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TOXICOLOGY AND APPLIED PHARMACOLOGY 96, 380-392 (1988) Hydrocarbon-Induced Hyaline Droplet Nephropathy in Male Rats during Senescence C. V. R. Murty,1'* M, J. Olson,2t B. D. GARG,t and A. K. Roy* *Departments ofObstetrics and Gynecology and Cellular and Structural Biology, University ofTexas Health Sciences Center, San Antonio, Texas 78284: and tBiomedical Science Department, General Motors Research Laboratories. Warren, Michigan 480903 Received May 12,1988: acceptedJuly 25,1988 Hydrocarbon-Induced Hyaline Droplet Nephropathy in Male Rats during Senescence. Murty, C. V. R., Olson, M. J., Garg, B. D., and Roy, A. K. (1988). Toxicol. Appl. Pharma col. 96,380-392. Male rats administered unleaded gasoline rapidly develop nephropathy charac terized by accumulation of hyaline droplets in cells of the proximal convoluted tubules (PCT). This acute response is implicated in development ofrenal carcinoma in male rats exposed chron ically to wholly volatilized gasoline. A major constituent of hyaline droplets is a,u-globulin, a protein ofhepatic origin for which the rate ofsynthesis declines during aging. Little information, however, is presently available on possible age-dependent susceptibility of male rats to hydrocar bon-induced nephropathy. In kidneys of untreated male Fischer 344 rats the number of consti tutive hyaline droplets declined progressively with increasing age. Electrophoresis ofrenal corti cal homogenates revealed a protein with M, about 18 X 10\ probably a2ll-globulin, in young (3.5 months old) male rats and total absence of this protein in aged (26 months old) males. RIA confirmed that constitutive levels ofrenal and hepatic 2u-globulin in old rats were less than 1.5% of those in young adults. Unleaded gasoline (0.4 ml/kg/day, po, 5 days) caused accumulation of hyaline droplets in renal PCT of3.5-month-old males accompanied by a marked increase (about twofold) in the renal content of a2,,-globulin, whereas the same treatment was without effect in 26-month-old rats. Finally, in the renal cortex ofyoung rats the activities ofthe lysosomal prote ases cathepsin B and D were increased following gasoline administration, presumably in re sponse to protein accumulation. However, in 26-month-old rats cathepsin B activity was un affected, while cathepsin D was increased by gasoline administration. Thus, we conclude that animal age is an important determinant in the development ofhydrocarbon-induced nephropa thy and only rats which produce large amounts of a2u-globulin are susceptible to development ofthis pathology. toss Academic Pus. Inc. INTRODUCTION Acute or subchronic exposure of rats to a va riety ofpure hydrocarbons and mixtures such 1 Present address: Wyeth Laboratories, Inc., P.O. Box 8299, Philadelphia. PA 19101. 2 To whom correspondence should be addressed: Mi chael J. Olson, Ph.D., Biomedical Science Dept., GM Research Labs., Warren. MI 48090. 3 The Research Biomedical Laboratory of GM Re search Laboratories is accredited by the American Asso ciation for the Accreditation of Laboratory Animal Care (AAALAC). The rationale and experimental protocol for as unleaded gasoline has been reported to in duce hyaline droplet nephropathy, a condi tion characterized by excessive accumulation of proteinaceous material in cells lining the renal proximal convoluted tubules (PCT), with minimal abnormality in renal function (Table 1). Such alterations in renal morphol ogy and function are largely specific to male the use ofan animal model in the experiments described in this publication have been reviewed by the GM Re search Laboratories' Animal Research Committee. 0041-008X/8813.00 Copyright (y 1988 by Academic Press, Inc. Alt rights of reproduction in any form reserved 380 SL 035525 it exas Health ence. irmatarao 1CT). iironlin, a ition. >r> of bout ct in rote11 ret unthat opancnt sorted to in>y, a condicumulation s lining the Jies (PCT), ial function tl morpholific to male ents described the GM Re- HYDROCARBON-INDUCED HYALINE DROPLET NEPHROPATHY TABLEI Chemicals Reported to Cause Hyaline Droplet Nephropathy in Male Rats Chemical and usage classification Reference Food constituents D-Limonene Fuels and fuel components Decahydronaphthalene (Decalin) Jet fuels (RJ-5, JP-4, JP-5, JP-10) Naphthas (various) Tetralin 2,2,4-Trimethylpentane Unleaded gasoline Solvents 60 Solvent. 70 solvent C10-C12 Isoparaffinic solvent Diisobutyl ketone Methyl isobutyl ketone Napthenic aromatic solvent Pentachloroethane Perchloroethylene Stoddard solvent VM & P naphtha Miscellaneous p-Dichlorobenzene (deoderant) Isophorone (wood preservative) Webb and Alden, 1987 Alden et at., 1984: Kanerva et al.. 1987 Bruner. 