Renal tubular epithelial cell apoptosis is associatedwith caspase cleavage of th - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:34 |只看该作者 |倒序浏览 |打印

作者：Karen L.Wu, ShenazKhan, SujataLakhe-Reddy, LimingWang, GeorgeJarad, R. TylerMiller, MarthaKonieczkowski, Arthur M.Brown, John R.Sedor,  Jeffrey R.Schelling,作者单位：Departments of Medicine and Physiology and Biophysics, and Rammelkamp Center for Education and Research, CaseWestern Reserve University School of Medicine, Cleveland 44109; and Louis B. Stokes Veterans Administration MedicalCenter, Cleveland, Ohio 44106


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      【摘要】5 e, H9 x+ P9 b2 b6 o$ A" F, Z
   Renal tubular epithelial cell(RTC) apoptosis causes tubular atrophy, a hallmark of renaldisease progression. Apoptosis is generally characterized byreduced cell volume and cytosolic pH, but epithelial cells arerelatively resistant to shrinkage due to regulatory volume increase,which is mediated by Na /H exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavageof NHE1. Staurosporine- and hypertonic NaCl-induced RTCapoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amilorideanalogs, suggesting NHE1 activity opposes apoptosis.NHE1-deficient fibroblasts demonstrated increased susceptibility toapoptosis, which was reversed by NHE1 reconstitution. NHE1expression was markedly decreased in apoptotic RTC due todegradation, and preincubation with peptide caspase antagonistsrestored NHE1 expression, indicating that NHE1 is degraded by caspases.Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmicdomain into five distinct peptides, identical in molecular weight toNHE1 degradation products derived from staurosporine-stimulated RTClysates. In vivo, NHE1 loss-of-function C57BL/6.SJL- swe/swe mice with adriamycin-induced nephropathy demonstrated increased RTCapoptosis compared with adriamycin-treated wild-type controls,thereby implicating NHE1 inactivation as a potential mechanism oftubular atrophy. We conclude that NHE1 activity is critical for RTCsurvival after injury and that caspase cleavage of RTC NHE1 may promoteapoptosis and tubular atrophy by preventing compensatoryintracellular volume and pH regulation.
      【关键词】 cell death nephropathy regulatory volume increase renal disease tubular atrophy Na /H exchanger
   INTRODUCTION. g1 c( g0 U$ {1 P! h* j6 n
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TUBULAR ATROPHY IS A HALLMARK of chronic renal diseases and is superior to glomerularpathology as a histological predictor of clinical outcomes( 41 ). We have previously shown that renal tubularepithelial cell (RTC) apoptosis is a mechanism of tubular atrophy ( 20, 43 ). In the original descriptions ofapoptosis morphology, Wyllie et al. ( 51 ) termedthe process "shrinkage necrosis" due to reductions in cell volumeobserved during apoptosis. Apoptotic cell shrinkage isachieved by net loss of intracellular osmoles and H 2 O, aswell as by caspase-dependent proteolysis of housekeeping and structuralproteins, which mediates cell disassembly. Many studies have alsodemonstrated that apoptosis is associated with a decrease incytosolic pH ( 16, 27, 28, 30, 36, 46 ), which is requiredfor activation of the caspase cascade ( 30, 45 ).

In contrast to neuronal cells and lymphocytes, epithelium-derived cellsare relatively resistant to apoptosis following exposure tohypertonic extracellular conditions ( 6, 34 ), due to an enhanced capacity to rapidly expand intracellular volume through regulatory volume increase (RVI) pathways ( 17, 26, 31, 33 ). RVI is achieved by activation of theNa /H exchanger isoform NHE1 and, depending onthe cell type, the anion exchanger (AE) 2 isoform of theCl /HCO 3 − exchanger and/or theNa /K /2Cl symporter ( 26, 31, 33 ). The net effect is ion and H 2 O influx, whichleads to intracellular volume re-expansion. If RVI-dependent transporters are robustly activated after initiation of anapoptotic stimulus, restoration of intracellular volume may preemptapoptosis ( 26, 33 ). Alternatively, for a cell toundergo apoptosis, RVI must be overcome or inhibited ( 26, 33 ). Neither AE2 nor the types 1 or 2 bumetanide-sensitivecotransporter (BSC-1 or BSC-2, respectively) isoforms of theNa /K /2Cl symporter areexpressed in proximal tubule ( 1, 15, 18 ), the nephronsegment that demonstrates the most abundant apoptosis in animalmodels of progressive renal disease ( 20, 43 ). However, NHE1 is ubiquitously expressed, including within the proximal tubule,suggesting that NHE1 may be critical to RTC survival by promotingresistance to apoptotic cell shrinkage.
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In addition to regulating cell volume via RVI, NHE1 mediates otherhousekeeping functions, such as intracellular pH (pH i ) regulation through electroneutral Na influx andH efflux. NHE1-dependentNa /H exchange has been linked to essentialcell functions, such as proliferation ( 4, 32 ), whereasdiminished NHE1 activity has been associated with lymphocyteapoptosis ( 27, 40 ). Moreover, NHE1 has recentlybeen recognized to function as a scaffold for binding with ezrin,radixin, and moesin (ERM) ( 14 ), adaptor molecules thatlink cytoskeleton to plasma membrane proteins, suggesting that NHE1 mayfacilitate apoptosis resistance by preserving cytoarchitectureand maintaining cell volume independent ofNa /H antiporter functions.

