Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641
Submitted 15 July 2003 ; accepted in final form 26 December 2003
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ABSTRACT |
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intestinal brush-border membrane vesicles; renal brush-border membrane vesicles; sodium-phosphate cotransport; phosphate reabsorption; 2'-phosphophloretin; 2'-phospho-4',4,6'-trimethoxy phloretin
Na+-phosphate cotransporter proteins are a diverse group of proteins, which appear to share few functional or structural characteristics other than coupling Na+ uptake and phosphate uptake into a variety of cell types. NaPi-IIa and NaPi-IIb differ in structure and transport properties. Structurally, the two proteins differ in molecular mass, I° structure (NaPi-IIa and NaPi-IIb share 55% homology), and the presence of a structurally and functionally essential disulfide bridge (21). Functionally, the substrate sites of the two proteins appear to differ. NaPi-IIa is thought to transport either H2PO4 or HPO4, with a preference for HPO4, has a substrate stoichiometry of 2 or 3, and increases its Na+ affinity with increasing pH (3). NaPi-IIb is thought to transport only H2PO4, may be inhibited by HPO4 at an internal regulatory site (10, 32), has a substrate stoichiometry of 2, and increases its Na+ affinity with decreasing pH (10, 32, 33, 41). NaPi-IIa is more sensitive to phosphites (foscarnet) and less sensitive to arsenate (43) than NaPi-IIb (3, 24).
NaPi-III (PiT-1 and PiT-2) subfamily members share little homology with either the NaPi-II or NaPi-I proteins (28). Although NaPi-III family members appear to resemble NaPi-IIb and NaPi-Ia in transport characteristics (substrate affinity, substrate selectivity, and pH dependence of transport), there is little common inhibitor sensitivity or structural similarity (28).
Based on functional characteristics, NaPi-IIb appears to have more in common with NaPi-Ia than NaPi-IIa. NaPi-Ia and NaPi-IIb have increased Na+ affinity with decreasing pH and appear to preferentially or exclusively transport H2PO4 (10, 25, 28, 32, 41, 44). Comparisons of the structures of the two proteins have not been reported.
Differential inhibitor sensitivity is one method commonly used to distinguish between members of a transport protein family. Inhibitor sensitivity differences have been used to distinguish between members of the Na+/H+ exchanger (NHE) family (8, 39), Cl- channel family (18), and anion exchange protein family (23). Previous studies suggested that the members of the NaPi family differ in their inhibitor sensitivity to phosphonoformic acid (PFA) and vanadate (24, 49). These inhibitors are not specific for the Na+-phosphate cotransporters.
The plant chalcone phloretin is an inhibitor of a variety of transport proteins. The aglucone phloretin is an inhibitor of AE-1 (Band 3, 12, 14), urea transport in the kidney (6) and erythrocytes (46), lactate and pyruvate transport in cardiomyocytes (48), and GLUT-4 (2, 12, 14). Phloretin has also been reported to be an inhibitor of NaPi-IIa and a weak inhibitor of PiT-1, NaPi-Ib (45), and NaPi-IIb (34). The 2'-glycosylated derivative phloridzin inhibits Na+ and glucose cotransport in renal and intestinal brush-border membranes (BBM) (12).
In vitro a phosphorylated chalcone 2'-phosphophloretin (2'-PP) has been reported to specifically inhibit NaPi-IIb in human (35), rat (34), and rabbit (34) intestinal BBM vesicles (BBMV). In vivo 2'-PP decreased serum phosphate in aged adult rats (34).
Phosphophloretins are of potential interest in the treatment of renal failure and as a tool for studying the NaPi family of proteins. Differential phosphophloretin sensitivity would provide a biochemical assay for the determination of NaPi family distribution in tissues. Phosphophloretins are also of potential use for the study of the phosphate sites of the NaPi family of proteins. We hypothesize that differential phosphate selectivity of the NaPi family of proteins is related to differences in phosphate site solvent exposure or depth in the membrane field. As a starting point, we compared the effect of a watersoluble derivative, 2'-phosphophloretin (2'-PP), to the effect of a poorly water-soluble derivative, 2'-phospho-4,4',6'-trimethoxy-phosphophloretin (PTMP), on Na+-dependent phosphate uptake into intestinal and renal BBMV. Methylation of 2'-PP was selected to alter the water solubility of 2'-PP without adding large bulky groups, which could alter inhibitor potency through steric hindrance. The results indicate that NaPi-IIb and NaPi-Ia were 2'-PP sensitive and that NaPi-IIa was PTMP sensitive. These results are consistent with 2'-PP competing with H2PO4 binding to NaPi-IIb and NaPi-Ia and PTMP competing with HPO4 for binding to NaPi-IIa.
