Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule.

The effects of pH on cotransporter kinetics were studied in renal proximal tubule cells. Cells were grown to confluence on permeable support, mounted in an Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, dinitrostilbene-disulfonate-sensitive current (DeltaI) was Na+ and HCO3- dependent and therefore was taken as flux through the cotransporter. When the pH of the perfusing solution was changed between 6.0 and 8.0, the conductance attributable to the cotransporter showed a maximum between pH 7.25 and pH 7.50. A similar profile was observed in the presence of a pH gradient when the pH of the apical solutions was varied between 7.0 and 8.0 (basal pH lower by 1), but not when the pH of the basal solution was varied between 7.0 and 8.0 (apical pH lower by 1 unit). To delineate the kinetic basis for these observations, DeltaI-voltage curves were obtained as a function of Na+ and HCO3- concentrations and analyzed on the basis of a kinetic cotransporter model. Increases in pH from 7.0 to 8.0 decreased the binding constants for the intracellular and extracellular substrates by a factor of 2. Furthermore, the electrical parameters that describe the interaction strength between the electric field and substrate binding or charge on the unloaded transporter increased by four- to fivefold. These data can be explained by a channel-like structure of the cotransporter, whose configuration is modified by intracellular pH such that, with increasing pH, binding of substrate to the carrier is sterically hindered but electrically facilitated.     

ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na⁺-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [¹⁴C]--methyl-D-glucopyranoside (-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10^–6 M) manner. ATP stimulated -MG uptake by increasing in V_max without affecting K_m. ATP-induced increase of -MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of -MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y₁ receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of -MG uptake was blocked by pertussis toxin (PTX, a G_i protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of -MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases -MG uptake via cAMP and p38 MAPK in renal PTCs. adenosine 5'-triphosphate; mitogen-activated protein kinase     

Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells.

Effect of epinephrine on alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells. Epinephrine has known to be a very important factor in the regulation of renal sodium excretion. However, the effect of epinephrine on Na+/glucose cotransporter was not fully elucidated. Thus, we examined effect of epinephrine on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signal pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). Epinephrine inhibited alpha-MG uptake in a time- and dose-dependent manner and also decreased SGLT1 and SGLT2 protein level. Both phentolamine and propranolol completely prevented epinephrine-induced inhibition of alpha-MG uptake. The epinephrine-induced inhibition of alpha-MG uptake was blocked by SQ-22536 or myristoylated PKA inhibitor amide 14-22 and epinephrine increased the intracellular cAMP content. In western blotting analysis, epinephrine increases phosphorylation of p44/42 and p38 MAPKs and PD 98059 or SB 203580 blocked the effect of epinephrine. In addition, epinephrine increased AA release and PGE2 production and effects of epinephrine on alpha-MG uptake and AA release were blocked by staurosporine and bisindolylmaleimide I or mepacrine and AACOCF3. Indeed, epinephrine translocated PKC or cPLA2 from cytosol to membrane fraction. In conclusion, epinephrine partially inhibits the alpha-MG uptake through PKA, PKC, p44/42, p38 MAPK, and cPLA2 pathways in the PTCs.     

Active ion transport in the renal proximal tubule. III. The ATP dependence of the Na pump

The dependence of the Na pump activity of intact renal tubules on the ATP concentration was investigated using a suspension of rabbit cortical tubules. Rotenone (an inhibitor of mitochondrial oxidative phosphorylation) was used in graded fashion to alter the cellular ATP, and the Na pump activity was measured when the pump was stimulated by adding KCl to tubules suspended in a K+-free medium. The K+ uptake into the tubule was measured using an extracellular K+ electrode, and the oxygen consumption (QO2) was measured using a Clark-type oxygen electrode. The Na pump activity was found to have a linear, nonsaturating dependence on the ATP concentration. However, the Na,K- ATPase hydrolytic activity (assayed biochemically) of lysed proximal tubule membranes demonstrated saturation and had a K0.5 value of 0.4 mM ATP. Presumably, unknown cytosolic factors present in the intact renal cell but not normally present in the biochemical assay accounted for the differences between the two measurements. The data suggest that an alteration in the intracellular ATP will result in a proportional change in active ion transport activity. Moreover, additional findings also suggest that the basal (non-transport-related) QO2 may be redirected to support the proximal Na pump activity when transport activity is stressed. Thus, basal respiration is not invariant under all conditions, and ion transport activity appears to be maintained foremost among cellular ATP-dependent processes.     

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.     

Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

Heterogeneity of pH in the aqueous cytoplasm of renal proximal tubule cells

Heterogeneity of cytosolic pH was studied with compounds that distribute between the cytosol and mitochondrial matrix in fundamentally different ways, i.e., according to the extent of ionization or according to the function of H+-coupled transport systems. Results show that the average cytosolic pH is considerably more alkaline than the region to which mitochondria are exposed. Because mitochondria are localized predominantly in the basal region, the results are consistent with a transcellular pH gradient within the cytosol of proximal tubule cells. Experiments analyzing the effects of inhibiting efflux of HCO3- at the basal surface and Na+-H+ exchange at the apical surface support the interpretation that the function of these systems contributes to the transcellular pH gradient. The existence of a heterogeneity in pH within the cytosol has important implications concerning the function and regulation of numerous cell processes.     

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.     

Synthesis of superoxide dismutase derivative that specifically accumulates in renal proximal tubule cells.

Protection of tissues from oxygen toxicity is one of the major prerequisites to aerobic life. Since a wide variety of xenobiotics with prooxidant activity is excreted by the kidney, renal tubule cells should be protected from hazardous oxygen species. Because intravenously injected Cu/Zn-type superoxide dismutase (SOD) is rapidly excreted in the urine in its intact form, effective dismutation of superoxide radicals cannot be achieved in vivo by intravenously administered SOD. To scavenge superoxide radicals and inhibit their toxic effects in and around renal tubule cells, a hexamethylene-diamine (AH)-conjugated SOD (AH-SOD) was synthesized. When injected intravenously into the rat, (125)I-labeled AH-SOD disappeared from the circulation with a half-life of 3 min and accumulated in the kidney. After 30 min of administration, more than 80% of the radioactivity derived from AH-SOD was found to localize in the kidney without being excreted in the urine. Immunohistochemical examination revealed that, 60 min after administration, the major part of AH-SOD localized in renal proximal tubule cells. Kinetic analysis using right-side-out-oriented renal brush border vesicles revealed that AH-SOD bound to their membrane surface by some mechanism which was inhibited by AH but not by heparin and albumin. These results indicated that AH-SOD rapidly underwent renal glomerular filtration, bound to apical plasma membranes of proximal tubule cells, and localized in these cells for a fairly long time without being excreted in the urine. Thus, AH-SOD might permit studies on the role of superoxide radicals in and around renal proximal tubule cells.     

Sulphate and phosphate transport in the renal proximal tubule

Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane.     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.     

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP_i cotransporter (NaP_i-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP_i-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaP_i-IIa from rat renal cortex tissue and HA-NaP_i-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP_i-IIa and truncation of the COOH-terminal TRL motif from NaP_i-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP_i-IIa interaction occurred directly between the two proteins. NaP_i-IIa activity is regulated by moderating its abundance in the apical membrane. High-P_i conditions induce NaP_i-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P_i conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP_i-IIa via PDZ interactions, and undergoes P_i-induced internalization. PDZ domains; endocytosis; degradation; epithelia     

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics

Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. This paper was resented 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 grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid of Research Award.