Long-term nitric oxide synthase inhibition in rat pregnancy reduces renal kallikrein.

This study was performed to test the hypothesis that long-term nitric oxide synthase (NOS) inhibition during pregnancy may alter the predominance of the vasodilator kallikrein system. Sprague-Dawley rats were treated with the competitive inhibitor of NOS N(omega)-nitro-L-arginine (L-NNA, 50 mg. kg(-1). d(-1), dissolved in water) from days 7 to 21 of pregnancy. Rats were studied before treatment (day 5), at days 11, 17, and 21 of pregnancy (during treatment), and at postpartum days 7 and 21 (after the drug was withdrawn at delivery). Each group (n=5 to 8) had its corresponding control group (C) that received only vehicle. Additional rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME) alone or with an excess of L-arginine. At each study day, we measured blood pressure, collected urine overnight, obtained blood samples, and processed the kidneys for conventional histology and immunohistochemistry. In L-NNA rats, fetal and placental weights were reduced at days 17 and 21. Blood pressure was higher at days 17 and 21, returning to normal after L-NNA was removed. Urinary kallikrein activity was lower at days 11 and 17 (L-NNA=1147+/-213 and C=2317+/-146 nmol/16 h, P<0.001). Plasma renin activity was reduced at day 21 (L-NNA=9.6+/-2.1 and C=25.9+/-5 ng x mL(-1) x h(-1), P<0.05) and remained lower at postpartum day 7 x L-NNA rats exhibited glomerular lesions and tubular atrophy, particularly of connecting tubules that displayed reduced kallikrein staining. Tubulointerstitial infiltrating macrophages (ED1+) were also observed. Renal lesions were present as early as day 11 and persisted at day 7 postpartum. L-NAME rats exhibited similar alterations that were attenuated with an excess of L-arginine. We postulate that the reduction in renal kallikrein may contribute to the hemodynamic alterations described in this model.

Maternal factors modulate the increase in vasoactive substances during rat pregnancy.

OBJECTIVE: To explore if the changes in vasoactive substances observed during early pregnancy in the rat are modulated by maternal or fetoplacental factors. METHODS: Urinary excretion of cGMP, 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), thromboxane B2 and kallikrein activity was measured in pregnant (P, n = 11), pseudopregnant (PSP, n = 12), and virgin (n = 13) rats and in ovariectomized virgin rats supplemented with slow-release pellets containing either progesterone (50 mg/pellet) or estradiol (0.5 mg/pellet) or a combination of both hormones, for 21 days. RESULTS: The cGMP excretion was higher in PSP rats than in virgin rats at day 5 (virgin = 82 +/- 7, P = 93 +/- 5, PSP = 110 +/- 8 nmol/24 h, p < 0.05); at day 10, values were significantly increased in P and PSP rats. 6-keto-PGF1 alpha excretion was similarly elevated in P and PSP rats at day 5 (virgin = 120 +/- 10, P = 160 +/- 10, and PSP = 174 +/- 14 ng/24 h, p < 0.01). This trend was still present at day 10. Thromboxane B2 excretion showed a nonsignificant increase in P and PSP rats in day 5; at day 10, values were significantly elevated in both experimental groups (virgin = 23 +/- 2, P = 32 +/- 4, and PSP = 32 +/- 2 ng/24 h, p < 0.05). Kallikrein excretion was significantly increased in PSP and P rats at days 5 and 10. Estradiol or progesterone administration caused a significant decrease in serum aldosterone and an increase in urinary kallikrein activity. CONCLUSIONS: These results indicate that during the first half of rat pregnancy, the increment in vasoactive substances is modulated by maternal and not by fetoplacental factors.

What causes pre-eclampsia?

Pre-eclampsia remains a leading cause of maternal and fetal morbidity and mortality. Despite extensive research, the mechanisms that cause pre-eclampsia are unknown and it has been considered to be the 'disease of theories'. Hippocrates wrote in one of his Aphorisms that 'convulsions take place from either repletion or depletion'. Since then, obstetricians have been divided on the question of which factor accounted for the convulsions observed during childbirth. Some considered that a sudden reduction in intra-abdominal pressure at delivery led to a pooling of blood diverted from the brain, causing collapse of the cerebral blood vessels and convulsions. Others postulated that cerebral congestion, secondary to compression of the abdominal organs by the large uterus, diverted blood to the brain, causing eclamptic convulsions. It is the purpose of this review to examine those theories about the cause of pre-eclampsia for which modern evidence is available. At present, it is believed that the pathological chain of events leading to pre-eclampsia is scheduled in two steps: an absolute or relative placental ischaemia is followed by a diffuse endothelial cell activation, which causes the clinical features of the disease.

