FILTRATION AND REABSORPTION OF PROTEIN BY THE KIDNEY* BY ALVIN L. SELLERS, M.D., NEILYN GRIGGS, JESSIE MARMORSTON, M.D., AND HOWARD C. GOODMAN, M.D.

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1 Published Online: 1 July, 1954 Supp Inf: Dwnladed frm jem.rupress.rg n August 21, 218 FILTRATION AND REABSORPTION OF PROTEIN BY THE KIDNEY* BY ALVIN L. SELLERS, M.D., NEILYN GRIGGS, JESSIE MARMORSTON, M.D., AND HOWARD C. GOODMAN, M.D. (Frm the Institute fr Medical Research, Cedars f Lebann Hspital, and the Department f Medicine, University f Suthern Califrnia Schl f Medicine, Ls Angdes) PLATEs 1 AND 2 (Received fr publicatin, February 26, 1954) The findings f Walker and Oliver (1) indicate that the glmerular filtrate in mammals may nt be entirely prtein-free. A large bdy f evidence, recently reviewed by Rather (2), makes it clear that the cells lining the prximal cnvluted tubule are able t reabsrb prtein frm the glmerular filtrate, and, in sme instances, at least partially t degrade it. Dck (3) and Gilsn (4) have labelled certain f the plasma prteins in viv by injecting the dye %1824 intravenusly and have fund the dye cncentrated in the cells f the prximal cnvluted tubule. Bth wrkers have interpreted this as evidence f glmerular filtratin and tubular reabsrptin f plasma prtein. It has recently been demnstrated (5) that the nt incnsiderable nrmal urinary prtein f the rat has the electrphretic and slubility characteristics f serum alpha and beta glbulin. If the glmerular membrane f the rat allws the passage f large serum glbulin mlecules, it wuld seem that the much smaller serum albumin mlecules wuld enter the glmerular filtrate even mre readily. This has been cnstrued as strng evidence that in this species, serum albumin is filtered thrugh the glmerular capillary and is cnstantly reabsrbed by the cells f the renal tubule. A small but cnstant prteinuria exists in man, and it has been shwn that the A/G rati f the urinary prteins is the reverse f that in the plasma (6). The cncentratin f prtein in glmerular filtrate is nt knwn with certainty. The nly direct measurements are thse f Walker, Btt, Oliver, and MacDwell (7). Using methds that culd nt distinguish prtein cncentratins f less than 3 rag. per cent with certainty, they bserved values ranging frm less than 3 nag. per cent t less than 2 rag. per cent. Dck (3), using Bickfrd and Wintn's technique f paralyzing tubular functin by perfusin f the rabbit kidney with ice-cld serum (8), btained prtein cncentratins f 15 t 22 rag. per 1 c. f "glmerular filtrate." The present study * This investigatin was supprted by a research grant frm the Natinal Heart Institute f the Natinal Institutes f Health, Public Health Service. 1

