Effects of phosphodiesterase V inhibition alone and in combination with BNP on cardiovascular and renal response to volume load in human preclinical diastolic dysfunction.

Preclinical diastolic dysfunction (PDD) results in impaired cardiorenal response to volume load (VL) which may contribute to the progression to clinical heart failure with preserved ejection fraction (HFpEF). The objective was to evaluate if phosphodiesterase V inhibition (PDEVI) alone or combination PDEVI plus B-type natriuretic peptide (BNP) administration will correct the impaired cardiorenal response to VL in PDD. A randomized double-blinded placebo-controlled cross-over study was conducted in 20 subjects with PDD, defined as left ventricular ejection fraction (LVEF) >50% with moderate or severe diastolic dysfunction by Doppler echocardiography and without HF diagnosis or symptoms. Effects of PDEVI with oral tadalafil alone and tadalafil plus subcutaneous (SC) BNP, administered prior to acute volume loading, were assessed. Tadalafil alone did not result in improvement in cardiac response to VL, as measured by LVEF, LV end diastolic volume, left atrial volume (LAV), or right ventricular systolic pressure (RVSP). Tadalafil plus SC BNP resulted in improved cardiac response to VL, with increased LVEF (4.1 vs. 1.8%, p = 0.08) and heart rate (4.3 vs. 1.6 bpm, p = 0.08), and reductions in both LAV (-4.3 ± 10.4 vs. 2.8 ± 6.6 ml, p = 0.03) and RVSP (-4.0 ± 3.0 vs. 2.1 ± 6.0 mmHg, p < 0.01) versus tadalafil alone. Plasma and urinary cyclic guanosine monophosphate (cGMP) excretion levels were higher (11.3 ± 12.3 vs. 1.7 ± 3.8 pmol/ml, 1851.0 ± 1386.4 vs. 173.4 ± 517.9 pmol/min, p < 0.01) with tadalafil plus SC BNP versus tadalafil alone. There was no improvement in renal response as measured by GFR, renal plasma flow, sodium excretion, and urine flow with tadalafil plus SC BNP compared to tadalafil alone. In subjects with PDD, tadalafil alone resulted in no improvement in cardiac adaptation, while tadalafil and SC BNP resulted in enhanced cardiac adaptation to VL. TRIAL REGISTRATION: ClinicalTrials.gov NCT01544998.

C-Type Natriuretic Peptide Analogue Therapy in Children with Achondroplasia.

BACKGROUND: Achondroplasia is a genetic disorder that inhibits endochondral ossification, resulting in disproportionate short stature and clinically significant medical complications. Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification. METHODS: In a multinational, phase 2, dose-finding study and extension study, we evaluated the safety and side-effect profile of vosoritide in children (5 to 14 years of age) with achondroplasia. A total of 35 children were enrolled in four sequential cohorts to receive vosoritide at a once-daily subcutaneous dose of 2.5 µg per kilogram of body weight (8 patients in cohort 1), 7.5 µg per kilogram (8 patients in cohort 2), 15.0 µg per kilogram (10 patients in cohort 3), or 30.0 µg per kilogram (9 patients in cohort 4). After 6 months, the dose in cohort 1 was increased to 7.5 µg per kilogram and then to 15.0 µg per kilogram, and in cohort 2, the dose was increased to 15.0 µg per kilogram; the patients in cohorts 3 and 4 continued to receive their initial doses. At the time of data cutoff, the 24-month dose-finding study had been completed, and 30 patients had been enrolled in an ongoing long-term extension study; the median duration of follow-up across both studies was 42 months. RESULTS: During the treatment periods in the dose-finding and extension studies, adverse events occurred in 35 of 35 patients (100%), and serious adverse events occurred in 4 of 35 patients (11%). Therapy was discontinued in 6 patients (in 1 because of an adverse event). During the first 6 months of treatment, a dose-dependent increase in the annualized growth velocity was observed with vosoritide up to a dose of 15.0 µg per kilogram, and a sustained increase in the annualized growth velocity was observed at doses of 15.0 and 30.0 µg per kilogram for up to 42 months. CONCLUSIONS: In children with achondroplasia, once-daily subcutaneous administration of vosoritide was associated with a side-effect profile that appeared generally mild. Treatment resulted in a sustained increase in the annualized growth velocity for up to 42 months. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov numbers, NCT01603095, NCT02055157, and NCT02724228.).

