CELL CONTOURS I N THE TWO SEGXESTS OF' THE

DOG KEPHRON PROXIMAL TUBULE I N THE CAT AND

J O H N J. POOTE AR'D ALLAN L. GRAFFLIN Department of Anatomy, Harcard Medical School, Boston, rllassnchusetts

N I K E PIGPRES

Previous papers from this laboratory have supplied quanti- tative measurements of the two differentiated segments of the proximal tubule in the cat and dog nephron (Foote, '36; Foote and Grafflin, '38), and have described and illustrated certain aspects of the complicated cell contours in the first segment of the proximal tubule in the cat (Grafflin and Foote, '38, '39). During the past 2 years some additional data have been obtained upon the problem of cell contours in the cat and dog kidney, and these data form the subject of the present report. The problem is beset with considerable difficulty from the technical point of view. Only a few specialized technics are available for impregnating the highly complicated cell boundaries of the renal epithelium, and even with these tech- nics the impregnation is invariably patchy, and consistently good results are never obtained. An intensive effort on our part to develop a specific and consistent technic for this pur- pose has so far been unsuccessful.

The evidence for segmental differeiitiatioii in the proximal tubule of the cat and dog nephron has been discussed else- where (Grafflin and Foote, '39). I n brief, the proximal tubule in these two animals regularly contains fat under normal

Fellow of the Henry Strong Denison Medical Foiindntion.

1

TIIE AXERICAN J O U R N A L O F ANATOMIT, YOIA 70, N O . 1 J A N U A R Y , 1942

2 J O H N J. FOOTE AKD ALLAN L. GRAFPLIN

conditions, and this fat is distributed in a characteristic manner for each species. In the cat the entire pars convoluta and the upper portion of the pars recta are normally fat-laden, while the terminal portion of the pars recta is fat-free. In the dog the reverse is true, the fat being confined to the terminal portion of the pars recta. The transition between the fat- laden and fat-free portions of the proximal tubule is charac- teristically abrupt in both cat and dog, and stands out verj- clearly in sections stained for fat and in tubules teased from acid maceration material (Foote and Grafflin, ’38). Zimmer- mann (’11) made an important advance when he succeeded in demonstrating that the abrupt transition with respect to fat content was likewise abrupt with respect to cell type. In both species the lumiiial cell-boundary (terminal bar) pattern in the terminal portion (Zimmerinann’s “ Schlussstiick”) of the pars recta - fat-free in the cat, fat-laden in the dog - is rectilinear. I n the remainder of the proximal tubule (i.e., from its origin at the glomerulus to the beginning of the “Schluss- stuck”)-fat-laden in the cat, fat-free in the dog-the luminal cell-boundary pattern is very complicated, showing in marked degree the interdigitations usually considered characteristic of the mammalian proximal tubule.2

These findings seem amply to justify the conclusion that we are dealing with two cytologically distinct segments of the proximal tubule in the cat and dog. It is important to empha- size the reversal in fat distribution in the two species; but still more important for the present problem is the fact that the cell type shows no such reversal. I n both cat and dog the first and second segments of the proximal tubule exhibit each a characteristic cell type which is entirely independent of the presence, or absence, of fat deposit. Observations upon the cell contours in these two segments will now be presented.

a That the cell type (i.e., shape) was different (i.e., simpler) in the pars recta as compared with the bulk of the proximal tubule in the dog dates back to Schachowa (1876), who did not, however, appreciate that there was an abrupt transition in this regard.

CELL CONTOURS IN PROXIMAL T U B U L E 3

OBSERVATIONS

Cat First segme?zt. The cell contours in the first segment of the

proximal tubule in the cat have been described, in chrome- silver preparations, by Grafflin and Foote ( ’39), who sum- marize their findings are follows : ‘‘At the luminal surface the cell outlines are extraordinarily wavy (interdigitated) in character. As one proceeds basally from the lumen, this extreme complexity disappears, and at an intermediate level the cell outlines are relatively simple, although still fa r from rectilinear. Proceeding still more basally, the cell outlines again become irregular, and this irregularity is progressive, being most marked at the level of the basement membrane. However, the pattern is here much coarser than a t the lumen.”