1984 Haider etal.. 1984 Serve etai, 1988 Stonard et at.. 1986: Short et at.. 1987 Haider etal., 1984; Olson ctal.. 1987 Phillips and Cockrell, 1984 Viau etal.. 1986 Dodd etal.. 1987 Phillips et al.. 1987 Phillips and Cockrell, 1984 Goldsworthy et at., 1987 Goldsworthy et at., 1987 Phillips and Cockrell. 1984 Phillips and Cockrell, 1984 Bomhard etal.. 1988 Strasser et at.. 1988 381 rats less than 12 months of age when treated with these hydrocarbon toxicants. A protein unique to the male rat, a2u-globulin, is a primary constituent of the hyaline droplets accumulated during hydrocarbon exposure (Stonard et al, 1986; Garg et al., 1987; Kanerva et al., 1987; Olson et al, 1987). The presence of a2u-globulin has been suggested, although not proven, to be the crit ical factor which renders male rats susceptible to chemically induced hyaline droplet ne phropathy (Alden et al., 1984; Gibson and Bus, 1988; Olson et al, 1987, 1988). a2uGlobulin is synthesized in the liver and the expression of this protein is strongly age de pendent as shown by decreasing hepatic syn thesis and urinary excretion of 2u-globulin with increasing age (Roy et al.. 1983). The de cline, and eventual cessation, of 2u-globulin production is due to development of hepatic androgen insensitivity during senescence (Roy et al, 1983; Sarkar et al, 1987). How ever, the toxicity studies conducted with chemicals shown to cause hyaline droplet ne phropathy in male rats have been performed with rats at ages when the synthesis and ex cretion of a2u-globulin is near maximal (Ta ble 1). Since <*2u-globulin synthesis declines during aging, we sought to determine whether a relationship existed between levels of 2u*globulin and susceptibility to hydrocarbon-induced nephropathy. Furthermore, since alterations of lysosomal morphology are known to accompany hydrocarbon-in duced a2u-globulin accumulation (Phillips and Cockrell, 1984; Garg et al, 1987; Kan erva et al, 1987; Short et al, 1987). we char acterized the response of several key renal ly sosomal enzymes involved in proteolysis to determine whether changes in the activity of these enzymes might be associated with gaso line-induced hyaline droplet nephropathy and how such changes might vary with ani mal age. The results show that, unlike the tox icity of certain therapeutic agents which is in creased in aged rats (reviewed by Goldstein et SL 035526 382 MURTY ET AL. al., 1988), the nephrotoxicity associated with hydrocarbon exposure is decreased with age, probably due to the decline in a^-globulin production. Thus, when male rats are used in the evaluation of nephrotoxic potential of chemicals, especially those agents which cause hyaline droplet nephropathy, animal age is an important consideration in experi mental design and interpretation of data. MATERIALS AND METHODS Animals and treatment protocol. Male Fischer 344 rats (CDF (F-344)Crl BR, VAF/Plus) obtained from Charles River Laboratories, Kingston. New York or the National Institute of Aging (strain F344/NHsd, maintained by Sprague-Dawley, Inc. Indianapolis, IN), were housed in the GMR Biomedical Science Department vivarium in conditions conforming to AAALAC recommendations with tap water and Purina 5012 chow freely available. Untreated rats of ages 3.5 (n = 6), 18 (n = 3), or 26 (n = 10) months were used for appraisal of renal morphol ogy as a function of age. Other male rats, ages 3.5 or 26 months received either commercially available unleaded gasoline (0.4 ml/kg/day, 5 days; six 3.5-month-old and six 26*month-old rats) or 0.9% sodium chloride solution (0,4 ml/kg/day, 5 days; four young and four old rats) by intragastric intubation and were euthanized 24 hr after the final dose. Tissues from each of these rats was used for light and electron microscopy of kidney, RIA deter mination of renal and hepatic a,,,-globulin content, elec trophoretic characterization of soluble renal proteins, and renal cathepsin activity measurements as described below. The chemical composition ofthe test material has been published previously (Olson et al. 1987). Except as specified, all chemicals and reagents were supplied by Sigma Chemical Co. (St. Louis. MO). Light and electron microscopic histology. While under pentobarbital anesthesia, whole body transcardiac perfu sion was performed with 0.9% sodium chloride solution, 4"C. Samples of renal conex were removed and fixed by immersion in 10% neutral buffered formalin. Tissues were processed for light microscopy by routine methods, embedded in glycol methacrylate, and stained with Lee's methylene blue-basic fuchsin method (Bennet et al.. 1976) after sectioning. Alternatively, samples of renal cortex were fixed in 2.5% glutaraldehyde, 0.1 M sodium cacodylate (pH 7.4). These tissues were postfixed in 1% osmium tetroxide in 0.1 M sym-collidine buffer (pH 7.4), embedded in Spurr's medium, sectioned to l-ym thick ness, mounted and stained with 1% toluidine blue (Rich ardson