Apical Proteins Research Articles

Phosphorylation of Podocalyxin (Ser415) Prevents RhoA and Ezrin Activation and Disrupts Its Interaction with the Actin Cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.

The Role of VAMP7/TI‐VAMP in Cell Polarity and Lysosomal Exocytosis in vivo

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

Expression and subcellular localization of apical junction proteins in canine duodenal and colonic mucosa

To examine the expression and distribution of tight junction (TJ) and adherens junction (AJ) proteins in canine duodenal and colonic mucosa. Mucosa obtained from 4 healthy Beagles. Biopsy specimens of the duodenum and colon were obtained via endoscopy from 4 healthy dogs. The expression patterns and subcelluar localization of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and β-catenin in the duodenum and colon were analyzed by use of immunoblotting and immunofluorescence microscopy. In the duodenum, there was clear expression of claudin-3 and -5, E-cadherin, and β-catenin proteins and weak expression of claudin-7 protein. In contrast, there was clear expression of claudin-2 and -3, E-cadherin, and β-catenin proteins and weak expression of claudin-5 and -7 proteins in the colon, as determined by use of immunoblotting. As determined by the use of immunofluorescence microscopy, the duodenum and colon had staining for claudin-3 and -5, E-cadherin, and β-catenin in the most apical region and staining for claudin-7 in the basolateral region. Staining for claudin-2 was also observed in the colon. Information was provided about the expression patterns of TJ and AJ proteins in the duodenum and colon of clinically normal dogs. These results may provide valuable information for use in evaluating the importance of these TJ and AJ proteins in the pathogenesis of inflammatory bowel disease in dogs.

Neurogenesis at the Brain–Cerebrospinal Fluid Interface

Cerebral cortical progenitor cells can be classified into several different types, and each progenitor type integrates cell-intrinsic and cell-extrinsic cues to regulate neurogenesis. On one hand, cell-intrinsic mechanisms that depend upon appropriate apical-basal polarity are established by adherens junctions and apical complex proteins and are particularly important in progenitors with apical processes contacting the lateral ventricle. The apical protein complexes themselves are concentrated at the ventricular surface, and apical complex proteins regulate mitotic spindle orientation and cell fate. On the other hand, remarkably little is known about how cell-extrinsic cues signal to progenitors and couple with cell-intrinsic mechanisms to instruct neurogenesis. Recent research shows that the cerebrospinal fluid, which contacts apical progenitors at the ventricular surface and bathes the apical complex of these cells, provides growth- and survival-promoting cues for neural progenitor cells in developing and adult brain. This review addresses how the apical-basal polarity of progenitor cells regulates cell fate and allows progenitors to sample diffusible signals distributed by the cerebrospinal fluid. We also review several classes of signaling factors that the cerebrospinal fluid distributes to the developing brain to instruct neurogenesis.

N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.

Arp2/3 promotes junction formation and maintenance in theCaenorhabditis elegansintestine by regulating membrane association of apical proteins

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.

Abelson tyrosine kinase is required for Drosophila photoreceptor morphogenesis and retinal epithelial patterning

Coordinated differentiation and morphogenesis transform the Drosophila retina from a layer of epithelial cells into a complex three-dimensional organ. In this study we show that the Abelson (Abl) tyrosine kinase localizes to the dynamically remodeling apical-junctional membrane domains of the developing photoreceptor cells. Analyses of abl mutant clone phenotypes demonstrate that abl is required for enriched localization of adherens junction and apical polarity complex proteins at photoreceptor-photoreceptor cell junctions and apical membrane domains, respectively, for rhabdomere generation and for spatial organization of ommatidial cells along the apical-basal axis of the epithelium. Loss of abl does not alter expression or localization of Enabled (Ena) nor does heterozygosity for ena dominantly suppress the abl phenotypes, suggesting the downstream effector mechanisms used by Abl in the eye may differ from those used in the embryo. Together our results reveal a prominent role for Abl in coordinating multiple aspects of photoreceptor morphogenesis.

David Bilder: Getting to know epithelia inside and out

Bilder explores epithelial form and function in Drosophila using forward genetic screens.

Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy

In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the timeline of CD46 protein expression changes across the GA transition zone, relative to control eyes, and relative to events in other chorioretinal layers. Eleven donor eyes (mean age 87.0 ± 4.1 yr) with GA and 5 control eyes (mean age 84.0 ± 8.9 yr) without GA were evaluated. Macular cryosections were stained with PASH for basal deposits, von Kossa for calcium, and for CD46 immunoreactivity. Internal controls for protein expression were provided by an independent basolateral protein, monocarboxylate transporter 3 (MCT3) and an apical protein, ezrin. Within zones defined by 8 different semi-quantitative grades of RPE morphology, we determined the location and intensity of immunoreactivity, outer segment length, and Bruch’s membrane calcification. Differences between GA and control eyes and between milder and more severe RPE stages in GA eyes were assessed statistically. Increasing grades of RPE degeneration were associated with progressive loss of polarity and loss of intensity of staining of CD46, beginning with the stages that are considered normal aging (grades 0–1). Those GA stages with affected CD46 immunoreactivity exhibited basal laminar deposit, still-normal photoreceptors, and concomitant changes in control protein expression. Activated or anteriorly migrated RPE (grades 2–3) exhibited greatly diminished CD46. Changes in RPE CD46 expression thus occur early in GA, before there is evidence of morphological RPE change. At later stages of degeneration, CD46 alterations occur within a context of altered RPE polarity. These changes precede degeneration of the overlying retina and suggest that therapeutic interventions be targeted to the RPE.

Immunolocalization of WNK4 in mouse kidney

Initial reports claim that WNK4 localization is mainly at intercellular junctions of distal convoluted tubules (DCT) and cortical collecting ducts (CCD) in the kidney. However, we recently clarified the major targets of WNK4 kinase to be the OSR1/SPAK kinases and the Na-Cl co-transporter (NCC), an apical membrane protein in the DCT, thus raising the question of whether the cellular localization of WNK4 is at intercellular junctions. In this study, we re-evaluate the intrarenal and intracellular immunolocalization of WNK4 in the mouse kidney using a newly generated anti-WNK4 antibody. By performing double immunofluorescence of WNK4 with several nephron-segment-specific markers, we have found that WNK4 is present in podocytes in glomeruli, the cortical thick ascending limb of Henle's loop including macula densa, and the medullary collecting ducts (MCD), in addition to the previously identified nephron segments, i.e., DCT and CCD. These results are consistent with the finding that WNK4 constitutes a kinase cascade with OSR1/SPAK and NCC in the DCT, and highlights a novel role for WNK4 in nephron segments newly identified as being WNK4-positive in this study.

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.

Ciliary diffusion barrier: The gatekeeper for the primary cilium compartment

The primary cilium is a cellular antenna that detects and transmits chemical and mechanical cues in the environment through receptors and downstream signal proteins enriched along the ciliary membrane. While it is known that ciliary membrane proteins enter the cilium by way of vesicular and intraflagellar transport, less is known about how ciliary membrane proteins are retained in, and how apical membrane proteins are excluded from the cilium. Here, we review evidence for a membrane diffusion barrier at the base of the primary cilium, and highlight the recent finding of a septin cytoskeleton diffusion barrier. We also discuss candidate ciliopathy genes that may be involved in formation of the barrier, and the role of a diffusion barrier as a common mechanism for compartmentalizing membranes and lipid domains.

Apico-basal polarity in polycystic kidney disease epithelia

Epithelial cell polarity is essential for the establishment and maintenance of morphological and functional asymmetries that underlie normal renal structure and function and are brought about by the appropriate delivery of growth factor receptors and ion and fluid transporters and channels to apical or basolateral cell membranes. The fundamental process of cellular polarization is established early during development and is controlled by sets of evolutionarily conserved proteins that integrate intrinsic and extrinsic polarity cues. Specialized structural domains between adjacent cells and cells with their matrix, termed adherens junctions (AJ) and focal adhesions (FA), respectively, are formed that contain specific components of multi-molecular complexes acting as sites to recruit proteins and to activate intracellular mechano-transduction pathways. Regulation of these processes results in tight spatio-temporal control of renal tubule growth and lumen diameter. Abnormalities in macromolecular polarization complexes lead to a variety of diseases in different organs, a common example of which is Polycystic Kidney Disease (PKD), where epithelial cysts replace normal renal tubules. Membrane protein polarity defects in Autosomal Dominant (AD) PKD include the mis-polarization of normally basolateral membrane proteins to apical, lumenal membranes, such as epidermal growth factor (EGFR/ErbB) receptors and Na +K +-ATPase-α1 subunit; mis-polarization of normally apical membrane proteins to basolateral membranes, including the Na +K +2Cl − (NKCC1) symporter; and the failure to traffic and insert proteins into membranes resulting in their intracellular accumulation, such as E-cadherin and the β1 subunit of Na +K +-ATPase. Abnormalities in structural AJ, FA and polarity complexes in ADPKD epithelia include loss of E-cadherin, and focal adhesion kinase (FAK), MALS-3, Crb and Dlg complexes as well as disruptions in Rab/sec and syntaxin trafficking and membrane docking pathways. Since proper polarization of epithelial cells lining renal tubules is essential for normal kidney development and differentiation to prevent abnormal cystic dilation, interventions to reverse polarity defects to normal would offer therapeutic opportunities for PKD. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.

