Statham P23Db pressure transducer connected to a chart recorder (model A8200, Anamed Instruments). After tracheotomy, the left jugular vein was catheterized with PE-50 tubing for infusion of homologous plasma to replace surgical losses and keep a euvolemic state.16 An amount of plasma equivalent to 1% body weight was given over about 45 minutes, followed by an infusion of 0.5 to 1.0 mL/h for the remainder of the experiment. PE-50 tubing was also placed in the right jugular vein for maintenance of a continuous saline infusion (1.5 mL/h) throughout the experiment. The right carotid artery was cannulated with PE-50 tubing for continuous BP monitoring at the late phases of the experiment (see below). The left ureter was catheterized with PE-10 tubing. About 2.5 hours after anesthesia, urine was collected during 20 minutes at baseline femoral arterial pressure for determination of flow and sodium excretion rates. Femoral arterial pressure (and hence renal perfusion pressure) was then lowered by 20 to 25 mm Hg with an inflatable cuff placed around the abdominal aorta just above the emergence of the renal arteries. After stabilization for 15 minutes, urine collection was repeated. The cuff was then deflated and the superior mesenteric and celiac arteries were ligated to raise renal perfusion pressure above baseline. Since urine flow always increased dramatically with this procedure, the saline infusion rate was raised at this time to 3 mL/h. After a new 15-minute stabilization period, a third timed urine sample was obtained. A bulldog clamp was then placed around the abdominal aorta below the renal arteries to further elevate renal perfusion pressure, now estimated by carotid pressure. After 15 minutes, a final urine sample was obtained. Urine samples were always obtained in this exact order. The experiment was discarded whenever the rat exhibited prolonged (>10 minutes) hemodynamic instability. Since renal mass reduction markedly aggravates injury in rats with chronic NO blockade,17 rats were not subjected to uninephrectomy before these experiments. Likewise, exogenous infusions of vasoactive compounds and renal denervation15 were not performed, because the pathogenesis of hypertension in this chronic model likely involves the participation of several vasoconstrictors1 18 19 20 as well as altered sympathetic output to the kidneys.21 22 23

Renal Functional Studies
For assessment of renal hemodynamics, 8 rats of groups LS0, 8 of group LS3, 8 of group LS25, 8 of group HS0, 8 of group HS3, and 8 of group HS25 were anesthetized at 10 to 15 days with thiobutabarbital and placed on a temperature-controlled surgical table for maintenance of rectal temperature at 37±0.5°C. The left femoral artery, jugular veins, trachea, and left ureter were cannulated as described above. Homologous plasma was infused through the left jugular as described above. Saline solution containing 14C-tagged inulin (2 µCi/mL) was infused through the right jugular at 1.5 mL/h in rats fed an LS diet and 3 mL/h in those given an HS diet. About 2.5 hours after anesthesia, urine was collected during 25 to 35 minutes for determination of flow rate and inulin clearance. Whole-kidney filtration fraction (FF) was determined by simultaneous collection of blood samples from the femoral artery and renal vein and measurement of the respective 14C activities in a scintillation counter (Beckman Instruments) for calculation of inulin extraction. Blood was obtained from the renal vein with a sharpened glass micropipette (about 40 µm OD). Renal plasma flow (RPF) was calculated as RPF=GFR/FF, where GFR is glomerular filtration rate. Renal total vascular resistance (RVR) was estimated by RVR=MAP(1-Ht)/RPF, where MAP is mean arterial pressure, Ht is arterial hematocrit, and RPF is renal plasma flow. At the end of the experiment, the renal tissue was perfusion-fixed at the observed BP with Dubosq-Brazil solution after a brief washout with saline.

Long-term Studies
Eight rats of each group were followed for up to 6 weeks of treatment. Mean BP, estimated by a tail-cuff method24 ; PRA, assayed in tail blood samples; and urinary albumin excretion rate were obtained at 3 and 6 weeks. At the end of 6 weeks, the rats were anesthetized with 50 mg/kg pentobarbital IP. The renal tissue was then perfusion-fixed in situ with Dubosq-Brazil solution after a brief washout with saline and examined by light microscopy for evaluation of glomerular and interstitial injury.