el al.. 1960) for light microscopy. Thin (ca. 60 nm) sections were cut and stained with uranyl acetate/ lead citrate prior to inspection by transmission electron microscopy. Electrophoresis of renal cortical proteins. Electropho retic separation of soluble renal cortical proteins was ac complished with SDS-containing polyacrylamide gels (SDS-PAGE) using a 15% resolving gel with a 3% acryl amide stacking gel (Laemmli, 1970). Kidneys, frozen in liquid Nj following whole body perfusion with 0.9% so dium chloride solution, were rapidly thawed and the me dulla was removed and discarded. A 12.5% homogenate of the cortex was prepared in 0.3 M sucrose buffer (pH 7.0) with a Tefion-glass tissue gnnder, A supernatant fraction (150g, 10 min, 4`C) ofhomogenized renal cortex was prepared and a portion added to 0.0625 m Tris-HCl (pH 6.8), 2% 2-mercaptoethanol, 2% SDS, 10% sucrose buffer and denatured at 100*C for 3 min. Samples (100 pg of supernatant protein) and heat-denatured marker proteins of known molecular weight were subjected to electrophoresis for about 5 hr at a constant current of 50 mA. Proteins were fixed in a methanol-acetic acid-water solution (5:5:2) and visualized by staining in 0.1% Coomassie blue made in the fixative solution (Sinclair and Rickwood. 1981). Radioimmunoassay of a^'globulin. Cytosolic frac tions were prepared from kidney or liver homogenates and assayed for a2u-globulin by double-antibody radio immunoassay (RIA) as described by Roy (1977). 2llGlobulin in the cytosolic fraction was expressed relative to total protein (Lowry etal.. 1951). Lysosomal enzyme activity determinations. Lyso somal enzyme activities were assayed in samples of the same supernatant fraction (PNS) prepared for electro phoresis. Determination ofprotein quantities used in ac tivity measurements of lysosomal enzymes was per formed using the BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL). Acid phosphatase activities were determined from the amount of inorganic phosphate (P,) liberated by the hy drolysis of /3-glycerophosphate (Trouet, 1974). Reagent blanks containing no PNS protein were used for correc tion of nonenzymatic phosphate production. The amount of P, released was established in absolute terms by comparison to an inorganic phosphate standard curve. Enzyme activity was expressed as nanomoles P, released from /3-glycerophosphate per minute per milli gram PNS protein. Cathepsin B activities were determined by fluorometric measurement of free /3-naphthylamine released from /V-carboxybenzoyl-L-alanyl-L-arginyl-L-arginyl-4-methoxy-/3-napthylamide 2 HCI (Kugler and Vomberger, 1986). Verification that hydrolysis was catalyzed enzy matically was obtained by analyzing random homoge nate samples m the presence of leupeptin (propionyl-Lleucyl-L-leucyl-L-argininaldehydc: 5 aM), an inhibitor of cathepsin B (Barrett, 1973). In all cases, addition of leu peptin resulted in greater than 90% inhibition of release of/t-napthylaminc from the peptidc-napthylamine sub strate. Cathepsin B activity was expressed relative to flu orescence values derived from standard concentrations of free d-naphthylamine and expressed as nanomoles product formed per minute per milligram PNS protein. SL 035527 Cathet maticalb soluble i protein i terminei tyrosine pressed: milligra. cubated ammo ammo-, cific mb which c Renai tret Mi renal the r meth diam the P1 or ov hetei plasr vioir quer was angv hyal line drop the t dull Ir rats trea few the (Fit bot pea mo the dis ind srr. terns. Electropholl proteins was aclyacrylaraide gels I with a 3% acrylCidneys, frozen in ion with 0.9% soawed and the me2.5% homogenate acrose buffer (pH -r. A supernatant mzed renal cortex 0625 M Tris-HCl iDS, 10% sucrose tin. Samples (100 lenatured marker were subjected to tant current of 50 acetic acid-water ing in 0.1% Cooion (Sinclair and Cytosolic fracver homogenates :-antibody radioRoy (1977). a2uxpressed relative motions. Lyson samples of the ared for electroltities used in aczymes was peragent kit (Pierce rmined from the crated by the hy. 1974). Reagent used for correcroduction. The n absolute terms sphate standard as nanomoles P, linute per milli- d by fluoromette released from arginyl-4-methnd Vomberger, catalyzed enzyndom homogein (propionyl-Li. an inhibitor of addition of leuintion of release ithylamine subJ relative to fluconcentrations I as nanomoles HYDROCARBON-INDUCED HYALINE DROPLET NEPHROPATHY 383 Cathepsin D activity was assayed by determining enzy matically catalyzed degradation of hemoglobin to acidsoluble peptides (Turk et al., 1984). After incubation, protein content of acidified and filtered samples was de termined relative to a standard curve prepared from ltyrosine (Turk et al.. 1984). Enzyme activity was ex pressed as micromoles L-tyrosine released per minute per milligram PNS protein. Random PNS samples were in cubated with pepstatin (isovaleryl-L-valyl-L-valyl-4amino - 3 - hydroxy - 6 - methylheptanoyl - L - alanyl - 4 amino-3-hydroxy-6-methylheptanoic acid; 5 iim), a spe cific inhibitor of cathepsin D activity (Turk et al., 1984), which caused near total loss of hemoglobin hydrolysis. RESULTS Renal Hyaline Droplets during Aging in Un treated Rats Methylene blue-basic fuchsia staining. In renal tissue of untreated 3.5-month-old rats, the methylene blue-basic fuchsin staining method identified numerous small (*(). 