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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 adducin- and ouabain-activated Na,K-ATPase, may protect podocytes from adducin- and ouabain-induced 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 α-adducin or β-adducin (NB) from Milan hypertensive rats and normal rat podocytes incubated with 10(-9) M ouabain were cultured with 10(-9) M 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 cultured podocytes 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.

Background and Purpose Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca2+ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity.

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, α1 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 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/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.

The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.

In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 microg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of alpha1, beta1, gammaa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat alpha1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 microg/kg/day in vivo or 10(-10)-10(-8) m in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.

PST 2238 is a new antihypertensive compound that is able to correct the molecular and functional alterations of the renal Na-K pump and the pressor effect associated with either alpha-adducin mutations or high circulating levels of endogenous ouabain (EO) in genetic and experimental rat models. Due to the close relationship between renal Na-K pump function and tubular Na reabsorption, PST 2238 was investigated to determine whether it is endowed with diuretic activity and consequently might trigger alterations of the renin-aldosterone system and the carbohydrate and lipid metabolism often associated with chronic diuretic therapy. In Milan hypertensive (MHS) rats, in which hypertension is genetically associated with alpha-adducin mutation, increased tubular Na reabsorption, and hyperactivation of the renal Na-K pump. PST 2238 reduced blood pressure and normalized the renal Na-K pump activity at oral doses of micro g/kg, but did not induce, either acutely or chronically, any diuretic activity or hormonal or metabolic alterations. In contrast, HCTZ, given to MHS rats orally at 40 mg/kg, although it displayed diuretic activity and reduced the renal Na-K pump activity, did not lower blood pressure and caused hyperactivation of the renin-aldosterone system, hypokaliemia, and hyperglycemia. The findings lead to the conclusion that PST 2238 is a new antihypertensive compound that normalizes the altered function of the renal Na-K pump associated with hypertension in rat models, but that it is devoid of diuretic activity and does not induce the diuretic-associated side effects.

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, by using congenic substrains of the Milan rats expressing either mutant α- or β-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.

Recent evidence of beneficial effects of ranolazine (RAN) in type II diabetes motivates interest in the role of the late sodium current (INaL) in glucose-stimulated insulin secretion. In the present work, we characterize INaL and its function in rat INS-1E cells and human islets cells. INaL was identified as steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM tetrodotoxin (TTX) (ITTX). Veratridine (VERA, 40 μM) was used as INaL enhancer. Baseline INaL was similar between INS-1E and human islet cells. In INS-1E cells, activated by glucose or tolbutamide, TTX or RAN hyperpolarized membrane potential (V m). VERA-induced depolarization was countered by TTX or RAN. ITTX and IRAN reversal potentials were negative to Na+ equilibrium one, but they approached it after Na+ substitution with Li+ or when K+ channels were blocked. This revealed INaL coupling with Na+-activated K+ current (IKNa); expression of IKNa channels (Slick/Slack) was confirmed by transcript analysis and Western blot. RAN or TTX blunted cytosolic Ca2+ response to depolarization. Long-term incubation in high (33 mM) glucose (CHG) constitutively enhanced INaL. VERA immediately increased glucose-stimulated insulin secretion. CHG increased glucose-independent secretion instead and abolished the secretory response to glucose. RAN or TTX countered VERA- and CHG-induced changes in insulin secretion. Our study demonstrated that (1) INaL was expressed in insulin-secreting cells and coupled to IKNa; INaL affected cytosolic Ca2+ but, unless enhanced, barely contributed to glucose-stimulated insulin secretion (GSIS); and (2) sustained hyperglycemic stress enhanced INaL, which contributed to the attending increase of glucose-independent insulin “leak” and GSIS impairment.

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose. Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension., Highlights • We identified urinary proteins differently excreted in hypertensive patients. • Nephrin 1 might predict salt sensitive phenotype and glomerular complications. • Uromodulin impacts salt homeostasis in hypertension. • We provide new insights into the pathogenesis of hypertension and salt sensitivity.