Analytic Methods
Urinary albumin excretion rate was determined by a radial immunodiffusion technique.25 PRA was measured by an enzymatic technique26 adapted for small samples. Urinary sodium concentration was determined by flame photometry.

Histological Techniques
After perfusion fixation, two midcoronal slices of the right kidney were embedded in paraffin, and 2- to 3-µm-thick sections were stained by the PAS reaction for examination by light microscopy. Additional sections were stained with Masson's trichrome for measurement of fractional interstitial area. Tissue was examined in a blinded fashion by light microscopy at x160 magnification. At least 300 glomeruli were examined for each rat. Glomeruli exhibiting global collapse or sclerosis were identified according to criteria described previously27 and detailed in "Results." The frequency of collapsed glomeruli was expressed as a percentage of the total number of glomeruli examined. For assessment of the extent of interstitial expansion, the fraction of renal cortex occupied by interstitial tissue staining positively for collagen was quantitatively evaluated in Masson-stained sections by a point-counting technique28 in 25 consecutive microscopic fields examined at a final magnification of x400 under a 110-point ocular grid.

Statistics
Differences among groups were compared by two-way ANOVA with pairwise Bonferroni comparisons.29 Pressure-natriuresis lines were obtained by least-squares regression analysis. Significance of the differences between curves was assessed by comparison of linear regression slopes and intercepts.30 A value of P<.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
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Pressure-natriuresis and pressure-diuresis lines obtained in groups SS0, SS3, and SS25 are shown in Fig 1Down. In all groups, urine flow and sodium excretion rate were best described as linear functions of renal perfusion pressure. Fitting to nonlinear functions such as two-phase exponential growth was also obtained, although the r values were lower and the differences between groups less apparent than with linear regression. In rats treated with low-dose L-NAME (group SS3), the slope of these regression lines was significantly lower than in untreated rats (group SS0). In rats receiving high-dose L-NAME (group SS25), the pressure-natriuresis and pressure-diuresis lines were both shifted to the right compared with SS0. However, their slopes were not significantly different compared with untreated rats. In rats receiving HS diet, the pressure-natriuresis and pressure-diuresis lines exhibited exactly the same behavior as in rats given an SS diet (data not shown).



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  		Figure 1. Pressure-natriuresis (left) and pressure-diuresis (right) lines at 2 weeks of L-NAME treatment in rats receiving SS (0.5% NaCl, wt/wt) diet. UNaV indicates urinary excretion of sodium; V, urine volume; and RPP, renal perfusion pressure.

Renal and systemic hemodynamic parameters measured at 10 to 15 days of treatment in LS and HS groups are shown in the TableDown. Body growth was similar among groups. Left kidney weight was not significantly altered by either L-NAME treatment or dietary salt intake, except for group HS3, which had slightly larger kidneys. Mean arterial pressure was not significantly affected by dietary sodium in the absence of L-NAME treatment. However, sodium intake was decisive for the establishment of arterial hypertension in rats receiving low-dose L-NAME. LS diet significantly attenuated arterial hypertension in rats receiving high-dose L-NAME, although it failed to normalize BP in these rats. Low-dose L-NAME (groups LS3 and HS3) did not significantly affect glomerular filtration rate. However, glomerular filtration rate was moderately decreased in groups LS25 and HS25, although only in the latter did the differences with the respective control achieve statistical significance. Filtration fraction was similar among groups, except for groups LS25 and HS25. Accordingly, renal plasma flow was lowered in these two groups compared with their respective controls. Renal vascular resistance was insensitive to salt intake in untreated rats. Low-dose L-NAME treatment increased renal vascular resistance by 41% in rats receiving an LS diet and by 48% in those given an HS diet. Vasoconstriction was more severe in rats receiving high-dose L-NAME.


