Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.     

Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells

Chronic exposure to cadmium causes preferential accumulation of cadmium in the kidney, leading to nephrotoxicity. In the process of renal cadmium accumulation, the cadmium bound to a low-molecular-weight metal-binding protein, metallothionein, has been considered to play an important role in reabsorption by epithelial cells of proximal tubules in the kidney. However, the role and mechanism of the transport of Cd(2+) ions in proximal tubule cells remain unclear. Zinc transporters such as Zrt, Irt-related protein 8 (ZIP8) and ZIP14, and divalent metal transporter 1 (DMT1) have been reported to have affinities for Cd(2+) and Mn(2+). To examine the roles of these metal transporters in the absorption of luminal Cd(2+) and Mn(2+) into proximal tubule cells, we utilized a cell culture system, in which apical and basolateral transport of metals can be separately examined. The uptake of Cd(2+) and Mn(2+) from the apical side of proximal tubule cells was inhibited by simultaneous addition of Mn(2+) and Cd(2+), respectively. The knockdown of ZIP8, ZIP14 or DMT1 by siRNA transfection significantly reduced the uptake of Cd(2+) and Mn(2+) from the apical membrane. The excretion of Cd(2+) and Mn(2+) was detected predominantly in the apical side of the proximal tubule cells. In situ hybridization of these transporters revealed that ZIP8 and ZIP14 are highly expressed in the proximal tubules of the outer stripe of the outer medulla. These results suggest that ZIP8 and ZIP14 expressed in the S3 segment of proximal tubules play significant roles in the absorption of Cd(2+) and Mn(2+) in the kidney.     

Transepithelial transport of vinblastine by kidney-derived cell lines. Application of a new kinetic model to estimate in situ Km of the pump

We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelia such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michaelis-Menten kinetics with an apparent Km = 1.1 microM. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 greater than MDCK greater than OK cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.     

Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia

We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.     

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.     

Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium

Summary A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cell were observed to retain in vitro a Na⁺-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na⁺-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitivep-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK acivity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by Grant 9 RO1 DK40286-07 from the National Institutes of Health, Bethesda, MD, and NIH Research Career Development Award 1 K04 CA 0088-01 to M.T.     

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.     

Polarity of neutral amino acid transport and characterization of a broad specificity transport activity in a kidney epithelial cell line, MDCK

The Madin-Darby canine kidney (MDCK) cell line was investigated with respect to the cellular polarity of amino acid transport in early confluent versus late confluent cultures. Early confluent cultures could take up amino acids from the apical and the basolateral sides of the cell layer via amino acid transport Systems A, ASC, and L. However, in late confluent cultures the activities of Systems A and L were clearly localized to the basolateral surface of the cell monolayer. In addition to the presence of systems A, ASC, and L, a novel activity, measurable under conditions used for quantitating System ASC, was found to be active in the apical membrane of these cells. This transporter, termed System G (for general), recognized basic and neutral amino acids with high affinity and acidic amino acids with lower affinity. System G exhibited broad substrate specificity, strict cation specificity, and a broad pH optimum with maximal activity at acidic pH. The activity of System G was relatively low after growth in serum-containing medium but was induced in a defined medium. Induction of System G activity was dependent upon the presence of prostaglandin E1. The broad substrate specificity, low pH optimum, and Na+ dependence suggest that System G may function in apical membranes as an energy-dependent transport route during reabsorption of amino acids from the kidney tubule lumen.     

A dileucine motif targets the sulfate anion transporter sat-1 to the basolateral membrane in renal cell lines

The sat-1 transporter mediates sulfate/bicarbonate/oxalate anion exchange in vivo at the basolateral membrane of the kidney proximal tubule. In the present study, we show two renal cell lines [Madin-Darby canine kidney (MDCK) and porcine proximal tubular kidney (LLC-PK1) cells] that similarly target sat-1 exclusively to the basolateral membrane. To identify possible sorting determinants, we generated truncations of the sat-1 cytoplasmic COOH terminus, fused to enhanced green fluorescence protein (EGFP) or the human IL-2 receptor -chain (Tac) protein, and both fusion constructs were transiently transfected into MDCK cells. Confocal microscopy revealed that removal of the last three residues on the sat-1 COOH terminus, a putative PDZ domain, had no effect on basolateral sorting in MDCK cells or on sulfate transport in Xenopus oocytes. Removal of the last 30 residues led to an intracellular expression for the GFP fusion protein and an apical expression for the Tac fusion protein, suggesting that a possible sorting motif lies between the last 3 and 30 residues of the sat-1 COOH terminus. Elimination of a dileucine motif at position 677/678 resulted in the loss of basolateral sorting, suggesting that this motif is required for sat-1 targeting to the basolateral membrane. This posttranslational mechanism may be important for the regulation of sulfate reabsorption and oxalate secretion by sat-1 in the kidney proximal tubule. enhanced green fluorescence protein; Tac; polarized cells; sorting; transport     

Principal and intercalated cells in primary cultures of rabbit renal collecting tubules revealed by monoclonal antibodies.

