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Strain-dependent renal toxicity of a nonsteroid antiinflammatory agent
TOXICOLOGY
AND APPLIED PHARMACOLCKiY19,
Strain-Dependent
D. L. BOKELMAN,
ill-124(1971)
Renal Toxicity of a Nonsteroid Antiinflammatory Agent W. J. BAGDON, P. A. MATTIS,
AND P. F. STONIER
Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486 Received October 29. 1970
Strain-DependentRenal Toxicity of a Nonsteroid Antiinflammatory Agent. BOKELMAN, D.L., BAGDON, W.J., MATTIS, P.A.,and STONIER, P.F. (1971), Toxicol. Appl. Pharmacol. 19, 111-124. Manor Farms SPF and CharlesRiver CD rats were given a nonsteroid antiinflammatory agent (1-p-chlorobenzylidine-5-methoxy-2-methyl-3-indenea~ticacid) po, at doselevelsof 3, 6, or 12mg,/kg/day,for periodsup to 29 wk. Hematuria wasobservedin all groups of male Manor Farms SPF rats, both control and drug-treated. The incidenceof hematuria was dose-dependent,and occurred 6, 12, or 24 timesmore often in male rats receiving 3, 6, or 12 mg/kg/day than in the controls. In contrast, hematuriawasnot observed in any control femalesand was infrequent in drug-treated females. The hematuriaobservedin the Manor FarmsSPFrats wasdue to lesions in the renal papilla. The initial lesionconsistedof focal hyperplasiaof the epitheliumcovering the renal papilla. Singlesmallfoci progressedto large, multiple, polypoid hyperplastic lesions. Areas of necrosis frequently observed within hyperplastic epithelium led to hemorrhage from the underlying vasarectae. Basically the lesionswere identical in the control and treated rats, but were more severein the latter. In contrast, renal lesionswere not observedin untreated CharlesRiver CD rats of either sex. When this strain of rat was treated with the same compoundat the samedoses,hematuriawasrarely seen,and no lesionsof the renal papillary epithelium were found. On the other hand, very slight interstitial edemaand necrosiswere observedin the interior of the papilla of a few drug-treatedrats. Theseobservationsdemonstratea type of problemthat can arisein the safety evaluation of a compounddue to strain-specificlesions. Lesions of the renal papilla have been produced experimentally by a variety of compounds, such as vinylamine (Levaditi, 1901; Mandel and Popper, 1951), tetrahydroquinoleine (Rehns, 1901), bromethylamine hydrobromide (Fuwa and Waugh, 1967), mono-N-methylaniline (White and Mori-Chavez, 1952), phenacetin (Fordham et al., 1965), mefenamic acid (Parke Davis Co., January, 1967), and human serum (Klavins et al., 1963; Patrick et al., 1964; Wizgird et al., 1965; Kroe and Klavins, 1965; Ljungqvist and Richardson, 1966; Gullbring et al., 1966). Other methods of producing lesions of the renal papilla have included fat-free diet (Borland and Jackson, 1931), ureteral ligation with or without bacterial infection (Mallory et al., 1940; Muirhead et al., 1950; Sheehan and Davis, 1959), and obstruction of the renal vein (Bestwick and Schatzki, 1960). 111
112
BOKELMAN
ET
AL.
The lesion most commonly produced by these various experimental procedures was necrosis of the renal papilla or associated changes, such as interstitial edema of the papilla. The incidence and severity of lesions have been variable, giving rise to numerous hypotheses to explain the pathogenesis of these lesions. Routine safety evaluation studies of a nonsteroid antiinflammatory agent in Holtzman rats for 4 wk and in Charles River CD strain rats for 16 wk showed no signs of renal toxicity. In a longer-term study, begun later in Manor Farms SPF rats, a low incidence of hematuria was noted 3 wk after the start of the study. The incidence of the hematuria was drug-related, but was noted also in a few control rats. The hematuria was traced to a series of lesions in the renal papilla. The present study was initiated to confirm these findings in another group of Manor Farms SPF rats, and also to investigate the responseof another strain of rats (Charles River CD) to this compound after prolonged treatment. This study points out the differences in responseof two different strains of rats to the samecompound and how the differences may influence the interpretation of an experiment. The study also illustrates a unique spontaneouslesion of the renal papilla of the Manor Farms SPF rat and how this condition was aggravated by the administration of the test compound. METHODS
Albino rats of two different strains, Manor Farms SPF’ and Charles River CD2, approximately 5-6 wk old, were given 1-p-chlorobenzylidine-5-methoxy-2-methyl3-indeneacetic acid, a nonsteroid antiinflammatory compound, the structure of which is shown in Fig. 1. CH,CO,H
C”3
CH
I-P-CHLOROBENZYLIDINE-5METHYL - 3- INDENEACETIC FIG.
