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Kidney International, Vol. 59 (2001), pp. 10241036

Gentamicin causes endocytosis of Na/Pi

cotransporter protein (NaPi-2)

VICTOR SORRIBAS,1 NABIL HALAIHEL,1 KRISHNA PUTTAPARTHI, THOMAS ROGERS,

ROBERT E. CRONIN, ANAISABELALCALDE, JOSEARAMAYONA, MANUELSARASA,

HUAMIN WANG, PAULWILSON, HUBERT ZAJICEK, and MOSHELEVI

University of Zaragoza, Zaragoza, Spain, and the Dallas VA Medical Center and The University of Texas SouthwesternMedical Center at Dallas, Dallas, Texas, USA

Conclusion. We conclude that gentamicin inhibits Na/PiGentamicin causes endocytosis of Na/Pi cotransporter proteincotransport activity by causing a decrease in the expression of(NaPi-2).

the type II Na/Pi cotransport protein at the level of the proxi-Background. Renal toxicity is a major side-effect of amino-mal tubular apical BBM and that inhibition of Na/Pi cotrans-glycoside antibiotics and is characterized by an early impair-port activity is most likely mediated by post-transcriptionalment in proximal tubular function. In a previous study, wemechanisms.have shown that gentamicin administration to the rat causes

an early impairment in sodium gradient-dependent phosphate(Na/Pi) cotransport activity. The purpose of our current studywas to determine the molecular mechanisms of the impairment Proximal tubular dysfunction is a central feature ofin Na/Pi cotransport activity, specifically the role of the proxi-

aminoglycoside nephrotoxicity. In humans and in exper-mal tubular type II Na/Pi cotransporter.imental animals treated with aminoglycoside antibiot-Methods. Rats were treated for one, two, and three days

with two daily injections of 30 mg/kg body weight gentamicin ics, the increase in the urinary excretion of glucose, aminoor the vehicle. acids, and luminal brush-border membrane (BBM)-bound

Results. Gentamicin caused a progressive decrease in super-enzymes occurs before a reduction in glomerular filtrationficial cortical apical brush-border membrane (SC-BBM) Na/Pirate (GFR) [14]. We have previously shown that treat-cotransporter activity (856 93 in control vs. 545 87 pmol/mgment of rats with gentamicin results in an early decreaseBBM protein in 3-day gentamicin, P 0.01). Western blot

analysis showed a parallel and progressive decrease in SC- in proximal tubular luminal BBM sodium gradient-BBM Na/Pi cotransporter protein abundance, a 50% decrease dependent phosphate transport (Na/Pi cotransport) andafter one day of treatment, a 63% decrease after two days of

pH gradient-dependent sodium transport (Na/H ex-treatment, andan 83%decreaseafter three days treatmentwithchange) activities, prior to a reduction in GFR [5].gentamicin. In contrast, gentamicin treatment had no effect

on Na/Pi cotransport activity or Na/Pi cotransporter protein The purpose of the present study was to characterizeabundance in BBM isolated from the juxtamedullary cortex the cellular and molecular mechanisms involved in the(JMC-BBM). Immunofluorescence microscopy showed a ma- gentamicin-induced decrease in renal proximal tubular

jor decrease in the expression of Na/Pi cotransporter proteinphosphate transport and specifically to determine a rolein the apical membrane of the proximal convoluted tubule,for the type II Na/Pi cotransport (NaPi-2) protein andwith progressive intracellular accumulation of Na/Pi protein.

Colocalization studies showed that in gentamicin-treated rats, mRNA in this process.Na/Pi protein was colocalized in the early endosomes and espe- Our results indicate that treatment of rats with genta-

cially in the lysosomes. Northern blot analysis of cortical RNA micin causes a progressive decrease in superficial corticalinterestingly showed no reduction in Na/Pi cotransporter mRNA(SC-BBM) but not juxtamedullary cortical (JMC-BBM)abundance even after three days of gentamicin treatment.Na/Pi cotransport activity, which is associated with aprogressive decrease in SC-BBM NaPi-2 protein abun-

1 Drs. Sorribas and Halaihel contributed equally to this study. dance but not NaPi-2 mRNA abundance. Furthermore,gentamicin causes a progressive translocation of NaPi-2

