Recent studies have demonstrated that a novel anion exchanger, pendrin, is expressed in the apical domain of type B intercalated cells in the mammalian collecting duct. The purpose of this study was 1) to determine the expression and distribution of pendrin along the collecting duct and connecting tubule of mouse and rat kidney and establish whether pendrin is expressed in the non-A-non-B intercalated cells and 2) to determine the intracellular localization of pendrin in the different populations of intercalated cells by immunoelectron microscopy. A peptide-derived affinity-purified antibody was generated that specifically recognized pendrin in immunoblots of rat and mouse kidney. Immunohistochemistry and confocal laser scanning microscopy demonstrated the presence of pendrin in apical domains of all type B intercalated cells in mouse and rat connecting tubule and collecting duct. In addition, strong pendrin immunostaining was observed in non-A-non-B intercalated cells. There was no labeling of type A intercalated cells. Immunoelectron microscopy demonstrated that pendrin was located in the apical plasma membrane and intracellular vesicles of both type B intercalated cells and non-A-non-B cells; the latter was identified by the presence of H(+)-ATPase in the apical plasma membrane. The results of this study demonstrate that both pendrin and H(+)-ATPase are expressed in the apical plasma membrane of non-A-non-B intercalated cells, suggesting that these cells are capable of both HCO and proton secretion. Furthermore, the presence of pendrin in both the apical plasma membrane and the apical intracellular vesicles of type B and non-A-non-B intercalated cells suggests that HCO secretion may be regulated by trafficking of pendrin between the two membrane compartments.

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

The anion exchanger pendrin is present in the apical plasma membrane of type B and non-A-non-B intercalated cells of the cortical collecting duct (CCD) and connecting tubule and is involved in HCO(3)(-) secretion. In this study, we investigated whether the abundance and subcellular localization of pendrin are regulated in response to experimental metabolic acidosis and alkalosis with maintained water and sodium intake. NH(4)Cl loading (0.033 mmol NH(4)Cl/g body wt for 7 days) dramatically reduced pendrin abundance to 22 +/- 4% of control values (n = 6, P < 0.005). Immunoperoxidase labeling for pendrin showed reduced intensity in NH(4)Cl-loaded animals compared with control animals. Moreover, double-label laser confocal microscopy revealed a reduction in the fraction of cells in the CCD exhibiting pendrin labeling to 65% of the control value (n = 6, P < 0.005). Conversely, NaHCO(3) loading (0.033 mmol NaHCO(3)/g body wt for 7 days) induced a significant increase in pendrin expression to 153 +/- 11% of control values (n = 6, P < 0.01) with no change in the fraction of cells expressing pendrin. Immunoelectron microscopy revealed no major changes in the subcellular distribution, with abundant labeling in both the apical plasma membrane and the intracellular vesicles in all conditions. These results indicate that changes in pendrin protein expression play a key role in the well-established regulation of HCO(3)(-) secretion in the CCD in response to chronic changes in acid-base balance and suggest that regulation of pendrin expression may be clinically important in the correction of acid-base disturbances.

Vascular endothelial growth factor A (VEGFA) production by podocytes is critical for glomerular endothelial health. VEGFA is also expressed in tubular epithelial cells in kidney; however, its physiologic role in the tubule has not been established. Using targeted transgenic mouse models, we found that Vegfa is expressed by specific epithelial cells along the nephron, whereas expression of its receptor (Kdr/Vegfr2) is largely restricted to adjacent peritubular capillaries. Embryonic deletion of tubular Vegfa did not affect systemic Vegfa levels, whereas renal Vegfa abundance was markedly decreased. Excision of Vegfa from renal tubules resulted in the formation of a smaller kidney, with a striking reduction in the density of peritubular capillaries. Consequently, elimination of tubular Vegfa caused pronounced polycythemia because of increased renal erythropoietin (Epo) production. Reducing hematocrit to normal levels in tubular Vegfa-deficient mice resulted in a markedly augmented renal Epo production, comparable with that observed in anemic wild-type mice. Here, we show that tubulovascular cross-talk by Vegfa is essential for maintenance of peritubular capillary networks in kidney. Disruption of this communication leads to increased renal Epo production and resulting polycythemia, presumably to counterbalance microvascular losses.