We have no further descriptive data on chrome-silver preparations to add to those previously published. However, an analysis of certain of the cell outline figures given in this earlier paper has yielded some very interesting results. This analysis was carried out in terms of circumference, area, and length of individual sides of the cells at different levels. The illustrations used for this study are reproduced in figures 1 and 2. The actual measurements, however, were carried out upon photographic enlargements of these figures to a magni- fication of about 12,000 x ; since it is the relative magnitudes which are important, the data are given here as measured, without conversion to the illustrated or actual size of the cells. Areas were measured planimetri~ally,~ lengths by a Keuffel and Esser map measurer. The area and circumference data are as follows:

PIOGRE CIRCUMFERENCE* ARE4“

cm. 84. cni.

1B 77.7 205.1 1c 133.3 296.1 2A 145.9 106.5 2B 60.8 145..?

* cn. x 1”,000. ~ ~ ~- ~ ~ _ _ -~ ~~~ ~. ~-~ -

For checking the accuracy of these plaiiimetric measurements we are indebted to the late Mr. Gustave S. Natapoff.

4 J O H N J. FOOTE AND ALLAN L. GRAFFLIR

For figure 1, both area and Circumference are greater at the basement membrane (C) thaii at the intermediate level (B), but it is to be noted that the circumference is dispropor- tionately greater than the area. For figure 2, while the area is smaller at the lumen (A) than at the intermediate level (B), the circumference of the cell is tremendously greater at A than at B. The wide disparity of the area and circumference at the two levels is strikingly illustrated in figure 3. In this figure both area and circumference of the cell at level B have been arbitrarily represented by circles of the same radius. Areas are compared to the left in the figure, the outer circle representing the area at level B, the inner white circle the area at the lumen (A). To the right in the figure, the inner solid circle represents the circumference of the cell at level B, the outer circle the circumference at level A. Such a marked disproportion between the circumference of the cell at the two levels refutes absolutely the idea that the markedly inter- digitated cell outlines observed at the luminal surface could possibly be attributed solely to a fixation (shrinkage) artifact.

The lengths of the individual sides of the cells at the differ- ent levels are likewise very interesting. These lengths were measured between successive three-rayed vertices on thc enlarged photographs described above, and the measurements, in centimeters, are summarized in the following tabulation :

1 A

a 45.9 b C

a c f g

1B 1c 2 A

25.0 64.9 13.3 4.6 4.0 12.5 9.1 15.8 14.2

18.2 22.6 42.2 20.8 26.0 2.0

25.4 36.3

2B

5.2 9.7 6.0

13.4 4.3 9.8

12.4

F o r figure 1 it will be noted that the length of a given side is in general considerably longer at the basement membrane than at the intermediate level, and may be markedly so (eg., side a ) . However, the length of one side (b) is slightly shorter at thc bascment membrane. For figure 2, a marked dispro-

CELL CONTOURS IN PnoxI3rAL TUBULE 5

portion in length between the intermediate level (B) aiid the lumen (A) is the rule, but here also, in one instance (side e ) , tlie length is somewhat shorter at the lumen.

e

Figs. 1 and 2 Tlwsc arc figurer 8 and 9 from Graffliii and Footc ( ’39). here reproduced with slight iiiodifications (greater reduction ; altered figure iiuiubers ; lcttering of individual cell sides added). They represent the outlines, a t different levels, of two cells in the first segment of the proxiiiial tubule in the eat. Fig. 1 : C-ontliiic a t bascnient membrane ; €3-outline at 10 p above basement iiiembraiic; A-partial outliiie at lumen (5 .5 to 7.5 p above level B). Fig. 2 : A-at lumen; €3-at approximately 6 p below lumen. See text aiid figure 3.

R few obsei.vations were made upon the appearance of the ccllls of the first segment of the proximal tubule iii teased preparations of material treated with ammonium chromate. This technic was used by R. Heidenhain (1874) in his classical paper, in which the complicated shape of some of the renal tubular cells was first clearly demonstrated. In our prepara- tions we arc able to observe not infrequently the highly irregu-

6 J O H N J. FOOTE AND ALLAN L. GBAFFLIN

lar “cel1s”pictured by Heidenhain (for the dog; see his fig. 8). Peripherally, the cellular processes appear torn or broken, as noted by Heidenhain. Some of the cells, with clearly recog- nizable brush border and well-preserved fat droplets, show unmistakable grooves or furrows on their lateral surfaces. This furrowing seenis to start in all instances at some dis- tance below the cell apex, the furrows becoming deeper (the intermediate crests higher) toward the base, giving a dis- tinctly flanged effect. I n some instances a single cytoplasmic

Fig. 3 Comparison of area and circumference of cell a t levels A and B in figure 2. See text.

column or crest is observed to divide into two at the extreme base of the cell. The picture is entirely similar to that de- scribed by Grafflin and Foote (’39) except for the absence of the delicate and complicated furrowing in the apical region. This aspect of the cells is never observed in the ammonium chromate preparations.

