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Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Shiga toxin-producing Escherichia coli is the main cause of post-diarrheal hemolytic uremic syndrome (HUS) which produces acute kidney injury mainly in children, although it can also affect adults. The kidneys are the organs most affected by Shiga toxin type 2 (Stx2) in patients with HUS. However, previous studies in pregnant rats showed that a sublethal dose of Stx2 causes severe damage in the uteroplacental unit and induces abortion, whereas produces mild to moderate renal damage. The aim of the present work was to study the progression of renal injury caused by a sublethal dose of Stx2, as well as renal recovery, in pregnant and non-pregnant rats, and to investigate whether pregnancy physiology may affect renal damage progression mediated by Stx2., Methods: Renal function and histopathology was evaluated in pregnant rats intraperitoneally injected with a sublethal dose of Stx2 (0.5 ng/g bwt) at the early stage of gestation (day 8 of gestation), and results in these rats were compared over time with those observed in non-pregnant female rats injected with the same Stx2 dose. Hence, progression of cell proliferation and dedifferentiation in renal tubular epithelia was also investigated., Results: The sublethal dose of Stx2 induced abortion in pregnant rats as well as a significant more extended functional and histological renal injury in non-pregnant rats than in pregnant rats. Stx2 also caused decreased ability to concentrate urine in non-pregnant rats compared to their controls. However, renal water handling in pregnant rats was not altered by Stx2, and was significantly different than in non-pregnant rats. The greatest renal injury in both pregnant and non-pregnant rats was observed at 4 days post-Stx2 injection, and coincided with a significant increase in tubular epithelial proliferation. Expression of mesenchymal marker vimentin in tubular epithelia was consistent with the level of tubular damage, being higher in non-pregnant rats than in pregnant rats. Recovery from Stx2-induced kidney injury was faster in pregnant rats than in non-pregnant rats., Conclusions: Adaptive mechanisms developed during pregnancy such as changes in water handle and renal hemodynamic may contribute to lessen the Stx2-induced renal injury, perhaps at the expense of fetal loss., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purposeof Review: Female sex hormones have systemic effects unrelated to their reproductive function. We describe experiences of different research groups and our own, on aspects related to the importance of female sex hormones on blood pressure (BP) regulation and salt-sensitivity-mediated BP response and salt sensitivity without alterations in BP, as well as renal sodium handling and interactions with the immune system., Recent Findings: Changes in sodium intake in normotensive premenopausal women cause more BP variations than in men. After menopause, women often develop arterial hypertension (HT) with a profile of sodium sensitivity. Besides, experimental results have shown that in adult rat models resembling the postmenopausal hormonal state induced by ovariectomy, controlling BP is not enough to avoid renal and other tissue infiltration with immune cells, which does not occur when sodium intake is low or normal. Therefore, excess sodium promotes an inflammatory state with the involvement of immune cells. The evidence of activation of adaptive immunity, besides changes in T cell subpopulations, includes changes in sodium transporters and receptors. More studies are needed to evaluate the particular sodium sensitivity of women and its meaning. Changes in lifestyle and sodium intake reduction are the main therapeutic steps. However, to face the actual burden of salt-sensitive HT in postmenopausal women and its associated inflammatory/immune changes, it seems reasonable to work on immune cell activity by considering the peripheral blood mononuclear cell phenotypes of molecules and transport proteins related to sodium handle, both to screen for and treat cell activation., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

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 underlying 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.

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., (© 2021 The Authors. Experimental Physiology © 2021 The Physiological Society.)

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.

