Effects of pathophysiological concentrations of albumin on NHE3 activity and cel - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：E. M. Lee, C. A. Pollock, K. Drumm, J. A. Barden,  P. Poronnik,作者单位：1 Department of Medicine, University of Sydney,Renal Research Group, Kolling Institute of Medical Research, Royal North ShoreHospital, St. Leonards, New South Wales 2065; Schoolof Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072; Department of Anatomy and Histology, Universi
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      【摘要】& g0 Y7 k- I1 `. U" n  f: y
   The progression of renal disease correlates strongly with hypertension andthe degree of proteinuria, suggesting a link between excessive Na reabsorption and exposure of the proximal tubule to protein. The present studyinvestigated the effects of albumin on cell growth and Na uptakein primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml)increased cell proliferation to 134.1 ± 11.8% ( P no change in levels of apoptosis. Exposure to 0.1 and1.0 mg/ml albumin increased total 22 Na uptake to 119.1± 6.3% ( P = 0.005) and 115.6 ± 5.3% ( P levels, respectively, because of an increase inNa /H exchanger isoform 3 (NHE3) activity. This wasassociated with an increase in NHE3 mRNA to 161.1 ± 15.1% ( P levels in response to 0.1 mg/ml albumin. Using confocalmicroscopy with a novel antibody raised against the predicted extracellular NH 2 terminus of human NHE3, we observed in nonpermeabilized cellsthat exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cellsurface to 115.4 ± 2.7% ( P and 122.4 ±3.7% ( P respectively. This effect wasparalleled by significant increases in NHE3 in the subplasmalemmal region asmeasured in permeabilized cells. These albumin-induced increases in expressionand activity of NHE3 in PTC suggest a possible mechanism for Na retention in response to proteinuria. % N8 t4 p9 \  x- m% q
      【关键词】 proteinuria sodiumhydrogen exchange sodium retention
   PROTEINURIA AND Na -dependent hypertension are wellknown to occur in the setting of renal disease. It has been considered thatproteinuria is primarily due to glomerular pathology and that excessivetubular Na reabsorption is a "normal" response toreduced plasma volume. However, in conditions such as diabetes mellitus( 34 ), microalbuminuria,hypertension ( 8 ), and increased proximal tubular Na reabsorption occur in the presence of volume expansion and are the initial manifestations of nephropathy. Similarly, inprimary proteinuric renal disease, Na retention, which can resultin edema and/or hypertension, may occur in the presence of a relatively normalserum albumin concentration. These observations suggest a more direct relationbetween proteinuria and Na reabsorption that is independent of thecirculating plasma volume.% V! B' {$ ]" @/ T

Under normal conditions, the kidneys filter 180 liters of filtrate andreabsorb 1.7 kg of NaCl per day( 21 ), a function principally performed by the Na /H exchanger (NHE) isoform 3 (NHE3)in the luminal membrane ( 3 ).Several lines of experimental evidence suggest that increases in NHE3 activityare linked to hypertension. Elevated levels of NHE3 protein and activity havebeen observed in freshly isolated tubular cells and isolated intact tubules from spontaneously hypertensive rats( 22 ). In NHE3 knockout mice,systolic and arterial blood pressures are reduced, suggesting a key role forNHE3 in maintaining these parameters( 27 ), whereas there is anincrease in NHE3 activity in puromycin aminonucleoside-induced nephrotic ratsthat may contribute to the increased Na retention in these animals( 4 ). Significantly, a study inhypertensive patients revealed that proximal tubule Na reabsorption was an independent determinant of the blood pressure response tosalt-induced hypertension ( 8 ).It is important to keep in mind that although animal models of diabetes maydevelop kidney disease that displays features in common with human diabeticnephropathy, no single animal model develops renal changes identical to thoseobserved in humans ( 37 ). As aresult, it is critical to determine the response of cells of human proximaltubular origin to exposure to conditions that mimic the proteinuric state.0 ]* ]" @* p* F: @) B' T

