Biochimica et Biophysica Acta, 446 (1976) 506-514

© Elsevier/North-HollandBiomedicalPress BBA 37463 BIOSYNTHETIC STUDIES WITH ISOLATED KIDNEY GLOMERULI

ISTVAN KRISKO" and W. GORDON WALKER Department o f Medicine, the Johns Hopkins Hospital, Baltimore, Md. 21205; Research Service, Veterans Administration Hospital, Houston, Texas 77211, and Department o f Pharmacology, Baylor College o f Medicine, Houston, Texas 77025 (U.S.A.)

(Received January 12th, 1976)

SUMMARY Glomeruli were isolated from rat kidneys and were found active in protein and glycoprotein synthesis in vitro. The incorporation of proline, galactose and fucose into macromolecules was linear for at least 8 h. The intracellular pool of free proline and lysine was 52 and 32 nmol/mg glomerular protein respectively. Vinblastin and cytochalasin B, two agents which interfere with normal cellular secretory processes, inhibited galactose and fucose incorporation, possibly by a feedback mechanism. Experimental nephrotic syndrome was induced in rats by the injection of puromycin aminonucleoside; the rate of proline and galactose incorporation was reduced in glomeruli isolated from the nephrotic animals. The system of isolated glomeruli is deemed suitable for future studies of selected kidney diseases which affect the glomeruli.

INTRODUCTION Much information has been accumulated on some aspects of injury to the glomerulus and its basement membrane [1-4], but in most of the previous work metabolic and biochemical features of the pathogenetic process, i.e., response to injury by the glomerulus itself, were not taken into consideration. Hence, studying the biosynthetic capabilities of the renal glomerulus is of special import when one considers that the glomerulus is the principal site of damage in many renal diseases and, in some, virtually the sole site of involvement until late stages of the disease [1]. Isolated glomeruli were deemed suitable for such biochemical studies in vitro. It was reasonable for us to employ this in vitro system because: (a) the early experience of one of us (W.G.W.) with isolated glomeruli [5]; (b) our preliminary results showing the feasibility of this approach [6]; (c) some recent reports by other groups. For instance, Brown and Michael [7] and Beisswenger [8] employed isolated glomeruli for investigations of basement membrane synthesis. Most recently, Grant et al. [9] * Correspondence should be addressed to Dr. Krisko at the V.A. Hospital, Research Service, 2002 HolcombeBlvd., Houston, Texas 77211, U.S.A. Abbreviation: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

507 and Cohen and Vogt [10] gave detailed accounts of collagen synthesis by rat glomeruli. We used a different approach from that of the last two groups of authors in that our primary aim was to further characterize the overall biosynthetic activity of isolated glomeruli. A preliminary report of part of this work has already appeared [6], and carbohydrate analysis of the newly-synthesized glomerular glycoprotein(s) is described in detail elsewhere [11, 12]. MATERIALS AND METHODS