5jmi diam) hyaline droplets in epithelial cells of the PCT (Fig. 1 A). These droplets were round or oval in cross section, uniform in size, and heterogeneously distributed within the cyto plasm of individual cells as described pre viously by Short et al. (1986, 1987). Infre quently, a single cell of the epithelial layer was observed which contained droplets with angular profiles or which was engorged with hyaline droplets. Macrodistribution of hya line droplets was observed with cords of droplet-containing PCT cells extending from the renal capsule to the outer stripe ofthe me dulla. In contrast to tissues from 3.5-month-old rats, examination ofkidney sections from un treated 18-month-old male rats revealed very few droplets which stained positively with the methylene blue-basic fuchsin method (Fig. IB). The absolute number of droplets both on a per cell and a tissue area basis ap peared to be reduced when comparing 18month-old to 3.5-month-old rats. In general, the droplets visible in 18-month-old rats were distributed randomly within the cytoplasm of individual PCT epithelial cells but were smaller in diameter (ca. 0.2 /im) than the droplets observed in 3.5-month-old rats. Ma crodistribution of PCT containing hyaline droplets within the kidney cortex was not ap parent in 18-month-old animals. The num ber of droplets in animals aged 26-months was reduced greatly compared to rats at either 3.5 or 18 months. In viewing many micro scopic fields of renal cortex from untreated 26-month-old rats it was impossible to iden tify any hyaline droplets with the methylene blue-basic fuchsin stain (Fig. 1C). Toluidine blue staining. To obtain en hanced resolution at light microscopic mag nification, 1 -jzm-thick sections of kidney cor tex embedded in Spurr's medium were exam ined after preparation with toluidine blue stain. Hyaline droplets observed in the renal tissue of 3.5-month-old rats prepared with this method were nearly identical in number, size, shape, and distribution to those de scribed in glycol methacrylate-embedded tis sues prepared with methylene blue-basic fuchsin staining (vide supra) (Fig. 2A). Furthermore, in renal tissues from salinetreated 26-month-old rats prepared in this manner, hyaline droplets were clearly visible in some PCT epithelial cells (Fig. 2C) al though the quantity of these droplets was re duced from that observed in young male rats. Unlike the droplets in young rats, those in old rats appeared to be heterogeneous in size and shape with numerous irregular, enlarged droplets apparent (Fig. 2C). In addition, the distribution of hyaline droplets throughout the cortex of old rats was more variable than in young rats; areas were visible in which nu merous small droplets of uniform size ex isted, other areas contained only a few large droplets of varying size. Effect of Unleaded Gasoline on Renal PCT Morphology and Ultrastructure Young rats. Renal PCT epithelial cells of saline-treated young rats contained nu merous phagolysosomes (secondary lysosomes) distinguishable by electron micros copy (Fig. 3A). These organelles were mem- SL 035528 384 MURTY ET AL. Fig. I. Age-dependent decrease in renal cortical hyaline droplet number in untreated male rats. (A) Tissue from a 3.5-month-old rat showing profiles of proximal convoluted tubules with prominent hyaline droplets ( -- ). (B) Male rat, 18 months old; (C) 26-month-old rat. Photomicrographs are typical of tissues examined. Methylene blue-basic fuchsin stain, glycol methacrylate embeddment, 50x, brane bound, round in cross section, larger than primary lysosomes, and possessed vari able electron density. Only rarely was an ir regularly shaped phagolysosome observed in the kidney of saline-treated rats. As described in previous reports from this laboratory (Garg et al., 1987; Olson et al, 1987), both the number and the size of hya line droplets in epithelial cells of the renal PCT were markedly increased in young rats SL 035529 tr d n a ti HYDROCARBON-INDUCED HYALINE DROPLET NEPHROPATHY 385 ale rats. (A) nent hyaline cal of tissues orts from this Olson ct at., ie size of hyai of the renal young rats Fig. 2. Effect ofgasoline administration on renal cortical morphology in young adult and senescent male rats. (A) Saline-treated 3.5-month-old rat; note