METHOXY-PACID
1. I-p-Chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic
acid.
The compound was given at the following approximate dose levels: 0 (control), 3, 6, or 12mg/kg/day. Twenty malesand 20 femaleswere included in each dosegroup in the Manor Farms SPF series,and 10 malesand 10 femalesmade up each dosegroup in the Charles River CD series.The drug was mixed in the food (milled Wayne Lab Rat Blox), and its concentration was adjusted periodically according to the results of food consumption and body weight measurements.Food consumption was determined weekly on 5 male and 5 female rats from each group. All surviving Manor Farms SPF animals at a doseof 3 or 6 mg/kg/day were treated for 17wk. Other rats in this experiment, given * Manor Farms Research Inc., Stattsburg, New York. 2 The Charles River Breeding Laboratories, Inc., North Wilmington,
Massachusetts.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
113
RATS
12 mg/kg/day, were treated for 20 wk, and then treatment was suspended for 4 wk followed by an additional 5 wk treatment period. Control rats of this strain were continued on test with the high dose group animals. All Charles River CD rats were treated continuously for 23 wk. Each rat was individually caged in air-conditioned quarters with food and water available at all times. All rats were selected for physical soundness and normal behavior and placed into groups so that average weights of groups by sex were similar. Physical signs were noted daily and weights were recorded weekly. Urine samples were obtained periodically by placing the animals in metabolism cages and the urine sediment was examined microscopically after centrifugation. Urine samples were collected from the Manor Farms SPF rats as follows : from 10 males and 10 females of each group in the 8th wk, from all surviving rats in the 12th wk, and from all surviving control and high dosage group animals weekly from the 19th through the 28th wk. Urine was examined from all Charles River CD rats three times prior to the experiment and during weeks 2,3,4,6,8,10,13,15, and 23 of the study. Urine of selected rats from both series showing hematuria was also examined at various additional times during the experiments. Hematologic, biochemical, and ophthalmic examinations were made at various intervals. All rats were necropsied, and sections of the various organs were fixed in 10 % aqueous formalin. Both kidneys of all animals were cut sagittally, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Semiserial sections were made of the kidneys of selected animals. Selected tissue sections were also stained with Masson (Goldner-Foot) Trichrome and Alcian Blue-periodic acid Schiff. RESULTS
Manor Farms SPF Rats Urinalysis. Grossly visible hematuria was first seenin the 3rd wk in 2 male rats receiving 12mg/kg/day. From this time on, the incidence of hematuria in the treated rats increased and the results of the urine examinations in the 8th and 12th wk are shown in Table 1. Blood was present in the urine of 2 control malesat 8 wk and in 1 control male TABLE 1 INCIDENCE OF HEMATURIAIN MANOR FARMS SPF RATSDURINGTHE 8TH AND 12TH WKS
Female0
Male”
Treatment bdWday)
8 wk
12 wk
8 wk
12 wk o/20 o/20 2120 o/20
Control
2/10
l/18
o/10
3 6 12
3110 4/10 5/10
8/20*b 8/20* 10/18**
O/l0 l/l0 o/10
a Number of animals with 10 or more erythrocytes per high power field/total animals examined. b Values marked with asterisks differ significantly from control values: *Pio.o5;**P
114
BOKELMAN
ET AL.
