Lack of a direct role for cyclic AMP in parathyrin action on phosphate reabsorption by the kidney

Isolated chick kidney proximal tubule cells have been used in a study of the mechanism by which PTH inhibits Na+-dependent Pi transport in the kidney. Treatment with PTH inhibits Pi uptake by the cells by 13% and stimulates cyclic AMP production by 77%. Forskolin, a potent activator of adenyl cyclase, brought about an 11-fold stimulation of cyclic AMP production by the cells, but in contrast to PTH, the drug had no effect on Na+-dependent Pi uptake. These results provide evidence that PTH action on phosphate transport is not mediated by cyclic AMP.     

The Renal Type II Na+/Phosphate Cotransporter

A sodium-dependent phosphate transporter (type II Na/Pi-cotransporter) was isolated which is expressed in apical membranes of proximal tubules and exhibits transport characteristics similar as described for renal reabsorption of phosphate. Type II associated Na/Pi-cotransport is electrogenic and results obtained by electrophysiological measurements support a transport model having a stoichiometry of 3 Na⁺/HPO₄ ⁼. Changes of transport such as by parathyroid hormone and altered dietary intake of phosphate correlate with changes of the number of type II cotransporters in the apical membrane. These data suggest that the type II Na/Pi-cotransporter represents the main target for physiological and pathophysiological regulation.     

Peculiarities of the Na+/d-glucose cotransport system in necturus renal tubules

The effects of d-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d-glucose produces $\text{10.5}\text{mV}\text{± 1.1}$ (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to $\text{2.1 ± 0.1}\text{mV}$. (3) The glucose-evoked depolarization, $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$, does not alter the intracellular K⁺ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na⁺ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li⁺ substituting for Na⁺) does not change the magnitude of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$; complete removal of sodium from the lumen lowers the value of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$ ($\text{3.2 ± 0.2}\text{mV}$) but the response is not abolished. This observation suggests that the d-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.     

Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport

Summary Activation of protein kinase C has been shown to cause both stimulation and inhibition of transport processes in the brush-border membrane and renal tubule. This study was designed to examine the dose-response nature and time-dependent effect of 4 -phorbol-12-myristate-13-acetate (PMA) on the rates of bicarbonate absorption (J _HCO3) and fluid absorption (J _v) in the proximal convoluted tubule (PCT) of rat kidney. Bicarbonate flux was determined by total CO₂ changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of PMA (10^–10 10^–5 M) within 10 min caused a significant increase ofJ _HCO3 andJ _v. A peaked curve of the dose response was observed with maximal effect at 10^–8 M PMA on both bicarbonate and fluid reabsorption, which could be blocked completely by amiloride (10^–3 m) and EIPA (10^–5 M). On the other hand, with an increase of perfusion time beyond 15 min, PMA (10^–8 and 10^–6 M) could inhibitJ _HCO3 andJ _v. Amiloride (10^–3 M) or EIPA (10^–5 M) significantly inhibitsJ _HCO3 andJ _v, while there is no additive effect of PMA and amiloride or EIPA on PCT transport. An inactive phorbol-ester, 4-phorbol, that does not activate protein kinase C, had no effects onJ _HCO3 andJ _v. Capillary perfusion of PMA (10^–8 M) significantly stimulate bothJ _HCO3 andJ _v; however, PMA did not affect glucose transport from either the luminal side or basolateral side of the PCT. These results indicate that activation of endogenous protein kinase C by PMA could either stimulate or inhibit both bicarbonate and fluid reabsorption in the PCT dependent on time and dose, and these effects are through the modulation of Na⁺/H^– exchange mechanism.     