Because predicted sequelae of NHE1 inhibition-cell shrinkage,intracellular acidification, and cytoskeleton collapse mimic theapoptotic phenotype, we investigated whether RTC apoptosis is regulated by NHE1 caspase cleavage. We find that RTCapoptosis is associated with caspase-dependent NHE1degradation. Furthermore, cell culture and whole animal datademonstrate that NHE1 loss-of-function mutations render RTC susceptibleto apoptosis. The data are consistent with a mechanism wherebyNHE1 degradation causes RTC apoptosis and tubular atrophy,which prevents RVI and promotes intracellular acidosis, an optimumcondition for caspase activity.' h1 y5 V2 ?: B. X3 {9 F1 e, T9 w
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MATERIALS AND METHODS& K: \1 c0 J3 h1 a, {& y- z# w
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Reagents. We used the following: amiloride, 4',6-diamidino-2-phenylindole(DAPI), ethyl- N -isopropylamiloride (EIPA),hexamethyleneamiloride (HMA), staurosporine (STS), adriamycinhydrochloride (Sigma, St. Louis, MO); z-VAD-fmk, z-DEVD-fmk,Ac-DEVD-CHO (Calbiochem, La Jolla, CA);2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Eugene, OR);anti-poly(ADP-ribose)polymerase (PARP) IgG, phycoerythrin(PE)-conjugated annexin V (Pharmingen, San Diego, CA); annexin V,anti-hemagglutinin (HA) IgG (Roche, Indianapolis, IN); horseradishperoxidase (HRP)-conjugated IgG (Santa Cruz Biotechnology, Santa Cruz,CA); green fluorescence protein (GFP) cDNA (Clontech, Palo Alto, CA);Red X-conjugated anti-mouse IgG, FITC-conjugated anti-mouse IgG (VectorLaboratories, Burlingame, CA); [ 35 S]methionine (ICN,Irvine, CA); C57BL/6.SJL  / , C57BL/6.SJL swe/ , and C57BL/6.SJL swe/swe mice (Jackson Laboratories, BarHarbor, ME); COOH-terminal, HA epitope-tagged rat NHE1 cDNA (a giftfrom Dr. J. Orlowski, McGill University); and KR/A and E266I NHE1mutant cDNAs (gifts from Dr. D. Barber, University of California at San Francisco). Rabbit polyclonal anti-NHE1 IgG was generated against anNHE1 cytoplasmic domain peptide as previously described( 23 ) and affinity purified.' B/ B8 T: w# N4 m* M% s- W7 W$ ^
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Cell lines. The human renal proximal tubule (HRPT) cell RTC line (gift from Dr. L. Racusen, Johns Hopkins University) has been extensively characterized( 20, 21, 39, 43 ). HRPT and HEK 293 (ATCC, Manassas, VA) cells were maintained in DMEM-F12 (Gibco-BRL, Rockville, MD) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1%penicillin-streptomycin-fungizone solution (Sigma). PS120 cells aregenetically deficient for NHE1 expression and were derived from controlCCL39 fibroblasts (gifts from Drs. D. Grall and J. Pouysségur,University of Nice).1 s8 _& C- k, H" w/ c% a, z
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Flow cytometry. Cells were incubated with STS, lifted with trypsin-EDTA (10 min,37°C), and washed twice in incubation buffer (10 mM HEPES/NaOH, pH7.4, 140 mM NaCl, and 5 mM CaCl 2 ) at 4°C. Washed cellswere incubated with BCECF-AM (1.6 µM, 30 min, room temperature),PE-conjugated annexin V (15 min, room temperature), and DAPI (2 µg/ml, 15 min, room temperature). Cytosolic pH was measured byBCECF-AM fluorescence, and relative cell volumes were determined fromforward vs. side light scatter characteristics with a Becton DickinsonFACS Vantage flow cytometer according to previously described methods( 21, 50 ). Apoptosis was measured by annexin Vbinding (see below).( p; d/ m6 K" f1 Q# b
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Apoptosis assays. Cells were plated on glass coverslips at 0.25 × 10 6 cells/ml density, grown to 80% confluence, and then maintained inserum-free medium combined with apoptotic stimuli. In someexperiments, cells were preincubated with NHE1 inhibitors for 2 hor caspase inhibitors for 1 h before apoptosis induction.Apoptotic cells were identified by simultaneous fluorescentlabeling of chromatin with DAPI and externalized phosphatidylserinewith annexin V as previously described ( 20, 21 ). Randomfields were viewed at ×40 magnification with a Nikon epifluorescencemicroscope (Tokyo, Japan), and the percentage of apoptotic cellswas separately determined by two blinded observers from a total of100-200 cells per experimental condition. Representative fieldswere photographed with a Spot Digital System camera and analyzed usingImage Pro software (Diagnostic Instruments, Sterling Heights, MI)./ I; y3 D! ~2 H5 x" U' ]

Plasmid transfections. Plasmids were transformed into DH-5 -competent bacterialcells according to manufacturer's protocol (Gibco-BRL), extracted using a Maxiprep kit (Qiagen, Valencia, CA), and amplified by culturein Luria-Bertani-ampicillin. GFP, HA-NHE1, KR/A (inhibits ERM binding),and E226I (inhibits Na /H exchange) mutantNHE1 cDNAs were transiently transfected into cells according topreviously described methods ( 21 ). Briefly, cells wereplated in six-well dishes (0.25 × 10 6 cells/well) andcultured overnight in DMEM-F12 plus 10% fetal bovine serum to achieve80% confluence. Cells were then washed and incubated with 100 µl ofserum-free DMEM (Gibco-BRL) containing 6 µl of Fugene 6 transfectionreagent (Roche) and 2.0-3.0 µg of plasmid DNA for 20 min at roomtemperature. Transfected cells were then cultured in complete mediacontaining DMEM-F12 and 10% fetal bovine serum for an additional24 h.  V$ j" K( C5 W2 K6 x5 D- {
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Immunoblot analysis. Methods have previously been described in detail ( 42 ).Whole cell lysates were prepared in boiling 2× SDS sample buffer (125 mM Tris, pH 6.8, 2% SDS, 5% glycerol, 1% -mercaptoethanol, and0.003% bromphenol blue). Samples were assayed for protein contentusing protein assay reagents (Bio-Rad, Hercules, CA). Proteins weredenatured by boiling for 5 min, and samples (60 µg/lane) wereresolved by 8 or 14% SDS-PAGE (Novex, San Diego, CA). Proteins weretransferred to polyvinylidene difluoride membranes, blocked with 5%nonfat milk, and incubated with either anti-PARP (1:2,000, 1 h,room temperature) or anti-HA (1:5,000, 1 h, room temperature)antibodies, followed by HRP-conjugated secondary antibody (1:5,000,1 h, room temperature). Band intensity was detected by enhancedchemiluminescence methods (Amersham Pharmacia Biotech, ArlingtonHeights, IL) and exposure to Kodak Biomax ML film. In someexperiments individual bands were digitized by phosphorimager (Molecular Dynamics, Sunnyvale, CA), quantified with Image Quant 5 software (Molecular Dynamics), and normalized to control values.

