High sodium, rather than high blood pressure, induces immune cell activation and renal infiltration in ovariectomized adult Wistar rats.

We used an animal model of salt-sensitive hypertension (SSH) in which ovariectomized (oVx) rats developed hypertension with high salt (HS) intake. Hypertension is accompanied by changes in the percentage of CD4+ T lymphocytes, immune CD45+ cell infiltration into renal tissue, and changes in Na+, K+- ATPase (NKA) expression in both renal tissue and peripheral blood mononuclear cells (PBMCs). To determine whether the observed changes resulted from HS intake, high blood pressure, or both, hydralazine (HDZ) was used to lower blood pressure. The oVx HS rats received two HDZ schedules either to prevent or to treat hypertension. NKA was overexpressed in the kidneys of all oVx groups and in PBMCs of oVx HS rats. This pattern was not altered with HDZ treatment. Changes in CD4+ T lymphocytes and renal infiltration of CD45+ cells were not reversed either. High salt, but not high blood pressure, induces immune cell activation and renal infiltration. Overexpressed NKA is the primary event, and HS is the perturbation to the system in this model of SSH, which resembles the postmenopausal state.

Obtención de nueva información sobre el síndrome urémico hemolítico mediante minería de texto / Obtaining new information on hemolytic uremic syndrome by text mining

Resumen El síndrome urémico hemolítico (SUH) está caracterizado por microangiopatía trombótica, anemia hemolítica, trombocitopenia e insuficiencia renal aguda. Puede causar desde secuelas permanentes hasta muerte, principalmente en niños. En este trabajo, utilizando minería de textos (MT), se analizó el texto explícito e implícito de 16 192 artículos científicos originales sobre SUH indexados en la base de datos de Europe PMC. Los objetivos fueron examinar comportamientos, realizar seguimiento de tendencias, hacer predicciones y cruzar datos con otras fuentes de información. Para el análisis se utilizaron -entre otras herramientas infor máticas- flujos de trabajo (FT) especialmente desarrollados en la plataforma KNIME. La MT sobre las palabras de los resúmenes de las publicaciones permitió: detectar asociaciones no descritas entre eventos relacionados con SUH; extraer información subyacente; hacer agrupamientos temáticos mediante algoritmos no supervisados; realizar predicciones sobre el curso de las investigaciones asociadas al tema. Tanto el abordaje como los FT desarrollados para realizar Ciencia de Datos sobre SUH pueden aplicarse a otros temas biomédicos y a otras bases de datos científicos, permitiendo analizar aspectos relevantes en el campo de la salud humana para me jorar la investigación, la prevención y el tratamiento de múltiples enfermedades.

Abstract Hemolytic uremic syndrome (HUS) is characterized by thrombotic microangiopathy, hemolytic anemia, thrombocytopenia and acute renal failure. It can cause from permanent sequelae to death, mainly in children. In this work, using text mining (TM), we analyzed the explicit and implicit text of 16 192 original scientific articles on HUS indexed in the Europe PMC database. The objectives were to examine behaviors, track trends, and make predictions and cross-check data with other sources of information. For the analysis we used -among other computational tools- specially developed workflows (WF) in the KNIME platform. The TM on the words of the abstracts of the publications made it possible to: detect undescribed associations between events related to HUS; extract underly ing information; make thematic clustering using unsupervised algorithms; make forecasting about the course of research associated with the topic. Both the approach and the WFs developed to perform Data Science on HUS can be applied to other biomedical topics and other scientific databases, making it possible to analyze relevant aspects in the field of human health to improve research, prevention and treatment of multiples diseases.

Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells.

