Pregnancy-Induced Hypertension in Rats With Adriamycin Nephropathy Is Associated With an Inadequate Production of Nitric Oxide
Abstract Hypertensive complications are relatively common in pregnancy, particularly in the presence of preexisting renal disease. Although the pathogenesis of such complications is still unknown, recent animal studies have suggested that it may be related to impaired synthesis of nitric oxide (NO). Rats with adriamycin nephropathy develop a “preeclamptic-type” pregnant state characterized by elevated blood pressure, lack of hyperfiltration, and enhanced proteinuria. Preliminary studies with this model have implicated inadequate NO synthesis in the development of preeclamptic-like pregnancy. The aim of the present study was to confirm this hypothesis. Pregnant rats, both normal (PREG) and those with adriamycin nephropathy (AN-PREG), received 100 mg/L Nω-nitro-l-arginine methyl ester PO from the middle of gestation to term (day 11, term approximately 22 days). One group of AN-PREG rats received either l-arginine or d-arginine (2 g/L) from midpregnancy. At term, systolic pressure, mean arterial pressure, urinary metabolites of NO, creatinine clearance, and urinary protein were assessed. At term, compared with virgin rats with adriamycin nephropathy, untreated AN-PREG rats had increased systolic pressure, mean arterial pressure, and proteinuria (mean arterial pressure, 124±2.5 versus 99.7±1.6 mm Hg [P<.05]; proteinuria, 434±58 versus 216±63 mg/d [P<.05]). Creatinine clearance did not change (1.68±0.23 versus 1.35±0.09 mL/min, P=NS). In PREG rats, urinary metabolites of NO increased approximately threefold at term pregnancy compared with control. By contrast, in AN-PREG rats, excretion of urinary metabolites of NO increased only by approximately 1.7-fold (P<.01) versus PREG rats. With the exception of AN-PREG rats, inhibition of NO synthesis with Nω-nitro-l-arginine methyl ester enhanced blood pressure and decreased creatinine clearance but did not influence proteinuria. Excretion of urinary metabolites of NO was similarly inhibited in all rats. In AN-PREG rats, l-arginine normalized blood pressure (91±2.15 mm Hg) and lowered proteinuria partially but significantly. d-Arginine had no effect. In summary, AN-PREG rats are unable to adequately increase NO synthesis when physiologically required. Correction of this deficit by l-arginine treatment induced a significant clinical improvement.
Development of hypertension in pregnancy occurs in about 4% to 8% of nulliparas. Preexisting renal injury increases the possibility of the development of such a complication.1 The pathophysiology is still incompletely understood. Experimental studies in rats and primates have shown that inhibition of NO synthesis during normal pregnancy induces a clinical picture similar to that of preeclampsia.2 3 NO was therefore suggested to contribute to the development of hypertension in pregnancy. However, further studies trying to precisely identify the part of NO in this setting have been hampered by the lack of an animal model of spontaneous preeclampsia. During the past few years, we have shown that in rats with underlying renal disease due to adriamycin nephropathy, pregnancy is complicated by the development of hypertension and increased UP excretion, which are clinical features similar to those of superimposed preeclampsia due to chronic renal disease.4 Administration of l-arginine improved the clinical picture, suggesting the existence of a deficit in NO synthesis.5 The aim of the present study was to further evaluate this possibility.
Age-matched (±3 days) female Wistar rats weighing 201 to 246 g (mean, 221 g) were fed a normal diet (0.35 g NaCl, 20 g protein, and 1.17 g arginine per 100 g food) and allowed tap water ad libitum. In some of the rats, adriamycin (Adriablastina, Abic) was injected at 3.5 mg/kg IV through a superficial femoral vein with rats under light ether anesthesia. Two weeks later, some of the rats were mated with a fertile male for 4 days. Day 1 of gestation was documented by the presence of spermatozoa in the vaginal smear.
Two experimental protocols were used. In the first, we assessed the effect of acute administration of L-NAME (20 μg/kg per minute) on MAP in five virgin and five pregnant rats with adriamycin nephropathy. In the second protocol, we studied the chronic effect of L-NAME (100 mg/L administered via the drinking water, changed every other day) from midpregnancy (day 11) to term in pregnant rats or for a comparable period in nonpregnant rats. Rats were divided into the following groups: group 1, control virgin rats: 1A, untreated (C, n=6) and 1B, treated with L-NAME (C-NAME, n=6); group 2, normal pregnant rats: 2A, untreated (PREG, n=9) and 2B, treated with L-NAME (PREG-NAME, n=6); group 3, virgin rats with adriamycin nephropathy: 3A, untreated (AN-V, n=7) and 3B, treated with L-NAME (AN-V-NAME, n=6); and group 4, pregnant rats with adriamycin nephropathy: 4A, untreated (AN-PREG, n=8) and 4B, treated with L-NAME (AN-PREG-NAME, n=10).