Protein degradation by [ 35 S]-labeled pulse chase. Cells were cultured to subconfluence, washed with PBS, and incubatedwith [ 35 S]methionine in methionine-free DMEM (0.1 mCi/ml,2 h, 37°C). Cells were washed with PBS and cultured in completemedia (0-6 h, 37°C) with or without STS and peptide caspaseinhibitors. Protein lysates (200 µg per sample) wereimmunoprecipitated with anti-HA (1 µg) or anti-NHE1 IgG (1 µg) andresolved by SDS-PAGE according to previously described methods( 42 ). Autoradiograms were developed from dried gels. Insome experiments, individual bands were digitized by phosphorimager(Molecular Dynamics), quantified with Image Quant 5 software (MolecularDynamics), and normalized to control values.
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Immunocytochemistry and fluorescence microscopy. Methods have previously been described in detail ( 20, 21, 43 ). Cells were maintained on sterile glass coverslips within six-well plates, fixed in paraformaldehyde (4%, 10 min, roomtemperature), and blocked with 5% low-IgG BSA and 0.2% Triton X-100(Sigma) for 30 min at room temperature. Cells were incubated withanti-HA IgG (1:200, 2 h, room temperature), followed by either redX-conjugated or FITC-conjugated anti-mouse IgG (1:200, 2 h,4°C). Negative controls were cells incubated with isotype-identicalIgG, which was immunoreactive with an irrelevant epitope. Coverslipswere mounted in antifade, aqueous media containing DAPI (Vectashield; Vector Laboratories) on standard microscope slides. Random fields wereviewed by two observers blinded to experimental condition, using aNikon epifluorescence microscope with appropriate fluorescence filters.Representative fields were photographed with a Spot Digital Systemcamera and analyzed using Image Pro software.% D/ \5 H0 d) s, T1 f

Assay for caspase-3 cleavage of in vitro-translated NHE1. The DNA template for in vitro translation was created by PCRamplification of the NHE1 cytosolic domain (cNHE1) from rat cDNA withupstream primer5'-CTACCGCTC- GAGCCACCATGCCCAAGGACCAGTTCATCATTGCC-3' that contains Xho I restriction endonuclease, Kozak and ATGstart sites, and downstream primer5'-TGCTCTAGACTAGCCCTGCCCTTTGGGGATGAAAGG-3' containing an Xba I restriction site and stop codon. The cNHE1 construct included theopen reading frame encoding amino acids 447-820, which correspondsto the 58 COOH-terminal amino acids within the transmembrane domain andthe entire cytosolic domain. The resulting DNA was digested with Xho I and Xba I (Gibco-BRL) and 1.1 kb productwas cloned into pTNT vector (Promega, Madison, WI). PCR-generated cNHE1nucleotide sequence was verified by automated sequencing (ClevelandGenomics, Cleveland, OH). [ 35 S]Met-labeled cytoplasmicNHE1 substrate was generated using the reticulocyte lysate-based TNTQuick T7-coupled transcription/translation system (Promega) accordingto manufacturer's instructions. Briefly, cNHE1 plasmid template (1 µg) was labeled with [ 35 S]Met (50 µCi, 90 min,37°C). Autoradiograms from dried gels yielded a single 45-kDa bandwithin 2- to 3-h film exposure (not shown). [ 35 S]Met-labeled, in vitro-translated cNHE1 (3 µl) wasincubated with or without Ac-DEVD-CHO (100 µM, 2 h, 37°C),followed by 1-2 µl of purified caspase-3 (6 h, 30°C

harmingen) in 5 µl of caspase buffer (100 mM HEPES, pH 7.5, 10%sucrose, and 0.1%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) according to published protocols ( 10, 22 ).Peptide products were resolved by 14% SDS-PAGE and examined forcleavage by autoradiography.! b8 o  Y) P! ?8 F

Animal models. C57BL/6.SJL swe/swe mice harbor an NHE1 A1639T pointmutation, which introduces a premature stop codon, resulting intruncation between the 11th and 12th NHE1 transmembrane domains andloss of NHE1-dependent Na /H activity( 13 ). Four-week-old swe/swe homozygotes have abrain phenotype that includes ataxia and seizures, but a gross renal phenotype was not observed, as determined by kidney histology and serumNa  , K  , Cl,HCO 3 −, urea nitrogen, creatinine, and albuminconcentrations. To identify the role of NHE1 in tubulointerstitial disease susceptibility, we induced nephropathy in 8-wk-old wild-type (C57BL/6.SJL  /  ), C57BL/6.SJL swe/ , andC57BL/6.SJL swe/swe mice by tail vein injection withadriamycin hydrochloride (10 µg/g) ( 49 ). Mice with aC57BL/6 genetic background were specifically chosen because C57BL/6.SJL swe/swe mice survive to adulthood, in contrast to SJL/J swe/swe, which die at ~3 wk ( 3, 13 ). Furthermore, in contrast to BALB/c mice, C57BL/6 mice do not develop adriamycin nephropathy ( 49 ), allowing for more robustcomparisons between potentially susceptible ( swe/swe, swe/  ) and resistant (  /  ) strains. Mice werekilled 10 days after adriamycin infusion; swe/swe, swe/ , and  /  animals were distinguished byimmunoblotting liver lysates (20 µg protein/lane) with anti-NHE1antibodies (1:1,000). RTC apoptosis from frozen kidney sectionswas determined by terminal deoxynucleotidyltransferase-mediated dUTPnick-end labeling (TUNEL) assays (Intergen, Purchase, NY) according topreviously described methods ( 21, 43 ). Three sagittalsections from two mice per genotype were scanned for TUNEL-positivecells by two observers blinded to experimental conditions. Kidney areawas determined with a Spot Digital System camera and Image Prosoftware, and data are expressed as apoptotic RTC/mm 2.The animal care protocol was approved by the Institutional Animal Careand Use Committee at Case Western Reserve University School of Medicine.

