Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Kidney International, Vol. 60 (2001), pp. 694–704

Glycosphingolipids modulate renal phosphate transport in potassium deficiency HUBERT K. ZAJICEK,1 HUAMIN WANG,1 KRISHNA PUTTAPARTHI, NABIL HALAIHEL, DANIEL MARKOVICH, JAMES SHAYMAN, RICHARD BE´LIVEAU, PAUL WILSON, THOMAS ROGERS, and MOSHE LEVI Department of Internal Medicine, The University of Texas Southwestern Medical Center and VA Medical Center, Dallas, Texas, USA; Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland, Australia; Department of Internal Medicine, The University of Michigan Medical Center, Ann Arbor, Michigan, USA; and Laboratoire d’Oncologie Mole´culaire, Universite du Que´bec a` Montreal, Quebec, Canada

that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency. Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the Vmax of brush-border membrane (BBM) Na/Pi cotransport activity (1943 ⫾ 95 in control vs. 1184 ⫾ 99 pmol/5 sec/mg BBM protein in K deficiency, P ⬍ 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 ⫾ 52 in control ⫹ PDMP vs. 1152 ⫾ 61 pmol/5 sec/mg BBM protein in K deficiency ⫹ PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms

1

Hypokalemia and/or potassium deficiency (KD) modulate a number of renal functional parameters including renal blood flow [1], glomerular filtration rate [2], proximal tubular Na/H exchange [3], Na/citrate [4], and Na/ sulfate [5] cotransport activities. Hypokalemia and/or KD have also been shown to be associated with hypophosphatemia and/or an increase in urinary phosphate excretion [6–21]. The cellular mechanisms showing how hypokalemia or KD regulates renal phosphate transport, however, have not been studied. The purpose of our study was to determine whether KD in the rat alters proximal tubular brush-border membrane (BBM) Na/Pi cotransport activity, and whether the regulation takes place at the level of the proximal tubular BBM Na/Pi cotransport system. Our results indicate that KD causes a significant increase in the urinary excretion of Pi and a parallel decrease in the Vmax of BBM Na/Pi cotransport activity. Surprisingly, the decrease in Na/Pi cotransport activity occurred despite an increase in the level of BBM type II Na/Pi cotransport protein (NaPi-2) and no change in cortical NaPi-2 mRNA abundance. In addition, the abundance of type I Na/Pi cotransport protein (NaPi-1) and type III Na/Pi cotransport protein (Glvr-1) was also increased. The decrease in BBM Na/Pi cotransport activity was associated with significant increases in BBM sphingomyelin, glucosylceramide and ganglioside GM3 content, and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in a significant increase of BBM Na/Pi cotransport activity back to normal levels, independent of any changes in BBM NaPi-2 protein or cortical NaPi-2 mRNA abundance. Our re-

Dr. Zajicek and Dr. Wang contributed equally to this work.

Key words: Na/Pi cotransport proteins, lipid fluidity, membrane lipid dynamics, hypokalemia, glucosylceramide, ganglioside GM3, brushborder membrane, proximal tubule. Received for publication December 12, 2000 and in revised form March 12, 2001 Accepted for publication March 15, 2001

 2001 by the International Society of Nephrology

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sults therefore indicate that hypokalemia inhibits Na/Pi cotransport activity by post-translational mechanisms that are mediated in part by an increase in glucosylceramide content. METHODS Experimental animals The experiments were performed in male SpragueDawley rats weighing 150 to 200 g (Harlan Industries, Indianapolis, IN, USA). Prior to study, the animals were stabilized on a control diet for five days in metabolic balance cages. The rats were then pair-fed a control or a K-deficient diet as previously described [4, 5]. On the seventh day, a 24-hour urine collection was obtained. On the eighth day, the rats were anesthetized with sodium pentobarbital. Blood was obtained from the inferior vena cava, and the kidneys rapidly removed and placed in an ice-cold buffer. Blood and urine samples were analyzed for inorganic phosphate by the method of Fiske and Subbarow and for creatinine by a creatinine autoanalyzer (Creatinine II Analyzer; Beckman, Fullerton, CA, USA) [22]. Total urinary phosphate excretion and tubular reabsorption of phosphate were calculated by standard clearance formulae. In experiments designed to determine the role of alteration in glucosylceramide content per se in the regulation of Na/Pi cotransport (Results section), rats fed a control or K-deficient diet were also treated with DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthase [23, 24]. Since previous studies have demonstrated that the circulating plasma levels of PDMP are prolonged in the presence of the cytochrome P450 inhibitor, piperonyl butoxide [23], the butoxide solution was administered before injection of PDMP. The piperonyl butoxide was prepared by dissolution in corn oil at a concentration of 150 mg/mL. On the first day of treatment, the butoxide solution was administered intraperitoneally four hours before PDMP treatment; on days 2 to 7 of treatment, it was administered just before the PDMP injection. PDMP was prepared as an emulsion with the detergent Myrj 52 in normal saline, buffered with sodium acetate. The final concentrations were PDMP 6 mg/dL, Myrj 52 12 mg/dL, and sodium acetate 8 mg/dL. The PDMP was given intraperitoneally as a dose of 100 mg/kg body weight. Brush-border membrane isolation The kidneys were placed in an ice-cold homogenizing buffer consisting of 300 mmol/L mannitol, 5 mmol/L egtazic acid (EGTA), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 16 mmol/L HEPES, pH 7.50, with Tris buffer. Thin slices were cut from the superficial cortex and homogenized with a Polytron homogenizer. Brushborder membrane vesicles were isolated from the resulting

