Renal Tubular Transport and Catabolism of Proteins and Peptides

Kidney International, Vol. 16 (1979), pp. 271 -2 78

Renal tubular transport and catabolism of proteins and peptides

FRANK A. CARONE, DARRYL R. PETERSON, SUZANNE OPARIL, and THEODORE N.PULLMAN

Departments of Pathology, Physiology and Medicine, Northwestern University Medical School and Veterans Administration Lakeside Hospital, Chicago, Illinois, and Department of Medicine, University of Alabama, Birmingham, Alabama

It is established that the kidney plays an impor- are not transported into tubular cells across the tant role in the metabolism of a number of protein, peritubular cell membrane, presumably due to the polypeptide, and small peptide molecules, including absence of an endocytic mechanism on this side of plasma proteins, growth hormone, L-chains of im- the tubular epithelium. munoglobulins, /32-microglobulin, lysozyme, in- Luminal uptake. Many morphologic, micro- sulin, proinsulin, parathyroid hormone, glucagon, puncture, microinfusion, and microperfusion stud- and small vasoactive peptides. Absorption, trans- ies have demonstrated that a wide variety of pro- port, and/or degradation of proteins or peptides are teins are absorbed across the luminal aspect of functions of the proximal tubule; there is little evi- proximal tubular cells [1, 2]. We quantified the up- dence that other segments of the nephron have the take of '251-labeled rabbit serum albumin micro- mechanisms for uptake or transport of these sub- perfused into isolated segments of the rabbit neph- stances. Indirect and direct studies indicate that a ron [3]. Proximal convoluted and proximal straight variety of proteins and polypeptides filtered at the segments accumulated 1251-albumin nearly linearly glomerulus are absorbed by the proximal tubule by as a function of time (Fig. 1), whereas cortical col- luminal endocytosis and hydrolyzed by lysosomal lecting segments did not accumulate measurable enzymes. Our recent studies suggest that small lin- amounts of protein. The rate of accumulation of al- ear peptides, consisting of eight to ten amino acids, bumin in the proximal convoluted tubule was 3.2 x are handled by the proximal tubule by a different 10-2 ng/mm/min, which was 2.6 times as great as mechanism. We have demonstrated that small lin- that in the proximal straight tubule. Assuming an ear peptides microinfused into proximal tubules are albumin concentration of 0.3 mg/dl in normal gb- hydrolyzed at the luminal surface of the brush bor- merular filtrate [4], we can calculate that the reab- der, which is rich in a variety of enzymes, by the sorptive capacity of the entire proximal tubule for process of membrane or contact digestion with albumin exceeds the amount of albumin filtered by reabsorption of most of the breakdown products. the normal glomerulus. The ultrastructural basis for reabsorption of la- Proximal tubular handling of proteins and large peptides beled albumin by proximal tubules was investigated Qualitative morphologic and indirect functional autoradiographically by Maunsbach with in vivo studies have demonstrated that proximal tubular microinfusion methods in the rat kidney [5] and in cells absorb protein from the luminal fluid and have our laboratory in isolated rabbit tubules micro- suggested a pathway by which protein undergoes perfused with '251-albumin [3]. Sequential studies intracellular digestion. These processes have been revealed that silver grains were initially located quantified directly. Although some findings suggest that certain proteins are transported intact across proximal epithelial cells, other investigations do not Received for publication March 13,1979 support this conclusion. Direct studies on isolated 0085-2538/7910016-0271$01.60 tubular segments indicate that albumin and insulin ©1979 by the International Society ofNephrology

271 272 Caroneet al

PCT

C, (0 PST C E1. -C

CCL 40 80 120 160 200 Durationof perfusion, mlvi Fig.1.Accumulationof iodinated albumin as a function of time in proximal convoluted (PCT) proximal straight (PST) and cortical collecting (CCT) segments of the rabbit nephron. All tubules were perfused with a 21-mg/dl solution of iodinated rabbit albumin at 18 to 20 nI/mm at 370 C. (Reprinted with permission of J Cell Biol [3])

- -'ç •A Fig. 3. Electron microscope radioautograph with radioactive label in dense bodies (arrows) in cells of the proximal tubule after 85 mm's perfusion with "51-albumin. (X15,200) (Reprintedwith S permission off Cell Biol [3])

over tubular invaginations at the base of the brush border and over small apical vesicles and later in larger membrane-bound apical vacuoles (Fig. 2). Fi- nally, grains becoming concentrated in cytoplasmic dense bodies (Fig. 3) were associated with acid phosphatase positive bodies, indicative of lysosomes. Similar ultrastructural findings were ob- served with labeled insulin in our laboratory [6] and with a number of other proteins in several experi- mental animals [1, 2]. There is evidence that the first step in endocytosis involves binding of protein to the luminal plasma membrane [2]. There is wide variation in the affinity of different proteins for the plasma membrane, which may be related to the number and chemical structure of membrane binding sites and

