Rat Renal Brush-border Membrane Research Articles

Absence of vacuolar H+-ATPases from rat small intestine brush-border membranes.

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.

Radiation-inactivation analysis of the oligomeric structure of the renal sodium/ d-glucose symporter

The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/ d-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294±40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84±25 kDa in the presence of a glucose gradient, and 92±20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225±49 kDa. The 294 kDa result supports earlier suggestions that the Na + gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.

Indomethacin and piroxicam inhibit Na+-adenosine transport in rat renal brush-border membranes

The effects of the cyclooxygenase inhibitors, indomethacin and piroxicam, were evaluated on Na + -dependent [ 3 H]adenosine transport in rat renal brush-border membranes of the outer renal cortex of the rat. Adenosine co-transport (1–10 μM) was estimated in the presence of 0.001–10 μM indomethacin and piroxicam. Both drugs inhibited the Na + -dependent transport in a dose-dependent manner with IC 50 of 3.5 μM and 0.1 μM, respectively. The Na + -independent transport was not modified. Preincubations carried out on the vesicles with 10–50 μM arachidonic acid increased transport in a dose-dependent manner up to 1.7 times. Whereas 50 pM to 5 μM prostaglandin E 2 in the presence of indomethacin did not change carrier activity, 5 μM prostaglandin E 2 increased the Na + -dependent transport 1.5 times. Other prostanoid synthesis pathways were investigated with 10 μM nordihydroguaiaretic acid (lipoxygenase inhibitor), and 17-octadecynoic acid and clotrimazole (leukotriene and cytochrome P 450 inhibitors). Our results demonstrated that the Na + -dependent adenosine transport in brush-border membranes was inhibited by indomethacin and piroxicam, suggesting that cyclooxygenase activity might modulate this co-transport.

Indomethacin and piroxicam inhibit Na +-adenosine transport in rat renal brush-border membranes

The effects of the cyclooxygenase inhibitors, indomethacin and piroxicam, were evaluated on Na +-dependent [ 3H]adenosine transport in rat renal brush-border membranes of the outer renal cortex of the rat. Adenosine co-transport (1–10 μM) was estimated in the presence of 0.001–10 μM indomethacin and piroxicam. Both drugs inhibited the Na +-dependent transport in a dose-dependent manner with IC 50 of 3.5 μM and 0.1 μM, respectively. The Na +-independent transport was not modified. Preincubations carried out on the vesicles with 10–50 μM arachidonic acid increased transport in a dose-dependent manner up to 1.7 times. Whereas 50 pM to 5 μM prostaglandin E 2 in the presence of indomethacin did not change carrier activity, 5 μM prostaglandin E 2 increased the Na +-dependent transport 1.5 times. Other prostanoid synthesis pathways were investigated with 10 μM nordihydroguaiaretic acid (lipoxygenase inhibitor), and 17-octadecynoic acid and clotrimazole (leukotriene and cytochrome P 450 inhibitors). Our results demonstrated that the Na +-dependent adenosine transport in brush-border membranes was inhibited by indomethacin and piroxicam, suggesting that cyclooxygenase activity might modulate this co-transport.

Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

Direct effect of inorganic mercury on citrate uptake by isolated rat renal brush border membrane vesicles.

Occupational exposure to mercury has long been associated with renal proximal injury and an increased incidence of proteinuria, as has such exposure to cadmium. Renal citrate excretion is very important with respect to acid-base balance since the metabolism of citrate generates three bicarbonate ions. In this study, we exposed isolated rat renal brush border membrane vesicles (BBMV) to mercury (Hg2+) and examined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration method. The preincubation of BBMV with 0.5 and 2 mM HgCl2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Hg preincubation. The analysis of the time course of citrate uptake during a 30-min preincubation of BBMV with 0.1 mM Hg2+ also revealed a significant reduction in the uptake compared with that of the control BBMV without preincubation. These findings indicate that the preincubation of BBMV with mercury results in a time- and concentration-dependent inhibition of citrate uptake.

Citrate uptake by isolated rat renal brush border membrane vesicles in cadmium-intoxicated rats.

Epidemiological Studies have reported that the formation of renal stones is often observed in workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. We previously reported that direct exposure to high-level cadmium impaired the citrate uptake by the brush border membrane vesicles (BBMV), which may mean that the formation of renal stones from exposure to Cd could be prevented. In the present study, to clarify the mechanism of Cd-induced renal stone formation, the characteristics of BBMV-induced citrate uptake were determined in Cd-intoxicated rats. Cd intoxication was induced by daily subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg body wt per day for 17 to 20 days. The BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. The citrate uptake per mg protein was not significantly different between the two groups. The volume of BBMV was significantly reduced (50%) in Cd-intoxicated rats compared with that of control rats. Percentages of the equilibrium value were also significantly inhibited in Cd-intoxicated rats.