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homogenate by differential centrifugation and Mg2⫹ precipitation, as previously described [25, 26]. To minimize the potential day-to-day variations in the BBM isolation procedure on the resultant measurements, each day BBM from control and K-deficient rats were isolated simultaneously. For each BBM sample, the kidneys from two rats from each experimental group were pooled (N ⫽ 1). A total of 24 rats were studied in each group, resulting in N ⫽ 12 BBM samples for each experimental group. The final BBM pellet was resuspended in a 300 mmol/L mannitol, 16 mmol/L HEPES, 10 mmol/L Tris, pH 7.50, buffer and was aliquoted for simultaneous measurements of (1) enzyme activity, (2) transport activity, (3) protein electrophoresis and Western blotting, (4) lipid composition, and (5) lipid fluidity. Brush-border membrane enzyme activity measurements The purity of each BBM preparation was determined by measurement of membrane-bound specific enzyme activity, including leucine amino peptidase (apical marker) and Na,K-ATPase (basolateral marker) in cortical homogenate and in BBM fractions as previously described [26]. Enzyme activities were expressed as ␮mol/h/mg cortical homogenate or BBM protein. Enrichment (specific activity in BBM/specific activity in homogenate) and recovery (total activity in BBM/total activity in homogenate) were determined using the previously mentioned enzymes as markers of apical and basolateral membranes. Protein was determined by the method of Lowry et al using crystalline BSA as standard [27]. Brush-border membrane transport measurements Transport measurements were performed in freshly isolated BBM vesicles by radiotracer uptake followed by rapid millipore filtration. To measure the Na⫹ gradientdependent 32Pi uptake (Na-Pi cotransport), 10 ␮L of BBM preloaded in an intravesicular buffer of 300 mmol/L mannitol, 16 mmol/L HEPES, 10 mmol/L Tris buffer, pH 7.50, was vortex-mixed at 25⬚C with 40 ␮L of an extravesicular uptake buffer of 150 mmol/L NaCl, 100 ␮mol/L K2H32PO4, 16 mmol/L HEPES, 10 mmol/L Tris, pH 7.50. Uptake after five seconds (representing initial linear rate) was terminated by an ice-cold stop solution consisting of 135 mmol/L NaCl, 10 mmol/L Na2 arsenate, 16 mmol/L HEPES, and 10 mmol/L Tris buffer, pH 7.50. All uptake measurements were performed in triplicate, and uptake was calculated on the basis of specific activity determined in each experiment and expressed as pmol 32 Pi/5 sec/mg BBM protein. To determine whether the differences in Na-Pi cotransport were specific for Pi, Na⫹ gradient-dependent glucose and proline uptake were also measured. BBM vesicles were preloaded with the 300 mmol/L mannitol, 10 mmol/L HEPES, 10 mmol/L Tris (pH 7.50) intravesicular buffer, and the uptake solution consisted of 150

