Physiological Functions and Regulation of the Na+/H+ Exchanger [NHE1] in Renal Tubule Epithelial Cells

The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival.Fil: Garramuño, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gil Lorenzo, Andrea Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Costantino, Valeria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Heat shock protein 70 and CHIP promote Nox4 ubiquitination and degradation within the losartan antioxidative effect in proximal tubule cells

Background: Angiotensin II/Angiotensin II type 1 receptor (AT1R) effects are dependent on ROS production stimulated by NADPH oxidase activation. Hsp70 regulates a diverse set of signaling pathways through their interactions with proteins. CHIP is a E3 ubiquitin ligase that targets proteins for polyubiquitination and degradation. Aim: We study whether Hsp70/CHIP contribute to the negative regulation of Nox4 after AT1R blockage. Methods/Results: Primary culture of proximal tubule epithelial cells (PTCs) from SHR and WKY were stimulated with Angiotensin II (AII) or treated with Losartan (L) or Losartan plus Angiotensin II (L+AII). Losartan decreased AT1R and Nox4 while enhancing caveolin-1 and Hsp70 protein expression in SHR PTCs. Immunoprecipitation and immunofluorescence proved interaction and colocalization of increased Hsp70/CHIP with decreased Nox4 in SHR PTCs (L) vs (All). Hsp72 knockdown resulted in enhanced Nox4 protein levels, NADPH oxidase activity and ROS generation in (L+AII) revealing that Losartan was unable to abrogate AII effects on Nox4 expression and oxidative activity. Moreover, MG132 exposed PTCs (L) demostrated blocked ubiquitinated Nox4 degradation and increased colocalization of Nox4/Ubiquitin by inmunofluorescence. Conversely, Hsp72 depletion reduced Nox4/Ubiquitin colocalization causing Nox4 upregulation due to proteosomal degradation inhibition, although Losartan treatment. Conclusion: Our study demonstrates that Hsp70 and CHIP mediates the ubiquitination and proteasomal degradation of Nox4 as part ofthe antioxidative effect of Losartan in SHR.Fil: Costantino, Valeria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gil Lorenzo, Andrea Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Lopez Appiolaza, Carlos Martìn. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Cacciamani, Valeria. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benardon, María Eugenia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; ArgentinaFil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Garramuño, Patricia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Patológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Urinary renin in patients and mice with diabetic kidney disease

In patients with diabetic kidney disease (DKD), plasma renin activity is usually decreased, but there is limited information on urinary renin and its origin. Urinary renin was evaluated in samples from patients with longstanding type I diabetes mellitus and mice with streptozotocin-induced diabetes mellitus. Renin-reporter mouse model (Ren1d-Cre;mT/ mG) was made diabetic with streptozotocin to examine whether the distribution of cells of the renin lineage was altered in a chronic diabetic environment. Active renin was increased in urine samples from patients with DKD (n=36), compared with those without DKD (n=38; 3.2 versus 1.3 pg/mg creatinine; P<0.001). In mice with streptozotocin-induced diabetes mellitus, urine renin was also increased compared with nondiabetic controls. By immunohistochemistry, in mice with streptozotocin-induced diabetes mellitus, juxtaglomerular apparatus and proximal tubular renin staining were reduced, whereas collecting tubule staining, by contrast, was increased. To examine the role of filtration and tubular reabsorption on urinary renin, mice were either infused with either mouse or human recombinant renin and lysine (a blocker of proximal tubular protein reabsorption). Infusion of either form of renin together with lysine markedly increased urinary renin such that it was no longer different between nondiabetic and diabetic mice. Megalin mRNA was reduced in the kidney cortex of streptozotocin-treated mice (0.70±0.09 versus 1.01±0.04 in controls, P=0.01) consistent with impaired tubular reabsorption. In Ren1d-Cre;mT/mG with streptozotocin-induced diabetes mellitus, the distribution of renin lineage cells within the kidney was similar to nondiabetic renin-reporter mice. No evidence for migration of cells of renin linage to the collecting duct in diabetic mice could be found. Renin mRNA in microdissected collecting ducts from streptozotocin-treated mice, moreover, was not significantly different than in controls, whereas in kidney cortex, largely reflecting juxtaglomerular apparatus renin, it was significantly reduced. In conclusion, in urine from patients with type 1 diabetes mellitus and DKD and from mice with streptozotocin-induced diabetes mellitus, renin is elevated. This cannot be attributed to production from cells of the renin lineage migrating to the collecting duct in a chronic hyperglycemic environment. Rather, the elevated levels of urinary renin found in DKD are best attributed to altered glomerular filteration and impaired proximal tubular reabsorption.Fil: Tang, Jeannette. Northwestern University; Estados UnidosFil: Wysocki, Jan. Northwestern University; Estados UnidosFil: Ye, Minghao. Northwestern University; Estados UnidosFil: Garramuño, Patricia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rein, Johannes. Northwestern University; Estados UnidosFil: Shirazi, Mina. Northwestern University; Estados UnidosFil: Bader, Michael. Charité Universitätsmedizin; AlemaniaFil: Gomez, Roberto Ariel. University of Virginia; Estados UnidosFil: Sequeira Lopez, Maria Luisa S.. University of Virginia; Estados UnidosFil: Afkarian, Maryam. University of California at Davis; Estados UnidosFil: Batlle, Daniel. Northwestern University; Estados Unido

