duannjarwin04 3/18/04 6:51 pm page 81 detection of injury ... · injury model in the rat,which...

The Journal of Applied Research • Vol. 4, No. 1, 2004 81

protein to creatinine ratio = 10 in con-trol compared to 125 in aminonucleo-side of puromycin-treated animals). At4 hours and on days 4 and 10 followingaminonucleoside of puromycin injec-tion, glomeruli were either microdissect-ed using a laser capture microdissectingmethod or were isolated by differentialsieving. Total RNA was prepared andanalyzed for Wilms’ tumor 1 gene levelsby RT-PCR. Because podocytes consti-tute a relatively small portion of totalglomerular cells, Wilms’ tumor 1 genemessenger RNA levels were factored bythose of podocin, a podocyte specificprotein found in the slit porediaphragms, whose messenger RNA lev-els do not change following aminonu-cleoside of puromycin induced injury. Inmicrodissected glomeruli there was a 43% decrement in Wilms’ tumor 1 geneexpression at 4 hours following adminis-tration of aminonucleoside ofpuromycin. This decrement was attenu-ated on days 4 and 10. These resultswere confirmed in glomeruli isolated bydifferential sieving in which the decre-

Detection of Injury-inducedChanges in Gene Expression of theGlomerular Epithelial Cell-specificMarker, Wilm’s Tumor-1, by LaserCapture MicrodissectionPu Duann, MD, PhDHideki G. Kawanishi, MDEugene J. Gross, BSPrasun K. Datta, PhDElias A. Lianos, MD, PhD

Department of Medicine, Nephrology Division, Robert Wood Johnson Medical School, NewBrunswick, New Jersey

KEY WO R D S : Wilms’ tumor, PA Nn e p h r o p a t h y, laser capture microdissec-t i o n , glomerular epithelial cell.

ABSTRACTThe Wilms’ tumor 1 gene is a transcrip-tion factor expressed in glomerularepithelial cells, where it regulatespodocyte cytoskeleton proteins that playa key role in glomerular capillary perm-selectivity to plasma proteins. It isunknown whether Wilms’ tumor 1 geneexpression changes in forms of glomeru-lar epithelial cell injury associated withproteinuria. In this study we employedthe aminonucleoside of puromycinmediated glomerular epithelial cellinjury model in the rat, which histologi-cally resembles human forms ofglomerular epithelial cell disease, toassess changes in glomerular Wilms’tumor 1 gene expression. A singleintraperitoneal injection of aminonucle-oside of puromycin (75 mg/kg) inSprague-Dawley rats induced protein-uria that peaked 10 days later (urine

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ment of Wilms’ tumor 1 gene was againmost pronounced (28.2%) at 4 hours.These observations indicate thatdecreased Wilms’ tumor 1 gene expres-sion is a very early molecular event inresponse to glomerular epithelial cellinjury.

INTRODUCTIONThe Wilms’ tumor 1 gene (WT-1) is atumor suppressor gene that plays a keyrole in the development of Wilms’tumor, an embryonic kidney cancer. Inaddition, it is crucial in kidney develop-ment during embryogenesis.1 Thus, WT-1knockout mice have no kidneys due tofailure of the nephric duct to grow outand apoptosis of the metanephricblastema. WT-1 may act as a transcrip-tion factor, transcriptional cofactor orpost-transcriptional regulator dependingon its spliced isoform and cell type. Askidney development and differentiationproceeds during embryogenesis, WT-1expression in the kidney is down-regu-lated, except in the visceral glomerularepithelial cells (podocytes) of themature glomerulus, where expressioncan be detected throughout life.Glomerular epithelial cells (GEC) linethe surface of glomerular capillaries fac-ing the urinary space. They play animportant role in maintenance of theglomerular basement membrane, andsupport the glomerular capillary tuft andcontrol glomerular filtration of protein.WT-1 was shown to be important inmaintaining normal GEC function. Thus,WT-1 is mutated in 94% of patients withDenys-Drash syndrome, in whom themost consistent finding is developmentof glomerular scarring. WT-1 mutationshave also been found in patients withnephrotic syndrome and in isolatedcases of glomerulosclerosis.2,3 ReducedWT-1 expression levels in transgenicmice result in glomerular lesions of pro-liferation or scarring. In these mice,there is also a striking reduction in 2

podocyte-specific genes that encode thecytoskeletal proteins, nephrin andpodocalyxin.4