Role of nitric oxide in maternal hemodynamics and hormonal changes in pregnant rats.

Normal pregnancy is characterized by a significant reduction in total peripheral vascular resistance and decreased pressor responsiveness to vasodilator agents. This review will consider whether nitric oxide (NO) contributes to these changes, and whether a deficiency of NO produces a preeclampsia like syndrome. The biosynthesis of NO increases in pregnant animals, as assessed by the raised plasma concentration, urinary excretion and metabolic production rate of guanosine 3',5'-cyclic monophosphate (cGMP), the second messenger of NO. In addition, urinary excretion of nitrate, the stable metabolites of NO, increases during pregnancy, paralleling the rise in cGMP. Several studies provide convincing evidence indicating that expression and activity of different NO synthases (NOS) are increased in gravid animals. Acute blockade of NOS causes a dose response increase in blood pressure and reverses the blunted vasopressor response to vasoconstrictor agents. Long-term NOS inhibition produces a pre-eclampsia like syndrome, characterized by maternal hypertension, proteinuria, thrombocytopenia, and renal damage, and lower litter size and fetal weight. Both acute and chronic responses are reduced when L-arginine, the substrate for NOS, is administered in high doses, indicating that these changes are specific to NO inhibition. In conclusion, present data suggest that a disturbance in NO release may contribute to the pathogenesis of pre-eclampsia.

[Reduced plasma volume and changes in vasoactive hormones in underweight pregnant women]. / Disminución del volumen plasmático y cambios en hormonas vasoactivas en embarazadas de bajo peso.

BACKGROUND: Pregnant women with low weight/height (wt/ht) have lower plasma volume and reduced birth weight than women with normal wt/ht. AIM: To explore the hormonal mechanisms involved in these alterations. PATIENTS AND METHODS: Plasma volume, and several hormones related to plasma volume regulation were determined in 24 near term pregnant women with low wt/ht and in 30 with normal wt/ht. RESULTS: Newborns's weight, height and ponderal index were reduced in the low wt/ht group. Plasma volume (3042 +/- 101 vs 3631 +/- 101 ml, p < 0.001); plasma renin activity (7.5 +/- 0.9 vs 11.1 +/- 0.9 ng/ml/h, p < 0.01) and aldosterone (428 +/- 47 vs 710 +/- 58 pg/ml, p < 0.001) were significantly reduced in the low wt/ht group. Similar reductions were observed in serum estradiol and progesterone levels. Urinary kallikrein activity (354 +/- 112 vs 824 +/- 134 nmoles/24 h, p < 0.05), 6-keto-prostaglandin F1 alpha (561 +/- 90 vs 1121 +/- 165 ng/24 h, p < 0.05) and thromboxane B2 (110 +/- 29 vs 280 +/- 29 ng/24 h, p < 0.05) were also reduced in low wt/ht women. CONCLUSIONS: We postulate that the reduced levels of vasoactive hormones observed in pregnant women with low wt/ht may interfere with plasma volume expansion and, in turn, cause low birth weight.

[Ethical problems in the biomedical research projects, presented to the Ethical Committee of the Medical School at the Pontificia Universidad Católica of Chile]. / Problemas éticos en los proyectos de investigación biomédica, presentados al Comité de Etica de la Escuela de Medicina de la Pontificia Universidad Católica de Chile.

BACKGROUND: The higher methodological complexity of either diagnostic or therapeutic procedures has raised concern about the ethical principles that should underlie the conduct of biomedical research involving humans. AIM: To evaluate ethical problems of research proposals submitted to an ethics committee. MATERIAL AND METHODS: All research proposals involving humans, submitted within a two years period to the ethics committee of the School of Medicine of the Catholic University, were retrospectively reviewed. "Ethical problem" was defined as any explicit disagreement with the ethical principles and guidelines for the protection of human subjects involved in biomedical research, according to the Helsinki declaration. RESULTS: In 20 of 44 reviewed projects, an ethical problem was identified. The most common problems were the absence or inadequacy of the informed consent, the justification of the use of placebo and problems related to the methodological aspects of the research, particularly the lack of an adequate control group when the potential benefits of a new drug were evaluated. CONCLUSIONS: According to the Nüremberg code, the Helsinki declaration and the International Principle of Ethics in Biomedical Research, we analyse ethical problems and suggest judgement elements for them.

Effects of long-term nitric oxide synthesis inhibition on plasma volume expansion and fetal growth in the pregnant rat.