Hypokalemic Distal Renal Tubular Acidosis

Distal renal tubular acidosis (DRTA) is defined as hyperchloremic, non-anion gap metabolic acidosis with impaired urinary acid excretion in the presence of a normal or moderately reduced glomerular filtration rate. Failure in urinary acid excretion results from reduced H+ secretion by intercalated cells in the distal nephron. This results in decreased excretion of NH4 + and other acids collectively referred as titratable acids while urine pH is typically above 5.5 in the face of systemic acidosis. The clinical phenotype in patients with DRTA is characterized by stunted growth with bone abnormalities in children as well as nephrocalcinosis and nephrolithiasis that develop as the consequence of hypercalciuria, hypocitraturia, and relatively alkaline urine. Hypokalemia is a striking finding that accounts for muscle weakness and requires continued treatment together with alkali-based therapies. This review will focus on the mechanisms responsible for impaired acid excretion and urinary potassium wastage, the clinical features, and diagnostic approaches of hypokalemic DRTA, both inherited and acquired.Fil: Garramuño, Patricia. Universidad del Aconcagua. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Battle, Daniel. Northwestern University; Estados Unido

Fisiología y trastornos del potasio

La regulación fisiológica del metabolismo del potasio se basa en mecanismos que van dirijidos a lograr una alta concentración de potasio en el citosol y en segundo término lograr gradientes de concentración de potasio a través de las membranas celulares requeridos para la excitabilidad nerviosa y contracción muscularFil: Exeni, Ramón Alfonso. No especifíca;Fil: Garramuño, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Cytoprotective role of nitric oxide associated with Hsp70 expression in neonatal obstructive nephropathy

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis. In this study, we postulated that the mechanism of apoptosis inhibition by NO would include stimulation of heat shock protein 70 (Hsp70) expression. Rats were subjected to unilateral ureteral obstruction (UUO) or sham operation, and kidneys were harvested 5 and 14 days after obstruction. After 14 days of obstruction, decreased endogenous NO and lower inducible nitric oxide synthase (iNOS) expression at mRNA and protein levels associated with downregulation of Hsp70 protein expression were shown in apoptosis induction, regulated by mitochondrial signal pathway, through the increased pro-apoptotic ratio Bax/BcL2 and consequently caspase 3 activity. Conversely, 5 days after kidney obstruction, increased Hsp70 expression linked to increase NO and iNOS expression at transcriptional and post-transcriptional levels with absence of apoptotic response, were demonstrated. In obstructed neonatal rats, in vivo administration of l-Arginine induced heat shock protein 70 (Hsp70) expression, which was associated with cytoprotection from apoptosis and transiently decreased nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity. Opposite effects were obtained after nitro l-Arginine methyl ester (l-NAME) treatment. The interaction between B-cell lymphoma 2 anti-apoptotic members (BcL2) and Hsp70 in the presence of l-Arginine and l-NAME, was determined by coimmunoprecipitation. Binding of BcL2 and Hsp70 increased after l-Arginine administration. These findings suggest that NO can produce resistance to obstruction-induced cell death by mitochondrial apoptotic pathway, through the induction of Hsp70 expression, in neonatal unilateral ureteral obstruction.Fil: Manucha, Walter Ariel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Garramuño, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentin

Severely ill pediatric patients with Shiga toxin-associated hemolytic uremic syndrome (STEC-HUS) who suffered from multiple organ involvement in the early stage

Background: Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is the main cause of pediatric acute kidney injury (AKI) in Argentina. Endothelial injury is the trigger event in the microangiopathic process. The host inflammatory response to toxin and E. coli lipopolysaccharide (LPS) is involved in disease pathophysiology. Methods: This retrospective study describes pediatric STEC-HUS patients with multiorgan involvement at the initial phase of disease. A retrospective study of critically ill HUS patients with evidence of E. coli infection was conducted through a period of 15 years. Results: Forty-four patients 35.4 ± 4.1 months were admitted to the intensive care unit for 21 ± 2 days. Mechanical ventilation was required in 41 patients, early inotropic support in 37, and 28 developed septic shock. Forty-one patients required kidney replacement therapy for 12 ± 1 days. Forty-one patients showed neurological dysfunction. Dilated cardiomyopathy was demonstrated in 3 patients, left ventricular systolic dysfunction in 4, and hypertension in 17. Four patients had pulmonary hemorrhage, and acute respiratory distress syndrome in 2. Colectomy for transmural colonic necrosis was performed in 3 patients. Thirty-seven patients were treated with therapeutic plasma exchange, and 28 patients received methylprednisolone (10 mg/kg for 3 days). Of the surviving 32 patients, neurological sequelae were seen in 11 and chronic kidney failure in 5. Conclusions: Severe clinical outcome at onset suggests an amplified inflammatory response after exposure to Shiga toxin and/or E. coli LPS. STEC-HUS associated with severe neurological involvement, hemodynamic instability, and AKI requires intensive care and focused therapy.Fil: Luna, Mariana. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Kamariski, Mariana. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Principi, Iliana. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Garramuño, Patricia. Universidad Nacional de Cuyo; Argentina. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Adiponectin in children on peritoneal dialysis: Relationship to insulin resistance and nutritional status

Aim: To study whether adiponectin and resistin serum concentrations in children on peritoneal dialysis (PD) were related to insulin resistance (IR) and anthropometric parameters of nutritional status, 11 PD patients, 9 chronic kidney disease (CKD) patients and 10 healthy children were studied. Methods: Glucose and insulin were measured during the oral glucose tolerance test. Levels of adiponectin and resistin were evaluated by ELISA, insulin by RIA. Results: In CKD patients, higher homeostasis model assessment-insulin resistance (HOMA-IR), fasting and 2-hour serum insulin levels were shown compared to control and to PD patients. Body mass index (BMI) and body fat content were severely decreased while serum adiponectin levels were significantly higher in PD patients relative to controls. No differences among groups were shown in resistin levels. On regression modeling, inverse independent associations were observed between adiponectin with percentile BMI, weight and height z-score, and with body fat content. In contrast, no relationship was found between adiponectin and IR parameters. In multiple regression analysis, adiponectin was negatively correlated with BMI. A negative association of adiponectin and resistin with glomerular filtration rate was also shown. Conclusion: A role for adiponectin in terms of its association with clinical wasting parameters in PD pediatric patients might be suggested.Fil: Kamariski, Mariana. Hospital Pediátricos H. Notti, Mendoza, Argentina; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Biscardi, Mónica. Hospital Pediátrico H. Notti, Mendoza, Argentina; ArgentinaFil: Cestino, Laura. Hospital Pediátrico H. Notti, Mendoza, Argentina; ArgentinaFil: Miatello, Roberto. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Guntche Enrique. Hospital Pediátrico H. Notti, Mendoza, Argentina; ArgentinaFil: Garramuño, Patricia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Role of endogenous nitric oxide in unilateral ureteropelvic junction obstruction in children