at 12 wk, but not in female controls at either period. The incidence of hematuria was significantly increased in the treated males at all dose levels, especially during the 12th wk, when 40% or more of all treated animals were affected. Hematuria was evident in only 1 female at 8 wk and 2 females at 12 wk. In each case, the animals were in the 6 mg/kg/day dose group. The incidence of grossly visible hematuria for the first 17 wk of the study was dosedependent (Table 2) in the male rats, occurring 6, 12, and 24 times more often in rats given 3,6, or 12 mg/kg/day than in the controls. No hematuria was evident in the control females and the incidence was low in the treated females. TABLE INCIDENCE
2
OF MACROSCOPIC HEMATURIA IN MANOR FARMS
DURING
No. of rats with hematuria Treatment b-WWday) Control 3 6 12
THE FIRST
17 WIG
SPF RATS
Male
Female
Incidence (days) of hematuria -___ Male Female
6
0
11 13
1 5
13 78 152
19
2
318
0 3 16 8
During the 21st wk, the surviving rats in the high dose and control groups were removed from drug and no drug was given again until the 25th wk. Urine samplesof these rats were examined weekly from the 19th through the 28th wk. The results of thesestudies (Table 3) indicated that the incidence of hematuria decreasedin the drugtreated male rats during the period of drug withdrawal, and did not significantly increase again when drug administration was reinstituted. TABLE RESULTS
OF URINE
STUDIES
3
PERFORMED FROM (MANOR FARMS
THE 19~~ WK SPF RATS)
THROUGH
THE 28TH
WK
Number of animalswith 10 or more erythrocytes/HPF/total animals No drug Treatment (mg/kg/day) Weeks: 19 Control $ 126 Control 9 12?
20
21
22
23
24
25
26
27
28
l/18 O/l8 O/18 2/18 l/l8 l/l8 O/l8 2/18 O/l7 O/18 S/16 7/16 2/16 3/16 3/16 4/16 4/16 6/16 5/16 2/11 o/20 o/20
o/20 l/20
o/20 o/20
o/20 o/20
o/20 o/20
o/20 l/20
o/20 l/20
o/20 l/20
o/20 2120
o/20 l/20
The duration of hematuria in individual rats varied from 1 day to several weeksbut was usually intermittent. Hematologic changes,body weight differences or changesin food consumption were not evident.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
115
Histologic observations. Focal hyperplasia of the epithelium of the renal papilla was first observed in 1 of 2 control male rats killed after 8 wk because of hematuria. This lesion consisted of a few well-localized foci of rounded polyhedral cells, 3-8 cells thick (Fig. 2). Two male rats in the 12 mg/kg/day dosage group were also killed for the same reason at this time and focal hyperplasia of the epithelium of the papilla was also evident. The lesion in these treated rats was identical to that found in the controls, except that areas of necrosis were evident in the hyperplastic epithelium (Fig. 3). Necrosis extended from the surface of the epithelium into the underlying vasa rectae causing hemorrhage into the pelvis. More advanced hyperplasia was evident in a high dose male rat sacrificed after 12 wk. The hyperplasia in this instance was polypoid, consisting principally of a central core of connective tissue covered by hyperplastic epithelium projecting into the renal pelvis (Figs. 4 and 5). Lesions occurred singly, but more often were multiple, forming clusters. Dilated collecting ducts and blood vessels were also frequently present below the hyperplastic epithelium. Lesions occurred most often near the junction (fornix) of the papillary and pelvic epithelium. The changes found in the kidneys of animals receiving 3 and 6 mg/kg/day and killed after 16 wk of drug administration are summarized in Table 4. Varying degrees of focal hyperplasia of the renal papillary epithelium occurred in approximately half the males in both groups, and foci of necrosis and hemorrhage accompanied the hyperplasia in about half of those affected. Lesions again were most prevalent near the fornix of