Regulation of gluconeogenesis in swine kidney proximal tubule cells

Summary Proximal tubule cells were isolated from swine kidney and cultured for periods of more than 30 days. The cells formed confluent monolayers after plating on a collagen surface and they were passaged more than 5 times on this matrix. The cells maintain several metabolic functions of proximal tubule cells, including gluconeogenesis and the ability to respond to epinephrine and parathyroid hormone. Gluconeogenesis, a principal metabolic pathway in proximal tubule cells, was examined as a function of days in culture. The isolated cells showed a nearly constant rate of gluconeogenesis from ¹⁴C-lactate, ¹⁴C-alkaine and 14C-glycerol with no significant loss of activity for at least 30 days in culture. Likewise, the activities of several cytosolic and membrane associated enzymes including, alkaline phosphatase, -glutamyltransferase, fructose-1,6-bisphosphatase and phosphofructokinase were nearly constant over the same time period.The cells responded to treatment with epinephrine and parathyroid hormone, and the rate of gluconeogenesis from ¹⁴C-lactate doubled in the presence of these hormones. The morphological and biochemical evidence obtained in these studies show that the proximal tubule cells isolated from swine kidney provide an excellent well defined system for studying the hormonal regulation of carbohydrate metabolism in this tissue.Abbreviations PTH Parathyroid Hormone - cAMP cyclic 3,5-adenosine Monophosphate     

Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.     

Bicarbonate permeability of the outwardly rectifying anion channel

Summary Single anion-selective channels have been studied in cultured human epithelial cells using the patch-clamp technique. Three cell types were used as models for different anion transport systems: (i) PANC-1, a cell line derived from the pancreatic duct, (ii) T₈₄, a Cl-secreting colonic cell line, and (iii) primary cultures of sweat duct epithelium. Outwardly rectifying anion-selective channels were observed in all three preparations and were indistinguishable with respect to conductance, selectivity and gating. Striking similarities between HCO₃- and Cl-secreting epithelia, and the high density of outward rectifiers in pancreatic cells prompted us to study HCO₃ permeation through this channels. HCO₃ permeability was significant when channels were bathed in symmetrical 150mm HCO₃ solutions, Cl–HCO₃ mixtures, and under bi-ionic conditions with outwardly and inwardly directed HCO₃ gradients. Permeability ratios (P _HCO3/P _Cl) estimated from bi-ionic reversal potentials ranged from 0.50 to 0.64, although conductance ratios greater than 1.2 were observed with high extracellular pH. Chloride did not inhibit HCO₃ permeation noticeably but rather had a small stimulatory effect when present on the opposite side of the membrane. The prevalence of outward rectifiers in PANC-1 and their permeability to bicarbonate suggests the channel may have a dual role in HCO₃ secretion; to allow Cl recycling at the apical membrane and to mediate some of the HCO₃ flux. Defective modulation of this channel in cystic fibrosis might provide a common basis for dysfunction in epithelia having very different anion transport properties (e.g., HCO₃ secretion, Cl secretion and Cl absorption).     

Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene

Summary The fluorescence enhancement of 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K _d =3.8 m) and rat (3.2 m); 100 m dihydro-4,4-diisothiocyano-2,2-disulfonic stilbene (H₂DIDS) blocked >95% of binding. H₂DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK _d doubled with 10mm SO₄, 50mm HCO₃ and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO₄, HCO₃ and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used³⁵SO₄ uptake to characterize several modes of rabbit BLM anion transport: H/SO₄ and Na/SO₄ cotransport, and Cl/SO₄ countertransport. Each transport process was electroneutral and was inhibited by H₂DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK _t 's for DBDS (3–20 m) were similar toK _d for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene.     