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MATERIALS AND METHODS |
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Reagents and solvents for the synthesis of 2'-PP and PTMP were purchased from Aldrich Chemical (Milwaukee, WI). Electrophoresis supplies were purchased from Bio-Rad (Hercules, CA). [32P]phosphate, [3H]glucose, and [35S]sulfate were purchased from New England Nuclear Life Sciences (Boston, MA). [3H]alanine was purchased from Pharmacia Life Sciences (Piscataway, NJ). HEPES, Tris, MES, MOPS, TAPS, and Percoll were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Fisher Chemical (Houston, TX) and were reagent grade or better. Peptide-specific affinity-purified antibodies were purchased from Alpha Diagnostics (San Antonio, TX). Texas red and fluorescein hydrazine derivatives were purchased from Molecular Probes (Eugene, OR).
Methods
Preparation of BBMV. Renal and intestinal BBMV were prepared by Ca2+ precipitation and differential centrifugation (42). Intestinal BBMV purification was determined using the enzyme markers sucrase (9), alkaline phosphatase (15), and leucine amino peptidase (19). Renal BBMV purification was assayed using the brush-border enzyme markers leucine amino peptidase, -glutamyl transpeptidase (30), and alkaline phosphatase. Protein was determined by the method of Lowry using bovine serum albumin as a standard (36).
Preparation of Distal Tubules and Proximal Tubules
Distal and proximal tubules from rabbit kidneys were isolated by Percoll gradient centrifugation (7). Briefly, rabbit kidneys were removed, perfused through the renal artery with 50 ml of PBS, and stored on ice. Kidneys were minced and passed through a 10-ml disposable syringe to normalize piece size. Minced kidney slices were digested with collagenase (Clostridium histolyticum, 1 mg/ml) in Krebs-Henseleit (KH; 138 mM NaCl, 3.8 mM KCl, 1.4 mM potassium phosphate, 1.4 mM MgSO4, 1.2 mM CaCl2, 25 mM NaHCO3, 60 mM mannitol, 1 mM pyruvic acid, 1 mM glutamate, 10 mM lactic acid, and 10 mM glutamine) media with 0.5 mg/ml BSA for 30 min at 37°C with gentle agitation (7). After digestion, ice-cold KH media was added (4-fold dilution) and the tissue was filtered through a Buchler funnel, followed by filtration through a nylon mesh with a pore diameter of 0.6 µm and then through a 0.2-µm filter. The effluent was centrifuged at 120 g for 10 min, and the pellets were resuspended in fresh KH media. This step was repeated three times.
After the final wash step, the pellets were resuspended in 150 ml of KH media and Percoll/rabbit (final Percoll concentration 40%) and gently mixed at 8°C for 15 min. Percoll gradient centrifugation was performed in a Beckman JA-17 fixed angle rotor spun at 28 kg for 30 min. The upper band (distal segments) and a second band in the bottom one-third of the tube were removed and washed three times by centrifugation (10,000 g for 15 min) to remove Percoll. Tubules were resuspended in 50 mM KCl and 1 mM Tris·HCl, pH 7, and stored at -80°C until needed. Typically, three tubule preparations were combined for isolation of BBMV.
Proximal tubule BBMV and distal tubule BBMV were prepared by Ca2+ precipitation and differential centrifugation. Fractions were assayed for protein (34, 36), leucine amino peptidase activity, alkaline phosphatase activity (15), succinate dehydrogenase activity (51), -glutamyl transpeptidase (30), phloridzin-sensitive Na+-dependent [3H]glucose uptake (35), and Na+-K+-ATPase activity (40). Proximal tubule BBMV and distal tubule BBMV enrichments are summarized in Tables 1 and 2.
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Phloretin Derivative Synthesis
2'-PP was synthesized from phloridzin as previously described (34); 171172°C, 1HNMR (d6-DMSO) 13.0 (s, 1H),
10.7 (br.s, 1H),
9.2 (br.s, 1H),
7.03 (d, J = 8.6 Hz, 2H),
6.64 (d, J = 8.4 Hz, 2H),
6.63 (dd, J = 1.2, 2.1 Hz, 1H),
2.77 (d, J = 7.6 Hz, 2H). 31P-NMR in D2O yielded a single peak at -4 ppm comprising 98% of the phosphorus signal. 31P-NMR in DMSO yielded a single peak at -4.3 ppm.
PTMP was synthesized from 2'-PP and dimethyl sulfate (27). Briefly, potassium carbonate (160 mg) was added to 2'-PP (50 mg in 25 ml acetone). Dimethyl sulfate (140 mg) was added and the mixture was stirred for 30 min at 23°C. Solvent was removed under reduced pressure. PTMP was purified by HPLC on a Waters reverse-phase C4 column eluted with 25% CH3CN and dried under reduced pressure.
PTMP was analyzed by 1HNMR in deuterated methanol (38) 7.13 (d, J = 8 Hz, 1H),
7.08 (t, J = 6 Hz, 12 Hz, 1H),
6.82 (d, J = 8 Hz, 1H),
6.79 (d, J = 8 Hz, 2H),
6.76 (d, J = 2 Hz, 1H),
6.3 (s, 1H),
6.27 (s, 1H),
6.2 (s, 1H),
3.8 (q, J = 9 Hz, 23 Hz, 3H),
3.7 (t, J = 7, 12 Hz, 3H),
3.68 (s, 2H),
3.56 (s, 2H).
