Relationships among alterations in renal membrane sodium transport, renin and aminopeptidase M activities in genetic hypertension.

Rats of the Milan Hypertensive Strain (MHS) may be considered a useful model for understanding the genetic molecular mechanism underlying a primary form of hypertension in at least a subgroup of patients. Many differences between MHS and its normotensive control strain (MNS) were found at the organ, cellular and biochemical level. In the present investigation renal cell membrane proteins (BBMV) were analysed by two-dimensional electrophoresis and a difference between MHS and MNS was shown in a polypeptide of 32 kDa, subsequently identified as the C-terminal fragment of aminopeptidase M (APM). The activity of the enzyme was higher in MHS. Genetic relationships between this enzyme and the other biochemical cellular abnormalities of MHS, namely sodium transport in BBMV and renin activity in kidney cortex were investigated in MHS, MNS and in two inbred recombinant strains. This analysis showed that faster sodium transport, low kidney levels of renin and hypertension, but not differences in two-dimensional electrophoretic pattern and in aminopeptidase M activity, cosegregated in recombinant strains. These results are consistent with the hypothesis that the faster sodium transport can be considered a primary cellular abnormality responsible for hypertension in MHS and that the aminopeptidase difference is not involved in the cellular abnormalities.

Bradykinin B2-receptor blockade facilitates deoxycorticosterone-salt hypertension.

The contribution of endogenous kinins to the regulation of blood pressure, urinary volume, and renal sodium excretion was evaluated in Wistar rats on high sodium intake by using the new bradykinin receptor antagonist Hoe 140 (D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin). Neither Hoe 140 (3 nmol/hr s.c. for 4 weeks) nor its vehicle altered systolic blood pressure (tail-cuff plethysmography) or renal function in rats given saline solution (0.15 mol/L NaCl) to drink ad libitum. Four-week administration of deoxycorticosterone (DOC), combined with high sodium intake and uninephrectomy, increased systolic blood pressure from 127 +/- 3 to 160 +/- 3 mm Hg (p < 0.01). When long-term infusion of Hoe 140 was combined with DOC, high sodium intake, and uninephrectomy, systolic blood pressure rose from 127 +/- 3 to 175 +/- 3 mm Hg (p < 0.01). The hypertensive effect was greater in the Hoe 140 group (48 +/- 4 versus 33 +/- 3 mm Hg in controls, p < 0.05). This difference was confirmed by direct measurement of mean blood pressure (Hoe 140 group, 154 +/- 4 mm Hg; vehicle group, 139 +/- 4 mm Hg; p < 0.05). The antagonist blunted the increase in urinary volume induced by salt load and DOC in uninephrectomized rats, whereas it did not alter the increase in urinary sodium excretion. These results suggest that endogenous kinins do not play a major role in the regulation of normal blood pressure in sodium-loaded rats, whereas they may attenuate the hypertensive effect induced by long-term administration of mineralocorticoids and salt in uninephrectomized rats.

Sequential changes in plasma luteinizing hormone and plasma prorenin during the menstrual cycle.

Prorenin, the enzymatically inactive biosynthetic precursor of renin, is secreted by the kidneys. However, the ovaries appear to be the source of the cyclical increase in plasma prorenin that occurs in the middle of the menstrual cycle. In this study we examined the temporal relationship between changes in plasma prorenin and LH in normal women to determine whether ovarian prorenin secretion might be stimulated by LH. Blood was collected from nine normal women daily for 7 days in the midcycle period and from six of them every 8 h on 6 of these days. Time zero was taken as the highest plasma LH value. The initial rise in LH (-24 h) preceded the initial rise in prorenin (-8 h) and the LH peak preceded the prorenin peak by 8-16 h. These sequential increases in plasma LH and prorenin occurred in the presence of high plasma estradiol levels. While LH fell in parallel with estradiol, the prorenin peak was more sustained and plasma prorenin remained above baseline at 40 and 48 h, at a time when both estradiol and LH had reached a new basal level. These results suggest that gonadotropins stimulate ovarian prorenin release. The timing of the changes in plasma prorenin and its presence in high concentrations in ovarian follicular fluid suggest that prorenin may be involved in the process of ovulation. The results also suggest that changes in plasma prorenin may determine the activity of an ovarian renin system that functions independently of circulating active renin.

