Increased urinary CD80 excretion and podocyturia in Fabry disease.

BACKGROUND: Certain glomerulopathies are associated with increased levels of CD80 (B7-1). We measured the urinary excretion of CD80, podocyturia and proteinuria in controls and in subjects with Fabry disease either untreated or on enzyme replacement therapy (ERT). METHODS: Cross-sectional study including 65 individuals: controls (n = 20) and Fabry patients (n = 45, 23 of them not on ERT and 22 on ERT). Variables included age, gender, urinary protein/creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), urinary uCD80/creatinine ratio (uCD80) and podocyturia. CD80 mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes. RESULTS: Controls and Fabry patients did not differ in age, eGFR and gender. However, UPCR, uCD80 and podocyturia were significantly higher in Fabry patients than in controls. As expected, Fabry patients not on ERT were younger and a higher percentage were females. Non-ERT Fabry patients had less advanced kidney disease than ERT Fabry patients: UPCR was lower and eGFR higher, but uCD80 and podocyturia did not differ between non-ERT or ERT Fabry patients. There was a significant correlation between uCD80 and UPCR in the whole population (r 0.44, p 0.0005) and in Fabry patients (r 0.42, p 0.0046). Lyso-Gb3 at concentrations found in the circulation of Fabry patients increased uCD80 expression in cultured podocytes. CONCLUSIONS: Fabry disease is characterized by early occurrence of increased uCD80 excretion that appears to be a consequence of glycolipid accumulation. The potential for uCD80 excretion to reflect early, subclinical renal Fabry involvement should be further studied.

Characterization of stx2 tubular response in a rat experimental model of hemolytic uremic syndrome.

BACKGROUND/AIMS: In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. The aim of our study was to analyze the early tubular response under the effect of Shiga toxin type 2 (Stx2) in a rat experimental model of HUS. METHODS: Adult male Sprague-Dawley rats were injected intraperitoneally with culture supernatant from recombinant Escherichia coli expressing Stx2. Functional, histological, immunohistochemical and Western blot studies were performed at 48 h postinoculation. RESULTS: Renal tubules showed the loss of the epithelial markers E-cadherin and ß-catenin, and an increase in transforming growth factor-ß1 expression. We detected the expression of α-smooth muscle actin in the interstitium and fibrosis in the periglomerular areas. CONCLUSION: Our results indicate that the early tubular response to the effects of Stx2 is related to an immunophenotype change of tubular cells and the presence of mild fibrosis in the interstitium.

In Acute IgA Nephropathy, Proteinuria and Creatinine Are in the Spot, but Podocyturia Operates in Silence: Any Place for Amiloride?

IgA nephropathy is the most frequent cause of primary glomerulonephritis, portends erratic patterns of clinical presentation, and lacks specific treatment. In general, it slowly progresses to end-stage renal disease. The clinical course and the response to therapy are usually assessed with proteinuria and serum creatinine. Validated biomarkers have not been identified yet. In this report, we present a case of acute renal injury with proteinuria and microscopic hematuria in a young male. A kidney biopsy disclosed IgA nephropathy. Podocyturia was significantly elevated compared to normal subjects. Proteinuria, renal function, and podocyturia improved promptly after steroids and these variables remained normal after one year of follow-up, when steroids had already been discontinued and patient continued on valsartan and amiloride. Our report demonstrates that podocyturia is critically elevated during an acute episode of IgA nephropathy, and its occurrence may explain the grim long-term prognosis of this entity. Whether podocyturia could be employed in IgA nephropathy as a trustable biomarker for treatment assessment or even for early diagnosis of IgA nephropathy relapses should be further investigated.

Functional and molecular expression of AQP9 channel and UT-A transporter in normal and preeclamptic human placentas.

Transcellular flux of urea across human placenta may be facilitated by aquaglyceroporins and/or by urea transporters (UT). Previously we have reported the presence of AQP3 and AQP9 in the syncytiotrophoblast of human term placenta (hST). In the present study, we detected a significant uptake of water, urea and mannitol sensitive to mercury and phloretin in explants from normal term placenta, which indicates a functional AQP9. In addition, we observed an increase of AQP9 expression in preeclamptic placenta with a lack of functionality of AQP9 for water and mannitol. Our data showed a molecular and functional expression of UT-A in hST from normal and preeclamptic placentas. In the last case, urea uptake sensitive to phloretin and mercury increased and the UT-A protein seems to be augmented. These results suggest that the increase of urea uptake in preeclamptic pregnancies could be attributed to an increase of expression of UT-A more than AQP9 proteins. In conclusion, our results provide new evidences that suggest the involvement of AQP9 and UT-A in the urea excretion mechanism across hST from mother to fetus in physiological conditions. Much further work is needed to define whether the overexpression of AQP9 plays a direct role either in the pathogenesis or in the adaptative response of preeclampsia.