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mmol/L NaCl and either 100 ␮mol/L D-[3H] glucose, or 100 ␮mol/L L-[3H] proline and HEPES-Tris, pH 7.50. Uptake was terminated by the stop solution, which consisted of 150 mmol/L NaCl, 0.25 mol/L phloridzin, and 16 mmol/L HEPES, 10 mmol/L Tris, pH 7.50. Brush-border membrane SDS-gel protein electrophoresis and Western blotting Brush-border membranes were denatured for two minutes at 95⬚C in 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.5 mmol/L ethylenediaminetetraacetic acid (EDTA), and 95 mmol/L Tris-HCl, pH 6.8 (final concentrations). Ten micrograms of BBM protein/lane were separated on 9% polyacrylamide gels according to the method of Laemmli [28] and were electrotransferred onto nitrocellulose membrane [29]. After blockage with 5% fat-free milk powder with 1% Triton X-100 in Trisbuffered saline (TBS; 20 mmol/L, pH 7.3), Western blots were performed with antiserum against the C-terminal amino acid sequence of NaPi-2, NaPi-1, or Glvr-1 at a dilution of 1:5,000. Primary antibody was visualized using enhanced chemiluminescence (ECL; Pierce, Bradford, IL, USA). The signals were quantitated in a Phosphor Imager with chemiluminescence detector and densitometry software (Bio-Rad, Richmond, CA, USA). Brush-border membrane lipid composition measurement Lipids from BBM were extracted by the method of Bligh and Dyer [30], as we have previously described [24, 26]. First, to determine free cholesterol content, an aliquot of the lipid extract was injected into a 530 ␮m 50% phenyl methyl silicone column in a Hewlett-Packard model 5890 gas chromatograph with a flame ionization detector, run isothermally at 280⬚C, with coprostanol serving as an internal standard. Area ratios were computed with a Hewlett-Packard 3392A integrator, and cholesterol was expressed as nmol/mg BBM protein [26, 31, 32]. Second, to determine individual phospholipid polar head-group species, an aliquot of the lipid extract was applied to thin-layer chromatography plates (Silica Gel 60; E. Merck, Darmstadt, Germany) and individual phospholipids, including sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, were separated by a two-dimensional solvent system [33], as we have previously described [24, 26]. Phospholipid content in total and individual phospholipids was determined by measuring phosphorus content by the method of Ames and Dubin [34]. Third, to determine the glycosphingolipid composition, an aliquot of the lipid extract was evaporated to dryness and subjected to alkaline methanolysis. Briefly, the dried lipids were dissolved in 2 mL of chloroform. The reaction was started by adding 1 mL of 0.21 N sodium hydroxide:methanol, was continued for one hour at 37⬚C, and terminated by adding 0.8 mL of 0.2 mol/L

acetic acid. The resultant mixture was centrifuged for five minutes at 800 ⫻ g. The upper layer was removed. The lower layer was washed with 1 mL of methanol plus 0.8 mL of water. After the same centrifugation, the lower layer was transferred into another glass tube and dried under a stream of nitrogen gas. The lipids were then chromatographed on high-performance thin-layer chromatography plates (HPTLC; E. Merck 5641). Glucosylceramide and ganglioside GM3 were separated with a solvent system consisting of chloroform:methanol:water (65:25:4) on plates which were pretreated with 2.5% borax in methanol:water (1:1). The lipid bands were visualized by impregnating the plates with a modified charring reagent (100 g of CuSO45H2O in concentrated H3PO4:water:methanol (100:750:400) [23, 24]. The charred thin-layer chromatography plates were scanned with a video densitometer. Comparing the density of each spot with the density of the corresponding standard curve quantitated the glucosylceramide and ganglioside GM3 bands. Brush-border membrane lipid fluidity measurements Brush-border membrane fluidity was determined by the fluorescence measurements of (1) 1,6-diphenyl-1, 3, 5-hexatriene (DPH) and (2) 6-dodecanoyl-2-diethylaminonaphthalene (Laurdan; Molecular Probes, Eugene, OR, USA). The steady-state emission spectra of Laurdan were measured in a spectrofluorometer (PC1, ISS, Champaign-Urbana, IL, USA). Excitation wavelength was 340 nm, and emission was measured at 440 and 490 nm. In phospholipid vesicles and in BBM the emission maximum for Laurdan is 440 nm in the gel phase and 490 nm in the liquid-crystalline phase [35–37]. The emission spectra of Laurdan are quantitated by the generalized polarization (GP) GPLaurdan ⫽

I440 ⫺ I490 I440 ⫹ I490

where I440 and I490 are the emission intensities at 440 nm and 490 nm, respectively [35–37]. The steady-state anisotropy of DPH (rDPH) was measured in the same spectrofluorometer equipped with excitation and emission polarizers. Excitation wavelength was 360 nm and emission was viewed through a KV 399 nm filter [31, 32]. rDPH is determined by rDPH ⫽

III ⫺ II III ⫹ 2II

where III and II represent the intensities of the parallel and perpendicular components of the emission respectively [31, 32]. RNA isolation Thin slices were cut at 4⬚C from the superficial cortex and homogenized with Polytron in a 4 mol/L guanidium