Statistics. Data are representative of three to five experiments per condition.Graphical results are expressed as means ± SE unless otherwise indicated. Comparisons between paired samples were made by the Student's t -test. Comparisons between groups containingmore than two samples were made by one-way analysis of variance withthe Bonferroni, Student-Newman-Keuls, or Kruskal-Wallis tests for multiple comparisons. Statistical significance is defined as P# p8 e- }8 N# M& @! \% P6 i! I' |
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RESULTS
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Apoptotic RTC are shrunken and acidic. To determine whether apoptotic RTC develop reduced cell volume andcytosolic pH, as has been described in leukocytes, we stimulated cultured RTC to undergo apoptosis with STS. Cell size and pHwere determined by flow cytometry. As shown in Fig. 1, a greater proportion of RTC incubatedwith STS displayed smaller cell volumes and lower cytosolic pH, similarto STS- and Fas-induced Jurkat T cell apoptosis ( 30 ). These data demonstrate that the apoptotic RTCphenotype includes shrinkage and acidification, consistent with a rolefor NHE1 inhibition.6 M: P2 x4 k, D. f4 c
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Fig. 1. Apoptotic renal tubular epithelial cells (RTC) areshrunken and acidic. Subconfluent monolayers of cultured RTCwere incubated with or without staurosporine (STS, 1 µM, 5 h,37°C). Apoptosis was determined by annexin V labeling(apoptotic cells are red, nonapoptotic cells are green).Relative cytosolic pH was measured by2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AMfluorescence ( y -axis) and relative cell volumes by lightscatter characteristics ( x -axis). Results are representativeof 5 separate experiments.7 v& K7 v3 \' \8 p

Hypertonicity stimulates RTC apoptosis. Epithelial cells are relatively resistant to apoptosisinduction by anisotonic conditions due to robust RVI ( 6, 34 ), which is mediated by multiple transporters ( 26, 31, 33 ). To determine whether cell stress imposed by hypertonicitycauses apoptosis, we exposed RTC to increasing extracellularconcentrations of the impermeant sugars mannitol and sucrose or NaCland then simultaneously assayed them for apoptosis by annexin Vlabeling of externalized phosphatidylserine and chromatin condensation. Figure 2 shows that hypertonicity causedRTC apoptosis in response to all three stimuli in aconcentration-dependent fashion, indicating that RVI was surmounted andRTC were susceptible to hypertonic stress-induced apoptosis.

Fig. 2. Hypertonicity stimulates RTCapoptosis. RTC were incubated in media supplementedwith mannitol, sucrose, or NaCl at indicated concentrations for 5 h. Apoptotic cells were identified by fluorescence microscopyanalysis of annexin V labeling of externalized phosphatidylserine( A ) and 4',6-diamidino-2-phenylindole (DAPI) staining ofcondensed chromatin ( B ). Data represent means ± SEfrom 3-5 separate experiments.
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Hypertonicity stimulates RTC apoptosis by an NHE1-dependentmechanism. To determine the role of NHE1 in apoptosis induced byhypertonic conditions, we examined the effect of NHE1 inhibitors on RTCapoptosis, assayed by DAPI labeling of condensed chromatin (Fig. 3 A ) and annexin Vlabeling of externalized phosphatidylserine (Fig. 3 B ). Thesestudies show that the Na /H inhibitoramiloride caused modest apoptosis but significantly potentiatedRTC apoptosis from hypertonic stress. Because amiloride, particularly at high concentrations, may inhibit multiple sodium transporters, including other NHE isoforms expressed in proximal tubule, NaCl-induced apoptosis was assayed followingpreincubation with NHE1-specific amiloride analogs EIPA and HMA( 35, 47 ). Figure 3, C and D,demonstrates that, like amiloride, EIPA and HMA caused modestapoptosis and both inhibitors enhanced hypertonic EIPA), suggesting thatNHE1 inactivation potentiates RTC apoptosis induced bystresses, such as hypertonicity.* S  H/ o, B5 @8 p3 Q
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Fig. 3. Hypertonicity stimulates RTC apoptosis by anNa /H exchanger (NHE) 1-dependent mechanism.RTC were preincubated with amiloride for 2 h at indicatedconcentrations ( A and B ) or NHE1 inhibitorsethyl- N -isopropyl-amiloride (EIPA, 5 µM, 2 h, 37°C)or hexamethylene amiloride (HMA, 100 µM, 2 h, 37°C; C and D ) and then exposed to hypertonic media(DMEM-F12 300 mM NaCl, 5 h, 37°C). Apoptosis wasdetermined by DAPI staining of condensed chromatin ( A and C ) and annexin V labeling of externalized phosphatidylserine( B and D ) by immunofluorescence microscopy. Dataare presented as means ± SE from 4 separate experiments.* P P

