Basolateral Membrane Of Type Research Articles

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit μ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85-91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.

Molecular and functional evidence for a role of a K,Cl cotransporter in the transepithelial secretion of fluid by mosquito renal tubules

In the mammalian renal collecting tubule, a K,Cl cotransporter (KCC4) recycles Cl− across the basolateral membrane of type A intercalated cells to permit the function of a basolateral Cl/HCO3 exchanger (AE1). Thus, it contributes to the transepithelial, V-type H+-ATPase-driven secretion of acid. In the renal (Malpighian) tubules of the mosquito Aedes aegypti, we have recently demonstrated that a basolateral, DIDS-sensitive AE contributes to the transepithelial, V-type H+-ATPase-driven secretion of urine during diuretic conditions. The presence of a KCC that regulates the AE is unknown. In the present study, we used RT-PCR on Malpighian tubules to clone a full-length cDNA (AeKCC) that encodes 1096 amino acids with ~55% identity to human KCCs. When expressed heterologously in Xenopus oocytes, the AeKCC mediates 86Rb+ uptake that is 1) activated by cell swelling and 2) inhibited by lowering external Cl− or adding DIOA. In isolated Malpighian tubules, adding peritubular DIOA reduced the spontaneous rates of fluid secretion by ~85%. In tubules secreting fluid at diuretic rates, adding peritubular DIOA returned secretion rates to unstimulated levels. The latter effect of DIOA resembles that of DIDS, which indicates that the KCC and AE may be functionally coupled. The former effect of DIOA indicates that the KCC also plays a role in tubule function when the AE is less active. Supported by NIH Grant K01-DK080194-01 to PMP.

Unraveling trafficking of the kidney anion exchanger 1 in polarized MDCK epithelial cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed at the basolateral membrane of type A intercalated cells in the kidney collecting tubule. Mutations occurring in the gene encoding this protein can give rise to distal renal tubular acidosis (dRTA), a disease characterized by an impaired urine acidification, nephrocalcinosis, and renal failure. Here we review how the study of dRTA mutants in polarized epithelial cells has shed light on the cellular mechanisms resulting in this renal disease.

Ultrastructural localization of UT-A and UT-B in rat kidneys with different hydration status

Urea transport in the kidney is mediated by a family of transporter proteins, including renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). We aimed to determine whether hydration status affects the subcellular distribution of urea transporters. Male Sprague-Dawley rats were divided into three groups: dehydrated rats (WD) given minimum water, hydrated rats (WL) given 3% sucrose in water for 3 days before death, and control rats given free access to water. We labeled kidney sections with antibodies against UT-A1 and UT-A2 (L194), UT-A3 (Q2), and UT-B using preembedding immunoperoxidase and immunogold methods. In control animals, UT-A1 and UT-A3 immunoreactivities were observed throughout the cytoplasm in inner medullary collecting duct (IMCD) cells, and weak labeling was observed on the basolateral plasma membrane. UT-A2 immunoreactivity in the descending thin limbs (DTL) was observed mainly on the apical and basolateral membranes of type I epithelium, and very faint labeling was observed in the long-loop DTL at the border between the outer and inner medulla. UT-A1 immunoreactivity intensity was markedly lower, and UT-A3 immunoreactivity was higher in IMCD of WD vs. controls. UT-A2 immunoreactivity intensities in the plasma membrane and cytoplasm of type I, II, and III epithelia of DTL were greater in WD vs. controls. In contrast, UT-A1 expression was greater and UT-A2 and UT-A3 expressions were lower in WL vs. controls. The subcellular distribution of UT-A in DTL or IMCD did not differ between control and experimental animals. UT-B was expressed in the plasma membrane of the descending vasa recta of both control and experimental animals. UT-B intensity was higher in WD and lower in WL vs. controls. These data indicate that changes in hydration status over 3 days affected urea transporter protein expression without changing its subcellular distribution.

Cl(-)-ATPase in rat brain and kidney.