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  		Table 1. Systemic and Renal Hemodynamic Parameters at 2 Weeks of L-NAME Treatment

Long-term treatment with L-NAME was associated with normal body growth in all groups except group HS25, in which body weight (262±9 g) was low compared with rats receiving either low-dose L-NAME and salt overload (HS3, 307±11 g) or high-dose L-NAME and salt restriction (LS25, 322±5 g). No limitation to body growth was seen in the other groups. The evolution of tail-cuff pressure during 6 weeks of treatment is shown in Fig 2Down. Tail-cuff pressure increased steadily in group HS3 compared with untreated controls, reaching 152±8 mm Hg after 6 weeks of treatment, compared with 124±4 mm Hg in group HS0 (P<.05). Salt restriction prevented BP elevation in group LS3, characterizing the development of hypertension at this L-NAME dose as salt dependent. Rats receiving high-dose L-NAME and HS intake (group HS25) developed more severe and rapidly progressive hypertension, reaching 170±10 mm Hg after 6 weeks of treatment. Since the mortality rate in this group was 20%, as opposed to 0% in the remaining groups, and since it is likely that the BPs of the rats that died would have been even higher at 6 weeks, the tail-cuff pressure measured at the end of the study may have underestimated the severity of the hemodynamic derangement occurring in this group. Salt restriction attenuated but did not prevent hypertension in group LS25 (145±4 versus 118±1 mm Hg in group LS0, P<.05).



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  		Figure 2. Tail-cuff pressure as a function of time in rats receiving no treatment, low-dose L-NAME (3 mg/kg per day), or high-dose L-NAME (25 mg/kg per day) and varying amounts of dietary sodium.

As expected, plasma renin levels were persistently higher in rats receiving an LS diet than in those given salt excess (Fig 3Down). Low-dose L-NAME treatment had little effect on PRA in rats receiving an HS diet but promoted a steady decline in renin levels in rats given an LS diet. High-dose L-NAME treatment depressed PRA even faster in rats receiving an LS diet. In rats receiving an HS diet, high-dose L-NAME further lowered circulating renin levels at 3 weeks but markedly increased PRA at 6 weeks of treatment.



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  		Figure 3. PRA as a function of time in rats receiving no treatment, low-dose L-NAME (3 mg/kg per day), or high-dose L-NAME (25 mg/kg per day) and either HS or LS diet. AI indicates angiotensin I.

Albumin excretion rate (Fig 4Down) was increased at 6 weeks of treatment in group HS3, reaching 30.2±18.3 mg/24 h, although this variation did not reach statistical significance compared with groups HS0 (1.3±0.3 mg/24 h) and LS3 (1.2±0.2 mg/24 h). Urinary albumin excretion rate was numerically increased in group SS25, reaching 19.9±7.0 mg/24 h, although this value did not differ statistically from those observed in groups LS25 (2.7±0.6 mg/24 h) and SS3 (1.3±0.3 mg/24 h). In group HS25, however, albuminuria rose steadily from the beginning of the study, reaching 168±22 mg/24 h at 6 weeks (P<.05 versus groups HS0, HS3, and LS25).



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  		Figure 4. Urinary albumin excretion rate as a function of time in rats receiving no treatment, low-dose L-NAME (3 mg/kg per day), or high-dose L-NAME (25 mg/kg per day) and varying amounts of dietary sodium.