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Effects of dual angiotensin type 1 receptor/neprilysin inhibition vs. angiotensin type 1 receptor inhibition on target organ injury in the stroke-prone spontaneously hypertensive rat.

OBJECTIVES: The combination of AT1 blocker/neutroendopeptidase neprilysin inhibition (ARNi) represents an interesting approach to reduce cardiovascular risk in hypertension. We assessed the efficacy of ARNi, compared with angiotensin II type 1 receptor blockade alone, on blood pressure (BP) and on protection from target organ damage development in the stroke-prone spontaneously hypertensive rat (SHRSP). METHODS: In high-salt fed SHRSP, we assessed plasma and tissue natriuretic peptides, urinary volume, BP and body weight over a short-term treatment (6 weeks) with either ARNi (sacubitril/valsartan 68 mg/kg per day) or valsartan (30 mg/kg per day), protection from stroke and renal damage (as documented by proteinuria) over 4 months of treatment with either sacubitril/valsartan or valsartan; the ability of either treatment to reduce progression of cerebrovascular and renal damage after 2 weeks of high-salt diet. RESULTS: Higher levels of plasma and tissue atrial natriuretic peptide, of urinary cyclic guanosine 3'5'monophosphate and urine volumes, along with lower BP levels, were found upon sacubitril/valsartan as compared with valsartan over the short-term treatment. Sacubitril/valsartan caused a significant reduction of both BP and proteinuria levels and complete prevention of stroke over the long-term treatment. Once organ damage was established, a significant delay of its progression was observed with sacubitril/valsartan. CONCLUSION: The dual angiotensin II type 1 receptor/neutroendopeptidase inhibition significantly increased atrial natriuretic peptide level and reduced BP. Complete prevention of stroke was achieved in this model. The ability of sacubitril/valsartan to reduce organ damage progression was superior to that of valsartan alone. ARNi may represent a highly effective therapeutic agent to protect from target organ damage development in hypertension.

Urinary cGMP predicts major adverse renal events in patients with mild renal impairment and/or diabetes mellitus before exposure to contrast medium.

BACKGROUND: The use of iodine-based contrast agents entails the risk of contrast induced nephropathy (CIN). Radiocontrast agents elicit the third most common cause of nephropathy among hospitalized patients, accounting for 11-12% of cases. CIN is connected with clinically significant consequences, including increased morbidity, prolonged hospitalization, increased risk of complications, potential need for dialysis, and increased mortality rate. The number of in-hospital examinations using iodine-based contrast media has been significantly increasing over the last decade. In order to protect patients from possible complications of such examinations, new biomarkers are needed that are able to predict a risk of contrast-induced nephropathy. Urinary and plasma cyclic guanosine monophosphate (cGMP) concentrations are influenced by renal function. Urinary cGMP is primarily of renal cellular origin. Therefore, we assessed if urinary cGMP concentration may predict major adverse renal events (MARE) after contrast media exposure during coronary angiography. METHODS: Urine samples were prospectively collected from non-randomized consecutive patients with either diabetes or preexisting impaired kidney function receiving intra-arterial contrast medium (CM) for emergent or elective coronary angiography at the Charité Campus Mitte, University Hospital Berlin. Urinary cGMP concentration in spot urine was analyzed 24 hours after CM exposure. Patients were followed up over 90 days for occurrence of death, initiation of dialysis, doubling of plasma creatinine concentration or MARE. RESULTS: In total, 289 consecutive patients were included into the study. Urine cGMP/creatinine ratio 24 hours before CM exposure expressed as mean±SD was predictive for the need of dialysis (no dialysis: 89.77±92.85 µM/mM, n = 277; need for dialysis: 140.3±82.90 µM/mM, n = 12, p = 0.008), death (no death during follow-up: 90.60±92.50 µM/mM, n = 280; death during follow-up: 169.88±81.52 µM/mM, n = 9; p = 0.002), and the composite endpoint MARE (no MARE: 86.02±93.17 µM/mM, n = 271; MARE: 146.64±74.68 µM/mM, n = 18, p<0.001) during the follow-up of 90 days after contrast media application. cGMP/creatinine ratio stayed significantly increased at values exceeding 120 µM/mM in patients who developed MARE, required dialysis or died. CONCLUSIONS: Urinary cGMP/creatinine ratio ≥ 120 µM/mM before CM exposure is a promising biomarker for the need of dialysis and all-cause mortality 90 days after CM exposure in patients with preexisting renal impairment or diabetes.