Key words:early endosomes, lysosomes, axial hetergeneity, phospha-protein from the apical BBM to the late endosomal/tidylinositol, renal toxicity, type II Na/Pi cotransport activity, nephro-

toxicity. lysosomal compartment. Our results therefore suggestthat gentamicin causes a progressive decrease in Na/Pi

Received for publication June 9, 2000cotransport activity by post-transcriptional mechanisms,and in revised form September 22, 2000

Accepted for publication September 28, 2000 most likely involving the progressive endocytosis ofNaPi-2 protein from the apical BBM.2001 by the International Society of Nephrology

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Sorribas et al: Gentamicin and NaPi-2 1025

METHODS Enzyme activity measurements

The activities of maltase (a marker of proximal convo-Study groupsluted tubule BBM) and -glutamyl transferase (a markerStudies were performed on male Sprague-Dawley ratsof proximal straight tubule BBM) were measured as pre-(Charles River Laboratories, Kingston, NY, USA). Theviously described [5, 6] in SC and JMC homogenatesrats were virus antibody titer-free and weighed betweenand SC and JMC BBM to determine the purity of BBM225 and 250 g. Experiments were started after animalsisolated from control and gentamicin-treated rats.were adapted on a control diet (0.6% Ca, 0.6% Pi, Teklad)

for seven days. The animals then received gentamicinBrush-border membrane transport

sulfate, 30 mg/kg body weight, administered subcutane-activity measurements

ously twice a day, or vehicle (0.9% NaCl) for a one-,Brush-border membrane Na/Pi cotransport activitytwo-, or three-day period. At the end of the treatment

measurements were performed in freshly isolated BBMperiod, eight rats in each experimental group were anes-vesicles by theradiotracer uptake of 100mol/LK2H

32PO4thetized with intraperitoneal pentobarbital. The kidneys(Dupont-NEN Research Products, Boston, MA, USA)

were then rapidly removed; one half of one kidney wasand an inwardly directed sodium gradient (150 mmol/L

used for RNA isolation, and the other three halves wereNaCl) followed by rapid filtration [8]. Uptake was ter-

used for BBM isolation. In additional experiments, fourminated after 10 seconds, representing the initial linear

rats in each experimental group were anesthetized andrate. Na/glucose and Na/proline cotransport measure-

perfused in vivo for immunofluorescence microscopy for ments were also performed in a similar manner by radio-NaPi-2 protein, as described later in this article.tracer uptake of [3H]-d-glucose or [3H]-l-proline (Du-pont-NEN), respectively.Isolation of cortical homogenate and

brush-border membranesBrush border membrane SDS-gel protein

Thin slices from the rat kidney SC and JMC were cut electrophoresis and Western blot analysisat 4C and homogenized with a Polytron homogenizer Brush border membranes were denatured for two min-in a buffer consisting of 300 mmol/L mannitol, 5 mmol/L utes at 95C in 2% sodium dodecyl sulfate (SDS), 10%ethylene glycol-bis (-aminoethyl ether) -N, N, N N- glycerol, 0.5 mmol/L ethylenediaminetetraacetic acidtetraacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, (EDTA), 95 mmol/L Tris-HCl, pH 6.8 (final concentra-16 mmol/L N-2-hydroxyethylpiperazine-N -2 ethane- tions). Ten micrograms of BBM protein/lane were sepa-sulfonic acid (HEPES), and 10 mmol/L Tris (hydroxy- rated on 9% polyacrylamide gels according to the

methyl) aminomethane (Tris), pH 7.50. Aliquots were method of Laemmli [9] and then electrotransferred ontosaved for (1) enzyme activity measurement, (2) Western nitrocellulose paper [10]. After blockage with 5% nonfatblotting, and (3) measurement of gentamicin content. milk powder with 1% Triton X-100 in Tris-buffered sa-From the resulting SC and JMC homogenates, apical line (20 mmol/L, pH 7.3), Western blots were performedBBM vesicles were prepared by differential centrifuga- with antiserum against the C-terminal amino acid se-tion after Mg2 aggregation as previously described [6]. quence of NaPi-2 at a dilution of 1:5000. Primary anti-Briefly, to each 15 mL of homogenate in isolation buffer, body binding was visualized using enhanced chemilumi-0.54 mL of 1.0 mol/L MgCl2 in 21 mL of water was nescence (Pierce, Bradford, IL, USA), and the signalsadded. After 20 minutes of shaking, the homogenate were quantitated in a Phosphor Imager with chemilumi-was centrifuged at 2790 g for 15 minutes. The pellet nescence detector and densitometry software (Bio-Rad,resulting from the first Mg precipitation step was saved Richmond, CA, USA). The protein blots were alsoand labeled as non-BBM membranes, and an aliquot probed with antiserum against -actin (Sigma).