Acute effects of two natural corticosteroids on the kinetics of the Na<SUP>+</SUP>/H<SUP>+</SUP> exchanger in rat renal brush-border membranes

We studied the effect of adrenalectomy and acute hormone replacement, using physiological doses of natural corticosteroids, on the kinetics of the Na+/H+ exchanger in brush border membrane vesicles. We collected the data using the acridine orange uptake technique. Adrenalectomized (ADX) rats presented a decreased maximal rate (Vmax) when compared with sham-operated animals (30,000 versus 41,000 fluorescent units/min, respectively). Administration of corticosterone (B) to ADX rats restored Vmax to values above control (up to 66,000 fluorescent units/min). Smaller doses of 18-OH-B led to similar results. K(m) (16 mM) remained the same for all the groups. Amiloride behaved as a pure competitive inhibitor, with a Ki = 0.02 mM and an I50 = 98 microM (in the presence of 50 mM sodium gluconate). The presence of sodium in the external buffer, before adding the vesicles, inhibited the exchange, with an I50 = 2 mM. We observed, a significant decrease in the Na+/H+ exchanger under non-acidotic conditions in response to adrenalectomy. Acute administration of physiological doses of natural occurring corticosteroids reversed the effect.

Cholesterol modulates organic cation transport activity and lipid fluidity in rat renal brush-border membranes

The role of cholesterol in organic cation transport was studied in rat renal brush-border membranes. H + gradient-dependent uptake of the organic cation tetraethylammonium in brush-border membrane vesicles was stimulated by cholesterol enrichment in a dose-dependent manner. The dissipation rate of the H + gradient, a driving force for organic cation transport in brush-border membranes, was reduced by cholesterol enrichment. Tetraethylammonium uptake in the absence of H + gradient was also stimulated by cholesterol enrichment. These findings indicate that cholesterol modulates tetraethylammonium uptake by affecting the intrinsic activity of the organic cation transporter and the H + gradient dissipation rate. Therefore, cholesterol content should be an important determinant for organic cation transport in renal brush-border membranes.

Effect of ethanol on folate binding by isolated rat renal brush border membranes

Deficiency of folic acid, an essential vitamin involved in critical metabolic pathways, occurs in several conditions, including alcoholism. In humans and animal models, chronic ethanol consumption leads to decreased plasma levels and increased urinary levels of folate. An isolated perfused rat kidney model has shown that ethanol produces increased urinary excretion of folate, suggesting a direct effect of ethanol on the kidney. Because the folate binding protein, located in the brush border membrane (BBM) of proximal tubule cells, is thought to be involved in renal folate reabsorption, the effects of ethanol on BBM binding of folate were assessed. Binding studies were conducted using isolated rat kidney cortex BBM preparations, incubated with 3H-labeled 5-methyltetrahydrofolate (5-CH 3H 4PteGlu) at varying concentrations (0.1–100 nM). Ethanol at 500 mg/dl did not significantly affect [ 3H]5-CH 3H 4PteGlu binding in BBM. The structural analogue, folic acid, decreased [ 3H]5-CH 3H 4PteGlu binding under similar conditions. Because of the lack of effect of ethanol on binding to isolated BBM, the effects of ethanol probably occur at other steps in the renal uptake and metabolism of folate.

Solubilization and reconstitution characteristics of the carrier protein(s) responsible for the transport of ceftibuten, a substrate for the oligopeptide transporters, in rat renal brush-border membrane

Optimal procedures for the reconstitution of the transport activity of ceftibuten, a dianionic ß-lactam antibiotic, from rat kidney brush-border membrane were developed. The uptake activity into reconstituted proteoliposomes appeared to be particularly sensitive to the extraction conditions, and to the lipid composition used for reconstitution. Changes in the concentration of octyl glucoside significantly affected the extraction of ceftibuten transport activity, and optimal extraction was achieved at a concentration of 60 mM. Optimal reconstitution was achieved using a lipid composition of asolectin, cholesterol and phosphatidylserine in a w/w percent ratio of 60:30:10, respectively, and with a lipid-to-protein ratio of 10. The uptake of ceftibuten into the resulting proteoliposomes showed temperature and pH dependency, was inhibitable by a range of cephem antibiotics, oligopeptides and the organic anion PAH, and was trans-stimulated by cephalexin and dipeptides. This reconstitution system will likely prove useful in future studies on the functional analysis of the peptide transport system in a purified form.

Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes

The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na +-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na + gradient-driven transport process. The absolute requirement for Na + was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na +-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 ± 0.03 μM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na + increased the affinity of the binding process for carnitine and the Na +/carnitine coupling ration for the binding process was 1. The dissociation constant for the interaction of Na + with the binding of carnitine was 24 ± 4 mM. This constitutes the first report on the identification of Na +-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na + gradient-driven carnitine transport but no Na +-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles possess neither Na + gradient-driven carnitine transport nor Na +-dependent carnitine binding.

Phosphate deprivation induces overexpression of two proteins related to the rat renal phosphate cotransporter NaPi-2

Polyclonal antibodies were raised in rabbits against the C-terminal portion of the rat renal brush-border membrane sodium/phosphate cotransporter NaPi-2. Antibody specificity and molecular sizes of proteins related to NaPi-2 were assayed by Western blot analysis. Proteins of 40 and 70–75 kDa (p40 and p70) were immunodetected in rat and mouse brush-border membranes and proteins of 72 and 82 kDa were detected in rabbit. The absence or presence of β-EtSH in the samples before electrophoresis greatly influenced the immunodetection profile of the rat proteins. Since the 40 kDa protein (p40) can only be detected under reducing conditions, it probably originates from reduction of disulfide bonds in p70. Tryptic cleavage of p40 and p70 revealed identical protein fragments showing the close structural identity of those proteins. Both proteins were more abundant in the outer cortex portion of the rat kidney than in the juxtamedullary portion. Furthermore, rats fed a low-phosphate diet for 24 h showed a 20- and 14-fold increase in the amount of p40 and p70, respectively, compared to control rats, showing that the adaptation to P i deprivation by increasing renal phosphate reabsorption is not only the result of overproduction of p70, as previously shown, but is also due to the novel p40 which most probably derives from p70.

Regulation of expression of taurine transport in two continuous renal epithelial cell lines and inhibition of taurine transporter by a site-directed antibody.

The renal tubular epithelium adapts to changes in the sulfur amino acid composition of the diet, particularly in terms of reabsorption of taurine. The adaptive response is expressed by enhanced or decreased NaCl-dependent taurine transport by rat renal brush border membrane vesicles (BBMV). Taurine transport activity in two cultured renal epithelial cell lines (MDCK and LLC-PK1) is up- or down-regulated by extracellular taurine concentration as the result of reciprocal changes in the Vmax of the transporter. In MDCK cells, abundance of taurine transporter mRNA (pNCT mRNA) was up- or down-regulated after incubation in media containing 0, 50, or 500 microM taurine. Decreased mRNA was observed in both cell lines after 12 h, and it was appreciably reduced after 72 h exposure to 500 microM taurine. Northern blot analysis of mRNA from LLC-PK1 cells using pNCT cDNA as a riboprobe showed that two transcripts, 9.6 kb and 7.2 kb, were expressed; the abundance of mRNA was increased or decreased after incubation in taurine-free or high taurine medium, respectively. Down-regulation was observed primarily in the 7.2 kb transcript after 24 h incubation. Rapid up-regulation occurred in the 9.6 kb transcript within 12 h of transfer from high to low taurine. Nuclear run-off assays showed that the gene for pNCT is induced at the transcriptional level by taurine. Regulation of expression of the taurine transporter was also studied by injection of pNCT cRNA into Xenopus laevis oocytes. Expression of transport activity was significantly reduced (64%) when oocytes were incubated in 50 microM taurine as compared to 0 microM taurine. Transport activity was totally blocked when pNCT cRNA-injected oocytes were exposed to an active phorbol ester, PMA (10(-6) M). Inhibition of uptake was reversed by staurosporine, an inhibitor of protein kinase C activity. An inactive phorbol ester, 4 alpha-phorbol, had no effect on taurine transport. A polyclonal antibody directed a highly conserved intracellular segment between homologous transmembrane domains VI and VII inhibited taurine transport activity in both pNCT cRNA-injected oocytes and BBMV. Incubation of oocytes with 10 micrograms/ml antibody (Ab) reduced taurine uptake to 46% of control, and 20-80 micrograms/ml Ab reduced uptake to 20% of control. In BBMV, active taurine uptake (10 microM) was inhibited approximately 30% by 10 pg Ab/mg protein, whereas none specific IgG had no significant effect. Proline uptake (20 microM) by BBMV was not inhibited by the Ab, nor was GABA uptake (50 microM). Two pNCT proteins, approximately 70 kD and approximately 30 kD, were detected by Western blot, and the abundance of both was regulated by medium taurine. (i) regulation of taurine transport activity in LLC-PK1 cells by medium taurine occurs at a level of mRNA transcription; (ii) regulation of pNCT occurs at both transcriptional and translational levels; (iii) pNCT expression is regulated by protein kinase C-dependent phosphorylation; and (iv) the intracellular segment between domains VI and VII may be required for activation of the taurine transporter; this segment may function as a gate in taurine transport.