Na+-dependent uptakes. [32P]phosphate, [3H]glucose, [3H]alanine, and [35S]sulfate uptakes into renal and intestinal BBMV were determined using 100 µg of BBMV protein and a rapid filtration set up at 23°C (34, 35, 42). Uptake was determined as filter-retained counts from media containing 100 mM NaCl or 100 mM KCl, 100 mM mannitol, 20 mM HEPES/Tris, pH 7.5, and 100 µM substrate {[32P]phosphate, [3H]glucose, [3H]alanine, or [35S]sulfate} and 5-s incubations with uptake media. In some experiments, uptakes were determined from 150 mM NaCl or 150 mM KCl, 20 mM HEPES/Tris, pH 7.5, and 100 µM substrate {[32P]phosphate, [3H]glucose, [3H]alanine, or [35S]sulfate}. Na+-dependent uptake was defined as retained counts in the presence of Na+ minus retained counts in the presence of K+. Results are expressed as picomoles per milligram per second and are means ± SE of three to five experiments. Results were corrected for filter-retained counts in the absence of protein. In some experiments, the effect of phosphophloretins on Na+-dependent uptakes was examined. In those experiments, 2'-PP was added in 10 mM borate buffer, pH 7.5, immediately before the start of the experiment. In experiments examining the effect of PTMP on uptake, PTMP was added in 1 µl of DMSO.
In some transport experiments, the effect of medium pH on Na+-dependent transport and the effect of phosphophloretins were examined. In these experiments, uptakes were determined from media containing 150 mM NaCl or 150 mM KCl, 100 µM [32P]phosphate, and 20 mM buffer (HEPES/Tris, pH 7.5; MES/Tris, pH 6.5 and 6.0; Pipes/Tris, pH 7; and TAPS/HCl, pH 8 and 8.5).
In some transport experiments, the effect of Na+ concentration on phosphophloretin inhibition of Na+-dependent [32P]phosphate uptake was examined. In these experiments, the Na+ concentration was varied between 50 and 250 mM, and the solution osmolality was maintained at 500 mosmol/kgH2O by addition of mannitol.
Western blotting. SDS-PAGE was performed on 10% polyacrylamide gels by the method of Laemmli (20). Protein was transferred to nitrocellulose paper by semidry electrophoretic blotting. Transferred gels were probed with primary antibodies at a 1:3,000 dilution and then with horseradish peroxidase-coupled anti-IgG secondary antibody according to the manufacturer's instructions. In some experiments, 25 µg of renal BBMV, BBMV from purified proximal tubules, and BBMV from purified distal tubules were used. In some experiments, applied BBMV protein was varied to titrate Npt antibodies.
Renal BBMV were probed with NaPi-IIa peptide-specific antibodies raised against the amino acid sequences SGILLWYPVPC (Npt 25) or PATPSPRLALPAHHNAC (Npt 21). NaPi-Ia peptide-specific antibodies were raised against the amino acid sequence CKWAPPLERGRLTSMS (Npt 11). Peptide sequences were confirmed by MSMS and amino acid sequencing at the Protein Chemistry Laboratory (UTMB, Galveston, TX). Specificity of the antibodies for the specified sequences was examined by preblot reaction of the antibody with the peptide used to generate the peptide sequence-specific antibodies.
Western blot and antibody sensitivity were determined by analysis of antibody binding to renal cortex BBMV protein (2.5 to 50 µg), distal tubule-enriched BBM protein (5 to 25 µg), and proximal tubule-enriched BBM protein (2.5 to 25 µg) separated by SDS-PAGE (20). Protein was transferred to nitrocellulose filters, and gels were stained with Ponceau S stain to determine protein transfer efficiency. NaPi-IIa and NaPi-Ia labeling were determined as described above. Standard curves for each antibody were generated as a function of protein concentration from 2.5 to 25 µg.
Immunohistochemistry
Antibodies specific for NaPi-Ia (Npt-11) and antibodies specific for NaPi-IIa (Npt-21) were labeled with fluorescent hydrazine derivatives at their carbohydrate moieties (31). The appropriate antibody (200 µg) was reacted with 100 mM sodium acetate, pH 5.5, and 10 mM NaIO4 for 30 min at 4°C. Carbohydrate oxidation was stopped by addition of 50 mM sodium sulfite and incubation for 1 h at 4°C. The antibody was dialyzed for 48 h against 100 mm sodium acetate, pH 5.5, with three buffer changes.
Antibodies were labeled with 5 mM Texas red hydrazide (Npt-11) or 5-{(2-[carbohydrazino]methyl)thio} acetyl aminofluorescein (Npt-21) in 100 mM sodium acetate, pH 5.5, for 2 h at 23°C. Antibodies were purified by gel filtration chromatography through Sephadex G-25 columns (5-ml syringe) eluted with PBS. Fractions containing antibodies were dialyzed for 1 wk against PBS. After dialysis, antibodies were assayed for protein (36) and lyophilized. Labeled antibodies were resuspended in PBS at a final concentration of 1 µg/µl.