Brain kinins are responsible for the pressor effect of intracerebroventricular captopril in spontaneously hypertensive rats.

The role of the brain kallikrein-kinin system in the regulation of arterial blood pressure of normotensive and spontaneously hypertensive rats was evaluated. Intracerebroventricular administration of the kinin antagonist [DArg0]Hyp3-Thi5,8[DPhe7]bradykinin caused no change in mean blood pressure in Wistar-Kyoto, Sprague-Dawley, or spontaneously hypertensive rats. The antagonist proved to be very potent in blocking the pressor effect of intracerebroventricular bradykinin (32 +/- 3 vs. 3 +/- 1 mm Hg, p less than 0.01). It was specific, as the pressor effect induced by other unrelated peptides was similar during the infusion of either vehicle or kinin antagonist (angiotensin II, 25 +/- 4 vs. 26 +/- 2 mm Hg; prostaglandin E2, 48 +/- 3 vs. 47 +/- 8 mm Hg; norepinephrine, 17 +/- 2 vs. 18 +/- 2 mm Hg; leucine-enkephaline, 15 +/- 2 vs. 16 +/- 1 mm Hg; neurotensin, 18 +/- 2 vs. 19 +/- 1 mm Hg; substance P, 19 +/- 2 vs. 19 +/- 2 mm Hg). Intracerebroventricular administration of 1 mg captopril, an inhibitor of kininase II (one of the enzymes responsible for kinin degradation), caused no change in mean blood pressure in normotensive rats, whereas it increased mean blood pressure by 44 +/- 9 mm Hg (p less than 0.01) in spontaneously hypertensive rats. This increase in mean blood pressure was blocked and then reversed into a hypotensive effect (22 +/- 6 mm Hg, p less than 0.05) during the infusion of kinin antagonist. Our data suggest that the pressor effect induced by intracerebroventricular captopril is due to a transient elevation in endogenous brain kinin levels, supporting the hypothesis that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure in spontaneously hypertensive rats.

The role of alpha-adducin polymorphism in blood pressure and sodium handling regulation may not be excluded by a negative association study.

The basic requirement for declaring an association study positive is that the "hypertension-favoring" allele is more frequent in hypertensive cases than in normotensive controls. However, both positive and negative associations with hypertension have been found for the same polymorphism when studied in different populations. In the present study, we addressed the question of the possible cause(s) of this discrepancy among populations by using the alpha-adducin polymorphism as a paradigm. Four hundred ninety hypertensives and 176 normotensives enrolled in Sassari, Italy, and 468 hypertensives and 181 normotensives enrolled in Milano, Italy, were genotyped for the alpha-adducin Gly460Trp polymorphism. The blood pressure response to 2 months of hydrochlorothiazide therapy could be evaluated in 143 (85 in Sassari and 58 in Milano) hypertensives with and without the 460Trp alpha-adducin allele. The alpha-adducin 460Trp allele was not significantly more frequent in hypertensives in the Sassari population but was more frequent in hypertensives than in normotensives in Milano (P=0.019). Basal plasma renin activity was lower and blood pressure fall after diuretic therapy more pronounced (P<0.01) in hypertensives carrying at least one 460Trp allele than in Gly460Gly homozygotes, irrespective of their membership in the Sassari or Milano cohort. The effect of alpha-adducin genotype in predicting basal plasma renin activity and blood pressure decrease with diuretic treatment is similar in Sassari and Milano, despite the lack of association of the alpha-adducin genotype with hypertension in Sassari.

Non-association of the thiazide-sensitive Na,Cl-cotransporter gene with polygenic hypertension in both rats and humans.

