Acute nephropathy induced by gold sodium thiomalate: Alterations in renal heme metabolism and morphology

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

91, l93-203(1987)

Acute Nephropathy Induced by Gold Sodium Thiomalate: Renal Heme Metabolism and Morphology JULIE L. EISEMAN,

Alterations

in

*"JORGE L. RIBAS,?,2E~~~~~~~~~~,*~~~A~~~~~P.A~~~~~~*23

Departments of *Pharmacology and TPathology, Uniformed Services University, Bethesda, Maryland 20814

Received March 9,1987: accepted July 6, 1987 Acute Nephropathy Induced by Gold Sodium Thiomalate: Alterations in Renal Heme Metabolism and Morphology. EISEMAN, J. L., RIBAS, J. L., KNIGHT, E., AND ALVARES, A. P. (1987). Toxicol. Appl. Pharmacol. 91, 193-203. Gold compounds are used clinically in rheumatoid arthritis therapy. Acute renal toxicity is observed in some patients receiving chrysotherapy. The present study addresses morphofimctional and biochemical changes in rat kidneys during the first 8 days following a single ip injection of gold sodium thiomalate (AuTM), one of the gold compounds presently in clinical use. Compared to controls, AuTM pretreatment resulted in increased urine output and elevated serum creatinine and urea nitrogen concentrations. Also, by Day 8, treated rats had decreased body weights and increased kidney weights. Postmortem examination on Day 1 showed pale and mottled kidneys and diffusely pale inner cortex. Microscopically, there was severe coagulative necrosis of the proximal tubular epithelium. Epithelial regeneration was prominent by Day 4 and was nearly complete by Day 8. The regenerating epithelium was hyperplastic with basophilic cytoplasm and pleomorphic nuclei. Alterations in renal heme biosynthesis and drug metabolism paralleled the morphologic changes. The activity of &aminolevulinic acid dehydratase and benzo[a]pyrene hydroxylase were inhibited on Days 1,2, and 4 following AuTM administration. Decreases in monooxygenase activity were accompanied by decreases in renal cytochrome P-450 levels. In contrast, renal microsomal heme oxygenase activity was elevated 9.5-fold on Day 1 and 2.5fold on Day 2. By Day 8, all renal enzymatic activities assayed for were similar to those obtained with untreated rats. o 1987 Academic Press. Inc.

sotherapy is often accompanied by transient proteinuria and, in l-3% of patients, there is also hematuria (see review by Blocka et al., 1986). Silverberg et al. (1970) observed the development of nephrotic syndrome in two out of 75 patients receiving gold therapy. Gold compounds inhibit numerous sullhydryl-containing enzymes (Persillin and Ziff, 1966; Ennis et al., 1968; Westwick et al., 1974) and the inhibition of lysosomal enzymes by gold may in part explain its therapeutic effcacy. Previous studies from our laboratory (E&man and Alvares, 1978) have shown that AuTM, administered acutely to rats, inhibits the sulfhydryl enzymes of the heme biosynthetic pathway, namely &aminolevulinic acid dehydratase and ferrochela-

Chrysotherapy has been used extensively in the treatment of rheumatoid arthritis, a chronic disease of unknown etiology. Gold compounds, primarily gold sodium thiomalate (AuTM) and auranofin, are presently used clinically as antiarthritic drugs. Gold-induced intoxication, which occurs in a significant number of patients receiving chrysotherapy, involves the skin and mucous membranes and, to a lesser degree, the kidney and hematopoietic tissues. Initiation of chry’ Present address: Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore,MD21201. * Present address: Armed Forces Institute of Pathology, Washington, DC 20305. 3 To whom reprint requests should be addressed. 193

