FIVE FIGIJRXS

I t has heen adequately tlrmonstixtcd that the proximal tubule *I of tlie cat and dog nepliron cliai~actcr.istical1~ contains fat iiiider normal conditions, and that this fat is regularly distributed in a particular manner in the two species (Peter, ’09 ; Zimmermann, ’1 1 ; Naliarnura, ’ 3 5 ) . I n the cat the eiitire pars convoluta and the upper portion of the pars recta are normally fat-laden, while tlie terminal portion of the pars

n is fat-free. In the dog the iw-ersc is true, the fat being confined exclusircly to the terminal portion of the pars recta. In tubules teased from acid mae~rat ion material the transi- tion between the fat-laden and fat-frcc portions is characdtpr- istieally abrupt (Peter, Nakamura). Zimmermann added greatly to 0111- knowledge by demonstrating illat the abrupt transition with respect to f a t content is liltewise abrupt with respect to cell type. In both species the luiniiial cell boundary pattern in tlw terminal portion of the pars recta-fat-free in the cat, fat-laden in the dog-is rcctilinear. In the remainder of the proximal tubule-fat-laden in the cat, fat-free in the dog-the luniiiial cell boundary pattern is very complicated,

’ A brief preliriiiiiary xecouiit of this study 118s k e n pnhlislied b y Foote ( ’36). Fcllon of the Henry Strong Denison Rledical P’oundation. ‘Prouinial tubule’ will be used in this paper to designate the entire portlon

of tlic nephroii probidcd with Ijrusli border, cvtendiiig fro111 the glomerulus t o thc liarisition to t h e thin segment.

lF9

170 J. J. FOOTE AND A. L. GlUYl,’L,TN

showing in mai-lied degrec~ tlic interdigitations i1s11~11y cot1 sidered ehaiacteristic of tlie mammrtlian proximal tubule. Despite tlie reversal in fat distribution in the tlvo species, these findings seem amply to <justify the conclusion thwl we are dealing here with two distinct segments of the proximal tuhule. TThetlier the bulk of t lie pi.oxinial tabulc (exclusivcl of tlie terminal port ion of tlie pars recta) can he fnrthcr sub- divided 011 cytological gr*oiinds mubt await further wor~k. A s is well kiiowm, Suzulii ( , l a ) has subdivicled the mammalian proximal tubule into Ihree, o r in thc rabbit four, segments, but no critical cytological 1)i.oof of his tlicsis has J-et been acldutwl. I n a re-investigation of the prohlem-the details of which will be published at a later. date-we liavt. up to the prcsmt time been forced to concluck that, in the cat and dog, there are only two cytologically distillcat segments in the proximal tuhiile, namely, the two established 11)- Zimrncrniann. Since, in both cat and clog, the alorupt shift in fat tlistrihution demarcates so acc*uralely tlic cytological transition hetween the u l~pe r and lower segments, we hare used isolated proxirnul tubule* stained f o r fat as a means of obtaining quaiitit a t‘ i re - measure- rneiits of the relative lengths of the two segments.

MATERIAL AND h1F:THIOOS

Tlip cats a i d (logs used for this study wcrc apparently health-\- adults, taken immediately before use from the animal farm, wlierc they had heen maiiitairied for varying lengths of time on the routine diet. Cats: no. 4, male, 3.6 kg.; no. 5, male, 3.8 kg.; no. 6, female, 2.5 kg.; no. 7, female, pregnant (near term), 3.7 kg. Dogs : no. 1, fernale, 6.2 kg. ; no. 2, male, 8.2 Bg.; no. 4, f ~ m a l e , 10.0 kg.; no. 5, female, 11.6 kg.; 110. 6, male, 9.7 kg. Tlie cats were killed instantaneously by meclian- ical means, and the dogs were Billed by illumiriatiiig gas. The kidneys were reiiiovecl at o~ic(: arid cut into thin ( 2 to 3 mm,f strips, which ~ w r e placed in 10% formalin and kept tliere uritil used. Trine aspirated from the intact blatldpr of all animals gave a negative test f o r protein.