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thiocyanate, 25 m mol/L sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 mol/L 2-mercaptoethanol denaturation solution. Sequentially, 0.1 volume 2 mol/L sodium acetate, pH 4.0, 1 volume water-saturated phenol, 0.2 volumes chloroform-isoamyl alcohol mixture (49:1) were added to the homogenate. Total RNA was isolated as previously described [38] and resuspended in water treated with diethyl pyrocarbonate (DEPC). Absorbency at 260 and 280 nm was obtained to quantify and assess the purity of the RNA fraction. Formaldehyde agarose gel electrophoresis and Northern blot analysis Following denaturation of RNA samples in formaldehyde, 20 ␮g total RNA per lane were size fractionated using 0.66 mol/L formaldehyde, 1% agarose (final concentration) gels (Bio-Rad). RNA size standards (GIBCO BRL, Gaithersburg, MD, USA) were run in parallel. After electrophoresis the gel was placed onto a vacuumblotting device (Bio-Rad) and vacuum of 60 cm H2O was applied for four hours using 20 ⫻ SSC (3 mol/L NaCl, 0.3 mol/L Na3 citrate, pH 7.0) as a blotting buffer, which resulted in the complete transfer of RNA. The RNA was blotted onto GeneScreen Plus nylon membranes (NEN/Dupont, Boston, MA, USA) and the RNA was immobilized by irradiation with ultraviolet (UV) light (UV crosslinker; Bio-Rad). Prehybridization (4 hours at 42⬚C) and hybridization (18 hours at 42⬚C) of the RNA blots were performed with a buffer (250 ␮L/cm2) consisting of 5 ⫻ SSPE (0.75 mol/L NaCl, 50 mmol/L NaH2PO4, 5 mmol/L EDTA, pH 7.40), 5 ⫻ Denhardt’s solution [0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% BSA (fraction V)], 0.1% SDS, 100 ␮g/mL denatured salmon sperm DNA, and 50% deionized formamide as previously described [24, 39, 40]. cDNA probes of NaPi-2 [41], GAPDH [42], and 18S [43], all full length, were labeled by random priming (Pharmacia) using [␣-32P] dCTP (NEN/DuPont). After hybridization, each blot was washed twice for 15 minutes in 2 ⫻ SSPE, 0.1% SDS at room temperature, twice for 15 minutes in 0.1 ⫻ SSPE, 0.1% SDS at 37⬚C, and twice for 15 minutes in 0.1 ⫻ SSPE, 0.1% SDS at 50⬚C. Autoradiography was performed at ⫺70⬚C with NEN/DuPont Reflection film using a DuPont intensifying screen (NEN/DuPont). Membranes were stripped (0.1 ⫻ SSC, 0.1% SDS at 95⬚C for 5 minutes) before another hybridization was performed. mRNA levels for NaPi-2 was quantitated by a Phosphor Imager (Bio-Rad) and the accompanying densitometry software and normalized to the density of the corresponding GAPDH or 18S bands. Isolation of poly Aⴙ RNA, injection into Xenopus laevis oocytes and uptake measurements Rat renal cortical poly A⫹ RNA (mRNA) was purified through an oligo dT column, and mRNA (0.2 ␮g/␮L) was

Table 1. Chronic potassiuim deficiency effects after seven days Control 24-hour urinary Pi excretion mg/24 h 21.6 ⫾ 2.8 BBM Na/Pi cotransport activity pmol/5 s/mg protein 646 ⫾ 51 BBM NaPi-2 protein abundance densitometry units 3.5 ⫾ 0.3 Renal cortical NaPi-2/18S mRNA abundance densitometry units 1.06 ⫾ 0.14 BBM NaPi-1 protein abundance densitometry units 3.6 ⫾ 0.1 Renal cortical Glvr-1 protein abundance densitometry units 5.7 ⫾ 0.4

K deficiency P value 32.8 ⫾ 1.3

⬍0.01

460 ⫾ 37

⬍0.02

10.3 ⫾ 1.2

⬍0.001

0.92 ⫾ 0.06

NS

9.8 ⫾ 2.1

⬍0.001

10.9 ⫾ 0.7

⬍0.001

BBM is brush-border membrane.