In addition to Na reabsorption, it is estimated that several grams of albumin enter the proximal tubules on a daily basis, yet the urinaryexcretion of albumin is normally this suggests constitutivereabsorption of albumin ( 28 ).This reabsorption occurs in the proximal tubule via receptor-mediated endocytosis involving the scavenger receptor megalin( 10 ). Albumin itself has beenshown to exert a number of effects on the proximal tubule. Exposure of opossumkidney (OK) cells to pathophysiological concentrations of albumin has beenshown to stimulate cellular proliferation( 12 ) and also to impair albumin endocytosis by reducing the number of binding sites ( V max ) at the plasma membrane( 19 ), whereas exposure toalbumin has been shown to induce apoptosis in LLC-PK 1 cells( 16 ).- a0 S1 n4 d$ ^! y1 B5 G- F7 F) y
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NHE3 has been recently demonstrated to play a critical role inreceptor-mediated albumin uptake in cell cultures. Studies in OK cells haveshown that failure to acidify the early endosome ( 18 ) or inhibition of NHE3with amiloride analogs significantly reduces albumin uptake( 17 ), an observation that issupported by the finding that albumin uptake is abolished in NHE3-deficient OKcells ( 20 ). Thus changes inthe regulation of endosomal pH may play a significant role in tubulardysfunction ( 29 ). In rabbitrenal cortical membrane fractions, a substantial fraction of the cellular poolof NHE3 has been shown to be associated with the scavenger receptor megalinvia interaction with its COOH-terminal tail( 6 ), and NHE3 in themegalin-associated pools in the rabbit brush border was inactive( 5 ). These data indicate a dualrole for NHE3 in Na reabsorption and albumin uptake. In contrastto the other proximal tubule NHE isoforms 1 and 2, NHE3 is known to existprimarily in endosomes, with only 15% of the total cellular pool of NHE3located in the plasma membrane( 2, 7 ). Rapid alterations in theactivity of NHE3 are accomplished by changes in the rates of deployment ofNHE3 from the endosomes or retrieval of NHE3 from the plasma membrane( 13 ). Furthermore, recentevidence shows that NHE3 exists in lipid rafts in rabbit ileal brush bordersand that stimulation with epidermal growth factor (EGF), which increases NHE3activity, results in a preferential increase in the amount of NHE3 in thelipid raft fraction ( 26 ).

From these data, it is clear that NHE3 exists in functionally distinctpools at the plasma membrane, for example, pools associated withNa reabsorption and others involved in receptor-mediated albuminendocytosis. Thus there may be different retrieval/insertion mechanisms to andfrom these pools, such that the rates of insertion of NHE3 into the plasmamembrane may occur independently of the rate of the NHE3 internalization inconjunction with receptor-mediated albumin endocytosis. This raises theintriguing possibility that increased tubular albumin may have two separate actions: 1 ) it may reduce the capacity of the albumin uptake pathway,thereby increasing levels of protein in the urine; and 2 ) if therates of insertion of NHE3 into the different pools are not directly linked tothe rate of receptor-mediated endocytosis, levels of functional NHE3 may beincreased, resulting in increased Na retention by the proximaltubule. Such a model may provide an explanation for the link betweenhypertension and proteinuria in diabetic nephropathy. The aims of the present study were, therefore, to determine the effects of albumin on the expression,distribution, and activity of NHE3 in human proximal tubule cells (PTC) and tocorrelate these effects with changes in cellular growth.

METHODS

Primary culture of human PTC. Segments of macroscopically and histologically normal renal cortex were obtained under aseptic conditions frompatients undergoing nephrectomy for small ( cm) tumors. Patients wereaccepted for inclusion into the study if there was no history of renal orsystemic disease known to be associated with tubulointerstitial pathology.Written informed consent was obtained from each patient before surgery, andethical approval for the study was obtained from the Royal North ShoreHospital Human Research Ethics Committee. The methods for primary culture ofhuman PTC are described in detail elsewhere ( 25 ). Briefly, tubularfragments were derived from segments of renal cortex by collagenase digestionand isolated by centrifugation in 45% Percoll (Pharmacia, Uppsala, Sweden).The PTC were resuspended in serum-free hormonally defined media consisting ofa 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's and Ham's F-12 media(DMEM-F-12; ICN Pharmaceuticals, Costa Mesa, CA) supplemented with 10 ng/mlEGF (Collaborative Research, Bedford, MA), 5 mg/ml human transferrin, 5 mg/mlbovine insulin, 0.05 mM hydrocortisone, 50 mM prostaglandin E 1, 50nM selenium, 5 pM triiodothyronine (all from Sigma, St. Louis, MO), 100 U/mlpenicillin, 100 µg/ml streptomycin, and 0.292 mg/ml L -glutamine(Invitrogen, New York, NY). At confluence, the cells were harvested usingdispase (Integrated Sciences, Sydney, Australia) and stored in liquid nitrogen. When required, the cells were thawed, and after reaching confluence,they were harvested using dispase and subcultured. These cells were designated passage 2. The experiments in this study were performed on primarycell cultures derived from the kidneys of 16 patients.3 g# |4 M# h  ~  Y