This work was aimed to determine the effect of 17β-estradiol (17βE) 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 17βE proliferative response. Treatment with 17βE (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 17βE activated the classic estrogen receptor alpha (ERα) but not ERβ. Treatment of HRTEC with the GPER-1 antagonist G-15, the ER inhibitor ICI182,780, or the β-catenin inhibitor iCRT14, completely abrogated the increase in BrdU uptake induced by 17βE. We also show that 17βE stimulated β-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 β-catenin activation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Postdiarrhea hemolytic uremic syndrome is the most common cause of acute renal failure in children in Argentina. Renal damage has been strongly associated with Shiga toxin (Stx), which binds to the globotriaosylceramide (Gb3) receptor on the plasma membrane of target cells. The purpose of the study was to evaluate the in vivo effects of C-9, a potent inhibitor of glucosylceramide synthase and Gb3 synthesis, on kidney and colon in an experimental model of hemolytic uremic syndrome in rats. Rats were i.p. injected with supernatant from recombinant Escherichia coli expressing Stx2 (sStx2). A group of these rats were orally treated with C-9 during 6 d, from 2 d prior until 4 d after sStx2 injection. The injection of sStx2 caused renal damage as well as a loss of goblet cells in colonic mucosa. Oral treatment with C-9 significantly decreased rat mortality to 50% and reduced the extension of renal and intestinal injuries in the surviving rats. The C-9 also decreased Gb3 and glucosylceramide expression levels in rat kidneys. It is particularly interesting that an improvement was seen when C-9 was administered 2 d before challenge, which makes it potentially useful for prophylaxis.

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.

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.

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK(1) cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys(8)]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 approximately 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 --> 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

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.

Background: Urea transport depends on the diffusion through cell membrane and the facilitated urea transport. Two groups of urea transporters (UT-A and UT-B) have been identified in mammals, and both are involved in intrarenal recycling of urea. The aim of our study was to examine the renal urea handling in rats with chronic renal failure (CRF)., Methods: CRF rats were induced by 5/6 nephrectomy followed by a high-protein (HP) diet to increase the progressive loss of renal function for 5 months. Functional studies on water and urea handling were performed. RT-PCR, immunoblotting and immunohistochemistry were used to identify UT-A proteins in remnant kidney., Results: A significant decrease in creatinine clearance consistent with development of CRF was observed. The remnant kidneys were hypertrophied, and total renal mass was increased. Urine production increased markedly, whereas urine osmolality and solute-free water reabsorption decreased significantly. Fractional urea excretion was increased reaching values of 105% -/+ 8%. UT-A protein was localized in pars recta by immunohistochemical studies, and it was identified as UT-A2 in outer medulla from remnant kidneys by RT-PCR and immunoblotting., Conclusion: In uremic rats, an urea transporter type UT-A2 was expressed in the pars recta, suggesting a possible relation with the fractional urea excretion increase. This expression may be a consequence of an adaptive mechanism in the handling of urea during development of CRF. Further studies will be necessary to elucidate the contribution of this mechanism to renal damage observed in the progression of CRF.

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, plaquetopenia and kidney damage. It is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure. Shiga toxin-producing Escherichia coli (STEC) is the first etiologic agent of HUS being its main reservoir cattle and transmitted via contaminated food. At present, there is no specific treatment to reduce the progression of HUS. The study of the mechanisms by which STEC infects and Shiga toxin induces HUS can help to find new strategies to prevent this disease.

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.

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.

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.

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.

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.

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water., (Copyright 2003 Wiley-Liss, Inc.)

We report the characterization of a frog (Rana esculenta) urea transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box urea transporter signature sequence were found in this amphibian urea transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thiourea, but the fUT-mediated urea transport was strongly inhibited by this compound. Thus, this amphibian urea transporter displays transport characteristics in between those of UT-A2 and UT-B.

The purpose of this study was to characterize patients who play tennis after undergoing hip arthroplasty in terms of their functional abilities and degree of satisfaction. A questionnaire was sent to all United States Tennis Association member associations in a tri-state area to identify players who had undergone a hip arthroplasty. The study group had 50 men and 8 women with a mean age of 70 years (range, 47 to 89). Only 14% of the patients' surgeons approved this tennis activity, with 34% of the surgeons recommending only doubles. Three patients required revision surgery after a mean of 8 years. One year after arthroplasty, players played both singles and doubles approximately three times per week. All tennis players were extremely satisfied with their hip arthroplasties and their increased ability to participate in their favorite sport. Because this study was confined to association members, further studies are needed to assess the general effect of tennis on total hip arthroplasty. Until future studies are performed, the authors would recommend that physicians advise caution in tennis activities and to carefully follow their patients yearly to see if osteolysis is occurring prematurely.

