Relationship between Para-aminohippurate Secretion and

Cellular Morphology in Rabbit Proximal Tubules

PHILIP B. WOODHALL,C. CRAIG TISHER, CHARLESA. SIMONTON, andROSCOER. ROBINSON, Division of Nephrology, Department of Medicine,and the Department of Pathology, Duke University Medical Center,Durham, North Carolina 27710

A B S T RA C T Previous studies in the mammalianproximal tubule have suggested that para-aminohip-purate (PAH) secretion is -threefold greater in thestraight segment, or pars recta, than in the convolutedsegment, or pars convoluta. However, the possibilitythat the site of maximal PAH secretion might be re-lated better to particular tubule segments as identi-fied by cell type had not been explored. In addi-tion, the presence or absence of differences in PAHsecretion between morphologically identical regionsof superficial (SF) vs. juxtamedullary (JM) proximaltubules has not been examined. These issues werestudied using a combination of' histologic methodsand measurement of' [3H]PAH secretion in isolatedperfused tubules. Measturements of microdissected SFand JM proximal tubules from young and adult rabbitsrevealed that SF proximal tubules were slightly butsignificantly longer than JM tubtules ([young rabbits:SF, 8.69+SE 0.14 mmvs. JM, 7.97±SE 0.13 mm; P< 0.01] [adult rabbits: SF, 10.61±SE 0.28 mm; JM,9.17±4SE 0.19 mm; P < 0.001]). Light and electronmicroscopy revealed three se(luential segments (Sl,S2, and S3) along the lenigth of' SF and JM proximaltubules as defined by cell type. PAH secretion wasmeasured in each of these three segments by the iso-lated perfused tuibule technique. Net PAH secretionin fmol/mm per min in SF proximal tubtules was:SI, 281±SE 21; S2, 1,508+SE 104; S3, 318±SE 46.Corresponding values in JM proximal tubules were353±SE 31, 1,391+SE 72, and 188±SE 23. Net PAH

This report was presenited in part before the Southern See-tion of the American Federation for Clinical Research, NewOrleans, La., 22 January 1976, and the combined session ofthe American Federation for Clinical Research and the Ameri-can Society for Clinical Investigation, Atlanitic City, N. J.,2 May 1976.

Dr. Woodhall was the recipient of a fellowship from theNorth Carolina Heart Association.

Received for publicationt 11 Julyl 1977 atnd itn revised form.5 December 1977.

secretion did not differ between comparable segmentsof SF and JM proximal tubules. It is concluded thatdifferences in PAHsecretion along the proximal tubulecorrelate best with cell type rather than the arbitrarydivision of' the proximal tubule into pars convolutaand pars recta according to its external configuration.Evidence of functional heterogeneity between com-parable segments of SF and JM proximal tubuleswas not observed.

INTRODUCTION

It is widely held that secretion of'the organic acid, para-aminohippurate, occurs principally in the straightportion, or pars recta, of the mammalian proximaltubule, whereas the contribution of the convolutedportion, or pars conivoluta, to this active transportprocess is relatively minor. Tune, et al. (1) were thefirst to directly measure the secretion of [3H]Jp-amino-hippurate in isolated perfused segments of rabbitproximal tubule and f0und that the rate of secretionwas approximately three times greater in the straightas compared to the convoluted segmenit. However,the division of the mammilalian proximal tubule into tworegions, the pars convoluta and the pars recta, de-fined by certaini gross and external macroscopic fea-tures, runs counter to other morphologic data. Light,fluorescence, and electron microscopic observations,particularly those of Suzuki (2), Sjdistranid (3), Mauns-bach (4), Rhodin (5) and Tisher, et al. (6) have re-vealed the presence of at least three imorphologicallydistinct segments in all mammalian proximal tubulesthus far studied in detail. Moreover, the distributionof the three major segments (S,, S2, and S3) definedaccording to cell type, does not conform to the divi-sion of' the proximal tubule into a conivolulted and astraight portion. For instance, in the rat (4), S, islimited to the beginning of the pars convoluta, S2includes the more distal portions of the pars convolutaand the beginning of the descendinig part of the

J. Clint. Invest. © Thle Ametrican Society for Clinical Investigation, Inc., 0021-97381T780501-132() $1.001320

proximal tubule, or pars recta, and S3 encom'lpassesthe remainder of the pars recta. Despite the extensiveuise of the rabbit kidney in the isolated perfulsedtulbule procedure (7), it has not yet been establishedwhether similar segmentation defined according tocellular morphology also exists in the rabbit proximaltubule.