Small blocks of tliesc formalin-fixed kidnrys, extending from the capsule well into the mcdnlla, were macerated in c*oricentrated hydrochloric acid (specific gravity 1.18 to 1.19) :

tlle length of time required varicd considerably wit11 diff creiit blocks, and estimation of the proper degree of maccratior~ had to be learned by experience. Tlie blocks were w-ashed free of acid with many changes of distilled mater, and thcn stair~erl with either. Sudan TI1 (saturated solution in 70% alcohol with 1% KOH) 01- Scharlach R (saturatcd solution iii TO‘/, alwhol with 2 % NaOIl) for periods of from 3 to 5 hours. The blocks were then retransfcrrcd to distilled water and allo~ved to ‘piiff’ until suitable for teasing. From the stained blocks cornplctc. proximal tubules, with thp attached glomei.ihs and witli a portion of the tliiri seffment o f Heiile’s loop, were teased and mounted under the dissecting microscope on cover slips smeared with a thin film of glycerine-albiimcri mixture. T h e w cover slips were then mounted on slides in glycerine diluted with water (for grcatcJ. ease in handlillg). Finally, within a short time after. the preparations wei*c made, since they do not last, the outlines of the entire proximal tubules ere drawn with the aid of the camera liwida, at a magnifica- tion varying between X 170 and x 180 (accuralcly de te rmind for each clrawing). The point of abrupt shift in f a t distribn- tion w a s rioted on the drawings, and from the latter the dc- sired measurements \WIT made with N Keuffcl and Esser map mcasurc~-. The figures given in tht. table:: were c+alcuIated from the measurements made on the clrawiiigs. The point of transition from tlic proximal tubule t o the thiil segnicr~t is sharply defined, and can be determinecl withoiit difficulty.

OBS KRVBTTOKS

In the following cliscussion the major portion-entire p a n coiivoluta aiid upper portion of pars recta-of the pmximal tuhuIe will be referred t o as the ‘ f i r n t sqmeiit ,’ and tho tcr- miiial portion of the pars rccta oi’ tlie pr*oximal tubule will he referred to as tlie ‘secoiid segment.’ As explaimcl in the introduction, the first segment is norma1ll)- fat-laden in the cat, fat-free in tlie dog; aiid the second segment is notmally fat-free in the cat, fat-laden in tlie dog.

Cat Ry the methods described above, sixty cwmplete proxi-

mal tubules-fifteen from each o f €0111‘ ca ts-were isolated,

172 .J. J. FOOTB ,4KU A. L. GRAFl~’L1P;

mounted, drawn and measured, tlie leugths of the first and second segments being determined by the fat distribution. The results are summarized in table 1. Tlie average percent- age length of the second segment in each of the four animals is 17.7, 15.5, 20.7 and 23.9% respectively. All of the tubules from cats nos. 4 am1 5 were isolated from thc ccntral portion of the kidney, i.e., the region dii*ectly opposite the hilns and approximately eyuiclistant from the poles. In tlie other two animals groups of tubules were isolated, for comparisoii, from llie ccintral and polar regions of the kidney oil the one lianti (cat no. 6 ) , arid from tlie central and Iatwal regions of the kidney 0x1 tlie other liarid (cat no. 7) . Since the measurements

‘l‘ABLh 1

Total lpngtk , and 7 ~ n g t h of first and secor~d s r y m e n t s , of pro.rimaZ t u b u l e of cat nephron; f i f t c t i z proszntul tubules f r o m each ca t ; 011

meaaurc i n ~ r i l s t n mzllzmetcrs

TOTAL LEYGTH LSNGTII O F PROXIXAL TUBULE 1ST SEGMENT EAT NO.