Hypertonicity-induced RTC apoptosis is mediated bycaspase-3 activation and decreased NHE1 expression. To determine whether NHE1 is linked to caspase activation inapoptosis due to hypertonic stimuli, we incubated RTC withhypertonic NaCl and amiloride and then probed for activation ofcaspase-3, which is the final downstream executioner caspase in manyapoptosis signaling cascades. Figure 4 A shows that amilorideaccentuated hypertonicity-induced cleavage of the caspase-3 substratePARP, suggesting that NHE1 activity opposes apoptosis, perhapsby inhibiting decreases in cytosolic pH, which enhance caspase-3activity ( 30, 45 ). Figure 4, B and C, demonstrates that hypertonic NaCl induction of caspase-3activity was associated with diminished NHE1 expression, indicatingthat NHE1 may represent a caspase-3 target, which is consistent withthe possibility that NHE1 dysfunction could contribute to the acidicand shrunken cell phenotype, as shown in Fig. 1.3 g. Z# ]7 S8 X) K+ v& w
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Fig. 4. Hypertonicity-induced RTC apoptosis is mediatedby caspase-3 activation and decreased NHE1 expression. A :RTC were preincubated with amiloride at indicated concentrations for2 h and then maintained in hypertonic media (DMEM-F12 300 mM NaCl, 5 h, 37°C). Whole cell lysates were probed forpoly(ADP-ribose)polymerase (PARP) expression by immunoblot analysis.Caspase-3 activation was determined by PARP cleavage, which isindicated by the arrow. Human renal proximal tubule (HRPT) cells( B ) or hemagglutinin (HA) epitope-tagged NHE1-transfectedHEK 293 cells ( C ) were incubated in serum-free media ( ) orin serum-free media plus 300 mM NaCl ( ) for 6 h at 37°C. Wholecell lysates were immunoblotted with anti-NHE1 IgG ( B, top ) or anti-HA IgG ( C, top ). Lysateswere blotted with anti-PARP IgG ( B and C, bottom ). The 85-kDa PARP cleavage product is marked by thearrow. Results are representative of 3 separate experiments. M r, molecular weight.4 c. B2 A  w# J: E1 \2 W4 l
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RTC apoptosis under isotonic conditions is associated withdecreased NHE1 expression. Because the data indicated that cell shrinkage-induced RTCapoptosis is mediated by NHE1 inhibition and caspase-3activation, we queried whether NHE1 could be a caspase-3 substrate. Toexplore this possibility, we transiently cotransfected HEK 293 cellswith an NHE1 cDNA construct containing a carboxy-terminal HA tag and GFP cDNA (to mark transfected cells), followed by STS incubation tostimulate caspase-3-dependent apoptosis. Nuclear morphology andNHE1 expression patterns were determined by standard, fluorescence microscopy. Figure 5, A and B, demonstrates representative, transfected, apoptotic, and nonapoptotic cells. Approximately 20% of allcells underwent apoptosis, but only a small percentage oftransfected, apoptotic cells expressed NHE1 on the cell surface(Fig. 5, C and E ). Conversely, almost alltransfected, nonapoptotic cells expressed NHE1 in a plasma membranedistribution (Fig. 5, C and E ). Similar resultswere observed in RTC (data not shown). The results from theseexperiments suggest that NHE1 is cleaved during apoptosis andthat loss of NHE1 expression renders cells susceptible toapoptosis.# n0 W: n3 t. Y6 w8 _" p: Y6 Z
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Fig. 5. RTC apoptosis under isotonic conditions is associated withdecreased NHE1 expression. HEK 293 cells were cotransfectedwith carboxy-terminal, HA-tagged NHE1 and green fluorescence protein(GFP) cDNAs (1 µg/well with each vector) then incubated with STS (1 µM, 5 h, 37°C) to induce apoptosis. Fluorescence (notconfocal) micrographs show transfected cells (green, A ),apoptosis [fragmented nuclei stained with DAPI (blue)]( B ), NHE1 expression by immunocytochemical staining withbiotinylated anti-HA IgG and Texas red-conjugated streptavidin (red, C ), and merged images ( D ) from A-C. Results are representative of 3 experiments. E : 200 GFP-positive cells per experiment were scored forapoptosis, as defined by DAPI labeling of condensed chromatinand plasma membrane NHE1 expression, by immunocytochemical staining, asdescribed in C. Quantitation of NHE1 expression inapoptotic vs. nonapoptotic cells is shown in E.Results represent means ± SE from 3 experiments.* P t -test.1 R6 S# J, P+ n' y
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NHE1 reconstitution promotes resistance to apoptosis. To further investigate the role of NHE1 in apoptosis, wecompared NHE1-deficient PS120 cells, which were derived from Chinese hamster ovary fibroblasts ( 37 ), and NHE1-expressingcontrol fibroblasts (CCL39 cells) for susceptibility to STS-inducedapoptosis. As shown by DAPI and annexin V assays in Fig. 6, A and B,respectively, apoptosis was observed in a significantly greaterpercentage of PS120 cells compared with the CCL39 cells, consistentwith a recent report in these two cell lines ( 2 ). NHE1function was subsequently addressed by add-back experiments, in whichPS120 cells were transiently transfected with increasing concentrationsof NHE1 then stimulated with STS and assayed for apoptosis.Figure 6 C demonstrates that NHE1 reconstitution in PS120cells conferred resistance to apoptosis. At the highest NHE1expression levels, apoptosis was equivalent to control CCL39cells (Fig. 6, A and B ).
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Fig. 6. NHE1 reconstitution promotes resistance toapoptosis. A and B :NHE1-deficient PS120 fibroblasts and NHE1-expressing CCL39 fibroblasts(control) were treated with STS (1 µM, 5 h, 37°C).Apoptosis was determined by annexin V labeling ( A )and DAPI staining of fragmented nuclei ( B ). Results aremeans ± SE from 3 experiments. * P t -test. C : PS120 cells were transfected with graded concentrationsof HA epitope-tagged NHE1 cDNA (expressed as [NHE1 cDNA] inµg/well) followed by STS stimulation (1 µM, 5 h, 37°C). NHE1expression is shown by immunoblot ( top ) andapoptosis of corresponding groups, as determined by annexin Vlabeling and quantitation by immunofluorescence microscopy( bottom ). Histograms are presented as means ± SE.* P D : PS120 cells were transientlycotransfected with GFP (to mark transfected cells) and empty vector(Mock), wild-type NHE1 (WT), an NHE1 mutant that does not bind ezrin,radixin, and moesin (ERM) proteins (KR/A), or aNa /H exchange-defective NHE1 mutant (E266I).Cells were then stimulated with STS (1 µM, 5 h, 37°C) toundergo apoptosis. %Apoptosis in the transfected cellsubpopulation was determined by annexin V labeling andimmunofluorescence microscopy. Data are presented as means ± SDfrom 3 experiments. * P
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To assess which NHE1 domains are required for apoptosisresistance, we transiently transfected PS120 cells with wild-type NHE1,an NHE1 mutant that does not bind ERM proteins due to multiple K/A orR/A substitutions in residues 553-564 (KR/A) or anNa /H exchange-defective NHE1 constructcontaining a point mutation in the third cytoplasmic loop (E266I).Significant differences in transfection efficiency were not observedbetween groups (not shown). Cells were then stimulated with STS andassayed for apoptosis. Similar to results in Fig. 6, A and B, PS120 cells were susceptible toapoptosis, which was reversed by transfection of wild-type NHE1expression (Fig. 6 D ). Expression of KR/A mutant NHE1partially restored cell viability (Fig. 6 D ), implying thatNHE1-ERM binding is not the sole determinant of apoptosisresistance. Importantly, E266I expression did not rescue cells fromapoptosis (Fig. 6 D ), which indicates thatNa /H exchange is critical forapoptosis resistance.5 d' l" o' L/ y" P  [, K