Inhaled nitric oxide increases urinary nitric oxide metabolites and cyclic guanosine monophosphate in premature infants: relationship to pulmonary outcome.

OBJECTIVE: Inhaled nitric oxide (iNO) has been tested to prevent bronchopulmonary dysplasia (BPD) in premature infants, however, the role of cyclic guanosine monophosphate (cGMP) is not known. We hypothesized that levels of NO metabolites (NOx) and cGMP in urine, as a noninvasive source for biospecimen collection, would reflect the dose of iNO and relate to pulmonary outcome. STUDY DESIGN: Studies were performed on 125 infants who required mechanical ventilation at 7 to 14 days and received 24 days of iNO at 20-2 ppm. A control group of 19 infants did not receive iNO. RESULTS: In NO-treated infants there was a dose-dependent increase of both NOx and cGMP per creatinine (maximal 3.1- and 2-fold, respectively, at 10-20 ppm iNO) compared with off iNO. NOx and cGMP concentrations at both 2 ppm and off iNO were inversely related to severity of lung disease during the 1st month, and the NOx levels were lower in infants who died or developed BPD at term. NOx was higher in Caucasian compared with other infants at all iNO doses. CONCLUSION: Urinary NOx and cGMP are biomarkers of endogenous NO production and lung uptake of iNO, and some levels reflect the severity of lung disease. These results support a role of the NO-cGMP pathway in lung development.

Vasotocin analogues with selective natriuretic, kaliuretic and antidiuretic effects in rats.

The aim of the present study was an investigation of mechanisms mediating selective effect of vasotocin analogues on water, sodium, and potassium excretion. We tested vasotocin analogues: Mpa(1)-vasotocin (dAVT), Mpa(1)-Arg(4)-vasotocin (dAAVT) and Mpa(1)-DArg(8)-vasotocin (dDAVT). The effects on water, sodium, and potassium transport were evaluated in experiments using normal and water-loaded Wistar rats. It was shown that all tested peptides exerted antidiuretic activity. Vasotocin and dAVT induced natriuresis and kaliuresis in rats. V1a agonist (Phe(2)-Ile(3)-Orn(8)-vasopressin) reproduced the renal effects of dAVT on sodium and potassium excretion but not on water reabsorption. dAAVT, dDAVT and V2 agonist (desmopressin) induced kaliuresis without any effect on sodium excretion. Natriuresis was associated with increase in cGMP excretion, whereas kaliuresis was correlated with rise of cAMP excretion. V1a antagonist (Pmp(1)-Tyr(Me)(2)-vasopressin) significantly reduced the dAVT-stimulated natriuresis and did not influence on urinary potassium excretion. V2 antagonist (Pmp(1)-DIle(2)-Ile(4)-vasopressin) significantly reduced the dAVT- and dAAVT-induced kaliuresis. It is assumed that effects of the nonapeptides on sodium and potassium transport are independent of their antidiuretic activity and mediated by different subtypes of V receptors (the V1a or V1a-like receptor for natriuretic effect and V2 or V2-like one for kaliuretic). In accordance to the data obtained, there is a possibility of selective regulation of renal water reabsorption and urinary sodium and potassium excretion with involvement of neurohypophysial hormones.

Suppression of the nitric oxide pathway in metastatic renal cell carcinoma patients receiving vascular endothelial growth factor-signaling inhibitors.

