Methods for radioactivity measurements in drinking water using gamma spectrometry.

In this study, three methods to measure activity concentrations of radionuclides through high resolution gamma spectrometry are developed, optimized, and tested on drinking water samples. Two pre-concentration methods (partial evaporation and ion-exchange resins) were optimized for accuracy, precision, detection limits, costs, preparation, and measurements times. A new sampling method for 222Rn was designed and optimized to directly sample water from the tap, reducing and minimizing losses of radon during the sampling. A total number of 85 water samples were collected between 2017 and 2019 in collaboration with two drinking water suppliers in a wide area (~2000 km2) of the Veneto region, northeast Italy. These are the first results of radionuclides activity concentration in drinking water concerning a large extension in the foothill Veneto region. Finally, this study provides a first attempt of determining the spatial distribution and seasonal variations of radon activity concentration in drinking water in the study area.

Proinflammatory/profibrotic effects of aldosterone in Gitelman's syndrome, a human model opposite to hypertension.

PURPOSE: Aldosterone proinflammatory/profibrotic effects are mediated by the induction of mononuclear leucocytes (MNL) to express oxidative stress (OxSt)-related proteins, such as p22phox, and by the activation of RhoA/Rho kinase pathway. Gitelman's syndrome (GS), an autosomal recessive tubulopathy, is an interesting opposite model to hypertension, being characterized by hypokalemia, activation of renin-angiotensin-aldosterone system yet normo/hypotension and lack of cardiovascular-renal remodeling. We aimed to evaluate the proinflammatory/profibrotic effect of aldosterone in MNL of 6 GS patients compared with 6 healthy subjects (HS). METHODS: p22phox expression and MYPT-1 phosphorylation status, a marker of RhoA/Rho kinase pathway activation, were evaluated in MNL of GS patients and HS at baseline and after incubation with aldosterone (1 × 10-8 M) alone or with canrenone (1 × 10-6 M). RESULTS: At basal condition, p22phox expression was significantly higher in HS than in GS patients (1.02 ± 0.05 densitometric unit (du) vs 0.40 ± 0.1 du, respectively). Aldosterone significantly increased p22phox expression in HS and this effect was reversed by coincubation with canrenone (1.4 ± 0.05 du and 1.09 ± 0.03 du, respectively). No significant change was reported in GS after incubation of MNL with aldosterone and/or canrenone compared with basaline. Even MYPT-1 phosphorylation was significantly higher in HS compared with GS patients at basal condition (1.16 ± 0.1 du vs 0.69 ± 0.07, respectively). Aldosterone significantly increased MYPT-1 phosphorylation only in HS (1.37 ± 0.1 du vs 0.83 ± 0.12 du in GS). CONCLUSIONS: GS patients seem to be protected by the OxSt status induced by aldosterone and revealed in HS. This human model could provide additional clues to highlight the proinflammatory/cardiovascular remodeling effects of aldosterone.

Gαq/p63RhoGEF interaction in RhoA/Rho kinase signaling: investigation in Gitelman's syndrome and implications with hypertension.

PURPOSE: Gitelman's syndrome (GS) presents normo-hypotension and absence of cardiovascular-renal remodeling despite high angiotensin II (Ang II), activation of renin-angiotensin-aldosterone system and is a human model of endogenous antagonism of Ang II signaling, opposite to hypertension. GS's clinical presentation leads to questions regarding what features might be responsible. One area of investigation involves Ang II signaling. In hypertensive patients, RhoA/Rho kinase (RhoA/ROCK) pathway activation by Ang II is involved in hypertension development/maintenance and induction of long-term consequences (cardiovascular-renal remodeling), while GS has reduced p63RhoGEF gene and protein levels and ROCK activity. Ang II signaling is mediated by Gαq, which interacts with p63RhoGEF via the α6-αN linker connecting p63RhoGEF's DH and PH domains acting as a conformational switch to activate RhoA/ROCK signaling. METHODS: We have investigated in GS patients, the presence of mutations in either p63RhoGEF's α6-αN linker domain and in Gαq's Ala253, Trp263, and Tyr356 residues, crucial for p63RhoGEF-Gαq interplay. RESULTS: No mutations have been found in specific aminoacids of p63RhoGEF α6-αN linker and Gαq, key for p63RhoGEF/Gαq interplay. CONCLUSIONS: Gitelman's syndrome normo/hypotension and lack of cardiovascular-renal remodeling are not due to mutations of p63RhoGEF α6-αN linker and Gαq interactions. This opens the way for investigations on different coding and no-coding regions (p63RhoGEF and Gαq promoters) and on altered transcriptional/post-transcriptional regulation. Clarification of how these biochemical/molecular mechanisms work/interact would provide insights into mechanisms involved in the GS's Ang II signaling fine tuning, in human physiology/pathophysiology in general and could also identify significant targets for intervention in the treatments of hypertension.