OBJECTIVE: Genes underlying renal regulation of sodium and water balances are a priori valid candidates for polygenic hypertension susceptibility genes. Having recently identified the association of alpha1 Na,K-ATPase (ATP1A1) and Na,K,2Cl-cotransporter (NKCC2) as interacting hypertension susceptibility loci in both a rat model and human hypertensives, we investigated whether the thiazide-sensitive Na,Cl-cotransporter (TSC) gene contributes to hypertension susceptibility in a rat F2 intercross and in a northern Sardinian human cohort for polygenic hypertension. SUBJECTS AND METHODS: The rat TSC (rTSC) gene was analyzed directly for cosegregation with salt-sensitive hypertension in an F2 (Dahl S x Dahl R) rat population (n = 102) characterized for blood pressure by radiotelemetry. The human TSC (hTSC) gene was analyzed for association with hypertension in a human hypertensive cohort from northern Sardinia that consisted of 220 unrelated normotensives and 254 unrelated hypertensives. The TSC gene was subjected to single locus and digenic (in combination with ATP1A1 and NKCC2 genes) analyses in both rat and human cohorts. RESULTS: In both rat model and human cohorts, the rTSC and hTSC genes did not show linkage or association with high blood pressure, respectively. Furthermore, interaction with either ATP1A1 or NKCC2 was not detected in both the rat F2 intercross and human hypertension cohorts. CONCLUSIONS: These data exclude a primary role of the TSC gene in hypertension pathogenesis in the hypertension cohorts studied.

Effects of Hoe 140, a bradykinin B2-receptor antagonist, on renal function in conscious normotensive rats.

1. The present study was designed to determine if endogenous kinins are involved in the regulation of arterial blood pressure and renal function in conscious rats given deoxycorticosterone enantate (DOC, 25 mg kg-1, s.c., weekly) or vehicle for two weeks. 2. The bradykinin B2-receptor antagonist, D-Arg[Hyp3,Thi5,D-Tic7,Oic8]- bradykinin (Hoe 140), at a dose of 300 micrograms kg-1, s.c., blocked the hypotensive effect of 300 ng kg-1 bradykinin i.a., but it did not alter the blood pressure lowering action of 300 ng kg-1 acetylcholine or prostaglandin E2. Inhibition of the response to bradykinin persisted up to 6 h after the administration of Hoe 140. 3. Administration of 300 micrograms kg-1 Hoe 140 s.c. four times a day did not alter mean blood pressure, renal blood flow, or renal function in rats given DOC-vehicle. However, it decreased urinary volume by 70% (from 48.2 +/- 3.8 to 14.3 +/- 3.7 ml 24 h-1, P less than 0.01) and urinary secretion of sodium by 54% (from 1.02 +/- 0.05 to 0.47 +/- 0.16 mmol 24 h-1, P less than 0.01) and potassium by 30% (from 2.93 +/- 0.15 to 2.04 +/- 0.15 mmol 24 h-1, P less than 0.05) in DOC-treated rats. Mean blood pressure, glomerular filtration rate and total renal blood flow remained unchanged. 4. Our results suggest that endogenous kinins play a role in the regulation of renal excretion of water and sodium in the presence of elevated levels of DOC.

Activation of human prorenin by lipidic constituents of the cell membrane.

Activation of semipurified human kidney prorenin was found to occur in vitro in presence of a mixture of lipids that mimics the composition of the inner human cell membrane. The lipid-dependent activation was indeed only partial (38 +/- 4%) when compared to that obtained by trypsin in liquid phase (100 micrograms/mL) used as a control of maximal activation (100%) under our experimental conditions (semipurified human kidney prorenin in presence of semipurified human plasma renin substrate at a concentration of 1400 ng/mL, at pH 7.2). The phenomenon was time-dependent up to 60 min whereas the angiotensin I generated after 120 min was virtually the same as that generated after 60 min thus indicating a possible reversible activation of human prorenin. We speculate that prorenin may be reversibly activated by contact with the lipidic portion of the cell membrane either inside or outside the cells thus allowing a limited angiotensin II-generating cascade at a local site initiated by prorenin independently from the presence of active renin.

Recognition of markers of response to potassium-canrenoate in essential hypertension.