We conducted the present study to investigate whether the vasodilator nitric oxide plays a role in plasma volume homeostasis during pregnancy. Pregnant Sprague-Dawley rats were randomly assigned to a control group (n = 18) or to groups receiving 0.69 mmol/L (n = 11) or 1.7 mmol/L (n = 14) N omega-nitro-L-arginine, a competitive inhibitor of nitric oxide synthetase, from gestational days 7 through 21. On day 20 systolic pressure was measured. On day 21 blood samples were taken for plasma volume, hematocrit, and hormonal measurements. Fetal and placental weights also were determined. Systolic pressure was significantly higher in experimental rats (101 +/- 6 and 115 +/- 6 mm Hg in the 0.69 and 1.7 mmol/L groups, respectively) than in controls (79.7 +/- 7.5 mm Hg), and plasma volume was lower (18.4 +/- 1.1 and 17.1 +/- 0.5 mL) than in controls (21.5 +/- 0.8 mL). Both experimental groups had increased hematocrit levels. Plasma renin activity was significantly lower in the experimental groups (11.5 +/- 3 and 7.2 +/- 1.5 ng angiotensin I/mL per hour) than in controls (21.9 +/- 2.7 ng angiotensin I/mL per hour); however, no changes were observed in aldosterone levels. Experimental groups had lower fetal weight (4.6 +/- 0.1 and 5.1 +/- 0.1 g) than controls (5.5 +/- 0.1 g). In addition, fetal hindlimb hypoplasia was observed in the experimental groups. In conclusion, the present data indicate that long-term N omega-nitro-L-arginine administration to pregnant rats leads to increased blood pressure, reduced plasma volume expansion, lower plasma renin activity, and fetal growth retardation.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal plasma volume expansion and hormonal changes in women with idiopathic fetal growth retardation.

OBJECTIVE: To explore the mechanisms underlying the reduced maternal plasma volume associated with idiopathic fetal growth retardation (FGR). METHODS: In 30 normotensive women with growth-retarded fetuses and 26 with normal-size fetuses, plasma volume was measured with a modified Evan's blue method. Plasma levels of atrial natriuretic peptide, plasma renin activity, aldosterone, estradiol, and progesterone, and urinary excretion of kallikrein, prostacyclin, and thromboxane A2 were measured at 34-40 weeks' gestation. RESULTS: Compared with controls, gravidas with growth-retarded fetuses had a reduced plasma volume expansion (P < .01), similar atrial natriuretic peptide and plasma renin activity levels, and lower serum aldosterone (P < .001) and placental steroids (P < .03). These women also had decreased urinary kallikrein activity and prostaglandin excretion (P < .05). When both groups were combined, maternal plasma volume correlated significantly with birth weight (r = 0.53) and placental weight (r = 0.66). CONCLUSION: Normotensive women with idiopathic FGR have reduced plasma volume expansion. Although the exact mechanisms of this change are unknown, we postulate that the lower maternal aldosterone levels and reduced levels of vasodilator substances, such as prostacyclin and kallikrein, may have a causal role.

Maternal hemodynamic adjustments in idiopathic fetal growth retardation.

The hemodynamic characteristics of 11 normotensive gravidas with idiopathic fetal growth retardation (FGR), were compared with 11 controls of similar age, parity and body size. At weeks 36-38 of gestation, plasma volume was 3,161 +/- 121 ml in controls and 2,624 +/- 95 ml in the FGR group (p < 0.003); cardiac output (CO) was 6,191 +/- 132 ml/min in controls and 5,483 +/- 186 ml/min in the FGR group (p < 0.01). Total peripheral vascular resistance (TPVR) was lower in controls than in FGR (1,031 +/- 33 vs. 1,306 +/- 62 dyn/s/cm5; p < 0.001). Birth weight was correlated with both plasma volume (r = 0.61; p < 0.01) and CO (r = 0.53; p < 0.02) and inversely correlated with TPVR (r = -0.69; p < 0.001). These results are in line with the hypothesis that a reduced plasma volume leads to a lower CO and, secondarily, to reduced uterine blood flow and FGR.

[N-methyl-Tyr1,N-methyl-Arg7-D-Leu8]-dynorphin-A-(1-8)ethylamide, a stable dynorphin analog, produces diuresis by kappa-opiate receptor activation in the rat.