Modulation of organic cation transport and lipid fluidity by benzyl alcohol in rat renal brush-border membranes.

Organic cations are actively transported in renal brush-border membranes (BBM) by the H+/organic cation antiport system. In the present study, we investigated the relationship between membrane fluidity and organic cation transport in the BBM. The effects of benzyl alcohol, a membrane fluidizing agent, on the organic cation tetraethylammonium (TEA) uptake were studied using renal BBM vesicles isolated from rat kidney. BBM fluidity was assessed by fluorescence polarization technique. H+ gradient-dependent uptake of TEA in BBM vesicles was inhibited by benzyl alcohol in a dose-dependent manner, with an apparent half inhibitory concentration of 18mM. The decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in BBM, which represents the increase in membrane fluidity, was correlated with the decrease in TEA transport activity. The dissipation rate of H+ gradient, a driving force for organic cation transport in BBM, was increased by benzyl alcohol. In addition, H+ gradient-independent TEA-TEA exchange was also inhibited by benzyl alcohol. These findings indicate that benzyl alcohol inhibits the uptake of TEA by affecting the intrinsic activity of the organic cation transporter and the H+ gradient dissipation rate. The membrane fluidity should be an important determinant for organic cation transport in renal BBM.

Micromolar Concentrations of Bromosulfophthalein Affect Transport of <i>D</i>-Glucose, <i>L</i>-Alanine and <i>p</i>-Aminohippurate in Rat Renal Brush-Border Membranes

The effect of bromosulfophthalein (BSP) on potential-stimulated p-aminohippurate (PAH) uptake as well as on Na+-D-glucose and Na+-L-alanine cotransport was investigated in rat renal brush-border membrane vesicles. All three transport mechanisms were inhibited by BSP in the following order: PAH > D-glucose > L-alanine. Half-maximal inhibition of initial uptakes was observed at approximately 10,75 and 250 µM BSP, respectively. The inhibition of initial Na+-D-glucose uptake could not be attributed to increased cation permeability, since equilibrium exchange was equally affected by BSP. The nonspecific short-term inhibition of unrelated transport systems by BSP suggests a more general effect, such as a change in membrane fluidity. In addition, long-term incubation with 0.5 mM BSP resulted in a loss of vesicle integrity.

Direct effect of cadmium on citrate uptake by isolated rat renal brush border membrane vesicles

High incidence of multiple kidney stone formation has been observed among workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. To study the direct effect of cadmium on citrate uptake by the renal brush border membrane, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cadmium and determined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. Preincubation of BBMV with 2 and 10 mM CdCl 2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Cd. Analysis of the time course of citrate uptake during 30-min preincubation of BBMV with 0.5 mM Cd also revealed significant reduction of the uptake compared with that of the control BBMV without preincubation. These findings indicate that preincubation of BBMV with cadmium results in time-dependent and concentration-dependent inhibition of citrate uptake.

Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity.

Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes

To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.

Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate

Previously, we isolated a cDNA (NaPi-1) related to a rabbit renal proximal tubular Na-Pi cotransporter (A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, and H. Murer. Proc. Natl. Acad. Sci. USA 88:9608-9612, 1991.). In this study, we isolated an additional (rabbit renal) cDNA (NaPi-6), which induces Na-dependent Pi uptake in Xenopus laevis oocytes. Substrate specificity and kinetic properties corresponded to those known for rabbit renal brush-border membrane (BBM) Na-Pi cotransport. NaPi-6 was cloned by homology using NaPi-2 cDNA, a rat renal BBM Na-Pi cotransporter (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979-5983, 1993). NaPi-6 encodes a protein of 642 amino acids, exhibiting at least eight transmembrane domains. NaPi-6 mRNA and protein in kidneys of rabbits fed a low-Pi diet (LPD; 0.11% Pi) for 1 wk were increased by 1.5- and 4-fold, respectively, compared with those of rabbits fed a high-Pi diet (HPD; 1.20% Pi). This effect was correlated with an increase in Na-Pi cotransport of BBM vesicles isolated from animals adapted to LPD (2.5-fold with respect to HPD). In contrast, NaPi-1 mRNA and protein were not altered in response to LPD. Thus rabbit proximal tubular BBMs contain two different Na-Pi cotransport systems: NaPi-1 (type I) and NaPi-6 (type II). Only the type II transport system seems to be under regulatory control in response to low-Pi dietary intake.