The purpose of the present investigation was three-fold. First, the rabbit proximal tubuile was examinedmorphologically to establish the presence or absenceof cellular segmentation. Second, after demonstrationof the presence of three morphologically distinct seg-menits in this species as well, para-aminohippturate(PAH)' secretion was measured in each segment bythe isolated perfused tubtule technique to determinewhether the site or sites of maximal secretion of' thisorganic acid have any relationship to cell type. Finally,to determine whether heterogeneity existed with re-spect to PAH secretion in comparable regions of proxi-mal tubules from different nephron populations, secre-tory rates in each of the three cellular segments weremeasured in both superficial and juxtamedullarynephrons.

METHODS

Anatomic studiesMicrodissection. Proximal tubules from superficial (SF)

and juxtamedullary (JM) nephrons were dissected to estab-lish the lengths of the convoluted and straight segments ofthe tubule. Five young, female, New Zealand white rabbits(mean age, 82 days; mean body weight, 2.1 kg) and fouradult female rabbits of the same species (mean age, 163days; mean body weight, 3.5 kg) were lightlv anes-thetized with i.v. pentobarbital sodium (20-30 mg/kg bodyweight). 2 ml of 10% (wt/vol) sodiun ferrocyanide solu-tion wvas then injected i.v. and 20 s after completion of theinjection the left or right kidney was removed. Sagittalslices of the excised kidney were snap-frozen in liqtuidnitrogen and incubated overnight in a cold (-18°C) solu-tion of ethanol (95 vol%) and concentrated hydrochloric acid(5 vol%) containing 50 g/100 ml of ferric chloride. The kid-ney slices were then digested in 20% (wt/vol) hydrochloricacid at 45°C for 20-40 min, at which time individual tubulescould be freely dissected.

The SF proximal tubules were defined as those with atleast one convolution on the kidney surface. The JM proxi-mal tubules were defined as those belonging to a nephronwhose glomerulus was situated immediately adjacent to thecorticomedullary junction. Only tubules in which theglomerulus and the thin descending limb were attachedwere included in the study. Approximately 10 SF and 10 JMproximal tubules were removed from one kidney of eachrabbit and transferred from the dissecting dish to a standardglass microscope slide. The exact transition from the con-voluted to the straight portion of the tubule was markedon the slide. The tubule was then uncoiled gently, keep-

'Abbreviations used in this paper: JM, juxtamedullary;PAH, para-aminohippurate, p-aminohippuric acid; PC, parsconvoluta; PR, pars recta; SF, superficial.

ing the transition point stationary. The sli(lde was theniplaced in a camera lucida (WN'ild Heerbrugg, Switzerland)and a tracing made of the tubule at 4(x. WX'ith a cali-brated planimeter, the lenigth of the entire tubule and itsconvoluted and straight segments wvere dletermined fromthe tracing.

Light and electrotn miicroscoptj. Tissue {or microscopicexamination was obtained from three, healthv, votiuig,female, New Zealand white rabbits. These animilals wereanesthetized lightly with pentobarbital so(liuiim (20-30mg/kg body weight) before intravascular perflision fixation ofthe left kidney was performed with Karnovsky's fixativediluted to one-half strength (osmolality: 960 mosmol/kg H2()as previously described (8, 9). Thin coronial slices of thesekidneys were immersed in the same fixative at room tempera-ture for 4 additional h, then stored in a potassiumiii phosphatebuffer (osmolality: 950 mosmol/kg H210).

Portions of both SF anid JMI proximal tubuiles wereidentified and chosen for microscopic examinlation. To ex-amine early proximal conivoluted tubules, blocks of tissuiewere selected from either the JM or outer cortical regions ofthe kidney slices and routinely processed and sectioned forlight and electron microscopy (8, 9). Earls proximal con-voluted tubules were so identified becallse they wverevisibly connected to the renal corpuiscle of their respectivenephrons. Late proximal convoluited tubules wvere idelntifiedin one of several ways. A kidney slice was placed flat in aPetri dish containing buffer and directly vistualized under astereomicroscope at 60-15Ox. The open luminia of well-preserved straight portions of SF and JM proximal tubuileswere clearly visible. VWith standard micropuincture tech-niqtues, latex (General Biological Supply Houise, Chicago,Ill.) wvas injected into each individual tubule unltil the lateconvolution was filled in a retrograde manner. Blocks oftissue containing late SF or JM proximal convoluted tubulesfilled with latex were cut and processed for microscopy(10). Alternatively, late convolutions of SF and JM proxi-mal tubules could occasionally be dissected directly fromfixed tissue without the aid of acid digestion. These tubulefragments were also examined by microscopy. Manual dis-section of straight portions of proximal tubules of SF and JMnephrons was also accomplished from the fixed tissue.These tubules usually contained either the final portion ofthe late proximal convolution at one end or the thin de-scending limb at the opposite end. Occasionally, the entiresegment from the last convolution to the thin limb could beobtained. These individual straight segments of proximaltubules were divided into sequential 1-mm lengths andprocessed for light and electron microscopy. With a combina-tion of these histologic techniqlues, an anatomic profile of theentire proximal tubule was obtained.

