Claudin-2-mediated cation and water transport share a common pore.

AIM: Claudin-2 is a tight junction protein typically located in 'leaky' epithelia exhibiting large paracellular permeabilities like small intestine and proximal kidney tubule. Former studies revealed that claudin-2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. This study analyses whether the diffusive transport of sodium and water occurs through a common pore of the claudin-2 channel. METHODS: Wild-type claudin-2 and different claudin-2 mutants were expressed in MDCK I kidney tubule cells using an inducible system. Ion and water permeability and the effect of blocking reagents on both were investigated on different clones of the mutants. RESULTS: Neutralization of a negatively charged cation interaction site in the pore with the mutation, D65N, decreased both sodium permeability and water permeability. Claudin-2 mutants (I66C and S68C) with substitution of the pore-lining amino acids with cysteine were used to test the effect of steric blocking of the claudin-2 pore by thiol-reactive reagents. Addition of thiol-reactive reagents to these mutants simultaneously decreased conductance and water permeability. Remarkably, all experimental perturbations caused parallel changes in ion conductance and water permeability, disproving different or independent passage pathways. CONCLUSION: Our results indicate that claudin-2-mediated cation and water transport are frictionally coupled and share a common pore. This pore is lined and determined in permeability by amino acid residues of the first extracellular loop of claudin-2.

Tight junction composition is altered in the epithelium of polycystic kidneys.

Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium.

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.

Transmission of hepatitis B infection from hepatitis B core antibody--positive liver allografts is prevented by lamivudine therapy.

Donor shortage has led to the use of hepatitis B core antibody (anti-HBc)--positive (anti-HBc(+)) liver allografts for patients in need of relatively urgent orthotopic liver transplantation (OLT). Because anti-HBc(+) allografts transmit hepatitis B virus (HBV) infection at a high rate, effective prophylaxis is required. We assessed the effectiveness of lamivudine in preventing HBV transmission by anti-HBc(+) allografts. Between March 1996 and March 2000 at Cedars-Sinai Medical Center (Los Angeles, CA), 15 of 169 patients (8.9%) received liver allografts from anti-HBc(+) donors. Six patients were hepatitis B surface antigen (HBsAg)(+) (group 1), and 9 patients were HBsAg negative (HBsAg(-); group 2) before OLT. All patients were administered lamivudine, 100 or 150 mg/d, orally after OLT. Patients who were HBsAg(+) before OLT also were administered hepatitis B immunoglobulin (HBIG) prophylaxis. Hepatitis B serological tests were performed on all patients, and HBV DNA was determined in liver tissues in 10 patients. All 15 patients remained HBsAg(-) at their last follow-up 2 to 40 months (mean, 17 months) post-OLT. All patients in group 1 had antibody to HBsAg (anti-HBs) titers greater than 250 mIU/mL post-OLT (mean follow-up, 20 months; range, 7 to 40 months). Of the 2 patients in group 1 who underwent liver biopsy after OLT, 1 patient had detectable hepatic HBV DNA despite being anti-HBs(+) and HBsAg(-). Among the patients in group 2, none acquired anti-HBc or HBsAg. Hepatic HBV DNA was undetectable in the 7 patients in group 2 who underwent liver biopsy after OLT. Anti-HBc(+) allografts can be safely used in patients who undergo OLT for chronic hepatitis B and susceptible transplant recipients if prophylaxis with combination HBIG and lamivudine or lamividine alone is administered after OLT, respectively. However, more data are needed to determine the efficacy of lamivudine monotherapy in preventing transmission of HBV infection from anti-HBc(+) liver allografts to susceptible recipients.

Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants.

Genetic mutations of the Cl(-) channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl(-) > I(-) anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.

Hyperfibrinolytic activity in hospitalized cirrhotic patients in a referral liver unit.