Therapies that target the vascular endothelial growth factor (VEGF) pathway cause hypertension, but the mechanism remains unknown. This cross-sectional study tested the hypothesis that VEGF inhibition causes hypertension by suppressing VEGF-mediated vasodilatory pathways. Urine was collected from 80 patients with metastatic renal cell carcinoma from 2002 to 2009, 40 at baseline and 40 while on VEGF inhibitors. Measured urinary biomarkers include albumin, metabolites of the nitric oxide (NO) pathway and its downstream effector cGMP, and prostaglandin pathway biomarkers prostaglandin E2, 6-keto prostaglandin F1α, and cAMP, all normalized to urinary creatinine. The mean age in both groups was 61.8 years, 76% were men, and urinary albumin was higher in patients receiving VEGF inhibitors (median: 18.4 versus 4.6 mg/g; P = 0.009). cGMP/creatinine was suppressed in patients on VEGF inhibitors (0.28 versus 0.39 pmol/µg; P = 0.01), with a trend toward suppression of nitrate/creatinine (0.46 versus 0.62 µmol/mg; P = 0.09). Both comparisons were strengthened when patients on bevacizumab were excluded, and only those receiving small molecule tyrosine kinase inhibitors were analyzed (cGMP/creatinine: P = 0.003; nitrate/creatinine: P = 0.01). Prostaglandin E2, 6-keto prostaglandin F1α, and cAMP did not differ between groups. These results suggest that hypertension induced by VEGF inhibitors is mediated by suppression of NO production. Prospective studies are needed to explore whether these biomarkers may be useful predictors of efficacy in patients receiving VEGF-targeted therapies.

Osmoregulation of natriuretic peptide receptors in bromoethylamine-treated rat kidney.

Extracellular osmolarity is known as an important factor for the regulation of natriuretic peptide receptors (NPRs). We investigated the intra-renal osmoregulation of NPRs using renal medullectomized rats with bromoethylamine hydrobromide (BEA, 200mg/kg). The administration of BEA caused the decreased food intake and body weight. Water intake was decreased on the first day and then increased from the second day. Urine volume was persistently increased from the first day and free water clearance was also increased from the second day. Urinary excretions of sodium and potassium were decreased on the second day and then recovered to control level. Plasma levels of atrial natriuretic peptide (ANP) and Dendroaspis natriuretic peptide (DNP) in BEA-treated rats were not different from control rats. The inactive renin was increased. The maximum binding capacities of (125)I-ANP as well as (125)I-DNP decreased in glomeruli and medulla of BEA-treated rat kidneys but the binding affinity was not changed. In renal cortex, the gene expressions of ANP, NPR-A, and NPR-B were not changed but that of NPR-C decreased. In renal medulla, the gene expressions of NPR-A, -B, and -C decreased without change in ANP mRNA. Both renal medullary osmolarity and sodium concentration by BEA treatment were lower than those in control kidney. The cGMP concentrations in renal medulla and urine in BEA-treated rats were higher than those in control rats. These results suggest that the increased cGMP production may be partly involved in the decrease in NPRs mRNA expression and their binding capacities by BEA-induced medullectomy.

Endogenous cardiotonic steroids and differential patterns of sodium pump inhibition in NaCl-loaded salt-sensitive and normotensive rats.

BACKGROUND: Endogenous sodium pump inhibitors promote sodium excretion in normotensives and contribute to vasoconstriction in NaCl-sensitive hypertension. Marinobufagenin (MBG), an endogenous bufadienolide inhibitor of alpha-1 sodium pump, contributes to hypertension in Dahl salt-sensitive rats (DS). We hypothesized that in NaCl-loaded DS and normotensive Sprague-Dawley rats (S-D), MBG would elicit different patterns of sodium pump inhibition. METHODS: We compared systolic blood pressure (SBP), renal sodium excretion, activity of the sodium pump in aorta and renal medulla, and levels of MBG, atrial natriuretic peptide (ANP), and cyclic guanosine monophosphate (cGMP) in salt-loaded DS and S-D (20% NaCl, 2.5 ml/kg, intraperitoneally). RESULTS: NaCl loading produced sustained elevations in renal MBG excretion in both DS (2.41 +/- 0.24 vs. 0.79 +/- 0.08 pmol/h/kg, P < 0.01) and S-D (1.97 +/- 0.37 vs. 0.60 +/- 0.07 pmol/h/kg, P < 0.01) vs. that at baseline (n = 10 for each group). In NaCl-loaded DS, SBP rose by 18 mm Hg (P < 0.01) and aortic sodium pump was inhibited by 22% (P < 0.05 vs. control), while in S-D, SBP and activity of aortic sodium pump did not change. NaCl-loaded S-D excreted twice as much sodium as DS; in S-D, renal sodium pump was inhibited by 24% vs. 14% inhibition in DS (P < 0.05). NaCl loading elicited increases in plasma ANP and in renal cGMP excretion in S-D but not in DS. CONCLUSIONS: Our present observations demonstrate that in NaCl-loaded S-D and DS, a comparable MBG response is associated with preferential inhibition of the sodium pump in the kidney and in vascular smooth muscle, respectively, resulting in an adaptive natriuresis in S-D but sodium retention and pressor response in DS.

