Settimo Milanese, Italy
Settimo Milanese, Italy

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Citterio L.,Vita-Salute San Raffaele University | Ferrandi M.,Prassis Sigma tau Research Institute | Delli Carpini S.,Vita-Salute San Raffaele University | Simonini M.,Vita-Salute San Raffaele University | And 10 more authors.
Hypertension | Year: 2013

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na + reabsorption via a direct action on basolateral Na-K ATPase and luminal Na-H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressurenatriuresis in patients. Naive hypertensive patients (n=574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na+ loading, and the slope of the pressure-natriuresis relationship between blood pressure and Na+ excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure-natriuresis curve (0.017±0.004 μEq/mm Hg per minute) compared with the ACC (0.0013±0.003 μEq/mm Hg per minute; P=0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r=0.83; P<0.001) or α1 Na-K ATPase (r=0.557; P<0.01) and between α1 Na-K ATPase and Na-H exchanger type 3 (r=0.584; P<0.01) or Src (r=0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n=23) but not ACC (n=14) variants. A functional signaling complex among PRKG1, α1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na + reabsorption, suggestive of a blunt pressure-natriuresis response. © 2013 American Heart Association, Inc.


Ferrandi M.,Prassis Sigma Tau Research Institute | Ferrandi M.,San Raffaele Scientific Institute | Molinari I.,Prassis Sigma Tau Research Institute | Molinari I.,San Raffaele Scientific Institute | And 6 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2014

Glomerulopathies are important causes of morbidity and mortality. Selective therapies that address the underlying mechanisms are still lacking. Recently, two mechanisms, mutant β-adducin and ouabain, have been found to be involved in glomerular podocytopathies and proteinuria through nephrin downregulation. The main purpose of the present study was to investigate whether rostafuroxin, a novel antihypertensive agent developed as a selective inhibitor of Src-SH2 interaction with mutant adducinand ouabain-activated Na,K-ATPase, may protect podocytes from adducin-and ouabain-nduced effects, thus representing a novel pharmacologic approach for the therapy of podocytopathies and proteinuria caused by the aforementioned mechanisms. To study the effect of rostafuroxin on podocyte protein changes and proteinuria, mice carrying mutant β-adducin and ouabain hypertensive rats were orally treated with 100 μg/kg per day rostafuroxin. Primary podocytes from congenic rats carrying mutant a-adducin or β-adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10-9M ouabain were cultured with 10-9M rostafuroxin. The results indicated that mutant β-adducin and ouabain caused podocyte nephrin loss and proteinuria in animal models. These alterations were reproduced in primary podocytes from NB rats and normal rats incubated with ouabain. Treatment of animals, or incubation of culturedpodocytes with rostafuroxin, reverted mutant β-adducin-and ouabain-induced effects on nephrin protein expression and proteinuria. We conclude that rostafuroxin prevented podocyte lesions and proteinuria due to mutant β-adducin and ouabain in animal models. This suggests a potential therapeutic effect of rostafuroxin in patients with glomerular disease progression associated with these two mechanisms. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.


Ferrandi M.,Prassis Sigma tau Research Institute | Molinari I.,Prassis Sigma tau Research Institute | Torielli L.,Prassis Sigma tau Research Institute | Padoani G.,Prassis Sigma tau Research Institute | And 5 more authors.
Science Translational Medicine | Year: 2010

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na+- and K+-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.


Ferrandi M.,Prassis Sigma tau Research Institute | Ferrandi M.,San Raffaele Scientific Institute | Molinari I.,Prassis Sigma tau Research Institute | Molinari I.,San Raffaele Scientific Institute | And 9 more authors.
Human Molecular Genetics | Year: 2014

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant β-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant β-adducin and ouabain, byusingcongenic substrains of the Milan rats expressing eithermutanta-orb-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant β-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of β-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans. © The Author 2014. Published by Oxford University Press. All rights reserved.


PubMed | Prassis Sigma Tau Research Institute, San Raffaele Scientific Institute and Renal Research Laboratory
Type: Journal Article | Journal: International journal of molecular sciences | Year: 2016

Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, 1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 g/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 g/kg/day rostafuroxin for four weeks. In group 1, 2-3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO.


Linde C.I.,University of Maryland Baltimore County | Karashima E.,University of Maryland Baltimore County | Raina H.,University of Maryland Baltimore County | Zulian A.,University of Maryland Baltimore County | And 5 more authors.
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2012

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na + reab-sorption. Recently we demonstrated that Ca 2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na +/Ca 2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca 2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca 2+ signals in response to 5 (xM PE or ATP in the absence and presence of extracellular Ca 2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca 2+ release and increased Ca 2+ entry, respectively. The increased SR Ca 2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ~70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca 2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca 2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. © 2012 by the American Physiological Society.