While evidence supporting the sig-nificance of WT-1 in genetic forms ofGEC defects resulting in nephrotic syn-drome is solid, studies assessing changesin WT-1 expression in response to non-genetic forms of GEC injury are lacking.Non-genetic forms of GEC injuryinclude drug-induced, immune (ie, mem-branous nephropathy) and viral (ie, HIVnephropathy). A well-established formof drug induced GEC injury is the oneinduced by the anti-metaboliteaminonucleoside of puromycin (PAN).GEC are uniquely vulnerable to PANand the ensuing lesion histologicallyresembles human forms of GEC dis-eases, such as minimal change diseaseand focal segmental glomerulosclerosis.5

This study examined changes inglomerular WT-1 expression levels atearly and late stages of PAN-inducedGEC injury.

METHODSExperimental Design and TissueProcessingMale Sprague-Dawley rats (130 to 150g) were anesthetized with intraperi-toneal injection of 0.1 cc of ketamine:xylazine (1:3 ratio) per 100 g bodyweight. They subsequently received asingle intraperitoneal injection of 75mg/kg of aminonucleoside of puromycin(Sigma-Aldrich, St. Louis, Mo) to induceglomerular epithelial cell (GEC) injury,as previously described.6 Control ani-mals were injected with equal volumesof normal saline.

A single single timed urine samplewas collected at 4 and 10 days followingthe PAN injection. Urine protein wasmeasured using a Bio-Rad Dc Proteinassay kit (Bio-Rad Laboratories,Hercules, Calif). Urine creatinine wasmeasured using a Sigma-Aldrich kit(Sigma-Aldrich, St Louis, Mo). The

Vol. 4, No. 1, 2004 • The Journal of Applied Research82


urine protein to creatinine ratio wasthen calculated to determine the extentof proteinuria. Following completion ofurine collections, rats were euthanisedand nephrectomies were immediatelyperformed at 4 hours, 4 days, and 10days after the PAN injection.

The kidneys were immersed in opti-mal cutting temperature compound(Tissue-Tek OCT compound, SakuraFinetek, Torrance, Calif) and placed inRNAse free cryomolds. The cryomoldswere placed in 2-methlybutane andcooled by immersion in liquid nitrogenuntil frozen. This process reduces archi-tectural distortion by shortening thefreezing time. Kidneys were then storedat -80˚C until processed for laser capturemicrodissection.

Tissue Sectioning and Staining for LaserCapture MicrodissectionUsing a Leica CM IS 50 Cryostat, kid-neys were sectioned at 6 µm and placedonto Arcturus HistoGene LCM Slides

(Arcturus, Mountain View, Calif). Theslides were then stained using theArcturus HistoGene LCM FrozenSection Staining Kit (Arcturus,Mountain View, Calif) according to themanufacturer’s protocol, and placed intoxylene to achieve dehydration andthereby, optimizing capture. The slideswere immediately used for laser capturemicrodissection (LCM).

Laser Capture Microdissection The Arcturus PixCell II Laser CaptureMicrodissection System (Arcturus,Mountain View, Calif) was employedunder RNase-free conditions to captureglomeruli. The stained sections wereremoved from xylene and allowed to airdry at room temperature for 5 minutesto evaporate xylene and was immediate-ly used for laser capture microdissection.Sixty glomeruli from each sample wereidentified and captured onto ArcturusCapSure HS LCM Caps. The laser set-tings were as follows: laser spot size 30


Figure 1. Proteinuria, expressed as urine pro-tein (Up) to urine creatinine (Uc) ratio, in PAN-treated rats at the points of study (days 4and 10) is shown. There was a markedincrease in urine protein excretion on day 10only. There was no detectable increase inurine protein excretion on day 4. Valuesshown are mean ± standard deviation (± SD).

Figure 2. Figure 2A shows a representativeglomerulus following laser dissection but priorto its capture on the vinyl film of the LCMcap. Figure 2B shows the empty space onthe remaining cortical section following cap-ture and removal of the glomerulus. Notethat, following capture and removal of theglomerulus, a number of glomerular cellsremained in the space previously occupiedby the glomerulus. In 2C, the capturedglomerulus on the LCM cap is shown. In 2D,the LCM cap surface following TRIzol lysis ofthe captured glomerulus is shown.