Metformin improves putative longevity effectors in peripheral mononuclear cells from subjects with prediabetes. A randomized controlled trial.

BACKGROUND AND AIMS: Prediabetes increases cardiovascular risk and is associated with excess mortality. In preclinical models, metformin has been shown to exert anti-ageing effects. In this study, we sought to assess whether metformin modulates putative effector longevity programs in prediabetic subjects. METHODS AND RESULTS: In a randomized, single-blind, placebo-controlled trial, 38 prediabetic subjects received metformin (1500 mg/day) or placebo for 2 months. At baseline and after treatment, we collected anthropometric and metabolic parameters. Gene and protein levels of SIRT1, mTOR, p53, p66Shc, SIRT1 activity, AMPK activation, telomere length, and SIRT1 promoter chromatin accessibility were determined in peripheral blood mononuclear cells (PBMCs). Plasma N-glycans, non-invasive surrogate markers of ageing, were also analysed. Compared to baseline, metformin significantly improved metabolic parameters and insulin sensitivity, increased SIRT1 gene/protein expression and SIRT1 promoter chromatin accessibility, elevated mTOR gene expression with concomitant reduction in p70S6K phosphorylation in subjects' PBMCs, and modified the plasma N-glycan profile. Compared to placebo, metformin increased SIRT1 protein expression and reduced p70S6K phosphorylation (a proxy of mTOR activity). Plasma N-glycans were also favourably modified by metformin compared to placebo. CONCLUSION: In individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. ClinicalTrials. gov identifier: NCT01765946 - January 2013.

Synovial colony-stimulating factor-1 mRNA expression in diffuse pigmented villonodular synovitis.

OBJECTIVES: To delineate the molecular mechanisms underlying the process of the diffuse-type giant cell tumours, also called pigmented villonodular synovitis, a rare, aggressive condition of the synovium, the knee synovial tissue expression of colony-stimulating factor-1 gene, as detected by real-time polymerase chain reaction, was compared between patients affected with pigmented villonodular knee synovitis and knee meniscal tears, or persistent gonoarthitis. METHODS: Multiple synovial biopsies of the knee were performed by arthroscopy in five consecutive patients affected by diffuse pigmented villonodular knee synovitis and in 12 patients affected by knee meniscal tears (n. 6) or persistent active gonarthritis (n. 6), recruited from the patients attending the Rheumatology Day Surgery Outpatient Clinic of the University of Padova Hospital. The ethics committee approved the study protocol and the participants signed consent statements after being informed about the content of the study. The diagnosis was made on the basis of a histological examination. The colony-stimulating factor-1 gene expression was assessed by reverse transcription followed by real-time polymerase chain reaction. RESULTS: The detection by RT-PCR of synovial colony-stimulating factor-1 mRNA showed a wide spectrum of expression in the three groups of distinct knee joint disease affected patients, with significantly higher level of colony-stimulating factor-1 mRNA expression in synovial tissue of pigmented villonodular synovitis, in comparison to that of knee meniscal injuries and persistent gonoarthritis patients. CONCLUSIONS: Our findings point out to an important role of colony-stimulating factor-1 in pigmented villonodular knee synovitis disease process and support the idea that colony-stimulating factor-1/colony-stimulating factor-1 receptor interaction may represent a potential therapeutic target of this disease.