Second segment. Numerous observations upon chrome- silver preparations (after Landauer, 1895 ; for technic see Grafflin and Foote, ’39) make it clear that the cell outlines in

CELL CONTOURS IN PROXIDIAL TUBULE 7

this segment are essentially rectilinear at the luminal surface. As one focusses toward the basement membrane, these out- lines gradually lose their rectilinear character, and a t the basement membrane the cells are usually interdigitated to a considerable degree. The pictures obtained in the cat exhibit no significant differences from those in the dog, and figure 9, for the dog, will serve adequately well for both species. The order of complexity of the cell outlines a t the basement mem- brane is quite variable; they may be somewhat more o r less complicated than those illustrated (fig. 9). However, it should be emphasized that a true rectilinear pattern is never observed.

Imtcrsegmeiztal trauzsitiom. Transitions between the first and second segments of the proximal tubule can be readily identified in these chrome-silver preparations. They are characteristically abrupt with respect to both fat content and cell contours, although the transition may occur at a sonie- what or even considerably lower level on one side of the tubule than on the other. Despite the fact that these transitions can be readily identified, it has so far proved impossible to find a single tubule in which an even moderately extensive cell- outline pattern, either at the lumen or at the basement mem- brane, could be accurately traced on both sides of the point of transition. This is understandable in view of the consider- able shrinkage in chrome-silver preparations, and the fact that the tubular lumina are regularly quite small in diameter. h striking illustration of the transition in the cat has been

supplied by Zimmermann ('11, fig. 7, iron hematoxylin prepa- ration), representing thc abrupt alteration in cell contours at the luiiiinal surface. The accompanying drawing (fig. 4) affords an interesting comparison with Ziminermann 's figure, since it illustrates the cell-outline pattern, insofar as it could be accurately traced, at a level about mid-way (in the mid- axial line) between the lumen and tlic basement membrane. The first segment is to the left, the second segment to the right, and the point of transition is quite evident. The cell outlines in the first segment at this level are entirely in keep-

8 J O H N J. FOOTE AND ALLAS L. GRAFFLIX

iiig with our earlier findings (Grafflin and Foote, '39; see above). The most distal cell of the first segment is wortliy of comineiit in that it extends straight across (almost half-way arouiid) the tubule without interruption. One would normally cspect to find an additional cross-bar subdividing this unusu- ally long cellular area into two, but none was present. Since the impregnation was otherwise sharp and complete, the illus- trated pattern was accepted as valid. Fat droplets of fair size, but few in number, were present in the cells of the first

f

Fig. 4 Cell-boundary pattern at traiisitioii from first to second seqinciit in prosirnal tubule of cat. The level of the drawing is about mid-way between the lumen and the basement membrane. Chrome-silver (Landauer) preparation. C:imera luriiln draiving, x 1190. See test.

segment, right up to the point of transition, while the second segment mas fat-free. The somewhat irregular cell outlines in the second segment at this level arc in agreement with the description given above.

D0.Y First seglumht. When fresh spreads of tlie dog kidney arc

made in physiological salt solution shortly after death, the first aiid second segments of tlie proximal tubule can be readily identified. I n the first segment, a t high magnification, one can

ix~l)catedl>- obseivc complex p i c tu iu of markedly irregular aiicl 1)rancliiiig lines, g1aii.y in appearance, d i c h seem to bc iiiterprctable only a s cell boundaries. On focussing froin a 1 1

