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.
•Methods to measure activity concentrations of radionuclides through gamma spectrometry in drinking water are proposed.•These methods allow the simultaneous determination of all gamma emitters and produce high precision results.•A new sampling method for 222Rn analysis in water with gamma spectrometry is presented.•The radionuclides activity concentrations in drinking water in the study area was measured in 85 samples.•Radon distribution in drinking water in North East Italy was studied on 85 samples.
OBJECTIVE:Evidence on cellular and molecular mechanisms that lead to atrial fibrillation (AF) are scanty. Increased expression of ROCK and myosin phosphatase target subunit 1 (MYPT-1), involved in ...ROCK signaling were shown in left atrial appendage biopsies of AF patients, which correlated with the expression of Connexin40 (Cx40), an integral membrane protein of heart gap junctions, fundamental for the rapid cell-cell transfer of action potential. AF is the most frequent cardiac arrhythmia in dialysis patients who, we demonstrated to present increased MYPT-1 phosphorylation, marker of ROCK activity, which correlated with their LV mass. Given the known role of ROCK in cardiovascular (CV)-renal remodeling together with its induction of impairment of cell-to-cell coupling and conduction of potential promoting AF initiation and perpetuation by reducing atrial wavelength, we evaluated in dialysis patients with AF, the phosphorylation state of MYPT-1, the expression of Cx40 and their relationship to support their mechanistic involvement in AF induction.
DESIGN AND METHOD:11 AF-dialysis patients (AFDP) (chronic bicarbonate dialysis 3 times/week), 8 males, 3 females, 49–91yo, 11 sinus rhythm-dialysis patients (DP), 7 males, 4 females, 53–91yo, and 11 healthy subjects (C), 7 males, 4 females, 26–38yo were enrolled. Mononuclear cells MYPT-1 phosphorylation and Cx40 expression were determined by Western blot. Data were expressed as mean ± SE and evaluated by unpaired data ANOVA.
RESULTS:In AFDP MYPT-1 phosphorylation was increased vs DP and C (1.57 ± 0.17 du vs 0.69 ± 0.04 vs 0.51 ± 0.05 respectively, p < 0.0001). MYPT-1 phosphorylation in DP was also higher vs C, p = 0.009. Cx40 was higher in AFDP (1.23 ± 0.12 vs 0.74 ± 0.03 vs 0.69 ± 0.03, p < 0.0001). MYPT-1 phosphorylation correlated with Cx40 expression (r = 0.67, p < 0.001) in all dialysis patients.
CONCLUSIONS:These data confirm at mechanistic level in dialysis patients a role for ROCK increased activity and Cx40 expression association as a possible mechanism leading to induction of AF. The correlation between MYPT-1 phosphorylation and Cx40 in all dialysis patients further strengthens this mechanistic relationship for AF induction. The clarification, on a mechanistic basis, of this relationship might give further insights for additional pharmacologic targeting of AF.
OBJECTIVE:Aldosterone has a known profibrotic and proinflammatory effect on cardiovascular system essentially via activation of oxidative stress.Gitelman syndrome (GS) is an autosomal recessive ...tubulopathy characterized by hypokalemia, activation of renin-angiotensin-aldosterone system with secondary hyperaldosteronism yet normal-hypotension and lack of cardiovascular remodeling, a picture opposite to hypertension.
DESIGN AND METHOD:In a previous study we have characterized mineralcorticoid receptor (MR) in mononuclear cells (MNL) and subsequently we have reported that healthy subjects’ MNL incubated with aldosterone increased oxidative stress in terms of protein expression of p22phox, which was blocked by co-incubation with canrenone.In the present study we evaluated the inflammatory-proliferative effect of aldosterone co-incubation (1 × 10–8 M) in MNL of 6 GS patients, a human model opposite to hypertension, compared with healthy subjects, via the protein expression of p22phox and phosphorylation of MYPT-1, a marker of RhoA/Rho kinase activation and its inhibition by canrenone (1 × 10–6 M).
RESULTS:In GS patients p22phox was unchanged by aldosterone (vehicle 0.33 ± 0.13 d.u. vs aldosterone 0.36 ± 0.17 p:ns; aldosterone + canrenone 0.45 ± 0.08 p:ns). Confirming prevoius data, in healthy subjects aldosterone increased p22phox (baseline 0.91 ± 0.06 vs aldosterone 1.11 ± 0.09 p = 0.02), which was reversed by canrenone (0.90 ± 0.12).In GS phosphorylation of MYPT-1 was not increased by aldosterone (vehicle 0.69 ± 0.07 vs aldosterone 0.83 ± 0.12 p:ns) while in healthy subjects it was increased (vehicle 1.16 ± 0.1 vs aldosterone 1.37 ± 0.1 p = 0.04)
CONCLUSIONS:Data in GS, confirming previous observations with angiotensin II, show that aldosterone does not induce oxidative stress/oxidative-stress signaling and associated inflammation/proliferation, which are shown in healthy subjects. These results in a human model with lack of cardiovascular remodeling opposite to hypertension as GS, further highlights the relevance of the proinflammatory/cardiovascular remodeling effects of aldosterone.