004 1-008X/87 $3.00 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved

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tase, in rat liver and kidneys. Further, the gold compound, like other heavy metal compounds (Maines and Kappas, 1976, 1977) decreased the levels of the heme protein cytochrome P-450 and cytochrome P-450-dependent enzymatic activities. In contrast to its inhibitory effects on heme biosynthesis and the functional capacity of cytochrome P-450, AuTM caused 1.5 and 8-fold inductions in the liver and kidney, respectively, of microsomal heme oxygenase activity, the rate-limiting enzyme in heme catabolism (Eiseman and Alvares, 1978). From the studies of Rosenberg et al. (1982) it appears that the ability to induce heme oxygenase is not restricted to the ionic forms of the metal, but can be extended to the organic form of the metals as well. The kidney, aside from being a major excretory organ, is an important site of extrahepatic drug metabolism. In the kidney, cytochrome P-450 content is highest in the cortex and appears to be localized in the epithelial cells of the pars recta of the proximal tubules (Orrenius et al., 1973). This region also appears to be a major target for metal-induced renal damage. The nephrotoxicity of metals, particularly of mercury compounds, is well documented (Maher, 1976). In the first 8 hr following administration of mercury, loss of brush border, dispersion of ribosomes, and formation of clumps of smooth membranes in the cytoplasm of the proximal tubules is observed. The earliest changes appear in the middle and terminal portions of the proximal tubules. Even though the nephrotoxicity of chrysotherapy has been known for several years, knowledge of the mechanisms underlying this toxicity is still lacking. Since the kidney is a target organ for gold deposition and toxicity, the present studies were conducted to compare changes in morphology and heme metabolism over time, following the administration of a single dose of AuTM to male rats. METHODS Male Sprague-Dawley rats weighing 100-I 50 g, purchased from Taconic Farms (Germantown, NY), were

ET AL. used in these studies. Animals were allowed I week to acclimatize to the animal facility prior to study, and food (RMH 3000) and water were provided ad libitum. AuTM, purchased from K & K Chemicals (Plainview, NY), was administered ip as an aqueous solution. Control rats received saline. At the appropriate time after treatment the animals were killed by guillotine. Tissue preparation. A kidney from each rat was excised, blotted, weighed, and homogenized in 1.15% KC1 to a concentration equivalent to 250 mg kidney, wet wt, per milliliter of solution. An aliquot of the homogenate was assayed for &aminolevulinic acid dehydratase activity and the remainder of the homogenate was centrifuged at 9000g for 20 min. The 9000g supematant was subsequently centrifuged at 105,OOOgfor 1 hr to sediment the microsomes. The 105,OOOg supematant was used as a source of biliverdin reductase for the heme oxygenase assay. The microsomal pellet was suspended in 0.1 M NaH2P04-K2HP04 buffer, pH 7.4, such that each milliliter contained microsomes derived from 200 mg wet wt kidney. Enzyme assays. &Aminolevulinic acid dehydratase activity was determined by the micromethod of Granick et al. ( 1972) using 5 gl of tissue homogenate. A millimolar extinction coefficient of 61 mrv-’ cm-r was used to calculate the amount of porphobilinogen formed. Benzo[a]pyrene hydroxylase activity was determined using the postmitochondrial fraction as described previously (E&man and Alvares, 1978) and the product formed was measured by the method of Nebert and Gelboin (1968). Four milligrams of tissue, wet weight, was used in the assay and an incubation time of 10 min was used to maintain the linearity of the reaction rate. Cytochrome P-450 content was determined using microsomes equivalent to 200 mg of tissue, wet wt. The hemeprotein content was assayed by the method of Omura and Sato (1964) using an extinction coefficient of 9 1 mM-’ cm-’ between 450 and 490 nm. Microsomal heme oxygenase activity was determined as described previously (E&man and Alvares, 1978). Bilirubin formed was calculated using an extinction coefficient of 40 mM-’ cm-’ between 468 and 530 nm. Protein content of the various cell fractions was determined by the method of Lowry et al. (195 1), using bovine serum albumin as a standard. Clinical chemistries. Creatinine and urea nitrogen levels were determined in serum and urine. Rats were placed in metabolism cages, and urine was collected for 24-hr periods. The urine volume was recorded and the samples were frozen until assayed. Blood was collected by cardiac puncture at the end of each 24-hr urine collection. Blood samples were stored on ice and allowed to clot. Serum was obtained by centrifugation at 5000 rpm for 5 min. Creatinine and urea nitrogen levels in urine and serum were determined using commercial kits purchased from Sigma Chemical Co. (St. Louis, MO). Creatinine and urea nitrogen clearances were calculated using the formula C = UJJP, where U,and P,are the concen-