'4- to the net charge on the protein molecules. Small amounts of certain proteins bind largely to the plasma membrane, whereas large amounts in tubu- Fig. 2. Electron microscopic radioautograph of a proximal tu- lar fluid appear mainly in endocytic vesicles un- bule perfused 10 mm with "51-albumin. Grains are located at the base of the brush border (small arrow), in small apical vesicles bound to the plasma membrane [2]. Thus, endo- (medium arrow), and in apical vacuoles (large arrow). (X26,510) cytosis may be largely specific when small quan- (Reprinted with permission of f Cell Biol [3]) tities of protein are reabsorbed due to membrane Tubular handling of proteins and pept ides 273 binding and less specific when large amounts of pro- 4.0 tein are reabsorbed. Several studies suggest that the membrane of apical invaginations and vesicles in 3.0 the endocytic process is replaced by a de novo syn- oxC c::i thesis of plasma membrane [7, 8] and not by down- contraIumnaI ward flow of brush border membrane [9]. Most evi- 2.0 dence favors the conclusion that protein is trans- ferred to lysosomes by fusion of endocytic vacuoles and preexisting lysosomes. 111.0 Lysosomes contain many hydrolytic enzymes which have been shown to digest a wide variety of 0 proteins. Lysosomal extracts isolated from renal PCT PST TAL CCT cortical homogenates have been used to quantify Fig.4. Comparison of luminal and contraluminal accumulation hydrolysis of albumin and other proteins. Hydroly- rates of albumin -in isolated renal tubules. Luminal uptake data is taken from Fig. I above. (Reprinted with permission of Am J sis of '251-albumin is maximal at a low pH, and the Physiol [16]) major labeled product of digestion is monoiodotryo- sine [10]. Other in vitro studies have demonstrated hydrolysis of absorbed protein within isolated intact 700 lysosomes [11] or within lysosomes of intact cells in kidney slice preparations [12]. Small-molecular- 600 weight metabolites of proteins diffuse out of lysosomes into the cell cytoplasm and interstitial fluid. 500 There is no evidence for nonendocytic reabsorption aC of protein on the luminal side of proximal tubular 400 cells or for release of intact reabsorbed protein from a lysosomes on the contraluminal side of the cells. .0300

Contraluminal uptake of protein. Although the C uptake of proteins from the luminal aspect of the 200 proximal tubules is well established, uptake from the contraluminal aspect is uncertain. Studies in intact kidneys, however, provide evidence for con- 100 traluminal uptake of certain proteins such as f32-mi- 0— croglobulin [13] and insulin [14]. Because the base- Luminal Contraluminal ment membranes of isolated perfused tubules are Fig.5. Luminal and contraluminal 1251-insulin accumulation by moderately permeable to albumin, as demonstrated isolated perfused proximal convoluted tubules. (Reprinted with by Welling and Grantham [15], it is possible that in- permission of Am J Physiol [6]) terstitial protein is in contact with the basilar membranes of tubular cells. We studied the con- due to the absence of a prominant endocytic mecha- traluminal uptake of albumin directly in isolated nism or the lack of specific receptor sites on the bas- perfused segments of proximal thick ascending limb ilar side of tubular cells. and cortical collecting tubules of the rabbit in- Intercellular and transcellular transport of intact cubated in '251-albumin [16]. After 2 to 90 mi con- proteins. In normal mature kidneys, intercellular traluminal uptake of albumin was negligible in all transport of proteins has not been demonstrated. In segments compared to luminal uptake in the proxi- the immature kidney [17] and in certain abnormal mal tubules (Fig. 4). In a companion study [6], we states, such as elevated tubular hydrostatic pres- also found that contraluminal uptake of '251-insulin sure [18], proteins may pass between cells through was negligible compared to luminal uptake (Fig. 5). cellular junctional complexes. On the other hand, It is known that albumin and insulin accumulate transcellular transport of intact protein by renal tu- within proximal tubular cells from the luminal side bules remains a controversial topic. A number of where endocytosis is prominent, but not in cells of studies provide evidence both for and against a collecting tubules, where endocytosis is not pro- mechanism for the transcellular transport of intact nounced. Absence of significant contraluminal up- proteins [2]. This problem may be resolved by di- take of albumin, or insulin, or both is presumably rect studies that quantify and characterize protein 274 Carone et a! and protein catabolites absorbed and released by peptide was microinfused into proximal nephrons tubular cells. with excess unlabeled L-lle, urinary recovery of 14C greatly exceeded that seen with 14C-AII alone and Renal handling of small peptides increased directly with distance of the infusion site The kidney has been shown to degrade circulat- from the glomerulus (Fig. 7). Because '4C-Ile ap- ing angiotensin II (All) rapidly. Although 40 to 70% peared as the predominant labeled material in urine of 14C-AII infused into the renal artery is extracted and only 5% of the labeled material excreted was in by the dog kidney, little labeled material appears in the form of intact All, these results suggest that ex- the urine. The high extraction ratio suggests that cess unlabeled Ile interfered with the reabsorption renal handling involves more than glomerular filtra- of labeled Ile derived from All. When it is consid- tion alone [19]. Similar studies have shown that 75% ered, however, that the total recovery of radio- of infused 14C-AII is metabolized in a single passage labeled material was much greater when isoleucine through the kidney, and 98.7% of injected radio- was infused with 14C-AII than when 14C-AII was in- labeled material is recovered in renal venous blood fused alone, the 5% of radioactivity as unaltered [20]. These findings indicate that extensive renal hy- All assumes greater prominence. Thus, suppres- drolysis occurs, and that tissue sequestration of '4C- sion of the reabsorption of labeled Ile by excess un- All or its metabolites is not prolonged. Thus, renal labeled Ile was the predominant effect and over- hydrolysis of All allows for the rapid return of shadowed the inhibition of hydrolysis of All by ex- cleavage-products to the general circulation. cess unlabeled Ile. Recently, we have assessed the role of individual From similar experiments and analogous reason- nephrons or isolated nephron segments in the trans- ing, we concluded that excess aspartic acid also af- port and hydrolysis of radiolabeled angiotensin I fected both hydrolysis and reabsorption of All but (Al), All, bradykinin (BKN), and oxytocin (OT). The techniques for in vivo microinfusion of surface tubules in rats [21], and in vitro microperfusion of ANGIOTENSIN 11 isolated rabbit nephron segments [22] were used. 1 2 3 4 6 7 Reabsorption of radiolabeled material was mea- 8 sured, and the intact peptide or its metabolites were H — Asp - Arg Vat - Tyr lieu — His - Pro - Phe - OH identified and quantified in urine, or bathing medium and collection fluid. In addition, peptides were "14C I incubated in the presence of isolated membrane Tyrpsin Chymotrypsir, Carboxypepticlase preparations to localize a probable cellular site of Fig.6. Angiotensin II labeled at fifth position with '4C. Digestion with trypsin, chyrnotrypsin, and carboxypeptidase A (arrows) hydrolysis. Characterization of labeled material yielded standard breakdown products. was accomplished by two-dimensional peptide mapping involving high voltage paper electrophoresis in combination with decending paper chroma- 14C Afl-isoleucine tography [23] or high-voltage paper electrophoresis 100 alone. Standard peptide fragments were generated by digesting labeled peptide in the presence of sev- 80 eral purified enzymes. . '4C-AII labeled in the fifth position on isoleucine -o •• 60 .. (Fig. 6) was microinfused in vivo into individual 0 .. surface nephrons of the rat kidney [23]. Following 0> proximal infusion, 11% of '4C was recovered in the 0C S.. 40 urine, and most (95%) of this was present as metab- C) . olites, including the carboxyl terminal tetrapeptide as the principal hydrolytic end-product. In contrast, 20 recovery of 14C was 95% when distal tubules were infused, and virtually all was intact octapeptide. , °c%ocP06o° The data suggest that the peptide is rapidly hydro- 10 20 30 40 50 60 lyzed and reabsorbed in the proximal tubule but not Proximal length, % in the distal tubule. Fig.7. Percent recovery of 14C-AII as a function of tubular Further in vivo studies in the rat suggested that length. Closed circles refer to infusion of '4C-AII plus unlabeled isoleucine in molar ratio of 1:2000. Open circles refer to infusion constituent free amino acids influence proximal of 14C-AII alone. (Reprinted with permission of Am J Physiol tubular handling of 14C-AIl [24]. When the labeled [24]) Tubular handling of proteins and peptides 275