BACKGROUND: Shiga toxin-producing Escherichia coli is responsible for post-diarrheal (D+) hemolytic uremic syndrome (HUS), which is a cause of acute renal failure in children. The glycolipid globotriaosylceramide (Gb3) is the main receptor for Shiga toxin (Stx) in kidney target cells. Eliglustat (EG) is a specific and potent inhibitor of glucosylceramide synthase, first step of glycosphingolipid biosynthesis, actually used for the treatment of Gaucher's disease. The aim of the present work was to evaluate the efficiency of EG in preventing the damage caused by Stx2 in human renal epithelial cells. METHODS: Human renal tubular epithelial cell (HRTEC) primary cultures were pre-treated with different dilutions of EG followed by co-incubation with EG and Stx2 at different times, and cell viability, proliferation, apoptosis, tubulogenesis, and Gb3 expression were assessed. RESULTS: In HRTEC, pre-treatments with 50 nmol/L EG for 24 h, or 500 nmol/L EG for 6 h, reduced Gb3 expression and totally prevented the effects of Stx2 on cell viability, proliferation, and apoptosis. EG treatment also allowed the development of tubulogenesis in 3D-HRTEC exposed to Stx2. CONCLUSIONS: EG could be a potential therapeutic drug for the prevention of acute kidney injury caused by Stx2. IMPACT: For the first time, we have demonstrated that Eliglustat prevents Shiga toxin 2 cytotoxic effects on human renal epithelia, by reducing the expression of the toxin receptor globotriaosylceramide. The present work also shows that Eliglustat prevents Shiga toxin 2 effects on tubulogenesis of renal epithelial cells. Eliglustat, actually used for the treatment of patients with Gaucher's disease, could be a therapeutic strategy to prevent the renal damage caused by Shiga toxin.

Ovariectomy and high salt increase blood pressure and alter sodium transport proteins in peripheral blood mononuclear cells of adult Wistar rats.

NEW FINDINGS: What is the central question of this study? In a model of salt-sensitive hypertension in ovariectomized (oVx) adult Wistar rats, what is the expression of proteins related to sodium transport in peripheral blood mononuclear cells (PBMCs), and how does the response of proteins to high sodium intake compare with changes in blood pressure in intact female rats? What is the main finding and its importance? Sodium transport proteins in PBMCs react to high sodium and blood pressure markedly differently in oVx versus intact female rats. Protein expression shows sodium and pressure sensitivity. Renal immune cells increase in oVx under high salt. ABSTRACT: Hypertension is a worldwide public health problem. High sodium consumption is associated with hypertension, and hypertensive mechanisms involve immunity cells. Peripheral blood mononuclear cells (PBMCs) are endowed with proteins related to sodium transport. We studied their abundance in PBMCs from intact (IF) or ovariectomized (oVx) adult Wistar rats under normal (NS) or high (HS) salt intake. Ovariectomy was performed at 60 days of life. At 145 days, one group of IF and oVx rats received NS or HS intake for 5 days. Another group of IF HS and oVx HS rats received hydralazine (HDZ) to reduce blood pressure (BP). Sodium balance and BP were recorded. Expression of Na+ ,K+ -ATPase (NKA), Na+ -K+ -2Cl- cotransporter 1 (NKCC1), serum/glucocorticoid-regulated kinase 1 (SGK1), dopamine D1 like receptor (D1DR), CD4+ and CD8+ were determined in PBMCs and CD45+ leukocytes in renal tissue. IF HS rats showed increased natriuresis and normal BP. NKA and CD4+ expression diminished in IF HS. Instead, oVx HS rats had sodium retention and high BP and increased the expression of NKA, NKCC1, D1DR, CD4+ and CD8+ in PBMCs. Renal CD45+ leukocytes increased in oVx HS rats. HDZ decreased BP in all rats. Upon HDZ treatment, NKA did not change, NKCC1 decreased in oVx HS rats, while SGK1 increased in both IF HS and oVx HS rats. Hormonal background determines BP response and the expression of proteins related to sodium transport in PBMCs and renal immune cells at HS intake. The analysis of NKA, NKCC1 and SGK1 expression in PBMCs differentiated salt-sensitivity from BP variations.

[Female hormones in renal physiology. Salt sensitivity and regulation of epithelial proliferation]. / Las hormonas femeninas en fisiología renal. Sensibilidad a la sal y regulación de la proliferación epitelial.

Female sex hormones participate in the regulation of blood pressure and renal epithelial proliferation, effects not related to their reproductive function. About one-third of the world's population has abnormally high levels of blood pressure, hypertension, which is responsible for almost 50% of deaths from stroke and coronary heart disease. Salt sensitivity is a risk factor for cardiovascular morbidity and mortality and other diseases as well. We reported a model of salt sensitive hypertension in adult ovariectomized (oVx) Wistar rats. oVx rats are normotensive under normal salt intake (NS, 0.24% NaCl), but upon a high salt intake (HS, 1% NaCl) oVx rats developed a blood pressure profile of salt-sensitive hypertension. Our studies on kidney molecules related to sodium balance found that the circuit dopamine D1-like receptor, cytochrome P450 4A and Na+, K+-ATPase is altered by the absence of ovary hormones which is accompanied by a reduced ability to excrete sodium. In oVx rats HS intake also promotes changes in the expression of proteins related to sodium transport in peripheral blood mononuclear cells, mainly peripheral lymphocytes. Therefore, sodium transport is modified at several levels of normal physiology. Lately, we described that estradiol increases the rate of renal epithelial cell proliferation in primary cultures developed from human renal cortex. Thus, salt sensitivity, adaptive immunity, blood pressure and renal cell proliferation are complex biological responses regulated by female sex hormones.