Physiologic studiesTransport of PAH. After completion of the anatomic

studies, the capacity of histologically defined segments of SFand JM proximal tubules to secrete PAH was determinedby the isolated perfused tubule techni(que. General methodsfor the isolation and perfusion of tubular segments werethose of Burg et al. (7). Animals utilized in the study werefemale New Zealand white rabbits similar in age and sizeto the younger animals used for the anatomic studies. Tubulesegments selected for study were anatomically defined andchosen in the following manner. In SF nephrons, early con-voluted segments (S,) were those either with the glomerulusattached or located more than 2 mmproximal to the be-ginning of the descending portion of the tubule. Late con-

Para-aminohippurate Secretion in Rabbit Proximal Tubule 1321

voluted segments (S2) were the 1-1.5 mmof tubule immedi-ately before the start of the descending portion of'the proximaltubule. Upper straight portions (S2) came from the outerone-half of the cortex, and lower straight portions (S3) con-sisted of the tubular segment starting at the corticomedullaryjunction and ending with the transition into the descendingthin limb of Henle's loop.

Proximal tubules of JM nephrons were located either bytheir attachment to a JM glomerulus or to a long, thin limbdescending into the inner medulla. Early segments of' JMproximal tubule (S,) were selected by their attachment to theglomerulus or their position greater than 3 mmproximal tothe beginning of the descending portion of the convolution(see Results for comment on the configuration of the JMproximal tubule). Late segments of JM convoluited tubule(S2) were taken from the 2-3 mmof tubule located justproximal to the straight portion. The straight portion (S3)was that segment between the last convolution and the startof the descending thin limb. This segment was rarely greaterthan 1.5 mmin length and lay almost entirely within theouter medullary region.

Each segment of tubule was perfused with an ultrafiltrateof rabbit serum prepared by pressure dialysis at 4°C withan XM-50( membrane (Amicon Corp., Lexington, Mass.) andwvas bathed in 2 ml of isosmotic rabbit serum from the sameserum lot. Perfuisate and bath were preequilibrated bybubbling with a 95% 02-5% CO2 gas mixture for 10-20 minjust before use. During the experiment the bath chamberwas bubbled continuously with the same gas mixture andmaintained at 37°C.

The secretion studies in all segments of proximal tubulewere performed in anl identical manner. After the tubulewas connected to perfusing and collecting pipettes, an initiale(luilibration period of 15 min in a bath of normal rabbitserum was allowed. The bathing fluid was then changed torabbit serum containing [3H]PAH (sp act, 130 mCi/mmol,New England Nuclear, Boston, Mass.) at a chemical concen-tration of 24 uM. After a second 15-min equilibration period,three consecutive timed sample collections were made underoil using a constant volume pipette. In most experimentsouabain (50 ,M) was added to the bath after completion ofthe third collection and 5 min later a final timed collec-tion was obtained. The perfusion rate ranged from 4-15nl/min in different experiments and was usually varied duringseparate collection periods of the same experiment by chang-ing the height of a fluid column that was hydraulicallyattached to the perfusion pipette.

Secretion of PAH was also examined in the same mannerin an additional group of JM and SF S3 segments bathedand perfused with a slightly hypertonic ultrafiltrate of rabbitserum (osmolality: 350-360 mosmol/kg H2O) to simulate themore hypertonic environmen-t of tubules in the outer medulla.The osmolality was increased by the addition of NaCl.

To rule out the presence of leakage due to mechanicaldamage, [1251]iothalamate (Abbott Laboratories, N. Chicago,Ill.) or [14C]inulin (New England Nuclear) was added tothe bath along with [3H]PAH. During each experimentalperiod the bath was sampled to assure that the chemicalconcentration of PAH remained constant during each experi-ment and was similar in all experimental groups. Radio-activity in all samples was measured by liquid scintilla-tion counting in Aquasol (American Cyanamid Co., PearlRiver, N. Y.) plus 4% water by volume using a dual-channelscintillation spectrophotometer (Isocap 300, Searle AnalyticInc., Des Plaines, Ill.) or by gamma counting (BeckmanBiogamma, Beckman Instruments, Inc., Fullerton, Calif:).

The (qtuantity of PAHpresent in each sample was calculatedfrom the observed counts, the counting efficieniey for appropri-ate [3H]PAH standard solutions, and the specific activity ofthe [3H]PAH.

Statistical comiparisons were made with iunipaired t testanalysis.

RESULTS

Anatomny

Gross observations obtained bty microdissection ofindividuial tubules. The term pars convoluta (PC)was applied to any portion of the proximal tubulethat was visibly convoluted, and the termii pars recta(PR), or straight portion of the proximal tubule, wasused specifically to designate that portion which wasvisibly straight, or approximately so. The charac-teristic external configuration of dissected SF and JMIproximal tubules is shown in Fig. la and b. As indicated,there was a marked disparity between the configura-tion of these two types of tubules as observed underthe dissectin-g microscope. Table I contains the meas-urements of dissected proximal tubules from bothyoung and adult animals. The length of each segmentfor individual animals represents the mean value forthe length of that segment in 9-12 tubtules from thesame rabbit. In younger animals the average totallength of the SF proximal tubule (8.69+SE 0.14) ex-ceeded that of the JM proximal tubule (7.97-+-SE 0.13;P < 0.01). The absolute length of the PCof JM proximaltubules greatly exceeded that of SF proximal tubules(P < 0.001), whereas the converse was true for the PRof JM proximal tubules (P < 0.001). The PC of SFnephrons represented -60% of the total length of theproximal tubule, and the PC represented -90% of thetotal length of JM nephrons. In the adult group therelative lengths of the PC and PR segments in the twosubpopulations of nephrons were nearly identical tothose observed in younger animals. Again, in adultrabbits, the average mean length of the SF proximaltubule (10.61±+-SE 0.28) exceeded that of the JMproximal tubule (9.17±+SE 0.19; P < 0.001). In JMnephrons the straight, or PR segment, representedonly the terminal 1 mmor less of the tubule. All seg-ments of the proximal tubule were longer in the adultanimals except the straight portion of JM proximaltubules, which remiiained essenitially unchanged (younig,0.81±+SE 0.05 vs. adult, 0.84+±0.06; P, NS).

Morphological observations obtained wvith light andelectron microscopy. Light and transmission electronlmicroscopy were employed to define the cellu-lar com-position of SF and JM proximal tubules. It was foundthat the proximal tubule of both SF and JM nephronscould be divided into three distinct regions as pre-viously demonstrated in the rat (3, 4), nmouse (5), and

1322 Woodhall, Tisher, Sintonton, and Robinson

rhestus monkey (6). The segiments wvill he referredto as S1, S., and S3 to conform Twith terminology ctir-reIntlIN in wvide usage to describe this region of thenephroin. Each of the three segments was idenitifiedlaccordiing to the appearaince of a partictilarly distinetanid unifiOrm cell type within eaclh segimieint. Cellilararchlitettiure in each segimlenit wvas comparable in SFanid JMI inephroins. The S, segimienit, which represenitedthe early proximilal convoluted tulbile in all niephroIns,begain after a coninectiing or "neck" regioin of variablelengthl oft up to 90 /im as described by Sperber (11) andSelhioil}nevIder anid Mauinsbach (12). Cells in the S, re-gionl were coluimlnar with a tall, thick brtush border,inuImlerouis elonIgated imlitochondria freq(IuenItly conI-tained within invaginations of' the basal plasimialeimmiiia,and an apical region which contaiined a well-developedendocytic apparatus (Fig. 2a). The basement meimi-brane in the S, region wvas thicker than that of othersegmeints. In SF nephrons, cells typical of the S, seg-meint were never located in late proximal convolutionsor the PR, nor were they seen in the late convolu-tions just preceding the short straight teriiminial portioniof' JI proximal tubules. However, the exact point oftranisition fronm S, to S., was not identified with certainty.

la

FIGURE la Photomiiicrographl illtustrating the gross appear-anee of a microdissected proximal tubule from a SF nieph)ron.MIagniification, x 42.

lb

FIGURE 1 b Photomicrograph illtustrating the gross appear-aice of a microdissecte(d proximal tubule from a JMI nephronl.Magnification, x42.

Para-aminohippurate Secretion itn Rabbit Proximal Tubule 1323

TABLE IResults of Microdissection in Kidneys of Young and Adult Rabbits

Total length ofproximal tubule Length of PC Length of PR

Animal KidneyGroup Age wt wt SF JM SF JM SF JM

days kg g mm mm mmYoung animals (n = 48) (n = 47) (n = 50) (n =47) (n = 49) (n = 47)

1 80 1.8 9.55 7.50 7.87 4.66 7.55 2.94 0.322 93 2.2 8.25 8.85 7.15 5.29 6.37 3.56 0.793 71 1.9 5.60 8.21 7.37 5.12 6.60 3.09 0.754 79 2.0 5.95 8.83 8.45 5.45 7.47 3.38 0.975 89 2.6 8.54 9.74 8.94 5.93 7.91 3.81 1.03

Mean 82 2.1 7.58 8.69 7.97 5.30 7.16 3.36 0.81SE 0.14 0.13 0.10 0.13 0.08 0.05P <0.01 <0.001 <0.001

Adult animals (n = 42) (n = 43) (n = 42) (n = 46) (n = 42) (n = 44)1 165 4.0 11.6 12.88 10.87 8.06 9.70 4.60 1.162 160 3.2 7.1 8.50 8.86 5.01 8.01 3.48 0.863 161 3.3 8.6 9.88 8.72 5.83 7.78 4.05 0.944 166 3.5 9.4 11.32 8.41 7.00 8.01 4.32 0.40

Mean 163 3.5 9.2 10.61 9.17 6.47 8.35 4.11 0.84SE 0.28 0.19 0.23 0.16 0.09 0.06P <0.001 <0.001 <0.001

Cells of the S2 segment of the proximal tubule weretall, cuboidal to low, columnar and had a relativelyshort brush border (Fig. 2b). Although mitochondrialprofiles were numerous, they were less prominentthan in the S, segment. Microbodies were more com-mon in this segment than in Si. The basement mem-brane was thinner than Sl, but thicker than S3. Thebasal plasmalemma was thrown into numerous in-vaginations, the height of which was less than in theS, region. Cells with these morphological characteris-tics formed the lining epithelium of the late convolu-tions of SF proximal tubules. In addition, serialsections of individually dissected PR revealed thatcells typical of the S2 region in SF proximal tubules ex-tended into the lower half of the PR to the vicinity ofthe corticomedullary junction. Distal to this transition,cells of the S3 type were found. In SF proximal tubulesthe transition from S2 to S3 was gradual and appearedto extend over a distance of -1 mm. In the JM proximaltubules it was found that the late convolutions werecomposed of S2 cells. The S2 segment ended -1 mmfrom the end of the proximal tubule, again in thevicinity of the corticomedullary junction. The terminalportion, or PR, of the JM proximal tubule, althoughnot as straight as that of SF nephrons, was composedentirely of S3 rather than S2 cells.

The S3 cell (Fig. 2c) was cuboidal and had a longerbrush border than the S2 cells, although not quite aslong as that described in the rat (4). The cell height

was distinctly less than that in either S, or S2. Pro-files of mitochondria were not as profuse as in S2, andinvaginations of the basal plasmalemma were infre-quent. Sections of tissue taken perpendicular to thepath of proximal tubules in the outer stripe of theouter medulla allowed dozens of tubules to be sur-veyed and indicated that the terminal segment of allproximal tubules was composed of S3 cells. Thus, allS, and S2 cells were found in the cortex, while S3cells were confined chiefly to the innermost regionsof the cortex and the outer stripe of the outer medulla.Fig. 3 indicates schematically the distribution of thethree segments of the proximal tubule in SF and JMnephrons. The PC is heterogeneous with respect tocell type in both SF and JM nephrons. The PR of SFproximal tubules also contains two cellularly discreteregions, (S2 and S3), whereas, the PR of JM nephronscontains S3 cells alone.

Physiologic observationsPAH secretion. The results of the PAH secretion

studies are shown in Fig. 4 and Table II. Fig. 4 de-picts the results of a characteristic group of measure-ments performed in S, segments dissected from fiveSF proximal tubules. The net secretory rate remainedrelatively constant with time and it was not alteredby varying the perfusion rate over a range of 4-15nl/min. The addition of ouabain at a concentration of

1324 Woodhall, Tisher, Simonton, and Robinson

50 ,uM markedly reduced net secretion within minutes.Net PAH secretory rates for each of the three histo-logically defined segments of the proximal tubulein both SF and JM nephrons are listed in Table IIand the results are summarized in Fig. 5. MaximumPAH secretion was limited strictly to the S2 segmentsof both SF and JM proximal tubules. In SF proximaltubules, the maximum net secretion of PAH was notconfined solely to the straight portion of the S2 seg-ment, inasmuch as three of the eight S2 segmentsexamined were entirely convoluted in character andexhibited a mean secretory rate of -1,500 fnmol/mm