Delivery of recombinant adeno-associated virus-mediated human tissue kallikrein for therapy of chronic renal failure in rats.

The tissue kallikrein-kinin system is important in regulating cardiovascular and renal function, and dysregulation of the system has been implicated in heart and kidney pathologies. These findings suggest that if balance can be restored to the kallikrein-kinin axis, then associated disease progression may be attenuated. To test this hypothesis, recombinant adeno-associated virus (rAAV)-mediated human tissue kallikrein (HK) expression was induced in a rodent model of chronic renal failure involving 5/6 nephrectomy (nephrectomy plus 70% reduction of remaining kidney). rAAV-HK treatment attenuated the rise in blood pressure, glomerular sclerosis, and tubulointerstitial injury observed in this model. rAAV-HK treatment also attenuated renal function decline as measured by urinary microalbumin, osmolarity, and cGMP levels. Reverse transcriptase-polymerase chain reaction analysis showed that rAAV-HK-treated rats had higher levels of bradykinin receptor-2 (B(2)R) and dopamine receptor-1 mRNAs. In contrast, angiotensin II receptor-1, endothelin receptor-A, and vasopressin receptor-2 mRNAs were markedly downregulated in kidneys from HK-treated rats. Bradykinin induced similar changes in receptor levels and prevented transforming growth factor-beta(1)-induced tubulointerstitial fibrosis. The effects of bradykinin could be reversed with the B(2)R antagonist HOE-140. Together, these findings suggest that restoration of the kallikrein-kinin system reduces kidney injury and protects renal function in 5/6-nephrectomized rats via changes in the expression and activation of G protein-coupled receptors including B(2)R.

Nitric oxide bioavailability and not production is first altered during the onset of insulin resistance in sucrose-fed rats.

Although the role of nitric oxide (NO) in peripheral glucose uptake has been thoroughly described, little is known regarding the alterations in NO metabolism during the early onset of insulin resistance. During this study we investigated the alterations in NO synthesis and bioavailability in a model for dietary modulations of insulin sensitivity. For 6 weeks, rats were fed a standard diet (C), a high-sucrose diet inducing insulin resistance (HS), or high-sucrose diets supplemented with cysteine, which endowed protection against the high-sucrose-induced insulin resistance (Ti). Several markers of NO synthesis and bioavailability were assessed and confronted with markers of insulin sensitivity. After 5 weeks, although urinary cGMP excretion did not differ between the groups, insulin resistance in HS rats was associated with both a significant increase in NO oxidation, as determined by plasma nitrotyrosine concentrations, and in the inducible NO synthase (iNOS)/endothelial NO synthase (iNOS/eNOS) mRNA ratio in skeletal muscle compared with C rats. These alterations were prevented in rats fed the cysteine-rich diets. NO production, as assessed by urinary 15NO3* excretion following a [15N2-(guanido)]-arginine intra-venous bolus, independently and significantly correlated with insulin sensitivity but did not significantly differ between C, HS, and Ti rats; neither did the aortic eNOS protein expression or skeletal muscle insulin-induced eNOS activation. Our results indicate that in this model of dietary modulations of insulin sensitivity (i) NO production accounts for part of total inter-individual variation in insulin sensitivity, but (ii) early diet-related changes in insulin sensitivity are accompanied by changes in NO bioavailability.

Growth hormone treatment improves markers of systemic nitric oxide bioavailability via insulin-like growth factor-I.

