Elena Naso

Increased rho kinase activity in mononuclear cells of dialysis and stage 3-4 chronic kidney disease patients with left ventricular hypertrophy: Cardiovascular risk implications.

AIMS 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 clinical condition opposite to hypertension, its downregulation associates with lack of CV remodeling. Information on ROCK activation-LVH link in CKD and DP is lacking. MATERIALS AND METHODS Mononuclear cells (PBMCs) MYPT-1 phosphorylation, a marker of ROCK activity, and the effect of fasudil, a ROCK inhibitor, on MYPT-1 phosphorylation were assessed in 23 DPs, 13 stage 3-4 CKD and 36 healthy subjects (HS) by Western blot. LV mass was assessed by M-mode echocardiography. KEY FINDINGS DP and CKD had higher MYPT-1 phosphorylation compared to HS (p<0.001 and p=0.003). Fasudil (500 and 1000μM) dose dependently 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). SIGNIFICANCE In DP and CKD, ROCK activity tracks with LVH. This ROCK activation-LVH link provided in these CVD high-risk patients along with similar findings in hypertensive patients and added to opposite findings in a human model opposite to hypertension and in type 2 diabetic patients, identify ROCK activation as a potential LVH marker and provide further rationale for ROCK activation inhibition as target of therapy in CVD high-risk patients.

Intensive Home Hemodialysis: An Eye at the Past Looking for the Hemodialysis of the Future

Multiple observational studies along with a limited number of randomized clinical trials suggest that intensive hemodialysis (IHD) not only improves outcomes for uremic patients undergoing chronic dialysis but does so with a more favorable cost/benefit ratio compared with conventional hemodialysis. As a result of this, there has been a rapid increase in the interest in home hemodialysis (HHD) as HHD represents the easiest means of implementing IHD. While HHD has generated increased interest given its association with better outcomes/reduced hospitalizations, there are very few randomized controlled trials comparing HHD with other hemodialysis methods. Reported HHD-associated increased survival benefits compared with in-center hemodialysis are from uncontrolled studies, which raise patient selection bias as underlying the differences found. Thus, while HHD draws increasing attention, studies that pay careful attention to the psychosocial, demographic, and clinical factors associated with patients selected to undergo HHD will be needed to ultimately demonstrate its benefits, clarify the clinical applications, and determine the limits of IHD use in dialysis patients.

Assessing the Relationship of Angiotensin II Type 1 Receptors with Erythropoietin in a Human Model of Endogenous Angiotensin II Type 1 Receptor Antagonism.

Hypothesis/Introduction: Angiotensin II (Ang II) has been shown to control erythropoietin (EPO) synthesis as Ang II type 1 receptor (AT1R) blockers block Ang-II-induced EPO oversecretion. To further explore the involvement of AT1R in processes controlling EPO levels, plasma EPO and mononuclear cell NADPH oxidase 4 (NOX4) - a NOX family member involved in oxygen sensing, which is a process central to controlling EPO levels - were assessed in Bartters/Gitelmans syndrome (BS/GS) patients, a human model of endogenous AT1R antagonism and healthy subjects. Heme oxygenase (HO)-1, antioxidant and anti-inflammatory factor related to NOX4 activation, and the relationship of EPO and NOX4 to HO-1 were also assessed. Materials and Methods: EPO was measured by chemiluminescent immunoassay, HO-1 by sandwich immunoassay and NOX4 protein expression by Western blot. Results: EPO was increased in BS/GS patients compared to healthy subjects (7.64 ± 2.47 vs. 5.23 ± 1.07 U/l; p = 0.025), whereas NOX4 did not differ between BS/GS and healthy subjects (1.76 ± 0.61 vs. 1.65 ± 0.54 densitometric units; p = n.s.), and HO-1 was increased in BS/GS patients compared to healthy subjects (9.58 ± 3.07 vs. 5.49 ± 1.04 ng/ml; p = 0.003). NOX4 positively correlated with HO-1 only in BS/GS patients; no correlation was found between EPO and either NOX4 or HO-1 in those two groups. Conclusions: The effect of the renin-angiotensin system on EPO cannot be solely mediated by Ang II via AT1R signaling, but rather, EPO levels are also determined by a complex interrelated set of signals that involve AT2R, nitric oxide levels, NOX4 and HO-1 activity.

Pathophysiology of Post Transplant Hypertension in Kidney Transplant: Focus on Calcineurin Inhibitors Induced Oxidative Stress and Renal Sodium Retention and Implications with RhoA/Rho Kinase Pathway

Post-transplant hypertension is a common occurrence during treatment with calcineurin inhibitors (CNIs) in kidney transplant population. The pathogenesis of vasoconstriction induced by CNIs involves vascular tone alterations and kidney sodium transport regulation. Among the factors involved a key role is played by the activation of intrarenal renin-angiotensin system with enhanced release of Angiotensin II (Ang II) and increase of oxidative stress. A common pathway between oxidative stress and hypertension induced by CNIs may be identified in the involvement of the activation of RhoA/Rho kinase pathway, key for the induction of hypertension and cardiovascular-renal remodeling, of the oxidative stress mediated increased nitric oxide (NO) metabolism and increased renal sodium retention via increased activity of thiazide-sensitive sodium chloride cotransporter (NCC) in the distal tubule. We examined literature data including those coming from our group regarding the role of oxidative stress and sodium retention in CNIs induced hypertension and their involvement in cardiovascular-renal remodeling. Based on the available data, we have provided support to the activation of RhoA/Rho kinase pathway as an important effector in the pathophysiology of CNIs induced post-transplant hypertension via activation of oxidative stress and sodium retention. Clarification of how the biochemical and molecular mechanisms that regulate the processes involved in CNIs induced post transplant hypertension work and interact, would provide further insights not only into the comprehension of the pathophysiology of CNIs induced post transplant hypertension but could also have a positive impact on the clinical ground through the identification of significant targets. Their specific pharmacologic targeting might have multiple beneficial effects on the whole cardiovascular-renal function. The demonstration that in kidney transplanted patients with CNIs induced post-transplanted hypertension, the treatment of hypertension with different antihypertensive drugs inducing a comparable blood pressure reduction but different effects for example on oxidative stress and oxidative stress related proteins and/or Rho kinase and sodium retention, could be helpful for the choice of the antihypertensive treatment in these patients which takes advantage from effects of these drugs beyond blood pressure reduction.

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

PurposeGitelman’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.MethodsWe 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.ResultsNo mutations have been found in specific aminoacids of p63RhoGEF α6–αN linker and Gαq, key for p63RhoGEF/Gαq interplay.ConclusionsGitelman’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.

[OP.5C.05] INCREASED RHO KINASE ACTIVITY IN DIALYSIS AND STAGE 3–4 CHRONIC KIDNEY DISEASE PATIENTS WITH LEFT VENTRICULAR HYPERTROPHY: IMPLICATIONS WITH CARDIOVASCULAR RISK

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 Bartters/Gitelmans 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.