Isolation of kidney glomeruli ; protein and glycoprotein synthesis in vitro Kidneys were obtained from male albino rats (80 to 150 g, Holtzman Co., Madison, Wisconsin) under ether anesthesia. All subsequent steps were carded out at 0 ° to 4 °C unless otherwise indicated. The procedure for the isolation of glomeruli was essentially as described by Spiro [13]. Briefly, the cortex was separated from the rest of the kidney tissue, minced coarsely with a razor blade, and homogenized in tissue culture medium (Medium 199, Microbiological Associates, Bethesda, Md.) utilizing a loose-fitting glass homogenizer (Kontes Glass Co., Vineland, N.J.). Then the glomeruli were isolated by trapping them on top of appropriate stainless steel screens. Preparations from younger rats (80-100 g) were consistently better with the 200 mesh screen (pore size 75 #m); from older rats, the 150 mesh screen (pore size 106 #m) gave better yields. The glomeruli were approximately 95 ~o pure, as estimated by phase-contrast microscopy [11]. The yield was 3-5 mg of glomerular protein [14] per 10 g of kidney tissue (wet weight). The isolated glomeruli were suspended in tissue culture medium and incubated in the presence of the desired labeled precursors. In a typical experiment, Medium 199 was modified, each flask containing in 2.0 ml total volume: isolated glomeruli (approximately 0.5 mg of glomerular protein); 30 mM HEPES (Calbiochem, La Jolla, Calif.) buffer, pH 7.2; 1.5 mM NaHCO3; 100 units/ml of penicillin; 100/~g/ml of streptomycin; and labeled amino acids (the corresponding unlabeled amino acids were omitted from the medium) or labeled carbohydrates (glucose was omitted) as described under Results. The incubation was in 25 ml Erlenmeyer flasks in an atmosphere of 5 ~ CO2/95~o 02 at 37 °C with continuous gentle lateral shaking. The reaction was stopped by adding 2.0 ml of 10 ~o trichloroacetic acid to each flask, and the precipitated material washed four times using 8.0-10.0 ml of 5 ~ cold trichloroacetic acid. The final pellet was hydrolysed in 1.0 N NaOH and aliquots were taken for determination of protein [14] and radioactivity (Nuclear-Chicago, Mark I; efficiency was approx. 25 ~o for aH and 70~o for t4C). In all incorporation experiments, except those presented in Fig. 1, the zero time count (which was identical with the dead tissue count; cf. Results, Fig. 1) was subtracted from the total count prior to any calculation. Cell fractionation and amino acid analysis The incubation was appropriately scaled up for this experiment. After incubation (37 °C for 3 h) using o-[1-3H]galactose, the glomeruli were centrifuged at 200 × g for 5 min and the pellets washed 5 times in large excess of ice-cold physiological saline (dialysis against saline gave identical results). All subsequent procedures were at 0--4 °C. The glomeruli were then suspended in a small volume of physiological

508 saline and the cells disrupted by sonication (4 × 1 min at 40 % power intensity; 10 mm probe; Biosonik IV, Bronwill-VWR Scientific, Baltimore, Maryland). The material was centrifuged first at 30 000 × g for 20 min, then at 177 000 × g for 2 h. The supernatant which remained in solution after the high-speed centrifugation was lyophilized, dissolved in 0.5 M NaC1, and analyzed as described in the legend to Fig. 3. Samples for amino acid analysis were hydrolyzed in 6 N HC1 at 110 °C in vacuo. Amino acids were determined using a Beckman Model 120C amino acid analyzer.

Materials The following radioactive compounds were purchased from New England Nuclear Corp., Boston, Mass.: o-[6-3H]glucosamine hydrochloride, 7.5 Ci/mmol; D- [ 1-3H]galactose, 2.04 Ci/mmol; L- [ 1,5,6-3H]fucose, 3.18 Ci/mmol; L- [G-SH]proline, 4.75 Ci/mmol; and L-[G-3H]lysine, 3.2 Ci/mmol. Vinblastin was from Sigma Chemical Co., St. Louis, Mo.; cytochalasin B from Imperial Chemical Industries, Alderley Park, England, and puromycin aminonucleoside* from ICN Nutritional Biochemicals, Cleveland, Ohio. RESULTS

Incorporation of labeled proline, lysine and galactose The time course of labeled proline incorporation into total glomerular protein is shown in Fig. 1. Incorporation was linear for 8 h. This experiment did not distin40

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* 6-Dimethylaminopurine-3-amino-( + )-ribose.

509 TABLE I INCORPORATION OF VARIOUS PRECURSORS BY ISOLATED RAT KIDNEY GLOMERULI IN VITRO The incubation was as described under Materials and Methods. Equimolar quantities of labeled proline and lysine (210 pmol each) and of labeled galactose and glucosamine (520 pmol each) respectively were used. Linearity of in vitro incorporation of these precursors has been established for the indicated periods of incubation. The values are mean 4- S.E. of three independent determinations, each in duplicate. Precursor

Duration of incubation (h)

Incorporated (pmol)