Potassium canrenoate (K-Can) prevents hypertension in Milan hypertensive strain (MHS) but not in spontaneously hypertensive rats (SHR). Essential hypertensive patients (HT) may have differential sensitivity to diuretics, since a subgroup of HT insensitive to hydrochlorothiazide (HCTZ) but sensitive to K-Can has previously been found. The aims of this study were: 1) to seek markers of response in essential hypertensive patients selectively sensitive to K-Can: and 2) to test whether selective sensitivity to furosemide may also be demonstrated. After 2 weeks of placebo (P) 50 uncomplicated, mild to moderate HT (46 +/- 9 yrs, mean +/- SD) received K-Can (50 mg/day) for 4 weeks. After 2 more weeks of P, patients received HCTZ (25 mg) and furosemide (25 mg) for 4 weeks each in a single blind crossover design, with 2 weeks P between each treatment. Dosages were doubled after 2 weeks if diastolic blood pressure (DBP) was > 90 mmHg. Responders (R) were those HT whose DBP was < or = 90 mmHg and/or at least 10 mmHg lower than before treatment. Systolic blood pressure (SBP)/DBP was measured every 2 weeks with plasma renin activity (PRA), red blood cell Na(+)-K(+)-Cl- cotransport (COT) and Na(+)-K+ ATPase pump activity measured at the end of the first P period, and serum electrolytes at the end of each period. Four HT dropped out because of low compliance, 6 because of reversible side effects, and 1 because blood pressure was not back to pre-treatment value after the second placebo period.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-activatable inactive renin in cat plasma and kidney.

Inactive renin has been identified in cat plasma and kidney extracts. It is identical to human plasma prorenin in all respects tested except that the pH optimum of activated cat inactive renin is slightly different from that of active renin. To further investigate whether cat inactive renin is indeed prorenin we examined whether it could be reversibly acid-activated, an unusual characteristic of human prorenin. We found that dialysis of partially purified, cat renal inactive renin to pH 3.3 caused reversible acid activation. In plasma, when acidification was followed by a second dialysis to pH 7.4 at 4 degrees C, irreversible activation took place, just like human prorenin. Unlike human angiotensinogen, cat angiotensinogen was apparently not affected by acid dialysis. These results suggest that cat inactive renin is most likely prorenin.

Plasma prorenin during early pregnancy: ovarian secretion under gonadotropin control?

To explore the time-course and the source of the changes in plasma prorenin that occur in early pregnancy we studied a normal subject (subject 1), an in vitro fertilization (IVF) patient (subject 2) and an ovarian failure patient who received a donor egg (subject 3). Day 0 was the luteinizing hormone (LH) peak (subject 1), the day of human chorionic gonadotropin (hCG) administration (subject 2) or 3 days before embryo transfer (subject 3). In subjects 1 and 2 prorenin increased transiently (three- and ninefold respectively) on days 0-4, then returned towards baseline and began to increase again around day 12 to a maximum (six- and 26-fold baseline) around day 20. Active renin was consistently less than 10% of total renin. In the ovarian-failure patient only small fluctuations occurred in total renin yet her hCG was 137 mlU/ml on day 15, proving that she was pregnant. These results suggest that the prorenin rise that occurs at mid-menstrual cycle and following conception may be due to ovarian prorenin secretion in response to stimulation by gonadotropic hormones.

Correlates of atrial natriuretic factor in chronic renal failure.

Plasma atrial natriuretic factor (ANF), blood pressure, age, plasma renin activity and creatinine were measured in 50 normal volunteers, 141 essential hypertensives, 35 patients with chronic renal failure who had never been dialysed and 27 patients with end-stage renal failure on constant haemodialysis. Plasma ANF was correlated positively with age in the normal group (r = 0.52, P less than 0.01) and with blood pressure in the essential hypertensives (r = 0.50, P less than 0.001), and negatively with renin in the normal and end-stage renal failure patients (r = -0.47, r = -0.34; P less than 0.01, P less than 0.05, respectively). When patients without left ventricular hypertrophy were matched for age and blood pressure, plasma ANF was significantly different between the essential hypertensives and the normal and end-stage renal failure patients (16 +/- 1, 38 +/- 6 and 148 +/- 24 pmol/l, respectively; P less than 0.001). Cardiac factors are therefore not the only determinant of circulating plasma ANF in humans with chronic renal failure.

Circulating prorenin and renin in response to intravenous adrenocorticotrophic hormone in essential hypertension.