µm, power 50 to 55 mW, and duration of4.5 to 4.8 micro seconds. The number ofpulses to capture each glomerulus variedaccording to the size of the individualglomerulus. On an average, 4 to 8 pulseswere used to capture each glomerulusexcluding cells comprising theBowman’s capsule. After completion ofthe capturing step, sections and capswere examined to ensure adequatetransfer of glomeruli to the cap. Thetime from removal of each slide fromxylene to capture onto a HS LCM capwas less than 45 minutes.

Isolation of Glomeruli by DifferentialSievingGlomeruli were isolated according to anestablished technique first described byMisra.7 Briefly, the kidneys wereremoved, and the cortex was separatedfrom the medulla and finely minced.The glomeruli were separated fromother cortical tissues by passing theminced cortices through 2 different sizessieves (106-µm and 75-µm sieves). Withrepeat flushing, using ice-cold non-serum RPMI 1640 media, the glomeruliwere retained on the 75-µm sieve whilethe non glomerular tissue, mainly corti-

cal tubules, were washed through thissieve. The purity of glomerular prepara-tions was determined microscopicallyand was routinely 95% to 98%.

Preparation of Total Glomerular RNAand RT-PCRTotal RNA was extracted either fromcaptured or isolated glomeruli, using theTRIzol reagent method (Gibco BRL,Carlsbad, Calif). To isolate total RNAfrom captured glomeruli, HS LCM capscontaining approximately 60 glomeruliwere attached to a 0.5 mL eppendorftube containing 200µL of TRIzol via anadaptor supplied by the manufacturer(Arcturus, Mountain View, Calif).Glomerular RNA was precipitated in–80˚C overnight. To optimize RNArecovery, 20 µg of glycogen (an inertRNA carrier molecule) was added dur-ing the overnight precipitation. TheRNA was subsequently re-suspended in5µL of 10 mM Tris-EDTA/DEPC bufferand stored at 70˚C until RT-PCR wasperformed.

RT-PCRMessenger RNAs (mRNA) for WT-1,podocin, and glyceraldehyde 3-phos-


Figure 3. Levels of WT-1 and podocin mRNA detected by RT-PCR performed on total RNA isolat-ed from microdissected glomeruli. The bar graph is densitometric analysis of the gel and showschanges in WT-1/podocin ratio. A decrement in this ratio was most pronounced at 4 hours.



phate dehydrogenase (GAPDH) weredetected and quantified by reverse tran-scription-polymerase chain reaction(RT-PCR). Podocin, a podocyte-specificgene, was used as a marker for presenceof podocytes in captured glomeruli.Detection of GAPDH RNA served as amarker of comparable extent of captureof glomeruli from each cortical kidneysection. Aliquots of 5 µL of RNA wereused for complimentary DNA (cDNA)synthesis using the iScript cDNASynthesis Kit (Bio-Rad Laboratories;Hercules, Calif) according to the manu-facturer’s instructions. Aliquots of 3 µLof cDNA were used for PCR amplifica-tion of WT-1, podocin, and GAPDHusing 500 nM of gene specific primersand iQ Supermix (Bio-RadLaboratories, Hercules, Calif) in a totalvolume of 25 µL.

WT-1 primers were: forward: 5’-AGCATCTGAAACCAGTGAGAA-3’;reverse: 5’-CAAAATCAGATTTG-GAAGCAGT-3’.

GAPDH primers were: forward: 5’-GTGCTGAGTATGTCGTGGA-3’;reverse: 5’-CACAGTCTTCGAGTG-GCA-3’.

Podocin primers were: forward: 5’-ATTTCCTTGTGCAAACCACTAT-GA-3’; reverse:5’-CCAAGGCAACCTTTGCATCT-3’.

The PCR cycling profile was 94˚Cfor 5 minutes to activate iTaq DNApolymerase followed by 1 minute at94˚C, 1 minute at 56˚C, 3 minutes at72˚C. Thirty-five-cycle amplificationswere performed followed by a finalextension at 72˚C for 10 minutes.Fifteen µL of PCR products was sepa-rated on a 2% pre-cast agarose gel(Invitrogen, Carlsbad, Calif).