Podocyturia: A Clue for the Rational Use of Amiloride in Alport Renal Disease.

No specific or efficient treatment exists for Alport syndrome, an X-linked hereditary disease caused by mutations in collagen type IV, a crucial component of the glomerular basement membrane. Kidney failure is usually a major complication of the disease, and patients require renal replacement therapy early in life. Microhematuria and subsequently proteinuria are hallmarks of kidney involvement, which are due to primary basement membrane alterations that mainly cause endothelial thrombosis and podocyte contraction and ulterior irreversible detachment. Commonly drug-based approaches include angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, which are employed to reduce proteinuria and thus retard kidney disease progression and cardiovascular morbidity and mortality. However, as any hereditary disease, it is expressed as early as in the intrauterine life, and usually an index case is helpful to detect family-related cases. As no specific treatment exists, pathophysiologically based approaches are useful. The present case illustrates the reduction rate of urinary podocyte loss and proteinuria after amiloride administration and suggests the molecular pathways involved in Alport renal disease. Finally, podocyturia rather than proteinuria should be considered as an earlier biomarker of kidney involvement and disease progression in Alport disease.

Impact of Infection Dose and Previous Serum Antibodies against the Locus of Enterocyte Effacement Proteins on Escherichia coli O157:H7 Shedding in Calves following Experimental Infection.

Escherichia coli O157:H7 is the main causative agent of haemolytic uremic syndrome. Cattle are the main reservoir of these bacteria, and have been shown to develop immune response to colonization. Our aim was to investigate the faecal shedding pattern of E. coli O157:H7 in calves challenged intragastrically with either 10(8) or 10(10) CFU, as well as the ability of specific preexisting antibodies to reduce shedding of the pathogen. Shedding was analysed by direct counting as well as enrichment of rectoanal mucosal swabs. Statistical analysis was performed using a linear model for repeated measures with and without the inclusion of preexisting antibodies against the carboxy-terminal fraction of intimin-γ (γ-intimin C280) as a covariable. Results suggest that there is a statistical difference in the area under the shedding curves between both doses for 14 as well as 28 days after challenge (p = 0.0069 and 0.0209, resp.). This difference is increased when the prechallenge antibodies are taken into account (p = 0.0056 and 0.0185). We concluded that the bacterial dose influences shedding on calves experimentally challenged and that preexisting antibodies against E. coli O157:H7 γ-intimin C280 could partially reduce faecal excretion.

Copious Podocyturia without Proteinuria and with Normal Renal Function in a Young Adult with Fabry Disease.

The time for starting a patient with Fabry disease on enzyme replacement therapy is still a matter of debate, particularly when no overt classical clinical signs or symptoms are present. With respect to Fabry nephropathy, a dual problem coexists: the reluctance of many nephrologists to start enzyme replacement infusion until signs of renal disease appear as the appearance of proteinuria or an elevation in serum creatinine and the lack of validated biomarkers of early renal damage. In this regard, proteinuria is nowadays considered as an early and appropriate marker of kidney disease and of cardiovascular morbidity and mortality. However, in this report we demonstrate that podocyturia antedates the classical appearance of proteinuria and could be considered as an even earlier biomarker of kidney damage. Podocyturia may be a novel indication for the initiation of therapy in Fabry disease.

Water channel proteins AQP3 and AQP9 are present in syncytiotrophoblast of human term placenta.

The syncytiotrophoblast of human term placenta (HST) is a continuous, multinucleated structure with minimal tight junctions, which results from the fusion of the underlying cytotrophoblast cells. Consequently, the transport of metabolites, ions and water from mother to fetus could take place primarily via transcellular routes. Transcellular water flux may be facilitated by aquaporins, membrane proteins functioning as water channels that are widely expressed in cells and tissues. Here, we report the presence of AQP3 and AQP9 in the apical membranes of HST using RT-PCR, immunoblotting and immunohistochemistry. Since AQP3 is not only a water channels, but also permits the rapid passage of both urea and glycerol, while AQP9 also mediates the passage of carbamides, polyols, purines, and pyrimidines, we have speculated that these proteins could be involved in the transport of water and solutes from mother to fetus.