FIGURE 2 Electron micrographs illustrating the chlaracteris-tic morphology of cells forming the a) S,, b) S2, atnd c) S3segments of the rabbit PT. See text for description. Nlagni-fications, (a) X6,000; (b) x6,000; and (c) x8,000.

per min, a value which corresponded (Ijiite closely tothat observed in five straight S2 segmiienits (1,350fmol/mm per min). In both SF anid JMI niephronis,the S3 segment possessed the lowest net secretoryrate, although the difference in net PAH secretionibetween the S, and S3 segments in SF nephronis wasnot statistically significant (P > 0.30). However, asmall but statistically significant difference in Imleanlsecretory rates was observed betweeni S, and(l S3 seg-ments of JM nephrons (P < 0.025). This differencewas largely due to the inclusion of one tubbule fromii aJM nephron with an extremely low rate of secretioni

Para-aminohippurate Secretion in Rabbit Proximiial Tubtilc 132.5

Both

Si

S2

Si

Co,t,co ,-od.llo5y S3Junct,on

52 I< .C

o Ec Eo '

-o

_ x4,z

S3 _

FIGURE 3 Schematic representation of the distribution of theS,, S2, and S3 segments defined by cell type in SF and JMproximal tubules. Transition regions are indicated betweenS, and S2 of both SF and JM proximal tubules anid betweenS2 anid S3 of SF tubules. See text for explanation.

(28 fmol/mm per min). Net secretory rates of S, and S3segments of JM nephrons did not differ statistically(P > 0.05) when this single tubule was excluded fromthe calculations. Importantly, net PAH secretion wasalso found to be comparable in morphologically identi-cal segments of SF and JM nephrons. Thus, withrespect to PAH secretion, evidence of functionalheterogeneity between SF and JM nephrons was notobserved when comparisons were made between seg-ments whose cellular morphology was identical.

In an attempt to exclude the possibility that low netPAH secretion by S3 segments was due to incubationof the segments in a bath which was less hypertonicthan the interstitial fluid which normally bathes thetubules in vivo, six S3 segments were studied in

400

300

200

I Serum + PAH

Ouaboin2 50M7

268±35 278 t33 299 t46 54 * 7

look1 2 3

Experimentol Period

4

FIGURE 4 PAH secretion in S, segments of SF proximal ttu-bules before (periods 1-3) and after (period 4) otlabain.

which the osmolality of the perfusate and bath was in-creased to 350-360 mosmol/kg H20 by the addition ofNaCl. The mean net PAHsecretion in this group of sixS3 segments obtained randomly from SF (it = 3) andJM (n = 3) proximal tubules was 281+SE 42 fmol/mnmlper min. This value was not statistically different from amean value of 253+SE 50 fmol/mm per min obtainedby pooling the data obtained from the JM and SF S3segments that were bathed and perfused in an isos-motic ultrafiltrate of rabbit serum.

DISCUSSION

The results of the present study provide a detailedstructural-functional analysis of PAHsecretion in prox-

TABLE IIPAH Secretion in Individual Segments of SF andJM Proximal Tubules

S, segmenit S, segment S3 segment

SF J m SF JM SF JM

Normal Oniabain Normal Ontabain Normal Ouabain Normal Ouabain Normal Ouabain Normal Ouabain

(n = 15)* (n = 5) (n = 15) (n = 5) (n = 27) (it = 6) (tl = 12) (n = 4) (n = 15) (t= 3) (n = 15) (n = 5)Net PAH secretion, 210 74 433 61 1,3261 76 1,772 122 311 - 189 57

fmol/mm 406 38 493 75 1,9101 120 1,180 126 181 - 243 64ttubtule/min 247 36 401 36 1,286t 118 1,211 121 639 67 28 14

331 64 231 59 1,507 - 1,402 133 167 60 266 82216 56 206 77 1,268 75 292 98 216 50

1,406 70)948 -

1,624 122

Mean-SE 281±21§ 54±7 353±31§ 62±7 1,508±104" 97±10 1,391±7211 126±3 318#46¶ 75±12 188±23¶ 53±11

Ttubtule length,* 1.23±0.11 1.13±0.14 1.06±0.11 1.22±0.09 1.23±0.11 1.13±0.12mm-SE

No. of periods of observation.I Late PC just preceding the descending portion of the ttubule.§ S, segment, SF vs. JM-P > 0.15.IS, segment, SF vs. JM-P > 0.45.¶ S3 segment, SF vs. JM-P > 0.10.** Length of perfised segment.