Semi-Quantitative AnalysisGels were visualized using a gel docu-mentation system (Bio-RadLaboratories, Hercules, Calif). The opti-cal density of the PCR products were

determined using a BioRad Gel Doc4.4.0 software program and a KODAKID image software. Because podocytesconstitute a relatively small populationof total glomerular cells, WT-1 mRNAlevels obtained by RT-PCR, as describedabove, were factored by those ofpodocin, a podocyte specific gene foundin the slit pore diaphragm. The observa-tion that mRNA levels of podocin donot change following PAN inducedinjury,8 makes podocin an appropriatemarker by which changes in WT-1 levelscan be factored.

Relative WT-1 expression wasdefined as WT-1 mRNA levels, deter-mined by densitometry, factored bythose of GAPDH. Relative podocinexpression was defined as podocinmRNA levels, determined by densitome-try, factored by those of GAPDH.Changes in WT-1 levels were expressedas changes in the relative WT-1/podocinratio.

RESULTSAminonucleoside-Induced ProteinuriaIn Figure 1, the degree of proteinuria(Up/Uc) in PAN-treated rats at thepoints of study (days 4 and 10) is shown.There was a marked increase in urineprotein excretion on day 10 only. Therewas no detectable increase in urine pro-tein excretion at 4 hours or on day 4.

Changes in Glomerular WT-1Expression Detected by LCMFigure 2A shows a representativeglomerulus following laser dissection butprior to its capture on the vinyl film ofthe LCM cap. Figure 2B shows theempty space on the remaining corticalsection following capture and removalof the glomerulus. Note that, followingcapture and removal of the glomerulus,a number of glomerular cells remainedin the space previously occupied by theglomerulus. In Figure 2C, the capturedglomerulus on the LCM cap is shown.


In Figure 2D, the LCM cap surface fol-lowing TRIzol lysis of the capturedglomerulus is shown.

The gel in Figure 3A shows levels ofWT-1 and podocin mRNA detected byRT-PCR performed on total RNA iso-lated from microdissected glomeruli.The bar graph in Figure 3B is the densit-ometric analysis of the gel in Figure 3A,and shows changes in WT1/podocinratio. A decrement in this ratio wasnoted at 4 hours. It returned to nearcontrol levels on days 4 and 10.

Confirmation of Changes in WT-1Expression in Glomeruli Isolated byDifferential SievingThe gel in Figure 4A shows levels ofWT-1 and podocin mRNA as deter-mined by RT-PCR performed on totalRNA of glomeruli, isolated by differen-tial sieving at 4 hours and on days 4 and10. The bar graph in Figure 4B showsthe densitometric analysis of the gel inFigure 4A, and shows the changes inWT-1/podocin ratio determined asdescribed in the methods section. Adecrement in WT-1/podocin ratio wasagain noted at 4 hours.

Magnitude of Decrement in WT-1 Levelsin Isolated Versus Captured GlomeruliThe bar graph in Figure 5 shows themagnitude of the decrement in WT-1levels in PAN treated animals expressedas the percent change compared to con-trol. The decrement in WT-1 level detect-ed in captured glomeruli was mostpronounced (43%) at 4 hours followingPAN treatment. The decrement in WT-1levels detected in glomeruli isolated bydifferential sieving was also most pro-nounced (28%) at 4 hours followingPAN treatment.

DISCUSSIONContact of glomerular epithelial cells(GEC) to the glomerular basementmembrane (GBM) involves a highly spe-cialized structure and signaling appara-tus in their foot processes and slitdiaphragms. Key protein constituents ofthis apparatus include nephrin, podoca-lyxin, podocin, and the cytoskeleton pro-tein synaptopodin, paxillin, actinin,vinculin, talin, a3/b1 integrin, andCD2AP. Nephrin, podocin, and a-actinin-4 are encoded by genes linked


Figure 4. Levels of WT-1 and podocin mRNA as determined by RT-PCR performed on total RNAof glomeruli isolated by differential sieving at 4 hours and on days 4 and 10. The bar graph is adensitometric analysis of the gel and shows the changes in WT-1/podocin ratio determined asdescribed in methods section. The number inside each bar is the actual ratio value. A decrement in WT-1/podocin ratio was most pronounced at 4 hours.