Un tercio de la población mundial tiene niveles anormalmente altos de presión arterial, hipertensión, responsable de casi el 50% de las muertes por accidente cerebrovascular y enfermedad coronaria. La sensibilidad a la sal es un factor de riesgo para la morbilidad y mortalidad cardiovascular y también para otras enfermedades. En estudios previos describimos un modelo de hipertensión sal sensible (HSS) en ratas Wistar ovariectomizadas (oVx) adultas. Las ratas oVx son normotensas con ingesta normal de sal (NS, 0.24% de NaCl), pero desarrollan un perfil de HSS con una ingesta elevada de sal (HS, 1% de NaCl). En los estudios en riñón encontramos que el circuito receptor D1 de dopamina, citocromo P450 4A y Na+, K+-ATPasa está alterado por la ausencia de hormonas ováricas, lo que se asocia a menor excreción de sodio e hipertensión arterial. La ingesta HS en ratas oVx también promueve cambios en la expresión de proteínas relacionadas con el transporte de sodio en células mononucleares de sangre periférica, principalmente linfocitos periféricos. Por lo tanto, el transporte de sodio se modifica en varios niveles de la fisiología normal. En estudios recientes observamos que el estradiol aumenta la proliferación y diferenciación de células epiteliales en cultivos de corteza renal humana. Sensibilidad a la sal, inmunidad adaptativa, presión arterial y proliferación de células epiteliales en riñón son fenómenos de gran importancia biológica regulados por estradiol.

Las hormonas femeninas en fisiología renal: Sensibilidad a la sal y regulación de la proliferación epitelial / Female hormones in renal physiology. Salt sensitivity and regulation of epithelial proliferation

Un tercio de la población mundial tiene niveles anormalmente altos de presión arterial, hipertensión, responsable de casi el 50% de las muertes por accidente cerebrovascular y enfermedad coronaria. La sensibilidad a la sal es un factor de riesgo para la morbilidad y mortalidad cardiovascular y también para otras enfermedades. En estudios previos describimos un modelo de hipertensión sal sensible (HSS) en ratas Wistar ovariectomizadas (oVx) adultas. Las ratas oVx son normotensas con ingesta normal de sal (NS, 0.24% de NaCl), pero desarrollan un perfil de HSS con una ingesta elevada de sal (HS, 1% de NaCl). En los estudios en riñón encontramos que el circuito receptor D1 de dopamina, citocromo P450 4A y Na+, K+-ATPasa está alterado por la ausencia de hormonas ováricas, lo que se asocia a menor excreción de sodio e hipertensión arterial. La ingesta HS en ratas oVx también promueve cambios en la expresión de proteínas relacionadas con el transporte de sodio en células mononucleares de sangre periférica, principalmente linfocitos periféricos. Por lo tanto, el transporte de sodio se modifica en varios niveles de la fisiología normal. En estudios recientes observamos que el estradiol aumenta la proliferación y diferenciación de células epiteliales en cultivos de corteza renal humana. Sensibilidad a la sal, inmunidad adaptativa, presión arterial y proliferación de células epiteliales en riñón son fenómenos de gran importancia biológica regulados por estradiol.