injected into Xenopus laevis oocytes as described previously [5]. Oocyte transport measurements were performed using K2H32PO4 (30 ␮Ci/mL) with 0.1 mmol/L K2HPO4, or K2 35SO42⫺ (30 ␮Ci/mL) with 0.1 mmol/L K2SO4, or L-[3H] leucine (20 ␮Ci/mL) with 0.1 mmol/L L-leucine, as described previously [5]. Immunofluorescence microscopy For these studies, six additional control and K-deficient rats were anesthetized with thiopental (Penthotal, 100 mg/kg body weight) and perfused retrogradely at a pressure of 1.38 hp through the abdominal aorta. The fixative consisted of 3% paraformaldehyde and 0.05% picric acid, in a 6:4 mixture of cacodylate buffer (pH 7.4, adjusted to 300 mOsm with sucrose) and 10% hydroxyethyl starch. After five minutes of fixation, the rats were perfused for an additional five minutes with the cacodylate buffer [40, 44, 45]. Coronal slices of fixed kidneys were snap frozen in liquid propane cooled by liquid nitrogen. Sections 3 ␮m thick were cut at ⫺22⬚C in the cryomicrotome, mounted on chromalum/gelatin-coated glass slides, thawed, and stored in cold PBS buffer until use. For NaPi-2 immunofluorescence staining, sections were preincubated for five minutes at room temperature with 3% milk powder in PBS containing 0.05% Triton X-100. They were then covered overnight at 4⬚C with the NaPi-2 antibody diluted 1:500 in the preincubation solution. The sections were rinsed three times with PBS prior to incubation for one hour at 4⬚C with the secondary antibody, swine anti-rabbit IgG conjugated to FITC (Dakopatts, Glostrup, Denmark). The actin microfilaments were stained directly with rhodamine-labeled phalloidin (Molecular Probes). After rinsing with PBS the sections were mounted using DAKO-Glycergel娃 (Dakopatts, Glostrup, Denmark) plus 2.5% 1,4-diazabicyclo- [2.2.2]-octane (DABCO; Sigma, St. Louis, MO, USA) as a fading retardant. They were then imaged with a laser scanning microscope (Zeiss LSM 410; Zeiss, Jena, Germany) by confocal fluorescence imaging.

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Fig. 1. Effect of K deficiency on kinetics of brush-border membrane Na/Pi cotransport activity. K deficiency caused a significant decrease in the Vmax of Na/Pi cotransport, while there were no changes in the Km for Pi or Na (data not shown). Symbols are: (䊉) control; (䊊) potassium deficiency (KD).

Data analysis The data are expressed as the mean ⫾ SE. The statistical significance of the results between samples obtained from control or K deficient rats was determined by the unpaired Student t test or one-way analysis of variance with the Student–Newman–Keuls analysis for multiple comparisons. Significance was accepted at the P less than 0.05. RESULTS Effects of chronic potassium deficiency on urinary Pi excretion and BBM Na/Pi cotransport activity Chronic K depletion of seven days’ duration caused a significant increase in the urinary excretion of Pi (Table 1). The increase in the urinary excretion of Pi was associated with a significant decrease in BBM Na/Pi cotransport activity (Table 1). As we have previously shown [4, 5] in parallel uptake measurements, there were no significant changes in BBM Na/glucose or Na/proline cotransport activities (results not shown). Effects of chronic potassium deficiency on BBM Na/Pi cotransport kinetics Kinetic studies varying the extravesicular Pi concentration revealed that the decrease in BBM Na/Pi cotransport activity in K depletion was caused by a significant decrease in the transport maximum (Vmax) of Na/Pi cotransport. There was no significant change in the affinity (Km) for Pi (Fig. 1). Similar studies varying the extravesicular Na concentration revealed no change in the Km for Na. Effects of chronic potassium deficiency on BBM NaPi-2, NaPi-1 and Glvr-1 protein abundance The next series of studies examined whether the decrease in BBM Na/Pi cotransport activity in K-deficient rats was mediated by a decrease in BBM type II Na/Pi

cotransport protein (NaPi-2) abundance. Western blotting of BBM proteins with a polyclonal antibody against the NaPi-2 protein actually showed a marked increase in BBM NaPi-2 protein abundance (Table 1). Although an equal amount of BBM protein was loaded, 10 ␮g BBM protein per lane, the blots for ␤-actin also were probed; there was no difference in the BBM ␤-actin abundance in control versus KD (results not shown). In contrast, when the BBM proteins were probed with an antibody against the sodium-sulfate cotransporter NaSi-1, the abundance of NaSi-1 protein decreased in parallel with the decrease in Na/Si cotransport activity in BBM isolated from K-deficient rats. In K-deficient rats an increase in proximal tubular apical membrane NaPi-2 protein expression was also seen by immunofluorescence microscopy (Fig. 2). The NaPi-2 protein colocalized with actin, which indeed confirms BBM localization and increased expression of NaPi-2 protein in K-deficient rats. Brush-border membrane proteins also were probed with a polyclonal antibody against type I Na/Pi cotransport protein (NaPi-1). Once again, the decrease in BBM Na/Pi cotransport activity in K-deficient rats was associated with a significant increase in BBM NaPi-1 protein abundance (Table 1), which was documented by immunohistochemistry as well (results not shown). Finally, the BBM proteins were probed with a polyclonal antibody against the type III Na/Pi cotransport protein (Glvr-1) [46]. In K-deficient rats, there was also a significant upregulation of the renal cortical Glvr-1 protein abundance (Table 1). Effects of chronic potassium deficiency on renal cortical NaPi-2 mRNA abundance and mRNA-induced oocyte Na/Pi cotransport activity We next determined whether the decrease in BBM Na/Pi cotransport activity in K-deficient rats was associ-