was saved for Western blotting. The supernatant wasMeasurement of brush-border membranesubjected to another round of Mg precipitation, and thelipid compositionresultant supernatant was centrifuged at 40,000 gfor

30 minutes. The resulting pellet corresponding to BBM Lipids from BBMs were extracted by the method ofwas resuspended in a buffer of 300 mmol/L mannitol, Bligh and Dyer [11]. To determine individual phospho-16 mmol/L HEPES, and 10 mmol/L Tris, pH 7.50, and lipid polar head group species, an aliquot of the lipidwas aliquoted for simultaneous measurements of (1) en- extract was applied to thin-layer chromatography plateszyme activity, (2) transport activity, (3) protein electro- (Silica Gel 60; E. Merck, Darmstadt, Germany). Individ-phoresis and Western blotting, (4) lipid composition, ual phospholipids, including sphingomyelin, phosphatidyl-and (5) gentamicin binding. Protein concentrations of the choline, phosphatidylethanolamine, phosphatidylserine,cortical homogenate, non-BBM, and BBM fractions were and phosphatidylinositol, were separated by two-dimen-

sional thin layer chromatography (TLC) [12]. Phospho-determined by the method of Lowry et al [7].

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Sorribas et al: Gentamicin and NaPi-21026

Table 1. Effects of three days of gentamicin administration on BBMenzyme activity

Control Gentamicin Pvalue

Superficial cortexMaltase

CH 16.50.7 16.91.1 NSBBM 183.819.0 177.26.1 NS

Enrichment 11.10.9 10.50.4 NSRecovery 32.02.6 30.20.4 NS

-Glutamyl transferaseCH 120.15.7 123.66.1 NSBBM 1627.178.1 1705.783.4 NSEnrichment 13.61.0 13.81.2 NSRecovery 39.22.9 39.83.4 NS

Justamedullary cortexMaltase

CH 9.90.6 10.10.8 NSBBM 107.910.4 112.112.1 NSEnrichment 10.90.7 11.10.8 NSRecovery 31.42.3 32.02.9 NS

-Glutamyl transferaseCH 258.711.8 255.912.6 NSBBM 3621.8173.8 3531.4184.9 NS

Enrichment 14.01.2 13.81.8 NSRecovery 40.33.4 39.83.1 NS

Values are means SE; N 12 rats in each group. Abbreviations are:CH, cortical homogenate; BBM, brush-border membrane. Enzyme activity isexpressedas micromolesper hourper milligramCH or BBMprotein.Enrichmentis defined as the ratio of specific activity in BBM fraction/specific activity inhomogenate. Recovery (%) is defined as the ratio of total activity in BBMfraction/total activity in homogenate.

capacity) and Kd (binding affinity) were calculated byFig. 1. Effect of gentamicin on sodium gradient-dependent phosphate Scatchard plot.(Na/Pi) cotransport activity in (A) superficial cortical brush-bordermembranes (SC-BBM) and (B) juxtamedullary cortical brush-border

RNA isolation, formaldehyde gel electrophoresis, andmembranes (JMC-BBM). NA/Pi cotransport activity was determinedby sodium gradient-dependent uptake of32Pi into BBM vesicles. Genta- Northern blot analysismicin caused a progressive decrease in SC-BBM but not JMC-BBM