Molecular pathways involved in synovial cell inflammation and tumoral proliferation in diffuse pigmented villonodular synovitis.

Diffuse-type tenosynovial giant cell tumors, also known as pigmented villonodular synovitis, are unique mesenchymal lesions that arise from the synovial tissue of the joints. They are predominantly intraarticular, aggressive, infiltrative processes, characterized by both inflammatory or neoplastic properties and local destructive progression. The pattern of synovial gene and protein expressions in pigmented villonodular synovitis, similar to those in activated macrophages in rheumatoid arthritis, and the phenotype of multinucleated giant cells, characteristic of osteoclasts, suggest that there is a common autocrine mechanism in osteoclast differentiation in both diseases and indicate the potential utility of tumor necrosis factor (TNF)-alpha blockade. High synovial colony stimulating factor 1 (CSF1) messenger RNA (m RNA) expression in pigmented villonodular synovitis, unrelated to a chromosomal translocation involving CSF1 locus, may indicate that there is a synergic paracrine loop mediated by TNF-alpha and CSF1, as shown in both inflammatory and neoplastic conditions. The effects of a new therapeutic approach consisting in intraarticular TNF-alpha blockade were studied in four pigmented villonodular synovitis knees. Knee injections produced a rapid reduction in clinical and sonographic indexes and immunohistological alterations, confirmed by arthroscopic synovectomy. A delayed relapse in one of the four knees and unaltered synovial CSF1 expression were other important findings. In the light of these observations, CSF1/CSF1R interaction probably represents a more sensible therapeutic target than TNF-alpha blockade in the diffuse form of pigmented villonodular synovitis.

Oxidative stress-related proteins in a Conn's adenoma tissue. Relevance for aldosterone's prooxidative and proinflammatory activity.

BACKGROUND AND AIM: Angiotensin II (Ang II) induces oxidative stress (OxSt), which is essential for cardiovascular remodeling. Aldosterone also induces fibrosis and remodeling through direct effect on non-classical mineralocorticoid (MR) target tissues. However, studies on the role of aldosterone on OxSt and related factors in humans are lacking. MATERIALS AND METHODS: We assessed gene and protein expression of p22phox (RT-PCR and Western blot), NAD(P)H oxidase subunit essential for superoxide production and gene expression of transforming growth fator (TGF) beta, plasminogen activator inhibitor (PAI)-1, and heme oxygenase (HO)-1, effectors of OxSt (RT-PCR), in a Conn's adenoma, removed from a patient with primary hyperaldosteronism. Ang II type 1 (AT1R) and MR receptors expression were also evaluated (RT-PCR). The normal adrenal tissue adjacent to the adenoma was used as control. RESULTS: p22phox gene and protein expression were higher (31% and 53%, respectively) in the adrenal adenoma. TGFbeta, PAI-1, and HO-1 gene expression were also higher (25%, 129%, and 25%, respectively) in the adrenal adenoma while AT1R gene expression was similar (8%). The expression of MR in the adenoma was documented. CONCLUSIONS: This report demonstrates in a human model that the increased aldosterone production has effects on enzyme systems related to OxSt, enhancing the systemic fibrogenic effects of aldosterone excess through TGFbeta and PAI-1 expression which was previously demonstrated only indirectly in vitro and in animal models. The presence of MR expression in the adenoma may link the hormone with the adenoma growth. Therefore, the results of this study derived from a single case might represent an important working hypothesis for further research in a larger number of cases to clarify the role of aldosterone overproduction on OxSt and its clinical relevance.

High angiotensin II state without cardiac remodeling (Bartter's and Gitelman's syndromes): are angiotensin II type 2 receptors involved?