CONTEXT AND OBJECTIVE: Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I. DESIGN, PATIENTS, AND RESULTS: Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment, we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P < 0.05). Likewise, plasma nitrate and nitrite levels were increased, whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly, IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P < 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly, blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability. CONCLUSIONS: GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus, GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases.

Urinary cyclic GMP after treatment of gynecological cancer. A prognostic marker of clinical outcome.

BACKGROUND: The search for biological markers to predict malignant disease and its recurrence, or to monitor the effectiveness of treatment is a continuous process in medicine. Several years ago, urinary excretion of cGMP in urine was found to be a sensitive predictor in the follow-up of ovarian cancer and of monitoring treatment of cancer of the uterine cervix. PATIENTS AND METHODS: In the present study, 27 patients with gynecological cancer, including cancer of the uterine cervix (n=13), cancer of the uterine corpus (n=8) and cancer of the ovaries (n=6), were monitored for 10 years. Blood and urinary samples were taken before primary treatment (baseline sample) and three months thereafter (three-month sample). The serum levels of CEA, CA-125 and PIIINP and urine excretion of cGMP and cAMP were determined. Creatinine levels in serum and urine were employed to determine renal clearance. RESULTS: After 10 years' observation of women with cancer of the uterine cervix, seven patients showed no relapse and cGMP levels in baseline samples and three-month samples were 36.8+/-4.1 and 24.9+/-4.4 nmol cGMP/micromol creatinine (mean+/-SEM, p<0.01), respectively. The levels in patients (n=6) with relapse after 10 years' observation were 32.8+/-4.0 (baseline sample) and 43.5+/-4.2 (three-month sample) nmol cGMP/micromol creatinine (mean+/-SEM, p<0.02). Among the patients treated for cancer of the uterine corpus (n=9), none showed recurrent disease within the observation period of 10 years. The cGMP levels fell from 37.9+/-6.3 (baseline sample) to 22.3+/-2.3 (three-month sample) nmol cGMP/micromol creatinine (p<0.005). In the patients with ovarian cancer (n=6), 4 patients relapsed during the observation period of 10 years. In these women the cGMP levels increased from 34.5+/-2.7 (baseline sample) to 46.3+/-4.7 nmol cGMP/micromol creatinine whilst in both patients without relapse the levels decreased from 31.8 (range: 26.5-37.1) to 27.3 (range: 25.7-28.8) nmol cGMP/micromol creatinine, respectively. The changes in levels of cAMP, CEA, CA-125 and PIINP did not show statistically significant differences. Early changes in cGMP levels appear to predict long-term prognosis in gynecological cancers.

Effect of chronic administration of sildenafil on sodium retention and on the hemodynamic complications associated with liver cirrhosis in the rat.

Previous studies demonstrated increased phosphodiesterase-5 (PDE5) activity and expression in the kidneys of rats with liver cirrhosis. Acute intravenous administration of PDE5 inhibitors enhanced sodium excretion in these rats. The aim of the present study was to examine the effects of chronic administration of sildenafil on renal sodium handling and hemodynamics in rats with liver cirrhosis. Male Sprague-Dawley rats underwent bile-duct ligation and excision or sham operation and were housed in metabolic cages throughout the study. Body weight, food intake, water intake and urine volume were measured daily, and plasma samples were obtained twice weekly. Fourteen days following surgery sildenafil or its vehicle (dimethylsulfoxide) were administered (20 mg/kg subcutaneously 3 times/day). Two weeks later, systemic hemodynamics were measured under general anesthesia. Sildenafil enhanced the systemic vasodilatation associated with liver cirrhosis and reduced the arterial pressure. There was no reduction in the glomerular filtration rate, however. Despite these hemodynamic changes, sildenafil prevented the decrease in sodium excretion observed in the bile-duct-ligated group receiving vehicle and markedly increased fractional sodium excretion relative to the other groups. These results suggest that chronic sildenafil administration may help prevent or ameliorate sodium retention in cirrhosis, but that hemodynamic adverse effects may ensue.

Role of PI3K and PKB/Akt in acute natriuretic and NO-mimetic effects of leptin.