Female sex hormones participate in the regulation of blood pressure and renal epithelial proliferation, effects not related to their reproductive function. About one-third of the world's population has abnormally high levels of blood pressure, hypertension, which is responsible for almost 50% of deaths from stroke and coronary heart disease. Salt sensitivity is a risk factor for cardiovascular morbidity and mortality and other diseases as well. We reported a model of salt sensitive hypertension in adult ovariectomized (oVx) Wistar rats. oVx rats are normotensive under normal salt intake (NS, 0.24% NaCl), but upon a high salt intake (HS, 1% NaCl) oVx rats developed a blood pressure profile of salt-sensitive hypertension. Our studies on kidney molecules related to sodium balance found that the circuit dopamine D1-like receptor, cytochrome P450 4A and Na+, K+-ATPase is altered by the absence of ovary hormones which is accompanied by a reduced ability to excrete sodium. In oVx rats HS intake also promotes changes in the expression of proteins related to sodium transport in peripheral blood mononuclear cells, mainly peripheral lymphocytes. Therefore, sodium transport is modified at several levels of normal physiology. Lately, we described that estradiol increases the rate of renal epithelial cell proliferation in primary cultures developed from human renal cortex. Thus, salt sensitivity, adaptive immunity, blood pressure and renal cell proliferation are complex biological responses regulated by female sex hormones.

Estradiol stimulates cell proliferation via classic estrogen receptor-alpha and G protein-coupled estrogen receptor-1 in human renal tubular epithelial cell primary cultures.

This work was aimed to determine the effect of 17ß-estradiol (17ßE) on cell proliferation in human renal tubular epithelial cells (HRTEC) isolated from kidneys from pediatric subjects, as well as the role of estrogen receptors involved in the 17ßE proliferative response. Treatment with 17ßE (10 nmol/L, 24 h) significantly stimulated cell proliferation, measured by 5-bromo-2-deoxyuridine (BrdU) uptake, in HRTEC primary cultures and in tubular structures obtained by 3D cultured-HRTEC. Incubation of HRTEC with the G protein-coupled estrogen receptor 1 (GPER-1) agonist G-1 increased BrdU uptake. Incubation of HRTEC with 17ßE activated the classic estrogen receptor alpha (ERα) but not ERß. Treatment of HRTEC with the GPER-1 antagonist G-15, the ER inhibitor ICI182,780, or the ß-catenin inhibitor iCRT14, completely abrogated the increase in BrdU uptake induced by 17ßE. We also show that 17ßE stimulated ß-catenin protein expression and translocation to the nucleus of HRTEC, effects that were abrogated by G-15 and ICI 182,780. In conclusion, estradiol stimulates cell proliferation in HRTEC primary cultures through both ERα and GPER-1 estrogen receptors and involves ß-catenin activation.

Diferencias de género en presión arterial, función renal y respuesta a la dieta hipersódica en ratas Wistar / Gender differences in blood pressure, renal function and response to a high-sodium diet in Wistar rats

Introducción: Es conocido que el sexo es un condicionante de la regulación renal de sodio y de la presión arterial. Material y métodos: Se estudiaron ratas Wistar machos y hembras a los 150 días de vida, con dieta normo o hipersódica (NaCl 1% v.o.) en los últimos cinco. Se determinaron presión arterial media (PAM), natriuresis, filtrado glomerular (VFG), flujo plasmático renal (FPR) y aldosterona plasmática. Se estudió la expresión Na+,K+-ATPasa total (t-NKA) y defosforilada (d-NKA), citocromo P4504A (CYP4A), cotransportadores Na+,K+,2Cl- tipo 2 (NKCC2) y Na+/Cl- (NCC) y por PCR el ARNm de la cadena α1 de NKA (Atp1a1) en corteza y médula renal. Resultados: La PAM fue mayor y la natriuresis menor en los machos bajo ambas dietas. Con ingesta hipersódica la aldosterona bajó en ambos sexos, el VFG fue menor en hembras y el FPR aumentó en machos (4,09 ± 0,17 vs 2,81 ± 0,12 ml/min/gR; p<0,01 vs dieta normosódica). La t-NKA, d-NKA y Atp1a1 en médula fue mayor en machos con ambas dietas. Con ingesta hipersódica, t-NKA en médula y d-NKA en corteza y médula disminuyeron en hembras y solamente d-NKA disminuyó en médula de machos. Asimismo, aumentó CYP4A y disminuyó NKCC2 y NCC en hembras, mientras que aumentó NKCC2, sin cambios en NCC, en machos. Conclusión: El sexo condiciona la presión arterial y el balance de sodio, disminuyendo su reabsorción en hembras y aumentando el FPR en machos. Esto sugiere posibilidades de estudio diferenciales según sexo en trastornos del metabolismo del sodio