Plasma prorenin and renin changes after a bolus injection of 25 U intravenous adrenocorticotrophic hormone (ACTH, synacthen) were studied in seven untreated uncomplicated essential hypertensives over a period of 24 h. Plasma prorenin did not change significantly during the study, whereas renin after 24 h was higher than at baseline (4.3 +/- 0.6 versus 2.3 +/- 0.9 ng angiotensin I (Ang I)/ml per h, P less than 0.01). We conclude that endogenous glucocorticoid stimulation induced by exogenous ACTH and ACTH itself seem to induce a secondary or tertiary rather than a primary effect on the human renin gene.

Conceptual basis and methodology of the SOPHIA study.

To clarify the role of gene polymorphisms on the effect of losartan and losartan plus hydrochlorothiazide on blood pressure (primary end point) and on cardiac, vascular and metabolic phenotypes (secondary end point) after 4, 8, 12, 16 and 48 weeks treatment, an Italian collaborative study - The Study of the Pharmacogenomics in Italian hypertensive patients treated with the Angiotensin receptor blocker losartan (SOPHIA) - on never-treated essential hypertensives (n = 800) was planned. After an 8 week run-in, losartan 50 mg once daily will be given and doubled to 100 mg at week +4 if blood pressure is more than 140/90 mmHg. Hydroclorothiazide 25 mg once daily at week +8 and amlodipine 5 mg at week +16 will be added if blood pressure is more than 140/90 mmHg. Cardiac mass (echocardiography), carotid intima-media thickness, 24 h ambulatory blood pressure, homeostatic model assessment (HOMA) index, microalbuminuria, plasma renin activity and aldosterone, endogenous lithium clearance, brain natriuretic peptide and losartan metabolites will be evaluated. Genes of the renin-angiotensin-aldosterone system, salt sensitivity, the beta-adrenergic system and losartan metabolism will be studied (Illumina custom arrays). A whole-genome scan will also be performed in half of the study cohort (1M array, Illumina 500 GX beadstation).

Pharmacogenomics of essential hypertension: are we going the right way?

Identifying the genetic predictors of the therapeutic response to drugs is the role of pharmacogenomics. Although polymorphisms in several genes have been associated with the blood pressure response to diuretics, beta-blockers and ACE-inhibitors, the pharmacogenomics of essential hypertension is still attempting to find satisfactory scientific evidence to be translated into clinical practice. The main reasons for this apparent failure are: the small sample sizes of the cohorts of patients analyzed, the methodological variability, the complexity of the biological organization, the context-dependency and the genetic heterogeneity. This review will summarize the available data on antihypertensive drugs and the criteria used for study design and conduction, focusing on their strong points and limitations.

Prorenin in the cells of the mature human ovarian follicle.

Angiotensin I generating capacity in presence of semi-purified plasma human angiotensinogen, before and after liquid phase trypsin activation, was found in the extracts of the cells retrieved from human hyperstimulated ovarian follicles in women undergoing in-vitro fertilization. More than 90% of this AI generating capacity was due to activated prorenin, whereas only less than 10% was due to active renin. Low levels of angiotensinogen were also present in the extracts (100 ng/ml). This AI forming capacity was completely abolished by pre-incubation with polyclonal anti-renin serum. More than 90% of the cells retrieved from the ovarian follicles were granulosa cells. An intracellular prorenin-renin system can exist in the human ovary than can modulate the ovarian function throughout the menstrual cycle and during pregnancy.

The cat: an animal model for studies of inactive renin.

Inactive renin, prorenin, is found in high concentrations in human plasma. We report herein the characteristics of trypsin-activated inactive renin from cat kidney and plasma. Cat and human plasma inactive renin were activated by similar concentrations of trypsin. As in humans, there was more inactive than active renin in cat plasma; also, inactive renin was low but detectable after nephrectomy. Trypsin-activated renal inactive renin, purified on Cibacron blue agarose and pepstatin-amino-hexyl-Sepharose chromatography, was inhibited by pepstatin and by a renin inhibitor similarly to cat and human active renins. The pH optimum of cat renin was biphasic: the higher peak of active renin was at pH 5.7, whereas that of activated inactive renin was at pH 7.5. As in humans, active and inactive plasma renin increased during sodium depletion and inactive renin increased during beta-adrenergic blockade, while active renin decreased. These results demonstrate that cat inactive renin is similar to human prorenin. Therefore, the cat may be a useful model for the study of prorenin.