Immunohistochemistry of purified distal tubules and proximal tubule preparations. Tubule-enriched fractions were immobilized on polylysine (50 µg/ml)-coated glass coverslips (50) and air-dried. Immobilized tubules were washed three times with PBS, two times with water, and finally with 70% ice-cold ethanol. Coverslips were reacted with fluorescently labeled antibodies at a 1:100 dilution (Ab:PBS) for 2 h at 23°C. Antibody was removed by suction, and the coverslips were washed three times with PBS and two times with water.
Tubule labeling was examined on a Nikon 800 fluorescence microscope, Optical Imaging Laboratory, Department of Physiology and Biophysics, UTMB. Coverslips were labeled with Npt-11, Npt-21, or double-labeled with Npt-21 followed by Npt-11. Due to fluorescence channel overlap, double-labeling with the Texas red-conjugated antibody (Npt-11) and the fluorescein-conjugated antibody (Npt-21) was performed by first labeling with the fluorescein-conjugated antibody, followed by the Texas red-conjugated antibody.
Analysis of antibody binding to coverslip-immobilized tubules was performed by epifluorescence on the Nikon 800 fluorescence microscope. Fluorescein- and Texas red-labeled structures were counted in 10 to 15 fields/coverslip and 8 to 10 coverslips/tubule preparation. Three tubule preparations were examined. Structures labeled with both Texas red and fluorescein were counted as proximal tubules, structures labeled with only Texas red were counted as distal tubules, and structures not labeled with either antibody were counted as nontubule distal segments.
Renal sections. Rabbit kidneys were removed, perfused with PBS, followed by 50 ml of 10% buffered formalin. Kidneys were placed in fresh formalin overnight at 8°C. Thick sections were prepared by the Immunohistochemistry Laboratory, Department of Human Biochemistry, and Cellular Genetics, UTMB. Sections were stained with hematoxylin and eosin or fluorescent antibodies at a 1:75 dilution (Npt-11) or 1:100 dilution (Npt-21). Sections were examined on a Nikon 800 fluorescence microscope at the Optical Imaging Laboratory (UTMB, Galveston, TX).
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RESULTS |
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The effect of phloretin and phloretin derivatives on rabbit renal BBMV Na+-dependent phosphate uptake is shown in Fig. 1. Phloretin (, broken line) inhibited Na+-dependent phosphate uptake 20% at 10 µM. PTMP (
, solid line) inhibited 70% at 10-7 M PTMP. 2'-PP (
, solid line) inhibited Na+-dependent phosphate uptake into renal BBMV <30% at pH 7.5.
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A similar series of experiments was performed with intestinal BBMV to compare the effect of phloretin derivatives on NaPi-IIb activity. The effect of 2'-PP and PTMP on Na+-dependent phosphate uptake into intestinal BBMV is shown in Fig. 2. 2'-PP inhibited Na+-dependent phosphate uptake into intestinal BBMV (, solid line) 94 ± 3% with an IC50 of 40 ± 4 nM (n = 5). PTMP (
, solid line) was a weak inhibitor of Na+-dependent phosphate uptake into intestinal BBMV. PTMP inhibition of Na+-dependent phosphate uptake into intestinal BBMV was similar to phloretin (
, broken line) accounting for <30% (29.6 ± 6%, n = 5) at inhibitor concentrations of 10-5 M.
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Phloretin is a nonspecific inhibitor of a variety of transport proteins including band 3 (14), the urea transporter (6), and pyruvate and lactate transporter of cardiomyocytes (48). The specificity of PTMP inhibition of NaPi transporters was examined by comparing renal cortex BBMV Na+-dependent phosphate uptake to PTMP inhibition of renal cortex BBMV Na+-dependent glucose, Na+-dependent alanine, and Na+-dependent sulfate uptake. The results are shown in Fig. 3.
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Figure 3 compares the effect of PTMP on Na+-dependent phosphate uptake (, dashed line), Na+-dependent glucose uptake (
, dashed line), Na+-dependent alanine uptake (
, solid line), and Na+-dependent sulfate (
, solid line) uptake into renal cortex BBMV. PTMP did not alter glucose, sulfate, or alanine uptakes into renal BBMV at PTMP concentrations up to 10 µM. These results are similar to the results seen with 2'-PP inhibition of intestinal Na+-organic substrate cotransport (34) and consistent with a selective inhibition of Na+-dependent phosphate uptake into renal cortex BBMV by PTMP.