In other groups (n=5 each) of AN-PREG rats, we also evaluated the effect of 2 g/L l-arginine and d-arginine from midpregnancy on SBP and MAP.
SBP, 24-hour UP, and UNOx were measured before mating and treatment (midpregnancy) and at the end of pregnancy. Gentamycin (6 mg per tube) was added to each test tube of collected urine to avoid bacterial contamination. On day 22, the usual day of delivery, blood samples were obtained for measurements of sodium, albumin, and creatinine. Serum l-arginine was measured in all untreated rats of each group.
Twenty-four-hour urine collections were obtained with the use of individual metabolic cages. SBP was measured in awake rats by tail-cuff manometry with an automated sphygmomanometer (Narco Biosystems). So that rats rested quietly during blood pressure measurements in the Plexiglas restraining cages, they were placed in the cages on at least two occasions before each measurement. SBP was measured eight times in each rat. Of the eight recordings, the first three were discarded, and the mean of the last five was taken as the result. For MAP determinations, the rats were briefly anesthetized with ether, and the tail artery was cannulated as previously described.4 The entire procedure was usually performed within 4 to 5 minutes, and rats were fully awake 3 to 4 minutes after the cessation of ether anesthesia. The rats were then placed in restraining cages for 3 hours in a quiet environment before any measurement. MAP was monitored continuously with a P231D Gould transducer and recorded with an MG Electronic recorder (model B2599).
Serum creatinine and albumin were measured by standard methods. Sodium was measured by flame photometry. Serum arginine was measured by the kinetic method described by Konings5 with slight modifications. Creatinine clearance was used as a marker of GFR. Previous studies have shown good correlation between inulin and creatinine clearances in both normal rats and rats with adriamycin nephropathy (References 44 -7 and unpublished observations, 1995). UP was measured by the sulfosalicylic acid method.6 UNOx was determined by an enzymatic, end point method with the use of nitrate reductase from Aspergillus sp. The decrease of absorbance at 340 nm as a result of the oxidation of β-NADPH was recorded. Flavin adenine dinucleotide was used as a supplementary electron carrier and added as an internal standard to avoid interference from possible inhibitors of the enzymatic reaction.7 9 All measurements were done with an automatic spectrophotometer.
Results are expressed as mean±SEM. Differences between groups were assessed by one-way ANOVA and multiple comparisons using the method of protected least significant differences and the Tukey test. Nonparametric tests and the t test for paired groups were used when appropriate.
Adriamycin administration was well tolerated by all the rats. The arginine content in the food was 11.7 mg/g. Considering their daily food consumption, the daily intake of l-arginine in virgin rats ranged between 0.58 and 0.85 g/kg, with no difference between normal rats or those with adriamycin nephropathy. Food intake increased by 20% to 30% during pregnancy. Accordingly, in pregnant rats, the daily intake of l-arginine increased, but as the rats’ weight also increased, the arginine intake in grams per kilogram of body weight varied only slightly, being 0.62 to 0.72 at day 1 to 2 of pregnancy and 0.70 to 0.80 at term. Arginine intake did not differ between PREG and AN-PREG rats.
The mean daily intake of L-NAME at the end of the study was 9.44±0.31 mg/kg in PREG rats versus 5.6±0.21 in control rats (P<.05) and 10.9±0.72 mg/kg in AN-PREG versus 6.90±0.56 in AN-V rats (P<.05). In AN-PREG rats, the l-arginine or d-arginine supply with water intake during the last 12 days of pregnancy ranged between 0.21 and 0.25 g/kg body wt.
The mean percentage increase in body weight during pregnancy was lower in rats with adriamycin nephropathy compared with normal rats (Table 1⇓). L-NAME treatment was associated with a lower increase in body weight only in PREG rats. The number of fetuses was diminished in L-NAME–treated rats (7±0.8 versus 13±0.3 in PREG rats, P<.01), whereas in AN-PREG rats, L-NAME had no effect (12±1.3 and 12±0.66 fetuses in AN-PREG and AN-PREG-NAME rats, respectively). No macroscopic fetal abnormality was found in rats treated with L-NAME.
Serum albumin levels were similar in PREG and AN-PREG rats, despite the heavy proteinuria of AN-PREG rats. Serum l-arginine levels were similar in normal and adriamycin-treated rats, whether or not they were pregnant (Table 2⇓).
Table 3⇓ shows SBP in the rat groups. Control, PREG, and AN-V rats remained normotensive throughout the study. L-NAME treatment markedly increased SBP in all rat groups.
In AN-PREG rats, SBP increased from 109±2.87 mm Hg at midpregnancy to 122±5.5 at the end of pregnancy (P<.01). These values were also significantly higher than in AN-V rats (115±3 mm Hg, P<.05). l-Arginine treatment prevented this increase in blood pressure, whereas neither L-NAME nor d-arginine had any effect on blood pressure.