4 5 6 7

Ziange Mean for Gn t 1 1 h u i ~

8.83-13.22 7 1.42-16.10

7.07-11.10 6.70- 9.92 6.70- 16.1 (I

10.81 mni.

6.61-11.89 9.62-1 3.70 5.37- 9.12 4.71- X.25 4.71-1 3.70

8.79 mni.

l i l N G T H ON 2ND SEGMENT

1.33-2.90 1.27-3.83 1.39-2..53 1 ..58-2.76 1.2 7-3.83

2.02 mm.

2 N D SEGXENT- PER, CENT O F

TOTAL LENGTH

13.3-25.0 9 23-28.7

14.8-2 7.5 16.9-31.8 9.8-3 1.8

19.5%

of the tubules in the different groups showed 110 significaiit cliffcrcnces dependent upon the location of the neplirons in the kidney suhstaiice, a11 of the tubules isolated from cats nos. li and 7 have heen grouped together in the tahle.

The distribution of fat in the proximal tubule o f the cat uephron was found to be relativcly constaut. The typical findings map be summarized as follows. At the glomerular end the fat begins abrupt\? mith no apparent neck segment. The fat droplets here are of both small a id medium size and are quite iiumcrous. For a short distance distally along the tubule thcj- gradually inerease in size, their nnmhers remain- ing essentially the same. After the fat deposit has bccomc

PROXIMAL ‘TCSLrLli O F CAT A S D DOG XEPHRON 173

heavy it remains so until 11cbar the distal end of the first scg nleut, where the fat gradually diminishes. At the end of the first segment the fat stops abruptly and completely; the second segment is wliolly f a t - f r e ~ . ~

Although most of the intersegmental ti.nnsitions observed ~ e r c abrupt (figs. 1 to 3 ) , occasional variations were eiicoun- teretl in each of the four cats. Tn some cases the cell type shifted at different levels on the two sides of the tubule, sev- eral fat-laden cells appearing opposite fat-free cells. In a few instances ‘double’ transitions (fig. 4) wvre noted: the re11 type cliaiiged abruptly-from that of tlic first to that of the secoad segment-arouncl the whole circumference of the tubule, then a short distance fartlicr on reverted to that of tlie first seg- ment, finally to changc again to that of the second segment, tliis type tlicri continuing on to the transition to the thin seg- ment. It should he emphasized, liort-ever, that tliesc varia- tions arc rare. In most instaiices, the proximal tubule exhibited a distinct,

though slight, increase in cliamder at the point of transition from the first to the second segment (fig. 1). 111 oiic animal (cat no. 5) this increase in diameter was verp marked (figs. 2 to 4). This animal likewise ~ h o w e d cliverticulu of tlic proxi- mal tubule as a constant feature. These diverticula regi~larly occurred in the distal third of the first segment, and usually numhered ahout fif n to each tubulc. Thcy ranged in size from slight irregularities in the contour of tlie tubule to struc- tures four o r five tubule diameters in lmgtli (fig. 5 ) . Occa- siorially in the aiiimal diverticula we1.e also noted in the sccond segment (fig. 3 ) . T;I.’hcn they appeared here thry were invari- ably close to the point of trailsition. The diverticula always exhibited the type of epithelium (with respect to fat content) characteristic of tlie segment of the proximal t u h l e from ‘ In id ca t (not oiir of the present srries) nhidr mas killed with chloioform and

cther instratl of by ineclianicsl rncwm, finme f a t droplets v c l e found in t h e cells at thc distal end of the srcoiid segment in about 90% of tllc proximal tnhules exsniincd. Thew clroplcts iiccaiiir fairly numernus tonard, but stopped shruptlg at, the transitiori t o the thin segment. ~ I I no jiistxnce in thr four cats reported in this sturlj H ~ S any f a t found in the second segment.