[3H]Proline [3H]Lysine [3H]Galactose [3H]Glucosamine

4 4 9 9

12.5 4- 0.3 16.5 + 0.5 9.6 -- 0.3 4.2 ± 0.2

guish whether the label, once incorporated into macromolecules, was present as proline or hydroxyproline. The relative rates of proline and lysine incorporation are given in Table I (the linearity of incorporation for the incubation time periods has been established independently). On amino acid analysis of total glomerular hydrolysates we found the following: proline, 54.9; 4-hydroxyproline, 11.1; 3-hydroxyproline, 1.5; lysine, 68.9; and hydroxylysine, 2.5 residues/1000 amino acids. When equimolar quantities of labeled proline and lysine were added to the incubation medium, more lysine was incorporated than proline. In the same type of experiments, approximately twice more labeled galactose was incorporated than labeled glucosamine.

Determination of intracellular pools of proline and lysine Using standard procedures [15], the intracellular pools of proline and lysine of glomeruli were determined by isotope dilution type experiments. When the reciprocal of cpm incorporated into protein was plotted against the amount of unlabeled amino acid added to the in vitro system, a straight line was obtained (Fig. 2). For considering the data in Fig. 2, the following notations were used: Cg = free amino acid in glomerular cells (i.e., that fraction available for protein synthesis under conditions of experiment) plus the added labeled amino acid Ca ---- amount of unlabeled amino acid added Ct = C~ q- C~ = total free amino acid in the system a = cpm incorporated into protein which is proportional to the fractional part of the glomerular free amino acid in Ct for a given amount of radioactive amino acid added, i.e., a = k

Cg C~ -- Cu

where k is a proportionality constant. Special points of the curve: when Cu ----0, 1/a = 1/k. This is the case of no dilution, designated as A in Fig. 2 (the intercept of the graph with the ordinate).

510 B = 2 × A, and since the ordinate is 1/cpm, this is the theoretical 50% inhibition. Bx is the intercept of the 50 % inhibition value with the graph. The corresponding value on the abscissa (A~) is the amount of unlabeled amino acid which, when added to the system, results in two-fold dilution of the labeled amino acid, or 50 % inhibition of incorporation. Hence, this value of added amino acid must be equal to the existing endogenous amino acid pool. By this method, we found the glomerular intracellular proline and lysine pools to be 52 and 32 nmol/mg protein, respectively.

Glycoprotein synthesis by isolated glomeruli and the effects of inhibitors Since we have shown recently that labeled carbohydrates are incorporated into macromolecules which are glycoprotein in nature [6, 11, 12], theoretically there should be soluble intracellular glycoproteins in isolated glomeruli. Applying such logic, glomerular cells were fractionated after in vitro incorporation experiments where labeled galactose was used. When the soluble intracellular macromolecules were chromatographed on Sephadex G-150, at least three peaks with radioactivity could be recovered (Fig. 3). It was shown by hydrolysis and subsequent analysis of

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Fig. 2. Estimation of proline and lysine polls in isolated kidney glomeruli by isotope dilution. Either 2.5 #Ci [3H]Pro [I] or 2.5/~Ci [3H]Lys(II) per flask was used with the corresponding amino acid omitted from the medium. Instead, known amounts of [1H]Pro (I) or [1H]Lys(II) were added to the incubation mixtures as shown on the abscissa. The ordinate is l/cpm, and cpm values have been corrected for 1 mg glomerular protein, as determined by the Lowry method [14]. Incubation was for 4 h at 37 °C, and specificactivity was determined as described in in Fig. 1. Each point represents the mean of two separate experiments. The dotted line parallel with the abscissa represents 50% inhibition of incorporation due to dilution of the 3H-labeled amino acid by the added unlabeled amino acid. The value on the abscissa corresponding to the intercept represents the estimated amino acid pool in glomeruli (with respect to 1 mg glomerular protein) for Pro and Lys, respectively (see text for further details).