Mean Arterial Pressure (Fig 1)
First protocol. In rats with adriamycin nephropathy, acute inhibition of NO synthesis with L-NAME enhanced MAP in both AN-V and AN-PREG rats. The percentage increase tended to be lower in AN-PREG rats (at 30 minutes, 6.5±1.3% versus 11.4±0.2%, P<.06; at 60 minutes, 14.4±2.9% versus 24±3.8%, P<.07, respectively).
Second protocol. In PREG rats, MAP was lower compared with control. Chronic L-NAME treatment increased MAP significantly in both control and PREG rats (control, 143±2 mm Hg; PREG, 154.5±4.4 versus 89.8±1.02 mm Hg in untreated PREG, P<.01).
In AN-V rats, L-NAME increased MAP in a manner similar to that in control rats.
In untreated AN-PREG rats, MAP was 124.25 mm Hg versus 99.7±1.6 in AN-V rats (P<.05). Treatment with either L-NAME or d-arginine had no effect on blood pressure. By contrast, l-arginine decreased MAP to 91±2.15 mm Hg (P<.01 versus untreated AN-PREG rats).
In PREG rats, creatinine clearance was 1.86±0.11 mL/min versus 1.4±0.14 in control rats (P<.05). L-NAME decreased creatinine clearance to 1.2±0.8 mL/min versus untreated PREG rats (P<.05). In AN-PREG rats, creatinine clearance was 1.68±0.23 versus 1.35±0.09 mL/min in AN-V rats (P=NS). L-NAME had no significant effect on creatinine clearance (1.58±1.1 mL/min, P=NS) versus untreated AN-PREG rats. A significant direct correlation was found between creatinine clearance and UNOx excretion (r=.77, P<.01) (Fig 2⇓).
Urinary Protein Excretion
Before pregnancy, UP was mild and equal in AN-V and AN-PREG rats. Until midpregnancy, daily UP remained similar. Later, in pregnant rats, UP increased significantly from 155±21 to 434±58 mg/d (P<.01 versus midpregnancy and versus AN-V), whereas in virgin rats at the same time period, UP increased from 134±42 to 216±63 mg/d (P=NS). L-NAME and d-arginine had no effect on UP, whereas l-arginine partially lowered it (Table 1⇑, Fig 3⇓). In AN-PREG-NAME rats, addition of l-arginine had no effect on UP.
Urinary Nitrate Excretion
UNOx excretion remained constant in control rats. UNOx may vary with diet, and as food intake was augmented by 20% to 30% during pregnancy, we assumed that any increase in UNOx above 20% to 30% represents increased NO synthesis.
In pregnant rats, a progressive increase in UNOx was observed during pregnancy, until at term it was 3-fold that of controls (Fig 4⇓). AN-V rats had a UNOx excretion rate similar to that found in control rats. In AN-PREG rats at the end of pregnancy, a 1.7-fold increase was observed compared with AN-V rats. The increase in UNOx in AN-PREG rats was significantly lower than that present in PREG rats (P<.01). NO blockade by L-NAME, as assessed by reduced 24-hour UNOx, was equally effective in all groups (Fig 5⇓).
The improvement observed with l-arginine treatment led us to measure in a new experiment the UNOx excretion in another four groups of AN-PREG rats (n=5 in each group): (1) untreated, (2) treated with l-arginine (2 g/L), (3) treated with L-NAME (100 mg/L), and (4) treated with L-NAME plus l-arginine. The drugs were given from midpregnancy. At the end of pregnancy, UNOx excretion was 32±3.8 μmol/d in AN-PREG rats and 42±1.98 in AN-PREG+l-arginine rats (P<.05). In AN-PREG-NAME rats, UNOx decreased to 3.38±1.1 μmol/d and increased to 7.55±2.1 (P=NS) when NAME and l-arginine were given simultaneously.
The hemodynamics of gestation are characterized by a high cardiac output, low-resistance state associated with a blunted response to angiotensin II.10 Recent studies have suggested a key role for NO in the homeostasis of this physiological condition.10 In both humans and animals,11 12 NO synthesis has been shown to be increased during pregnancy. Blockade of NO synthesis resulted in the elevation of blood pressure and onset of proteinuria, features characteristic of preeclampsia. The usual increase of GFR during gestation was absent11 ; in fact, GFR has even been reported to decrease (unpublished data, 1996). These findings implied the loss of the vasodilator effect of pregnancy, which is probably in large part mediated by NO. Our results in normal rats confirm these data. The decrease in blood pressure and increase in creatinine clearance in PREG rats were associated with a threefold increase in NO production. L-NAME treatment induced hypertension and a reduction of creatinine clearance that were similar in control and pregnant rats. In contrast to other reports, no increase in proteinuria was observed upon L-NAME administration. However, whereas these other studies used L-NAME for 14 to 17 days, in our study protocol, the drug was given only during the last 10 to 11 days of pregnancy. Therefore, it is possible that the duration of treatment with L-NAME may be an important factor in the development of proteinuria. The above changes in blood pressure and creatinine clearance in PREG rats with L-NAME were highly correlated to inhibition of NO synthesis, as reflected by the reduced UNOx excretion.