Effect of pH on PTMP Inhibition of Renal BBMV Na+-Dependent Phosphate Uptake
The presence of two Na+-dependent phosphate cotransporter pathways into renal cortex BBMV limits analysis of phosphophloretin derivative inhibition of NaPi-mediated phosphate uptake into renal cortex BBMV. Alkaline pH may be used to minimize NaPi-Ia activity, or acid pH at low-Na+ concentrations may be used to minimize NaPi-IIa-mediated Na+-dependent phosphate uptake (28). To examine the effect of phosphophloretins on NaPi-IIa-mediated and NaPi-Ia-mediated Na+-dependent phosphate uptake into renal cortex BBMV, the effect of pH on PTMP and 2'-PP inhibition of Na+-dependent phosphate uptake was examined between pH 5.5 and 8.5. The results of these studies are summarized in Fig. 4.
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PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake (, broken line) was maximal at alkaline pH. At 100 mM NaCl, the apparent pKa was 7.5 ± 0.3 pH units (n = 3). The IC50 for PTMP inhibition of Na+-dependent phosphate uptake was slightly affected by pH (IC50 = 23 ± 7 nM between pH 5.5 and 8.5) in 150 mM NaCl (results not shown). Alkaline pH sensitivity of PTMP inhibition of Na+-dependent phosphate uptake was similar to the effect of pH on NaPi-IIa activity (3, 44). In contrast, 2'-PP inhibition of renal cortex BBMV Na+-dependent phosphate uptake (
, solid line) was decreased at alkaline pH. At medium pH values above pH 7, 2'-PP inhibition of Na+-dependent phosphate uptake was <10% (5 ± 3%). The apparent pKa for 2'-PP inhibition of Na+-dependent phosphate uptake into renal cortex BBMV was 6.2 ± 0.4 (n = 3). The effect of pH on phosphophloretin inhibition of renal cortex BBMV Na+-dependent phosphate uptake is consistent with 2'-PP inhibition of NaPi-Ia activity and PTMP inhibition of NaPi-IIa activity.
The selectivity of PTMP for NaPi-IIa-mediated Na+-dependent phosphate uptake was tested by comparing the effect of pH on phosphophloretin inhibition of Na+-dependent phosphate uptake into renal cortex BBMV as a function of Na+ concentration. The results of these studies are shown in Fig. 5.
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If PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake was Na+ concentration sensitive at pH 6, then the low-PTMP inhibition of Na+-dependent phosphate uptake might be the result of low-NaPi-IIa Na+ affinity at acid pH and not differential inhibition of NaPi-IIa and NaPi-Ia. However, if Na+ concentration did not increase PTMP inhibition at acid pH, then PTMP has little effect on NaPi-Ia activity. Figure 5 shows PTMP inhibition at pH 6 (, solid line) was unaffected by increasing Na+ concentration. At pH 8, PTMP inhibition of Na+-dependent phosphate uptake was Na+ concentration sensitive (
, broken line). This result suggests that PTMP inhibition of phosphate uptake at acid pH was not limited by reduced Na+ affinity of NaPi-IIa. A similar series of experiments substituting 2'-PP for PTMP was more difficult to interpret due to very low 2'-PP-sensitive uptakes at alkaline pH. At acid pH and 50 mM NaCl, 2'-PP inhibition was 45 ± 10% (n = 3) of the Na+-dependent phosphate uptake and unaffected by increasing the Na+ concentration to 250 mM. These results are consistent with PTMP being a potent inhibitor of NaPi-IIa, with little effect on NaPi-Ia, and 2'-PP being a potent inhibitor of NaPi-Ia, with little effect on NaPi-IIa activity.
Effect of Phosphophloretins on Na+-Dependent Phosphate Uptakes into Distal Tubule-Enriched BBMV and Proximal Tubule-Enriched BBMV
Phosphophloretin derivative inhibition of NaPi-Ia- and NaPi-IIa-mediated Na+-dependent phosphate uptakes into BBMV purified from proximal tubule-enriched and from distal tubule-enriched fractions was examined to further define inhibitor sensitivity. Proximal tubule-enriched fractions and distal tubule-enriched fractions were purified by collagenase digestion and a combination of differential sieving and Percoll gradient centrifugation. Table 1 summarizes biochemical analysis of collagenase digested and Percoll gradient centrifugation purification of proximal tubules and distal tubules. Table 2 summarizes biochemical analysis of BBMV isolated from purified tubule fractions. Table 1 indicates that distal tubule fractions were three- to fourfold deenriched in the brush-border enzyme markers, alkaline phosphatase, leucine aminopeptidase, and -glutamyl transpeptidase compared with the renal cortex. Compared with the renal cortex total homogenate, proximal tubule fractions were enriched two- to threefold. Table 2 indicates that distal tubule-enriched BBMV were slightly enriched in BBM marker enzymes compared with the renal cortex homogenate, whereas proximal tubule-enriched BBMV were 12-fold enriched in BBM marker enzyme activities. These results are consistent with previous studies indicating that distal segments contain alkaline phosphatase and
-glutamyl transpeptidase (37) at lower expression levels than proximal tubule apical membranes.