Transimmortalized proximal tubule and collecting duct cell lines derived from the kidneys of transgenic mice

This review summarizes the strategy of cellular immortalization based on the principle of targeted oncogenesis in transgenic mice, used to establish models of transimmortalized renal proximal tubule cells, referred to as PKSV-PCT and PKSV-PR-cells, and collecting duct principal cells, referred to as mpkCCD_cl4 cells. These cell lines have maintained for long-term passages the main biochemical and functional properties of the parental cells from which they were derived. Proximal tubule PKSV-PCT and PKSV-PR cells have been proved to be suitable cell systems for toxicological and pharmacological studies. They also permitted the establishment of a model of multidrug-resistant (MDR) renal epithelial tubule cells, PKSV-PR_col50, which have served for the study of both MDR-dependent extrusion of chemotherapeutic drugs and inappropriate accumulation of weak base anthracyclines in intracellular acidic organelles. The novel collecting duct cell line mpkCCD_cl4, which has maintained the characteristics of tight epithelial cells, in particular Na⁺ absorption stimulated by aldosterone, has been extensively used for pharmacological studies related to the regulation of ion transport. These cells have permitted the identification of several aldosterone-induced proteins playing a key role in the regulation of Na⁺ absorption mediated by the epithelial Na⁺ channel ENaC. Recent studies have also provided evidence that these cell lines represent valuable cell systems for the study of host–pathogen interactions and the analysis of the role of renal tubule epithelial cells in the induction of inflammatory response caused by uropathogens that may lead to severe renal damage.     

The effects of acute phosphate depletion on isolated chick kidney tubule cells

Isolated tubule cells from chick kidney respond to a short period of phosphate deprivation with increased phosphate uptake and a resistance to parathyroid hormone. During phosphate depletion a considerable amount of phosphate may be released from the cells, but intracellular inorganic phosphate levels are maintained by the hydrolysis of organic phosphate esters. It is suggested that the concomitant changes in metabolism might act as the signal causing the onset of the changes in phosphate handling associated with phosphate deprivation.     

Molecular Mechanisms of Electrogenic Sodium Bicarbonate Cotransport: Structural and Equilibrium Thermodynamic Considerations

The electrogenic Na⁺-HCO₃^– cotransporters play an essential role in regulating intracellular pH and extracellular acid-base homeostasis. Of the known members of the bicarbonate transporter superfamily (BTS), NBC1 and NBC4 proteins have been shown to be electrogenic. The electrogenic nature of these transporters results from the unequal coupling of anionic and cationic fluxes during each transport cycle. This unique property distinguishes NBC1 and NBC4 proteins from other sodium bicarbonate cotransporters and members of the bicarbonate transporter superfamily that are known to be electroneutral. Structure-function studies have played an essential role in revealing the basis for the modulation of the coupling ratio of NBC1 proteins. In addition, the recent transmembrane topographic analysis of pNBC1 has shed light on the potential structural determinants that are responsible for ion permeation through the cotransporter. The experimentally difficult problem of determining the nature of anionic species being transported by these proteins (HCO₃^– versus CO₃^2–) is analyzed using a theoretical equilibrium thermodynamics approach. Finally, our current understanding of the molecular mechanisms responsible for the regulation of ion coupling and flux through electrogenic sodium bicarbonate cotransporters is reviewed in detail.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Cystine-glutamate transport interactions in rat renal cortical tubules,brushborder vesicles,and cultured renal tubule cells

Glutamate had no significant effect on the uptake of 0.025 mM cystine by isolated rat renal cortical tubules and brushborder membrane vesicles in contrast to lysine which significantly inhibits cystine transport. Glutamate, however, markedly inhibited cystine uptake by rat renal tubule cells grown in a serum-free, hormonally defined media for 5 days. Lysine also inhibited cystine transport in these cultured renal tubule cells.     

Cell pH and luminal acidification inNecturus proximal tubule

Summary Cellular potential and pH measurements (pH _i ) were carried out in the perfused kidney ofNecturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50mm, pH _i rose to 7.44 and 7.56, respectively. These changes in pH _i were fully reversible. Under all conditions intracellular H⁺ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires active H⁺ extrusion across one or both of the cell membranes. The observed rise in pH _i and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.     

Sequence- or Position-Specific Mutations in the Carboxyl-Terminal FL Motif of the Kidney Sodium Bicarbonate Cotransporter (NBC1) Disrupt Its Basolateral Targeting and α-Helical Structure

The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013–1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant α-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the α-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.