Effect of the HMG-CoA reductase inhibitors on blood pressure in patients with essential hypertension and primary hypercholesterolemia.

Certain hydroxymethylglutaryl coenzyme A reductase inhibitors, ie, statins, may cause vasodilation by restoring the endothelial dysfunction that frequently accompanies hypertension and hypercholesterolemia. Several studies have found that a blood pressure reduction is associated with the use of statins, but conclusive evidence from controlled trials is lacking. After an 8-week placebo and diet run-in period, 30 persons with moderate hypercholesterolemia and untreated hypertension (total cholesterol 6.29+/-0.52 mmol/L, systolic and diastolic blood pressure 149+/-6 and 97+/-2 mm Hg) were randomized in a double-blind manner to placebo or pravastatin (20 to 40 mg/d) in a crossover design. In 25 participants who completed the 32-week trial, pravastatin decreased total and LDL cholesterol (both -1.09 mmol/L, P=0.001), systolic and diastolic blood pressure (-8 and -5 mm Hg, both P=0.001), and pulse pressure (-3 mm Hg, P=0.011) and blunted the blood pressure increase caused by the cold pressor test (-4 mm Hg, P=0.005) compared with placebo. It also reduced the level of circulating endothelin-1 (P=0.001). The blood pressure results were virtually unchanged in stratified analyses according to gender and age and in intention-to-treat analyses that included the 5 patients who dropped out of the study. When the participants were taking either placebo or pravastatin, blood pressure was not significantly correlated with total or LDL cholesterol or with circulating endothelin-1. Pravastatin decreases systolic, diastolic, and pulse pressures in persons with moderate hypercholesterolemia and hypertension. This antihypertensive effect may contribute to the documented health benefits of certain statins.

Changes in plasma atrial natriuretic factor during sequential fluid removal and biochemical correction in end-stage chronic renal failure patients.

Plasma immunoreactive atrial natriuretic factor (ANF) levels, their chromatographic profiles (high-performance liquid chromatography; HPLC) and changes during sequential ultrafiltration (UF; 1 litre/h) and biochemical correction without fluid removal (BC; 3 h) were studied in 8 end-stage chronic renal failure patients on intermittent haemodialysis (greater than 1 year). Patients entered randomly the UF-BC or BC-UF protocols that were reversed after 1 week. HPLC showed a single peak of ANF immunoreactivity in plasma of end-stage chronic renal failure patients before dialysis sessions. ANF at the end of fluid removal fell by 31 +/- 2% (p less than 0.01) during UF-BC and by 30 +/- 2% (p less than 0.01) at the end of BC during BC-UF. In both sequences a further slight reduction in plasma ANF was observed during the second phase: it was 8.5 +/- 5% (n.s.) during BC of the BC-UF and 12.5 +/- 2% (p less than 0.05) during fluid removal of BC-UF. Plasma ANF was not significantly removed by the machinery. BC did not modify the microhaematocrit in the BC-UF sequence while the microhaematocrit was significantly increased by UF (13 +/- 1 and 14 +/- 1%, p less than 0.005 vs. basal, respectively), and decreased by BC in the UF-BC sequence (-5 +/- 2% vs. end UF, p less than 0.05). Serum creatinine and urea decreased significantly during BC in both protocols while they were unmodified during UF. No significant changes were seen in PRC during either protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a kinin antagonist on renal function and haemodynamics during alterations in sodium balance in conscious normotensive rats.

1. To evaluate whether sodium intake can modulate the action of endogenous kinins on renal function and haemodynamics, a receptor antagonist of bradykinin was infused in conscious normotensive rats maintained on either a normal or a low sodium diet. 2. The antagonist inhibited the hypotensive effect of exogenously administered bradykinin. It did not change the vasodepressor effect of acetylcholine, dopamine or prostaglandin E2. 3. The antagonist did not affect mean blood pressure, glomerular filtration rate, renal blood flow or urinary sodium excretion, in rats on sodium restriction. It did not change mean blood pressure, glomerular filtration rate or urinary sodium excretion, but decreased renal blood flow, in rats on a normal sodium intake. 4. The kallikrein-kinin system has a role in the regulation of renal blood flow in rats on a normal sodium diet.