to diseases involving GEC and charac-terized by proteinuria. For example, thecongenital nephrotic syndrome gene wasmapped to chromosome 19q13, whichencodes nephrin, a 185 KD protein thatlocalizes to the GEC slit diaphragm andplays a role in regulating signaling path-ways.9,10 The steroid resistant nephroticsyndrome (autosomal recessive inheri-tance) gene was mapped to chromosome1q25-31 which encodes podocin, a 383amino acid protein that also localizes tothe slit diaphragm and interacts directlywith nephrin.11 Identification of factorsthat regulate podocin and nephrin and

characterization of changes in expres-sion of such factors following GECinjury may enhance our understandingof the biology of diseases involvingGEC. Such factors may also serve asmarkers of the extent of injury.

The transcription factor WT-1 wasshown to regulate GEC proteins such asthe aforementioned nephrin andpodocalyxin.4 WT-1 was cloned based onits role in the development of Wilm’stumor. Two clinical syndromes, theDenys-Drash and the Fraiser syndrome,are caused by WT-1 mutations1 and both


Figure 5. Percent decrement in WT-1 levels in isolated versus captured glomeruli. The decre-ment in WT-1 levels in PAN treated animals is expressed as percent change compared to con-trol. The decrement in WT-1 level detected in captured glomeruli was most pronounced (43%)at 4 hours following PAN treatment. The decrement in WT-1 levels detected in glomeruli isolatedby differential sieving was also most pronounced (28%) at 4 hours following PAN treatment.



are characterized by proteinuricglomerular disease. The glomerularlesion in Fraiser syndrome is focal andsegmental glomerular sclerosis (FSGS)while that in Denys-Drash is diffusemesangial sclerosis.1 This apparent regu-latory significance of the WT-1 transcrip-tion factor, prompted us to explorechanges in its expression at very earlyand advanced stages of a GEC injurymodel, induced by the anti-metaboliteaminonucleoside of puromycin andresembling human forms of GEC injury,such as minimal changes disease andFSGS.5 We employed the laser capturemicrodissection (LCM) method as it canbe used in human kidney biopsies, inwhich a small number of glomeruli areusually available for evaluation.

Although expression of the house-keeping gene GAPDH was detected byRT-PCR in as few as 5 glomeruli, detec-tion of the less abundant WT-1 andpodocin genes required capture of atleast 60 glomeruli. A technical pitfall ofthe LCM method is the apparentlyincomplete capture of all glomerularcells. Thus, as shown in Figure 2A, a con-siderable number of cells were leftbehind, following capture of the repre-sentative glomerulus shown. This mayimpact on recovery of GEC genes thatare expressed in low abundance. Thispitfall and the fact that GEC constituteonly a fraction of the glomerular cellu-larity necessitated factoring of the WT-1expression values obtained by those ofpodocin, a GEC specific gene.

Our results indicate that, followingPAN-induced GEC injury, there is adecrease in WT-1 expression and thatthis occurs very early and is transient.Thus, the decrease in WT-1 occurred asearly as 4 hours following PAN injectionand returned toward control levels bydays 4 and 10. Therefore, this event farproceeded proteinuria. Moreover, pro-teinuria had no further effect on WT-1expression when urine protein excretionbecame pronounced (day 10).

A number of studies have demon-strated structural changes in GEC thatoccur much earlier than the onset ofproteinuria. The earliest ultrastructuralchanges identified at the electronmicroscopy level in cultured rat GECare cell rounding, surface blebbing andmarked increase in number of microvilli.These changes are observed within 3hours of exposure to PAN.12 The earliestultrastructural change identified at theelectron microscopy level in vivo (ratkidney) is a reduction in the number offoot processes and filtration slits withloss of the normal arrangement of inter-digitating podocytes and displacementof the slit diaphragm.5,13 These changesare observed within 2 days following asingle dose of PAN, a protocol of PANtreatment similar to the one used in thepresent studies. Changes in GECcytoskeleton proteins whose expressioncould be linked to the WT-1 transcrip-tion factor also occur prior to onset ofproteinuria. This has been shown for a-actinin, whose levels of expressiontransiently increase within 1 day postPAN injection and return to control lev-els by day 3 (prior to onset of protein-uria). a-Actinin expression remains atcontrol levels on day 10 when protein-uria is pronounced,14 a pattern similar tothe one observed with WT-1 expressionin the present studies.