Zulian A.,University of Maryland Baltimore County | Baryshnikov S.G.,University of Maryland Baltimore County | Linde C.I.,University of Maryland Baltimore County | Hamlyn J.M.,University of Maryland Baltimore County | And 2 more authors.
American Journal of Physiology - Heart and Circulatory Physiology | Year: 2010

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca2+ signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca2+ signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 ± 2 vs. 113 ± 1 mmHg, P < 0.05). Resting cytosolic free Ca2+ concentration (measured with fura-2) and ATP-induced Ca2+ transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba2+ entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na+/Ca2+ exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by ∼13-fold), whereas the expression of plasma membrane Ca2+-ATPase was not altered. Ca2+ entry via the reverse mode of NCX1 evoked by the removal of extracellular Na+ induced a rapid increase in cytosolic free Ca2+ concentration that was significantly larger in MHS arterial myocytes. The expression of α1/α2 Na+ pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca2+ signaling. We suggest that the increased Ca2+ signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats. Copyright © 2010 the American Physiological Society.


Pulina M.V.,University of Maryland, Baltimore | Zulian A.,University of Maryland, Baltimore | Baryshnikov S.G.,University of Maryland, Baltimore | Linde C.I.,University of Maryland, Baltimore | And 5 more authors.
Advances in Experimental Medicine and Biology | Year: 2013

Arterial smooth muscle (ASM) Na+/Ca2+ exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na+ pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na+ metabolism, Ca2+ signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention. © Springer Science+Business Media New York 2013.


Bignami E.,Vita-Salute San Raffaele University | Casamassima N.,Vita-Salute San Raffaele University | Frati E.,Vita-Salute San Raffaele University | Lanzani C.,Vita-Salute San Raffaele University | And 13 more authors.
Critical Care Medicine | Year: 2013

OBJECTIVES: Acute kidney injury is a frequent complication of cardiac surgery and increases morbidity and mortality. As preoperative biomarkers predicting the development of acute kidney injury are not available, we have tested the hypothesis that preoperative plasma levels of endogenous ouabain may function as this type of biomarker. RATIONALE AND DESIGN: Endogenous ouabain is an adrenal stress hormone associated with adverse cardiovascular outcomes. Its involvement in acute kidney injury is unknown. With studies in patients and animal settings, including isolated podocytes, we tested the above mentioned hypothesis. PATIENTS: Preoperative endogenous ouabain was measured in 407 patients admitted for elective cardiac surgery and in a validation population of 219 other patients. We also studied the effect of prolonged elevations of circulating exogenous ouabain on renal parameters in rats and the influence of ouabain on podocyte proteins both "in vivo" and "in vitro." MAIN RESULTS: In the first group of patients, acute kidney injury (2.8%, 8.3%, 20.3%, p < 0.001) and ICU stay (1.4±0.38, 1.7±0.41, 2.4±0.59 days, p = 0.014) increased with each incremental preoperative endogenous ouabain tertile. In a linear regression analysis, the circulating endogenous ouabain value before surgery was the strongest predictor of acute kidney injury. In the validation cohort, acute kidney injury (0%, 5.9%, 8.2%, p < 0.0001) and ICU stay (1.2±0.09, 1.4±0.23, 2.2±0.77 days, p = 0.003) increased with the preoperative endogenous ouabain tertile. Values for preoperative endogenous ouabain significantly improved (area under curve: 0.85) risk prediction over the clinical score alone as measured by integrate discrimination improvement and net reclassification improvement. Finally, in the rat model, elevated circulating ouabain reduced creatinine clearance (-18%, p < 0.05), increased urinary protein excretion (+ 54%, p < 0.05), and reduced expression of podocyte nephrin (-29%, p < 0.01). This last finding was replicated ex vivo by incubating podocyte primary cell cultures with low-dose ouabain. CONCLUSIONS: Preoperative plasma endogenous ouabain levels are powerful biomarkers of acute kidney injury and postoperative complications and may be a direct cause of podocyte damage. Copyright © 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins.


PubMed | Prassis Sigma Tau Research Institute, Karolinska Institutet, Osaka National Research Institute, Karolinska University Hospital and 3 more.
Type: Journal Article | Journal: PloS one | Year: 2014

Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of -adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, -adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that -adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2+/+ but not in sik2-/- mice. Thus LVH triggered by HS or the presence of a genetic variant of -adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.

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