the pelvis. Grading of the lesions was arbitrary (trace to marked), depending upon the number of lesions and the amount of the papillary epithelium affected. Both isolated low-grade (trace to very slight) foci of hyperplastic epithelium and polypoid hyperplasia (usually very slight or greater) were evident. In all kidneys where hyperplasia was severe it was polypoid. Scars were evident in the subepithelial areas of hyperplastic foci in many instances. These were undoubtedly sites of previous necrosis and hemorrhage which had healed. Mineralization of scar tissue was also commonly found. Inflammatory cell infiltration was not commonly associated with the lesions and was never pronounced. The severity of these changes was similar in males of both treatment groups, but females of both groups were markedly less affected. Trace amounts of epithelial hyperplasia were evident in the papilla of one low dose female animal, and similarly, traces of hyperplasia were found in the papilla of 6 females in the 6 mg/kg/day dose group. The incidence of necrosis and hemorrhage in the hyperplastic areas was also considerably less in females; only traces of each were found in 1 rat in the 6 mg/kg/day dose group. Scarring of the renal papilla was not evident in female rats. Trace amounts of focal hyperplasia of the renal papillary epithelium were evident in 2 of 18 male and 3 of 20 female control rats after 29 wk (Table 5). A tiny scar was also evident in the subepithelial area of the epithelium of one of the affected males. A higher incidence of hyperplasia was evident in the treated animals, especially the males. Hyperplasia of the renal papillary epithelium was evident in 6 of 12 high dose males and in 7 of 20 high dose females. The severity of this change in high dose males was also much greater than in the control males and also significantly greater than that seen in the high dose females. Marked hyperplasia of the renal papillary epithelium was found in 1 high dose female, and a large calculus (magnesium, ammonium, and phosphate ions) and pyelonephritis were also present.
116
BOKELMAN
ETAL.
FIG. 2. Focus of hyperplasia in epithelium covering the renal papilla of an untreated Manor Farms SPF rat. Sacrificed after 8 wks because of hematuria. Hematoxylin and eosin. :<100. FIG. 3. Focus of hyperplasia in epithelium covering the renal papilla with necrosis and hemorrhage. From a Manor Farms SPF rat sacrificed because of hematuria after 8 wks treatment, 12 mg/kg/day. Hematoxylin and eosin. x40.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
FIG. 4. Polypoid hyperplasia of epithelium covering the renal papilla of a Manor Farms SPF male rat treated for 12 wks with 12 mg/kg/day. Hematoxylin and eosin. ~40. FIG. 5. Pronounced polypoid hyperplasia of epithelium covering the renal papilla of a Manor Farms SPF rat treated with 12 mg/kg/day for 12 wks. Note dilated ducts and necrosis and debris in stroma below epithelium. Hematoxylin and eosin. x40.
14
10
9 19
8 9
12 12
16 17 6 13
18
20 12 20
0
1 (0-I)
0
1 (01.)
0
0
6 (O+-++)
4 (0-l -+)
0 5 (O+-++) 2 (+-++>
1 CO+)
Scarring
OF TREATMENT
Hemorrhage
3 CO+) 6 CO+-++ ) 7 (O+-i--t-ti-)b
Necrosis
2 CO+)
Hyperplasia
PAPILLARY EPITHELIUM OF RATS SACRIFICED (MANOR FARMS SPF)
Normal
IN THE RENAL
No. rats examined
OF LESIONS
’ Symbols: Of = trace; + = very slight; +-+ = slight; +++ = moderate; ++++ = marked. ’ Calculus in pelvis and pyelonephritis in animal with ++++, others 0+ or +.
8 ?
Control Control
SEVERITY
Sex
AND
Treatment (mdkdday)
INCIDENCE
TABLE 5
= marked.
AFTER 29 WKS
6 (Oi--++) 1 co+)
1 @+I
6 CO+-++)
6 CO+)
5 (0+---t+)
5 (O+-++)
Scarring
16 WKS OF TREATMENT
Hemorrhage
AFTER
11 (O+-++) 1 (Oi-) 10 (0+-i-+)
SACRIFICED
Necrosis
OF RATS
Hyperplasia
’ Symbols: Of = trace; + = very slight; ++ = slight; +++ = moderate; ++++
20 20 20 20
8 ? d ?