Fig 5Down shows the three most frequently occurring modalities of renal injury encountered in L-NAME–treated rats.27 Collapsed glomeruli (Fig 5Down, top) appeared reduced in size, with prominent thickening of the basement membrane and extreme narrowing of capillary loops, without adhesions to Bowman's capsule. Glomerulosclerosis (Fig 5Down, middle) was defined as tuft deposition of PAS-positive material with loop occlusion and/or adhesions to Bowman's capsule. Interstitial expansion (Fig 5Down, bottom) consisted of focal enlargement of interstitial tissue, with infiltration by fibroblasts and deposition of collagen-like material, frequently associated with tubular atrophy and vacuolization. Confluence of these areas was seen in more severely affected rats. Fig 6Down, left, depicts the frequency of glomerular collapse in the groups studied. In group SS25, 15.9±4.3% of glomeruli presented with tuft collapse (P<.05 versus SS0 and SS3), whereas sodium excess increased the frequency of these glomeruli in group HS25 (28.5±3.4%, P<.05 versus groups HS0 and HS3). Sodium restriction attenuated glomerular collapse in group LS25 (7.0±1.4%, P<.05 versus HS25 and P>.1 versus LS0). Parallel results were obtained regarding glomerulosclerosis (Fig 6Down, middle) and interstitial expansion (Fig 6Down, right). Focal and segmental necrosis of the glomerular tuft appeared in nearly 1.2% of glomeruli examined in group HS25 but was virtually absent in the other groups. Arteriolar wall thickening was observed in group HS3 and, more often, in group HS25. Fibrinoid necrosis of the arteriolar wall was seen only in group HS25.



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  		Figure 5. Top, Global collapse of the glomerulus, with widespread occlusion of capillary loops and thickening of basement membrane. PAS in 3-µm-thick section, magnification x160. Middle, Segmental sclerosis of the glomerular tuft, consisting of deposition of PAS-positive material with occlusion of capillary loops and adhesion to Bowman's capsule. PAS in 3-µm-thick section, magnification x160. Bottom, Focal expansion of the interstitial area. Fibroblast nuclei, matrix expansion, and tubular atrophy are readily visible. Masson's trichrome in 3-µm-thick section, magnification x100.



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  		Figure 6. Bar graphs show frequency of structural abnormalities in rats receiving L-NAME treatment for 6 weeks. Open bars represent LS diet; shaded bars, SS diet; and solid bars, HS diet. Left, collapsed glomeruli; middle, glomerulosclerosis; and right, relative interstitial area. The following comparisons were statistically significant: left, glomerular collapse: HS25 vs HS0, SS25 vs SS0, HS25 vs SS25, HS25 vs LS25, SS25 vs LS25, HS25 vs HS3, and SS25 vs SS3; middle, glomerulosclerosis: HS25 vs HS0, HS25 vs SS25, HS25 vs LS25, and HS25 vs HS3; right, interstitial area: HS25 vs HS0, HS25 vs SS25, HS25 vs LS25, and HS25 vs HS3.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present observations indicate a rather complex pathogenetic interaction between salt intake and chronic NO inhibition. At low L-NAME doses, the development of hypertension depended entirely on concomitant dietary salt overload, an observation in agreement with those made by Salazar and coworkers,3 who showed induction of salt-dependent hypertension in dogs by chronic infusion of subpressor doses of L-NAME, and by Tolins and Shultz,4 who showed marked aggravation of hypertension induced by low-dose L-NAME in rats also receiving dietary salt excess. In addition, these results are consistent with those reported by Chen and Sanders,6 who suggested that spontaneous NO deficiency may underlie the salt-dependent hypertension observed in Dahl rats. Collectively, these studies support the view that low-grade NO inhibition subtly impairs the renal ability to excrete sodium, predisposing the animals to the development of hypertension on salt overload. This effect was predicted by the pressure-natriuresis and pressure-diuresis relationships observed in the present study. Regardless of salt intake, the slopes of the regression lines were decreased compared with untreated rats, indicating that (1) relatively small reductions in salt intake would be expected to promote substantial BP lowering and (2) salt overload is necessary for hypertension to become manifest. Previous studies showed that the pressure-natriuresis relationship is blunted in rats and dogs acutely treated with NO inhibitors.7 8 The present observations indicate that this functional abnormality can persist for at least 2 weeks of NO inhibition and contribute to the maintenance of hypertension in salt-loaded animals.