2 FILTRATION AND REABSORPTION OF PROTEIN BY KIDNEY is an attempt t measure quantitatively the amunt f prtein reabsrbed daily by the renal tubules f the rat kidney. The dye T-1824 frms a stable blue cmplex with the plasma prteins. When the dye is injected int the bld stream in lw cncentratins, it attaches preferentially t plasma albumin. As the cncentratin f dye is increased, plasma albumin becmes saturated, and the dye begins t attach t the plasma glbulins, selecting first alpha glbulin. Rawsn (9) has shwn that the cncentratin f free dye in the equilibrium system between dye-prtein cmplex, plasma prtein, and free dye is infinitesimally small. Allen and Orahvats (1) have described the frces binding T-1824 t plasma prtein and cnsider the renal clearance f T-1824 a measure f the clearance f plasma prtein. We have previusly demnstrated that when 25 rag. f T-1824 is injected intravenusly int 2 gin. rats, n measurable amunt f free T-1824 exists in the circulating bld, and when prteinuria is prduced, all f the T-1824 fund in the urine is similarly prtein-bund (11). It was reasned therefre that T-1824 present in the lumen f the nephrn is prtein-bund and that the cmplex T-1824-prtein is reabsrbed by the cells f the prximal cnvluted tubule. Since T-1824 is carried int the tubule cell attached t prtein, and since it is seen t accumulate in the renal cells in the frm f intensely blue drplets, by measuring the ttal T-1824 cntent f the tubule cells, and the mean rati T-1824/prtein per unit time in the plasma, ne can calculate the milligrams f prtein reabsrbed by the kidney during any desired time interval. This has been dne in the present wrk. Methds Animals.--Femaie rats f the Slnaker-Addis strain weighing 2 t 21 gm. were used in all the experiments. Technique f Injectin.--AU animals received 2 nag. f T-1824 in 1. ml. f.89 per cent sdium chlride slutin intravenusly. Animals were bled while under ether anesthesia, by severing the abdminal arta with a sharp blade. Bld was cllected directly int a centrifuge tube held just belw the severed vessel. Chemical Prcedures.--Ttal serum prteins were determined by the micr Kjeldahl methd. T-1824 in serum, and kidney extracts were determined with the Cleman spectrphtmeter at a wave length f 62 m/~. Prcedure fr Determining T-1824 Cntent f K/dney.--Animals were injected at time zer with 2 nag. f T At the end f the desired time interval the animals were anesthetized with ether, the abdmen was pened and the arta and inferir vena cava expsed. Ligatures were placed arund the arta just eephaiad t the renal arteries, and apprximately ~ inch caudad t the renal arteries. The mesenteric vessels were ligated. A 2 gauge needle was inserted int the arta between the caudad artic tie and the renal arteries. An incisin was made in the inferir vena cava, and the kidneys were perfused thrugh the needle with 5 ml. f.89 per cent sdium chlride slutin, at a pressure f 125 cm. f water. At the end f this time, the perfusate frm the inferir vena cava was free f visible dye, and the bladder cntained several milliliters f clrless fluid. Unstained frzen sectins f these kidneys shw that dye had been cmpletely remved frm the renal bld vessels

3 SELLERS~ GRIGGS, MARMORSTON, AND GOODMAN and tubular lumen system. The kidneys were remved, and their capsules stripped. Each kidney was hmgenized in a mrtar with 5 gln. f sea sand and 6 ml. f distilled water. Twenty-five ml. f 1. per cent aersl OT was added, and the mixture allwed t stand with frequent agitatin fr 3 minutes. The T-1824 passed frm the tissue int the liquid phase. Fifty ml. f acetne was added t precipitate the tissue prteins and the mixture was shaken frequently fr 15 minutes befre centrifuging. Mter centrifugatin, the blue supernatant slutin was decanted and placed int a 25 ml. vlumetric flask. The remaining precipitate which had lst its blue clr was resuspended in 5 ml. f a mixture f 6 parts water, 25 parts aersl OT, and 5 parts acetne, and recentrifuged. The supernatant, which usually had a faint blue tinge, was added t the previus supernatant in the 25 ml. vlumetric flask. The precipitate was then free f blue dye, and additinal washing f this material revealed n additinal T-1824 n spectrphtmetric determinatin. The material in the 25 ml. vlumetric flask was made t vlume with the mixture f aersl, acetne and water. An aliqut was taken fr the spectrphtmetric determinatin f T Determinatin f Rati T-18g4/Prtein in Serum.--Animals were injected with 2 rag. f T-1824 at time zer. At frequent time intervals ranging frm 3 minutes t 48 hurs after injectin, grups f rats were bled frm the abdminal arta. The cncentratin f T-1824, and ttal prteins in the serum were determined by the methds utlined abve. RESULTS The T-1824/prtein rati is pltted against time in Text-fig. 1. Each pint n the curve represents the mean f determinatins made n three animals. In rder t determine the mean T-1824/prtein rati in the serum fr any desired time interval, planimetric integratin was perfrmed by measuring the area under the curve during this time interval with a cmpensating plar planimeter. Unstained frzen sectins f the kidney f rats taken 15 minutes after the intravenus administratin f 25 rag. f T-1824 revealed blue dye in the peritubular capillaries, and t sme extent in the capillary frming the glmerular tuft. Sectins taken 3 and 6 minutes after injectin were clrless. At 9 minutes, a few small blue drplets were seen in cells lining the prximal cnvluted tubules, and were fund clse t the luminal brder f the cell (11). Sectins taken 3, 7, 19, and 24 hurs after injectin shwed prgressively mre dye in these cells, and mre f the cells were seen t cntain the dye. The drplets appeared t becme larger, and t ccupy the entire cell frm luminal t capillary brder. Dye was nt seen elsewhere in the kidney, except fr ccasinal staining f the elastic layer f the larger arteriles (Figs. 1 t 3). There was usually less dye in the cells f the prximal tubule in sectins taken 48 hurs after injectin (Fig. 4), while 72 hurs later the amunt f dye was always reduced. Frm these sectins, it is clear that when T-1824 is extracted frm the perfused kidney by the methds described abve, it can be said t cme entirely frm the cells f the prximal cnvluted tubule. The ttal T-1824 cntent f the tw kidneys f the rat is pltted against time after injectin in Text-fig. 2. Each pint is the mean f determinatins made n the kidneys frm three rats.