Introduction: It is known that sex is a determinant of renal sodium regulation and blood pressure. Methods: Male and female Wistar rats, which were 150 days old and a diet with normal or high levels of sodium (NaCl 1% v.o.), were studied for the last five days. Mean blood pressure (MBP), natriuresis, glomerular filtration rate (GFR), renal plasma flow (RPF) and plasma aldosterone level were established. The following were studied: expressions of total Na+,K+,-ATPase (t-NKA); dephosphorylated NKA (d-NKA); cytochrome P4504A (CYP4A); Na+K+-2Cl- (NKCC2) and Na+/Cl- (NCC) cotransporters. The mRNA expression of the NKA α1 (Atp1a1) chain was examined through PCR analysis in the renal cortex and marrow. Results: Male rats having both types of diet showed higher MBP and lower natriuresis. High sodium intake triggered lower aldosterone levels in both sexes; GFR was lower in females and RPF was higher in males (4.09 ± 0.17 vs. 2.81 ± 0.12 ml/min/gr; p<0.01 vs. diet with a normal sodium level). Marrow t-NKA, d-NKA and Atp1a1 were higher in males on both diets. High sodium intake caused lower marrow t-NKA as well as lower cortex and marrow d-NKA in females. In the case of males, only marrow d-NKA decreased. Furthermore, females showed a higher level of CYP4A and lower levels of NKCC2 and NCC, whereas males showed higher levels of NKCC2 and no variations in NCC. Conclusion: Sex conditions blood pressure and sodium balance, reducing resorption in females and increasing RPF in males. This suggests the possibility of studying sodium metabolism disorders differently according to sex

Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

Epithelial cells activate plasmacytoid dendritic cells improving their anti-HIV activity.

Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.

Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells.

Clostridium perfringens epsilon toxin (ETX) is responsible for a fatal enterotoxemia in different animal species, producing extensive renal damage, neurological disturbance and edema of lungs, heart and kidneys. However, there is no information about the susceptibility of humans to ETX. Here, we report that primary cultures of human renal tubular epithelial cells (HRTEC) exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The incubation of HRTEC with ETX produced a reduction of cell viability in a dose- and time-dependent manner. The CD(50) after 1-hour and 24-hour incubation were 3 µg/mL and 0.5 µg/mL, respectively. The pulse with ETX for 3 min was enough to produce a significant cytotoxic effect on HRTEC after 1-hour incubation. ETX binds to HRTEC forming a large complex of about 160 kDa similar to what was found in the Madin-Darby canine kidney (MDCK) cell line. The HRTEC could be a useful cell model to improve the understanding of the mechanisms involved on the cell damage mediated by ETX.

Síndrome urémico hemolítico inducido por Escherichia coli enterohemorrágica / Hemolytic uremic syndrome caused by enterohaemorrhagic Escherichia coli

El síndrome urémico hemolítico (SUH) se caracteriza por anemia hemolítica microangiopática, plaquetopeniay daño renal. Constituye la primeracausa de insuficiencia renal aguda en la edad pediátrica y la segunda de insuficiencia renal crónica.Escherichia coli productor de toxina Shiga (STEC,por su sigla en inglés) es el primer agente etiológico de SUH; su principal reservorio es el ganado bovinoy la vía de transmisión, los alimentos contaminados.Hasta el presente no existe un tratamiento específicopara disminuir la progresión del SUH.El estudio de los mecanismos por los cuales STEC infecta y la toxina Shiga induce SUH puede ayudar a desarrollar nuevas estrategias para impedir estaenfermedad.

Inhibition of water absorption in human proximal tubular epithelial cells in response to Shiga toxin-2.

Postdiarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children in Argentina. It is well established that Shiga toxin type 2 (Stx2) causes direct damage to glomerular endothelial cells and tubular epithelial cells, leading to a reduction in the water handling capacity of the kidney. In this study, we demonstrate that Stx2 and its B subunit (Stx2B) were able to inhibit water absorption across human renal tubular epithelial cell (HRTEC) monolayers without altering the short circuit current and the (3)H-mannitol permeability. Quantitative evaluation of (14)C-inulin transport across HRTEC monolayers showed a similar transport rate both before and after HRTEC treatment with Stx2 that confirmed the integrity of the paracellular pathway. Furthermore, Stx2 produced significant protein synthesis inhibition of HRTEC at concentrations as low as 0.001 ng/ml and 1 h of incubation, whereas Stx2B did not modify it at concentrations as high as 10,000 ng/ml and 6 h of incubation. Our findings suggest that whereas the action of Stx2 appears to be caused mainly by the inhibition of protein synthesis mediated by the A subunit, the binding of Stx2B subunit to the Gb3 receptor may affect the membrane mechanisms related to water absorption. We speculate that inhibition of water absorption may occur in proximal tubular cells in vivo in response to Stx2 and may contribute to the early event of HUS pathogenesis.