To confirm results on changes inWT-1 expression obtained by LCM, weemployed the well-established methodof glomerular isolation by differentialsieving. Purity of typical preparation ofglomeruli isolated by this method is90% to 95%.7 As shown in Figure 4, adecrement in WT-1 expression was againnoted at 4 hours following administra-tion of PAN. This decrement was againtransient with expression values return-ing towards control levels on days 4 and10.

The significance of the very earlydecrease in WT-1 expression in GEC

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injury remains to be established and tobe linked to changes in key cytoskeletonproteins that regulate glomerular capil-lary permeability to protein. Whetherassessment of WT-1 expression is ofvalue in human forms of GEC injury,such as minimal change disease andFSGS, also remains to be elucidated inkidney biopsy material from patientswith nephrotic syndrome resulting fromthese diseases. In this regard, LCMapplied to kidney biopsy material mayprove to be a valuable tool.

ACKNOWLEDGMENTSHideki G. Kawanishi was a recipient ofthe Kidney-Urology Foundation ofAmerica Fellowship. This work was sup-ported in part by a Paul TeschanResearch Fund grant from DialysisClinic Inc. awarded to Prasun K. Datta,PhD.

REFERENCES1. Pitchard-Jones K. The Wilm’s tumor gene,

WT-1, in normal and abnormal nephrogene-sis. Pediatr Nephrol. 1999;13:620-625.

2. Ito S, Ikeda M, Takata A, Kikuchi H, Hata J,Honda M. Nephrotic syndrome and end-stage renal disease with WT1 mutationdetected at 3 years. Pediatr Nephrol.1999;13:790-791.

3. Yang Y, Jeanpierre C, Dressler GR, LacosteM, Niaudet P, Gubler MC. WT1 and PAX-2podocyte expression in Denys-Drash syn-drome and isolated diffuse mesangial sclero-sis. Am J Pathol. 1999;154:181-92.

4. Guo JK, Menke AL, Gubler MC, et al. WT-1is a key regulator of podocyte function:reduced expression levels cause crescenticglomerulonephritis and mesangial sclerosis.Hum Mol Genet. 2002;11:651-659.

5. Caulfield JP, Reid JJ, Farquhar MG.Alterations of the glomerular epithelium inacute aminonucleoside nephrosis. Evidencefor formation of occluding junctions andepithelial cell detachment. Lab Invest.1976;34:43-59.

6. Koukouritaki SB, Tamizuddin A, Lianos EA.Enhanced expression of the cytoskeletal-associated protein, paxillin, in experimentalnephrotic syndrome. J Investig Med.1998;46:284-289.

7. Misra, R. P. Isolation of glomeruli frommammalian kidneys by graded sieving. Am JCell Pathol 1972;58:135-139.

8. Kawachi H, Koike H, Kurihara H, Sakai T,Shimizu F. Cloning of rat homologue ofpodocin: expression in proteinuric states andin developing glomeruli. J Am Soc Nephrol.2003;14:46-56.

9. Holzman LB, St John PL, Kovari IA, VermaR, Holthofer H, Abrahamson DR. Nephrinlocalizes to the slit pore of the glomerularepithelial cell. Kidney Int. 1999;56:1481-1491.

10. Huber TB, Kottgen M, Schilling B, Walz G,Benzing T. Interaction with podocin facili-tates nephrin signaling. J Biol Chem.2001;276:41543-41546.

11. Boute N, Gribouval O, Roselli S, et al.NPHS2, encoding the glomerular proteinpodocin, is mutated in autosomal recessivesteroid-resistant nephrotic syndrome. NatGenet. 2000;24:349-354.

12. Fishman JA, Karnovsky MJ. Effects of theaminonucleoside of puromycin on glomerularepithelial cells in vitro. Am J Pathol.1985;118:398-407.

13. Ryan GB, Rodewald R, Karnovsky MJ. Anultrastructural study of the glomerular slitdiaphragm in aminonucleoside nephrosis.Lab Invest. 1975;33:461-468.

14. Smoyer WE, Mundel P, Gupta A, Welsh MJ.Podocyte alpha-actinin induction precedesfoot process effacement in experimentalnephrotic syndrome. Am J Physiol.1997;273(1 Pt 2):F150-F157.


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