uniform size and distribution of hyaline droplets ( - ). (B) Gasoline-treated 3.5-month-old rat; hyaline droplet number is increased and droplet shape and size are altered. (C) Saline-treated 26-month-old rat. Note variability in number, size, and distribution of hyaline droplets in saline-treated senescent rats compared to those of young rats. (D) Gasoline-treated 26-monthold rat; failure of gasoline administration to alter hyaline droplet number or size. Treatment protocol described under Materials and Methods. Photomicrographs are typical oftissues examined. Toluidineblue stain, tissue in Spurr's medium, 50x. treated with gasoline (0.4 ml/kg/day, po, 5 days) (Fig. 2B). In severely affected tubules, apical blebs were apparent and some cells un dergoing lysis were observed (not shown). Consistent with earlier reports, hyaline drop lets were seen to be phagolysosomes when ex amined by electron microscopy (Fig. 3R). Following repeated gasoline administration some phagolysosomes were still morphologi cally similar to those of controls (Fig. 3B). SL 035530 386 MURTY ET AL. Fig. 3. Representative transmission electron micrographs of renal proximal tubules from 3.5-monthold (A,B) and 26-month-old (C.D) male rats. Electron-dense phagolysosomes (-*) appear in the cytoplasm of animals at both ages. (A,C) Taken from rats treated with saline and illustrate the variable size and shape of phagolysosomes of aged control rats compared to those in young animals. (B.D) Gasoline-treated rats. Gasoline causes no change in phagolysosome number or conformation in 26-month-old male rats relative to age-matched saline-treated controls. 2400X. However, most phagolysosomes were angu lar in profile with conspicuous electron-dense inclusions, some with crystalline structure, in their matrices (Fig. 3B). Infrequently, clear areas of cytoplasm without obvious delimit ing membranes and containing phagolyso somes, electron-dense bodies and cellular de bris were seen indicating the occurrence of autolysis (not shown). Small phagolysosomes were occasionally seen abutting each other, suggesting fusion to form the large phagolyso somes characteristic of gasoline intoxication. There appeared to be a slight increase in the number and size of endocytotic vesicles in gasoline-treated rats. However, other por tions of the endocytotic apparatus (e.g., mi crovilli) appeared unaffected by administra tion of gasoline. Alterations in other cellular SL 035531 organe to rats Sen. croscc somes treatei dimin lation highls large. 3C). I conta sions younj from some mark the b other line days) alteri phagi ther mien Bioe lei El proh (SD! nize mor neni On t this nate 4) a tein moi pro you gas< h tati of> ora >m 3.5-monthi the cytoplasm size and shape ne-treated rats, ale rats relative Tng each other, large phagolysone intoxication, increase in the otic vesicles in ver, other portratus (e.g., miby administra- other cellular HYDROCARBON-INDUCED HYALINE DROPLET NEPHROPATHY 387 organelles were not apparent in comparison to rats receiving saline treatment only. Senescent rats. As suggested by light mi croscopy (Fig. 2C), the number of phagolyso somes visible with electron microscopy in un treated or saline-treated aged male rats was diminished (Fig. 3C). The constitutive popu lation of phagolysosomes in old animals was highly variable in size and shape with many large, irregular phagolysosomes obvious (Fig. 3C). Frequently these large phagolysosomes contained flocculent electron-dense inclu sions (Fig. 3C) of a type not observed in young control or gasoline-treated rats. Aside from the age-related alteration in phagolyso somes, PCT of 26-month-old rats were marked by thickening and reduplication of the basal laminae. Age-related changes in other organelles were unremarkable. Gaso line administration (0.4 ml/kg/day po, 5 days) to 26-month-old rats caused no obvious alteration in number or morphology of phagolysosomes in PCT epithelial cells at ei ther light microscopic (Fig. 2D) or electron microscopic magnification (Fig. 3D). Biochemical Characteristics ofHyaline Drop lets in Young and Old Male Rats Electrophoretic separation ofrenal cortical proteins. Polyacrylamide gel electrophoresis (SDS-PAGE) of soluble proteins in homoge nized renal cortices from saline-treated 3.5month-old rats showed a prominent compo nent of MT approximately 18 X 103 (Fig. 4). On the basis ofthe dramatic intensification of this protein band in renal cortical homoge nates from gasoline-treated young males (Fig. 4) and apparent molecular weight, this pro tein was presumed to be a2u-globulin. In 26month-old rats no band corresponding to the prominent low-molecular-weight protein of young rats was observed in either saline- or gasoline-treated animals. (Fig. 4). Renal content ofa:,,-glohulin. RIA quanti tation of u2u-globulin content in the kidney of young and aged male rats (Table 2) corrob orated the observations made by SDS-PAGE 12 3 4 UG S UQ S Fig. 4. SDS-PAGE of renal cortical proteins from male rats treated with saline (S) or gasoline (UG) demon strating gasoline-induced accumulation of low-molecular-weight protein only in young adult rats. Lanes 1 and 2, 26-month-old rats; lanes 3 and 4. 