Role of NHERF1, cystic fibrosis transmembrane conductance regulator, and cAMP in the regulation of aquaporin 9.

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.

Postnatal expression of aquaporins in epithelial cells of the rat epididymis.

The mammalian aquaporins (AQPs) are a family of 13 transmembrane channel proteins that are involved in the transport of water in numerous organs. In the male excurrent duct, the movement of fluid and solutes across the epithelium is essential for establishing the proper luminal environment in which sperm mature and are stored. AQP9 is abundantly expressed in the efferent ducts, the epididymis, and the vas deferens, where it could represent an important apical pathway for transmembrane water and solute movement. However, other organs in which water transport is critical, including the kidney, the lung, or the eye, express several different AQPs with a cell-specific pattern. To undertake a systematic analysis of the expression of known AQPs in the postnatal and adult rat epididymis, we examined the expression of their respective mRNAs in epithelial cells isolated by laser capture microdissection (LCM), and we determined their corresponding protein expression pattern by immunofluorescence and Western blotting. Our data show that, whereas AQP9 is the main AQP of the epididymis, the mRNA specific for Aqp2, 5, 7, and 11 are also expressed in epididymal epithelial cells. AQP5 protein colocalizes with AQP9 in the apical membrane of a subpopulation of principal cells in the corpus and cauda regions. Aqp2 mRNA was detected in epithelial cells after the second postnatal week and the amount significantly increased up to adulthood. However, AQP2 protein was detected only in the distal cauda of young rats (between the second and fourth postnatal week). No AQP2 protein was detected in the adult epididymis, indicating that posttranscriptional mechanisms are involved in the regulation of AQP2 expression. In addition, epididymal epithelial cells express significant amounts of the mRNAs coding for AQP7 and 11. No mRNA or protein for AQPs 0, 4, 6, and 8 were detectable in epithelial cells, and Aqp1 was detected in whole epididymal samples, but not in epithelial cells. Thanks to the recent development of microdissection technologies, our observations suggest that epididymal epithelial cells express several members of the AQP family with a region-specific pattern. AQPs may be involved not only in the transepithelial transport of water in the epididymis but also in the postnatal development of this organ, as suggested by the differential expression of AQP2.

Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells.

Shiga toxin-producing Escherichia coli produces watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS). In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 holotoxin) and its B subunit (Stx2 B subunit) on human renal tubular epithelial cells (HRTEC), in the presence and absence of inflammatory factors. Cell morphology, cell viability, protein synthesis and apoptosis were measured. HRTEC are sensitive to both Stx2 holotoxin and Stx2 B subunit in a dose- and time-dependent manner. IL-1, LPS and butyrate but not TNF, IL-6 and IL-8, increased the Stx mediated cytotoxicity. The effects of Stx2 B subunit appear at doses higher than those used for Stx2 holotoxin. Although the physiological importance of these effects is not clear, it is important to be aware of any potentially toxic activity in the B subunit, given that it has been proposed for use in a vaccine.

Papel de la Toxina Shiga en el sindrome urémico hemolítico / Role of the Shiga toxin in the hemolytic uremic syndrome

In the last years, infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolitic-Uremic Syndrome (HUS) became relevant as a public health since it was considered as one of the most important emergent patogen present in the food contaminated by cattle feces. STEC infection may be asymptomatic or begins with a watery diarrhea that may or may not progress to bloody diarrhea (hemorrhagic colitis) and HUS. In Argentina, HUS is the most common pediatric cause of acute renal insufficiency and the second cause of chronic renal failure. Up to now, STEC infection lacks of known effective treatment strategies that diminish risk of progression to HUS. The mechanisms by which Shiga toxin (Stx) induce HUS may help to find strategies to prevent or ameliorate HUS. In this article, recent progress that has contributed to understanding the disease pathogenesis of STEC is reviewed. New strategies to prevent further uptake of Shiga from the gut, either during the diarrheal phase or once HUS has developed are discussed.

Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells.

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.