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.

Mechanistic approach to the pathophysiology of target organ damage in hypertension from studies in a human model with characteristics opposite to hypertension: Bartter's and Gitelman's syndromes.

INTRODUCTION: Extensive studies using Bartter's/Gitelman's syndrome patients have provided insights into the angiotensin II (Ang II) signaling pathways involved in the regulation of vascular tone and cardiovascular-renal remodeling. The renin-angiotensin-aldosterone system is activated in these syndromes, however, patients do not develop hypertension and cardiovascular remodeling and clinically manifest conditions opposite to hypertension. The short- and the long-term signaling of Ang II remains an important matter of investigation to shed light on mechanisms responsible for the pathophysiology of hypertension and its long-term complications. The long-term signaling of Ang II is involved in the pathophysiology of cardiovascular-renal remodeling and inflammatory responses in which the balance between RhoA/Rho kinase pathway and NO system plays a crucial role. METHODS AND RESULTS: In this brief review, the results of our studies in Bartter's and Gitelman's syndromes are reported on these processes. CONCLUSIONS: The information obtained from these studies can clarify, confirm or be used to extend the biochemical mechanisms responsible for the pathophysiology of hypertension and its long-term complications and could offer further chances to identify additional potential significant targets of therapy.

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.

Revisiting essential hypertension--a "mechanism-based" approach may argue for a better definition of hypertension.

Several major overarching themes have recently emerged in our understanding of the pathophysiology of hypertension which may allow to revisit essential hypertension with an eye towards the possibility of adopting a more rational "mechanistic-based" definition of hypertension and moving away from the unsatisfactory "essential" label for hypertension from unknown cause. As our understanding of the biochemical and physiological mechanisms that control blood pressure rapidly evolves, the "essential" label of hypertension is losing both value as well as utility as it will describe an increasingly small number of hypertensive patients. This paper uses some recently identified pathways central to hypertension and uses this understanding of pathophysiology to argue for a better definition of hypertension.

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.

Myocardial perfusion defects in Bartter and Gitelman syndromes.

BACKGROUND: Normotensive hypokalaemic tubulopathies (Bartter and Gitelman syndromes (BS/GS)) are genetic diseases that are considered benign. However, QT prolongation, left ventricular dysfunction and reduction of cardiac index upon exercise leading to arrhythmias and sudden cardiac death have been reported in these patients. Hence, we aimed to verifying whether an isometric exercise could represent a useful tool for the identification of patients at risk for future cardiac events. PATIENTS AND METHODS: Myocardial function (MF) and perfusion, evaluated as myocardial blood flow (MBF) of 10 BS/GS patients and 10 healthy controls, were investigated at rest and during isometric exercise. MF and MBF were evaluated using quantitative two-dimensional and myocardial contrast echocardiography. RESULTS: BS/GS patients had normal baseline MF and MBF. During exercise in BS/GS patients, corrected QT (QTc) was prolonged to peak value of 494 +/- 9.1 ms (P < 0.001). In controls, MF increased from resting to peak exercise (left ventricular ejection fraction: 65 +/- 4% to 78 +/- 5%, P < 0.003) while in seven BS/GS patients (Group 1) it declined (64 +/- 5% to 43 +/- 9%, P < 0.001). Myocardial perfusion increased upon exercise in controls as shown by changes of its markers: beta (a measure of myocardial flow velocity; 0.89 +/- 0.12 vs. 0.99 +/- 0.12, P < 0.001) and myocardial blood volume (14.4 +/- 2 vs. 20.2 +/- 0.25, P < 0.001), while in Group 1 BS/GS it decreased (0.87 +/- 0.15 vs. 0.67 +/- 0.15, P < 0.001; and 14.5 +/- 1.9 vs. 8.3 +/- 0.22, P < 0.001, respectively). CONCLUSIONS: Our results document for the first time that exercise induce coronary microvascular and myocardial defects in BS/GS patients. Therefore, this may challenge the idea that BS/GS are benign diseases. In addition, the diagnostic approach to these syndromes should include an in-depth cardiac assessment in order to identify patients at higher risk.

Intravenous thrombolysis in the emergency department for the treatment of acute ischaemic stroke.

BACKGROUND AND AIMS: Thrombolytic therapy with intravenous recombinant tissue plasminogen activator (rt-PA) improves outcome in patients with ischaemic stroke treated within 3 h of symptom onset, but its extended implementation is limited. A pilot study was designed to verify whether evaluation of patients with acute ischaemic stroke and their treatment with intravenous rt-PA in the emergency department (ED), followed by transportation to a semi-intensive stroke care unit, offers a safe and effective organisational solution to provide intravenous thrombolysis to acute stroke patients when a stroke unit (SU) is not available. METHODS: After checking for inclusion and exclusion criteria, ED doctors contacted the stroke team with a single page, located family members and urgently obtained computed tomography scan and laboratory tests. A stroke team investigator clinically assessed the patient, obtained written informed consent and supervised intravenous rt-PA in the ED. After treatment, the patient was transferred to the SU for rehabilitation and treatment of complications, under supervision of the same stroke team investigator. RESULTS: 52 patients were treated with intravenous rt-PA within 3 h of symptom onset. 20 patients (38%) improved neurologically after 24 h, the number increased to 30 (58%) after one week. At 3 months 22 patients had a favourable outcome (43%). The 3-month mortality rate was 12%. Symptomatic cerebral haemorrhage was observed in two patients (4%). CONCLUSIONS: Intravenous rt-PA administration in the ED is an effective organisational solution for acute ischaemic stroke when an SU is not established.

A new approach to the evaluation of hyperphosphatemia in chronic kidney disease.

AIMS: Hyperphosphoremia, main contributor to cardiovascular calcifications, has a major impact on the morbidity and mortality of chronic renal failure (CRF) patients. Phosphate binders and dietary phosphate limitation are not effective enough to abolish hyperphosphoremia-induced cardiovascular abnormalities, therefore, the identification of other and more timely approaches for serum phosphorous reduction is necessary. Salivary fluid contains phosphate which, if related to the daily salivary secretion (1,000 - 1,800 ml), deserves attention as a marker for an earlier start of pharmacologic treatment for phosphorous removal. In ESRD patients under dialysis we have shown increased salivary phosphate closely to be related with serum phosphorous and interpreted as compensatory. This study evaluates salivary phosphate secretion in 77 nondialyzed CRF compared with healthy subjects and its relationship with renal function. METHODS: Saxon's test confirmed normal salivary function in patients and controls. Serum phosphorous, creatinine and GFR were also measured. RESULTS: Salivary phosphorous was significantly higher in CRF patients compared with controls: 38.60 mg/dl (range 12.20 - 95.60) vs 16.30 (10.30 - 27.10), p < 0.0001; serum phosphate was also significantly higher: 3.70 (2.10 - 6.80) vs 3.50 (2.3 4.6), p = 0.013. In CRF patients, salivary phosphorous positively correlated with serum phosphorous (r - 0.45, p < 0.0001) and with serum creatinine (r = 0.72, p < 0.0001), while negatively correlated with GFR (r = -0.72, p < 0.0001). CONCLUSIONS: The results of our study show also in CRF patients increased salivary phosphate secretion, which is related with renal function. On this basis the use of salivary phosphate secretion as a marker for an earlier start of the abnormal phosphate, metabolism pharmacologic treatment could be proposed.