Background. Obstructive nephropathy leads to tubulointerstitial fibrosis and loss of renal function. Nitric oxide has been shown to have antifibrotic properties. We examined nitric oxide synthase (NOS) activity and expression in kidneys from children who underwent surgery release of unilateral ureteropelvic junction (UPJ) obstruction in relation to clinical and histologic parameters. Methods. NOS activity and the expression of NOS isoforms measured at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay were determined in tissue obtained by biopsy from obstructed kidneys of 18 children at the time of pyeloplasty. Tissue from kidneys removed because of various malignancies were issued as control. Results. A significant increase in calcium/calmodulin-independent NOS activity (iNOS) and iNOS mRNA expression was found in the medulla of obstructed kidneys. Calcium/ calmodulin-dependent NOS activity (cNOS) and endothelial (eNOS) mRNA, by contrast, were increased in the cortex from obstructed kidneys. A role of tumor necrosis factor-α (TNF-α) on enhanced iNOS was suggested by the finding of increased urine levels in obstructed pelvis. Increased interstitium macrophage number, by immunolabeling of CD68, was related to the delay in obstruction release and to decreased glomerular filtration rate (GFR) at surgery. A positive linear relationship was found between cNOS activity in cortex and creatinine clearance. The degree of interstitial fibrosis correlated negatively with cNOS activity in cortex. Conclusion. In kidneys from children with UPJ obstruction an increased activity and expression of iNOS in medulla and cNOS-dependent eNOS in cortex were demonstrated. A role of cNOS in modulating GFR and interstitial fibrosis can be suggested. Prolonged UPJ obstruction would lead to a worsened prognosis on renal injury.Fil: Garramuño, Patricia. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Pascual, Luis. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Fisiología; ArgentinaFil: Manucha, Walter Ariel Fernando. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Carrizo, Liliana. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Fisiología; ArgentinaFil: Rüttler, Maria. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Fisiología; Argentin

The renal antioxidative effect of losartan involves heat shock protein 70 in proximal tubule cells

Angiotensin II exerts a cardinal role in the pathogenesis of hypertension and renal injury via action of angiotensin II type 1 (AT1) receptors. Local renin-angiotensin system (RAS) activity is essential for the mechanisms mediating pathophysiological functions. Proximal tubular angiotensinogen and tubular AT1 receptors are augmented by intrarenal angiotensin II. Caveolin 1 plays an important role as a regulatory molecule for the compartmentalization of redox signaling events through angiotensin II–induced NADPH oxidase activation in the kidney. A role for the renin-angiotensin system in the development and/or maintenance of hypertension has been demonstrated in spontaneously hypertensive rats (SHRs). Many effects of angiotensin II are dependent on the AT1 stimulation of reactive oxygen species (ROS) production by NADPH oxidase. Angiotensin II upregulation stimulates oxidative stress in proximal tubules from SHR. The NADPH oxidase 4 (Nox4) is abundantly expressed in kidney proximal tubule cells. Induction of the stress response includes synthesis of heat shock protein 70, a molecular chaperone that has a critical role in the recovery of cells from stress and in cytoprotection, guarding cells from subsequent insults. HSP70 chaperones function in part by driving the molecular triage decision, which determines whether proteins enter the productive folding pathway or result in client substrate ubiquitination and proteasomal degradation. This review examines regulation of losartan-mediated antioxidative stress responses by the chaperone HSP70 in proximal tubule cells of spontaneously hypertensive rats.Fil: Garramuño, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Bocanegra, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Costantino, Valeria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gil Lorenzo, Andrea Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Benardon, María Eugenia. Universidad Nacional de Cuyo; ArgentinaFil: Cacciamani, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin