Functional Profile of the Isolated Uremnic Nephron

ROLEOF COMPENSATORYHYPERTROPIIY IN THE CONTROLOF

FLUID REABSORPTIONBY THE PROXIMALSTRAIGHTTUBULE

LEONG. FINE, WALTERTRIZNA, JACQUESJ. BOURGOIGNIE, andNEAL S. BRICKER, Department of Medicine, Divisioni of Nephrology,University of Miami School of Medicine, Miami, Florida 33152

A B S T RA C T An in vitro approach to the study ofsingle nephron function in uremia has been employedin evaluating the control of fluid reabsorption by therenal superficial proximal straight tubule (PST). Iso-lated segments of PSTs from the remnant kidneysof uremic rabbits (stage III) were perfused in vitroand their rate of fluid reabsorption comiipared withnormal PSTs and with PSTs derived from the remnantkidneys of' nonuremic rabbits (stage II). All segmentswere exposed to a peritubular bathing medium of'both normal and uremic rabbit serum thereby per-mitting a differentiation to be made between adapta-tions in function which are intrinisic to the tubularepithelium and those which are depenident upon auremic milieu.

Compared with normal and stage II PSTs, there wassignificant hypertrophy of the stage III tubules asevidenced by an increase in leingth aind internaldiameter, and a twofold increase in the dry weightper unit length. Fluid reabsorption per unit lengthof tubule was 70% greater in stage III than in normaland stage II PSTs, and was closely correlated withthe increase in dry weight. Substitutions between nor-mal and uremic rabbit serum in the peritubularbathing medium did not affect fluid reabsorptionsignificantly in any of the three groups of' PSTs. Per-fusioin of' the tubules with an ultrafiltrate of' normal vs.uremic serumn likewise failed to influence the rateof' net fluid reabsorption.

It has previously been observed that net fluiid secre-tion mlay occur in nonperfused or stop-flow per-fused normal rabbit PSTs exposed to human uremicserum. Additional studies were thus performed on

Dr. Fine is the recipient of a Research Career Develop-ment Award from the National Institutes of Health. Dr. Bour-goignie is an investigator of the Howard Hughes MedicalInstitute.

Receivedfor publication 8 August 1977 and in revisedform30 January 1978.

normiial and( stage III PSTs to evaluate whether netsecretioni occurs in the presence of' rabbit uremiiieserumii. No evidence for net secretioni was fi)und.

These std(lies (elenoiustrate that fltuid reabsorptioniis greatly inerease(d in the superficial PST of' theuiremiiie remnanlilit kidney and that this functionial adapta-tion is closelv correlated with compensatory hvper-trophy of the segmiienit. Humoral f'actors in the peri-tubular en-vironmiiienit do not appear to be importanitmedliators of the enhlan-ced fluid reabsorption.

INTRODUCTION

The progressive destruetion of' nephrons which char-acterizes miany fturms of' chronic renal disease leads tospecific adaptations in the surviving nephrons whichenable the organiismii to survive despite f;ar advanicedrenial failure. Such f'actors as compensatory hyper-trophy (1-3), alterations in the rate and distributionof intrarenial blood flow (4-5), the accumulatioin inthe extracellular fluid of' organic anions (6-8), and theelaboration of' hunoral factors which modify renaltubular transport (9-11) have been implicated in theadaptive responses of' the diseased kidney. Tubularfunctioin under these circumstances may be alteredeither by adaptations which are intrinsic to the tubularepithelium or to influences which are essentiallyextrinsic to the nephron. The respective roles of'intrinsic vs. extrinsic changes in determining a givenifunctionial patterni have hitherto been undefined andit is probable that a delicate interplay between thetwo elements exists. Conse(luently, further unider-standing of nephron function in uremia re(quires theability to identify and characterize each of' thenumerous influences which are exerted upon the sur-viving nephrois.

The present paper describes a new experimental ap-proach to the study of the functional characteristicsof the nephrons of the uremic kidney. Isolated seg-

J. Clin. Invest. C) The American Society for Clinical Investigation, Inc., 0021-9738178/0601-1508 $1.001508

milenlts of sinigle proximail straight tubules obtainedfroim (a) noriiail rabbits with tw\o intact kidneys,(b) noniureiic ral)b)its with one "reminilanit" andcl olleinitact kidney, and(I (c) uremic animiluals with a solitary"reminiiant" kidney were perfuisedl in vitro to ev/aluaitecliages in the initrinsic fuinctionial capacity of theuremic tubules. Each group of nephron segments wasstuidied in ani exteriial bathing imiediumiil of niorimial aiiduremilic seriium in anl effort to examiniiie the effectof a niorimial vs. uiremilic environment on their patterin offunction. The sttudies included an estimate of the de-gree of' hypertrophy of' the uremiiic nephronis as well asthe influence of uremiiic serumiii on fluid secretioni innoniperfuised neplhronis.

All of the data were obtaine(d on stuperficial proximiialstraight tubules (PST).1 Informiiation about the PST inuremiiia is of' particular interest in that this segmiienitcannot be evaluated ade(quatelv by clearance teeh-niq(uies and is inaccessible to micropuneture. Becauisecompensatory hypertrophy is prestumiied to ocecur in thePST of the remniiiant kidney, anl effort %vas imiade toestablish whether a correlation exists between fluidreabsorption anid structurial growth wvhich is sillilar tothat described for the proximal convoluted tubtule(1, 12). The studies were also (lesignied to dletermiiinexvhether fluiid reabsorptioni was influieniced by the bath-ing solution. Finally, in view of recent stuidies wvhichhave shown that hlutmiiian uremiiie serumiii canl convert nietfluid reabsorption to net secretioni in the PST ofnormal rabbits (8), the presenit studies evaluatedwhether rabbit uiremiiie serumiii had a sim-iilar effect.

The restults of the studv inidicate thalt PSTs ofuremic rabbits vith single remiiniiant kidnevs reabsorbfluid at a rate which is approximately 70% greatter perunit length thani that of nornmal PSTs. This augmiienitedreabsorptive rate is directly correlate(d withl the processof' compensatory hypertrophv of this segmenit anid isnot observed in PSTs from nionihpertrophied remniiianitkidneys. Fltuid reabsorption in both niormal anld uiremllicPSTs appeared to be an intrinisic property of' thetubtules anid was not altered by the presenee or ab-sencee of humiiioral factors in uremiiie seruml. Finally, noevidence for net fluid secretioni couild be demiionistrated.

METHODS

Experitmzetital groupsThree groups of' animiicals were uised as the souirec of the

PSTs sttudied. PSTs were obtained fromll (a) the left kidneyof' normiial rabbits (stage I animals), (b) the remnliaint kidl-nev of' nonuremic ralblits with ain i ntacet conitralaterall ki hlnev

'Abbreviationts used in thzis paper: BUN, blood urea nitro-gen; Jv, net fluid reabsorption; PD, potential differenice; PST,proximal straight tubule; SNGFR, sinigle niephroni glomerullarfiltration rate; UF, ultrafiltrate.

(stage II animals), and (C) the solitary remnant kidney ofuremic rabbits (stage III animals). Remnant kidneys wereprepare(d by exposinig the posterior surface of the pedicleof' the left kidney retroperitoneally via a flank incision andligating branches of the renal artery so as to infarct approxi-mately 70-85% of the kidney. In the stage III animalsthe contralateral kidney was removed during the same proce-dture. All animilals were mainitained on a diet of'standard rabbitchow and tap water for 1-4 mo before study. Body weightwas 3-4 kg at the time of study and was similar in all threegroups.

Dissection of tubulesThe animals were killed by a blow to the back of the neck,

the kidneys to be studied were rapidly removed, and a 1-2-mmcross section of' cortex was transf'erred to a bath of chillednormal rabbit serum. Cortical wvidth wvas measured to thenearest 0.5 mmni1. Superficial proximal straight tubules were ob-tainied by grasping the tubtules close to the corticomedullaryjunction vith fine forceps and dissecting them free in thedirectioni of' the outer cortex. A superficial PST was definedas one which joined a proximal convoluted segment in theoutermiiost 0.5 mmof the cortex. PSTs were dissected withrelative ease f'rom both stage II and stage III animals.

Preparation of sera and perfusion solutions

Normal rabbit serum was obtained commercially (Microbio-logical Associates, Inc., Walkersville, Md.). Sera from stageII and stage III rabbits were prepared by cannulating thefPemioral arterv at the time of removal of the kidney for dis-sectioni and collecting blood into a sterile glass beaker at roomtemiiperature. After clotting and centrifugation the serum wasdecanted and stored at -4°C. Before each experiment serawere bubbled with 95% 02-5%CO2 and the pH adjusted to7.4. The osmolality of' the normal and stage II sera and ofthe perfuisate (see below) were adjusted to that of the stageIII (uremic) serum by addition of urea so that all solutionsusedl in a given experiment were isosmolal. Final osmolalityvaried f'rom experiment to experiment between 295-330mosmlol/kg H20. Osmolality was measured with a vapor pres-.sure osmiiomiieter (Wlescor Inc., Logan, Utah) and was adjustedimimiediately before each experiment.

An artificial perfusate was designed to simulate end-proxi-mal convoluted tubular fluid (low bicarbonate and free of glu-cose and amino acids).2 This was used in the majority of ex-perimenits. The composition of the perfusate was as follows:(in millimiioles per liter) NaCl, 130; NaHCO3, 5; NaAcetate,10; Na2HP04, 3; KCl, 5; MgSO4, 1.2; and CaAcetate, 1.8. Thep1i wvas adjusted to 6.7. In certain experiments an ultrafiltrateof uremic (stage III) serum was used as the perfusate. Thiswas prepared by ultrafiltering the serum through a DiafloXM150 memibrane in an ultrafiltration cell (Amicon Corp., Lex-ingtoni, Mass.) at 4°C. The pH of this fluid was also adjustedto 6.7 with HCl.

2 Micropuncture data on the composition of end-proximaltutbular fluiid are not available for the rabbit. Since fluid re-absorption in the rabbit proximal convoluted tubule (13) ap-pears to be quantitatively similar to that observed in othermammiiiials, the assumptioni is made that quantitative changesin tubuilar fluid composition are similar in the two species.The perfusate composition is thus based on data obtained inthe rat (14) and the dog (15).

Flu id Reabsorption bil Uremic Proximal Straight Tubules 1509

Perfusion studies

Free-flow perfusion. Seven normal, eight stage II, andnine stage III PSTs were studied by this method. PST seg-ments 1.1-3.2 mmin length were transferred to a bath ofserum (volume 1.4 ml) maintained at 37°C and bubbled con-tinuously with 95%02-5% CO2 and viewed through an in-verted microscope at x40-400. The method of perfuision hasbeen described previously from this laboratory (10, 16). Bothends of the tubule were insulated with Sylgard 184 (DowCorning Corp., Midland, Mich.) [carboxyl4C]inulin wasadded to the perfusate (50 gCi/ml) as an impermeable volumemarker. Assuming a normal single nephron glomerular filtra-tion rate (SNGFR) for the rabbit of' 20 nl/min (13) and re-absorption of 60% of the tubular fluid by the end of the proxi-mal convoluted tubule, the perfusion rate f'or the PST shouldbe approximately 8 nl/min for PSTs from normal kidneys and16 nl/min for those f'rom stage III remnant kidneys in whichSNGFRwould be expected to be approximately twice normal,based on rat micropuncture data (17, 18). Acordingly in eachexperimental group approximately one-half the experinmentswere conducted at the slower perfusion rates (6-10 nl/min)and one-half at the more rapid rate (14-20 nl/min). Sinceperfusion rate did not influieniee the results (see Tables I-III)the data for each experimental group have been pooled.

In each experiment three sets of observations were madese(Iuentially by changing the type of serum bathing the tubuile.Two diff'erent se(quences used were (a) normal serum, uremieserum, normal serum or (b) uremiie serum, normal serumil,uremie serum. The meani of the results fromii the first andthird periods were compared with the seconid period so thatany bias resulting froIm clhange in the preparationi as a funcltionof time was elimiiinatedl. Stage II PSTs were studliedI in normal,uremiiie, anid stage II serumii with the order being v!aried rani-domly.

Additional experiments were conducte(d on five normiialPSTs bathed in normal serumil and four ureiniic- PSTs bathedin uremie serum. Each tubtule was perfused se(tuenitially withan ultrafiltrate of' normal serumii ancl an ultrafiltrate of uremiiieserum. The order was varied randomly for eaclh experimenit.The osmolality of the two ultrafiltrations aiud of the bath wasideintical in each experiment.

In each of the experimental periods a 15-min period of equil-ibration was allowed before sample collections were begun.Four-five consecutive timed collections ofttubular fluid weremaade under mineral oil into a constrictioni pipette wvhich wascalibrated at the terminiiation of' the experimenit.

Samples of tubular fluiid were pipettedl directly into A(Iuasolli(uid scinitillation fluid (Nev England(i Nuclear, Bostoni,Mass.) and total counlts per minute measuredl with a PackardlTricarl) li(uid scinitillationi couniiter (Packard Inistrtumnenit Co.,Inc., Downers Grove, Ill.). Perfutsioni rate (V,) was calculatedas V0, = SlI(So)t wlvere SL is the total amiiouniit of isotope col-lected, (S,,) is the coneniitration of isotope in the perftisate,and t is the time. Net fluiid imovemiienit, Jv, expressed as nianio-liters per miiillimneter ttubule per mmiin, is thuts (V(-VL)/L wvhereV, is the collection rate and L is the length of the tuibule.Tuibuile lenigth and(l internial diamiieter were measuired dulrinigperftusioni with a calibrated reticle in the ocuilar of the mnicro-scope.

rhe perfiusion pipette vhiclh served as a luminial electrodewas connecte(l to a calomel halfLcell throuiglh a 0.16-M NaCl-4% agar bridge. The hallf' cell wvas connected to the inputof 'an imiipedaniee converter and the signial displayed on a Grassmodel 7 polygraplh with a model 7 P1 A lowv level DC pre-amllplifier (Gra.ss Inistrumiienit Co., Quii ncy, Miass.). The eircuiitwas comnpleted throuigh a 0.16-M NaCl-4% agar bridge con-niecti ng the bathl to a reference calomiiel lalc-cell connected

to ground through a precision millivolt reference source (W-PInstruments, Inc., New Haven, Conn.). Any small potentialdifference due to asymetry of electrodes was nulled beforethe experiment by means of a variable potentiometer in theimpedence converter. Transepithelial potential difference(PD) was monitored continuously throughout the experiment.A correction of +0.9 mVwas made for the liquid junctionpotential (19). This value was added to the recorded PD toderive the true transtubular PD.

The mean (+SE) length of PSTs perfused was 1.83+±0.18mmin normal rabbits, 1.70±0.20 mmin stage II, and 1.79+0.26 mmin stage III rabbits.

Stop-flotw perfusiont. 10 PSTs from stage III rabbits werestudied by this method. The arrangement for perfusion fol-lowed the method described by Grantham et al (8). The per-fusion pipette had a tip diameter of 12-15 gnm; approximately1 mnm f'rom the tip the internal diameter of the pipet lumenwvas exactly 75 gium over a length of 5 mm. A drop of' xylenewas sucked into the tip of the perfusion pipette followed bya smiiall amount of perfusate. The tubule was cannulated andperfused for approximiiately 30 s. A knuiekle of' tubule nearits f'ree end was then sucked into a collecting pipette so asto occlude the lumen. The perfusion pressure was adjustedto 10 cm H2Oand remained constant throughout the experi-ment. Sylgard 184 was used to insure a water-tight seal atthe perfusion end. Movement of the oil droplet toward thetubuile indicated net fluid reabsorption and movement awayfromii the tub)ule net secretion.

Net fluid movement was calculated as:

Tx ( ID 2Jv =

tL 2)'

where X is the distance traveled by the oil droplet in time t,L is the lengtlh of the tubule, acnd ID is the innier dianmeteroftthe pipette. Tubuile length anid pipette diamiieter were ineas-tired with a calibrated reticle in the ocular of'the microscope.

Notperrfised tuibtiles. Grantlhamii and co-workers havedemonistrated the plheniomiienioni of lu-meni expansion in non-perfUsed PSTs f'rom normncal rabbits exposed to huiman uiremicseruim (8). The teclhnii(qute (lescribedl by these workers wasused to study fltuid secretioni in nioniperfiused tuibuiles. Five-sixsegments of superficial PSTs from normal and stage III kidneyswere dissected f'rom the cortex anled tranisferred in approxi-miately 5 gl of normnal rabbit serumiii to eaclh of six empty wellsof a Terasaki Microtest tissuie etltuire plate (Falconi Plastics,Div. of Bio0Quest, Oxnard, Calif.). The tubules were observedat x440-400u)(nler an iniverted microscope. Oinly tubuleswvhiicl were comipletely collapsed and(I without a visible lulmewere sttIdiedl (see Restults).