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AuTM. On Day 1, body weights of the controls and gold-treated rats were not significantly different from each other. However, on Day 8 the control rats weighing 156 f 3 g 2 were significantly (p < 0.05) heavier than the f gold-treated rats, 134 ? 11 g. Liver weights z 2o were not significantly different throughout I 3 the g-day experimental period. The kidney g weights of AuTM-treated rats were similar to Y 5 IOthose of controls on Day 1; however, by Day 8 the kidney weights of the gold-treated rats were about twofold greater (p < 0.05) than those of the corresponding control rats. The mean kidney weights of the controls and 2 4 6 a AuTM-treated rats were 0.76 and 1.53 g/100 DAYS AFTER TREATMENT g body wt, respectively, on Day 8. FIG. 1. Comparison of 24-hr urine volume from conThe renal functional capacity was assessed trol and AuTM-pretreated rats. Male Sprague-Dawley by determining the urea nitrogen and creatirats were administered AuTM, 75 mg/kg, ip as a single nine concentrations in serum and urine and injection on Day 0. Rats were placed in metabolism cages and urine collected over a 24-hr period was recorded. using the resulting values to calculate their Each value is the mean for at least six rats and represents clearance rates. When compared to control the urine volume collected over the previous 24-hr pe- rats, urine output of AuTM-treated rats was riod. Each asterisk represents a value significantly differsignificantly increased when measured at ent from its respective control value (p < 0.05). Days 1 and 4 following a single dose of the gold compound (Fig. 1). AuTM-treated rats were also uremic throughout the study. Setrations of either creatinine or urea nitrogen, expressed rum creatinine and urea nitrogen levels were as mg/ IO0 ml, in urine and plasma, respectively, and V, consistently elevated (Fig. 2) and creatinine is the urine volume expressed as milliliters per minute and urea nitrogen clearances were concomiper kilogram. 30-

Statistical analysis. The Student t distribution was used as a test of the null hypothesis, using a level of significance ofp equal to or less than 0.05. Histopathology. Kidneys were fixed by immersion in either 10% buffered formalin or in a fixative made of 1% glutaraldehyde and 4% formalin (McDowell and Trump, 1976). Tissues were then dehydrated and embedded in paraffin or epon/araldite. The paraffin sections were cut at 4 or 6 pm on a A0 rotary microtome and the plastic sections were obtained at 1 pm in a Reichert OMU-3 ultramicrotome. Deparaffinized sections were stained with hematoxylin and eosin. Plastic sections were stained with toluidine blue.

RESULTS Male Sprague-Dawley rats were administered 75 mg AuTM/kg, ip. Control rats received saline, 0.2 ml/100 g body wt. Body, liver, and kidney weights were determined on Days 1 and 8 following the single injection of

5.0

-- 100

4.0

-80

I

0

3

E v1 1 I8 3.0

-60

3m M z :: s =: P

s

z 2.0 E

-40

5 25 f

-20

2”

E” 1.0

CDlltNil

DAY 1

DAY 4

DAY 7

FIG. 2. The effect ofAuTM treatment on serum creatinine and urea nitrogen concentrations. Serum creatinine and urea nitrogen concentrations were determined on Days 1, 4, and 7 following a single ip dose of 75 mg AuTM/kg. Each value is the mean + SE for at least six rats. Asterisks represent values significantly different from the control value (p < 0.05).