RTC apoptosis leads to diminished NHE1 expression byprotein degradation. Because NHE1 has a long (~24 h) half-life ( 9, 12 ) and isnot significantly regulated by membrane cycling ( 38 ),rapid NHE1 disappearance with STS incubation suggests an NHE1degradation mechanism, rather than suppressed synthesis. To determinemore definitively whether STS-induced decreases in cell surface NHE1 were due to protein degradation, we conducted 35 S-labeledpulse-chase experiments in HEK 293 cells transfected with HA-NHE1 cDNAand then induced them to undergo apoptosis with STS.Immunoprecipitation with anti-HA IgG revealed diminished 35 S-labeled NHE1 beginning 2.5 h after STS incubation,which was markedly more pronounced at 5-6 h (Fig. 7 ), indicating that STS stimulates NHE1degradation. 35 S-labeled NHE1 levels did not appreciablychange in unstimulated cells from 0 to 4 h, consistent with thelong NHE1 half-life (not shown). More importantly, 35 S-labeled NHE1 levels were significantly greater inunstimulated compared with STS-stimulated cells at 4 h (notshown), further indicating that NHE1 is degraded withapoptosis. Because STS is a known caspase-3 activator, the dataalso suggest a caspase-3 mechanism of NHE1 degradation.# }7 J2 p0 K% V" w; t! B

Fig. 7. RTC apoptosis leads to diminished NHE1 expressionby protein degradation. A : HEK 293 cells weretransiently transfected with HA-tagged NHE1 cDNA, metabolically labeledwith [ 35 S]methionine, and then treated with 1 µM STSfor indicated times. Cell lysates (200 µg/sample) wereimmunoprecipitated with anti-HA IgG and resolved by SDS-PAGE. Imagerepresents autoradiogram from a dried gel. B : individualbands were digitized by phosphorimager, quantified, and normalized tocontrol (time = 0) values. Results are means ± SE from 3 separate experiments. * P
! w1 ^; q2 {$ e& ?; h
Although HA-tagged proteins have been successfully employed as caspasesubstrates ( 8, 11, 48 ), because the HA epitope sequencecontains a putative caspase-3 cleavage site (YPY DPVD YA), pulse chase experiments were also conducted in untransfected RTC, followed by immunoprecipitation of endogenous NHE1 with rabbit polyclonal anti-human NHE1 IgG raised against the membrane-proximal cytoplasmic domain ( 23 ). These studies also revealed NHE1degradation (Fig. 9 A ), with a similar kinetic pattern as inFig. 7, demonstrating that NHE1 is degraded during apoptosis.

NHE1 is degraded by caspase-3. To determine whether apoptosis-associated NHE1 degradation isdue to caspase cleavage, we transiently transfected RTC with carboxy-terminal HA, epitope-tagged NHE1, and stimulated them with STSto undergo apoptosis in the presence or absence of the cell-permeable, broad-spectrum peptide caspase inhibitor z-VAD-fmk orthe peptide caspase-3 inhibitor z-DEVD-fmk. Whole cell lysates wereprobed for NHE1 expression and PARP cleavage by immunoblot analysis.Figure 8 A demonstrates thatSTS induced concomitant caspase-3 activity and NHE1 degradation in HEK293 cells, and both caspase-3 activity and NHE1 degradation werepartially inhibited by z-DEVD-fmk or z-VAD-fmk preincubation. Similarresults were observed in RTC (Fig. 8 B ). These data stronglysuggest that NHE1 is cleaved by caspase-3 during apoptosis.( [6 F+ {# J8 z" @* A, }

Fig. 8. NHE1 is degraded by caspases. HEK 293 cells( A ) or cultured RTC ( B ) were transientlytransfected with carboxy-terminal, HA-tagged NHE1, preincubated withthe caspase-3 inhibitor z-DEVD-fmk (100 µM, 1 h, 37°C) orthe broad-spectrum caspase inhibitor z-VAD-fmk (100 µm, 1 h, 37°C), and then incubated with STS (1 µM, 5 h, 37°C).Whole cell lysates were probed for NHE1 expression by immunoblottingwith anti-HA antibodies. Individual bands were digitized byphosphorimager, quantified, normalized to control = 100, andshown in histograms beneath the corresponding blots. Results aremeans ± SE from 3 separate experiments. Note that y -axes are discontinuous. Blots were stripped andre-probed for PARP, with PARP cleavage product designated by thearrows. * P' J% i4 Y# l3 g

To further define whether NHE1 is a caspase substrate, we metabolicallylabeled RTC, induced them with STS to undergo apoptosis, andanalyzed anti-NHE1 immunoprecipitates for potential caspase cleavageproducts. Figure 9 A revealssimultaneous NHE1 degradation and appearance of several bands rangingin size from M r ~15 to ~32 kDa, suggestingthat NHE1 is cleaved by caspase-3. To directly assess whether NHE1 is acaspase target, we incubated in vitro-translated cNHE1 with recombinantcaspase-3 in the presence and absence of the caspase-blocking peptideAc-DEVD-CHO and then evaluated it for degradation. As shown in Fig. 9 B, in vitro degradation of cNHE1 by caspase-3 resulted inthe generation of peptides identical in M r compared with NHE1 peptides derived from STS-stimulated whole celllysates (Fig. 9 A ) in the absence, but not presence, of thepeptide caspase inhibitor. A 37-kDa band was observed in controlconditions (Fig. 9 B ), which was not inhibited byAc-DEVD-CHO, suggesting that this product was nonspecifically generatedby caspase buffer alone. However, the other DEVD-inhibitable bands (shown by arrows) represent true caspase cleavage products. Together, data from Figs. 8 and 9 provide compelling evidence that NHE1 is acaspase-3 substrate.