1326 Woodhall, Tisher, Simonton, and Robinson

n-27 El Superficialj, 12 El Juxtamedullary

SeCBment of p l tubule uabainFGR1250 naloof observationsE

E

I750-

.2 500-n=15 ,1

250- "15z4

Si S2 S3Segment of proximal tubule

FIGURE 5 Summiary of PAH secretion in all three segmientsof SF and JM proxiimal tubules. Malximal secretory rates wereobserved in S2 segments of both nephron populationls.

imal tubules of SF and JM nephrons of the rabbitkidney. On the basis of cellular morphology alonie, itwas possible to subdivide the proximal tubule intothree distinct segments (Si, S2, and S3) and to estab-lish that the segmentation was similar in both SF andJNl proximal tubules. PAH secretion was found to bemaximal in the S2 segment and similar secretory pat-terns were noted in both SF and JM proximal tubules.

Although previous morphological studies in mouse(5), rat (4), and the rhesuis monkey (6) have providedevidence of cellular segmentation within the proximaltubule, the presence of at least three distinct segmentshas not been docuimented previously in the rabbit prox-imal tubule. In addition, the present observations pro-vide evidence in the mammalian kidney that both theSF and JM proximal tubules are composed of at leastthree anatomic segments. Although the division ofthe proximal tuibuile into the PC and the PR on thebasis of external gross and macroscopic anatomic fea-tures has been found convenient for purposes of exam-ining a large variety of functional events in this regionof the nephron, examination of Fig. 3 reveals that inmany instances two cellularly distinct segments maybe included in a particular functional analysis whenthe proximal tubule is subdivided in this manner. Forexample, most mieropuncture studies are commonlyconducted along the initial 60% of the proximal tubuleand, in many instances, fiunctional data are undcoubt-edly obtained from either the S, or S2 segments, orboth. It is likely that punctures of "late" SF proximalconvoluted tubules most often involve the S2 seglllelt.Indirect evidence in support of this conclusion can bemustered by comparing the results of recent micro-puncture observations in the rabbit with certain mor-

phological data in the presenit study. Chonko et al. (13)measuired the length of the proximal tul)ule accessib)leto mieropuneture in 12 SF nephronis obtained fromrabbits weighing 2-2.8 kg by the latex cast methlod.Their valuie of 5.40 mmll is essenitially identical to thatof 5.30+ SE 0. 10 mnm for the PC of SF proximlal tuibulesof younig animals those wveight averaged 2. 1 kg in thepresent stuidy. If, as is genierally assumed, the portioni ofthe proximal tubule that is accessible to miiciroptuiicturerepresenits -60% of its enitire length, it Couldlbe esti-mated that the lengthl of the entire proximal tubulewould -9 mm. This latter value com)pares (quite fa-vorably with our measure(l value of 8.69+SE 0.14 mimin SF proximal tubules of young animcals. Thlus, at leastin the rabbit, the punicture of "late" SF proximal con-voluted tubules would, in all likelihood, fall wellwithin an S2 segment as definied in the p)reseint study.The problem of physiological samplinig fromii morpho-logically heterogeneous tubule segmenits may also existin stuidies which involve the use of isolated perfuse(dtul)ules. This isstue will be addressed in greater (letailin subse(lqtent sections.

The results of the PIAH secretory stt(lies reveal theexisteniee of f'tunetional heterogeneity along the lengthiof the proximal ttubuile in both SF and(l JMI neephlrons.The highest rate of' PAH secretioni was notedl in theS2 segnmenit of'both SF andl JX nephronis, whereas theS, anci S3 segnments exhibited a lower capacity for PAHsecretioni. At first glanee these findinigs appear to beat variance with earlier measturemiienits of PAH secre-tion in the isolated perfulsed rabbit proximal tubtule(1). The mean net PAHsecretion by the PC' wvas 235+38fmol/mm per min, a value very similar to that obtainedin SI segments of SF andcl jAI proximiial tubules in thepresent stuidy. The restults suggest that Tuine et al. (1)were studying relatively earrly regions of the PC wfhiel-iwould correspond to the S, segment. These samle work-ers reported a meani valtue for net PAH secretioni of869±52 fimiol/imm per min for the straight portion orPR of' proximlal tubules wvhose nephron of origin, thatis, SF or JMI, was not identified. Basecl on our owvnfindiings, it is readily apparent that if PAH secretioiuis examined in the PR of proximal ttubtules fromi SFnephrons without regard to the cellular compositionof the tubule under studv, segmnenits of higlh (S,) aswell as low (S3) PAH secretion nmav be inieluded inthe preparation. Their value of' 869+52 f'Mol/mm pernmiii obtained from straight segments is approximilately,midway betwveen the meain net PAH secretory ratesobtained in the S2 aind S3 segments in the present in-vestigationl and may reflect inielusioni of tullliles rel)re-senitinig a mixtture of' the two segments. This potentialpitfall was intentionally avoided in the presenIt stulvybv the selection of short tubtule segmenits of uniiif'ormiicell type from well within the boundries of the tlhreesegmiienits as definied initially via liglht anid electronl