Cl(-)-stimulated ATPase/ATP-dependent Cl(-) pump (Cl(-)-ATPase/pump) has been found as a candidate for an active outwardly directed Cl(-) transporter in brain neurons. (1) A 520-kDa protein complex with Cl(-)-ATPase/pump activity was isolated from rat brain. It consisted of four protein subunits (51, 55, 60, and 62 kDa proteins), the 51-kDa protein being a covalent phosphorylenzyme subunit. (2) An antiserum against the 51-kDa protein inhibited Cl(-)-ATPase/pump activity. Western blot analysis showed an immunoreactive 51-kDa protein in the brain, spinal cord, and kidney. By enzyme histochemistry and immunohistochemistry, Cl(-)-ATPase-like activity or immunoreactivity was observed on the plasma membranes of brain neurons, and on the baso-lateral membranes of type A intercalated cells of renal collecting ducts. (3) Reconstituted Cl(-)-ATPase/pump activity was highest in liposomes with phosphatidylinositol-4-monophosphate. LiCl, an inhibitor of inositolphosphatase, reduced Cl(-)-ATPase activity and increased intracellular Cl(-) concentrations in cultured rat hippocampal neurons with increased phosphatidylinositol turnover. (4) In the brains of patients with Alzheimer's disease (AD), where phosphatidylinositol 4-kinase activity is reduced, Cl(-)-ATPase activity was also reduced. Thus, Cl(-)-ATPase is likely an outwardly directed ATP-dependent Cl(-) transporter that consists of four subunits and is regulated by phosphatidylinositol-4-monophosphate. Changes in Cl(-)-ATPase activity may be related to the pathophysiology of human neurodegenerative diseases. J. Exp. Zool. 289:224-231, 2001.

Immunohistochemical demonstration of Cl- pump in type A intercalated cells of rat kidney.

In order to demonstrate the localization of an ethacrynic acid-sensitive Cl- pump in the rat kidney, immunohistochemical analysis was performed using an anti-Cl- pump antibody raised against rat brain Cl- pump protein with confocal laser scanning microscopy. The antibodies against Na+,K+-ATPase, aquaporin 2 and a type B intercalated cell marker, 43-kDa protein, were also used for comparison. Anti-Cl- pump antibody recognized a 51-kDa renal protein of the same size as that in the brain on Western blots. Cl- -pump-like immunoreactivity was observed on the basolateral membranes of 42+/-3% of cortical collecting duct (CCD) cells and of 38+/-1% of outer medullar collecting duct (OMCD) cells. Such immunoreactivity in CCD was sometimes co-localized with Na+,K+-ATPase, but in OMCD, the Cl- pump-like immunoreactivity co-existed with neither Na+,K+-ATPase, aquaporin 2 nor the type B intercalated cell marker 43-kDa protein. Thus, the Cl- pump was demonstrated to be localized on the basolateral membranes of type A intercalated cells of cortical and medullary collecting ducts in the rat kidney.

Autosomal dominant distal renal tubular acidosis and the AE1 gene.

Autosomal dominant distal renal tubular acidosis and the AE1 gene.