Experimental protocol. All experiments were carried out on passage 2 PTC. Cells were made quiescent by incubation in DMEM-F-12 containing 5 µg/ml human transferrin and 5 mM D -glucose withoutgrowth factors for 48 h. The cells were then cultured for a further 48 h inmedium containing 5 mM D -glucose (control) or 5 mM D -glucose containing 0.1 or 1.0 mg/ml delipidated bovine serumalbumin (Sigma Chemical)./ m/ ^$ J8 F( o5 b  v( V  ~9 x
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Growth studies. Experiments to measure cell number and cell protein were performed in parallel, with the results obtained for totalprotein being adjusted to the total cell number in each of the treatments.Cell numbers in response to each treatment were determined by manual cellcounts on trypsinized cells using a hemocytometer. The total protein contentof cells was determined as a marker of cellular hypertrophy. Cells were solubilized with 0.2 M NaOH, and the protein was measured using a proteinassay (Bio-Rad, Hercules, CA).

22 Na uptake. 22 Na uptake into cells was measured on the basis of themethod of Rindler et al. ( 35 ).PTC were grown to confluence in 48-well culture plates, quiesced for 48 h, andthen exposed to 0.1 or 1.0 mg/ml albumin or control conditions for 48 h. Cellswere washed twice in HEPES-buffered saline (in mmol/l: 136 NaCl, 5.4 KCl, 1.2CaCl 2, 0.8 MgCl 2,10 acidic-HEPES, and 5 glucose, pH 7.4)and incubated with Na -free solution (HEPES-buffered saline withNaCl replaced by N -methyl- D -glucamine) for 15 min todeplete intracellular Na  . All uptake solutions contained thecorresponding amounts of albumin. The cells were preincubated with 100 µMouabain with or without the NHE blocker ethyliso-propylamiloride (EIPA, 100µM) in Na -free medium for a further 30 min. The Na solution was replaced with the uptake solution containing 22 Na tracer (1 µCi/ml; New England NuclearGeneworks, Boston, MA) in glucose-free HEPES-buffered saline for 20 min. Atthe end of the 22 Na uptake period, the cells werewashed rapidly three times with ice-cold 0.1 M MgCl 2 andsolubilized in 0.1 M NaOH. Cell lysate was mixed with scintillation fluid andcounted in a beta scintillation counter (LKB Wallac, Turku, Finland). Parallelcell counts were performed, and the total 22 Na uptakeswere adjusted to cell number in each treatment and expressed as a percentageof the control values.

Cell cycle analysis. PTC were grown to confluence in six-well plates and exposed to 0.1 or 1.0 mg/ml albumin or control conditions for 48 h.The cells were then trypsinized, placed in 1.5-ml tubes, washed in 0.5 ml ofphosphate-buffered saline (PBS), centrifuged (1,000 rpm, 10 min, 4°C), andfixed in 70% (vol/vol) ethanol at -20°C for 3 h. Cells werewashed in PBS and permeabilized in 0.5 ml of PBS with 0.1% Triton X-100 on icefor 30 min. Cells were then centrifuged, and the pellet was resuspended in 0.5ml of fluorochrome solution [propidium iodide (50 µg/ml), RNase (1 mg/ml),and Triton X-100 (0.1% vol/vol)] in PBS and incubated for 1 h at 4°C. Cellcycle analysis was performed using the FACSVan-tage SE flow cytometry system(Becton Dickinson, San Diego, CA). The propidium iodide fluorescence ofindividual nuclei and the forward and side scatter were measured usingidentical instrument settings with 20,000 events.

Competitive RT-PCR. Competitive RT-PCR was performed to determine the changes in NHE3 expression level induced by exposure to albumin. PTC weregrown to confluence in six-well plates and exposed to 0.1 mg/ml albumin for 48h before RNA extraction. Total RNA was extracted using TRIzol reagent (GIBCOBRL, Gaithersburg, MD) according to the manufacturer's instructions. RNA wasreverse transcribed using the Superscript II reverse transcriptase kit (GIBCOBRL).

