Elsa Zotta

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Functional characterization and localization of AQP3 in the human colon.

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

CFTR May Modulate AQP9 Functionality in Preeclamptic Placentas

UNLABELLED Preeclampsia (PE) is a hypertensive disorder unique to human pregnancy. Although its causes remain unclear, it is known that altered placental villous angiogenesis and a poorly developed fetoplacental vasculature can affect the transport functions of the syncytiotrophoblast (hST). We have previously observed that in preeclamptic placentas there is an increase in AQP9 protein expression, with a lack of functionality. Up to now, the mechanisms for AQP9 regulation and the role of AQP9 in the human placenta remain unknown. However, there is strong evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) regulates AQP9 functionality. OBJECTIVE Here, we studied CFTR expression and localization in hST from preeclamptic placentas in order to investigate if alterations in CFTR may be associated with the lack of activity of AQP9 observed in PE. METHODS The expression of CFTR in normal and preeclamptic placentas was determined by Western Blot and immunohistochemistry, and CFTR-AQP9 co-localization was determined by immunoflurescence. Water uptake experiments were performed using explants from human normal term and preeclamptic placentas treated with CFTR inhibitors. RESULTS We found that CFTR expression significantly decreased in preeclamptic placentas, and that the hST apical labeling almost disappeared, losing its co-localization with AQP9. Functional experiments demonstrated that water uptake diminished in normal term explants incubated with CFTR inhibitors. CONCLUSIONS These results suggest that CFTR expression decreases in preeclampsia and may thus be implicated in the regulation of AQP9 activity.

Interaction between Lysophosphatidic Acid, Prostaglandins and the Endocannabinoid System during the Window of Implantation in the Rat Uterus

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids’ receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins’ synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids’ levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.

A Glucosylceramide Synthase Inhibitor Protects Rats Against the Cytotoxic Effects Of Shiga Toxin-2

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.

Clade 8 and clade 6 strains of Escherichia coli O157:H7 from cattle in Argentina have hypervirulent-like phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

Effect of Shiga toxin 2 on water and ion transport in human colon in vitro

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 Jw with no effect on the basal Isc 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.

Inhibition of water absorption and selective damage to human colonic mucosa are induced by subtilase cytotoxin produced by Escherichia coli O113:H21.

ABSTRACT Shiga toxin-producing Escherichia coli O157:H7 (STEC) is by far the most prevalent serotype associated with hemolytic uremic syndrome (HUS) although many non-O157 STEC strains have been also isolated from patients with HUS. The main virulence factor of STEC is the Shiga toxin type 2 (Stx2) present in O157 and non-O157 strains. Recently, another toxin, named subtilase cytotoxin (SubAB), has been isolated from several non-O157 strains and may contribute to the pathogenesis of HUS. Here, we have demonstrated that an O113:H21 STEC strain expressing SubAB and Stx2 inhibits normal water absorption across human colon and causes damage to the surface epithelium, necrosis, mononuclear inflammatory infiltration, edema, and marked mucin depletion. This damage was less marked, but nevertheless significant, when purified SubAB or E. coli O113:H21 expressing only SubAB was assayed. This is the first study showing that SubAB may directly participate in the mechanisms of diarrhea in children infected with non-O157 STEC strains.

Evidence for insulin-mediated control of AQP9 expression in human placenta

UNLABELLED The AQP9 gene contains a negative insulin response element, suggesting that it may be modulated by insulin. Previously, we reported AQP9 overexpression in preeclamptic placentas but a lack of functionality of AQP9 in water and mannitol transport. We also observed high serum levels of insulin and TNF-α in preeclamptic women. OBJECTIVE To evaluate whether AQP9 expression is regulated by insulin in the human placenta, and whether the dysregulation of AQP9 observed in preeclamptic placentas may be related to the inability to respond to insulin stimuli. METHODS Explants from normal and preeclamptic placentas were cultured at different concentrations of insulin. Treatment with TNF-α was used to induce phosphorylation of insulin receptor substrate (IRS), which may desensitize insulin action. AQP9 molecular expression and water uptake was determined. RESULTS Insulin decreased the molecular expression of AQP9 exclusively in explants from normal placentas in a concentration-dependent manner. Treatment with TNF-α previous to insulin addition prevented these changes. Moreover, insulin treatment did not modify water uptake neither its sensitivity to HgCl(2.) CONCLUSION AQP9 water permeability seems to be independent of its molecular expression, strongly suggesting that AQP9 might not have a key role in water transport in human placenta. We also propose another mechanism of down-regulation of AQP9 molecular expression mediated by insulin in a concentration-dependent manner in human placenta and provide new evidence that in preeclamptic placentas the mechanisms of insulin signaling may be altered, producing an overexpression of AQP9 that does not correlate with an increase in its functionality.