Forty-two team orthopedists representing all 28 major league baseball teams were surveyed to ascertain their definitive treatment for a hypothetical starting rotation pitcher who had sustained a grade III acromioclavicular (AC) separation to his throwing arm 1 week before the season. Twenty-nine (69%) of the physicians would treat the injury nonoperatively, while 13 (31%) would operate immediately. Twenty-five (60%) of the orthopedists had actually treated a pitcher or position baseball player with a grade III AC separation in the throwing arm, the 25 treating a total of 32 patients. Twenty (63%) of these injuries were treated nonoperatively, and 12 (37%) were treated operatively. The physicians reported that 16 (80%) of the patients treated nonoperatively regained normal function and achieved complete relief of pain, while 18 (90%) had normal range of motion after treatment; of those treated operatively, 11 (92%) regained normal function, achieved complete relief of pain, and had normal range of motion after surgery.

Problems with the gastrocnemius-soleus muscle group can severely impair the gait of children with cerebral palsy. Treatments, including bracing, muscle lengthening, neurectomy, or a combination, have been used with mixed results. Soleus neurectomy was performed as the primary treatment for ankle clonus in 38 legs of 21 children with a variety of cerebral palsies. Concurrent heel cord or muscle lengthening was performed if needed. Patients were followed for an average of 9 years (range, 2 to 14 years). Clonus recurred in 4 treated ankles. In 2 cases, this was due to a nerve anomaly. Postneurectomy Achilles tendon lengthening was required in 8 of the treated ankles. Neurectomy was beneficial for 19 of 21 children. Functional improvements included better control of stopping, better balance, and less toe walking. The greatest improvement was seen in those patients who did not also have heel cord contractures.

This article describes the scintigraphic appearance of both a symptomatic and asymptomatic retroisthmic cleft in two athletes with low back pain. This lesion, which involves the lamina, is the least common of the neural arch defects of which spondylolysis is the most common. The anatomy of the lesion is discussed and illustrated. The literature about the possible cause of these lesions is reviewed. The report emphasizes the valuable role of radionuclide bone imaging in patients who have pain of potentially osseous origin, and who have a lesion of uncertain physiologic significance seen on an anatomic study such as a plain x-ray, CT scan, or MRI.

Therapeutic communities (TCs) are residential self-help programs for recovering addicts. Traditional TCs are staffed by recovered addicts. Recently the TC model has been modified for the treatment of chemical abusers with severe mental illness. These programs are to varying degrees staffed by mental health professionals. Furthermore, they are often housed in institutions whose model for care is based on service to patients rather than self-help. There are inherent conflicts in the modified TC which is a hybrid of these two treatment approaches. This paper explores issues related to the roles of patients, mental health professionals, and the parent institution in the TC modified for the treatment of the mentally ill chemical abuser.

The objectives of this study were to investigate HIV-1 seroprevalence and risk factors, disease progression, and awareness of HIV-1 serostatus in a population of inner city, substance using, psychiatric inpatients. To pursue these goals, we tested 118 (103 M, 15 F) dually diagnosed, acute care inpatients for HIV-1 antibodies and administered structured interviews. Twenty-seven (23%, including 24 M and 3 F) of the subjects were HIV-1 seropositive. Seropositivity was twice as great among intravenous drug users and men who had sex with other men as among patients not belonging to either of these two groups. Logistic regression analysis among male subjects revealed a significantly elevated HIV-1 risk associated with a primary diagnosis of depression (odds ratio adjusted for age, race, and presence of an AIDS risk behavior = 4.2, 95% confidence interval = 1.1, 16.5; p = 0.04). Less than half of the seropositives knew their HIV-1 status prior to this study, one had AIDS and four had two or more constitutional symptoms of AIDS. The high rate of seropositivity in this indigent, dually diagnosed population presents challenges to the health-care community. That few individuals had HIV-1 related symptoms may have implications for other treatment settings.