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Mar 1998https://doi.org/10.1152/ajprenal.1998.274.3.Fa1Original articleMoreSectionsPDF (9 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 273, October 1997 Volume 42, October 1997 Pages F601–F614: S. L. Alper, A. K. Stuart-Tilley, D. Biemesderfer, B. E. Shmukler, and D. Brown. “Immunolocalization of AE2 anion exchanger in rat kidney.” Some panels of Figs. 5 and 11 were misidentified. In the results, on p. F606 and F607 (inAE2 in cell surface membranes of inner medulla), the single reference to Fig. 5a in the second sentence should refer to Fig. 5b, and the two references to Fig. 5b should refer to Fig. 5a. The corrected text should read as follows: AE2 in cell surface membranes of inner medulla. Figure 5 shows double immunostaining of an SDS-pretreated transverse section through the upper one-third of the inner medulla. In Fig. 5b, monoclonal anti-AE1 again detected AE1 in the basolateral membranes of type A IC (arrows) and in retained red blood cells. In Fig. 5a, anti-AE2 aa 1224–1237 detected weaker basolateral staining in the IMCD cells, in addition to AE1 staining in the same type A IC (arrows) and red blood cells. The narrow diameter AE2-positive profiles in Fig. 5a likely represent thin limbs.Also, on p. F611, in the legend to Fig. 11, the reference to panelsG–L in the first sentence should refer to panelsJ–L. Therefore, the first sentence of this legend should read as follows:Specificity of immunostaining in rat kidney cortex with anti-AE1 aa 917-929 (A–C) and with anti-AE2 aa 1224–1237 (D–L) with (D–I) or without SDS pretreatment (J–L).This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated articlesImmunolocalization of AE2 anion exchanger in rat kidney 01 Oct 1997American Journal of Physiology-Renal Physiology More from this issue > Volume 274Issue 3March 1998Pages Fa1-Fa1 Copyright & PermissionsCopyright © 1998 the American Physiological Societyhttps://doi.org/10.1152/ajprenal.1998.274.3.Fa1History Published online 1 March 1998 Published in print 1 March 1998 Metrics Downloaded 46 times

Autosomal Dominant Distal Renal Tubular Acidosis Is Associated in Three Families with Heterozygosity for the R589H Mutation in the AE1 (Band 3) Cl−/HCO3−Exchanger

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

Sodium chloride cotransport at the basolateral membrane of type II pneumocytes.

Current evidence indirectly supports the hypothesis that Na+/Cl- cotransport occurs at basolateral membranes of type II pneumocytes. To test this hypothesis, enriched apical and basolateral plasma membrane vesicles were prepared from adult bovine type II pneumocytes. Uptake of 22Na+ or 36Cl- by these vesicle populations was monitored over time. Using enriched basolateral vesicles, substitution of formate- for Cl- nearly eliminated 22Na+ uptake, and substitution of Tris+ for Na+ significantly reduced 36Cl- uptake. These observations of Cl(-)-dependent 22Na+ uptake and Na(+)-dependent 36Cl- uptake demonstrated coupling between Na+ and Cl- absorption at the basolateral membrane. Na+/Cl- cotransport inhibitors, furosemide (1 mM) and bumetanide (0.1 mM), also reduced 22Na+ and 36Cl- uptakes by enriched basolateral vesicles. By contrast, furosemide and bumetanide did not affect 22Na+ and 36Cl- uptakes by enriched apical vesicles. Collectively, these observations support localization of Na+/Cl- cotransport to the basolateral membranes of mature type II pneumocytes.

Colocalization of GAPDH and band 3 (AE1) proteins in rat erythrocytes and kidney intercalated cell membranes

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.2.12) (GAPDH) is a multifunctional protein that associates with the cytoplasmic face of intact human erythrocyte membranes. This association has been postulated to be critically dependent on the interaction of GAPDH with the highly acidic NH2-terminal domain of the principal integral membrane protein of the erythrocyte plasma membrane, the band 3 anion exchanger (AE1). This domain is not conserved in murine erythrocyte AE1 and is fully deleted in the alternatively spliced AE1 isoform that is expressed in the kidney. The lack of conservation of this domain has been proposed to explain the reported absence of GAPDH association with rodent erythrocyte membranes. To determine whether GAPDH could be associated with AE1 proteins in rodent cell membranes, specific rabbit antibodies to peptide sequences of rat GAPDH and mouse AE1 were used to immunolocalize these proteins in sequential semithin sections of rat erythrocytes and kidney medulla. In rat erythrocytes, GAPDH immunoreactivity was predominantly membrane associated and colocalized with AE1. In the kidney medulla, GAPDH was concentrated in the basolateral membrane of type A intercalated cells, where it colocalized with the alternatively spliced kidney form of AE1. GAPDH immunoreactivity was not detected in the plasma membrane of any other cell type in the kidney, indicating its predominant association with AE1-rich membranes. If this membrane interaction occurs via AE1 binding, then GAPDH must have binding sites in addition to those previously described for such binding in human AE1.