Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.     

Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti-annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non-confluent or confluent cells which remained attached to the collagen-coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen-coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley-Liss, Inc.     

Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.     

Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney.

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO(3)(-) absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40-45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H(+)-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO(3)(-) absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.     

Identification of the cDNA for xlcaax-1, a membrane associated Xenopus maternal protein.

xlcaax-1 is a cDNA coding for a CAAX box containing protein in Xenopus laevis that undergoes isoprenylation and palmitoylation. Here we report on the confirmation that this clone (formerly xlgv7) codes for a 110 kDa membrane associated protein and not an 80 kDa nuclear protein as originally believed (1). The reason for the misidentification was the presence of a common epitope on these two proteins recognized by the monoclonal antibody 37-1A9. We clarified the discrepancy by raising polyclonal antibodies against the xlcaax-1 protein produced in a bacterial expression system and demonstrating that these antibodies only recognize the 110 kDa protein on western blots of oocyte extracts. During early development xlcaax-1 protein starts reaccumulating from the neurula stage. In the adult frog both the xlcaax-1 protein and its cognate mRNA are highly enriched in the kidney. Consistent with the presence of CAAX box at the C-terminus this protein is associated with the membranes in Xenopus tissue culture cells (XTC).     

Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.     

Immunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium

We examined the localization of fodrin in epithelial cells of rat uriniferous and collecting tubules by immunofluorescence and immunoelectron microscopy of frozen sections. In the uriniferous tubule, fodrin was found along the cell membrane and in the well-developed terminal web, as previously reported in other epithelial cells: in the terminal web and along the basolateral cell membrane in the proximal tubule; all around the cell surface in the thin limb of Henle; along the basolateral surface in the thick limb of Henle's thick segment and the distal tubule. In the intercalated cells of the collecting tubule, fodrin was found not only along the basolateral cell membrane but also in the apical cytoplasm. The most peculiar labeling was obtained in the principal cells of the collecting tubule. In addition to labeling in the basolateral cell membrane, fodrin was found diffusely in the cytoplasmic matrix. Association of fodrin with any particular structure could not be identified, but the Golgi area was apparently free of labeling. Cytoplasmic labeling was more conspicuous in the principal cells of the medulla than in those of the cortex. The present results show that fodrin need not always exist in association with the cell membrane or the cytoskeleton but can occur in the cytoplasmic matrix, at least in epithelial cells. We discuss the possible physiological significance of the latter distribution.     

Ultrastructural localization of human complement C3b receptors in the human kidney as determined by immunoperoxidase staining with the monoclonal antibody C3RTo5

The precise localization of human complement C3b receptors in human kidneys was investigated by means of indirect immunoperoxidase staining of frozen kidney sections with the monoclonal antibody C3RT05, which is specific for human C3b receptors. Transmission electron microscopy indicated that C3b receptors are located on the entire cell membrane of the visceral epithelial cells of the glomeruli.     

Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney.

The multidrug resistance-associated protein (MRP) that is involved in drug resistance and the export of glutathione-conjugated substrates may not have the same epithelial cell membrane distribution as the P-glycoprotein encoded by the MDR gene. Because intestinal and kidney epithelial cells are polarized cells endowed distinct secreting and absorptive ion and protein transport capacities, we investigated the tissue and cell distribution of MRP in adult mouse small intestine, colon, and kidney by immunohistochemistry. Western blot analyses revealed the 190-kD MRP protein in these tissues. MRP was found in the basolateral membranes of intestinal crypt cells, mainly Paneth cells, but not in differentiated enterocytes. All the cells lining the crypt-villous axis of the colon wall contained MRP. MRP was found in the glomeruli, ascending limb cells, and basolateral membranes of the distal and collecting tubule cells of the kidney but not in proximal tubule cells. Cultured mouse intestinal m-ICcl2 cells and renal distal mpkDCT cells that have retained the features typical of intestinal crypt and renal distal epithelial cells, respectively, also possess MRP in their basolateral membranes. The patterns of subcellular and cellular distribution indicate that MRP may have a specific role in the basolateral transport of endogenous compounds in Paneth, renal distal, and collecting tubule cells.     

Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.     

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.     

Human copper transporter hCTR1 mediates basolateral uptake of copper into enterocytes: implications for copper homeostasis

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.     

Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway

Basolateral efflux is a necessary step in transepithelial (re)absorption of amino acids from small intestine and kidney proximal tubule. The best characterized basolateral amino acid transporters are y+LAT1-4F2hc and LAT2-4F2hc that function as obligatory exchangers and thus, do not contribute to net amino acid (re)absorption. The aromatic amino acid transporter TAT1 was shown previously to localize basolaterally in rat's small intestine and to mediate the efflux of L-Trp in the absence of exchange substrate, upon expression in Xenopus oocytes. We compared here the amino acid influx and efflux via mouse TAT1 in Xenopus oocytes. The results show that mTAT1 functions as facilitated diffusion pathway for aromatic amino acids and that its properties are symmetrical in terms of selectivity and apparent affinity. We show by real-time RT-PCR that its mRNA is highly expressed in mouse small intestine mucosa, kidney, liver, and skeletal muscle as well as present in all other tested tissues. We show that mTAT1 is not N-glycosylated and that it localizes to the mouse kidney proximal tubule. This expression is characterized by an axial gradient similar to that of the luminal neutral amino acid transporter B0AT1 and shows the same basolateral localization as 4F2hc. mTAT1 also localizes to the basolateral membrane of small intestine enterocytes and to the sinusoidal side of perivenous hepatocytes. In summary, we show that TAT1 is a basolateral epithelial transporter and that it can function as a net efflux pathway for aromatic amino acids. We propose that it, thereby, may supply parallel exchangers with recycling uptake substrates that could drive the efflux of other amino acids.     

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

A main pathway for energy ATP production inhuman body is by tricarboxylic acid cycle (Krebs cy-cle). Sodium-dependent dicarboxylate co-transporterprotein (SDCT, NaDC, NaC) is an organic aniontransporter protein family responsible for trans-mem- for 30 s, and extension 72℃ for 2 min; followed bybrane transport of Krebs cycle intermediate metabolite final extension 72℃ for 7 min. PCR products weresuch as succinate and citrate. They predominantly lo- …     

Site-specific Expression of IQGAP1, a Key Mediator of Cytoskeleton, in Mouse Renal Tubules

IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell–cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1–F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions. (J Histochem Cytochem 56:659–666, 2008)