Thin slices from kidney superficial cortex were cut onNa/Pi transport activity. The results are presented as mean SEM. N6 in each group. an ice-cold glass dish and homogenized with a Polytron

homogenizer in a denaturazation solution containing 4mol/L guanidium thiocyanate, 25 mmol/L sodium citrate,pH 7.0, 0.5% sarcosyl, and 0.1 mol/L of 2-mercaptoetha-

lipid content of total and individual phospholipids was nol. Sequentially, 0.1 volumes of 2 mol/L sodium acetate,determined by measuring phosphorus content by the pH 4.0, 1 volume water-saturated phenol, and 0.2 vol-method of Ames and Dubin [13]. umes chloroform-isoamyl alcohol mixture (49:1) were

added to the homogenate. Total RNA was isolated asCortical homogenate gentamicin content measurement

previously described [8, 15, 16], and 10g of each sampleGentamicin content of SC and JMC homogenate sam- were size fractionated by agarose-formaldehyde gel

ples obtained from gentamicin-treated rats was mea- electrophoresis and transferred to nylon membranes bysured by radioimmunoassay (Rainen gentamicin RIA a vacuum-blotting device (Bio-Rad). Prehybridizationkit; DuPont, Billerica, MA, USA) [14]. (4 hours at 42C) and hybridization (18 h at 42C) of the

RNA blots were performed with a buffer consisting ofBrush-border membrane 5 SSPE (0.75 mol/L NaCl, 50 mmol/L NaH2PO4,gentamicin-binding measurements 5 mmol/L EDTA, pH 7.40), 5 Denhardts solution

Binding of [3H]-gentamicin to SC-BBM and JMC- [0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, and 0.1%BBM isolated from control (that is, nongentamicin bovine serum (albumin fraction V)], 0.1% SDS, 100treated) rats was performed at 4C. Specific binding was g/mL denatured salmon sperm DNA, and 50% deion-calculated as the difference between total gentamicin ized formamide [8, 16, 17]. A full-length cDNA probebinding and nonspecific binding in the presence of 100- of NaPi-2 [18] was labeled by random hexamer priming

(Pharmacia) using [8-32P] dCTP (Dupont-NEN). Blotsfold excess nonradioactive gentamicin. Bmax (binding

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Fig. 2. Effect of gentamicin on NaPi-2 protein abundance, as determined by Western blotting in ( A) SC-BBM, (B) SC homogenate, (C) SC other,non-BBMs, and (D) JMC-BBM.Gentamicin caused a progressive decrease in SC-BBM NaPi-2 protein abundance, no change in SC-homogenate

(that is, total) NaPi-2 protein abundance, and a reciprocal increase in SC other membrane (that is, non-BBM) NaPi-2 protein abundance. Incontrast, gentamicin had no effect on JMC-BBM NaPi-2 protein abundance. The results are presented as mean SEM. N 6 in each group.

were washed twice for 15 minutes in 0.1 SSPE with0.1% SDS at 37C and twice for 15 minutes each timein 0.1 SSPE with 0.1% SDS at 50C. Hybridizationsignals were quantitated by a phosphor imager analyzingsystem (BioRad, Richmond, CA, USA) and normalizedfor loading using full-length rat glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) [19] and 18S rRNAoligonucleotide probes synthesized as described [20].

Immunofluorescence microscopy

Rats were anesthetized with thiopental (Penthotal, 100mg/kg body weight) and perfused retrograde at a pres-sure of 1.38 hp through the abdominal aorta. The fixativeconsisted of 3% paraformaldehyde and 0.05% picric acidin a 6:4 mixture of cacodylate buffer (pH 7.4, adjusted to300 mosmol with sucrose) and 10% hydroxyethyl starch.After five minutes of fixation, the rats were perfused for

Fig. 3. Effect of gentamicin on NaPi-2 mRNA abundance, as deter-an additional five minutes with the cacodylate buffer [21].mined by Northern blotting, in SC. Gentamicin caused no changes in

Coronal slices of fixed kidneys were snap frozen in NAPi-2 mRNA abundance. The results are presented as mean SEM.N 6 in each group.liquid propane cooled by liquid nitrogen. Sections 3 m

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Fig. 4. Effect of gentamicin on proximal tubular expression of NaPi-2 (fluorescein, green), as determined by immunofluorescence microscopy.Gentamicin caused a progressive decrease in proximal tubular apical brush-border membrane NaPi-2 protein abundance, while causing a progressiveincrease in intracellular NaPi-2 accumulation. The results are representative of four different rats studied in each group.

thick were cut at 22C in the cryomicrotome, mounted NaPi-2 antibody [22] diluted 1:500 in the preincubationsolution. The sections were rinsed three times with PBSon chromalum/gelatin-coated glass slides, thawed, and

stored in cold phosphate-buffered saline (PBS) buffer prior to incubation for one hour at 4C with the second-ary antibody, swine anti-rabbit IgG conjugated to FITCuntil use.