BACKGROUND/AIMS: While Angiotensin II (Ang II) is a major factor in the development of cardiomyocyte hypertrophy and a pivotal role for Ang II signals via ERK1/2 has been identified, mechanism(s) responsible are still unclear. As Bartter's and Gitelman's syndrome patients (BS/GS) have increased Ang II, and yet normo/hypotension, hyporesponsiveness to pressors and blunted Ang II signaling via type 1 receptors (AT1R), this study assesses BS/GS's left ventricular (LV) mass and structure as well as Ang II induced ERK1/2 phosphorylation compared with essential hypertensive patients (EH) and normotensive healthy subjects (C) to gain insight into Ang II mediated processes. METHODS: Indices of cardiac hypertrophy were determined by M-mode, two-dimensional echo Doppler and ERK phosphorylation by Western blot. RESULTS: None of BS/GS exhibited LV remodelling; LV mass, LV end-diastolic volume and mass/volume ratio were unchanged vs C (60+/-14 g/m2 vs 64+/-12, 64+/-12 ml/m2 vs 60+/-8 and 0.95+/-0.2 vs 1.0+/-0.2, respectively) and reduced vs EH (119+/-15, p<0.001, 78+/-9, p<0.05 and 1.52+/-0.15, p<0.01). Despite BS/GS's higher plasma renin activity and aldosterone and unchanged level of AT1R, Ang II induced ERK1/2 phosphorylation was reduced vs both C and EH: 0.64 d.u.+/-0.08 vs 0.90+/-0.06 in C, p<0.006, and vs 1.45+/-0.07 in EH, p<0.001. CONCLUSION: The data point to a direct cardioremodeling role for Ang II and support a role of Ang II type 2 receptor (AT2R) signaling as involved in the lack of cardiovascular remodeling in BS/GS. However, further studies using more direct approaches to demonstrate the effects of AT2R signaling must be pursued.

Effect of canrenone and amiloride on the prooxidative effect induced by aldosterone in human mononuclear leukocytes in vitro.

Clinical studies have demonstrated that aldosterone receptor antagonists do improve the survival of patients with chronic heart diseases and in vitro studies have shown that canrenone blocks the proinflammatory effect of aldosterone in mononucler leukocytes (MNL). The aim of the study was to compare, in the model of human MNL, the effect of potassium-sparing diuretics amiloride and canrenone, on the protein expression of p22phox, a NADPH-oxidase system subunit, that is a principal marker of production of superoxide anions. MNL were isolated from 10 informed healthy volunteers (5 males and 5 females, age range 24-36 yr) and the proteins extracted. p22phox protein expression was evaluated by Western blot and quantified using a densitometric semiquantitative analysis. The experiments showed that aldosterone (10(-8) M) enhances the protein expression of p22phox and that its effect is reversed by co-incubation with canrenone (10(-6) M), while incubation with amiloride (10(-6) M) reduced the prooxidative effect of aldosterone at a significantly lower extent than canrenone. Co-incubation with canrenone, amiloride, and aldosterone together produced the same effect as aldosterone plus canrenone. Incubation with cortisol (40(-8) M) was not effective. These data confirm the prooxidative effect of aldosterone in MNL. The addition of aldosterone-receptor antagonist canrenone produced a higher inhibition than sodium channel blocker amiloride on the effect of aldosterone on p22phox protein expression.

[Antioxidants, carnitine and erythropoietin]. / Gli antiossidanti, la carnitina e l'eritropoietina.

Patients with renal disease possess an excess of cardiovascular (CV) risk factors, which predispose these patients to a high rate of morbidity and mortality due to CV disease. Oxidative stress and oxidative stress induced apoptosis play an important role not only in the pathophysiology and progression of renal disease, but also in the induction of myocardial damage and heart failure with important implications concerning CV morbidity and mortality in general, and particularly for patients with renal disease. This has stimulated the realization of studies aimed at evaluating therapies with antioxidants, given the evidence that patients with renal disease and heart failure have an imbalance towards pro-oxidant factors. The correction of anemia with erythropoetin (EPO) is another important aspect related to oxidative stress and apoptosis, which has also revealed positive effects for the improvement in heart failure. EPO cellular mechanisms are not completely known, and the identification of close biochemical and molecular relationships between EPO and heme oxygenase-1 (HO-1), which has potent antioxidant and anti-apoptotic properties, could provide the rationale for the beneficial effect of carnitine having been shown to possess antioxidant, anti-apoptotic and erythropoietic activities and to induce HO-1 expression, not only in dialysis patients who fail to respond adequately to EPO, but also in patients with heart failure. The identification of these relationships between EPO, HO-1 and carnitine and their biochemical mechanisms could contribute to the opening of new perspectives in the improvement of CV mortality in these patients, which remains the most important cause of death in patients with end-stage renal disease.