Apart from controlling energy balance, leptin, a peptide hormone secreted by white adipose tissue, is also involved in the regulation of cardiovascular function. Previous studies have documented that leptin stimulates natriuresis and nitric oxide (NO) production, but the mechanism of these effects is incompletely elucidated. We examined whether phosphoinositide 3-kinase (PI3K) and its downstream effector, protein kinase B/Akt are involved in acute natriuretic and NO-mimetic effects of leptin in anaesthetized rats. Leptin (1 mg/kg i.v.) induced a marked increase in natriuresis and this effect was abolished by pretreatment with either wortmannin (15 microg/kg) or LY294002 (0.6 mg/kg), two structurally different PI3K inhibitors. Moreover, leptin increased plasma concentration and urinary excretion of NO metabolites, nitrites+nitrates (NO(x)), and of NO second messenger, cyclic GMP. In addition, leptin increased NO(x) and cGMP in aortic tissue. The stimulatory effect of leptin on NO(x) and cGMP was prevented by PKB/Akt inhibitor, triciribine, but not by either wortmannin or LY294002. Triciribine had no effect on leptin-induced natriuresis. Leptin stimulated Akt phosphorylation at Ser(473) in aortic tissue but not in the kidney. These results suggest that leptin-induced natriuresis is mediated by PI3K but not Akt, whereas NO-mimetic effect of leptin results from PI3K-independent stimulation of Akt.

Effect of renal injury-induced neurogenic hypertension on NO synthase, caveolin-1, AKt, calmodulin and soluble guanylate cyclase expressions in the kidney.

Single injection of a small quantity of phenol into the cortex of one kidney in rats results in development of persistent hypertension (HTN) which is thought to be mediated by activation of renal afferent and efferent sympathetic pathways and sodium retention. Nitric oxide (NO) plays a major role in regulation of renal vascular resistance, tubular Na(+) reabsorption, pressure natriuresis, and thereby systemic arterial pressure. The present study was performed to test the hypothesis that chronic renal injury-induced HTN may be associated with dysregulation of NO system in the kidney. Accordingly, urinary NO metabolite (NO(x)) and cGMP excretions as well as renal cortical tissue (right kidney) expressions of NO synthase (NOS) isoforms [endothelial, neuronal, and inducible NOS, respectively (eNOS, nNOS, and iNOS)], NOS-regulatory factors (Caveolin-1, phospho-AKt, and calmodulin), and second-messenger system (soluble guanylate cyclase [sGC] and phosphodiesterase-5 [PDE-5]) were determined in male Sprague-Dawley rats 4 wk after injection of phenol (50 mul of 10% phenol) or saline into the lower pole of left kidney. The phenol-injected group exhibited a significant elevation of arterial pressure, marked reductions of urinary NO(x) and cGMP excretions, downregulations of renal tissue nNOS, eNOS, Phospho-eNOS, iNOS, and alpha chain of sGC. However, renal tissue AKt, phospho-AKT, Calmodulin, and PDE-5 proteins were unchanged in the phenol-injected animals. In conclusion, renal injury in this model results in significant downregulations of NOS isoforms and sGC and consequent reductions of NO production and cGMP generation by the kidney, events that may contribute to maintenance of HTN in this model.

A randomized, double-blind, placebo-controlled, parallel-group study to assess the efficacy and safety of dual ACE/NEP inhibitor GW660511X in mild-to-moderate hypertensive patients.

This multicentre, double-blind, placebo-controlled, parallel-group study determined the efficacy and safety of GW660511 200 mg, a dual inhibitor of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), in mild-to-moderate hypertensive patients (diastolic blood pressure (DBP), > or =90 and < or =109 mm Hg; systolic blood pressure (SBP), > or =150 and < or =180 mm Hg). After a single-blind 2- to 4-week placebo run-in period, 123 patients (aged 18-65 years) were randomized to either placebo (n=62) or to active treatment (n=61) consisting of two consecutive 3-day dose titration periods of GW660511X 50 mg once daily and 100 mg once daily followed by GW660511X 200 mg once daily for 14 days. GW660511X 200 mg significantly lowered (baseline and placebo-corrected) both trough mean cuff SBP (-8.00 mm Hg, P=0.002) and DBP (-5.38 mm Hg, P=0.003). GW660511X 200 mg significantly reduced placebo-corrected mean 24-h and daytime but not night-time ambulatory SBP and DBP. Over the 0-24 h time period following GW660511X 200 mg, there were significant (P<0.001) reductions in serum ACE activity and significant (P<0.001) increases in plasma ANP concentration compared with placebo in terms of both peak and trough effects. In addition, treatment with GW660511X 200 mg significantly (P=0.003) increased (placebo-corrected, 1.52-fold) urinary excretion of cGMP over the 0-24 h interval. Treatment-related adverse events were experienced by 43% of the patients administered GW660511X 200 mg and 44% of those dosed with placebo with headache the most commonly reported. In conclusion, GW660511X 200 mg is an effective antihypertensive in mild-to-moderate hypertensive patients with potent effects on biological markers of ACE and NEP inhibition.