Salt sensitivity was less pronounced at higher L-NAME doses. Although massive salt overload did elevate final BP by 25 mm Hg compared with rats receiving an SS diet, reduction of salt intake to very low levels had little effect on BP. These results are in keeping with those reported previously in rats treated with relatively high doses of L-NAME10 11 12 and characterize a relatively salt-insensitive hypertension in this setting. Again, these effects of salt intake were predicted by the pressure-natriuresis/diuresis relationship obtained in this group. As in rats treated with low-dose L-NAME, the regression lines were shifted to the right, indicating that higher BPs were required for excretion of a given amount of sodium and water. However, the slopes of these lines were indistinguishable from those of control, indicating a resetting of the pressure-natriuresis relationship and predicting (1) no more salt sensitivity in these rats than in untreated controls and (2) little BP effect of salt deprivation compared with SS intake, as effectively observed in this group.

Although the analysis of the pressure-natriuresis/diuresis lines helps the understanding in phenomenological terms of the complex interplay between salt intake and different levels of NO inhibition in the generation of hypertension, the mechanistic aspects involved in this setting are unclear. Administration of low-dose L-NAME in the present study promoted a 40% increase in renal vascular resistance, whereas higher-grade NO inhibition increased renal vascular resistance by more than twofold. Although these hemodynamic effects may contribute to the discordant responses of the pressure-natriuresis relationship to differing doses of L-NAME, they may also reflect simple autoregulation. On the other hand, previous studies showed that selective NO production at the inner medulla in response to variations in renal perfusion pressure may strongly influence sodium excretion by modifying local blood flow9 31 32 and/or by directly influencing tubular sodium transport.33 Further investigation is necessary for determination of whether L-NAME has a dose-dependent effect on these mechanisms, thus explaining the differing functional effects of high and low doses of the inhibitor. Additional limitation to renal sodium excretion may have originated from an enhanced renal sympathetic influx,21 22 23 as well as from activation of the renin-angiotensin system.10 19

Of particular interest was the behavior of plasma renin levels in these experiments. As expected, PRA was depressed by sodium excess and augmented by sodium restriction in rats not receiving L-NAME, compared with levels previously observed in rats receiving an SS diet.1 27 34 Low doses of L-NAME promoted a progressive decline in PRA in both salt-depleted and salt-loaded rats, whereas the initial depression of renin levels was even faster with high L-NAME doses, suggesting that whatever the level of salt repletion, renin production at least partially depends on NO availability. These results are consistent with several recent studies from this laboratory and elsewhere in which chronic NO inhibition, at least in the initial weeks of treatment, was reported to lower circulating renin levels.1 10 11 27 34 They are also in accordance with the observed depression of renin levels by acute L-NAME treatment in vivo35 36 and in isolated perfused rat kidneys37 38 and by the observation that endothelium-derived NO enhances renin secretion in cultivated juxtaglomerular cells.39 Later in the course of treatment, renin levels were markedly and paradoxically elevated in rats receiving high-dose L-NAME and dietary sodium overload, confirming previous observations of this laboratory.1 27 In these rats, hyperreninemia is unlikely to reflect a primary effect of NO on renin production. Rather, enhanced renin production appears to result from the profound structural derangement of the renal parenchyma, especially the extensive glomerular ischemia associated with this model. Hyperreninemia is likely to have contributed to aggravation of hypertension in this group at the late stages of the study. However, the initiation and maintenance of hypertension at the initial phase of this process appear to bear no direct relationship to the circulating renin levels, which were elevated in the face of normal BPs in LS rats receiving low-dose L-NAME and depressed in HS rats given the same dose of the inhibitor that nevertheless were always hypertensive. Since administration of renin-angiotensin antagonists has been shown to largely prevent hypertension in this model,1 27 40 the beneficial effects of these drugs must depend on some aspect of the renin-angiotensin system not directly related to circulating renin levels.