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tantly decreased (Table 1). Control clearance values were similar to those previously reported for normal untreated rats (Mitruka and Rawnsley, 1977). Creatinine clearance rates were decreased to 27, 40, and 36% of control rates on Days 1,4, and 7, respectively, following a single injection of 75 mg AuTM/ kg. Urea nitrogen clearances (Table 1) in the AuTM-treated rats decreased to 16, 22, and 28% of control values on Days 1, 4, and 7, respectively. To assess the metabolic capacities of kidneys following AuTM pretreatment, rats were administered ip 75 mg AuTM/kg and killed 1,2,4, or 8 days later. Changes in heme metabolism enzymes observed at these time periods are shown in Table 2. The heme biosynthetic pathway enzyme, &aminolevulinic acid dehydratase, was significantly inhibited on Days 2 and 4 by 32 and 5 l%, respectively. Cytochrome P-450 content of kidney microsomes was decreased by 68% on Day 1, by 55% on Day 2, and by 50% on Day 4. Benzo[a]pyrene hydroxylase activity was decreased concomitantly by 43% on Day 1, by 55% on Day 2, and by 32% on Day 4. Microsomal heme oxygenase, the rate-limiting enzyme of heme catabolism, was increased 9.5fold on Day 1 and 2.5-fold on Day 2 following gold administration. However, on Day 4 this activity in AuTM-treated rats was not different from the mean control value. By Day 8, no renal enzymatic activities determined in AuTM-treated rats were significantly different from the control values. In data not shown, pretreatment of rats with sodium thiomalate, instead of AuTM, did not significantly alter renal heme metabolic enzyme activities. The gross photographs of the whole kidney and of longitudinal sections of each kidney obtained from controls and from rats killed on Days 1 and 7 following a single ip injection of AuTM, 75 mg/kg, are shown in Fig. 3. By Day 1, the kidneys were swollen and pale. The hemisected surface disclosed a white-gray coarsely granular inner cortex and a sharply demarcated red medulla. By Day 7,

ET AL. TABLE 1 CHANGES IN CREATININE AND UREA NITROGEN CLEARANCESIN RATS TREATED WITH AuTM Treatment

Day 1

Day I

Day 4

Creatinine clearance (ml/min/kg) Control AuTM

4.10+ 0.25 4.03kO.23

4.35kO.24

1.25+0.16”

1.56kO.20”

1.59kO.35”

Urea nitrogen clearance (ml/min/kg) Control AuTM

5.21 f 0.87 0.81 kO.14”

5.34 f 0.87 1.15+0.11”

4.32 + 0.49 1.20+0.11”

Note.Male Sprague-Dawley rats were administered 75 mg/kg AuTM as a single ip injection on Day 0 and killed on Day I, 4, or 7. Control rats received an equivalent volume of saline. Each value is a mean + SE for at least six rats. ’ Value is significantly different from the control value (p-cO.05).

the kidney remained enlarged but the hemisected surface was of normal color. Photomicrographs of the kidney cortex from an untreated rat and a rat killed 2 days after a single injection of AuTM are shown in Figs. 4 and 5, respectively. On Day 2, throughout the inner cortex there is acute coagulation necrosis of the epithelium with the major damage localized to the third segment of the proximal convoluted tubules. Coagulative necrosis is also present in a few proximal tubules in the outer cortex. The necrotic cells have a glassy homogeneous appearance and an intensely eosinophilic cytoplasm, and most of the nuclei have disappeared. In some areas, sloughing of necrotic epithelium has left the basement membrane denuded. Macrophages and other mononuclear cells are observed in the peritubular interstitium. Some of the distal tubules contain casts. By Day 4 the damage is apparent in all segments of the proximal tubules and the disintegration of the necrotic epithelium continues, exposing the basement membrane of more tubules (Fig. 6A). In other areas, there is a regeneration of the tubular epithelium with occasional mitotic figures. The regenerating epithelial cells are irregular and flattened and

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TABLE 2 CHANGES IN HEME METABOLISM ENZYMES IN KIDNEYS OF AuTM-TREATED

RATS

Days following treatment Assay

Day 0

Day 1

Day 2

Day 4

Day 8

ALA dehydratase (nmol PBG/mg protein/hr)

0.937 I!I 0.105

0.700 Zro.190

0.634” kO.155

0.456” kO.115

0.690 I? 0.140

Cytochrome P-450 (nmol/mg protein)

0.044 f 0.004

0.014” * 0.002

0.020” f 0.006

0.022” + 0.003

0.044 xk0.028

Benzo[a]pyrene hydroxylase (nmol OHBP/mg protein/hr)

0.402 -c 0.03 1

0.231” f 0.043

0.182” f 0.018

0.273” iz 0.010

0.29 1 + 0.025

Heme oxygenase (nmol bilirubin/mg

0.301 f 0.025

3.196” f 0.589

0.643” + 0.047

0.262 ?I 0.04 1

0.301 + 0.015

protein/hr)