Sera f'roml five tiremiiie rabbits were tested f'ur their abilityto stimuit late fluiid secretioni on the ttubular segmiienits of eachanimal. 25 ,lI of eachl test serumiii was added to eaclh well. 25gl of'norm-iial rabbit serumiii cmotaining 1 imlMIp-aminohippuratewas uised as a con-trol inl eachi experiment.

The tissuie eultuire plates were tlhen incubated at 37°C( ini ahlumlidifie(d atln(osp)here with ani ambient gas mlixtture of 95%02 + 5% (()2. After 15 mimh of inictubation the PST were in-sp)ectedl for evidence of luiniial expansion. Failture to observeexpanisioni ini the control segimienits bathed inI mMp-amino-hippurtiate was takeni as evidence of ionnresponsiveness of theparticuilar kid(l(ey tiuiler stlldvy ainid the experiment discardled.The appearanice of' a visible Ilumen in at least two tubules,whlethier this occtirred aloig the whole lenigth ot the ttibuleor ini a segimienit of it, was taken ats p)ositive evidenice for secre-tory activity.

Measiireimceiit oj tuibtulc dIrop uceiglbt. PSTs obtainied f'romthe same kidnievs as were sttidie(l in the f'ree-flow p)ertilsion

1510 L. G. Fine, W. Trizna, J. J. Bourgoignie, and N. S. Bricker

experiments wvere employed for measurement of dry weight.A 1-2-mmll cross section was transferred to a dish of chilledp)rotei n-free Ringers' b)icarbonate solution. Stuperficial PSTswere dissected as described above. A combined lengtlh of8-14 mm-ni was transferred in a droplet of the dissecting m11e-dium to single wells of a tissue culture plate. The tubuleswere photographed through an inverted microscope at x54and(I were then transferred with finie forceps to a tare(l p)ieceof aluminuim foil and dried ftor 60 min in a dry oxen at 80'C.They were theni weighed on a Perkini Elmner model AD2 Pre-cisioni Electronie balance (Perkin-Elmer Corp., InistrumilenitDiv,., Norwalk, Conn.) (resoltution 0.1 ,ug). The total length ofthe weighed segments was measured from the photograph.Dry wveight wvas expressed in micrograms per millimiieter. Du-plicate measuremiienits made on tubuiles from the same kidney(fiour observations) varied by less thani 10%.

Analytic methodsSodiiumii and potassitum concenitrations of' bath and perflisate

soluitionis were measured by flame photometry (Instrumllenlta-tion Laboratory, Ine., Lexington, Nlass.). Chloride conieentra-tion wvas measured with a Radiometer CMT10 chloride titra-tor (Radiometer Co., Copenhagen, Denmark). Blood ureanitrogen was measured by the Berthelot method (20).Creatinine concentration was measured with an AutoAnalyzer(Technicon Instruments Corp., Tarrytown, N. Y.). Serumhippurate concentration was measured according to themethod of Smith et al. (21). This method detects a numberof hippuric acid derivatives (including mn- and p-amino-hippuric acids, p-ammophenaceturic acid, p-aminophenyl-stuceinutiric acid, p-aminobenzoic acid, and p-aminomandelicacid). Sinee the p)urpose of the assay was to compare hippluratelevels betveen groups of sera rather than to (iilantitate theabsoluite levels of any given suibstancee, p-aminohipurate wasuised to set tup a standard curve, the colorimetric determiina-tioin being miiade at 10 min.

StatisticsResuilts of perfusion experimaenits are expressed ats the mleani

± SE of four-five collectionis in each experimential period. Themiiean of the iniitial plus finial periods vas comppared with themiiddle period xvith paired t anialysis. WVhen dlifferenit groulpsof tul)ules were compared, ani uinipaired t test was used1.

RESULTS

MorphologyComparably sized remnant kidneys were created in

the stage IL and the stage III animals. 1-4 mo laterthe stage II remnant appeared unchanged in sizewhereas the stage III remnant kidneys had increasedin size. The estimated incremenit was approximatelytwofold. In comparing the glomeruli and the PSTs ob-tained from stage III vs. stage I kidneys, both strtuctureswere grossly enilarged (Figs. 1 and 2) and occasionallythe ttubtules were distorted in shape with localized areasof' dilatation.3

3 In isolated cases the surgical procedure appeared to haveled to a minor degree of internal hydronephrosis often vithsmiiall vellovislsb-browvn calculi lying within the pelvi-calyceal

Serum analysesSodium, potassium, and chloride concentrations

were not significantly different between sera obtainedfrom normal (150+2; 5.7+0.5; 102+1.0 meq/liter), stage11 (152±2; 5.9±0.7; 102+1.2 me(l/liter), or stage IIIrabbits (151±2; 5.9±0.7; 100± 1.2 me(l/liter). Bloodurea nitrogen (BUN) results are listed in Tables 1-111.Serum creatinine concentrations in the three groupswere 1.1±0.4, 1.2±0.6, and 3.4+±0.4 mg/100 ml. Mean±SE hippurate concentrations were 0.037+.01 mg/100ml in five normal sera and 0.07+.01 mg/100 ml in sixstage III (uremic) sera.

Tubule dry weight atnd sizeDry weights of the PSTs for each experiment are

included in Tables I-III and a composite plot of thedata is shown in Fig. 3. No diflference in weight perunit length was demonstrable in comparing normalwith stage II tubules. However, values for the stageIII PSTs were significantly greater than either the nor-mal (P < 0.001) or the stage II (P < 0.005) segments.

The superficial PSTs in all three groups of kidneyswere observed to extend f'rom the corticomedullaryjunction to the outermost cortex. Thus, f'rom measure-ment of cortical width an approximation of the totallength of these segments in each group could be ma(le.Cortical width averaged 2.9±0.1 mmn in the normnal kid-neys, 3.3+0.1 mmin the stage II kidneys, and 4.6±0.2mmin the stage III kidneys. PSTs from the latter grouipof kidneys were thus greatly increased in length. Anadditional assessment of the relative size of'the tubuleswvas obtained from measurements of'the internal diamn-eter during perfusion. The values were as follows: nor-mal 22.3+0.5 ,m; stage 11, 24.1±1.5 gm; and stage III,31.5±0.8 ,UIll. The internal diameter of stage III PSTswas significantly greater than the former two groups(P < 0.005). Thus, all parameters of' tubule size (dryweight, length, and internal diameter) indicate markedhypertrophy.

Net fluid reabsorptiotFREE-FLOWPERFUSION

Normal PSTs (Table I). Net fluid reabsorption was0.41+0.05 nl/mm per min in a bath of' normal serumand 0.37±0.07 nl/mm per min in uremic serum. These

system. In these kidneys the PSTs often had patent lumilensat the time of dissection. After incubation in niormiial rabbitserumii for 30 min, the lumens remainied patenit; stop-flow per-fusioni did not reveal evidence of net fluiil secretioni and thepateniey, vhich was never observed in normal PSTs, is pre-sumablv attributable to a combination of struettural hyper-trophy and long standing dlistentioni. Apart from three tubulesused in stop-floy perftusion experiments, nonie of the PSTswith a patent lumenivas used for functionial studies.

Fluid Reabsorptiotn by Uremic Proxinmal Straight Tubules 1511

:::SiR. v- * -* @b: .t .:

* B *: ;, R '. ',.'. o.

**'''.. . <w...-F --q .*

k **. "*......' ... tff -s ........ *--.R "^-_ +.'+* * i:> k Xl o, M,$ *.* _|!_}X_:1 r_F. .........:.X 4i.*...:.: *i: x. ...

E 7 ....

.. ,**:

....* .. ..... . s ** w: . : ,#.....

* * ';i l__ |

,w,_w,,..x x

..... V24ii..; '& ..

FIGURE 1 Isolated glomeruli from (a) a normal rabbit kidney and (b) a uremic remnant kidneyphotographed at the same magnification, showing marked hypertrophy of the latter.

values are not significantly different from each other.Transepithelial PD averaged + 1.5+±0.3 mV (lumenpositive) in normal serum and + 1.5 +0.2 mVin uremicserum.

Stage II PSTs (Table II). Net fluid reabsorption was0.45+0.55 nl/mm per min in normal serum, 0.41+0.05nl/mm per min in uremic serum, and 0.37+0.06 nl/mmper min in stage II serum. There is no significant dif-ference between any of these values. TransepithelialPDwas +0.3+0.2 mV(lumen positive) in normal serumand +0.6±0.2 mVin uremic serum and 0.5+0.2 mVinstage II serum.

Stage 111 PSTs (Table III). Net fluid reabsorptionwas 0.67+0.04 nl/mm per min in a normal serum bathand 0.73+0.05 nl/mm per min in a uremic serum bath.These results are not significantly different from eachother. Transepithelial PD was -0.4+0.3 mV (lumennegative) in normal serum and -0.4+±0.3 mVin uremicserum. The values for net fluid reabsorption wherean ultrafiltrate of uremic serum was used as the per-

fusate (see asterisks in Tables I-III) were not distin-guishable from the results with the artificial perfusate.

A comparison of net fluid reabsorptive rates betweenthe three experimental groups is shown in Fig. 4. Itmay be seen that the stage III PSTs absorbed fluidat a rate which was approximately 70% greater thanboth normal and stage II PSTs.

The data from all tubules studied indicated that therewas a significant correlation between net fluid reab-sorption and dry weight per unit length (r = 0.6958;P < 0.0005).

Influence of normal vs. uremic serum ultrafiltrate(UF) as perfusate. An ultrafiltrate of both normal anduremic serum was used to perfuse each of five normaland four uremic PSTs. In normal PSTs, Jv was 0.40+0.02 nl/mm per min with normal UF and 0.39+0.03nl/mm per min with uremic UF. In uremic PSTs, Jvwas 0.66+0.02 nl/mm per min with normal UF and0.70+0.05 nl/mm per min with uremic UF. Neither ofthese differences is significant.

1512 L. G. Fine, W. Trizna, J. J. Bourgoignie, and N. S. Bricker

ig-, ip,.

I

.a/

I

I #~~~

*.-.

/f 1///z e

/;7y~~~~I I/

FIGURE 2 Nonperfused superficial proximal straight tubules from (a) a normal kidney and (b)a uremic remnant kidnev photographed at the same magnification. The uremic segment is grosslyhypertrophied and has an irregular outer border.

TABLE ISize, Dry Weight, Transtubular PD, and Net Fluid Reabsorption of Superficial PSTs of Normal Rabbits

Net fluidKidney PD reabsorptioncortical Internal Dry Perfusion

BUN width diameter weight rate NS US NS US

mng/100 ml mm JAm pg/mm nl/min mV nil/mm/min

15.8 2.5 22.0 0.67 6.73 +2.4 +2.1 0.67 0.7815.0 2.5 22.4 0.56 7.21 +0.7 +0.7 0.40 0.2515.7 3.0 22.2 0.58 7.22* +1.5 + 1.0 0.31 0.2517.3 3.0 24.1 0.62 9.90 +1.1 +1.6 0.47 0.2016.8 3.0 20.8 0.41 14.36 +1.1 +1.1 0.31 0.3517.0 2.5 24.1 0.60 16.31 +2.5 +2.5 0.33 0.3815.8 3.5 20.6 0.58 18.24* +1.2 +1.4 0.36 0.34

Mean 16.2 2.9 22.31 0.57 11.42 +1.5 +1.5 0.41 0.37±SE 0.3 0.1 0.53 0.03 1.82 0.3 0.2 0.05 0.07

Potential diff'erence is corrected for calculated= uremic serum bath.* Ultrafiltrate of' uremic serum used as perfusate.

liquid junction potential. NS = normal serum bath; US

Fluid Reabsorptioni by Uremic Proximal Straight Tubules

b

1513

TABLE IISize, Dry Weight, Transtubular PD, and Net Fluid Reabsorption of Superficial PSTs of

Remnant Kidneys of Stage II (Nonuremic) Rabbits

Duration Kidney PD Net fluid reabsorptionpost- cortical Internal Dry Perfusion

surgery BUN width diameter weight rate NS US St II S NS US St II S

mo mg/100 ml mm JAm jigImm nl/min mV nil/mm/min

2.5 25.5 3.0 24.4 0.62 6.01 +0.4 +0.7 +0.5 0.36 0.39 0.373.3 22.1 3.5 22.8 0.62 6.78 +0.6 +0.6 +0.6 0.49 0.25 0.132.5 24.9 3.5 28.9 0.62 9.00* +1.2 +1.9 +1.9 0.39 0.43 0.372.3 21.4 4.0 24.4 0.64 10.22 +0.4 +0.5 +0.5 0.44 0.41 0.381.6 22.9 3.0 22.2 0.75 14.10 -1.1 -0.4 -0.6 0.75 0.69 0.690.8 24.3 3.5 31.1 0.65 16.44* +0.4 +1.1 +0.4 0.35 0.481.3 22.9 3.0 20.8 0.46 17.38 +0.8 +0.8 +0.8 0.28 0.37 0.342.5 23.5 3.0 17.8 0.69 18.54 +0.3 +0.1 +0.1 0.56 0.24 0.31

Mean 2.1 23.4 3.3 24.1 0.63 12.31 +0.3 +0.6 +0.5 0.45 0.41 0.37±SE 0.3 0.5 0.1 1.5 0.03 1.74 0.2 0.2 0.2 0.05 0.05 0.06

Potential difference is corrected for calculated liquid junction potential. NS = normal serum bath; US = uremic serum bath;St II S = stage II serum bath.* Ultrafiltrate of uremic serum used as perfusate.

STOP-FLOWPERFUSION

The effects of rabbit uremic serum on net fluid reab-sorption in proximal straight tubules from nine uremicrabbits is illustrated in Fig. 5. The experimental se-quence for each tubule was normal serum, uremic se-rum, normal serum. In four experiments, 1 mMp-ami-nohippurate was then added to the bath. Three of thetubules had patent lumina before perfusion. All tu-bules, including those with patent lumina, demon-

strated net fluid reabsorption when bathed in normalrabbit serum (0.22±0.2 nl/mm per min). Net fluid reab-sorption in uremic serum was 0.15±0.04 nl/mm per minand the postcontrol value after replacement of the ure-mic serum by normal serum was 0.20±.03 nl/mm permin. There is no significant difference between anyof these values. The use of an ultrafiltrate of uremicserum as the perfusate in two experiments did not yieldresults which differed from the remainder of the experi-ments. Net fluid secretion in uremic serum was ob-

TABLE IIISize, Dry Weight, Transtubular PD, and Net Fluid Reabsorption of Superficial PSTs of the Solitary

Remnant Kidney of Stage III (Uremic) Rabbits

Net fluidDuration Kidney PD reabsorption

post- cortical Internal Dry Perfusionsurgery BUN width diameter weight rate NS US NS US

mo mg/100 ml mm mm jigImm ni/min mV nl/mm/min

2.3 71.0 4.5 32.0 1.81 7.66 +0.1 -0.3 0.79 0.873.8 97.1 4.5 29.2 1.06 7.88* +0.1 +0.5 0.60 0.750.8 28.6 5.0 31.1 1.36 7.42 +0.1 +0.3 0.87 0.893.9 180.0 5.0 31.1 0.75 8.63 -1.5 +0.5 0.71 0.633.8 35.0 4.0 32.7 1.49 10.12 -1.9 -1.9 0.72 0.641.6 35.0 4.5 24.0 16.54 -0.3 -0.5 0.53 0.950.8 20.0 5.0 26.4 0.89 17.22* +0.2 -1.6 0.71 0.744.0 71.9 5.0 33.3 0.79 19.91 +0.1 +0.1 0.70 0.591.6 63.2 4.0 34.2 1.25 20.02 -0.5 -0.7 0.46 0.53

Mean 2.5 66.9 4.6 31.5 1.18 12.37 -0.4 -0.4 0.67 0.73-+SE 0.5 16.4 0.1 0.8 0.13 1.77 0.3 0.3 0.04 0.05

Potential difference is corrected for calculated liquid junction potential. NS = normal serum bath; US = uremic serum bath.* Ultrafiltrate of uremic serum used as perfusate.

1514 L. G. Fine, W. Trizna, J. J. Bourgoignie, and N. S. Bricker

1.6k

1.4kEE

ID

b_

L..

EE

-a

CD

I-

1.2k

1.0

08

0.6

NORMAL STAGE II STAGE IIIREMNANT REMNANT

FIGURE 3 A comparison of the dry weight/unit length of su-perficial proximal straight tubules from normal rabbit kidneys,remnant kidneys of nonuremic rabbits (stage II), and remnantkidneys of uremic rabbits (stage III).

served in only one experiment. 1 mMp-aminohip-purate in the bath resulted in net fluid secretion inall four experiments in which it was used.

NONPERFUSEDTUBULES

The results of incubation of PSTs from the five nor-mal and five uremic rabbits in rabbit uremic serumare shown in Table IV. PSTs from each animal wereincubated in five different uremic rabbit sera and onenormal serum containing 1 mMp-aminohippurate. Ineach experiment lumen expansion was induced by 1mMp-aminohippurate. Uremic rabbit serum had noconsistent secretory effect. Lumen expansion was ob-served in only 4/25 assays on normal tubules and 3/25assays on uremic tubules.

DISCUSSION

A logical requirement for the further elucidation of themultitude of factors which influence renal tubular func-tion in uremia is an experimental model which is ableto discriminate between intrinsic tubular functions andinfluences which are essentially extrinsic to the neph-ron. The model described in this study utilized iso-lated nephron segments from normal and chronicallydiseased kidneys, each being exposed to both a nor-mal and a "uremic" milieu. With this new experimentalapproach, fluid reabsorption was studied in PSTs ofuremic rabbits with single remnant kidneys. The func-tion of this nephron segment in uremia has not beenpreviously characterized largely due to its inaccessi-bility to micropuncture.

STAGE 1I STAGE Ill

REMNANT REMNANT

FIGURE 4 A comparison of net fluid reabsorption per milli-meter of superficial proxim-al straight tubule from normal rab-bit kidneys, remnant kidneys of nonuremic rabbits (stage II),and remnant kidneys of uremic rabbits (stage III) showingthe effect of normal and uremic rabbit serum. * P < 0.005 vs.normal and stage II.

The requirements for fluid reabsorption in the PSTare best viewed in the light of observations made onthe proximal convoluted tubule in uremia. In non-glomerular diseases such as pyelonephritis (22, 23),partial nephrectomy, i.e., "remnant" kidneys (3, 17, 18)and uninephrectomy (1, 3) in which the SNGFRof

NORMAL UREMIC NORMALRABBIT RABBIT RABBITSERUM SERUM SERUM

I-

zw

o E.

> cw.z-

0.1

mMPAH

FIGURE 5 Net fluid movement in uremic proximal straighttubules perfused by stop-flow methods and exposed to a peri-tubular bathing medium of normal rabbit serum, uremic rab-bit serum, and normal rabbit sertum containing 1 mMv-amino-hippurate (PAH). * Indicates experiments in which an ultra-filtrate of uremic serum was used as the perftisate.

Fluid Reabsorption by Uremic Proxinmal Straight Tubules

1 81

v

1515

TABLE IVNet Fluid Secretion in Nonperfused Superficial PSTs of Normal and Uremic

Rabbits Incubated in Uremic Rabbit Sera

Normal PSTs Uremic (Stage III) PSTsRabbit no. Rabbit no.

BUN N, N2 NJ N4 N, U, U, U, U4 U,

mg/i OOml

53.7 - - + - - - - + - -

70.1 - + - - -66.1 - -

62.6 - - - - + - + - - -

50.8 - - - + - + - -

p-aminohip-purate,lmM + + + + + + + + + +

Lumen expansion indicates net secretion of fluid.+, Lumen expansion; -, no visible lumen.

the surviving nephrons consistently increases, absolutefluid reabsorption by the accessible portion of the prox-imal convoluted tubule also increases (1, 18). Asso-ciated with this functional adaptation are the structuralchanges of compensatory hypertrophy (1, 12). In thesedisease states, delivery of tubular fluid to the PSTsis increased because fractional reabsorption by theproximal convoluted tubule is normal or decreased (1,17, 22-24). Thus, if the adaptive phenomena are con-sistent throughout the length of the proximal tubule,the rate of fluid reabsorption should increase in thePSTs of the uremic remnant kidney.

The enhanced reabsorptive capacity of the proximalconvoluted tubule does not appear to be a response toan increased tubular flow rate per se since acute re-duction in nephron mass results in an increase inSNGFRwith no change in absolute reabsorption bythe proximal tubule (25, 26). It appears, therefore, thatthe phenomenon is related either to the chronicity ofthe elevation in SNGFRor more probably to the de-velopment of compensatory hypertrophy of the tubule.

In none of the above-mentioned studies have ob-servations been extended to the pars recta. This seg-ment of the proximal tubule not only has a reabsorptivefunction but an important secretory one (27), and thecontrol of net fluid movement may well be determinedby the balance between these processes.

In the present studies PSTs from the remnant kid-neys of uremic rabbits were perfused in vitro and theirfunction compared with the same segments derivedfrom normal kidneys and from remnant kidneys of non-uremic animals.

Net fluid reabsorption per unit length of tubule wasapproximately 0.7 nl/mm per min in stage III PSTs vs.approximately 0.4 nl/mm per min in normal and stageII PSTs. Associated with this increment of approxi-mately 70% in reabsorptive rate was a marked degree

of structural hypertrophy such that a significant correla-tion was found between fluid reabsorption and the dryweight per unit length of tubule. The latter increasedby nearly twofold in the stage III segments. The valuesfor net fluid reabsorption in the normal PSTs closelyapproximated those observed by other workers (28-30)as did those of the PSTs derived from remnant kid-neys of nonuremic animals. The data from this lattergroup of nephron segments, which did not undergocompensatory hypertrophy, provides clear evidencethat the alteration in reabsorptive function was not afeature of the remnant kidney or the surgical procedureper se but was related to the uremic state and(or) struc-tural hypertrophy.