BACKGROUND: enhancement of the sustained component of the sodium current (INaL) is a major factor contributing to myocardial damage. A beneficial effect of the INaL blocker Ranolazine (RAN) on glycemic control was recently described in the MERLIN-TIMI 36 trial in diabetic patients. However, the mechanism underlying this finding hasn't been elucidated yet.AIM: To characterize INaL in insulin-secreting cells (INS-1E) and evaluate its role in glucose-stimulated insulin secretion (GSIS).METHODS: INaL, identified as the steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM TTX (ITTX), and its impact on membrane potential (Vm) were assessed by patch clamp. Veratridine (VERA) was used as INaL enhancer. INaL-dependent intracellular Ca2+ changes were detected by Fluo-4AM. To simulate hyperglycemic stress (CHG), INS-1E cells were exposed to 33 mM glucose for 24 hrs. GSIS was measured by HTRF assay.RESULTS: Glucose- and tolbutamide-triggered action potentials were suppressed by TTX, but not by RAN. VERA caused depolarization, countered by INaL blockade. The reversal potentials of ITTX and IRAN were negative to Na+ equilibrium potential, but they approached it when K+-channels were blocked. This revealed INaL coupling to Na+-activated K+ current (IKNa); expression of IKNa channels (Slo2.1/2.2) was confirmed by transcript analysis. INaL blockade blunted cytosolic Ca2+ response to depolarization. CHG enhanced INaL. Whereas acute INaL enhancement (VERA) increased GSIS, chronic one (CHG or VERA) depressed GSIS, which was partially restored by RAN.CONCLUSIONS: INaL is expressed in insulin-secreting cells, is coupled to IKNa , affects Ca2+ signalling and, when enhanced acutely, increases GSIS. However, constitutive INaL enhancement, induced by chronic hyperglycemic stress, and leads to GSIS depression instead. Overall, chronic INaL enhancement may represent a mechanism of damage progression common to myocardial and insulin-secreting cells.

Adducins are cytoskeletal actin-binding proteins (α, β, γ) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates α- and β-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of α- and β-adducin on glomerular function and disease using β-adducin null mice, congenic substrains for α- and β-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of β-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, α-actinin, ZO-1, Fyn). The introgression of polymorphic MHS β-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, α-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS α-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic β-adducin (ADD2, 1797T, rs4984) with a significant interaction with α-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00109-009-0549-x) contains supplementary material, which is available to authorized users.

A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K-ATPase function both in vivo and in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na-K pump selectively in those rat models carrying the adducin mutation (MHS) and/or increased EO levels (OS) at oral doses of 0.1-10 micro g/kg. In NRK cells either transfected with mutated adducin or incubated with 10(-9) M OU, PST normalizes the Na-K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10% increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 micro g/kg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU-dependent activation of growth-related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na-K pump function.

The Milan hypertensive strain (MHS) of rats, in addition to having hypertension, is also characterized by a genetic deficiency in calpastatin, the endogenous inhibitor of calpain. Since this protease has been implicated in long-term potentiation (LTP), we have investigated whether induction of this form of plasticity was altered in this strain of rats as compared to control animals (Milan normotensive strain, MNS). Progressive induction of LTP by increasing numbers of high frequency trains resulted in a greater degree of potentiation measured with all inducing protocols in MHS as compared with MNS animals. This difference was not related to the hypertension, since another hypertensive strain (the SHR strain) and a segregated Milan hypertensive strain, expressing only the hypertension but not the calpastatin deficiency (the MHNE strain), exhibited an LTP indistinguishable from control rats. Treatment of MHNE rats for 2 months with isovalerylcarnitine, a compound that increases calpain activity, also resulted in a greater amount of LTP induced by high frequency trains. These effects were not related to an enhancement of the NMDA receptor dependent component of responses to burst stimulation. These results are consistent with the idea that conditions under which calpain activation is facilitated are associated with a greater degree of synaptic potentiation.

We studied calpastatin activity in erythrocytes of Milan hypertensive and prehypertensive rats, in their normotensive controls, in F1 and F2 hybrids, and in two inbred strains derived from F2, one hypertensive and the other normotensive. Our results show that the decrease in calpastatin activity observed in Milan hypertensive rats was not caused by hypertension, it was transmitted in a recessive way in heterozygous, and it was not correlated to hypertension.

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 pressure-natriuresis 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 r =0.557; P r =0.584; P r =0.691; P + reabsorption, suggestive of a blunt pressure–natriuresis response.