The renal parenchymal injury observed at 6 weeks in L-NAME–treated rats not subjected to salt restriction may have contributed additionally to the development of hypertension. As described previously,1 27 the most frequent structural abnormality encountered in these rats was a global collapse of the glomerular tuft, consistent with severe glomerular ischemia. Correa-Rotter and coworkers41 reported evidence that these glomeruli, also found in the renal ablation model,41 42 may release excessive amounts of renin, which in turn might promote the development of both systemic hypertension43 and renal structural injury.42 Exacerbation of this abnormality by salt overload coincided with aggravation of systemic hypertension, especially at later stages, suggesting a pathogenetic role for inadequately "high" circulating and/or local renin levels. Sodium restriction prevented both hypertension and glomerular collapse, further suggesting a pathogenetic association between these two phenomena. The mechanisms whereby salt intake levels might influence the development of glomerular collapse are unclear. NO inhibition and sodium overload might interact to promote severe afferent constriction, an abnormality shown in this and previous studies27 to associate with glomerular collapse. Sodium overload has been shown to increase cytosolic free calcium concentrations in erythrocytes.44 Although direct experimental evidence in this regard is lacking, excess sodium might influence smooth muscle cells similarly, thus favoring vasoconstriction. Conversely, sodium restriction might promote smooth muscle relaxation by lowering cytosolic calcium concentration, thus exerting a protective effect akin to that previously obtained in this model with chronic nifedipine treatment.45 Alternatively, glomerular collapse may have resulted directly from systemic hypertension. Although this hypothesis would also explain the observed prevention of glomerular collapse by salt restriction, it must be noted that hypertension as severe as that observed in this study is not associated with glomerular collapse in models such as the spontaneously hypertensive rat,46 renal ablation,47 or the Milan hypertensive rat.48

The present study confirms previous observations of this laboratory that renal interstitial fibrosis is a prominent feature of the structural derangement observed in rats receiving chronic L-NAME treatment and that this process is worsened by simultaneous sodium overload.27 In addition, sodium restriction largely prevented renal interstitial expansion in L-NAME–treated rats, regardless of the dose used. Given the antimitogenic properties of NO,49 50 51 renal interstitial enlargement in this model may be related at least in part to abnormal cell proliferation. Sodium overload has been shown to elevate cytosolic calcium concentration,44 whereas the latter has been consistently linked to activation of mitogenesis.52 53 54 Thus, it is possible that in the chronic NO blockade model, sodium overload aggravates interstitial expansion by enhancing cell proliferation, whereas sodium restriction has the opposite effect. In the absence of direct experimental evidence, however, this possibility must remain speculative.

In summary, the present observations indicate that chronic NO inhibition promotes salt-dependent hypertension. However, salt dependence can be demonstrated only at a relatively low dose of the inhibitor. Hypertension resulting from higher doses of the inhibitor is aggravated by dietary salt overload but cannot be prevented by salt restriction. Whatever the dose of NO inhibitor used, sodium restriction largely prevents the renal parenchymal injury associated with this model. Elucidation of the intimate mechanisms dictating the pathogenetic interaction between chronic NO inhibition and salt intake levels awaits further investigation.


*    Selected Abbreviations and Acronyms
 
BP = blood pressure
HS = high salt
L-NAME = N{omega}-nitro-L-arginine methyl ester
LS = low salt
NO = nitric oxide
PAS = periodic acid–Schiff
PRA = plasma renin activity
SS = standard salt


*    Acknowledgments
 
This work was supported by grant No. 92/3496-9 from the São Paulo Foundation for Research Support (FAPESP). During these studies Dr Zatz was the recipient of a Research Award (No. 326.429/81) from the Brazilian Council of Scientific and Technologic Development (CNPq). We are thankful to Cláudia Ramos de Sena and Marinete Miristeni dos Santos for expert technical assistance.


*    Footnotes
 
Parts of this study were presented at the 28th Congress of the American Society of Nephrology, San Diego, Calif, November 5-8, 1995, and published in abstract form (J Am Soc Nephrol. 1995;6:635).

Received November 20, 1995; first decision December 18, 1995; accepted January 22, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Ribeiro MO, Antunes E, De-Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension. Hypertension. 1992;20:298-303. [Abstract/Free Full Text]
  2. Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest. 1992;90:278-281.
  3. Salazar FJ, Alberola A, Pinilla JM, Romero JC, Quesada T. Salt-induced increase in arterial pressure during nitric oxide synthesis inhibition. Hypertension. 1993;22:49-55. [Abstract/Free Full Text]
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