We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na ؉ /H ؉ exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.

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 ...

Abstract. Chronic allograft nephropathy (CAN) is due to both immunologic and non-immunologic factors and results in the development of nonspecific pathologic features that may even be present in long-term well-functioning renal allografts. To investigate the natural history of CAN and potential risk factors associated with progression of these histologic lesions, this study evaluated the of histologic alterations of 124 sequential protocol biopsies performed at 2, 3, and 5 yr after transplantation in 46 patients who exhibited histologic evidence of CAN in the 1-yr biopsy.

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg 2؉ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl ؊ revealed high nitrate permeability even at pH 7.4; P NO 3 /P Cl > 9.8. Sitedirected mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO 3 ؊ /Cl ؊ selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO 3 ؊ /Cl ؊ mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan. Nanogels e¡ectively prevented protein aggregation (i.e. carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation. Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins. The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins. The nanogels acted as a host for the trapping of refolded intermediate proteins. Cyclodextrin is an e¡ector molecule that controls the binding ability of these host nanogels to proteins. The present nanogel system was also e¡ective at the renaturation of inclusion body of a recombinant protein of the serine protease family.

No abstract

Urinary exosomes, secreted into urine from renal epithelial cells, are known to contain many types of renal functional membrane proteins. Here, we studied whether renal ischemia-reperfusion (I/R) affects urinary exosomal aquaporin-1 (AQP1) excretion in rats subjected to renal I/R and patients who underwent renal transplantation. Immunoblotting studies demonstrated reduction of the urinary exosomal AQP1 level even at 6 h after renal I/R, and the level continued to be low over 96 h after I/R. Renal AQP1 mRNA and protein analyses revealed that the decreased excretion of urinary exosomal AQP1 is associated with renal AQP1 protein retention in the early phase and with a decreased expression level of renal AQP1 in the later phase of renal I/R injury. Decreased abundance of urinary exosomal AQP1 in a recipient patient was also observed at 48 h after renal allograft transplantation. No significant decrease in urinary exosomal AQP1 was observed in a rat model of nephropathy or in patients with proteinuria. Our studies suggest that the renal AQP1 expression level is possibly controlled by its urinary exosomal excretion and indicate that urinary exosomal AQP1 is a novel urinary biomarker for renal I/R injury.

This paper is concerned with the blowup phenomena for initial value problem of semilinear wave equation with critical space-dependent damping term, f and g are compactly supported smooth functions and ε > 0 is a small parameter. The main result of the present paper is to give a solution of (DW:V 0 ) and to provide a sharp estimate for lifespan for such a solution when N N−1 < p ≤ p S (N + V 0 ), where p S (N) is the Strauss exponent for (DW:0). The main idea of the proof is due to the technique of test functions for (DW:0) originated by Zhou-Han (2014, MR3169791). Moreover, we find a new threshold value V 0 = (N−1) 2 N+1 for the coefficient of critical and singular damping |x| −1 . (2010): Primary: 35L70. Mathematics Subject Classification