4 ZILTRATION AND REABSOEPTION Ol ~ PROTEIN BY KIDNEY Frm the data belw (Table I), we have calculated the milligrams f prtein reabsrbed by the cells f the prximal tubule (Text-fig. 3). Fr the first 4 hurs after dye injectin, prtein is reabsrbed at a rate f 4 t 6 rag. per hur per rat. As time ges n, this rate appears t fall and level ff at a value f abut 2 rag. per hur per rat. $. 2.5,5 i z ~ 4 5 s 7 TIME ~ (hurs) T~xT-F~. l. The rate f fal~ f the T-1824/prtein rati in rat serum fllwing the intravenus injectin f 2 rag. T DISCUSSION In studying the rate f prtein reabsrpdn by the kidney, ne aims in s far as pssible t study the reabsrptin f native, unaltered prtein. We have fund it necessary t tag native rat plasma prtein with dye T While it des nt seem unreasnable that certain prperties f the serum prteins under investigatin culd be altered by this prcedure, all available evidence indicates that tagged and unaltered plasma prtein behaves in an identical manner. Rawsn (9) has shwn that T-1824 albumin has the same electric mbility as untagged albumin. It has been demnstrated (12) that tagging plasma albumin with T-1824 des nt alter the highly specific immunchemical char-

5 SELLERS~ GRIGGS~ MARMORSTON~ AND GOODMAN acteristics f this substance. Others have shwn that curves describing the disappearance f T-1824-albumin frm the circulatin are nt significantly different frm thse f albumin tagged with I m (13, 14). On the basis f these bservatins, we have assumed that T-1824-prtein underges glmerular filtratin and tubular reabsrptin in a manner identical with that f untagged plasma prtein G ".65 c~.55 T ~.45 I I-- ~.35 I h.1,, I I I I I TIME (hurs) TExT-FI. 2. The rate f uptake f T-1824 in the ttal renal tissue fllwing a single intravenus injectin f 2 rag. f the dye. Once T-1824-prtein is reabsrbed int the cells f the prximal cnvluted tubule it appears t be cncentrated int drplets. Here, it is mst likely that "1"-1824 is split ff and remains in the drplet, while the prtein miety f the cmplex is mre r less degraded, and the degradatin prducts are discharged int the peritubular venus capillary. The drplets cntaining T-1824 accumulate in the cells fr at least 24 hurs, and after 48 t 72 hurs sectins f the kidney reveal less dye than was bserved previusly. Rather (15) has shwn that reabsrbed hemglbin disappears frm the cells f the cnvluted tubules in a matter f hurs, while Oliver (16) states that it takes 2 t 3 days fr re- 5O