Para-ainot1ohippurate Secretiotn in Rabbit Proximlal Tubule 1327

microscopy. Thus, we conclude that it is the S2 seg-ment, and not the PR of the proximal tubule, wheremaximum PAH secretion is principally located.

The present investigation represents one of the fewstudies in which a functional comparison has beenmade between morphologically identical segments ofSF and JM nephrons. In the present study SF andJM proximal tubules were divided initially into spe-cific segments carefully definied accordling to charac-teristic morphologic features and then, on the basisof these features, examined functionally to determinewhether heterogeneity did exist between comparableregions of different nephron populations for a particularphysiologic event. Although striking differences in netPAH secretion were observed from one cellular seg-ment to another along the proximal tubule, evidence offunctional heterogeneity between different nephronpopulations was not found. It should be noted, how-ever, that an apparent difference in PAH secretion be-tween straight segments of SF and JM proximal tubulesmight well have been observed if the usual gross ana-tomic criteria that have been employed in many stud-ies had been followed in the present experiments.This is because, of course, virtually all of the PR ofJM proximal tubules is composed of S3 cells, whereasmuch of' the PR of SF proximal tubules is cortical inlocation and composed principally of S2 cells. Thus,any functional comparison betweeni the PR of' JM andSF proximal tubules will most likely include a com-parison of' JM S3 segments with either SF S2 segments,if' the latter are dissected exclusively from the outercortex, 6r a combination of' SF S2 and SF S3 segmentsif both cortical and outer medullary SF straight seg-ments are studied.

Although our studies f:ailed to reveal the presenceof functional heterogeneity between diff'erent nephronpopulations, other investigators employing the isolatedperfused tubule techni(lue have described apparentfunctional differences between SF and JM proximaltubules. Kawamura et al. (14) examined the sodiumand chloride permeabilities of SF and JM straight seg-ments, the former derived from both the cortex andthe outer medulla. The results of' both electrophysio-logical and isotopic estimates of chloride to sodiumpermeability suggested that SF straight segments wereapproximately two times more permeant to chloridethan to sodium. The converse was true of JM straightsegments. In a later study, Jacobson and Kokko (15)compared the relative chloride and sodium permeabil-ities in SF and JM proximal convoluted tubules byelectrophysiological techni(ues only. Sodium permea-bility was founid to exceed chloride permeability inthe early portion of the PC (probably the S, segment)in both SF and JM proximal tubules. However, therelative sodium permeability continued to exceed the

chloride permeability throughout the remainder of theJM proximal convoluted tubule (probably correspond-ing to the late S, and S2 segments), whereas the con-verse was true in the more distal regions of the SF prox-imal convoluted tubule (S2 segment). In contrast to theresults obtained for PAH secretion, these data suggestthat fulnctional heterogeneity between anatomicallysimilar segments of different nephron populationsexists for chloride aind sodium permeabilities along theproximal tubule.

Recently, Wlarnock and Burg (16) reported a differ-ence in permeability and transport rate of CO2betweenisolated perfused proximal straight tubules of SF andJM nephrons of the rabbit. Although a definite attemptwas made by these investigators to study proximalstraight tubules from two distinct nephron populations,it is not possible to ascertain from the published ac-count of the methodology that was employed whethermorphologically identical segments based on the cellui-lar composition were actually examined.

ACKNOWNILEDGMENTSThe authors gratefully ackniowledge the technical assistanceof' Mrs. Helen Parks and Mrs. Kathy Blake and the secretarialcontribution of' Mrs. Greta Owens. Mr. William Boyarsky andlMrs. Jessie Calder prepared the illuistrative material.

This work was supported in part by U. S. Public HealthService grants AMI 13845, HL 05848, and AM 17195.

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