Fig. 9. A : [ 35 S]methionine-labeled RTC,treated with 1 µM STS for indicated times, were immunoprecipitatedwith affinity-purified rabbit polyclonal anti-NHE1 IgG. B :[ 35 S]methionine-labeled, in vitro-translated cNHE1protein (3 µl) was preincubated with or without the peptide caspaseinhibitor Ac-DEVD-CHO (DEVD, 100 µM, 2 h) and then withrecombinant caspase-3 (casp-3, 1-2 µl, 6 h, 30°C) asdescribed in MATERIALS AND METHODS. The reaction productwas resolved by 14% SDS-PAGE, and the dried gel was exposed to filmfor 3 h. The most prominent, common bands from A and B are demarcated by arrows. Results are representative of 3 separate experiments. cNHE1, NHE1 cytosolic domain.' x; `! u6 q* ?# h& F7 f
! f! a/ \$ r; N9 _5 N. J) \# E
NHE1 regulates RTC apoptosis in vivo. To test the role of NHE1 inactivation as a mechanism of RTC deletion inan in vivo model of progressive renal disease, wild-type (C57BL/6.SJL  /  ), NHE1 loss-of-function mutant (C57BL/6.SJL swe/swe ), and heterozygote (C57BL/6.SJL swe/  ) mice underwent adriamycin infusion to inducenephropathy ( 49 ). Kidneys removed 10 days postinfusionrevealed little histopathological damage in any of the genotypes (datanot shown). RTC apoptosis was rarely observed in wild-typecontrols (Fig. 10 A ),consistent with a failure of adriamycin to cause nephropathy in micewith a C57BL/6 genetic background ( 49 ). However,significant increases in RTC apoptosis were observed in C57BL/6 swe/swe and C57BL/6 swe/  compared with C57BL/6  /  mice (Fig. 10, B and C ). Becauseneither C57BL/6 mice infused with adriamycin nor NHE1 loss-of-functionmutant C57BL/6 swe/swe mice demonstrate histological orfunctional kidney abnormalities ( 13, 49 ), the data suggestthat NHE1 inhibition unmasked a renal phenotype in adriamycin-treatedC57BL/6 mice. In accordance with our previous reports demonstratingthat RTC apoptosis precedes and contributes to tubular atrophy( 20 ), these studies indicate that NHE1 conferscytoprotection, whereas loss of RTC NHE1 function is associated withapoptosis and tubular atrophy.+ h; ?- ~* `" R/ I; c9 R

Fig. 10. NHE1 regulates RTC apoptosis in vivo. Wild-type(C57BL/6  /  ), mutant C57BL/6 swe/swe, andC57BL/6 swe/  mice were injected by tail vein withadriamycin (10 µg/g) and killed 10 days later. Mice were genotyped byimmunoblotting liver lysates (20 µg protein/lane) with anti-NHE1antibodies ( C, top ). RTC apoptosis wasassayed by immunofluorescence terminaldeoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)labeling of frozen kidney sections. Representative fields (×20magnification) are shown for C57BL/6  /  ( A ) and C57BL/6 swe/swe ( B ) kidneys. Quantitative measures ofapoptosis for each genotype are expressed as means ± SEand shown in C, bottom. * P swe/swe and swe /  groups byANOVA.; q$ J3 v2 s$ ?, ^: T/ o1 ^( L
* c, B1 _! p7 C" y
DISCUSSION
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NHE1 is ubiquitously expressed, and activation has been linked tovital housekeeping functions such as cell volume regulation ( 31, 33 ) and growth factor-dependent proliferation ( 4, 32, 44 ). Decreased NHE1 activity has been associated with lymphocyteapoptosis ( 27, 40 ), but a specific mechanism of NHE1 inhibition in apoptosis has not previously been described. A major finding in our study is that RTC NHE1 is inhibited by caspasecleavage. The results have potentially broad implications to diseasepathogenesis, inasmuch as the data were generated in both epithelialand mesenchymal cells and in response to apoptosis induction byhypertonic stress or STS.
! l4 }7 _3 O) @+ a+ f9 p  x
Many epithelium-derived cell lines were previously considered to beresistant to apoptotic cell volume reduction due to robust expression and function of RVI pathway components ( 6 ).However, our data demonstrate that RTC RVI can be overcome, permitting apoptosis to proceed. Of the transporters that mediate RVI, we focused on NHE1 partly because it is responsible for intracellular volume regulation, and an anticipated consequence of NHE1 inhibition, cell shrinkage, is a characteristic apoptotic feature. Indeed, wefound that RTC apoptosis is associated with diminished cell volume, consistent with NHE1 inhibition. Our data do not exclude rolesfor regulatory volume decrease pathways, which can be activated inapoptosis ( 33 ). In addition, other transporters( 7, 29, 52 ) may participate in RTC RVI triggered byapoptotic stimuli. For example, inhibition of NHE3, which isexpressed on the apical proximal RTC membrane, could conceivablycontribute to RTC shrinkage and acidosis. However, because hypertoniccell shrinkage suppresses NHE3 activity ( 19 ), we reasonedthat apoptotic cleavage of NHE3 was unlikely to result insignificant further suppression of transporter activity. Inhibition ofother transporters that have been implicated in RVI, such as the AE2Cl /HCO 3 − exchanger or the BSC-1 andBSC-2 isoforms of the Na /K /2Cl cotransporter, could also be involved in RTC apoptosis, byimpeding intracellular volume expansion. However, prominent functionsfor these proteins in proximal RTC apoptosis are unlikely,since none are abundantly expressed in the proximal tubule ( 1, 15, 18 ).( q. Q6 M2 H; z