Effect of doxazosin on oxidative stress related proteins in essential hypertensive patients.

The role of oxidative stress in the pathophysiology of hypertension has stimulated the investigation of strategies to reduce oxidative stress via antioxidant defenses. Using a molecular biology approach, we report, in essential hypertensive patients, the effect of doxazosin treatment on the mononuclear cell gene and protein expression of two major elements in the oxidative stress and vascular remodeling-related pathways: p22(phox) and PAI-1. Ten essential hypertensive patients were treated with Doxazosin (4 mg/day) for two weeks (EH + D) and compared with ten untreated hypertensive patients (EH) and ten normotensive subjects (C). In EH p22(phox) and PAI-1 mRNA and protein level was increased compared with C. In EH + D, doxazosin reduced p22(phox) and PAI-1 gene and protein expression, which was similar to that of C. These results demonstrate for doxazosin an inhibitory effect on oxidative stress related proteins at gene and protein level, which confirms at molecular level a powerful antioxidant potential for this agent that could translate, in the long term, into a powerful antiatherosclerotic effect.

[Intra-articular treatment with the TNF-alpha antagonist, etanercept, in severe diffuse pigmented villonodular synovitis of the knee]. / Trattamento intra-articolare con l'antagonista del TNFalpha etanercept nella sinovite villo-nodulare pigmentosa diffusa del ginocchio.

Pigmented villonodular synovitis (PVNS) is a rare pre-malignant disease that require aggressive treatment as surgical synovectomy, eventually followed by radiosynovectomy. Nevertheless, the disease often reoccurs after these treatments. To determine the safety and efficacy of intra-articular (IA) TNFalpha blockade with etanercept (ETN), before extended arthroscopic synovectomy, in severe PVNS of the knee, two patients, (a 26-year-old man with B27+ undifferentiated spondylarthropathy and a 32-year-old femal with seronegative oligoarthritis), affected by diffuse knee PVNS (diagnosis made by histological examination), resistant to IA corticosteroid injections and to repeated arthroscopic synovectomy, were submitted, after protocol approval by human research committee and patient's written informed consent to intra-articular etanercept (IA-ETN) treatment with a different dosage schedule: 12.5 mg weekly IA-ETN injection for 4 weeks, followed by extended arthroscopic synovectomy and of 25 mg IA-ETN injection for 4 weeks, respectively. Previous DMARDs treatment was continued in stable appropriate doses. Any adverse events were recorded throughout the study. The following parameters were considered as clinical endpoints: 1) Knee Joint Index (KJI: range 0-14); 2) Thompson index (THI: range 0-9) At the study entry and at the end of follow-up, high frequency ultrasound grey scale synovial thickening (US-ST) was also assessed. No adverse events were observed due to IA-ETN and to arthroscopic synovectomy. Marked improvement of knee disease activity over time and sustained functional recover was obtained. US-ST evaluation before treatment initiation and at the end of follow-up confirmed the regression of knee joint synovial proliferation.

Vitamin E-coated dialyzers reduce oxidative stress related proteins and markers in hemodialysis--a molecular biological approach.