Competitive RT-PCR was performed using primers specific to the COOH-terminal tail of human NHE3 (GenBank accession no. U28043  ). The primersequences were 5'-GTTCTTCACCGTCATCTTCCA (sense, bp 1345-1365) and5'-CTGAGAGAAAATGTCAGCGCT (antisense, bp 1769-1789), and thecompetitor primer sequence was 5'-CTGAGAGAAAATGTCAGCGCTGCCATCAGCTACGTGGCC (bp 1637-1655). Theseprimers produced products of 445 and 311 bp, respectively. Products weresequenced and confirmed to be human NHE3. Competitive RT-PCR against -actin was used as a control. Primers were designed against the human -actin gene (GenBank accession no. M10277  ). The primer sequences were5'-CATGTACGTTGCTATCCAG (sense, bp 2058-2076) and5'-CGCAACTAAGTCATAGTCC (antisense, bp 3003-3021), and thecompetitor primer sequence was 5'-CGCAACTAAGTCATAGTCCATGGAGCCGCCGATCCAC (bp 2889-2906). These primers produced products of 964 and 849 bp,respectively.# J5 n0 [! k6 I) t8 q& X
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PCR were performed on cDNA using the sense and competitor primers, and theproducts were gel purified and quantitated. For the PCR, the competitor cDNAwas used at 0.2, 0.5, 1.5, 4.6, and 13.7 fM. The reactions were for 35 cycleswith an annealing temperature of 60°C using the Expand High-Fidelity PCR System (Roche, Mannheim, Germany). The products were run on a 2% agarose gelstained with ethidium bromide and photographed. The photograph was thenscanned into a computer, and the relative intensities of the individual bandswere quantitated using NIH Image software (version 1.60). To normalize thedata to the levels of the housekeeping gene -actin, the ratios of the concentration of competitor primer for NHE3 to the concentration of the -actin primers at the equivalence point were determined. The equivalencepoint is defined as the point at which the ratio of the intensity of the bandsfor the competitor to native cDNA is unity, that is, the point at which theconcentration of the competitor is equivalent to the concentration of the message for the target gene.  l" K* y9 W6 h

Polyclonal antibody to the extracellular domain of human NHE3. Thepeptide sequence of human NHE3 (GenBank accession no. NP_004165  ) was screenedfor potential extracellular epitopes. The polypeptide GGVEVEPGGAHGESGGF wasselected. This peptide corresponds to amino acids 26-42 of the predictedhuman sequence, a region that is within the predicted first extracellularloop. The SignalP algorithm was used to predict the position of any putativesignal peptide cleavage site in the NH 2 -terminal region of humanNHE3 ( 31 ). The peptide wassynthesized, and an NH 2 -terminal cysteine was added to the epitopefor conjugation via diphtheria toxin usingmaleimidocaproyl- N -hydroxy-succinimide (Chiron Mimotopes, Clayton,Victoria, Australia). Polyclonal antibodies were raised in rabbits, and theimmune serum IgG was affinity purified.' v; K! c# b# v. |9 d, s6 S' Q# P

Confocal immunofluorescence. Confocal microscopy was performed onPTC grown on Cell-Tak (BD Biosciences, Bedford, MA)-coated coverslips asfollows. PTC were fixed with 4% paraformaldehyde in PBS for 2 min. Whererequired, cells were permeabilized with 0.1% DMSO in 2% normal horse serum,0.1% Triton X-100, and 0.1% bovine serum albumin in PBS for 2 min.Permeabilized and nonpermeabilized cells were blocked with 20% normal horse serum in PBS for 20 min. The cells were then incubated with the anti-NHE3antiserum (1:100) for 2 h at room temperature, washed, and incubated with aCy3 anti-rabbit antibody (1:200; Jackson Immunochemicals) for a further 45min. Slides were sealed with mounting medium (Dako, Carpenteria, CA) andvisualized under a Leica TCS NT laser confocal microscope (Leica, Solms, Germany) with excitation at 488 nm and emission at 570 nm.
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To demonstrate the specificity of the polyclonal anti-NHE3 antibody, 80µM epitope peptide was preincubated with the antibody for 10 min to blockthe binding of the antibody-binding sites. Surface NHE3 distribution wasdetermined on PTC quiesced in 5 mM glucose medium for 48 h and then exposed to0.1 or 1.0 mg/ml albumin for 48 h. Cells were nonpermeabilized to determine surface levels of NHE3 or permeabilized to determine total NHE3 in the sameapical plane used to determine the surface levels. Images were processed withAdobe Photoshop (version 5.02), and pixel densities at the apical pole of thecells were quantitated using NIH Image software (version 1.60).

Statistical analysis. Experiments were performed at least in triplicate on a minimum of four different cell culture preparations. Unlessotherwise stated, results are expressed as percentage of control values (cellsgrown in the absence of albumin for the experimental period). Statisticalcomparisons between groups were made by analysis of variance or paired t -tests where appropriate. Analyses were performed using the softwarepackage Statview (version 4.5, Abacus Concepts, Berkley, CA). P considered significant.2 |: g# J  K4 e