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Fig. 2. Effect of K deficiency (KD) on proximal tubular NaPi-2 protein expression, as determined by confocal immunofluorescence microscopy. The NaPi-2 protein is stained with FITC-labeled secondary antibody. In spite of a marked decrease in Na/Pi cotransport activity, KD is associated with an increase in proximal tubular apical BBM NaPi-2 protein expression.

ated with a decrease in renal cortical NaPi-2 mRNA abundance. Northern blotting of RNA from the renal cortex with full-length NaPi-2 cDNA probe revealed no change in NaPi-2 mRNA abundance in K-deficient rats when compared with control rats (Table 1). This was true even when the mRNA signal was normalized to 18 S. mRNA isolated from the renal cortex of K-deficient and

control rats was injected into Xenopus laevis oocytes, and the induced Na/Pi cotransport activity measured. No difference in the induced Na/Pi cotransport activity by mRNA from K-deficient versus control rats was found (Fig. 3), and there was no significant difference in Na/ leucine cotransport activity. However, as we have recently reported [5], there was a significant decrease in

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Fig. 3. Effect mRNA isolated from control and K-deficient rat renal cortex on oocyte Na/Pi cotransport activity. There was no difference in the induced Na/Pi cotransport activity, indicating that changes in mRNA levels or activity do not mediate the decrease in Na/Pi cotransport activity in K deficiency.

Na/Si cotransport activity that correlated with the decreases in BBM Na/Si cotransport, BBM NaSi-1 protein and renal cortical NaSi-1 mRNA abundance. Effects of chronic potassium deficiency on BBM lipid composition and lipid fluidity Since the decrease in BBM Na/Pi cotransport activity in K-deficient rats occurred independently of changes in BBM NaPi-2 protein abundance, we questioned whether there were alterations in lipid composition and lipid fluidity that could potentially modulate BBM Na/Pi cotransport activity in K-deficient rats. After determining that BBM lipid fluidity was significantly decreased in K-deficient rats, as shown by the increases in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan (Fig. 4), there were significant alterations in BBM phospholipid and glycosphingolipid composition. In K-deficient rats, there was a significant decrease in BBM phosphatidylcholine content and a significant increase in BBM sphingomyelin content, resulting in a significant increase in BBM sphingomyelin to phosphatidylcholine mole ratio (Fig. 5); additionally, in these rats there were significant increases in the renal cortical homogenate as well as BBM glucosylceramide and ganglioside GM3 content (Fig. 6). Other researchers as well as our group have shown previously that these lipid compositional changes are associated with changes in BBM lipid fluidity and modulation of BBM Na/Pi cotransport activity [24, 31, 32, 47]. Role of increased glucosylceramide content in modulating Na/Pi cotransport activity in potassium-deficient rats In dexamethasone-treated rats, we have previously shown that the increase in glucosylceramide content

plays an important role in the modulation of Na/Pi cotransport activity [24]. To determine whether the increase in glucosylceramide content also modulated Na/Pi cotransport in K-deficient rats, control and K-deficient rats were treated with the glucosylceramide synthase inhibitor PDMP. As previously demonstrated in dexamethasone-treated rats [24], PDMP caused significant decreases in renal cortical homogenate and BBM glucosylceramide content in control as well as in K-deficient rats (results not shown). Treatment with PDMP caused significant increases in BBM Na/Pi cotransport activity in both control rats and in K-deficient rats. In fact, the resultant BBM Na/Pi cotransport activity in K-deficient rats was identical to the Na/Pi cotransport activity in control rats (Table 2). The effect of PDMP to increase and in fact normalize BBM Na/Pi cotransport activity in K-deficient rats occurred independent of changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance (Table 2). DISCUSSION Hypokalemia and/or potassium deficiency have been reported to be associated with decreases in serum Pi concentration and increases in the urinary Pi excretion and Pi depletion [6–21]. Studies examining the cellular mechanisms in the regulation of renal phosphate transport in K deficiency, however, have been lacking. We used a model of dietary K deficiency in the rats that was previously shown to be associated with an increase in BBM Na/citrate [4] and a decrease in BBM Na/Si cotransport activities [5]. Our study shows that K deficiency results in a significant increase in urinary phosphate excretion and a parallel decrease in BBM Na/Pi cotransport activity; also, the decrease in BBM Na/Pi cotransport activity is mediated by a decrease in the Vmax of Na/Pi cotransport. In previous studies in adaptation to alterations in dietary Pi [40, 44], parathyroid hormone administration [45, 48], metabolic acidosis [49], aging [50], glucocorticoids [24], thyroid hormone [51], and epidermal growth factor [52], we have shown that the alterations in the Vmax of Na/Pi cotransport activity are associated with parallel changes in BBM type II Na/Pi cotransport protein (NaPi-2) abundance as determined by Western blotting and/or immunofluorescence microscopy. Interestingly, the current study shows that the decrease in the Vmax of BBM Na/Pi cotransport activity was actually associated with an increase in BBM NaPi-2 protein abundance. These effects were not due simply to differences in the quality or purity of the BBM preparation, as the increase in the abundance of the type II Na/Pi cotransport protein was determined both by Western blotting and immunofluorescence microscopy. Double staining for actin and NaPi-2 by immunofluorescence microscopy further documented that in K deficiency, the increased