3 3 6 6
4
PAPILLARY EPITHELIUM (MANOR FARMS SPF)
Normal
IN THE RENAL
No. rats examined
OF LESIONS
Sex
AND SEVERITY
Treatment bx/kdday)
INCIDENCE
TABLE
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
119
The degree of hyperplasia in other affected females was very slight in 1 and trace in 5 others. Foci of necrosis and hemorrhage were found in I high dose male, but were not seen in either of the control groups or females in the high dose group. Areas of scarring in the edges of the papilla adjacent to the hyperplastic epithelium were found in all but one affected high dose male and in 2 of 7 affected females in this group. All the lesions of the papilla, i.e., hyperplasia, necrosis, hemorrhage, and scarring in the most severely affected individuals were bilateral, but were very frequently unilateral in the less severely affected animals. Both kidneys were also not always affected to the same degree. The overall incidence of renal papillary lesions in male rats is summarized in Table 6. Blood-tinged urine was found in the urinary bladder of 6 high dose rats sacrificed after 8-27 wk of treatment, but was not present in any other animals. The usual incidence of chronic nephritis was observed equally in the control and treated rats and was not considered to bear any relationship to the lesions described. One rat in the high dose group died of diffuse peritonitis resulting from a perforated small bowel ulcer, but no other changes attributable to treatment were observed in any other organs in any of the animals. Charles Riaer CD Rats Urinalysis. Slight hematuria was grossly visible during the study in 7 drug-treated males: 4 receiving the low dose, 1 receiving the middle dose, and 2 receiving the high dose. Hematuria was not observed more than 4 times during the study in male rats, except in I low dose rat in which it was frequently observed from the 18th through the 20th wk. One high dose female had hematuria once in the 10th wk. The incidence of urine samples with 10 or more erythrocytes per high power field was very low. Three rats had a large number of erythrocytes in the urine; 1 high dose female had 13-40 cells per high power field during the pretest observations, 1 high dose male had 70-80 cells per high power field during the 8th wk of drug administration, and 1 low dose male had 20-30 cells per high power field. Histologic obsermtions. Microscopic changes attributable to treatment were observed in the kidneys of rats in the 3 and 12 mg/kg/day dose groups, but no changes were evident in the kidneys of animals in the 6 mg/kg/day dose group or controls. Focal interstitial edema of the renal papilla was observed in 5 ratsin the high dosegroup and 1 animal in the low dose group. This was characterized by minimal accumulation of finely granular eosinophilic material in the interstitium and was always confined to the central part of the papilla near the tip, but never bordering on the papillary epithelium. Interstitial edema of the papilla accompanied by focal necrosis of the papilla was found in 2 rats in the high dose group and papillary necrosis alone was evident in another high dose animal (Figs. 6 and 7). The location of necrosis was exactly the same as the interstitial edema described earlier. The papillary epithelium was never involved. The cells of the necrotic tubules were absent or ill-defined, and if nuclei were visible they were pyknotic. Empty spaces were found where many tubules were necrotic. In some instances, the collecting ducts were slightly dilated proximal to the lesion in the papilla. Trace amounts of hemorrhage at the site of edema or necrosis were also present in 2 of the high dose animals. A very small amount of blood was evident in the renal pelvis of 1 high dose animal, but no kidney lesions were evident in this rat.
BOKELMAN
ET AL.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
121
FIGS . 6 and 7. Focal interstitial edema with minimal necrosis of the interior of the renal papi lla lofa Charle s River CD rat treated for 24 wk with 12 mg/kg/day. Hematoxylin and eosin. Fig. 6: x40; Fi lg. 7: x100.
The:se lesions were usually unilateral and focal. Inflammatory cells were not associated with this lesion and thrombosis of regional vesselswas not evident. Other PIarts of the nephron were not affected.
122
BOKELMAN
ET
AL.