open lumeii into the epitlielial wall of the tubule, one eiicoun- t e w the highly inteidigitated pattern a t orice. This pat tern usually appears to remain of appl*oxiiiiatcly tlic same oi*dci* of coniplexity :is one proceeds from tlie cell apices to thc baseniciit iiiernl~ranc, but may hccoiiie considerably simplei. at tlie iiuclcar Icvc~l, again attaining marked complexity at tlie basciiicwt iiicmbrane. The pictures arc extrenicly confnsing, atid it is impossi1)le to determine the outlirics of ail iritliridual cell with even t i sernblancc of accuracy. These p iqa ra t ions arc best studied with the iris diaphragm strongly contracted : tlai.1t-field illmiiiiiation is of littlc or no assistance. Tlie appear- m c c ’ s of tlic cell bounclarics observed in tlicsc fi’esli spreads seem to he cwtiix~ly analogous to tliose seeii in irori-hematox>-- lin prepiratioiis of ki l ter-foimol material ( s e e heiow), nltlioagli tlic glairy lilies a i ~ oil the wliolc soiiicwliat thinnei- iii tlie fresh tliaii iii the pernianent preparations.

111 frozen scctioiis of m a t o i d fixed in 10% i icu t~ .d foriiialin, the cell outline patterns obsei-vecl in the first segmeitt are cssctitiallj- tlic sanic a s tliose in fresh sprca(ls of the ltidnep. No significant differences in tlic complexity of the pattern caii he detected at the luniiiial, inter-mediate and 1)asal levels of the cells iii a. given portion of the tubulc ; in soilit’ itistaiices, however, tlie patter11 is sonicwliat 01’ even considerably simplei. tlian in ot1iei.s. In one instance an extraordinarily coml)licated pattcrii, 1xisisting from the lunicn to tlic baseiiient mcmbi’anc, was ol)sci.vccl in the first segment just distal to its coiiiicction ivitli llic gloiiieimtar capsule.

Ti1 iron liciiiatox~lin preparations of bicliromate-iiioi.(l~iiitclcl Zenker-formol a very striking picture of the coni- plicatcd cell 1)omidarics in this segment is obtained. The cyt o-

’ Small blocks of kidney were h’xed immediately after dcatli in Zenlter-f orinol (Zeirkcr stock 9 Imits, ncutial fonn;iliii 1 lmrt) for 24 hour$, t2lc.n nrordantcd i n 3% pot;rssiuin diclrroniate for 34 hours a t 40°C.

plasm, with its abundant mitochonclrial suhstamx’, is \\-ell stained; tlie cell boundaries, on tlie other hand, take no staiii whatever a id stand out in sharp relief. A carefully executed (Imwiiig of such a preparation is given in figure 5. A iiiicro- photograph of tlic same preparation, f o r purposcs of orienta- tion, is supplied in figure 6, in which tlie illustrated tic1 1 cl ~ can lie readily identified. The drawing represents approxiiiiatelj- a middlc focus of the section, though it was necessary to vary the focus slightly from place to place in order to establish the exact coiiliections and c o n i w of tlic cell houridai.ic~s. Tlicrc

Fig. 2 Cell bountl:trics iii the first seyiiieiit of tlie prosiiiial tubule (in pars recta) of tlie dog. The brusli Iiorder, with w r y indistinct striation, is see11 :ibovc mid to tliv Icf t . Paraffin section, 5 f i . Heidcnhaiii’s iron Iieiuatosgliii, orange G. C:mic~rn lucidn drawing, X 2080. Sce text.

Fig. G I’liotoi~iicrogrnph, documentary for figure .5. x 660. See text.

CELL CONTOURS I N PROXIXMAL TUBULE 11

is a marked variation in the thickness of the boundaries, and the finer lines seen in the drawing are in some cases still more delicate in the original preparation. Even with the highest niagnifications, the boundaries appear entirely structureless and homogeneous.

It is clear from the drawing, and abundantly confirmed from study of the preparations, that the cell contours are extremely complicated, and that a marked degree of interdigitation is present at all levels, from the lumen to the basement mem- brane. The pictures are usually so complicated that accurate tracing of the boundaries is impossible, and it was very diffi- cult to locate a field of any size in which the pattern could be reliably reproduced (fig. 5). Repeated attempts to determine the exact contours of a single cell at several levels from the lumen to the basement membrane have so fa r failed. Our most successful attempt is illustrated in figure 7. The cell is taken from the first segment of the proximal tubule, in the upper portion of the pars recta. The long axis of the cell corre- sponds approximately to the long axis of the tubule, and the cell was located in about the mid-axial position. The drawings represent the cell outline at three successive levels: A-at the basement membrane; B-about 3 p above the basement mein- brane ; and C-about 8 p above the basement membrane. The cell was studied in serial sections a t 5 v. I n the first section, from which A was drawn, the cell is incomplete, and only the portion given can be accurately traced. B and C represent the highest (most basalward) and deepest (most luminal- ward) levels in the second section at which the cell boundaries can be sharply focussed. The plane of the third section passes through the lunieii of the tubule and cuts through the apical, projecting portions of the cells to give a confusing picture, in which the outlines of the particular cell under consideration cannot be followed.