3.5-month-old rats. Molecular weight markers are identified by numbers cor responding to A1, X 10_i. The position of ^-globulin is indicated (--). of renal cortical proteins (Fig. 4). Specifically, gasoline administration (0.4 ml/kg/day, 5 days) to 3.5-month-old male rats increased renal 2u-globulin content by 173% when de termined 24 hr after the final dose (Table 2). Larger increases in the content of this protein are possible in young rats at higher gasoline doses (Olson et al., 1987), however, the dose administered was chosen to ensure survival of the old rats. The constitutive level of a2uglobulin in the kidney of 26-month-old rats was only 1.4% of that in young rats (Table 2). The low level of renal 2u-globulin was likely due to the greatly diminished hepatic synthe sis of this protein in aged rats; hepatic 2uglobulin content in 26-month-old rats was 0.4% of that in 3.5-month-old rats. Adminis tration of gasoline to old rats was without effect on the renal or hepatic content of a2uglobulin (Table 2). SL 035532 388 MURTY ET AL. TABLE 2 let Effect of Old Age on Gasoline-Induced Renal Accumulation of o^-Globulin in Male Rats this ere ^-Globulin content nui (Mg/mg total protein) , ---------------------------------------------------------------------------------------------------------- hy; Treatment n Kidney Liver Young adult males (3.5 month) Saline Gasoline Senescent males (26 month) Saline Gasoline 2 2 4 6 36.5(38.0.35.1) 99.5(83.0,115.9) 0.5 0.2 0.5 0.1 1.3 (1.2, 1,4) 1.5 0.7,1.3) 0.005 0.002 0.003 0.000 rat of s|n tin Note. Data are expressed as means SEM of triplicate determinations on each of n animals per treatment group. For groups with n = 2, mean values from triplicate determinations on tissues from each animal appear in parentheses. Details oftreatments and analytical technique under Materials and Methods. Lysosomal peptidase enzyme activity. In young rats the activity of the lysosomal proteolytic enzymes cathepsin D (a carboxyl endopeptidase), and cathepsin B (a thiol endopeptidase) was nearly doubled when assayed 24 hr after discontinuing administration of gasoline (Table 3). Conversely, the activity of acid phosphatase, which acts on phosphoric monoesters such as those in nucleic acids, was unaffected by gasoline. The basal levels of both acid phosphatase and cathepsin B were reduced in saline-treated 26-month-old rats compared to those of similarly treated 3.5- month-old rats (Table 3). In gasoline-treated senescent male rats the activities of renal cortical acid phosphatase and cathepsin B were not different from those of age-matched controls (Table 3). However, cathepsin D activity in gasoline-treated old rats was increased to 232% of the value observed in saline-treated age-matched controls. DISCUSSION Two previously unreported observations relevant to chemically induced hyaline drop- mt fa' ati dr m (F ni 2( in P' 11 o' ci P 0 c g d TABLE 3 Effect of Gasoline Administration on Renal Lysosomal Hydrolase Activities in Young and Aged Male Fischer 344 Rats Lysosomal enzyme activities Treatment Acid phosphatase Cathepsin B n (nmol/min/mg protein) Cathepsin D (umol/min/mg) Young adult males (3.5 month) Saline Gasoline Senescent males (26 month) Saline Gasoline 4 6 4 6 5.5 0.2 5.4 0.1 4.0 0.3** 4.0 0.2 206.6 20.5 336.3 6.4* 61.3 4.6** 64.5 5.7 27.7 0.9 45.3 5.4* 34.5 12.5 80.0 6.6* Note. Data are expressed as means SEM of triplicate determinations on each of n animals per treatment group. Details of treatments and analytical technique under Materials and Methods. * Different from age-matched saline-treated group, p < 0.05 (one-way analysis of variance and Dunnett's test). ** Different from value for saline-treated 3.5-month-old rats, p < 0.05. a I i < t SL 035533 n Male Rats it i) Liver .3(1.2, 1.4) .5(1.7, 1.3) 1.005 0.002 '003 0.000 er treatment group, pear in parentheses. 