RESULTS
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Growth studies. Micropuncture experiments in animals yielded concentrations of albumin in the postglomerular filtrate of 10-200µg/ml ( 28 ). We thereforeused albumin at 0.1 mg/ml to reflect an upper level of normal and 1.0 mg/ml toreflect a pathological level in the proximal tubule. We found that exposure ofPTC to 0.1 mg/ml albumin for 48 h had no significant effect on cell growthparameters. Cell number was 104.0 ± 4.7% ( n = 12; Fig. 1 A ) and cellularprotein content 102.7 ± 5.1% ( n = 12; Fig. 1 B ) of controlvalues. In contrast, exposure of PTC to 1.0 mg/ml albumin for 48 h caused apronounced increase in cell number to 134.1 ± 11.8% ( n = 9) ofcontrol values ( P Fig. 1 A ), consistent with a proliferative response. This was accompanied by a significant reductionin protein per cell (76.9 ± 5.8%, n = 9, P = 0.0005)compared with control values ( Fig.1 B ).
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Fig. 1. A : effects of 48 h of exposure of proximal tubule cells (PTC) toalbumin at 0.1 mg/ml ( n = 12) and 1.0 mg/ml ( n = 9) on cellnumber. B : effects of 48 h of exposure of PTC to albumin at 0.1 mg/ml( n = 12) and 1.0 mg/ml ( n = 9) on levels of protein percell. Total protein values were adjusted to cell numbers determined inparallel samples. Results are standardized to control levels (100%), i.e.,cells grown in the absence of albumin. , albumin concentration.Values are means ± SE. * P 0.0005.

Apoptosis. Fluorescein-activated cell sorter analysis revealed that, under control conditions, 0.77 ± 0.16% of cells were in thepre-G 1 peak, which represents apoptotic cells. No significantchanges were observed in the numbers of cells in the pre-G 1 peakafter exposure to 0.1 or 1.0 mg/ml albumin for 48 h: 0.85 ± 0.37 and0.71 ± 0.25%, respectively. These data indicated that exposure toalbumin did not increase the levels of apoptosis of PTC.# `! r1 V9 z% z) L+ ]9 ?, n5 N
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22 Na uptake. Total 22 Na uptake was significantly increased in PTC afterexposure to albumin for 48 h at 0.1 and 1.0 mg/ml: 119.1 ± 6.3%( n = 13, P = 0.0005) and 115.6 ± 5.3% ( n =11, P ( Fig. 2 ). Incubation of thecontrol cells with 100 µM EIPA reduced 22 Na uptaketo 42.4 ± 2.8% ( n = 13) of baseline levels( Fig. 2 ). Similarly, incubationof the albumin-treated cells with EIPA reduced 22 Na uptake to the level observed in control cells: 38.6 ± 4.3% ( n = 13) in 0.1 mg/ml albumin and 47.9 ± 3.7% ( n = 11) in 1.0mg/ml albumin ( Fig. 2 ). Thusthe increase in total 22 Na uptake was abolished bytreatment with EIPA, and these data show that albumin exposure resulted insignificant increases in the EIPA-sensitive component of 22 Na uptake to 131.0 ± 6.3% ( P =0.0001) for 0.1 mg/ml albumin and 124.7 ± 8.6% ( P mg/ml albumin compared with control cells not exposed to albumin (100%). Importantly, 1.0 µM EIPA had no effect on 22 Na uptake (data not shown), indicating that thiscomponent of 22 Na uptake was not occurring via NHEisoform 1 ( 32 ). Furthermore, no effects were observed on 22 Na uptake in cellsexposed to lower concentrations of albumin (0.001 or 0.01 mg/ml) for 48 h(data not shown)., J. ~+ d* d9 _: f
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Fig. 2. Effects of 48 h of exposure of PTC to albumin at 0.1 mg/ml ( n =13) and 1 mg/ml ( n = 11) on 22 Na uptake.Ethylisopropylamiloride (EIPA) was used to block Na /H exchange isoform 3 (NHE3). Results are standardized to control levels (100%),i.e., total 22 Na uptake in cells grown in the absenceof albumin. Values are means ± SE. * P P
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Competitive RT-PCR. Competitive RT-PCR was performed to determine whether the increases in 22 Na uptake observed inresponse to exposure to 0.1 mg/ml albumin were paralleled by an increase inNHE3 mRNA expression levels. A representative gel of the products of thecompetitive RT-PCR is shown in Fig.3 A, and logarithmic plots of the ratio of the target tocompetitor bands against competitor concentration for NHE3 and thehousekeeping gene -actin are shown in Fig. 3 B. There is asmall but significant shift in the equivalence point for NHE3, whereas nochange is observed with -actin. The NHE3 ratios were standardized to the -actin ratios to account for any changes in total mRNA. Overall, therewas a significant increase in the levels of mRNA for NHE3 to 161.4 ±15.1% ( n = 6, P relative to the total mRNA pool(as reflected by -actin) in PTC exposed to albumin for 48 h( Fig. 3 C ). These dataconfirm that exposure of PTC to 0.1 mg/ml albumin, a concentration that increases NHE3 activity, also results in a parallel increase in NHE3 messagelevels.' x" D  k) u- r' w
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Fig. 3. Effects of 48 h of exposure of PTC to 0.1 mg/ml albumin on levels of NHE3and -actin mRNA. A : representative gels of products fromcompetitive RT-PCR. Lanes 1-4, control cells; lanes5-8, treated cells. B : logarithmic plots of concentrationsof competitor vs. ratio of intensity of target to competitor band. There is aclear shift in equivalence point for NHE3 mRNA in response to albumin( n = 6); there is no effect on -actin mRNA levels ( n =3). Vertical dashed lines represent equivalence points. C : graphicalrepresentation of data in B showing the relative increase in NHE3competitor concentration standardized to -actin competitor concentrationin the presence and absence of albumin (0.1 mg/ml). Results are standardizedto control levels (100%), i.e., cells grown in the absence of albumin. Valuesare means ± SE. * P
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Confocal immunofluorescence. Surface labeling was performed onnonpermeabilized and permeabilized PTC. When a confocal x-y scan wastaken through the upper (subapical) part of the cells, nonpermeabilized cellsexhibited a punctate distribution of labeling, principally at the cellperiphery ( Fig. 4 A ), whereas permeabilized cells also exhibited significant levels of intracellularstaining ( Fig. 4 B ). z -Axis scans on nonpermeabilized and permeabilized cells revealedthat NHE3 was primarily localized to the apical domain of the cells( Fig. 4, C and D ). These staining patterns are consistent with the knowndistribution of NHE3 in other cell types( 2, 9, 33 ). To confirm the antibody specificity, the antiserum was preincubated with the antigenic peptide beforethe cells were labeled. This resulted in a dramatic decrease in thefluorescent signal ( Fig. 4, E and F ), indicating that the antibody staining was indeedspecific for the epitope on NHE3. A similar decrease in fluorescence was alsoobserved with a lower peptide concentration (10 µM; data not shown).- N$ I/ Z5 e* B2 @$ f# W