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Fig. 3. Gel filtration of soluble glomerular proteins after [aH]galactose incorporation. The reaction mixture was sealed up ten-fold and contained 25/~Ci of [Sill-Gal. The glomeruli and all their cellular elements were disrupted by sonication at the end of the incubation, which was for 3 h at 37 °C. The supernatant which remained in solution after centrifugation at 177 000 × g for 2 h was lyophilized, dissolved in 0.5 M NaCI, and dialyzed against 3 changes of the same. Approximately 1.0 mg protein was applied on a 52 × 1.2 cm Sephadex G-150 column and was chromatographed using 0.5 M NaCI as eluent. e a c h p e a k u s i n g t h i n - l a y e r c h r o m a t o g r a p h y [11] t h a t t h e l a b e l w a s still in g a l a c t o s e . The kinetics of galactose incoroporation into macromolecules by glomeruli are s h o w n in Fig. 4A. I n c o r p o r a t i o n was l i n e a r f o r at least 9 h a n d c y t o c h a l a s i n B a n d v i n b l a s t i n i n h i b i t e d i n c o r p o r a t i o n , w h i c h is s i m i l a r to t h e findings o f E h r l i c h

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Fig. 4. (A) Effect of vinblastin and cytochalasin B on incorporation of [3H]galactose by isolated rat kidney glomeruli. Glucose was omitted from the incubation mixture and 3/~Ci of [3H]Gal was added to each flask. 0 - - 0 , no inhibitor; O---O, 5" 10 -5 M vinblastin; x --- x , 5/~g/ml of cytochalasin B. Each time point is the mean 4- S.E. of three separate experiments, each in duplicate. (B) Effect of cytochalasin B on fucose incorporation. Glucose was omitted and 5/~Ci [aH]fucose was added to each flask. 0 - - - 0 , no inhibitor; x - - x , cytochalasin B, 5/~g/ml added at zero time. Each time point is the mean 4- S.E. of two separate experiments, each in triplicate. (C) Effect of puromycin aminonucleoside treatment of rats on [3H]galactose incorporation by glomeruli in vitro. Glucose was omitted from the incubation mixture and 2.5/zCi of [aH]Gal was added to each flask. O - - O , control (10 rats); x --- ×, treated (7 rats). Treatment schedule is described in the text. Each time point represents the mean ± S.E.

512 and Bornstein [16]. General protein synthesis in the glomeruli was not inhibited by these agents (data not shown). The carbohydrate moiety of glomerular basement membrane glycoprotein contains one residue of fucose [17, 18]. Its incorporation into glomerular macromolecules was also linear for at least 9 h (Fig. 4B). Cytochalasin B inhibited incorporation, but to a somewhat lesser degree than that of galactose. The experiments to be presented next were conceptually different from those just described. Whereas cytochalasin B and vinblastin were used in vitro, puromycin aminonucleoside was administered to rats (i.e., employed in vivo) and the glomeruli were subsequently isolated for biochemical studies. Male rats weighing 80--90 g were given daily subcutaneous injections of the drug in a dose of 1.5 mg/100 g body weight for ten days. Control animals received equivalent amounts of physiological saline. Urinary protein concentration was tested on the third, seventh and tenth days using Labstix, and, on the tenth day, 24 h urine was collected for total protein determination by the method of Lowry et al. [14]. Proteinuria appeared in the treated animals on the seventh day. On the tenth day the control group excreted 11.3 mg protein/h/rat (average). A pronounced ascites was present in all treated animals by the tenth day. The animals were sacrificed on the eleventh day, and the glomeruli were isolated and tested in the in vitro assay. Fig. 4C is the time course of galactose incorporation showing that glomeruli from rats treated with puromycin aminonucleoside incorporated galactose less efficiently than those from the control animals. DISCUSSION We have reported previously [11, 12] that isolated kidney glomeruli are suitable for biosynthetic studies, and shown that glomeruli from young rats and piglets synthesize protein in vitro. The incorporation of labeled amino acids or monosaccharides into glomerular macromolecules was linear for 6 to 10 h. We have presented evidence that glycoprotein macromolecules were synthesized and postulated that a portion of the newly-synthesized glycoprotein(s) was glomerular basement membrane. This paper is an extension of the above work. Employing the same system, but using only rat glomeruli, we have: (a) determined the pool sizes of proline and lysine; (b) further characterized the system with additional precursors; (c) examined the effects of vinblastin and cytochalasin B; and (d) employed the system to study glomeruli from rats with puromycin aminonucleoside-induced nephrosis, an experimental kidney disease. The usefulness of isolated glomeruli for biosynthetic studies is now well appreciated [6-12, 19, 20]. For example, Burlington and his colleagues [19] succeeded in culturing goat glomeruli which lemained viable for several months and apparently synthesized erythropoetin; Fish et al. [20] reported secretion of protein consistent with glomerular basement membrane by glomerular cells from human kidneys. Most recently, two groups of authors [9, 10] published evidence of collagen synthesis by rat glomeruli. Cohen and Vogt [10] labeled glomerular proteins with lysine, following which they determined lysine and hydroxylysine in the medium, intracellularly and in partially purified basement membrane. In contrast to Cohen and Vogt [10], we have made no attempt to separately analyze that fraction of the newly-synthesized material which is secreted into the medium. However, in those experiments where no