Note. AuTM, 75 m&kg, was administered on Day 0 to male Sprague-Dawley rats. Rats were killed on Day 1,2,4, or 8, and kidneys were removed and assayed for the various enzyme activities as described under Methods. Each value is the mean f SE of at least three rats. ALA, &aminolevulinic acid, PBG, porphobilinogen: OHBP, hydroxybenzo[a]pyrene. ’ Value significantly different from its respective control value (p < 0.05).

have basophilic cytoplasm, pleomorphic nuclei, and prominent nucleoli. Their cytoplasm insinuates between the necrotic debris and the basement membrane. Many of the regenerating tubules are dilated and lined by squamous to low columnar epithelium (Fig. 6B). There is dystrophic calcification in a few of the proximal tubules, and occasional distal tubules are occluded with necrotic debris or tubular casts. Peritubular capillaries are moderately congested. The interstitial inflammatory cell infiltrate is still present around tubules and capillaries, and now there is a mild multifocal increase in fibroblasts but no collagen deposition. By Day 8 (Fig. 7) most proximal tubules have regenerated. However, unlike the epithelium of the proximal convoluted tubule of control kidneys (Fig. 4) the regenerated cells are crowded together with many cells exfoliating into the lumen. Tubular casts are multifocally present in distal tubules. A moderate monocytic cell infiltrate and a mild fibroblastic proliferation persists in the interstitium. DISCUSSION AuTM and auranofin are gold compounds which appear to be efficacious in the treat-

ment of rheumatoid arthritis. Clinically, auranofin is administered orally, whereas AuTM is administered intramuscularly. Although the pharmacokinetics and tissue distribution of the two gold compounds show marked differences (Walz et al., 1982), recent studies demonstrate that when these two compounds are administered acutely to rats they have similar effects on heme metabolism (E&man and Alvares, 1978; Gondal et al., 1987). A toxic manifestation of gold therapy in humans is renal damage, manifested as proteinuria. Albuminuria is more commonly seen, but the nephrotic syndrome and glomerulitis do occasionally occur. The intracellular distribution of gold has been reported after AuTM injection (Penneys et al., 1976). In rat kidney, gold concentrates primarily in the mitochondria of the proximal tubule cell, while in rat liver it predominates in the lysosomal fraction. Following the acute administration of AuTM, dose-response studies have shown decreases in liver cytochrome P-450 and ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities with maximal decreases occurring in rats treated with 75 mg AuTM/kg (Gondal et al., 1987), the dose used in the present studies. Changes in heme

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ET AL.

Control

1 Day

7 Days

FIG. 3. Gross longitudinal sections of the whole kidney from untreated rats and AuTM-treated rats. AuTM, 75 mg/kg, was administered ip as a single injection and rats were killed 1 and 7 days later. Kidneys were removed and the right kidney was bisected longitudinally. The right side of each panel shows the gross appearance of the left kidney.

metabolism were of the same magnitude whether AuTM was administered by the im route or by the ip route. In the present studies, a time-course study of the effects of a sin-

gle dose of AuTM on heme metabolism showed that the sulfhydryl enzyme, &aminolevulinic acid dehydratase, was significantly decreased up to 4 days following drug admin-

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FIG.

4. Photomicrograph

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199

of kidney cortex from an untreated rat. H & E stain: X200; paraffin section.

&ration. This inhibition paralleled the significant decreases in renal cytochrome P-450 and aryl hydrocarbon hydroxylase activity. By Day 8, these enzyme activities were not significantly different from the control values. The well-known effect of heavy metals on heme oxygenase (Maines and Kappas, 1976, 1977) was observed in the kidney microsomes of the gold-treated rats. However, the time course of the induction of this enzyme of heme catabolism did not parallel the inhibitory effects of AuTM on heme biosynthesis. A 9.5-fold increase was observed 1 day following AuTM administration, and a 2.5 fold increase in heme oxygenase was observed on Day 2. The renal heme oxygenase activity returned to control values by Days 4 and 8 following AuTM injection. These