membrane of the proximal tubule, followed by reabsorption of hydrolytic products [27]. Upon microperfusion of isolated rabbit nephron segments for 30 mi tritium was rapidly reabsorbed (Fig. 8). Elec- trophoretic analysis of the collection fluid and bathing medium revealed that tritiated Leu appeared as the predominant labeled material. Incubation of tn- tiated AT directly in the presence of isolated membranes from renal brush border yielded tritiated Leu as the major labeled hydrolytic end-product. Thus, hydrolysis at the brush border was directly related I to reabsorption of a hydrolytic end-product. There is considerable evidence that renal handling of bradykinin (BKN) is similar to that of Al and All. Surface nephrons in the rat kidney were microinfused in vivo with tritiated BKN labeled in

10 20 30 the second position on proline [28]. After proximal Time, rn/n infusion, urinary recovery of tritium was 24%, 85% Fig.8. Reabsorption of tritium from proximal straight tubular ofwhich was in the form of metabolites (81% Pro2 segments microperfused with tritia ted Al as a function of time. and 4% Arg1-Phe5). Distal infusion resulted in re- Reabsorption is expressed as picograms tritiated Al per millimeter of tubular length. (Reprinted with permission of Am J Physiol covery of 98% of tritium, all of which appeared in [27]) the urine as intact tritiated BKN. In addition, tn- tiated BKN and 14C-inulin were simultaneously in- to a different degree than isoleucine. These data are fused into proximal and distal surface nephrons, consistent with the observation that aspartic acid is and recovery of the respective labels was plotted as a much stronger aminopeptidase inhibitor than is a function of time at 30-second intervals following isoleucine [25]andsuggest that the effect of these microinfusion (Fig. 9). For both proximal and distal constituent amino acids on hydrolysis of All may tubules, the urinary concentration-time curves for be accomplished by amino-peptidase suppression. tritium derived from tritiated BKN did not differ ap- In vitro microperfusion of rabbit proximal preciably from those for '4C-inulin (Fig. 9). Because straight nephron segments with 14C-AII provided di- tritium represents both intact tritiated BKN and hy- rect evidence for proximal hydrolysis of the pep- drolytic products in varying proportions depending tide, accompanied by rapid and extensive reabsorp- upon the site of microinfusion, tubular transit time tion of 14C-labeled material across the tubular epithelium [26]. Approximately 30% of perfused '4C was reabsorbed per millimeter of tubular length Proximal Distal over a broad range of delivery rates. Most of the labeled material was rapidly transported across the 30 30 .....c3 H tubular epithelium into the bathing medium, and less than 1% of perfused label remained sequestered by the tubular cells following the 35-mm perfusion period. Electrophoresis of collected perfusate dem- 20 20 0 onstrated that '4C-AII was hydrolyzed to 14C-Ile. 4- The foregoing data indicate that All is quickly de- 0 graded upon passage through the renal proximal tubule and suggest that cleavage occurs at the level of 10 10 the tubular luminal membrane. This hypothesis was confirmed by incubations of 14C-AII directly in the presence of isolated membranes from rabbit renal brush border [27]. Hydrolysis of the peptide yielded i 234 2:34 '4C-Ile as the only labeled end-product appearing in Time, rn/n Time, rn/n the incubation medium. Fig.9. Appearance of tritium and 14C in urine plotted against In vitro studies with tritiated Al labeled in the time. Tritium and '4C are quantified as percents of total excre- tions of each isotope, respectively. Isotopes were simultaneous- 10th position on leucine have provided further evi- ly microinfused into rat tubules as tritiated bradykinin and 14C- dence that angiotensin is degraded at the luminal inulin. (Reprinted with permission of Am J Physiol [28]) 276 Carone et al for the metabolites of tritiated BKN is essentially as minal aspect of the tubular cells. The mechanism rapid as that of 14Cinulin or intact tritiated BKN. for tubular handling appears to involve enzymatic These data suggest that cleavage of tritiated BKN in hydrolysis at the brush border, followed by rapid the proximal tubule is a rapid process and favor the reabsorption of metabolites (Fig. 11). It is reason- interpretation that tritiated BKN, like tritiated AT or able to believe that amino acids released by hydrol- 14C-AII, is hydrolyzed at the luminal brush border ysis at the luminal membrane are actively reab- membrane. Indeed, Ward et al [29] have demon- sorbed by amino acid pumps known to exist there. strated kininase activity in rat brush border mem- The distal tubule appears to lack this property. branes. The concept of membrane or contact diges- Furthermore, the mechanism of this process ap- tion has been developed to explain the hydrolysis pears to differ from that for the proximal reabsorp- and absorption of proteins, peptides, and other sub- tion of proteins and large peptides, which involves strates by cell membranes, particularly the mucosa endocytosis and lysosomal digestion (Fig. 11). It re- of the small intestine [30]. mains to be determined whether these larger mole- The mechanism for proximal reabsorption of cules are partially degraded at the luminal mem- BKN and metabolites appears to be of high capacity brane of the proximal tubule prior to or during the but not high specificity, and the processes of hy- endocytic process. drolysis and reabsorption may be characterized by Our studies indicate that the proximal tubules of different capacities and specificities [31]. Upon si- the mammalian kidney possess a high capacity multaneous microinfus ion of tritiated BKN with ex- mechanism for the rapid hydrolysis of small linear cess unlabeled BKN or AT, urinary recovery of triti- peptides. This mechanism may be important biolog- um was increased to the same extent. Only unla- ically to (1)conserveamino acids, (2)inactivate beled BKN, however, and not unlabeled Al, toxic peptides, and (3)helpregulate the circulating effectively inhibited the hydrolysis of tritiated levels of small peptide hormones because there is BKN, as determined by identification and quan- evidence that serum levels of these hormones are tification of products appearing in the urine. determined more by rates of degradation than they A nonlinear molecular configuration may restrict are by synthesis. the hydrolysis and uptake of small peptide hormones at the luminal membrane of the proximal tu- Lumen bule. Oxytocin (OT) (Fig. 10) and vasopressin both contain a disulfide bridge. Although the kidney has Residual digestive been implicated in the hydrolysis of both peptides body