513 further fractionation was done, our incorporation data are directly comparable (noting, of course, the different precursors used) to the total counts of Cohen and Vogt [I0]. This is so because we stopped biosynthesis by adding trichloroacetic acid to the total reaction mixture; and it is known that under these conditions trichloroacetic acid precipitates all macromolecules in question, including those secreted into the medium and which are, in their experiments, nondialyzable. The intracellular pool size was 52 nmol/g and 32 nmol/mg of glomerular protein for proline and lysine, respectively. We are unaware of prior information in the literature on lysine or proline pools in cells of the kidney glomerulus. By definition, our experiments measured that intracellular pool size for the two amino acids which is immediately available for protein biosynthesis under the conditions of the experiment. In the experiment reported here, the incorporation of radiolabeled proline, galactose and fucose was linear for 8 h or longer. Since these two monosaccharides are constituents of glomerular basement membrane glycoprotein(s), they can be viewed as potential precursors of glomerular basement membrane in this system [11 12]. Because of the several chemical similarities between vertebrate collagen and glomerular basement membrane [13, 17, 18], we deemed it logical to consider biosynthetic information available on collagen as valid guidelines for designing experiments. By such logic we used cytochalasin B (a fungal metabolite which inhibits a variety of cellular processes such as cytokinesis, phagocytosis and sugar transport into cells; cf. refs. 16, 21) and vinblastin (an inhibitor of microtubular functions; cf. 16). Both compounds inhibited glomerular glycoprotein synthesis without any appreciable effect on general protein synthesis. These results are conceptually in agreement with those of Ehrlich and Bornstein [16], who found that cytochalasin B and vinblastin inhibited collagen, but not general protein synthesis in cranial bones. They attributed this to a feedback inhibition (the two compounds interrupted secretion of collagen and procollagen). Assuming that the macromolecules whose synthesis was inhibited in our system were secretory glycoproteins, we hypothesize that our results are also due to a similar feedback inhibition. Administration of puromycin aminonucleoside induces the nephrotic syndrome [22, 23]. Farquhar and Palade [23] concluded that alterations of the basement membrane and of epithelial cells were responsible for protein leakage in this experimental nephroses. Further, it has been reported [24] that hydroxyproline, hydroxy!ysine and galactose contents of the glomerular basement membrane was significantly less in treated rats than in the control animals. If so, one might expect that the rate of incorporation of certain amino acids or carbohydrate precursors would be decreased (assuming no change in catalytic rate). We found decreased rate of proline (which in these experiments reflects the sum of proline and hydroxyproline; data not included) and galactose (Fig. 4C) incorporation. Therefore, our findings are conceptually in agreement with those obtained previously by a different approach [24]. The molecular mechanisms by which puromycin aminonucleoside (an adenosine analog) damages the kidney, the glomerulus and its basement membrane in particular, are still unclear [25-27]. Our experiments with puromycin aminonucleoside are included as examples for the potential utility of such an approach to gain better understanding of specific kidney diseases in both experimental animals and naturally occurring diseases of man.