Fig. 4. Representative images of confocal immunofluorescence microscopy showingdistribution of NHE3 in PTC. Images are representative of 6 separateexperiments. A : nonpermeabilized cells; x-y scan through thesubapical pole of the cells shows punctate distribution of NHE3 at the cellperiphery. B : corresponding view on permeabilized cells showingintracellular distribution. C : x-z scan of nonpermeabilizedcells showing NHE3 at the apical pole of the cells. D : correspondingview on permeabilized cells showing that NHE3 is primarily located in thesubapical region of the cell. E : antibody specificity shown ascontrol permeabilized cells labeled with NHE3 antibody. F : cellslabeled with NHE3 antibody preincubated with 80 µM epitope peptide showinga pronounced reduction in staining, thereby confirming the antibodyspecificity. Images are representative of 3 separate experiments. Scale bars,5 µm.: A6 S- t6 x( Y( S3 |- G3 T# f

After exposure to albumin for 48 h, there was a significant increase in thelevels of cell surface NHE3 ( Fig. 5, A and B ). Similarly, when x-y scanswere performed on permeabilized cells viewed through the same optical plane,albumin also induced an increase in total NHE3( Fig. 5, C and D ). Measurements of relative pixel intensities inindividual cells revealed a significant increase in the level of fluorescenceintensity with both concentrations of albumin. In cells exposed to 0.1 mg/mlalbumin, the level of fluorescence at the apical surface was 115.4 ±2.7% ( n = 20, P control levels( Fig. 6 A ). A similarincrease in fluorescence (118.1 ± 3.2%, n = 17, P was observed in cells exposed to 1.0 mg/mlalbumin ( Fig. 6 A ). Asimilar analysis of pixel density at the apical pole was performed onpermeabilized cells to determine the amounts of NHE3 on the cell surfacerelative to the intracellular pool in the immediate vicinity of the apicalmembrane. In response to albumin, there was an increase in total NHE3 at theapical membrane increasing to 118.7 ± 2.4% ( n = 19, P 0.0001) and 109.8 ± 2.7% ( n = 20, P ( Fig.6 B ). When the pixel intensities for the nonpermeabilized cells were standardized to the permeabilized cells, these data revealed that76.1 ± 2.1% of the total apical NHE3 pool was located at the cellsurface ( Fig. 6 C ).Significantly, although the total amount of NHE3 at the cell surfaceincreased, the proportion of NHE3 at the cell surface relative to the total subapical pool did not change with albumin exposure: 74.0 ± 1.7% and81.9 ± 2.2% of total NHE3 for 0.1 and 1.0 mg/ml albumin, respectively( Fig. 6 C ). These datasuggest that the increase in NHE3 activity we observed in response toprolonged exposure to albumin resulted from an increase in total NHE3 thattranslated into a parallel increase in the plasma membrane, rather thanaltered rates of insertion from submembrane stores., v- ^9 G- b8 X3 p6 X