[32, 33], in vitro microperfusion of tritiated OT (la- Endocytic vesicle beled in second position on tryosine) (Fig. 10) Phagosome through rabbit proximal straight nephron segments Lysosome resulted in no detectable hydrolysis, and the reab- Phagolysosome sorption rate of labeled material was low compared to corresponding measurements for 54C-AII under similar conditions [26]. In vivo microinfusion of 525J argininevasopressin in rat nephrons yielded similar results [34]. Collectively, these studies demonstrate that small Amino acids Amino acids linear peptides are cleaved in the proximal tubule of FIg.11. Schemata comparing the cellular mechanisms of the proximal tubule for reabsorption and catabolism of protein or the rat and rabbit kidney when presented to the lu- large polypeptide molecules to that described in our laboratory for small, linear peptides. The left figure illustrates that protein is taken up by endocytic vesicles, which fuse to form phagosomes into which primary lysosomes empty their hydrolytic enzymes. OXYTOCIN Enzymatic cleavage of proteins occurs in the phagolysosomes; liberated amino acids diffuse into the interstitium and are re- turned to the renal circulation. The right figure depicts tubular handling of small, linear peptides. Hydrolysis occurs at the site of enzymes associated with the brushborder of the proximal tu- Cys- Tyr- Tie -Gin- Asn - Cys— Pro— Leu —Gly —NH2 bule. Liberated amino acids are rapidly transported across the epithelium, probably involving active amino acid pumps located Fig.10.Molecular structure qf oxytocinlabeled with tritium in at the apical cell membrane. Partially hydrolyzed peptide frag- the second position (asterisk) demonstrating the disulfidebridge ments may be reabsorbed intact or undergo further intracellular betweencysteines intheoneand six positions. cleavage prior to reabsorption. Tubular handling of proteins and peptides 277

Summary 10. MAUNSBACH AB: Ultrastructure and digestive activity of The kidney plays an important role in the metabo- lysosomes from proximal tubular cells. Proc4th Cong Neph- rol,Stockholm, Basel, Karger, 1970, Vol. 1, p. 102 lism of proteins and peptides. Current evidence in- 11. DAVIDSON SJ: Protein absorptionby renal cells: II. Very dicates that only the proximal tubule possesses the rapidlysosomal digestion of exogenous ribonuclease in vit- mechanism for degradation or transport of these ro. J Cell Biol 59:213—222, 1973 substances and reabsorption of metabolic products. 12. CHRISTENSEN El, MAUNSBACH AB: Intralysosomal diges- Proteins and large polypeptides filtered at the gb- tion of lysozyme in renal proximal tubule cells. Kidney mt 6:396—407, 1974 merulus are absorbed from proximal tubular fluid 13. RAVNSKOV U, JOHANSSON BG,GoTHLINJ:Renal extraction by luminal endocytosis into apical vacuoles. These of $2-microglobulin. 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