These results raise the question of whether an impaired NO generation might be involved in the pathogenesis of pregnancy-induced hypertension. Several researchers have assessed changes in UNOx excretion in normal and preeclamptic human pregnancies. The results are controversial.12 13 Human urine samples sometimes contain interfering factors, such as vitamin C, that disturb the technical measurement of UNOx.14 Furthermore, UNOx may vary as much as 56%15 according to the diet. In the majority of human studies, diets were not controlled. In an in vitro study of the vascular response of maternal vessels to acetylcholine or an NO donor, McCarthy et al16 found no differences between normal and preeclamptic pregnancies. In contrast, a case report documented a beneficial effect of an NO donor in the treatment of HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count).17
Evaluation of the role of NO in the development of hypertension in pregnancy has been problematic, principally because of the lack of a suitable animal model. In fact, in most of the experimental models studied thus far, whether in hypertensive models or those of animals with underlying renal disease, blood pressure during pregnancy either did not change or decreased. GFR was shown to increase in a manner similar to that of normal pregnancies. An exception in this regard is the decrease in single-nephron GFR in late pregnancy recorded by Baylis et al18 in rats with membranous nephropathy. However, blood pressure in their study was unchanged. We have previously shown that pregnant rats with early adriamycin nephropathy develop elevated blood pressure at term associated with enhanced proteinuria and an absence of hyperfiltration.4 In the present study, with this model, NO production was seen to increase during pregnancy but was significantly less than that found in normal pregnant rats. These data suggest an impaired ability to adequately increase NO synthesis to meet physiological requirements. In contrast to control and AN-V rats, in which NO blockade was seen to aggravate blood pressure, no such effect was demonstrated in AN-PREG rats. However, stimulatory NO synthesis in late pregnancy by l-arginine administration lowered blood pressure, whereas untreated AN-PREG or AN-PREG-NAME rats with mild or lower UNOx values remained mildly hypertensive. It is therefore possible that although high levels of NO are necessary for the maintenance of lower blood pressure values during pregnancy, beyond a certain threshold level the hemodynamic influences of NO are minor. Supportive of our findings is the recent work of Deng et al (unpublished data, 1996), in which blood pressure was significantly increased in L-NAME–treated pregnant rats despite the fact that their NO synthesis was similar to that in normotensive virgin rats.
The reason or reasons for the relatively decreased NO synthesis in AN-PREG rats are as yet unclear. Pregnancy may be associated with a low argininemia state caused by increased transfer of amino acids to the fetus.19 However, in the present study, the serum arginine levels were similar in normal and adriamycin-treated rats; thus, the decreased NO synthesis is probably not due to a lack of substrate. l-Arginine treatment increased NO synthesis, indicating an intact intracellular biochemical pathway for NO synthesis. Furthermore, in vitro studies in our laboratory have shown that even in a milieu with no exogenous arginine, small mesenteric arteries of AN-PREG rats vasodilate well in response to acetylcholine, indicating an intracellular reserve of arginine.20 Other possibilities may be an impairment in the synthesis of NO synthase isoforms observed during pregnancy21 or an impairment in the signal transmission for NO synthesis, such as a lesion in the membrane stretch receptors.
In summary, this model of chronic renal disease, characterized by increased blood pressure, enhanced proteinuria, and lack of hyperfiltration during gestation, is associated with insufficient levels of NO (as assessed by UNOx). Although seemingly high, the mild increment in NO production, compared with that in virgin rats, is probably inadequate to offset the effect of vasoconstrictor hormones, such as angiotensin II and endothelin, which are increased in pregnancy. The improvement observed with chronic oral treatment with l-arginine, similar to that observed in the “preeclampsia-like syndrome” induced by administration of lipopolysaccharides to normal pregnant rats,22 supports the concept that higher NO levels are required during pregnancy for blood pressure maintenance at the accepted physiological level. The reported normal levels of NO production therefore may be inadequately low. The net result would be the development of pregnancy-induced hypertension.
Selected Abbreviations and Acronyms
|GFR||=||glomerular filtration rate|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|MAP||=||mean arterial pressure|
|SBP||=||systolic blood pressure|
|UNOx||=||urinary nitric oxide metabolites|
|UP||=||urinary protein excretion|
- Received March 18, 1996.
- Revision received April 16, 1996.
- Accepted September 30, 1996.
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