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Fig. 4. Effect of K deficiency (KD) on BBM lipid fluidity as determined by the (A) fluorescence polarization (P) of 1,6-diphenyl-1, 3, 5-hexatriene (DPH) and (B) generalized polarization (GP) of Laurdan. Increases in PDPH and GPLaurdan indicate that K deficiency causes a decrease in lipid fluidity.

Fig. 5. Effect of K deficiency (KD) on BBM phospholipid composition. K deficiency causes an increase in sphingomyelin and a decrease in phosphatidylcholine mole content.

Fig. 6. Effect of K deficiency (KD) on BBM glycosphingolipid composition. K deficiency causes an increase in glucosylceramide and ganglioside GM3 content.

Table 2. Effects of DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) in control (Con) and potassium-deficient (KD) rats

BBM Na/Pi cotransport activity pmol/5 s/mg BBM protein BBM NaPi-2 protein abundance densitometry units Renal cortical NaPi-2/18S mRNA abundance densitometry units P ⬍ 0.05 compared to control P ⬍ 0.001 compared to control P ⬍ 0.05 compared to control d P ⬍ 0.01 compared to KD e P ⬍ 0.01 compared to control ⫹ PDMP a

b c

Con

KD

Con ⫹ PDMP

KD ⫹ PDMP

875 ⫾ 55 3.6 ⫾ 0.4 0.98 ⫾ 0.12

692 ⫾ 59a 10.5 ⫾ 1.2b 0.84 ⫾ 0.09

1120 ⫾ 54c 4.8 ⫾ 0.4c 0.99 ⫾ 0.08

1116 ⫾ 48d 10.4 ⫾ 0.8e 0.90 ⫾ 0.12

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abundance of NaPi-2 protein was localized to the apical membrane of the proximal tubule. Most interestingly, the decrease in Na/Pi cotransport activity and the increase in NaPi-2 protein abundance were associated with no change in renal cortical NaPi-2 mRNA abundance. Furthermore, when mRNA isolated from control and K-deficient rats were injected into oocytes, there were no differences in the resultant Na/Pi cotransport activity. These findings suggest that there is either a post-translational modification of the Na/Pi cotransport protein or that K deficiency may modulate Na/Pi cotransport activity through modulation of another Na/Pi cotransport protein. Recent studies using Npt2 knockout mice have demonstrated that 85% of proximal tubular Na/Pi cotransport activity is mediated by the type II Na/Pi cotransport protein [53]. Furthermore, mice deficient in the type II Na/Pi cotransport protein are not able to adapt to a low Pi diet and develop significant hypophosphatemia and phosphaturia, in spite of adaptive increases in the expression of NaPi-1 and Glvr-1 protein [54]. Indeed, the amounts of type I Na/Pi cotransport protein (NaPi-1), and type III Na/Pi cotransport protein (Glvr-1) were increased significantly. Therefore, these results suggest that in K-deficient rats the decrease in BBM Na/Pi cotransport activity most likely is mediated through posttranslational mechanisms that modify the activity of the type II Na/Pi cotransport proteins, the most abundant and physiologically most important Na/Pi cotransport protein in the proximal tubule. Potential post-translational mechanisms that we have identified include a decrease in BBM fluidity and increases in BBM sphingomyelin, glucosylceramide, and ganglioside GM3 content. In previous studies, these lipid alterations have been shown to cause alterations in lipid fluidity and lipid dynamics [55–57] and to modulate intestinal membrane Na/H exchange activity [58, 59]. In addition, we have shown that perturbations in lipid fluidity and/or lipid composition (cholesterol, sphingomyelin, and/or glucosylceramide) also modulate BBM Na/Pi cotransport activity [24, 37, 36, 47]. The mechanisms of how changes in lipid fluidity and/or lipid composition modulate Na/Pi cotransport activity are not known, but may include physical effects on the protein, including alterations in lateral or rotational mobility. In addition, we have recently provided evidence for the presence of lipid microdomains or lipid rafts in BBM that are modulated further by changes in membrane lipid composition [37]. The marked changes in BBM sphingomyelin and ganglioside lipid composition would be expected to further modulate the expression of lipid rafts and microdomains, which may in turn regulate the activity of the Na/Pi cotransport proteins [60, 61]. Furthermore, the possibility exists that also there may be some direct chemical modification of the protein by the lipids.