Although it is tempting to relate the enhanced re-absorptive rate of the hypertrophied tubules to the ob-served increase in luminal surface area, such a correla-tion may lead to erroneous conclusions because thePST is lined by a brush border epithelium, the sur-face area of which greatly exceeds the measured diam-eter as determined by light microscopy. Nevertheless,it is of interest to note that stage III PSTs show anincrease in Jv of 70%, an increase in dry weight of100%, and an increase in luminal area of 40%, all param-eters being factored for length. Since these observedincrements are not identical it is possible that someselectivity of adaptation occurs which is determinednot only by tubular mass but by other unidentifiedfactors.

An additional factor of importance and one whichstrengthens the close relationship between fluid reab-sorption and tubular size in the uremic PSTs was theobservation that the reabsorptive rate of the PSTs wasnot influenced by the humoral environment of the tubule.In all three groups, reabsorptive rates and transepithe-lial PD remained constant despite differences in theperitubular bathing medium. Uremic sera had no influ-

1516 L. G. Fine, W. Trizna, J. J. Bourgoignie, and N. S. Bricker

ence on the reabsorptive rates of normal PSTs and theenhanced reabsorption by the uremic PSTs was like-wise unaffected by normal serum. These observationsexclude an important role for undefined constituentsof uremic serum on fluid reabsorption by the PST. Theydo not, however, exclude the modifying role of otherextratubular factors, such as alterations in peritubularStarling forces which may result from changes in thedistribution of intrarenal blood flow in the remnantkidney (4).

Fluid reabsorption was not affected by differencesin perfusion rates or by the use of an ultrafiltrate ofuremic serum as the perfusate. These observationswould suggest that changes in tubular fluid flow ratein the physiological range and in the range anticipatedto occur in the remnant kidney (see Methods) are notimportant modulators of fluid reabsorption in this seg-ment. Likewise unidentified constituents of uremic se-rum which are filtered at the glomerulus (i.e., compa-rable to the ultrafiltrate used) do not play an importantrole in this process.

The role of organic anions in altering fluid transportby the PST has been recently reviewed by Grantham(31) and the influence of uremic serum on the renaltransport of hippurates has been well documented (6-8). The PST secretes substances such as p-aminohip-purate at a rate which is three times as great as thatof the proximal convoluted tubule (27) and significantisosmolal fluid secretion may be coupled to this trans-port process (32). Human uremic serum applied to theperitubular surface of nonperfused PSTs or PSTs per-fused by stop-flow will convert a process of net fluidreabsorption to one of net secretion (8, 33). Sincethe maximal rate of tubular transport of PAHper GFRis elevated in animals with chronic renal failure (34), itwas important to evaluate the contribution of this factorin the present studies.

Net fluid secretion was evaluated not only in non-perfused tubules and under stop-flow perfusion as de-scribed previously (8), but also under the more physio-logical conditions of free-flow perfusion. In these ex-periments uremic rabbit PSTs were exposed to uremicrabbit serum. The importance of examining uremicserum from an animal of the same species stems fromthe observation in the studies of Grantham et al. thatnormal human serum significantly depresses the rateof fluid reabsorption by rabbit PSTs, a phenomenonwhich may be related to different levels of organicanions in human serum or to some other undefinedinterspecies incompatibility (32). In the present studiesconducted under the same stop-flow conditions as de-scribed by Grantham et al. (32) net secretion was con-sistently absent. Indeed, no difference in reabsorptiverate was evident whether the tubule was exposed tonormal or to uremic serum. The difference betweenthis observation and that described for human uremic

serLim may lie in diff'erences in the levels of organicanions (e.g., hippurates) or of other unidentified sub-stances which exist in higher concentrations in humanuremic serum. Weare led to conclude, however, thatat least for the rabbit, organic anion secretion plays lit-tle if any role in modulating fluid reabsorption by thePST of the uremic animal.

The transepithelial potential differences of stage IIIPSTs were lower than those by the normal PSTs (i.e.,less lumen positive). Although no information is pro-vided by this study as to the reason for this difference,the observation would suggest that fluid reabsorptionmay be driven by different mechanisms in the stageIII tubules or that differential permselectivity to ionicspecies may be altered in these segments.

The results of the present studies conclusively dem-onstrate that fluid reabsorption by the PST of the rem-nant kidney of uremic rabbits is markedly increased.This adaptation is due to an intrinsic alteration in thefunction of the tubular epithelium and is closely linkedto the process of compensatory hypertrophy. The en-hanced reabsorptive rate presumably facilitates the re-absorption of the increased load of tubular fluid de-livered to this nephron segment by the convoluted por-tion of the proximal tubule in remnant kidneys withelevated values for SNGFR. It should not be construedthat this functional adaptation is universal to all formsof' chronic renal disease and the conclusions drawnfrom this study must obviously relate to the remnantkidney until further observations on other forms of re-nal disease become available. In experimental chronicglomerulonephritis, for example, SNGFRis normal ordiminished (35-37) and a parallel reduction in absolutefluid reabsorption by the proximal convoluted tubuleoccurs (35, 36). By analogy, fluid reabsorption in thePST should remain constant or diminish in parallelwith the change in SNGFRin these diseased states,but these data are not available.

Simplistically it would seem, therefore, that the samestimulus which leads to hypertrophy of glomerularfunction leads to a comparable increase in tubular func-tion, and whether this relationship holds throughoutthe nephron remains to be evaluated.

ACKNOWLEDGMENTS

These studies were supported by grants 7 R01 AM19822 andIT 32 AM07205 from the National Institutes of Health.

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1518 L. G. Fine, W. Triznca, J. J. Bourgoignie, and N. S. Bricker