Fig. 5. Representative images of confocal immunofluorescence microscopy showing theincrease in NHE3 in PTC after 48 h of exposure to albumin. All images weretaken from equivalent planes of x-y scans at the cell surface. A : nonpermeabilized control cells not exposed to albumin. B :nonpermeabilized cells exposed to 0.1 mg/ml albumin for 48 h showing anincrease in NHE3 at the cell surface. C : permeabilized control cellsnot exposed to albumin. D : permeabilized cells exposed to 0.1 mg/mlalbumin for 48 h. Comparison of A and C and comparison of B and D provide an estimate of the relative amount of NHE3at the surface compared with the total amount in the same optical plane.Images are representative of 3 separate experiments. Scale bars, 5 µm.

Fig. 6. Graphical representation of images in Fig. 5. A : pixelintensity analysis of cell surface NHE3 showed a clear increase in NHE3 inresponse to exposure to 0.1 and 1.0 mg/ml albumin ( n = 20 and 17,respectively). B : pixel intensity analysis of total NHE3 inpermeabilized cells labeled with NHE3 antibody and viewed through the sameoptical plane as in A ( n = 19 and 20, respectively). Datashow a clear increase in total NHE3 after exposure to albumin. C :proportion of surface NHE3 relative to total NHE3 in the same optical plane.Data show that changes in cell surface expression parallel changes in totalNHE3 ( n = 12, 19, and 20 for control and 0.1 and 1.0 mg/ml albumin,respectively). Results are standardized to control levels (100%). Values aremeans ± SE. * P P

DISCUSSION# z/ M* {; ?5 T- `! Y6 e

This study describes the effects of exposure of primary cultures of humanPTC to pathophysiological concentrations of albumin on cell growth and NHE3expression and activity. In addition to demonstrating altered cell growthparameters, we have shown that exposure of PTC to pathophysiological levels ofalbumin results in significant increases in Na reabsorptionthrough an increase in NHE3 activity. This occurs in association with increases in NHE3 gene and protein expression at the apical membrane of thePTC.

Our demonstration that albumin at 1.0 mg/ml resulted in a pronounced proliferative effect is consistent with findings in OK cells, where exposureto albumin increased cell number and total thymidine uptake, with a maximaleffect at 1.0 mg/ml albumin( 12 ). The fact that weobserved no effects on cell growth at the lower concentration of albumin (0.1mg/ml) may simply reflect a difference in the cell types studied. These datashow that delipidated albumin alone, at a concentration in the highpathophysiological range and in the absence of growth factors, is able toinduce cell proliferation. It has been shown that, after induction ofexperimental diabetes in a rat model, nephromegaly is preceded by an initialhyper-plastic phase over the first few days ( 23 ). Consequently, thisinitial hyperplasia may, in part, be accounted for by the proliferative effectof elevated albumin. 0.5 mg/ml) has been shown to protect cultured murinePTC from apoptosis by a mechanism involving the scavenging of reactive oxygenspecies ( 24 ). In the presentstudy, we observed no significant changes in the levels of apoptosis in thepresence and absence of albumin; thus our data are consistent with arenoprotective effect of albumin. These findings contrast with those reportedin LLC-PK 1 cells, where exposure to albumin induced apoptosis;however, in these studies, much higher concentrations 5 mg/ml) ( 16 ). An in vivostudy in protein-overload rats showed an increase in the number of proliferating cells as determined by in situ hybridization for histone mRNAs.However, this increase was counteracted by an even greater increase in thenumber of apoptotic cells, with tubular atrophy being the net result( 36 ). Furthermore, it has beenshown in LLC-PK 1 cells( 42 ), OK cells( 14 ), and primary cultures ofrat PTC ( 38 ) that exposure topathophysiological levels of albumin results in an activation of NF- Bthat results in enhanced cytokine/chemokine production. Thus the increases inPTC number that we observe in culture may result in vivo in the increasedcytokine/chemokine production that underlies the inflammatory phase oftubulointerstitial pathogenesis ( 41 ).