Distal tubule and proximal tubule Na+/phosphate cotransporter expression was performed by Western blot analysis of antipeptide antibodies to examine tubule expression of NaPi-IIa and NaPi-Ia and BBMV purification. Figure 6 is a Western blot analysis of BBMV purified from distal tubules (lanes 2-4) and BBMV purified from proximal tubules (lanes 5-8). Peptide-blocked Npt-11 antibody (lane 1) and peptide-blocked Npt-25 antibody (lane 9) are also shown. Varying amounts of distal tubule BBMV protein were added to the gel (lane 2 contained 5 µg, lane 3 contained 10 µg, and lane 4 contained 25 µg of protein) to examine blot sensitivity as a measure of proximal tubule contamination of the distal tubule-enriched BBMV. Variable amounts of proximal tubule BBMV protein were added to the gel (lane 8 contained 2.5 µg, lane 7 contained 5 µg, lane 6 contained 10 µg, and lane 5 contained 25 µg of protein) to examine the sensitivity of Npt-25 as a reporter NaPi-IIa expression. BBMV were probed with Npt-11 (NaPi-Ia-specific polyclonal antibody) followed by Npt-25 (NaPi-IIa-specific polyclonal antibody). Npt-25 (top arrow) recognized an 87-kDa polypeptide in proximal tubule-enriched BBMV (lanes 5-9) but not in distal tubule-enriched BBMV (lanes 1-4). Npt-11 (bottom arrow) recognized a 65-kDa polypeptide in both proximal tubule-enriched BBMV and distal tubule-enriched BBMV. Similar results were seen when blots were probed with Npt-25 followed by Npt-11 (results not shown). These results indicate that NaPi-Ia is expressed in the distal tubule-enriched fractions and suggest that Npt-25 is a sensitive reporter of NaPi-IIa and proximal tubule contamination of distal tubule fractions to a lower limit of 5% contamination of the distal tubule-enriched BBMV.
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Immunohistochemistry was performed on coverslips containing polylysine-immobilized, distal tubule-enriched fractions and proximal tubule-enriched fractions. Antibodies were directly labeled at their carbohydrate moieties to simplify washes and to improve reporter group access. Npt-11 labeled 70 ± 8% (n = 4) of the structures on polylysine-coated coverslips containing distal tubule-enriched fractions. Npt-11 and Npt-21 (NaPi-IIa-specific antibody) labeled 6 ± 3% of the structures, and neither antibody recognized 19 ± 7% of the structures. Labeling experiments were also performed on renal sections with fluorescently labeled Npt-11. The results from one experiment are shown in Fig. 7.
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Figure 7 shows Texas red-conjugated Npt-11 labeling of both proximal tubules (PT) and distal tubules (DT) at their luminal membranes. There was no labeling of the glomerulus or connecting tubules. Figures 6 and 7 and Tables 1 and 2 indicate that BBMV purified from distal tubule-enriched fractions isolated from rabbit renal cortex are at least 90% distal nephron and contain 5% proximal tubule contamination. Na+-dependent phosphate uptake experiments using BBMV purified from distal tubule-enriched, Ca2+-precipitated BBMV and into Ca2+-precipitated, proximal tubule-enriched were performed to test the suitability of BBMV for phosphophloretin inhibition experiments. Na+-dependent phosphate uptake into distal tubule-enriched BBMV and proximal tubule enriched BBMV as a function of time was examined. Phosphate uptake into distal tubule-enriched BBMV and into proximal tubule-enriched BBMV as a function of time is shown in Fig. 8.
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In the presence of NaCl, distal tubule-enriched BBMV phosphate uptake (, solid line) was maximal at 60 s reaching an internal concentration 2x equilibrium values. In the presence of NaCl, phosphate uptake into proximal tubule-enriched (
, solid line) BBMV was maximal at 120 s, reaching an internal concentration 3x equilibrium phosphate concentration. These results indicate that BBMV prepared from distal tubule-enriched fractions and proximal tubule-enriched fractions show a modest overshoot consistent with Na+-coupled phosphate uptake. On the basis of these experiments, 5-s uptakes were chosen for phosphophloretin inhibition studies using initial rates.
Phosphophloretin inhibition of Na+-dependent phosphate uptake into distal tubule-enriched BBMV was examined to test the hypothesis that NaPi-Ia is 2'-PP sensitive and not PTMP sensitive. Na+-dependent phosphate uptake into distal tubule-enriched BBMV and proximal tubule-enriched BBMV was examined. The results of these experiments are shown in Fig. 9.
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Na+-dependent phosphate uptake into distal tubule-enriched BBMV was sensitive to 2'-PP in a concentration-dependent manner. At pH 6 (, solid lines), the IC50 for 2'-PP inhibition of phosphate uptake was 35 ± 8 nM (n = 3). At pH 8.5 (
, solid line), the IC50 was 48 ± 6 nM (n = 3). Na+-dependent phosphate uptake into distal tubule-enriched BBMV was not sensitive to PTMP at pH 6 (
, dashed line) or at pH 8.5 (
, dashed line).