6 6 FILTRATION AND REABSORPTION OF PROTEIN BY KIDNEY absrbed egg white drplets t nearly cmpletely disappear, and Smetana (17) finds that diaztized serum prteins persist indefinitely within the renal epithelium. We have assumed that all f the 2"-1824 reabsrbed by the tubules ver a given time perid remains in the cell and that we recver this by ur extractin prcedure. It is entirely pssible that much f the T-1824 accumulates int drplets, but that sme traverses the cell in a highly dispersed frm and passes directly int the renal vein bld. Oliver and Lund (18) have described this srt f dual transprt f dye during the tubular secretin f neutral TABLEI Time after injectin Mean rati Mg. T-1824 Mg. prtein X 1-2 Mean ttal T-1824 cntent f kidneys Prtein re, absrbed hrs mg O reg./ms hr red. In s far as dye is reabsrbed and leaves the cell befre the experimental time has expired, ur figures will be in errr and it must be realized that the figure btained represents a minimal figure fr prtein reabsrptin. It is pssible that the fall in prtein reabsrptin rate described in Text-fig. 3 can be explained n this basis. During the first several hurs f the experiments, the rate f prtein reabsrptin ranges frm 4 t 6 mg. per hur per rat. After this, the rate falls and appears t level ff at apprximately 2 mg. per hur per rat. If T-1824 des leave the cell, nce it has been reabsrbed, this must be a prcess that takes time, else the dye wuld nt accumulate in the cells as it is shwn t d in Fig. I t 4. Hence, less T-1824 will have left the ceils during the first hurs

7 SELLERS~ GRIGGS; MARMORSTON~ AND GOODMAN..a..= ; "~ e~ I I I I I I to U~,l.O,Jl.mOLl / "BI61, NOI/d~OS8V3~I N 13,LOEId ~4 d

8 8 FILTRATION AND KEABSOP~I~TION OF PROTEIN BY KIDNEy f the experiment, and the fall in rate f prtein reabsrptin seen in later hurs may well be an errr due t lss f T-1824 int renal venus bld. Fr these reasns, we cnsider the figures btained during the first 3 t 4 hurs as the mst reliable index f the rate f prtein reabsrptin, and will take 5 reg./rat hur as being the mean rate f prtein reabsrptin fr this perid. The best available figure fr the rate f glmerular filtratin in the rat is.6 ml./1 gin. bdy weight/rain. (19) r fr animals weighing 2 gin., 1.2 ml./min. This is equivalent t 72 ml./hur. Female rats weighing 2 gm. excrete prtein in their urine at a rate f abut.5 rag. per hur. Hence, if prtein is being reabsrbed at a rate f 5 rag. per hur, the glmerular filtrate wuld cntain 5.5 rag. f prtein in 72 ml. f fluid r wuld cntain prtein t the extent f 7.6 rag. per cent. This cncentratin is well belw the limits discernible by the methds emplyed by Walker et al. and is smewhat lwer than the values btained by Dck in the perfused rabbit kidney. The circulating bld vlume f the 2 gm. rat is abut 1S ml. and the hematcrit value abut 6 per cent (2). If the cncentratin f plasma prtein is 6 gin. per cent, there is a ttal f 36 mg. f circulating plasma prtein. Cnsequently, the rat filters and reabsrbs 12 mg. f prtein per day, r apprximately 33 per cent f its circulating plasma prtein. If the reabsrbed prtein is at least partially degraded in its passage thrugh the renal tubule cell, the kidney can be said t metablize 33 per cent f the circulating prtein per day and may be cnsidered an imprtant rgan in regulating the rate f prtein synthesis. It is nt held that the calculated rates f prtein reabsrptin represent precisely what takes place in the kidney. We d believe, hwever, that it is a firstapprach t btaining a minimal value fr prtein reabsrptin in the intact animal. As such, it is permissible t reprt these data, inexact thugh they may be, in the hpe that frm here, mre precise methds may be develped and the figure may cnstantly be revised until smething appraching actual values will be btained. SUMMARY AND CONCLUSIONS Plasma prteins f the rat have been labelled by the in ~v injectin f the dye T Frm a study f the rate f disappearance f T-1824 frm the circulating bld, and the ttal T-1824 cntent f the perfused kidney the rate f prtein reabsrptin frm the glmerular fluid by the cells f the renal tubule has been calculated. rt is cncluded that prtein reabsrptin by the cells lining the prximal cnvluted tubule f the rat kidney prceeds at a rate f at least 5 rag. per hur, equivalent t a daily filtratin and reabsrptin f 33 per cent f the circulating plasma prtein.