For NaPi-2 immunofluorescence staining, sections (Dakopatts, Glostrup, Denmark).To perform NaPi-2/early endosome (EEA-1) andwere preincubated for five minutes at room temperature

with 3% milk powder in PBS containing 0.05% Triton NaPi-2/lysosome (lgp-120) double immunofluorescencestaining, the sections were covered overnight with theX-100. They were then covered overnight at 4C with the

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Fig. 5. Effect of gentamicin on proximal tubular expression of (a) NaPi-2 and (b) EEA-1, an early endosome marker protein, as determined byimmunofluorescence microscopy.Following treatment with gentamicin, apical membrane NaPi-2 expression is decreased, while there is increasedcolocalization with EEA-1.

antibody against NaPi-2 protein at 1:500 dilution, and tests was used to compare results between control andeither a mouse monoclonal antibody against early endo- gentamicin-treated rats. Significance was accepted atsomal antigen (EEA-1, Transduction Labs, Lexington, P 0.05.

KY, USA) at 1:500 dilution [23], or a mouse anti-ratantibody against the lysosomal membrane glycoproteinRESULTSlgp-120 at 1:200 dilution [24], in PBS/milk powder.

After rinsing with PBS, the sections were mounted us- Gentamicin causes a progressive decrease ining Dako-Glycergel (Dakopatts) plus 2.5% 1,4-diaza- superficial cortical brush-border membranebicyclo-[2.2.2] octane (DABCO; Sigma, St. Louis, MO, Na/Pi cotransport activityUSA) as a fading retardant. They were then imaged

Compared with vehicle-treated control rats, gentami-with a laser scanning microscope (Zeiss LSM 410, Jana,cin treatment for one, two, and three days caused a sig-Germany) by confocal fluorescence imaging.nificant and progressive decrease in Na/Pi cotransport

Statistical analysis activity in BBM vesicles isolated from the superficialcortex (SC-BBM; Fig. 1A). In contrast, as we have pre-Data are expressed as mean SE. A one-way analysis

of variance with Student-Newman-Keuls multiple range viously shown [5], gentamicin treatment had no effect on

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Fig. 6. Effect of gentamicin on proximal tubular expression of (a) NaPi-2 and (b) lgp-120, a lysosomal marker protein, as determined byimmunofluorescence microscopy.Following treatment with gentamicin, apical membrane NaPi-2 expression is decreased, while there is no changein the expression of lgp-120.

SC-BBM Na/glucose or Na/proline cotransport activities BBM NaPi-2 protein abundance (Fig. 2A). Interestingly,(results not shown). The differences in BBM Na/Pi co- gentamicin had no significant effect on superficial corti-transport activity were not due to differences in the pu- cal homogenate (total) NaPi-2 protein abundance (Fig.

rity of the BBM isolated from control versus gentamicin- 2B), while gentamicin caused a significant and progres-treated rats, as the enrichment for the BBM enzyme sive increase in superficial cortical non-BBM NaPi-2 pro-markers maltase and -glutamyl transferase were identi-

tein abundance (Fig. 2C). This suggests that gentamicincal in the two groups (Table 1).

causes translocation of NaPi-2 protein from the apical

BBM to inside the cell and contained in an intracellular

compartment in which they are not degraded, as thereGentamicin causes a progressive decrease inwas no change in total NaPi-2 protein abundance, evensuperficial cortical brush-border membrane

NaPi-2 protein abundance after three days of treatment with gentamicin. In contrast

to the effect on SC-BBM NaPi-2 protein abundance,The effect of gentamicin on SC-BBM Na/Pi cotrans-gentamicin had no effect on -actin protein abundanceport activity was paralleled by a similar effect of gentami-

cin to cause a progressive decrease in superficial cortical (results not shown).