Abstract 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.
OBJECTIVE:Cardiovascular disease (CVD) is the leading cause of excess mortality in chronic kidney disease (CKD) and dialysis patients (DP) who have higher prevalence of left ventricular hypertrophy ...(LVH), the strongest predictor of CV events. Rho kinase (ROCK) activation is linked in hypertensive patients to cardiac remodeling while ROCK inhibition suppresses cardiomyocyte hypertrophy and, in a human model opposite to hypertension such as Bartterʼs/Gitelmanʼs syndromes patients, its downregulation associates with lack of CV remodeling. Information on ROCK activation-LVH link in CKD and DP is lacking.
DESIGN AND METHOD:Mononuclear cells (PBMCs) MYPT-1 phosphorylation, a marker of ROCK activity, and the effect of fasudil (500 and 1000 uM), a ROCK inhibitor, on MYPT-1 phosphorylation were assessed in 23 DP 42 and 75 years, 16 males and 7 females, 13 stage 3–4 CKD, 8 males and 5 females, 45–70 years and 30 healthy subjects (HS), 31–65 years, 20 males and 10 females, by Western blot. LV mass was assessed by M-mode echocardiography.
RESULTS:DP and CKD had higher MYPT-1 phosphorylation compared to HS (p < 0.001 and p = 0.003). Fasudil (500 and 1000 uM) reduced MYPT-1 phosphorylation in DP (p < 0,01). DP had higher LV mass than CKD (p < 0.001). MYPT-1 phosphorylation was higher in patients with LVH (p = 0.009) and correlated with LV mass both in DP and CKD with LVH (p < 0.001 and p = 0.006, respectively).
CONCLUSIONS:This study provides evidence linking the activation of ROCK, as reflected by the increased phosphorylation state of MYPT-1, to cardiac hypertrophy in dialysis and stage 3–4 CKD patients, a human clinical population at high risk for cardiovascular morbidity and mortality. The results of this study join those provided in another high risk for cardiovascular disease patients such as hypertensive patients and receive indirect support from data provided by a human model opposite to hypertension and type II diabetic patients identifying ROCK activation as a potential LVH marker and providing further rationale for ROCK activation inhibition as target of therapy in patients at high risk for cardiovascular disease such as CKD, dialysis, hypertensive and diabetic patients.
OBJECTIVE:Gitelmanʼs syndrome (GS) is a rare autosomal disease characterized by electrolytic alterations. Clinical features are hyperkalemia, hypomagnesemia and hypocalciuria, and hypo- or ...normotension notwithstanding marked activation of the renin-angiotensin-aldosterone system. These abnormalities are due to mutations affecting the thiazide sensitive Na-Cl cotransporter (NCC), which reabsorbs sodium in the distal convoluted tubule of the nephron. Over 100 mutations are known, but the molecular mechanisms underlying the impaired function remained unclear.In our cohort of GS a young woman presented with a NCC point mutation (c.1204G > A) never reported before, which causes an aminoacid exchange (Gly394Asp). Therefore, we used a molecular biology approach to investigate how this mutation affected NCC functionality.
DESIGN AND METHOD:We created different expression vectors containing either the wild type or the mutated NCC (SLC12A3) coding sequence. The DNA was then transfected into HEK 293 cells and RNA into Xenopus laevis oocytes. We then assessed the expression, maturation, and the trafficking of the protein by western blot, immunohistochemistry (IHC), and confocal microscopy, and the NCC functionality by Na22+ uptake.
RESULTS:Western blots show more expressed glycosylation bands in wild type than in mutated NCC at 24 (p = 0,003 NCC wt mean + SEM 0,233 ± 0,046; NCC mut mean + SEM 0,0268 ± 0,008) and 48 hours after transfection (p = 0,0003 NCCwt 0,524 ± 0,084; NCC mut 0,059 ± 0,012), suggesting impaired maturation of the NCC mutated protein. This result was confirmed with confocal microscopy in transfected oocytes that showed apical NCC staining in wild type and NCC retention in the inner portion in mutated oocytes. Furthermore, preliminary Na22+ uptake experiments prove impaired function of the mutated NCC compared to wild type NCC.
CONCLUSIONS:These results suggest that this novel GS mutation leads impaired NCC glycosylation with ensuing retention in the endoplasmic reticulum, compromised trafficking to the plasma membrane, and diminished Na reabsorption.
Purpose
Aldosterone proinflammatory/profibrotic effects are mediated by the induction of mononuclear leucocytes (MNL) to express oxidative stress (OxSt)-related proteins, such as p22
phox
, 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
p22
phox
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, p22
phox
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 p22
phox
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.
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.
Abstract 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)-α 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-α and CSF1, as shown in both inflammatory and neoplastic conditions. The effects of a new therapeutic approach consisting in intraarticular TNF-α 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-α blockade in the diffuse form of pigmented villonodular synovitis.
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