9 SELLERS, GRIGGS~ MARMORSTON, AND GOODMAN" BIBLIOGRAPHY 1. Walker, A. M., and Oliver, ]., Am. Y. Physil., 1941, 134, Rather, L. J., Medicine, 1952, 31, Dck, W., New England J. Med., 1942, 9.27, Gilsn, G. B., Prc. Sc. Exp. Bil. and Med., 1949, 72, Sellers, A. L., Rberts, S., Rask, I., Smith, S., Marmrstn, J., and Gdman, H. C., J. Exp. Med., 1952, 95, Rigas, D. A., and HeUer, C. G., J. Clin. Inv., 1951, 3, Walker, A. M., Btt, P. A., Oliver, J., and MacDwell, M. C., Am. J. Physil., 1941, 134, Bickfrd, R. G., and Wintn, F. R., J. Physil., 1937, 89, Rawsn, R. A., Am. J. Physil., 1943, 138, Allen, T. H., and Orahvats, P. D., Am. J. Physil., 1948, 134, Sellers, A. L., Smith, S., Marmrstn, J., and Gdman, H. C., J. Exp. Med., 1952, 96, Campbell, D. H., persnal cmmunicatin. 13. Schultz, A. L., Hammersten, ]. F., I-Idler, B. I., and Ebert, R. V., J. Clin. In% 1953, 32, Freinkel, N., Schreiner, G. E., and Athens, J. W., J. CUn. Inv., 1953, 32, Rather, L. J., J. Exp. Med., 1948, 87, Oliver, J., J. Mr. Sinai Hsp., 1948, 15, Smetana, H., Am. J. Path., 1947, 23, Oliver, ]. R., and Lurid, E. M., J. Exp. Med., 1933, 67, Smith, It. W., The Kidney, New Yrk, Oxfrd University Press, Lippman, R. W., Prc. Sac. Exp. Bil. and Med., 1948, 67, 193.

10 1 FILTRATION AND REABSORPTION 1' PROTEIN BY KIDNEy EXPLANATION OF PLATES PT.AT~. I FZG. 1. Unstained frzen sectin f rat kidney remved 3 hurs after the intravenus injectin f 2 rag. T Nte a few scattered dark drplets cntaining T in sme f the prximal cnvluted tubule ceils. These drplets are actually bright blue in clr. X 535. FIG. 2. Seven hurs after the intravenus injectin f 2 rag. T There is a sharp increase in the number d drplets ver that seen in Fig. I. X 535.

11 THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. 1 PLATE 1 (Sellers et al.: Filtratin and reabsrptin f prtein by the kidney)

12 PLATE 2 Fie,. 3. Twenty-fur hurs after T-1824 injectin. The number f drplets and the number f tubule cells cntaining drplets are maximal at this time. X 535. FI. 4. Frty-eight hurs after T-1824 injectin. There is a marked decrease in the number f T-1824 drplets since the 24 hur sectin. X 535.

13 THE JOURNAL OF EXPERIMENTAL MEDICINE VOL, 1 PLATE 2 (Sellers et al.: Filtratin and reabsrptin f prtein by the kidney)