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Fig. 7. (A) Effect of gentamicin on proximal tubular expression of NaPi-2 (fluorescein, green) and EEA-1 (rhodamine, red), an early endosomemarker protein, as determined by immunofluorescence microscopy. Following treatment with gentamicin, apical membrane NaPi-2 expression isdecreased, whilethere is increasedco-localization with EEA-1 (yellow color), suggesting accumulation of NaPi-2 in an early endosomal compartment.(B) Effect of gentamicin on proximal tubular expression of NaPi-1 (fluorescein, green) and lgp-120 (rhodamine, red), a lysosomal marker protein,as determined by immunofluorescence microscopy. Following treatment with gentamicin, apical membrane NaPi-2 is decreased, while there isincreased co-localization with lgh-120 (yellow color), suggesting accumulation of NaPi-2 in lysosomes.

Gentamicin does not cause a decrease in superficial caused a progressive decrease in NaPi-2 specific immu-cortical NaPi-2 mRNA abundance nostaining in apical luminal membranes of the proximal

convoluted tubule (Fig. 4). Thedecrease in luminal mem-In spite of the effect of gentamicin to cause a progres-

brane immunostaining was associated with a progressivesive decrease in superficial cortical BBM Na/Pi cotrans-increase in intracellular staining (Fig. 4).port activity and BBM NaPi-2 protein abundance, genta-

Additional immunofluorescence studies were per-micin had no effect on superficial cortical NaPi-2 mRNAformed to determine the intracellular compartment inabundance (Fig. 3). This suggests that the effect of genta-which the NaPi-2 immunostaining was localized, includ-micin to inhibit Na/Pi cotransport activity is mediateding the early endosomal compartment (Fig. 5) and lyso-by post-transcriptional mechanisms.somes (Fig. 6). The co-localization studies using immu-

Gentamicin causes enhanced internalization of nofluorescence microscopy revealed that gentamicinNa/Pi cotransport protein to endosomal and initially caused translocation of NaPi-2 protein from thelysosomal compartments apical BBM to the early endosomal compartment, as

evidenced by significant co-localization (yellow; Fig. 7A)Similar to the Western blot results (Fig. 2A), immuno-fluorescence microscopy also revealed that gentamicin of NaPi-2 immunostaining (green) with early endosomal

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in JMC-BBM PI content (Fig. 10A). The increase in PIcontent would result in further enhancement of gentami-cin binding to SC-BBM and result in increased gentamicinaccumulation in the superficial cortex (Fig. 8).

In agreement with our earlier results [5], gentamicinalso caused a significant decrease in SC-BBM sphingo-

myelin, an increase in phosphatidylcholine, and a de-crease in sphingomyelin to phosphatidylcholine mole ra-tio (Fig. 10B). In contrast, gentamicin had no significanteffects in sphingomyelin and phosphatidylcholine con-tent in JMC-BBM (Fig. 10B).

DISCUSSION

We have previously shown that treatment of rats withgentamicin causes an early decrease in proximal tubularapical BBM Na/Pi cotransport activity, prior to a reductionin GFR [5]. In the present study, we have determined thatFig. 8. Effect of gentamicin treatment on gentamicin levels in SC ()

and JMC (

) homogenates, as determined by radioimmunoassay. the decrease in BBM Na/Pi cotransport activity is medi-Treatment with gentamicin causes significantly higher gentamicin levels ated by a decrease in BBM NaPi-2 protein expression.in SC homogenate compared with JMC homogenate. The results are

One interesting and intriguing finding of our study isexpressed as mean SEM.N 6 in each group.that the decrease in BBM NaPi-2protein occurs indepen-dent of a decrease in NaPi-2 mRNA. In previous studies,we have shown that acute regulation of NaPi-2 by alter-ations in dietary phosphate [8, 21], parathyroid hormone

antigen EEA-1 immunostaining (red). There was also[25, 26], and metabolic acidosis [22] can indeed occur

evidence of progressively strong co-localization (yellow;prior to changes in NaPi-2 mRNA. However, usually

Fig. 7B) of NaPi-2 immunostaining (green) with lyso-after six hours of the experimental perturbation, there

somal immunostaining (red).is a parallel change in NaPi-2 mRNA as well. Interest-ingly, in this study, even after three days of gentamicin

Gentamicin has no effect on juxtamedullary cortical

administration, there is no change in NaPi-2 mRNA lev-brush-border membrane Na/Pi cotransport activityels. This suggests that gentamicin modulates NaPi-2 pro-