When frozen sections of formalin-fixed (10% neutral) material are treated according to Do1 Rio Hortega’s second modification of Ach6carro’s method (Romeis, ’32, p. 430), a

12 J O H N J. FOOTE AND ALLAN L. GEAFFLIS

very delicate impregnation of the cell boundaries, as a rom of fine black dots, is sometimes obtained in the first segment of tlie proximal tubule. In our hands the method has given patchy and variable results, and uniformity has nevcr been obtained.

I %t

C

Fig. 7 Outlines, a t three different levels, of a cell from the first segiiieiit of the proximal tubule in the dog, in the upper portion of the pars recta. Bichromate- inordanted Zenker-formol material; paraffin sections, 3 p ; iron hematoxylin. Camera lucida drawings, X 2225. See text.

However, in those tubules in which the impregnation is suc- cessful it is quite sharp, and reveals an extremely complicated cell-boundary pattern, so complicated, in fact, as to be almost uninterprctable. I n all instances which we have observed, the pattern seems to exhibit approximately the saiiic order of complexity from tlie luniinal surface of tlic cells to the base- ment inembrane.

CELL COFTOURS I N PROXIMAL T U B U L E 13

In chrome-silver (Landauer ) preparations, the marked coni- plexity of the cell contours in this segment is again demon- strated. Here, as in previous preparations, the order of coinplexity is such that accurate tracing of the boundaries of individual cells is extremely difficult. Figure 8 illustrates the outlines of two adjacent cells at the basement membrane. The cells are from the first segment of the proximal tubule, in the

1

Fig. 8 Outliiies, at the basement membrane, of two cells in the first segment of the proximal tubule in the dog, located in the pars convoluta. Chrome-silver (Landaner) preparation. Camera lucida drawing, X 2290. See text.

pars convoluta. The long axis of the tubules corresponds with the long axis of the figure, and the cells are located in the mid-axial line. The pattern affords an interesting comparison with figure 7A, in which the cell processes a t the basement membrane are shorter, more numerous, and more delicate. It should be pointed out that figure 8 represents a negative image of the original preparation, in which the cell boundaries are specifically impregnated.

14 J O H N J. FOOTE AND ALLAN L. GRAFFLIN

Second segmed. In fresh spreads of the kidney, cell out- lines can seldom be observed in the fat-laden second segment of the proximal tubule. I n those instances in which they can be visualized, the cell boundaries appear as thin, glairy lines forming an essentially rectilinear pattern which seems to persist from the luminal surface of the cell to the basement membrane.

In frozen sections of formalin-fixed material, the cell boun- daries are always sharply rectilinear at the luminal surface of the cells. As one focusses more deeply into the cell, one of two pictures may be observed. (1) The rectilinear pattern may persist, without detectable waviness or irregularity at any point, all the way to the basement membrane. This picture is less frequently observed than (21, in which, on focussing, the rectilinear pattern persists for a variable distance-usually to a level about mid-way between the apical pole and the equatorial plane of the nucleus, sometimes to the equatorial plane itself-and then rather abruptly “disappears”. I n the more basal portion of the cell, no trace of the cell boundaries can be visualized.

When frozen sections of the formalin material are treated according to Del Rio Hortega’s second modification of AchG- carro ’s method, successful impregnations are only seldom obtained, and the pictures observed are quite variable. (1) The luminal cell boundaries may be delicately impregnated as a row of fine black granules ; this pattern persists, on deepening the focus, to a point about mid-way between the apical pole and equatorial plane of the nucleus, and then rather abruptly “dis- appears ” in a manner similar to that observed in the untreated frozen sections (see above). ( 2 ) The cell boundaries may be impregnated only at the lumen, where they are rectilinear, and at the basement membrane, where they exhibit a somewhat, though not markedly, serrated or wavy pattern, analogous to that illustrated in figure 9. (3) The impregnation of the wavy cell-boundaries at the basement membrane may persist, as one focusses toward the lumen, to the level of the equatorial