OBJECTIVE: Increased frequency of hyperfibrinolytic activity was reported in patients with cirrhosis. However, the incidence, clinical presentation, and the parameters related to hyperfibrinolysis remain largely unknown in these patients. By utilizing euglobulin lysis time (ELT) and other clinical coagulation tests, the present study investigated the incidence of and clinical parameters related to hyperfibrinolytic activity, and assessed predicting factors to epsilon-aminocaproic acid (EACA) treatment in cirrhotic patients with hyperfibrinolysis in a liver unit. METHODS: The study included 86 consecutive patients who were referred and admitted to a referral liver unit for various liver diseases. The mean age was 50.0 yr, with a male: female ratio of 60:26. Sixty-six patients (76.7%) were Hispanic and 75 (87.2%) were cirrhotic. The etiologies of liver diseases included alcoholic liver disease (n = 68, 79.1%), hepatitis B (n = 2, 2.3%), hepatitis C (n = 6, 7.0%), autoimmune hepatitis (n = 3, 3.5%), cryptogenic liver disease (n = 4, 4.7%), and hepatocellular carcinoma (n = 3, 3.5%). Coagulation studies included ELT, PT, PTT, fibrinogen, D-dimer, and fibrin degradation product levels. RESULTS: Hyperfibrinolytic activity as reflected by shortened ELT was present in 27/75 cirrhotic (31.3%) but 0/11 noncirrhotic patients, which was significantly correlated with higher Child-Pugh (C-P) class, abnormal levels of PT, PTT, fibrinogen, platelet count, and total bilirubin. Shortened ELT was more frequently seen in patients with hepatic decompensation and mucocutaneous bleeding, although these relationships were not statistically significant. In 27 patients with hyperfibrinolysis, five (18.5%) required EACA treatment for progressive mucocutaneous bleeding and/or hematoma. EACA treatment was significantly associated with higher C-P scores; greatly shortened ELT (< or =50% of normal value); and abnormal levels of fibrinogen, total bilirubin, and PT, indicating that these factors may serve as predictors for EACA treatment. CONCLUSION: Hyperfibrinolytic activity was seen in 31.3% of patients with cirrhosis, which is correlated with higher C-P scores; abnormal PT, PTT, fibrinogen level, and platelet count; and hyperbilirubinemia. Patients who received EACA treatment usually have a more severe hyperfibrinolytic activity as indicated by shortened ELT and low level of fibrinogen, and more severe liver disease as indicated by higher C-P scores and hyperbilirubinemia.

Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct.

Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471-6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function.

Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in transporting epithelial cells.

Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

Hepatolithiasis and biliary parasites.

Hepatolithiasis, or the presence of intrahepatic stones, is prevalent in East Asia and is characterized by the finding of stones within the intrahepatic bile ducts proximal to the confluence of the right and left hepatic ducts. Bile stasis and bacterial infection have been incriminated as the major aetiopathogenic factors. Clinical features include recurrent pyogenic cholangitis, multiple liver abscesses, secondary biliary cirrhosis and cholangiocarcinoma. The goals of management include accurate localization of pathologies, control of biliary sepsis and the elimination of stones and stasis. Ultrasonography, computed tomography and direct cholangiography complement each other in defining the stones, strictures and degree of liver damage. Non-operative biliary decompression by endoscopy and interventional radiology is effective in controlling the infection, but surgery remains the mainstay for the treatment of stones and strictures. Intra-operative ultrasound and flexible choledochoscopy, combined with percutaneous transhepatic cholangioscopy and intraductal lithotripsy, facilitate stone removal. Balloon dilatation and biliary stenting serve to open the bile duct strictures. The creation of a hepaticocutaneous jejunostomy after conventional surgery allows atraumatic access to the biliary system for the removal of recurrent stones. The management of biliary parasites begins with conservative measures, including analgesics and anti-helminthic therapy. In refractory cases or patients with acute cholangitis, endoscopic biliary drainage and the extraction of worms may be necessary. Improvement in sanitation plays a crucial role in the epidemiological control of these biliary diseases.

T helper cells in immune mice amplify the primary anti-tumor cytotoxic response.

In this report we examine the influence of splenic helper cells in the primary cytotoxic T lymphocyte (CTL) response against syngeneic murine leukemia virus-(MuLV) induced tumor cells. We identify an Lyt-1+ 800 R radiation-resistant helper T cell that will amplify the in vitro generation of CTL against syngeneic tumor cells from nonimmune spleen cells.