174 .J. J . FOOTF: .4NI) A. L. GI:AFFT,TN

> 1 - 3 1

Figures 1 to 4 (froin left l o riglit). Trartsilioits f r o m fat-laden ( f i y i t ) ’LO fa t - f ter (second) segment of pro~11iia1 tubule of cat rirpliron ; tcaied 1)re1~aiatioith from inaceration niaterial.

Fig. 1 Chaixctcristic transitioii, diowing ahinpt sliift in f a t riistii1)ution :ind

Fig. 2 Tiansition shon ing marked dilatation of second srgment a t p i n t of

Pig. 3 Transition slioiring sinall dixerticiilum of first poi t ion of spconrt ocg-

Fig. 1 ‘Douhle’ tr:iiisition (fat-laden, fa t - f rw, fat-laden, fat-frec.) ; ore nest.

FiguIe 1, f rom cat no. 1, stained with Sndnn 111; figures 2, 3 and 4. from cat

sotiieirliat l a g e r diameter of second segment.

transition. x 200.

ment. X 200.

x 200.

no. 5 , stained with Sc1i:irl:rch X.

X 350.

which thcy arose. Jn no other animal studied were these diverticula encountered.

I n tlie course of diss iiig the proximal tubules under the observation microscope, it was iiotcd that the timisitioii be- tween the first uncl second sc.gments typically occurs within the medullary ray. Tlic~re is no defiiiite level at which it occurs iii all tubules. In geiiei*al it map he said tliat tlie tnhnles origiiiatiiig from glomeidi near the periplierp of the cortex hax7~ their transition nearer. the upper end of thc ray than those oi-igiiiating from lo\.vcr-lyiiig glomeruli. It was further noted that the ‘pars recta’ of some of the pr*oxirnal tubules

Fig. 5 L)ivrrticnl:r of the distal third o f the first ( f a t laden) segmcnt of the Thefie diierticula ncrr among tllr lnigest

From cat no. 6 ; teased preparation from maceration materiid, stainrd proximal tuhiile of the cat nephron. observed. with Scharlach R. X 200.

does not run a straight course, hut is of a spiral or corkscrew shape, as originally emphasized by Schachowa (1876). Proxi- mal tubules originating from glomeruli lying near the niechdla almost iiivariahly show this arrangemcnt of their ‘pars recta.’ All variations from slight helids to a marlied corkscrew shape a re encountered, the most markccl distortion ocnxring in those tuhules originating from the lowest-lying glomeruli.

Ilog For the dog, as for the cat, sixty complete pi~osirnal tubules

-fifteen from each of four dogs-wei*e isolated, mounted, drawn and measured. The results are summarized in table 2. The average percentage length of thc sccoiid segment iii each of the four animals is 13.9, 10.0, 9.1 aiid 10.3%) i*espe.ctively.

176 J . J. FOOTE A N D A. 1.. CR$FFLIN

Most of the tubules mere isolated from tlie central portion of tlie kidiiey j the remainder were isolated from other region?. Ai l of the tubules from a given animal have been gi-oupd together in the tahle, s iwe in the dog-as in the cat-the loca- tion of the nephron iii the kidney substance is apparently without particular significance fo r this aspect of the problem.

As discussed in tlie introduction, the first segment o f the dog 's proximal tubule is typically fat-free, the second segment typically fat-laden, and the traiisition between the two seg- ments is abrupt. In dogs I anti 4 these findings were the rule, the second segment showing an accumulation of large

TABLE 2

Total Zengl l~, arcd lcnglh of firs1 and stcoird segments, of proxinial tubule of dog

ineavvremcats in, rd l t inc~ ter s ncphroic; fifteen proxiinal (ubitles f rom each dog; all

DOG KO.

1 4 5 6

Range Mean for 60 tubules

TOTAL LEGGTH 'BO\lMAl, TL'UUlrl

16.68-21.60 13.02-16.61 22.00-36.80 26.70-33.0n 13.02-36.80

22.50 mm.