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.

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.

Physiopathological effects of Escherichia coli O157:H7 inoculation in weaned calves fed with colostrum containing antibodies to EspB and Intimin.

Escherichia coli O157:H7 is responsible for severe intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. Cattle are the primary reservoir for E. coli O157:H7 and the main source of infection for humans. In this study, we evaluated the ability of transferred maternal colostral antibodies against γ-Intimin C280 and EspB, to protect young weaned calves from E. coli O157:H7 infection. Hyperimmune colostra were obtained by immunization of pregnant cows with a mix of the mentioned antigens. All vaccinated cows mounted a significant IgG response against γ-Intimin C280, and EspB in sera and colostra. Colostrum-fed calves also exhibited high serum IgG titers against γ-Intimin C280 and EspB along with a rise in mucosal γ-Intimin C280-specific IgG antibodies at recto-anal junction and ileum. Additionally, 70 day-old calves received a challenge with E. coli O157:H7 but no reduction in total bacterial shedding or frequency of E. coli O157:H7 excretion from these calves was observed. Most tissue samples showed granulocyte focal infiltrations of the lamina propria and enterocyte erosion. In conclusion, up to the 70th day, the passively acquired γ-Intimin-C280 and EspB-IgG antibodies present in sera and recto-anal mucosa reached a titer insufficient to reduce EHEC O157 shedding and damages of experimentally inoculated young calves.

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.

Intraperitoneal administration of shiga toxin type 2 in rats in the late stage of pregnancy produces premature delivery of dead fetuses.

Infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolytic-Uremic Syndrome (HUS) have become relevant in public health since STEC is considered as one of the most important emergent pathogens. STEC infection may either be asymptomatic or begin with watery diarrhea associated with hemorrhagic colitis and HUS. The major virulence factor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2) although strains that express only Stx2 are highly prevalent. Up to now, it has not been established whether STEC infection affect pregnant women. In this study, we evaluated the effect of Stx2 on maternal lethality, fetal status and delivery time by injecting Stx2 in rats in the late stage of pregnancy. Stx2 induced fetal resorption, placental abruption, intrauterine hemorrhage and fetal death at 1-2 days post-injection in a dose-dependent manner. With 2ng Stx2/g body weight, placentas and fetuses presented extensive necrotic areas, while uteri and kidneys showed normal histology. Immunolocalization of Stx2 was observed in placentas and fetuses. With 4 and 6ng Stx2/g body weight maternal death was also observed. Those rats that survived after Stx2-treatment were able to become pregnant and deliver normal pups at term. Our results show, for the first time, that the preterm labor with fetal death observed in treated rats may be a consequence of the action of Stx2 on the feto-maternal unit. Although there are no reports of Stx2 effects in human pregnancy, we speculate that STEC infections could be one of the causes not yet determined of fetal morbimortality.

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.

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 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.

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.

Efecto citotóxico de la toxina Shiga tipo 2 y su subunidad B en células epiteliales tubulares renales humanas en cultivo / Cytotoxic effect of Shiga toxin type 2 and its B subunit on human renal tubular epithelial cell cultures

Escherichia coli enterohemorrágica productora de toxina Shiga (Stx) causa diarrea acuosa, colitis hemorrágica y síndrome urémico hemolítico (SUH). En Argentina, el SUH es la principal causa de insuficiencia renal en niños. El objetivo de este trabajo fue estudiar la toxicidad de Stx tipo 2 (Stx2) y su subunidad B (Stx2B) en células epiteliales tubulares renales humanas (CERH), en presencia y ausencia de factores inflamatorios. Los efectos citotóxicos se evaluaron como alteración de la funcionalidad del epitelio; daños histológicos; viabilidad celular; síntesis de proteínas y apoptosis celular. Los resultados muestran que Stx2 regula el pasaje de agua a través de CERH a tiempos menores de 1h de incubación. A tiempos mayores, hasta 72 hs, el estudio de la morfología, la viabilidad, la síntesis de proteínas y la apoptosis demostró que las CERH fueron sensibles a la acción citotóxica de Stx2 y Stx2B de una manera dosis y tiempo dependiente. Estos efectos fueron potenciados por lipopolisacáridos bacterianos (LPS), IL-1, y butirato.

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.

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 percent 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