Similar experiments in proximal tubule-enriched BBMV were similar to phosphoploretin inhibition experiments with BBMV isolated from renal cortex. 2'-PP inhibition of Na+-dependent phosphate uptake into BBMV isolated from proximal tubule-enriched fractions decreased with increasing pH. Maximum inhibition was 34% (%Imax = 34 ± 8%, n = 3) at pH 6 to 3% (3.4 ± 2%, n = 3) of the Na+-dependent phosphate uptake into proximal tubule-enriched BBMV at pH 8.5. PTMP was a potent inhibitor of Na+-dependent phosphate uptake into proximal tubule-enriched BBMV at all pH values studied. The IC50 for PTMP inhibition of Na+-dependent phosphate uptake into proximal tubule-enriched BBMV was 22 ± 6 nM (n = 4).
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DISCUSSION |
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Previous studies suggested a correlation between vanadate and PFA sensitivity and cotransporter preference for HPO4 (24, 43, 45). Based on these studies with phosphate site analogs, we decided to examine NaPi family sensitivity to phosphophloretin derivatives. In addition, solvent polarity has a marked effect on phosphate dissociation state (26). We hypothesized that differences in phosphate valence state preference within the NaPi family of proteins may be related to substrate site polarity. To test this hypothesis and to develop selective phosphophloretin derivatives, the effect of water-soluble and poorly water-soluble phosphophloretins on three members of the NaPi family of proteins with different phosphate valence state preferences was examined.
Na+-dependent phosphate uptakes into BBMV prepared from rabbit kidney cortex were sensitive to 2'-PP and PTMP (Fig. 1). The order of inhibitory potency followed the sequence PTMP >> 2'-PP > phloretin. 2'-PP inhibition of Na+-dependent phosphate uptake into renal cortex BBMV was variable but always significantly greater than phloretin sensitivity. In contrast, intestinal BBMV Na+-dependent phosphate uptake (Fig. 2) was sensitive to 2'-PP and insensitive to PTMP. Intestinal BBMV Na+-dependent phosphate uptake was inhibited more than 90% at 500 nM 2'-PP with an IC50 of 40 ± 4 nM (Fig. 2). 2'-PP inhibition of intestinal BBMV Na+-dependent phosphate uptake was greater at acidic pH than at alkaline pH. The pH dependence of 2'-PP inhibition of intestinal Na+-dependent phosphate uptake was consistent with the pH dependence of Na+ binding to the cotransporter (10, 28, 33, 41). These results are similar to previous studies (28, 34, 35). Based on these studies, phloretin derivative sensitivity of NaPi-IIb was 2'-PP >> PTMP phloretin. Further studies attempted to define NaPi-IIa and NaPi-Ia phloretin derivative sensitivity.
Assigning inhibition in rabbit renal cortex BBMV is complicated by the presence of two Na+-phosphate cotransporters in BBM, NaPi-IIa and NaPi-Ia. To examine Na+-phosphate cotransporter inhibitor sensitivity, conditions were altered to minimize each cotransporter's contribution to Na+-dependent phosphate uptake in membrane vesicles. NaPi-IIa activity was selectively examined at alkaline pH. NaPi-Ia activity was selectively examined at acidic pH and in BBMV purified from distal tubule-enriched fractions.
The pH dependence of PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake suggested PTMP inhibition of NaPi-IIa activity (Fig. 4). The IC50 of PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake was independent of pH (results not shown). The magnitude of PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake (Fig. 4, , broken line) was greater at alkaline pH than at acidic pH, consistent with Na+ activation of NaPi-IIa-mediated phosphate uptake and Na+ activation of a Na+-dependent inhibitor of phosphate uptake.
The decreased inhibition of Na+-dependent phosphate uptake at acid pH was not due to reduced NaPi-IIa Na+ affinity at acid pH reducing the effect of a Na+-dependent inhibitor like PTMP. Increasing the Na+ concentration to about three times the KM (Na+) did not increase PTMP inhibition of Na+-dependent phosphate uptake into renal cortex BBMV at acid pH (Fig. 5). This result is consistent with PTMP inhibition of renal cortex Na+-dependent phosphate uptake being limited to NaPi-IIa and not due to reduced NaPi-IIa Na+ affinity at acidic pH (Fig. 5).
Renal cortex BBMV was less sensitive to 2'-PP than to PTMP. 2'-PP inhibition of renal BBMV Na+-dependent phosphate uptake was 45% at acid pH and decreased with increasing pH to 5% at alkaline pH (Fig. 4,
, solid line). The effect of pH on 2'-PP inhibition as a function of pH is inconsistent with inhibition of NaPi-IIa, consistent with the pH dependence of NaPi-Ia activity, and consistent with 2'-PP inhibition of NaPi-Ia.
The results from our renal cortex BBMV Na+-phosphate cotransporter inhibitor studies were consistent with NaPi-IIa being PTMP sensitive and NaPi-Ia being 2'-PP sensitive. To define NaPi-Ia inhibition of Na+-dependent phosphate uptake in the kidney, distal tubules and proximal tubules were prepared from renal cortex by collagenase digestion, molecular sieving, and Percoll gradient centrifugation.