In contrast to the inhibitory effect of gentamicin on tein expression by post-transcriptional mechanisms thatSC-BBM Na/Pi cotransport activity, gentamicin was are independent of changes in mRNA.without an inhibitory effect on JMC-BBM Na/Pi cotrans- Another interesting and intriguing finding of our studyport activity (Fig. 1B). In agreement with the lack of is that although gentamicin causes a progressive decreaseinhibitory effect of gentamicin on JMC-BBM Na/Pi co- in the expression of apical membrane NaPi-2 protein,transport activity, gentamicin had no effect on JMC- the NaPi-2 protein accumulates in an intracellular com-BBM NaPi-2 protein abundance (Fig. 2D). partment and is not degraded. In fact, even after three

To determine the potential mechanisms for the differ- days of treatment with gentamicin, the level of total kid-ential effect of gentamicin on SC and JMC-BBM Na/Pi ney (that is, cortical homogenate) NaPi-2 protein is nottransport activity, we measured gentamicin levels in SC altered. Therefore, following gentamicin administration

and JMC homogenates. Gentamicin levels were much NaPi-2 is endocytosed and accumulates in an intracellu-higher in SC homogenate compared with JMC homoge- lar compartment, most likely the lysosomes. Followingnate (Fig. 8). BBM gentamicin-binding studies indicated treatment with a high Pi diet [8, 21] and/or parathyroidthat this was associated with increased binding of genta- hormone (PTH) [25, 26], NaPi-2 is also endocytosed frommicin to SC-BBM compared with JMC-BBM (Fig. 9). the apical BBM and is eventually accumulated in theFinally, since gentamicin binding to BBM is enhanced lysosomes where it is degraded. In thecase of gentamicin,by negatively charged phospholipids, including phospha- however, gentamicin is rapidly targeted to the lysosomestidylinositol (PI), we measured PI content in SC- and [14, 2731] and is an inhibitor of lysosomal enzymesJMC-BBM. We found that PI levels are markedly higher [3238], and therefore, the NaPi-2 protein is accumulatedin SC-BBM (Fig. 10). In addition, following treatment in the lysosomes without degradation. A similar picturewith gentamicin, there is a further significant increase in is seen when rats are first pretreated with leupeptin, an

inhibitor of lysosomal enzymes, and then the rats areSC-BBMPI content, whilethere is no significant alteration

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Fig. 9. Gentamicin binding to SC-BBM andJMC-BBM in BBMisolatedfrom control, thatis, nongentamicin treated rats, as determinedby Scatchard plot. Symbols are: () control;() gentamicin. Bmax(binding capacity) is sig-nificantly higher in SC-BBM compared withJMC-BBM. The results are expressed asmean SEM.N 6 in each group.

fed a high Pi diet and/or infused with PTH, in which the creases in membrane fluidity would enhance the activityinternalized NaPi-2 protein is localized in the lysosomes of the Na/Pi cotransporter proteins [16, 43, 44], the in-[21, 26, 39]. crease in SC-BBM lipid fluidity may in part explain the

Gentamicininterestingly has differential effectson Na/Pi leveling of the decrease in SC-BBM Na/Pi cotransportcotransport activity and NaPi-2 protein abundance in activity in spite of a further progressive decrease in SC-SC-BBM versus JMC-BBM. Gentamicin causes a pro- BBM NaPi-2 protein abundance.

gressive decrease in SC-BBM Na/Pi cotransport activity Megalin or glycoprotein 330 [45] plays an importantand NaPi-2 protein abundance, whereas it has no effect role in binding and internalizing gentamicin in the renalon JMC-BBM Na/Pi cotransport activity or NaPi-2 pro- proximal tubule [46, 47]. In addition, megalin has alsotein abundance. The differential effects of gentamicin been shown to play an important role in the gentamicin-are associated with differential accumulation of gentami- induced inhibition of rat renal homotypic endosomalcinin SC versus JMC homogenate. Wepropose that differ- fusion [48]. Megalin has also been shown to mediate theential gentamicin accumulation in SC versus JMC is most

endocytosis of albumin in the proximal tubule [49] andlikely mediated by an increased capacity of SC-BBM to

to be associated with the Na/H exchanger isoform NHE3bind gentamicin, which is mediated by increased PI levels

in the proximal tubule [50]. Therefore, megalin may playin SC-BBM, which is further increased after treatment

a role in gentamicin-induced internalization of NaPi-2.with gentamicin. Previous studies have indeed shown