Caenorhabditis elegansSNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells

It is generally accepted that soluble N-ethylmaleimide-sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein-tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29-mediated fusion is required to maintain proper organellar morphology and functions.

The Hippo pathway and apico–basal cell polarity

The establishment and maintenance of apico-basal cell polarity is a pre-requisite for the formation of a functioning epithelial tissue. Many lines of evidence suggest that cell polarity perturbations favour cancer formation, even though the mechanistic basis for this link remains unclear. Studies in Drosophila have uncovered complex interactions between the conserved Hpo (Hippo) tumour suppressor pathway and apico-basal polarity determinants. The Hpo pathway is a crucial growth regulatory network whose inactivation in Drosophila epithelial tissues induces massive overproliferation. Its core consists of a phosphorylation cascade (comprising the kinases Hpo and Warts) that mediates the inactivation of the pro-growth transcriptional co-activator Yki [Yorkie; YAP (Yes-associated protein) in mammals]. Several apically located proteins, such as Merlin, Expanded or Kibra, have been identified as upstream regulators of the Hpo pathway, leading to the notion that an apical multi-molecular complex modulates core kinase activity and promotes Yki/YAP inactivation. In the present review, we explore the links between apico-basal polarity and Hpo signalling. We focus on the regulation of Yki/YAP by apical proteins, but also on how the Hpo pathway might in turn influence apical domain size as part of a regulatory feedback loop.

Regulation of apical membrane enrichment and retention of plasma membrane Ca2+ ATPase splice variants by the PDZ-domain protein NHERF2

The localization of plasma membrane calcium ATPase (PMCA) isoforms in specified membrane compartments is crucial for their function in local Ca2+ handling. PMCA2w/b is present in the apical membrane whereas alternative splice variants PMCA2x/b and 2z/b reside in the basolateral membrane in polarized epithelial cells. Here we found that the apical scaffolding protein NHERF2 greatly enhances the apical concentration of PMCA2w/b by tethering the pump to the underlying actin cytoskeleton. The interaction requires the C-terminal PDZ binding sequence in PMCA2b and results in increased membrane retention and decreased lateral mobility of the pump. In contrast, PMCA2x/b remains exclusively basolateral even when NHERF2 is overexpressed. Our results suggest that the alternatively spliced intracellular loop in PMCA2 imposes dominant membrane targeting information. NHERF2-mediated recruitment may be an effective means for polarized cells to regulate the abundance of PMCA2w/b in the apical membrane to meet an increased demand for local Ca2+ extrusion.

Transport of Free and Peptide‐Bound Glycated Amino Acids: Synthesis, Transepithelial Flux at Caco‐2 Cell Monolayers, and Interaction with Apical Membrane Transport Proteins

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Validation of the histological localization and subcellular polarity of the Oatp4c1 transporter in the rat kidney

Recently, the organic anion transporting polypeptide 4c1 (4c1) was identified as a novel uptake transporter predominantly expressed on the basolateral membrane of rat kidney proximal tubules. However, in‐vitro studies with MDCKII cells showed that upon transfection 4c1 polarizes to the apical membrane. We used multiple antibodies (N=4) to validate the subcellular localization of 4c1 in rat tissues and in MDCKII and LLCPK1 cells expressing rat 4c1. Antibodies amenable to immunohistochemistry were generated using N‐ and C‐terminal peptide sequences. Apical and basolateral protein markers were used for colocalization studies. Immunofluorescence studies showed that 4c1 polarizes to the apical surface in the in‐vitro cell models. In rat kidney, colocalization of 4c1 with aquaporin 1 but not calbindin in the subcortical region suggest that apical 4c1 expression is localized in proximal straight tubules. 4c1 also colocalizes with P‐glycoprotein and breast cancer resistance protein, but not with multidrug resistance protein 4 in rat kidney. These results were verified by western blot analysis of brush‐border membrane vesicles isolated from the kidney cortex and conclusively demonstrate the apical expression of 4c1 in the rat kidney. To investigate the physiological role of 4c1, future studies will focus on identifying substrates and the driving force for 4c1 mediated transport.