The distribution of NaPi-IIa, NaPi-Ia, and brush-border marker enzymes along the nephron is controversial. NaPi-IIa and NaPi-Ia are present in the apical membrane of the proximal tubule (28). However, there is little agreement concerning the presence of phosphate reabsorption, -glutamyl transpeptidase, and leucine aminopeptidase at more distal sites.
Four lines of evidence suggest that NaPi-Ia is present in distal tubule fractions purified from rabbit renal cortex. Tables 1 and 2 indicate that following Percoll gradient centrifugation and Ca2+ precipitation, distal tubule-enriched BBMV are four-fold deenriched in the BBM markers, leucine aminopeptidase, and -glutamyl transpeptidase relative to the renal cortex. Based on marker enzymes and transport activities, the distal BBMV is at least 10-fold deenriched in proximal tubule activities.
Western blot analysis shown in Fig. 6 indicates that NaPi-IIa was not detected in BBMV purified from the distal tubule-enriched fractions (Fig. 6, lanes 2-4). Western blot analysis of serial dilutions of renal cortex BBM protein indicated that lower limit of Npt-25 sensitivity was <2.5 µg or approximately a 10-fold dilution of NaPi-IIa (results not shown). Similar studies with Npt-11 indicated that the lower limit for Npt-11 recognition of NaPi-Ia in renal cortex or proximal tubule-enriched BBMV was 10 µg. These results indicate that a 10% proximal tubule contamination of distal tubule BBMV would be detected with our antibodies. Immunocyto-chemistry of polylysine-coated coverslips with immobilized distal tubules and proximal tubule renal sections indicated that 70% of the structures were labeled with the NaPi-Ia-specific antibody (Npt-11) and 6% of the structures were labeled with the NaPi-IIa-specific antibody and the NaPi-Ia-specific antibody.
Immunohistochemistry of renal slices were consistent with NaPi-Ia localization in both proximal tubules and distal segments (Fig. 7). The apical membranes of distal tubules contained NaPi-Ia and lacked NaPi-IIa (4, 5, 22, 28, 44). These results are in agreement with previous reports (1, 4, 5, 16, 17, 22, 28) of phosphate handling in distal nephron segments. These results indicate that at least 70% of distal tubule-enriched BBMV were from distal tubule-derived structures.
Finally, phosphate uptake into BBMV isolated from distal tubule-enriched fractions displayed an overshoot of equilibrium phosphate uptake in NaCl media (Fig. 8). The presence of an overshoot in Na+ is characteristic of Na+-dependent organic solute cotransporters, indicating active accumulation of phosphate above intervesicular equilibrium due to continued uptake of Na+ (47). The presence of Na+-dependent phosphate uptake in BBMV from distal tubule-enriched fractions (Fig. 8) and apparent absence of NaPi-IIa in distal tubule-derived BBMV (Fig. 6) strongly suggests that NaPi-Ia is present in the distal tubule-enriched BBMV and that these membrane vesicles may be useful in studies of phosphophloretin sensitivity of NaPi-Ia.
PTMP inhibition of Na+-dependent phosphate uptake into distal tubule-enriched BBMV was not statistically different from phloretin inhibition (Fig. 9). PTMP inhibited <10% of the Na+-dependent phosphate uptake into these membrane vesicles. 2'-PP inhibited Na+-dependent phosphate uptake into BBMV purified from distal tubules in a concentration-dependent manner with an IC50 of 38 nM at pH 6 and 48 nM at pH 8.5 (Fig. 9). These results are consistent with 2'-PP inhibition of NaPi-Ia in renal BBMV. The results indicate that NaPi-IIa is PTMP sensitive and NaPi-Ia and NaPi-IIb are 2'-PP sensitive. Based on our studies, we suggest that PTMP and 2'-PP may be useful tools for the analysis of NaPi protein expression.
With regard to the molecular mechanism responsible for differential phosphophloretin sensitivity in renal and intestinal BBMV, we hypothesize that differences in the phosphate sites of the cotransporters are the most likely cause of phosphophloretin sensitivity differences within the NaPi family. Phosphate valence state preference appeared to correlate with cotransporter phosphophloretin derivative sensitivity. Na+/phosphate cotransporters preferring H2PO4 (NaPi-Ia and NaPi-IIb) were sensitive to 2'-PP. A preference for HPO4 was associated with a greater sensitivity to PTMP.
Differences in phosphophloretin derivative sensitivity were consistent with differences in the degree of phosphate site-solvent accessibility within the NaPi family. 2'-PP is water soluble and an acid in water. PTMP is poorly water soluble and slightly basic in water. PTMP sensitivity of NaPi-IIa may indicate a more hydrophobic phosphate site. In this regard, changes in solvent polarity, such as mixed organic solvent: water solutions, have been shown to alter phosphate dissociation (26). A less polar phosphate site may contribute to cotransporter preference for HPO4, and a more polar phosphate site may contribute to a preference for H2PO4 transport. The selective inhibition by phosphophloretin derivatives is consistent with this interpretation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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