differences in time-course studies demonstrate that the regulation of heme metabolism is only partly controlled at the level of heme oxygenase, the rate-limiting enzyme in the heme degradative pathway. The heavy metal nephropathy produced by several metals such as cadmium, lead, tin, and mercury was also observed in the kidneys of rats treated with a single ip dose of AuTM. Severe coagulative necrosis of the proximal tubular epithelium was apparent as early as 24 hr after the administration of gold and was more prominent at the cortico-medullary junction. By Day 4 regeneration of the epithelial cells with occasional mitotic figures was observed. The regenerating epithelium of the proximal tubules showed pleomorphic nuclei and prominent nucleoli. The morpho-

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ET AL.

FIG. 5. Photomicrograph of a kidney cortex from a rat killed 2 days following a single injection of AuTM. There is coagulation necrosis of the epithelial cells of the proximal convoluted tubules. The distal convoluted tubular epithelium and the glomerulus are not affected. A few inflammatory cells are in the interstitium. Toluidine blue; plastic section; X250.

logic findings during the first 4 days following AuTM pretreatment are similar to those reported by Payne and Rhodes ( 1978). By Day 8, the proximal tubular epithelium was almost completely regenerated but a few mitotic figures were still observed. Renal function, as measured by creatinine and urea nitrogen clearance rates, was compromised and the rats showed polyutia. At all time periods studied, namely 1,4, and 7 days following the administration of AuTM, the serum levels of creatinine and urea nitrogen were significantly higher than the levels of those compounds in control rats. Although renal heme and drug metabolism was nearly

normal and tubular regeneration was almost complete by Day 8, the functional capacity of the kidney was partially restored, as evidenced by the normal urine output and declining serum creatinine and urea nitrogen levels. Since the latter parameters were measured on Day 7 instead of Day 8, it is possible that they may not accurately represent functional restoration by Day 8. Ultrastructural studies are underway to determine if glomerular lesions are induced by a single dose of AuTM. The twofold increase in renal weight observed on Day 8 may reflect the diffuse epithelial hyperplasia of regenerating tubules seen also on Day 8. Additionally, it may also

FIG. 6. Photomicrograph of kidney cortex from rat killed 4 days after a single injection of AuTM. (A) Regenerated epithelium lining proximal tubules has pleomorphic nuclei and prominent nucleoli. In other tubules, the necrotic epithelium has sloughed off leaving naked but intact basement membranes. (B) Regenerating tubules are lined by squamous to low cuboidai epithelium. Tubular casts and necrotic debris are in the htmina of distal and convoluted tubules. Toluidine blue; plastic section. (A) X250, (B) X630. 0”

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ET AL.

FIG. 7. Photomicrograph of kidney cortex from rat killed 8 days after a single injection of AuTM. The epithelial lining of the proximal tubules is crowded with regenerated cells, some of which have exfoliated into the lumen. There is moderate peritubular and perivascular inflammation. Toluidine blue; plastic section; X250.

reflect an expansion of the interstitium brought about by a mild but diffuse inflammatory cell infiltrate and by interstitial edema. In summary, this study describes the time course of morphofunctional changes in the kidneys after the administration of a single dose of AuTM. The dose dependency of this nephropathy has not been experimentally demonstrated, although in clinical practice nephrotoxicity is clearly dose dependent. Since acute nephrotoxicity was also observed after a single dose of 192 mg AuTM/kg (Payne and Rhodes, 1978), it would be of interest to determine if similar morphological

and biochemical changes are observed after administration of dosages of AuTM below 75 mg/kg. Further, it would be of interest to examine the effects of auranofin on renal morphology and biochemical functions, since this new orally administered gold compound differs from AuTM in both its physical and pharmacokinetic properties. ACKNOWLEDGMENTS The authors thank Jeane McWilliams for her secretarial assistance in the preparation of this manuscript. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official

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GOLD-INDUCED

or reflecting the views of the U.S. government. The experiments reported herein were conducted according to the principles set forth in the Guide to the Care and the Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Publication (NIH) 85-23. These studies were supported in part by a grant from the National Institutes of Health AM 34696.

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