NHE3 has been shown to exist primarily in subapical endosomal pools and ajuxtanuclear compartment in OK cells( 2 ) and AP-1 cells transfectedwith NHE3 ( 15 ). It has beendemonstrated that as little as 15% of the total cellular NHE3 is present inthe plasma membrane ( 1, 2, 7 ). To directly monitor changes in levels of NHE3 at the cell surface, we developed an antibody to a predictedextracellular epitope in the first extracellular loop of human NHE3 on thebasis of the model of NHE3 with 12 transmembrane loops and intracellularNH 2 and COOH termini. The epitope recognized by our antibodycorresponds to amino acids 26-42 of the published human NHE3 sequence(GenBank accession no. NP_004165  ). A recent topological analysis of rabbit NHE3, however, showed that the first 30 amino acids of NHE3 form a signalpeptide that is cleaved during processing, resulting in an extracellularNH 2 terminus ( 40 ).We found a predicted signal peptide cleavage site between Gly27 and Val28 ofthe human sequence comparable to the predicted cleavage site in rabbit NHE3between Gly29 and Ala30 ( 40 ).Thus our antibody recognizes the first 16 amino acids of the NH 2 terminus of human NHE3 after signal peptide cleavage, and the binding of ourantibody to the surface of nonpermeabilized PTC confirms that this region ofhuman NHE3 is extracellular." m8 H0 d8 w8 I5 _* ~
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Surface labeling of PTC revealed a punctate distribution of NHE3 similar tothat observed in OK cells ( 2, 39 ). In permeabilized PTC,there was considerably more NHE3 in the cytosol, and this intracellular NHE3appeared to be associated largely with endosomal compartments in the subapicalregion of the PTC ( Fig. 4 ). Therefore, these data obtained from confocal sections taken through the cellbody are in agreement with the overall cellular distribution of NHE3 in othercell types, e.g., PS120 and OK cells( 2, 15 ), where only 10-15%of the total cellular NHE3 pool was reported to be inserted at the cellsurface ( 1, 2, 7 ). Interestingly, our data,derived from confocal sections taken in the plane of the apical cell surfaceon permeabilized and nonpermeabilized cells (Figs. 5 and 6 ), suggest that a significantproportion ( 70%) of the total NHE3 in the immediate direct vicinity ofthe cell membrane is present at the cell surface. However, because of thedifficulties in precise quantitation of relative intensities due tooverlapping stores of NHE3 visualized under permeabilized and nonpermeabilizedconditions, it is possible that this method may overestimate the proportion of total NHE3 at the cell surface. Nevertheless, our data support the study in OKcells showing that NHE3 exists in different functional domains and is presentin large complexes at the apical surface of OK cells, with the intracellularpools acting as reservoirs for membrane recruitable NHE3( 2 ). It has been reported thatNHE3 exists as an active oligomer in the microvillar domain and as an inactivemegalin-associated form in intermicrovillar domains( 5 ) and that NHE3 levelsincrease preferentially in lipid rafts after stimulation with EGF( 26 ). Thus the increase insurface staining for NHE3 and increased NHE3 activity that we observed afterexposure to albumin may reflect an enhanced association with the plasmamembrane domains involved in mediating Na reabsorption. Thismodel, on the basis of the present data, is consistent with a recent findingin nephrotic rats, where the increase in NHE3 activity was associated with ashift from the inactive megalin-associated pool to the active pool in the brush border of proteinuric rats( 4 ), potentially contributing to increased Na retention. Our findings are also in agreement witha report that albumin increased NHE3 activity and immunoreactivity in OKcells, although higher concentrations of albumin ( 5 mg/ml) were requiredto elicit a response ( 30 ).2 y6 k! ~% s; q" U8 ~

The increase in NHE3 expression and activity suggests that exposure toalbumin increases the Na reabsorptive capacity of the human proximal tubule. Significantly, albumin induced increases in Na reabsorption at concentrations below which it exerted its proliferativeeffect. High concentrations of albumin are reported to enhance theproliferation of PTC ( 11 );however, the differential effects we observed at lower concentrations indicatea specific role of albumin in regulating Na reabsorption.& O/ Q8 F2 i* B* R: ]. o
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In summary, the present study demonstrates the concentration-dependent uptake of albumin by primary cultures of human PTC and shows that albuminuptake is associated with an increase in the activity of NHE3. A sustained10-20% increase in the transcription and activity of NHE3 in response toelevated albumin may lead to a significant increase in Na retention, contributing to the development of hypertension, whereas aconcomitant reduction in the tubular absorption of albumin in the presence ofelevated albumin would result in the increased excretion of albumin manifest as proteinuria. Thus the data in the present study present a possiblemechanism to explain the link between reduced albumin uptake and increasedNa retention as observed in diabetic nephropathy.! k( X; Q% D1 J2 H% O9 K2 Y" n

DISCLOSURES
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This work was supported by grants from the National Health and MedicalResearch Council of Australia and the Juvenile Diabetes Research Fund (P.Poronnik and C. A. Pollock).* a7 L) {! u9 w6 p* m4 V; @( N3 c* S
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ACKNOWLEDGMENTS) S7 [9 K; t$ w& m

We thank Prof. David Cook for helpful discussions.
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