CELL CONTOURS I N PROXIMAL TUBULE 15

plane of the nucleus or to a level about mid-way between this plane and the apical pole of the nucleus, where it rather abruptly “disappears ’,. In these instances one can usually observe that the waviness or serration of the boundaries be- comes progressively less marked as one proceeds from the basement membrane toward the lumen. Furthermore, at the level a t which the impregnation ceases, it is frequently quite obrious that this serrated pattern is directly continuous with an essentially rectilinear pattern which, even though unim- pregnated, can be readily visualized and followed to the lumi- iial surface of the cells.

In chrome-silver (Landauer ) preparations, the cell boun- daries in the second segment are seen to be quite similar to those described above for the cat. The pattern is essentially rectilinear at the lumen, and gradually loses its rectilinear character as one focusses toward the basement membrane, where the cells are usually interdigitated to a considerable degree. The pictures obtained are fairly well typified by the accompanying illustrations (fig. 9), which represent the out- lines of two adjacent cells at five different levels: a-at the lumen; e-at the basement membrane; b, c and d-at three intermediate levels, equally spaced. The cell side marked with asterisks is shared in comiiion by the two cells, and lies essen- tially in the same perpendicular plane as seen a t the lumen and at the basement membrane. Variations in the contour of this side as drawn at comparable levels is explained by the fact that, although adjacent, the two cells are somewhat dif- ferent in height (cell 1, 9.4 p ; cell 2, 13.2 p ) ; and the outlines, which were drawn independently for each cell, are differently spaced accordingly. The long axis of the tubule runs parallel with the long edge of the page, and the cells, as reproduced, are carefully oriented with respect to this axis. The cell- outline pattern at the basement membrane may be somewhat more or less complicated than in the cells illustrated, but a true rectilinear pattern is never observed.

16 J O H N J. FOOTE AKD ALLA4N L. G R A F F L I N

Cell 1 Cell 2

Fig. 9 Outlines, a t fiw different levels from the lumen t o the basement inem- brane, of two cells from the second segment of the proxiiiial tubule in the dog. Chrome-silver (Landauer) preparation. Camera lucida drawings a t X 2400 ; as reproduced, X 1200. See text.

CELL CONTOURS I N PROXIMAL TUBULE 17

Iuterscgiizeiotal tvamnsitions. I n fresh spreads of the kidney, abrupt transitions between the fat-free first segment and the fat-laden second segment can be readily identified. This is likewise true in frozen sections of formalin-fixed material, particularly so when the preparations are stained with Sudan 111. I n both types of preparation, the fundamental difference in the character of .the luminal cell-boundary pattern in the zone of transition-markedly interdigitated in the first seg- ment, rectilinear in the second-can usually be established, although shar.ply defined pictures are not obtained. A curious finding in the Hortega-Achharro preparations was the fact that not infrequently the cell boundaries of the first segment were particularly well impregnated in the region just proxi- mal to the transition. I n these instances, although the recti- linear boundaries of the second seamelit (identifiable by fat content) were not impregnated, it could be clearly demon- strated that the markedly interdigitated pattern of the first segment persists right up to the point of transition.

DISCUSSION

First segnzeizt. The cell contours in the first segment of the proximal tubule in the cat have already been described and discussed in some detail by Grafflin and Foote ( '39), whose findings are summarized above. The present observations upon the cell contours in the first segment of the dog may be summarized as follows. The cell-boundary pattern at the lumen shows a marked degree of serration or interdigitation, in a manner entirely analogous to that observed in the cat. In some iiistnnces the pattern may exhibit approximately the same order of complexity all the way from the luiiicn to the basement membrane. I n other instances the complex pattern at the lumen undergoes a gradual simplification toward the

which persists is always greater than a t a coniparable level in the cat. In the more basal portions of the cells the pattern again becomes pi.ogressirely more complex. At the basement

intermediate levels, although the degrees of interdigit a t' 1011

18 J O H N J. FOOTE AND ALLAX L. GHAFFLIX

membrane the cell outlines are always markedly interdigi- tated, but the degree of interdigitation is quite variable. Even in its simplest form the pattern is considerably more compli- cated than in the cat. I n the most extreme instances, the order of complexity is suggestive of that observed a t the lumen in both cat and dog. Zimmermann ( '11) implies a progressive simplification of the cell contours, from the lumen to the base- ment membrane, in the first segment of the dog, but in our experience this is not the case. Del Rio Hortega ('24) states only that tlie cell contours in the convoluted tubules are extra- ordiiiarily complicated, and gives no specific data upon their appearance at different levels.