Fig. 7. Simplified outline of glycosphingolipid synthetic pathways. In K deficiency, both sphingomyelin and glucosylceramide levels are increased. PDMP is an inhibitor of glucosylceramide synthesis.

Although alterations in lipid fluidity and/or lipid composition do modulate the activity of the existing Na/Pi cotransporters, it is still not clear why there is actually an increase in the number of Na/Pi cotransporters. Interestingly, K deficiency has been shown to inhibit clathrinmediated endocytosis [62–66]. Although the mechanisms that mediate the endocytosis of the Na/Pi cotransport protein have not been fully elucidated, a recent immunogold microscopy study showed that, in response to parathyroid hormone-induced endocytosis of the type II Na/Pi protein, the Na/Pi protein was localized in clathrincoated vesicles [67]. It is therefore possible that K deficiency may cause inhibition of endocytosis of the type II Na/Pi cotransport protein, resulting in an increased BBM expression of this protein. Interestingly, in osteoblasts, acute depletion of cell K has been shown to induce an increase in Na/Pi cotransport activity [68]. However, at this time, the cellular mechanisms of how K deficiency causes an increase in the brush-border membrane localization of all the three types of Na/Pi cotransporter proteins remain unknown. In our model of chronic K deficiency, the increases in BBM sphingomyelin, glucosylceramide, and/or ganglioside GM3 content and the decrease in BBM fluidity ultimately impair the activity of the BBM Na/Pi cotransporters, resulting in a decrease in BBM Na/Pi cotransport activity and an increase in urinary phosphate excretion. Indeed, when K-deficient rats were treated with PDMP, an inhibitor of glucosylceramide synthase, there was a significant increase (in fact normalization of BBM Na/Pi cotransport activity) in the absence of further changes in BBM NaPi-2 protein abundance. The mechanisms of how K deficiency causes parallel increases in BBM sphingomyelin and glucosylceramide content are not known (Fig. 7 for a simplified outline of sphingomyelin and glucosylceramide synthesis), but may include hormonal mechanisms as well as alterations in cell metabolism that result in the enhanced synthesis of both sphingomyelin and glucosylceramide [69–71]. In fact,

Zajicek et al: Glycosphingolipids in K deficiency

in K deficiency adenosine 5⬘-triphosphate (ATP) citrate lyase activity is increased [72]. ATP citrate lyase plays an important role in the regulation of fatty acid synthesis [73, 74], which can then result in increased synthesis of both glucosylceramide and sphingomyelin (Fig. 7). In summary, our results indicate that K deficiency causes a decrease in BBM Na/Pi cotransport activity and an increase in urinary phosphate excretion. The inhibition of BBM Na/Pi cotransport activity, in spite of increases in BBM Na/Pi cotransport protein abundance, most likely occurs via novel post-translational mechanisms that include significant alterations in BBM lipid fluidity and BBM glycosphingolipid composition. ACKNOWLEDGMENTS This work was supported by grants from the Department of Veterans Affairs Merit Review (M.L. and J.S.), American Heart Association (M.L.), National Kidney Foundation (M.L.), Juvenile Diabetes Foundation (M.L.), NRSA fellowship grant from NIH (1 F32 DK09689-01 to H.Z.), and Department of Veterans Affairs Minority Grant Initiative (K.P.). The authors thank Ms. Teresa Autrey for secretarial assistance, Drs. Heini Murer and Jurg Biber (Zurich, Switzerland) for providing the NaPi-1 antibody and NaPi-2 cDNA probe, the Library Service at DVAMC for literature search, and the Medical Media Department at DVAMC for the illustrations. Reprint requests to Moshe Levi, M.D., V.A. Medical Center, 4500 South Lancaster Road, MC 151, Dallas, Texas 75216, USA. E-mail: [email protected]

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