In addition to cell shrinkage, RTC NHE1 degradation was also associatedwith intracellular acidification ( 16 ). The data are inagreement with studies in leukocyte cell lines, which demonstrate thatapoptosis is preceded by decreased pH i ( 16, 27, 36 ). Importantly, NHE1 stimulation prevented intracellularacidification and abrogated apoptosis ( 16, 36 ),indicating NHE1 activation opposes apoptosis. Rich et al.( 40 ) demonstrated that human leukemia cells exhibit asignificantly higher pH i compared with normal leukocytelineage cells. Moreover, decreased pH i was associated withincreased apoptosis, which was exacerbated by exposure to theNHE1 inhibitor HMA. Barrière et al. ( 2 ) recentlyreported that apoptosis was also associated with intracellularacidification in Chinese hamster lung fibroblasts and that NHE1-inducedincreases in pH i were sufficient to preventapoptosis. All of these findings are consistent withobservations that the optimum pH for endonuclease and caspaseactivation is 6.3-6.8 and support the notion that intracellularacidification is critical for apoptosis execution ( 16, 30 ). Grinstein's group ( 5, 25 ) has shown thatexpression of 566 NHE1 mutants (cytoplasmic domain deletion membranedistal to Met566) resulted in impaired osmoregulation, as well as aconstitutively decreased resting pH i, suggesting thatapoptotic NHE1 cleavage upstream from this domain would yield asimilar phenotype. Together, our data in RTC are consistent with NHE1inhibition contributing to both apoptotic acidification andintracellular volume dysregulation.
0 @' r# Q/ ?  v
Although the role of NHE1 as a Na /H exchangeris applicable to apoptotic cell volume decrease, a number ofstructural proteins must also be cleaved to achieve a shrunken cellmorphology. Denker et al. ( 14 ) recently demonstrated thatNHE1 is tethered to the plasma membrane and cytoskeleton through directinteraction with actin-binding ERM proteins. On the basis of thisdiscovery, NHE1 would appear to function as a cytoarchitecture scaffoldand require disassembly during apoptosis, consistent with ERMprotein dissociation from plasma membrane during apoptosis( 24 ). We reasoned that NHE1 may therefore conferapoptosis resistance by anchoring the actin cytoskeleton to theplasma membrane via interactions with ERM proteins. However, expressionof NHE1 constructs with point mutations (KR/A) that abolish ERM binding( 14 ) resulted in partial rescue of RTC fromapoptosis, whereas expression of E266I mutants with intact ERMbinding domains had no effect on apoptosis. Results from thesestudies suggest that mechanisms in addition to NHE1-ERM interactionsmust be required for apoptosis resistance. Furthermore, ourdata do not exclude the possibility that interactions between otherNHE1 domains and the cytoskeleton may be important for maintenance ofcell volume and resistance to apoptosis. Because the NHE1 KR/A mutant has normal Na /H exchange activity( 14 ), we conclude that Na influx and/orH efflux are critical NHE1 functions for apoptosis resistance.; K! c& c6 t1 F. }. e. |

Evidence to support the hypothesis that NHE1 is cleaved by caspasesincludes apoptosis-dependent loss of NHE1 expression due toprotein degradation, rescue of NHE1 expression by preincubation withcell-permeable peptide caspase inhibitors, and direct cleavage of invitro-translated NHE1 by caspase-3. Although consensus caspase cleavagesites are identified in the carboxy-terminal human NHE1 cytosolic tail(e.g., 755-DEED-758), deletion mutation studies predict that cleavageat this site would not result in Na /H exchange-dependent osmoregulatory dysfunction ( 5 ).Furthermore, cleavage at distal carboxy-terminal site(s) would resultin generation of very small fragments and, therefore, would not accountfor the peptide band pattern observed in Fig. 9, indicating thatcleavage at additional membrane-proximal, noncanonical sites isrequired. Further degradation of NHE1 and/or NHE1 caspase cleavageproducts by other protease pathways is also possible, althoughpreliminary studies revealed that STS-induced changes in NHE1expression are not altered by pretreatment with the proteosomeinhibitors lactacystin, MG132, and PS-1 (Wu KL and Schelling JR,unpublished observations).* ~1 a; v8 f, t

Although neither in vitro stimulus of apoptosis (STS,hypertonicity) is encountered by RTC in vivo, these agents wereemployed to mimic cell stresses that result in RTC apoptosis invivo. We have previously shown that RTC apoptosis and tubularatrophy are caused by the in vivo stresses hypoxia and Fas activationin murine models of progressive renal disease ( 20, 43 ). Inthe current studies, the in vivo role of NHE1 was established bydemonstration of increased RTC susceptibility to apoptosis inNHE1-deficient mice with adriamycin-induced nephropathy. To maximizethe likelihood of detecting apoptotic RTC before obliteration oftubulointerstitial architecture by renal scarring, we killed mice afteronly 10 days. Because of the short observation interval, animals didnot develop tubulointerstitial pathology, although we predict that thenatural history of enhanced RTC apoptosis is tubular atrophyand interstitial fibrosis.( r3 u  ?5 J6 d$ q6 g+ y2 O- n7 d

NHE1 is commonly referred to as a housekeeping protein, implying thatit is pedestrian and unregulated. To the contrary, NHE1 has been shownto mediate vital cell functions, and the current studies establish anew role for NHE1 as a defender against RTC death. We speculate that ininitial stages of RTC apoptosis in vivo, e.g., due toinflammation or uremia, NHE1 is likely to be activated in response tocell volume reduction cues. NHE1-dependent RVI may then be sufficientto prevent further cell volume shrinkage and perhaps even promote cellsurvival, provided the apoptotic stimulus is not too robust.However, once NHE1 is cleaved by caspases, the combined sequelae ofNHE1 inhibition, cytosolic acidification, and cell shrinkage promoteinexorable RTC apoptosis by optimizing pH i forfurther caspase activity and by bringing caspases in proximity tosubstrates via cell shrinkage.0 t$ v8 D. T+ J2 o# A
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ACKNOWLEDGEMENTS

We are grateful to Drs. Barber, Grall, Orlowski, Pouysségur,and Racusen for donation of reagents and to Dr. Eleanor Lederer forthoughtful observations and comments.$ |, F/ m5 {0 D* g
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