In this study we describe the production and characterization of two monoclonal antibodies (mAb 503 and mAb 703) raised against the apical membrane of rabbit cortical collecting tubule (CCT) cells. The specificity of the two monoclonal antibodies was studied by immunoelectron microscopy on kidney sections. These antibodies were used to identify principal and intercalated cells in primary cultures of CCT. To assess the maintenance of the basic characteristics of the cortical collecting cells during the growth process we determined the biochemical and electrophysiological properties of cultured CCT. Of the monoclonal antibodies produced mAb 503 was specifically directed against the luminal membrane of intercalated cells as shown by immunoelectron microscopy. mAb 703 bound specifically the apical membrane of the principal cells. In primary cultures of CCT mAb 503 and mAb 703 bound antigens present on the apical membrane of different cells and permitted the study of the distribution of the two cell types. Results showed the maintenance of the epithelial polarity of cultured CCT and the expression of specific antigens.     

Porcine myosin-VI: characterization of a new mammalian unconventional myosin

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.     

The fine structure of the mesonephros of the lamprey,Entosphenus japonicus Martens

Summary The fine structure of the mesonephric kidney of the lamprey, Entosphenus japonicus Martens, has been investigated with the electron microscope and discussed from the viewpoint of comparative morphology of the mesonephros.The structure of the capillary wall of the glomerulus essentially coincides with that of higher vertebrates, though its basement membrane is remarkably thick (300–400 m) because of a dense accumulation of fibrillar material between the endothelium and the basal lamina of epithelial cell. No obvious fenestration of the endothelial cell has been observed in the glomerulus or capillaries in any part of this organ.The kidney tubule is divided into three segments: 1. neck segment composed of ciliated cells with numerous mitochondria and glycogen particles, 2. proximal tubule composed of brush bordered cells provided with extensive pinocytotic vesicles and lysosomal granules in the apical cytoplasm and with lamellar membranes in the basal, and 3. distal tubule characterized by cells which, with their abundant mitochondria and branched tubular endoplasmic reticulum (about 500 Å diameter) with a central core, closely resemble the chloride cells in the gill filament of some teleosts. The possibility that the lamellar membranes in the proximal tubule cells correspond to basal infoldings is discussed.The extensive development of the tubular reticulum and of the mitochondria in the distal tubule cells is believed to reflect the active absorption of urine chloride in the urinary tubule of lamprey mesonephric kidney evidenced by physiologists. The proximal tubule is suggested to take a part also in the urinary transport of water and ions, as the lamellar membranes found in the cells of this portion likely correspond to the basal infoldings in more advanced forms of the kidney.The epithelial cells of the ureteric duct are characterized by granules suggesting a mucous secretion. No fine structure implying an absorptive activity in this duct has been observed.     

A discussion of Ambystoma kidney tubule ion channels, transporters, and pH regulation.

This paper explores the role and biophysical expression of the equivalent electrical circuit model as it applies to ionic conductances across the paracellular shunt, apical membrane, and basolateral membrane of the Ambystoma renal proximal tubule. Information about such conductances may be experimentally determined through transepithelial voltage and intracellular voltage measurements. The equivalent electrical circuit model has been applied extensively by investigators to define ion channels and transport mechanisms in the salamander proximal tubule. A comprehensive discussion of all known ionic conductance and transport pathways as well as pH-regulatory functions of contributory symports/antiports is examined in the Ambystoma proximal tubule. This paper explores renal physiological principles and serves as a companion to: Bock JF, Boulpaep EL: Bicarbonate transport mechanisms in the Ambystoma kidney proximal tubule: Transepithelial potential measurements.     

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.     

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.     

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

Intravital two-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate Texas red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate fluorescein, was seen only on the apical surface of proximal tubule cells, suggesting that internalized folate conjugates are localized to acidic compartments. The majority of the FTR conjugate internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of FTR were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, which is known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the subapical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Together, these data support the argument that folate conjugates (and, by extrapolation, physiological folate) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments. proximal tubule cell     

Phosphate transport by reconstructed monolayers from cultured mouse kidney cells

A reconstructed monolayer was formed using epithelial cells from normal mouse kidney to investigate the hormonal effect on phosphate transport by the renal cells. The cells, when cultured on a Millipore filter, formed a monolayer with an apical negative transepithelial potential of 8.4 +/- 0.4 mV. When radioactive phosphate was added onto the apical surface of the monolayer (corresponding to the luminal surface of a renal tubule), the phosphate was transported through the cell layer to the basolateral surface (corresponding to the peritubular surface of a renal tubule). This transport process was saturable, energy-dependent, and inhibited by 2,4-dinitrophenol or ouabain. Dose-dependent parathyroid hormone-induced inhibition (73% of the control) was also evident in this system. Similar inhibition (69% of the control) was observed with DBcAMP. Thus, monolayers reconstructed from cultured mouse kidney cells show characteristics similar to those of renal tubules.