13.82-1 8.41 11.68-15.21 19.21-34.50 23.90-28.93 1 l.fiX-.31.RO

20.14 mm.

LENGTH O F 'LNI) SEGnll.N'I'

1.86-3.30 1.01-1.67 1.36-3.14 2.3 0-4.3 0 1 ~ 1 4 . m

2.36 min.

9.30-17.1 0 6.84-11.66 4.27-13.90 6.85-13.00 4.47-13.911

10.870

fa t droplets just below the point of transition. T n some in- staiiccs this initial heavy deposit shaded off graduallp as the transition to the thin segment ~ v a s approached, tlie terminal portion of the second segment being at times fat-free. I n other instances, the initial heavy deposit xas not continuous, being interrupted at intci-vals by groups of cclls coiitaiiiing little or no fat. The filldings in clog 6 were esseiitially the same as in dogs 1 and 4, with the additional observation that some of the tnbnles showed a heavy accumulation of re ry finc --rather than large -fat droplets at the beginning of the second segment. Tlie tubules of clog 5 were at)-pical in that the first segment-over appi-oYimatciy one-quartel- of its length, beginning a t the g.lomcrL~lus-slio~-~~ very fine fat

droplets, appearing uiider the dissecting microscope a> a tlif- fuse staining. These droplets gimlually climiriislied in number as oiie proceeded distally, and tlic remaining three-quarters of the first segment wire w1ioll~- fat-free. The second seg- ment showed a lieavp deposit of fat droplets throughout. These droplets w w e piwloniinaiitly small, hiit iittersperscd among them were larger. o~ies , occurriiig either singly o r in small groups (up to ten in number). In all four of the ani- nials the two segments under consideration stood out quite dear ly and could he measured it-ithout difficulty.‘

In the dog, as in the cat, tlie ‘pars recta’ of the proximal tubule was not infrequently of a spiral or corkscrew shape. Where marked distortion was found, it could lie established that these tubules regularly origimtecl from glomeruli lying deep in tlic cortex.

I)IHCUSSION

As is well known, the effects of the acid maceration technic. upon tissues-particularly upon R tissue such as tlie kidney-- are liiglily \TiIr*iable with rrspect to the degree of swellixig obtained. This being true, we should like to emphasize that the absolute lengths of tlie proximal tubules rec*orclccl iii tablcs 1 aiicl 2 are iiot to be consiclerwl in any sense representative of the actual ltmgths of the tubules in the living, o r even in the fised, organ. On the other hand, we hare no reason to suspect that tlie two segments of the proximal tubule are clis- proportionatelg- altered in length by the rnaceration to any significant extent. lTiicler the circumstaiicc~s, wc believe that the percentage lengths of the two segments as obtil ind by this method arc valid.

If we accept this conclusion, there is an additional fact to be derived from the data in tables 1 aiid 2 wliiclt is worthy of

’ I n anotlrrr dog (no. 2 ) n o f a t vab dernon\tlablc a n p l i e r e in the prnximnl tubule. T h a t such a fat-free condition of tlie pars recta C R T I be attained, c.g., on a deficient diet, was emphasized long ago by Scli:tc.bow:t ( IdTA) , wlro delibrr- ately produced thr condition 111 0 1 dcr to study, under more ideal conditions, the shapes of the w l l a in this portion of tlir nephron.

178 J. J. FOOTF, AhTJ) A. L. GLZBEPLIN

mention. The averagc percentage length of the second seg- ment is almost twice as great in the cat (19.55gI) as i t is in tlie dog (10.8%) ; on the other hand, tlic average total length of the proximal tubule is roughly twice as great in the dog- (22.5 mm.) as in the cat (10.8 mm.). Thus, despiie the dis- p~~opor t ion in arerage percentage length of the second seg- rrieiit in the two species, thc average lengtb (in millimeters) of this sccorid segment is nearly the same fo r both animals (2.02 mm., cat ; 2.36 mm., dog). TVliat significaiice this oh- servatioii may have must remain purely speculative f o r the present, but the fact is clear.