However, the studies by Biemesderfer et al [50] andthat gentamicin preferentially binds to anionic phospho-

preliminary studies in our laboratory have not shown anlipids, including PI [4042], and the higher PI levels in association between megalin and NaPi-2 in the proximalSC-BBM are compatible with increased gentamicin bind-

tubule. Thus, a role for megalin in mediating the effectsing to this segment of the proximal tubule. In additionof gentamicin on NaPi-2 trafficking remains unknown.to Na/Pi cotransport activity, differential effects of genta-

In summary, our study indicates that gentamicin causesmicin on SC versus JMC are also demonstrated by the facta progressive decrease in SC-BBM Na/Pi cotransport ac-that gentamicin causes a decrease in sphingomyelin andtivity and NaPi-2 protein abundance by post-translationalan increase in phosphatidylcholine in SC-BBM but not inmechanisms. The effect of gentamicin to preferentiallyJMC-BBM. The effects of gentamicin on SC-BBM lipidinhibit SC-BBM rather than JMC-BBM is associated withcomposition are in agreement with our earlier study [5].increased gentamicin binding to SC-BBM and accumula-Interestingly, the gentamicin-induced decrease in sphin-tion in SC-homogenate, due to increased levels of thegomyelin to phosphatidylcholine mole ratio is associated

with an increase in SC-BBM lipid fluidity [5]. Since in- negatively charged phosphatidylinositol in SC-BBM.

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Fig. 10. Lipid composition of SC-BBM andJMC-BBM in vehicle () or gentamicintreated() rats. (A) Phosphatidylinositol (PI)levels are higher in SC-BBM compared withJMC-BBM. Furthermore, treatmentwith gen-tamicin causes a further increase in SC-BBMPI content. (B) Gentamicin causes a decreasein sphingomyelin (SM) andan increasein pho-sphatidylcholine (PC) levels in SC-BBM butnot in JMC-BBM. The results are expressedas mean SEM.N 6 in each group.

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of human and rat renal cortex Na/Pi cotransport. Proc Natl AcadACKNOWLEDGMENTSSci USA 90:59795983, 1993

19. Fort PL, Marty L, Piechaezyk M, et al: Various rat adult tissuesThis work was supported by grants from the Department of Veter-express only one major RNA species from the glyceraldehyde-2-ans Affairs Merit Review and National Kidney Foundation (Moshephosphate-dehydrogenase multigenic family.Nucleic Acids Res 13:Levi), Diputacion General De Aragon, Spain (Victor Sorribas PO78/14311442, 198599), an NRSA fellowship grant from National Institutes of Health

20. Moe OW, Miller RT, Horie S, et al: Differential regulation of(Hubert Zajicek, 1 F32 DK09689-01), and Department of VeteransNa/H antiporter by acid in renal epithelial cells and fibroblasts.Affairs Minority Initiative Grant (Krishna Puttaparthi). The authors

J Clin Invest88:17031708, 1991thank Drs. Heini Murer and Jurg Biber (Zurich) for providing the 21. Lotscher M, Kaissling B, Biber J, et al: Role of microtubules inNaPi-2 c-DNA probe, Teresa Autrey for secretarial assistance, andthe rapid regulation of renal phosphate transport in response tothe Medical Media Department at DVAMC for the illustrations.acute alterations in dietary phosphate content. J Clin Invest99:13021312, 1997Reprintrequests to Moshe Levi, M.D., TheUniversity of Texas South-

22. Ambuhl PM, Zajicek HK, Huamin W, et al: Regulation of renalwestern Medical Center at Dallas, Nephrology Section, VA North Texasphosphate transport by acute and chronic metabolic acidosis inHealth Care System, 4500South Lancaster Road, MC 151, Dallas, Texasthe rat. Kidney Int53:12881298, 199875216, USA.

23. Gruenberg J, Griffiths G, Howell KE:Characterization of theE-mail: [email protected] endosome and putative endocytic carrier vesiclesin vivoandwith an assay of vesicle fusion in vitro. J Cell Biol108:13011316,

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