Secoizd seginozf . I n both cat and dog the luminal cell- bouiidary pattern in the second segment of the proximal tubule is essentially rectilinear in a11 cases. In sonie instanccs in tlie dog, this rectilinear pattern appears to persist all the way from the lumen to the basement membrane. I n tlie great majority of instances in both cat and dog, however, the pattern gradually and progressively loses its rectilinear character, particularly in the more basal portions of the cells, and a t thc lcvel of the basement membrane may exhibit a considerablc degree of interdigitation. Zimmermann ( '11) indicates that the rectilinear pattern regularly persists, in both species, t o the basement membrane, but thisewe have been able to observc only occasionally in fresh spreads and frozen sections (forma- lin) of the dog kidney.

It should be emphasized that the cat and dog are the only two niaminaliaii species in which a sharp subdivision of the proximal tubule into two cytologically distinct segments has so far been clemonstrated.

SUMMARY

The proximal tubule of the cat and clog nephron is coni- posed of two distinct segnients, the transition between wliich is typically abrupt with respect to both cell shape and fat content. Although fat deposition is reversed in the tx-o species

CELL CONTOURS IN PROXIMAL TUBULE 19

(first segment of cat, second segment of dog), the cell shape exhibits no such reversal and is presumably entirely inde- pendent of fat content.

I n the first segment the cells are highly irregular in shape, with a marked degree of interdigitation. The cell contours are most complicated at the lumen, less so at the basement membrane, and (regularly in the cat ; at tinies in the dog) still less so a t intermediate levels. The cell shapes are on the whole far more irregular in the dog than in the cat.

In the second segment the cell contours at the lumen are essentially rectilinear in both species. For the most part these contours gradually and progressively lose their rectilinear character, particularly in the more basal portions of the cells, ,

and at the basement membrane may exhibit a considerable degree of interdigitation. However, in some instances (dog) the rectilinear pattern is apparently retained all the way from the lumen to the basement membrane.

LITERATURE CITED

DEL RIO HORTEGA, P. 1924 Sur la configuration et la structure des nhphrocytes Compt. rend. 8oc. de Biol., d a m les divers segments du tube urinifhre.

T. 91, pp. 831-833.

FOOTE, J. J. 1936 Segmental differentiation in the proximal couroluted tubule of the inammalian nepliron. Proc. SOC. Exp. Eiol. and Med., rol. 34,

Quantitative irieasureineiits of the f a t - laden and fat-free segments of the proximal tubule in the iiephron of the cat and dog.

pp. 196-198.

FOOTE, J. J., AND A. 11. GRAFFLIN 1938

Anat. Rec., vol. 72, pp. 169-179.

GRAFFLIN, A. L., AND J. J. FOOTE 1938 The Bodian technic as applied to thr Complicated cell bouiidaries of the renal epithelium in mammals. Anat. Rec., vol. 73, pp. 115-118.

__- 1939 Epithelial cell shapes in the first segment of the proximal titbule of the cat nepliron, as demonstrated by the chrome-silver method. Am. J. Anat., vol. 65, pp. 179-198.

HEIDENHAIN, R. 187.2 Mikroskopische Beitrage zur Aiiatouiie und Physiologic der Nieren. Arch. f . mikr. Aiiat., Ed. 10, S. 1-50.

LaNoarER, A. 1895 ITeber die S t luk tur des Nierenepithels. hnat. hnz., Bd. 10, S. 645-653.

20 J O H N J. FOOTE A N D ALLAN L. G R A F F L I N

ROMEIS. B. 1932 Tascheiibueh der mikroskopisclieii Teehiiik. 13th ed. Miinchen

ScIracHowA, S. 1876 Uiitersucliungen iiber die Nicren. Iiiaug. Dissertation,

and Berlin, R. Oldenbourg.

Bern.

ZINMERNANN, K. W. 1911 Zur Morpliologie der Epithelzelleii der Suge t i e r - niere. Arch. f . mikr. Aiiat., Bd. 78, S. 199-231.