'asoline-treated ies of renal corthepsin B were '-matched conpsin D activity h increased to i saline-treated d observations i hyaline drop- ITIES Cathepsin D (Mmol/min/mg) 27.7 0.9 45.3 5.4* 34.5 12.5 80.0 6.6* treatment group. Junnett's test). HYDROCARBON-INDUCED HYALINE DROPLET NEPHROPATHY 389 let nephropathy of male rats were made in this study: the spontaneous age-related de crease in PCT epithelial cell hyaline droplet number, and the failure of gasoline to cause hyaline droplet nephropathy in aged male rats. The apparent decrease and eventual loss of spontaneously occurring hyaline droplets which react with methylene blue-basic fuchsin stain (Fig. 1) was unexpected and not en tirely consistent with observations made on tissue prepared with toluidine blue staining (Fig. 2). The methylene blue-basic fushcin method stains proteins nonspecifically; there fore, the nature of the age-dependent alter ations in protein composition of hyaline droplets cannot be determined by this method alone. However, electrophoretic (Fig. 4) and RIA data (Table 2) from the kid neys of saline-treated old rats indicate that by 26 months of age a2u-globulin is absent. This information is consistent with that reported previously by Roy and co-workers (Roy et al., 1983) and suggests that decreased production of 2u-globulin contributes directly to the de crease in hyaline droplet number and age-dependent alteration of the staining properties of the remaining droplets. The precise stoi chiometry between the declining rates of a2uglobulin synthesis and the decrease in hyaline droplet number remains to be defined. After puberty male rats excrete large amounts of protein in the urine (Gray and Purmalis, 1965; Neuhaus and Flory, 1978). Thus, the renal proximal tubules of these ani mals are continually stressed by the require ment to reabsorb and process large quantities of proteins. However, no comprehensive bio chemical characterization of proteins in con stitutive or hydrocarbon-induced hyaline droplets in rats of various ages has been pub lished. Nor have detailed publications on agerelated alterations in renal histology of male rats mentioned changes in the number or characteristics of hyaline droplets (phagoly sosomes) found in PCT epithelial cells of rats older than 12 months (Andrew and Pruett, 1957; Gray el at., 1974; Coleman et al., 1977; Haley and Bulger, 1983). On the other hand, reports of increased glomerular permeability and altered urinary protein composition with increasing age (Gray and Purmalis, 1965; Neuhaus and Flory, 1978) point to the possi bility that hyaline droplet number and com position may change as a function of the pro teins present in the urine. A gradual age-dependent decline in the rate of synthesis of a2u-globulin by the liver can by noted as early as 150 days of age and beyond 750 days he patic synthesis of 2li-globulin is almost unde tectable (Roy et al.. 1983). Furthermore, at 5-7 months of age, the amount of albumin and other large proteins passing the renal glo merular filtration barrier of rats begins to in crease such that, as early as 9 months of age, the relative amount ofa2u-globulin in the uri nary filtrate has been greatly reduced (Neu haus and Flory, 1978). Since renal proximal tubules resorb individual proteins on the ba sis of both ionic properties and mass contri bution to total urinary protein, it seems likely that the majority of resorbed protein in rats at 1 year of age or more is not a2u-globulin. Considering this point and the age-dependent changes reported here, we conclude that the chemical composition of renal hyaline drop lets is altered markedly in the kidneys ofaged rats. a2u-Globulin is the primary urinary pro tein of young adult male rats (Roy and Neu haus, 1966) and the toxic effects of many hy drocarbons are unique to male rats of ages at which a2u-globulin excretion is high (Table 1). 2u*Globulin has also been shown to be a principal component of hyaline droplets ac cumulated during gasoline intoxication in rats about 3.5 months of age (Garg et al.. 1987; Olson et al.. 1987, 1988). Conversely, the failure of gasoline to alter hyaline droplet number in aged male rats (Figs. 2, 3) and the absence of a2u-globulin in kidneys of gaso line-treated old rats (Fig. 4, Table 2) suggest strongly that the presence of this unique pro tein contributes directly to the susceptibility of young postpubescent rats to hyaline drop let nephropathy. Furthermore, the age-re lated differential induction ofrenal lysosomal peptidase enzymes after administration of gasoline (Table 3) suggests that the contents SL 035534 390 MURTY ET AL. of phagolysosomes in gasoline-treated young rats differ from those in old rats. In young rats which are competent to produce (^-globu lin, the accumulation of this protein by renal phagolysosomes in response to gasoline (Fig, 4, Table 2) probably causes a compensatory increase in the activity of cathepsins B and D (Table 3). Substrate-mediated induction of lysosomal proteolytic enzymes is known to occur in many tissues (Helminen et ai, 1968; Salminen, 1985). We have shown previously that specific inhibition of cathepsin B by ad ministration of leupeptin in vivo results in rapid accumulation of a2u-globulin in the kidney of young male rats (Olson et al., 1988). Thus, cathepsin B activity appears to be intimately linked to the renal metabolism of 2u-gIobulin. In contrast, aged rats, which lack 