BACKGROUND: Hemodialysis patients (HD) are exposed to oxidative stress which contributes to cardiovascular disease and accelerated atherosclerosis, major causes of mortality in these patients. A new dialysis membrane coated with vitamin E has been proposed against oxidative stress and atherosclerosis due to their ability to inhibit lipid peroxidation by interacting with scavengers. The mechanisms however are not completely clarified. This study evaluated, using a molecular biology approach, the effect of 6 months treatment with vitamin E-modified dialyzers, CL-E, on the gene expression of oxidative stress related proteins and markers. PATIENTS AND METHODS: To this end, the gene expression of p22phox, a NAD(P)H oxidase subunit closely linked with the generation of superoxide anions and of Heme oxygenase-1 (HO-1), induced by and protective from oxidative stress, were evaluated by RT-PCR in mononuclear cells from 5 patients under 3 times a week chronic bicarbonate dialysis. Hydroperoxide (HPO) and total antioxidant power (AOP) plasma levels were evaluated at 3 and 6 months of treatment. HPO was also evaluated in 8 patients under CL-E treatment for 1 year and compared with 8 patients treated with cuprammonium-ryon filter (TAF). RESULTS: p22phox mRNA decreased from 0.61 +/- 0.05 d.u. to 0.48 +/- 0.03, p < 0.01 while HO-1 increased from 0.55 +/- 0.04 d.u. to 0.62 +/- 0.03, p < 0.01. HPO decreased in CL-E treated patients: from 2.72 +/- 0.26 microM to 1.45 +/- 0.27 at 3 months (p < 0.001) to 0.87 +/- 0.11, p < 0.001 at 6 months, while AOP increased: from 752 +/- 90 mmol/L to 1057 +/- 105, p < 0.001 at 6 months. HPO was also reduced in 1 year Excebrane CL-E treated patients compared with cuprammonium treated patients: 2.25 +/- 0.3 vs. 1.42 +/- 0.11 microM, p < 0.001. CONCLUSION: The reduced expression of oxidative stress related proteins and markers gives further support to the efficacy of the use of Vitamin E coated dialysers for the prevention or slowing progression of cardiovascular disease and atherosclerosis, major complications and causes of mortality in these patients in which oxidative stress plays a pivotal role.

Effect of epoetin on HO-1 mRNA level and plasma antioxidants in hemodialysis patients.

OBJECTIVE: Patients with renal failure and undergoing hemo- (HD) or peritoneal dialysis are under oxidative stress which is thought to contribute to the long-term complications noted in this patient population. One effect of HD-induced oxidative stress is via red blood cell (RBC) membrane lipid peroxidation leading to RBC destruction and anemia. Interaction of this oxidative stress with epoetin (EPO) treatment to increase RBC number and Hb concentration remains unexplored. PATIENTS AND METHODS: This preliminary study used RT-PCR as well as colorimetric based assay approaches to evaluate the effect of EPO-alpha treatment on markers of oxidative stress in hemodialysis patients. Eighteen patients (12 males, 6 females, age range 45 - 68), were treated with EPO-alpha (Eprex) 50 UI/kg thrice weekly over an 8-month study period. Monocytes were isolated at baseline, then monthly thereafter, monocyte heme-oxygenase-1 (HO-1) and plasma Hb and antioxidant power (AOP) were determined. RESULTS AND CONCLUSIONS: Treatment with EPO increased Hb (9.4 +/- 0.7 g/dl to 10.9 +/- 0.5, mean +/- SD p < 0.001). In addition, both monocyte HO-1 mRNA (0.34 +/- 0.08 vs. 0.59 +/- 0.02 d.u. p < 0.001) and plasma AOP (1,379.8 +/- 175 micromol/l to 1,624 +/- 170, p < 0.04) increased. While AOP changes showed no correlation with other indices, increases in HO-1 and Hb were positively correlated using 2 different measures: delta Hb (peak Hb - baseline Hb) vs. delta HO-1 (peak HO-1 mRNA - baseline HO-1 mRNA) as well as delta Hb(5 months-baseline) vs. delta HO-1 (5 months - baseline) mRNA (r = 0.81, p < 0.001 and r = 0.76, p < 0.001; respectively). In conclusion, the increases upon EPO treatment of both HO-1 gene expression and plasma AOP as well as the significant correlation between delta Hb and delta HO-1 mRNA suggest that EPO treatment reduces oxidative stress via a combination of effects. These could potentially include effects on oxidative stress directly as well as effects on the levels and types of antioxidants present in plasma.