The i.v. administration of E-2078 ([N-methyl-Tyr1-N-methyl-Arg7-D-Leu8]-dynorphin-A-(1-8) ethylamide) to conscious animals in doses of 15, 50 or 200 micrograms/rat caused a dose-related diuretic response associated with a significant in crease in glomerular filtration rate (GFR) and in blood pressure. The overall excretion of Na+ was not modified by the opioid, whereas it reduced K+ output and its fractional excretion. Time course studies demonstrated that the increase in GFR and in blood pressure were transient and did not parallel the changes in urine outflow. Pretreatment of the animal with 1 mg/kg of naltrexone or of naloxone reduced the pressor response but did not reduce the renal action of E-2078. Doses of naltrexone 10 times larger (10 mg/kg) were required to attenuate the diuretic effect and abolish completely the changes in K+ excretion; however, the increase in GFR was not antagonized by 10 mg/kg of naltrexone. Consonant with the studies in conscious rats, perfusion of isolated rat kidneys with 0.2 to 1.8 microM E-2078 increased urine flow in a dose-dependent manner, and this effect was prevented by the simultaneous perfusion of 2 microM naltrexone with the peptide. In pentobarbital-anesthetized animals, E-2078 elicited a diuretic response that was not parallelled by changes in GFR or electrolyte excretion. In addition, E-2078 caused a long lasting decrease in blood pressure which was blocked completely by pretreatment of the animal with 1 mg/kg of naltrexone. The diuretic effect of E-2078 was not modified by pretreatment of the animals with beta-funaltrexamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic changes in underweight pregnant women.

Twelve normal-weight and 12 underweight women were compared to test whether fetal growth retardation in underweight gravidas is related to inadequate maternal hemodynamic adjustments. Plasma volume (+/- standard error) was 3227 +/- 103 mL in normal-weight and 2731 +/- 84 mL in underweight women (P less than .002). Cardiac output was 6340 +/- 167 mL/minute in controls and 5689 +/- 213 mL/minute in underweight women (P less than .03). Total peripheral vascular resistance was lower in controls than in underweight subjects (1025 +/- 31 versus 1198 +/- 58 dyne/second/cm5). Mean birth weight was 2837 +/- 125 g in underweight women and 3362 +/- 106 g in controls (P less than .005). Similarly, placental weight was reduced in the underweight group. All infants delivered by control mothers had a normal birth weight, whereas six infants from underweight gravidas were growth-retarded. In all cases combined, maternal plasma volume correlated significantly with both birth weight (r = 0.6, P less than .002) and placental weight (r = 0.56, P less than .01); total peripheral vascular resistance also correlated significantly and inversely with newborn weight and placental weight. Cardiac output correlated only with placental weight (r = 0.54, P less than .02). These results are consistent with the hypothesis that underweight mothers are at higher risk of fetal growth retardation because of a smaller plasma volume and lower cardiac output.

Heterogeneous binding sites for alpha-atrial natriuretic peptide in human umbilical cord and placenta.

The distributions of atrial natriuretic peptide (ANP) clearance receptors in human umbilical cord and placenta were investigated by using des[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4-2 3) (C-ANP) as a specific ligand of this receptor to displace bound alpha-125I-labeled ANP. alpha-125I-ANP bound reversibly to umbilical venous and arterial intima and to fetal placental and maternal decidual tissues, with dissociation constants of 1.24 +/- 0.51, 0.58 +/- 0.19, 1.86 +/- 0.51, and 1.07 +/- 0.25 mM, respectively. Binding was reversed by 1 microM unlabeled alpha-ANP but not by unrelated peptides such as gastrin. The 1 microM C-ANP displaced bound alpha-125I-ANP from the intima of umbilical artery but not that of the vein. The specific reversible binding of alpha-125I-ANP to placental decidua but not to fetal placenta was also displaced by 1 microM C-ANP. Therefore high-affinity binding sites for alpha-ANP on the intima of the umbilical artery and on placental decidua differ from those on the umbilical vein and on fetal placental tissue; the binding sites of the intima of the umbilical artery and of the decidua in humans are consistent with clearance receptors for alpha-ANP.

Renal atrial natriuretic peptide receptor subtypes in spontaneously hypertensive rats.