2u'globulin, when treated with gasoline show an increase in only cathepsin D activity. Whether this is due to accumulation of pro teins other than <*2u-globulin is unknown. Therefore, the primary response of the rat kidney to gasoline, i.e., a2u-globulin accumu lation, as well as a secondary or compensa tory response to protein overloading caused by gasoline differs between young and aged male rats. Chronic exposure to several of the hyaline droplet-inducing nephrotoxic hydrocarbons is associated with an increased incidence of renal carcinoma in male rats (National Can cer Institute, 1977; National Toxicology Pro gram, 1983; MacFarland et al., 1984; Na tional Toxicology Program, 1988). In these animals, necrosis of individual cells in the PCT epithelium, perhaps due to phagolysosomal protein overloading, has been ob served following short-term hydrocarbon ex posure (Short et al., 1986, 1987). Repeated rounds of cell death and regeneration are sug gested to contribute to renal carcinogenesis by promoting clonal expansion of a small number of spontaneously occurring premalignant cells, increasing the number of cells becoming premalignant or both (Short et ai, 1987; Gibson and Bus, 1988). Such an indi rect mechanism of tumorigenesis is consis tent with both the failure to demonstrate ge- netic toxicity of hyaline droplet-inducing nephrocarcinogens and the relatively low in cidence with which renal tumors appear in gasoline-exposed male rats (MacFarland et ai, 1984). With respect to the mechanism of renal carcinogenesis just detailed, the failure to in duce hyaline droplet nephropathy in gaso line-treated old animals is especially signifi cant since this suggests that a2irglobulin is an essential component of the hydrocarbon-in duced nephropathic response. Alden and coworkers (1984) have shown previously that prepubertal male rats, which have neither a2u-globulin nor spontaneously occurring hy aline droplets, are resistant to nephropathy caused by Decalin. The findings with senes cent rats, however, extend the prepubertal rat paradigm significantly because we show that even in rats which have a spontaneously oc curring population of hyaline droplets and are presumably albuminuric, the absence of "2u-globulin is sufficient to cause the animals to become refractory to the toxicity of gaso line. Thus, 2u'globulin is essential and neces sary for the occurrence of the acute conse quences of gasoline exposure. Furthermore, if the mechanism proposed to explain gaso line-induced renal carcinogenesis in male rats is operative, these results imply that a2li-globulin is essential for the occurrence of nephrocarcinogenesis during chronic gasoline expo sure. The final extension of this reasoning then suggests that species which lack a2uglobulin, or at least high levels of structurally very similar proteins, will be refractory to both the acute and the chronic effects ofgaso line demonstrated in male rats. ACKNOWLEDG M ENTS The authors are grateful to J, T. Johnson, L. C. Li, A. N. Brady, S. R, Webb, and Dr. K. Smiler, Biomedical Science Department. GM Research Laboratories, for their contributions to this project. We also thank Dr. B. Chatterjee, Department of Cellular and Structural Biol ogy. University of Texas Health Sciences Center, San Antonio, for her cooperation. Some of the research equipment used in radioimmunoassay expenments was provided by funds from N1H Grant DK. 14744. SL 03553s Alden, C. L.C. <1` of volati Effects o C. P. Hr Eds.), pi Princeto Andrew. in the ki 1.51-7` Barrett. tion anc 131,80l Bennet, 1 NEEL.J for ligh methac Techno Bomharl Loesef phropa 433-4? Bruner. animal' terest. (M. A. and N. entific Colema G. W.. logical 344 m. Dodd, E andT male F tyl ket GaRG. F A. K.. accurr rats, t GaRG, F and F locali. gasoli Meeti (G. D San F Gibson lives ` nephi GOLDS' (198" tcin <and I Eroc  droplet-inducing e relatively low intumors appear in ts (MacFarland cl ;chanism of renal 1, the failure to inhropathy in gasoespecially signifi: a2u-globulin >s an e hydrocarbon-inlse. Alden and con previously that rich have neither usly occurring hyt to nephropathy \clings with seneshe prepubertal rat mse we show that pontaneously ocline droplets and , the absence of Imse the animals toxicity of gasosential and neces- the acute consere. Furthermore, to explain gasonesisin male rats ply that <*2u-globrence of nephroic gasoline expo>f this reasoning which lack a2uls of structurally be refractory to ic effects ofgasoits. IENTS Johnson, L. C, Li, Smiler, Biomedical h Laboratories, for 'e also thank Dr. B, ind Structural Bioliences Center, San ne of the research I experiments was ll. 14744. HYDROCARBON-INDUCED hyaline DROPLET NEPHROPATHY 391 REFERENCES '.lden, C. L., Kanerva, R., Ridder, G., and Stone, L. C. (1984). 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