Oxidative stress and TGFbeta in kidney-transplanted patients with cyclosporin-induced hypertension. Effect of carvedilol and nifedipine.

Cyclosporin is a powerful stimulator of oxidative stress signaling, leading to TGFbeta production, NO degradation, endothelial dysfunction, hypertension and post-transplant nephropathy. Carvedilol, alpha1-beta-blocker with strong antioxidant activity, may interfere with this chain of events. Therefore, we measured monocyte ecNOS, TGFbeta and heme oxygenase-1 (HO-1) mRNA level and plasma nitrite/nitrate, 3-nitrotyrosine, an estimate of peroxynitrite, and total plasma antioxidant power in kidney-transplanted patients with post-transplant hypertension, before and after treatment with carvedilol, 25 - 50 mg o.d. orally for 4 months (n = 15). The dihydropyridine calcium channel blocker nifedipine (n = 10) was used as comparator antihypertensive drug. Blood pressure fell to a similar extent with both drugs. Carvedilol increased plasma antioxidant power and HO-1 mRNA and reduced 3-nitrotyrosine and TGFbeta mRNA levels, while the same was not observed with nifedipine. Monocyte ec NOS mRNA levels and plasma nitrite/nitrate were higher in the patients than in a normotensive healthy control group and were unaffected by either treatment. In conclusion, carvedilol reduces the oxidative stress and corrects the altered cellular signaling mediated by oxidative stress in CsA-induced post-transplant hypertension. Therefore, it may prevent long-term complications, such as endothelial dysfunction, fibrogenesis and post-transplant nephropathy by decreasing NO degradation and production of TGFbeta, a key fibrogenic cytokine, and by activating HO-1 production.

[Arterial hypertension and oxidative stress induced by cyclosporin. Effect of carvedilol]. / Ipertensione arteriosa e stress ossidativo indotti dalla ciclosporina. Effetto del carvedilolo.

Cyclosporin-induced hypertension and endothelial dysfunction have been attributed to the effects of cyclosporin on factors controlling vasomotor tone. Endothelial nitric oxide (NO) regulates basal vasodilation, and an NO-mediated protective mechanism from cyclosporin-induced vasoconstriction has been proposed. In transplanted patients with cyclosporin-induced hypertension, we have recently demonstrated upregulation of the NO system and superoxide and free radical overproduction, which, by increasing NO metabolism, could induce hypertension, vascular remodeling and chronic rejection. In the present work, we have evaluated endothelial constitutive NO synthase (ecNOS), transforming growth factor beta and heme oxygenase-1 (protective against oxidative stress), mRNA production and plasma NO metabolites, peroxynitrite and antioxidant power in 15 kidney transplanted patients before and after 4 months of treatment with carvedilol alpha 1-beta-blocker and potent antioxidant. Our aim was to study the efficacy of the reduction of oxidative stress on complications such as endothelial dysfunction and fibrogenesis. Monocyte ecNOS and plasma NO metabolites remained higher versus those of control subjects and were unchanged by carvedilol, while antioxidant power and heme oxygenase-1 mRNA production increased. Peroxynitrite, as well as transforming growth factor beta mRNA, were reduced by carvedilol. It also normalized blood pressure. In conclusion, carvedilol reduces oxidative stress and normalizes blood pressure; ecNOS remains upregulated while mRNA for transforming growth factor beta, a key fibrogenic cytokine, is reduced by carvedilol, which seems to preserve protective mechanisms such as NO and heme oxygenase-1 against long-term complications of oxidative stress, e.g., endothelial dysfunction, fibrogenesis and chronic rejection.

Abnormalities of Gq-mediated cell signaling in Bartter and Gitelman syndromes.