Angiotensin II AT2 receptors inhibit proximal tubular Na+-K+-ATPase activity via a NO/cGMP-dependent pathway.

Angiotensin II AT2 receptors act as a functional antagonist for the AT1 receptors in various tissues. We previously reported that activation of the renal AT2 receptors promotes natriuresis and diuresis; however, the mechanism is not known. The present study was designed to investigate whether activation of AT2 receptors affects the activity of Na+-K+-ATPase (NKA), an active tubular sodium transporter, in the proximal tubules isolated from Sprague-Dawley rats. The AT2 receptor agonist CGP-42112 (10(-10)-10(-7) M) produced a dose-dependent inhibition of NKA activity (9-38%); the inhibition was attenuated by the presence of the AT2 receptor antagonist PD-123319 (1 microM), suggesting the involvement of the AT2 receptors. The AT1 receptor antagonist losartan (1 microM) did not affect the CGP-42112 (100 nM)-induced inhibition of NKA activity. The presence of guanylyl cyclase inhibitor ODQ (10 microM) and the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) abolished the CGP-42112 (100 nM)-induced NKA inhibition. ANG II (100 nM), in the presence of losartan, significantly inhibited NKA activity; the inhibition was attenuated by PD-123319. CGP-42112 also, in a dose-dependent manner, stimulated NO production (approximately 0-230%) and cGMP accumulation (approximately 25-100%). The CGP-42112 (100 nM)-induced NO and cGMP increases were abolished by the AT2 receptor antagonist PD-123319, ODQ, and L-NAME. The data suggest that the activation of the AT2 receptor via stimulation of the NO/cGMP pathway causes inhibition of NKA activity in the proximal tubules. This phenomenon provides a plausible mechanism responsible for the AT2 receptor-mediated natriuresis-diuresis in rodents.

Urinary cyclic guanosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate changes in spontaneous and induced onset active labor.

BACKGROUND: The aim of this prospective, randomized study was to investigate the changes in urinary cyclic guanosine 3',5'-monophosphate (cGMP) and cyclic adenosine 3',5'-monophosphate (cAMP) between the latent and the active phases of spontaneous and prostaglandin E(1) (PGE(1))-induced labor. METHODS: Seventy singleton pregnant women at 36-41(+) weeks' gestation without signs of fetal distress were enrolled. The first group consisted of 35 pregnant women in whom labor was induced by PGE(1) applied intravaginally. The second group consisted of 35 women who had spontaneous active labor. Clinical data of the two groups were assessed as labor progressed. RESULTS: After the onset of active labor, urinary cGMP/creatinine (U cGMP/Cr) decreased in both groups with the percentage decline of 35.2 and 9.7, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.033). After the onset of active labor, urinary cAMP/creatinine (U cAMP/Cr) decreased in both groups with the percentage decline of 36.5 and 15.6, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.001). The duration of the latent phase was significantly shortened in the PGE(1)-induced group compared with the spontaneous labor group (P<0.05). CONCLUSIONS: Decreased U cGMP/Cr and U cAMP/Cr may be a transition from the latent to the active phase in PGE(1)-induced labor. Our results suggest that U cGMP/Cr and U cAMP/Cr can serve as easily obtained secondary messenger markers of myometrial contractility and cervical ripening at the onset of active labor. The NO-cGMP system and the G-protein alpha-cAMP system in the human uterus may concomitantly contribute to uterine quiescence during pregnancy and show downregulation in U cGMP/Cr and U cAMP/Cr at the initiation of active labor.