514 ACKNOWLEDGEMENTS W e t h a n k D r . W i l l C o u l t e r for p e r f o r m i n g some o f the a m i n o acid analyses, M r . R i c h a r d G i n s b e r g for assistance in the a m i n o n u c l e o s i d e experiments, a n d Mrs. J u d y H e r m a n a n d Miss K a t h e r i n e Lewis for excellent technical help. This w o r k was s u p p o r t e d in p a r t b y G r a n t - i n - A i d f r o m the A m e r i c a n H e a r t Association, the O ' N e i l l R e s e a r c h F u n d , a n d b y Veterans A d m i n i s t r a t i o n Research. REFERENCES 1 Strauss, M. D. and Welt, L. G. (1971) Diseases of the Kidney, Vol. I, Second Edition, Little, Brown and Company, Boston 2 Koffler, D., Schur, P. and Kunkel, H. (1967) J. Exp. Med. 126, 607-624 3 Lemer, R. A., Glassock, R. J. and Dixon, F. J. (1967) J. Exp. Med. 126, 989-1004 4 Wilson, C. B. and Dixon, F. J. (1973) Kidney Int. 3, 74-89 5 Walker, W. G. (1967) in Acute Glomerulonephritis (Metcoff, J., ed.), pp. 261-269, Little, Brown and Company, Boston 6 Krisko, I. and Walker, W. G. (1972) Clin. Res. 20, 599 7 Brown, D. M. and Michael, A. F. (1973) Fed. Proc. 32, 650 8 Beisswenger, P. J. (1974) Clin. Res. 22, 460A 9 Grant, M. E., Harwood, R. and Williams, I. F. (1975) Eur. J. Biochem. 54, 531-540 10 Cohen, M. P. and Vogt, C. A. (1975) Biochim. Biophys. Acta 393, 78-87 11 Krisko, I. and Walker, W. G. (1974) Proc. Soc. Exp. Biol. Med. 146, 942-947 12 Krisko, I. and Gyorkey, F. (1974) in Lipmann Symposium: Energy, Biosynthesis and Regulation in Molecular Biology (Richter, D., ed.), pp. 345-358, de Gruyter Company, Berlin and New York 13 Spiro, R. G. (1967) J. Biol. Chem. 242, 1915-1922 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 15 Wang, C. H. and Willis, D. L. (1965) Radiotracer Methodology in Biological Science, Prentice Hall, Inc., Englewood Cliffs, New Jersey 16 Ehrlich, H. P. and Bornstein, P. (1972) Nat. New Biol. 238, 257-260 17 Kefalides, N. A. (1972) Adv. Nephrol. 2, 3-24 18 Hudson, B. G. and Spiro, R. G. (1972) J. Biol. Chem. 247, 4239-4247 19 Burlington, H., Cronkite, E. P., Reincke, U. and Zanjani, E. D. (1972) Proc. Natl. Acad. Sci. U.S., 69, 3547-3550 20 Fish, A. J., Michael, A. F., Vernier, R. L. and Brown, D. M. (1975) Lab. Invest. 33, 330-341 21 Estensen, R. D. and Plagemann, P. G. W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1430-1434 22 Vernier, R. L., Papermaster, B. W. and Good, R. A. (1959) J. Exp. Med. 109, 115-125 23 Farquhar, M. G. and Palade, G. E. (1961) J. Exp. Med. 114, 699-716 24 Kefalides, N. A. and Forsell-Knott, L. (1970) Biochim. Biophys. Acta 203, 62-66 25 Dickie, N., Norton~ L. F., Derr, R. F., Alexander, C. S. and Nagasawa, H. T. (1966) Biochim. Biophys. Acta 129, 288-293 26 Michael, A. F., Blau, E. and Vernier, R. L. (1970) Lab. Invest. 23, 649-657 27 Cholon, J. J., and Studzinski, G. P. (1974) Science (Wash. D.C.) 184, 160-161