Receptor subtypes for atrial natriuretic peptide (ANP) were characterized in kidneys of 18-wk-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) by in vitro autoradiography through use of des[Gln18, Ser19, Gly20, Leu21, Gly22] ANP-(4-23) (C-ANP) and ANP-(5-25) as subtype-selective ligands. alpha-125I-ANP (100 pM) bound reversibly but with high affinity to glomeruli, to stripes in outer medulla, and to inner medulla of both WKY and SHR. C-ANP (10 microM) inhibited approximately 70% of the glomerular binding but none of the medullary binding in either strain. All high-affinity specifically reversible binding sites for alpha-ANP that bound C-ANP were also bound by 10 microM ANP-(5-25). However, the specifically reversible binding of alpha-125I-ANP that was not inhibited by 10 microM C-ANP behaved differently in each strain. In WKY, this binding was weakly inhibited by ANP-(5-25), so that even the presence of 10 microM ANP-(5-25) did not inhibit some glomerular binding and greater than 40% of the specifically reversible medullary binding of alpha-125I-ANP. In SHR, this binding was inhibited by ANP-(5-25) with a significantly higher affinity so that all specifically reversible binding of alpha-125I-ANP was inhibited by 10 microM ANP-(5-25). SHR also showed higher affinities but lower maximum binding capacities for alpha-ANP in their outer cortical glomeruli and medullas. These results suggest that the preponderant medullary ANP receptor differs between WKY and SHR. Differences in glomerular subtypes of ANP receptor may also distinguish WKY and SHR.

Autoradiographic localization of atrial natriuretic peptide receptor subtypes in rat kidney.

The distribution of atrial natriuretic peptide (ANP) clearance receptors in rat kidney was investigated by in vitro autoradiography using des[Gln18,Ser19,Gly20,Leu21,Gly22]-ANP-(4- 23) (C-ANP) and 125I-Tyr0-ANP-(5-25) as relatively specific ligands of this receptor. Alpha-125I-ANP (100 pM) bound reversibly but with high affinity to glomeruli, outer medullary vasa recta bundles, and inner medulla. C-ANP (10 microM) inhibited greater than 60% of this glomerular binding but did not inhibit the binding of alpha-125I-ANP to medullary tissues. Alpha-125I-ANP also bound reversibly to the renal arteries up to the glomerulus. This arterial binding was only partly inhibited by 10 microM C-ANP. In the presence of 10 microM C-ANP, increasing concentrations of alpha-125I-ANP bound to a residue of glomerular sites with apparent dissociation constants of 0.82 +/- 0.16 to 2.73 +/- 1.20 nM at different cortical levels. 125I-Tyr0-ANP-(5-25) bound significantly to glomeruli and intrarenal arteries but not to vasa recta bundles or inner medulla. This glomerular binding also occurred with nanomolar dissociation constants. It was completely inhibited by 1 microM alpha-ANP and 10 microM C-ANP, but not by unrelated peptides such as gastrin. These results suggest that renal ANP clearance receptors are restricted in vivo to the glomeruli and renal arterial system of the rat.

Diuretic effect of bremazocine, a kappa-opioid with central and peripheral sites of action.

Intracerebroventricular or i.p. injections of bremazocine produced a dose-dependent diuretic response and increased glomerular filtration rate in hydrated as well as in nonhydrated rats. The potency and magnitude of the bremazocine-induced diuresis were more pronounced in the nonhydrated group of rats. That bremazocine has a central component of action is deduced from the fact that 0.1 microgram of the opioid administered centrally caused a significant increase in urine output; proportionally, larger doses of bremazocine were required to produce the same diuretic effect when the drug was administered parenterally. Bremazocine did not change the total amount of urinary Na+ and K+ as compared to the saline controls; it increased significantly the free water clearance. The bremazocine-induced diuresis was antagonized in a competitive fashion by 10 mg/kg of naloxone giving further support to the notion that the mechanism of action of bremazocine involves activation of kappa-opioid receptors. Bremazocine injected i.v. to nonanesthetized rats increased mean systemic blood pressure in a dose-dependent manner; the pressor action of the opiate was blocked and prevented by 1 mg/kg of naloxone. In contrast, i.c.v. administration of bremazocine did not change mean systemic blood pressure but produced a dose-related increase in urine output. To determine whether in addition to a central site bremazocine also activates a renal mechanism, experiments were performed in the isolated perfused rat kidney. Bremazocine (0.15-2.5 microM) caused a dose-dependent diuretic response and a significant rise in perfusion pressure as well as in glomerular filtration rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary kallikrein activity during rat pregnancy.

Changes in urinary kallikrein activity and its possible correlation with changes in blood pressure and renal excretory function during pregnancy were studied in the rat. To establish a possible physiological role of kallikrein in this condition aprotinin, which inhibits kallikrein as well as other serine protease was administered to pregnant rats. Urinary kallikrein activity was markedly increased during pregnancy and correlated positively with urine volume and electrolytes excretion, but not with blood pressure. Aprotinin administration almost completely inhibited kallikrein activity, however, blood pressure levels, urine volume and electrolytes were not changed after one day of aprotinin treatment. In conclusion, although renal kallikrein is highly enhanced during pregnancy, its physiologic role in this condition remains elusive.