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to G scoupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb-and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominantnegative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb-and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization timeof-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32 P incubation of the inner medulla followed by autoradiography of twodimensional gels demonstrated increased 32 Vasopressin regulates the water permeability of the renal collecting duct epithelium in part by inducing translocation of aquaporin-2-containing intracellular vesicles to the apical region of the principal cells, where they fuse with the apical plasma membrane (1). The resulting increase in water permeability accelerates water reabsorption from the tubule lumen to the blood. Regulation of the water permeability of the collecting duct is a key element of the homeostatic process whereby vasopressin regulates body water balance.Vasopressin acts in collecting duct principal cells by binding to the V 2 receptor located in the basolateral membrane. Ligand binding activates adenylyl cyclase VI (2) via the heterotrimeric G-protein G s , resulting in an increase in cAMP production. In addition, V 2 receptor occupation by vasopressin elicits an increase in intracellular calcium (3-7). The increase in intracellular calcium is seen at physiological concentrations of vasopressin and can be elicited in response to exposure of the cells to cell-permeant forms of cAMP (7), suggesting that vasopressin-induced calcium mobilization is triggered by cAMP, presumably via protein kinase A activation. Recent confocal imaging studies of the inner medullary collecting duct (IMCD) 1 have shown that the vasopressin-induced increase in intracellular calcium is oscillatory in nature (8). The calcium release is mediated by ryanodine-sensitive intracellular calcium channels in the collecting duct cells (7).Isolated perfused tubule studies have demonstrated that the ability of vasopressin to increase water permeability in the IMCD is blocked by intracellular Ca 2ϩ chelation with BAPTA or by blockade of ryanodine-sensitive Ca 2ϩ channels by ryanodine, indicating that vasopressin-induced Ca 2ϩ mobilization is important for translocation of aquaporin-2 and the water permeability response in the IMCD (7). Furthermore, inhibitors of calmodulin, viz. W-7 and trifluoroperazine, cause a reversible inhibition of vasopressin-d...

The electroneutral Na + -dependent HCO 3 − transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up-regulated during NH 4 + -induced metabolic acidosis. Here we used in vitro perfusion and BCECF video-imaging of mTAL tubules to investigate functional localization and regulation of Na + -dependent HCO 3 − influx during NH 4 + -induced metabolic acidosis. Tubule acidification was induced by removing luminal Na + (∆pH i : 0.88 ± 0.11 pH units, n = 10). Subsequently the basolateral perfusion solution was changed to CO 2 /HCO 3 − buffer with and without Na + . Basolateral Na + -H + exchange function was inhibited with amiloride. Na + -dependent HCO 3 − influx was determined by calculating initial base flux of Na + -mediated re-alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min −1 mm −1 . A 2.4-fold increase of base flux to 21.8 ± 3.2 pmol min −1 mm −1 was measured in NH 4 + -treated animals (11 days, n = 11). Na + -dependent re-alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n = 10). In addition, Na + -dependent HCO 3 − influx was of similar magnitude in chloride-free medium and also up-regulated after NH 4 + loading. Na + -dependent HCO 3 − influx was not inhibited by 400 µm DIDS. A strong up-regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT-PCR analysis revealed no evidence for the Na + -dependent HCO 3 − transporter NBC4 or the two Na + -dependent CI − /HCO 3 − exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS-insensitive NBCn1. Function and protein are strongly up-regulated during NH 4 + -induced metabolic acidosis. We suggest that NBCn1-mediated basolateral HCO 3 − influx is important for basolateral NH 3 exit and thus NH 4 + excretion by means of setting pH i to a more alkaline value.

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.