Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.Aquaporins (AQPs) are a family of membrane proteins that facilitate the transport of water and small solutes (8,15,21). They are distributed widely in nature from bacteria to animals. Eleven aquaporins (AQP0 to AQP10) have been identified and functionally characterized in humans. We reported the most recent AQP, AQP10 (11, 13). Their physiological importance is documented by the targeted disruption in mice (knockout mice) and by the discovery of humans and mice with nonfunctioning mutations. Of nine AQPs disrupted in mice and humans (AQP0 to AQP7 and AQP10), only AQP2-null mice die due to massive polyuria from nephrogenic diabetes insipidus (23). The milder phenotypes in AQP disruptions in general are surprising, since water is vital for organisms. Therefore, AQPs seem to be not critically essential for the survival of mammals but seem to be involved in the quality of their lives.The completion of human genome projects has revealed two more aquaporin-like genes, which we have deposited in GenBank under the names of AQPX1 and AQPX2 (9). They are renamed AQP11 and AQP12 with the approval of the Human Gene Nomenclature Committee. Rat AQP11 (AQPX1) is highly expressed in the testis and moderately expressed in the kidney, liver, and brain. On the other hand, rat AQP12 (AQPX2) is selectively expressed in the pancreas. They share similar genome structures with three exons, which are distinct from other AQPs: AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6 have four exons; AQP3, AQP7, AQP8, AQP9, and AQP10 have six exons. In humans, AQP11 is mapped to chromosome 11q14 and AQP12 to chromosome 2q34-37, to which no diseases have been mapped. Moreover, we were not able to express them functionally in Xenopus oocytes. Therefore, their functions and physiological significance remain to be clarified.Previous AQPs have two highly conserved, short sequences named NPA (asparagine-proline-alanine) boxes. Each one of the NPA boxes has ...

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 ؋ 10 4 copies per well and 100% at >3.5 ؋ 10 4 copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.Hepatitis E, an acute viral hepatitis caused by infection with hepatitis E virus (HEV), is a globally distributed human disease. In developing countries of Asia, Africa, and Latin America, where sanitation conditions are not well maintained, HEV infection is transmitted via the fecal-oral route through viruscontaminated water or food, with substantial mortality in pregnant women (7, 33). In industrialized countries, autochthonous hepatitis E is far more common than previously recognized and has a predilection for older men, in whom it causes substantial morbidity and mortality (5,13,31,36,44). HEV is the sole member of the genus Hepevirus within the family Hepeviridae (6). It is a single-stranded, positive-sense, polyadenylated RNA molecule of approximately 7.2 kb in size, with short 5Ј-and 3Ј-untranslated regions (53). The genomic RNA contains three open reading frames (ORFs). ORF1 encodes nonstructural proteins involved in virus replication and virus protein processing. ORF2 and ORF3 overlap, and the ORF2 and ORF3

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2 −/− ). The Cldn2 −/− mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2 −/− kidney. Ultrathin section and freezefracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2 −/− mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na + , Cl -, and water was significantly decreased in Cldn2 −/− mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na + ) selectivity and therefore relative anion (Cl -) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na + and Cl -excretions in Cldn2 −/− mice were similar to those in wild types, but both were greater in Cldn2 −/− mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na + )-selective paracellular channels within tight junctions of mouse proximal tubules. mouse proximal tubule | tight junction | paracellular transport | Na/Cl transport | water transport T ight junctions (TJs) are circumferential seals around cells that selectively modulate paracellular permeability between extracellular compartments (1-3). On ultrathin-section electron microscopy, TJs appear as foci where the plasma membranes of neighboring cells make complete contact (4). On freeze-fracture electron microscopy, TJs appear as a continuous and anastomosing network of intramembranous particle strands (TJ strands) (5). These strands are mainly composed of linearly polymerized integral membrane proteins called claudins with molecular masses of ∼23 kDa (2, 3, 6). The claudin gene family contains more than 20 members in humans and in mice (2, 3, 7). The expression pattern of claudins varies considerably; most cell types express more than two claudins in various combinations to constitute mosaic TJ strands.Through the formation of TJ strands, claudins are directly involved in creating a primary barrier to the paracellular diffusion of solutes and water across epithelia (8). However, TJs are not a simple barrier: the barrier varies in tightness, measured by the transepithelial electrical resistance (R T ), and charge selectivity. Furuse et al. (9) reported that, when canine claudin-2 cDNA was transfected into high-resistance Madin-Darby canine kidney (MDCK) I cells primarily expressing claudins-1 and -4, the R T decreas...

Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0610 7 copies ml "1 , genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4610 4 copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1610 5 copies ml "1 on day 60. When 8.6610 5 copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6610 7 copies ml "1 on day 60. HEV incubated at temperatures higher than 70 6C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 6C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [(3)H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-beta receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 microM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 microM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 microM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 microM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-beta receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.