No attempt will be rnade here to review the exteasire litera- ture upon tlie occuri-ence of fat ill the kidneys of the cat and dog. The more important references (’an he foiincl in r o n Mollendorff (’30) and Xakamara ( ’35). I n his recent paper Modell ( ’33) coiicludes that “large arviourits of l i p i d droplets arc dcmonstrahle in the convoluted portions of the renal tubiile of normal adult cats, and are absent in the other por- tions of the tuloulc.” Tliis statcmcnt is obviously iricomplelc as concerns the proximal tubule, since the ripper portion of tlie pars recta is likcwise fat-laden LIJI~CI* normal conditions.

SUhIhIARP

l ’ h c proximal tubule of the cat arid clog iiephron character- istically contailis fat under normal conditions, and this fat is regiilarly distributed in a particular manner in the two species. In the cat the entire pars convoluta aiid the upper portion of tlie pars recta are no~-mullp fat-laden, while the terminal por- tioii of the pars recta is fat-free. In the dog the wverse j s true, the fat being corifiried solely to the terminal portioii of the pars recta. The transition between tlic fat-laden and fat- free portions is abrupt, not only with respect to fat content, hiit also with respect to cell type (Zimrnermann). Despite tlie reversal in fa t distribution in the t ~ 7 0 species, the fiiidirigs seem to justify the coriclusioii that ITT are dealing with two cytologically distinct segments of the proximal tubulc.

In the present study the attempt has been made to olntain a qnantitative estimate of the relative lengths of these two segments in the adult. From each species sixty complete proximal tubules--fift cen from each of four animals-hare beell isolated from maceration material arid measnred. The tubules were stained fo r fat, aiid by this means the p i n t of traiisitioii he t~ -cen the two segments could l)e readily identi- fied. Tn percentage of the total length of the p i w k i a l tnhule, the average lcngth of the secwiirl segment is 19.5% in the cat, 10.8% in the dog.

T, I T E RA wr R P: (XTE I )

FOOTE, J. J. 1936 Segnieritdl differentiation in the proximal eonrolutcci tubule Proe. Soe. Exp. Kiol. and Med., vol. Ji.

~ I G D E L L , \V. 1033 Ohservations on the l i p i d s in the rrn:il t u h l c of thr c i t .

VON MOLLENDORFF, \V. 19:W Der E:xc.retionsapp:trat. In: voii Mollendorfl 'R

of thc iiianimaliari nrphroii. pp. 196-198.

Anwt. Rw., vol. 57, pp. 13-27.

Haiidhiicli der nlikrOSPOpiSChen Anatomie des >feiischen, Ed. 7! Ttil R. 1-328.

S A I ~ A N L I K %, 8 Beoh:tclitiiiigen uber die Fette in drn T1arnkun;tlehc.n tol l Katze iind Hund.

I'FTER, K. 1901) Die KierenkanLlchen des Yeiisrhen und einiger Saiigetiere. 111 : Untcrsuclruiigeii iiber E a u und Elitwicklung dcr Nicre, herausgrgeberi voii T h r l Prter. Jrna. Erstcls Heft , S. 1-358.

SC'HAfTIOn A , 8. 1576 TTntrrsiicliungrn iiher die l i e r r n fiiwi~g. Di Kern.

R c z u ; r ~ , T. pathologisc.hen Bedingungm. Jeria.

ZIMMERMAXN. K. W. 1911 Zur Rlorplrologie der Eyithel~ellen dci Sdugctier nieic. Aitl i . f . mikros. Anat., Bd. 78, 8. 199-231.

1935 P'olia Annt. Japon., Rd. 13, R. 45-54.

191% Zur Movpholugie cfrr Nicrc~iisekrctiori uiiter phj siologisclien und