BACKGROUND: The constitutive endothelial isoform of nitric oxide synthase (ecNOS) and nitric oxide (NO) production are increased in patients with Bartter syndrome (BS) and Gitelman (GS) syndrome and may reduce vascular tone. Moreover, these patients present an abnormal cell signaling [reduced stimulated intracellular calcium ([Ca(2+)](i)) and inositol-1,4,5,triphosphate ([IP(3)](i)) in neutrophils], suggesting the presence of a generalized reduction of protein kinase C (PKC) and cell reactivity. Since PKC regulates ecNOS gene expression, we evaluated the signal transduction system involving Gq protein, PKC, and ecNOS in circulating nucleated cells from patients with BS/GS. METHODS: Nucleated blood cells from 2 BS and 7 GS and from 10 controls (C) were used. PKC activity was evaluated in neutrophils by radioenzymatic assay; PKC alpha concentration was evaluated in monocytes by Western blot analysis. ecNOS and G alpha q mRNA production was evaluated in monocytes by reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers and quantitated by PCR-based semiquantitative analysis of ecNOS and G alpha q mRNA expression. RESULTS: Cytosol and membrane basal PKC activity were similar in neutrophils from BS/GS and C (70 +/- 3 vs. 80 +/- 2; 37 +/- 3 vs. 46 +/- 2 pmol/min/mg protein, respectively), while fMLP-stimulated membrane PKC activity increased to a lower extent in BS/GS (from 43 +/- 2 to 53 +/- 3 vs. 38 +/- 2 to 66 +/- 3 pmol/min/mg protein, P < 0.05 for the difference). Membrane PKC alpha expression was similar in basal conditions (8.5 +/- 1.5 vs. 12.4 +/- 4.0 densitometric units), but increased after fMLP was reduced in BS/GS (4.5 +/- 1.4 vs. 9.5 +/- 2.1, P < 0.01). In BS/GS, PKC stimulation with PMA dose dependently reduced ecNOS gene expression (from 0.80 +/- 0.05 to 0.78 +/- 0.03 densitometric units; PMA 50 nmol/L, P = NS; to 0.55 +/- 0.07, PMA 100 nmol/L, P < 0.001) to an undetectable expression (PMA 200 nmol/L). Qualitatively similar effects were seen in monocytes from control subjects. Incubation of monocytes from patients and controls with the PKC inhibitor GF109203X increased ecNOS mRNA, with no difference between patients and controls. G alpha q mRNA was reduced in BS/GS versus controls (0.87 +/- 0.013 vs. 0.98 +/- 0.005 densitometric units, P < 0.0004). CONCLUSION: An abnormal G alpha q expression blunts cell signaling and PKC production in BS/GS. A reduced PKC up-regulated NO system may contribute to the vascular hyporeactivity of BS/GS.

Analysis of Gq protein alpha subunit mRNA expression in human monocytes: relevance of the purification step.

BACKGROUND: Galphaq is a member of the Gq family of G proteins, which by stimulating the phospholipase Cbeta (PLCbeta)-IP(3)-Ca(++) mediated intracellular signaling systems controls some of the most fundamental cardiovascular cellular processes. The study of Galphaq is complicated by the presence of a pseudogene in human DNA, with signficant homology to the mRNA encoding the alpha subunit of Gq protein. The presence of this pseudogene will cause problems when the analysis of the Galphaq gene expression is based solely on RT-PCR. In this study, we report a simple method for avoiding unwanted amplification of the Galphaq pseudogene and the use of human monocytes as a readily available source for examining Galphaq. METHODS: RT-PCR was carried out on RNA extracted from peripheral blood monocytes of 10 normal subjects using specific primers for Galphaq. RESULTS: When several subjects' Galphaq was examined, the authentic Galphaq mRNA amplification product levels, as a ratio to unpurified pseudogene containing amplification products, declined by up to approximately 70%. CONCLUSION: Given the importance of Gq protein in cardiovascular signal transduction, it is fundamental to provide a reliable assessment of G alpha q gene expression. In addition to accurately assessing Galphaq levels, the use of circulating human monocytes as a useful source of Galphaq for investigating mechanisms involved in the regulation of vascular tone is shown.