DISCUSSION
The pathologic effects of I-p-chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic acid on the kidneys of 2 different strains of rats were manifested in 2 different ways. In both instances, lesions were confined to the papilla. In one strain, Manor Farms SPF, lesions were present in the epithelium covering the papilla; in the other strain, Charles River CD, the changes were evident only in the inner zone of the papilla near the tip. A few control rats of the Manor Farms SPF strain developed an intermittent hematuria early in the study, but hematuria of a greater incidence and severity was found in animals treated with the drug. As the experiment progressed, the incidence of hematuria in Manor Farms SPF control animals remained at a very low incidence and was extremely sporadic. On the other hand, the incidence and duration of hematuria in treated males of this strain increased steadily and was dose related. The lack of hematuria in control females and the low incidence of hematuria in treated females was also significant. The hematuria evident in the Manor Farms SPF animals was directly attributable to lesions of the epithelium of the renal papilla. These observations suggest that the Manor Farms SPF animals developed spontaneous renal papillary lesions of undetermined etiology and that the administration of l-p-chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic acid increased the incidence and severity of this change. The renal papillary changes observed in both control and treated Manor Farms SPF animals in the early part of the experiment were identical. The earliest change observed was the occurrence of focal areas of hyperplasia of the renal papillary epithelium, most often located near the fornix. Tiny foci of necrosis occurred within these hyperplastic areas and eroded into the vulnerable underlying vasa rectae causing hemorrhage into the pelvis. These lesions subsequently healed, and the necrotic tissue was replaced by fibrous scar tissue, and hematuria no longer occurred from that site. The intermittence of the hematuria may be explained by the healing of these lesions followed by necrosis and hemorrhage at another site in the hyperplastic epithelium. The incidence and severity of the hyperplasia in drug-treated animals, especially males, increased markedly during the experiment. The polypoid type of epithelial hyperplasia became more evident and in some instances the pelvis was studded with these projections. As the degree of hyperplasia increased, the incidence of necrosis and hemorrhage did also. The cause of the renal papillary lesions in the control animals is unknown. Changes of this nature have never been observed in control rats in our laboratory before. Our experience with the Manor Farms SPF strain of rats was, however, limited to experiments with this compound. Very tiny insignificant foci of hyperplasia, 2-3 cells thick, have occasionally been evident in the epithelium of the papilla of control rats of other strains used by us, but in no instances have they progressed in severity or led to necrosis and hemorrhage as was observed in this study. The reason for the increased incidence and severity of renal lesions in treated rats is not known. Metabolism experiments (unpublished data) in rats have demonstrated that the compound is well absorbed and the major route of excretion is in the feces. Tissue distribution studies (unpublished data) have likewise demonstrated that the concentration of the drug in the kidneys is “low.” The increased incidence of the condition in the treated animals may be related to the potent antiinflammatory activity of the
STRAIN-DEPENDENT RENAL TOXICITY IN RATS
123
compound, but this has not been investigated. The lesion was not due to precipitation of crystals in the urine since these were never evident in microscopic observations of urine sediment. White and Mori-Chavez (1952) described similar changes in the epithelium of the renal papilla of rats after oral administration of mono-N-methylaniline. However, in their experiments the primary renal lesion was hyaline thrombosis of smaller vessels resulting in necrosis of the entire tip of the papilla. In addition, they noted marked edema and mucoid degeneration of the interstitium of the papilla. In Manor Farms SPF rats, lesions were evident only in the surface epithelium of the papilla and its immediately adjacent tissue. Vascular lesions were not evident in these animals. Hyperplasia of the pelvic epithelium was also described in rats given a fat-free diet (Borland and Jackson, 1931). The diet was not balanced, however, and deficiencies of one or several vitamins may have contributed to this lesion. Lesions noted in Charles River CD animals coincide more with those described in the literature as renal papillary necrosis, but the pathogenesis of this change is equally obscure. Vinylamine (Levaditi, 1901; Mandel and Popper, 1951) and tetrahydroquinoleine (Rehns, 1901) have caused papillary necrosis in animals due to thrombosis of the vasa rectae. Extensive papillary necrosis occurred in rats after injection of human serum (Patrick et al., 1964), but not bovine or rabbit serum, suggesting an antigenantibody reaction. Fuwa and Waugh (1968) pointed out that localization of the necrosis in the inner medulla and papilla (similar to our observations in Charles River CD rats) of rats fed 2-bromethylamine hydrobromide (BEA) corresponded with the area of maximal osmolality as demonstrated by micropuncture studies conducted by Gottschalk and Mylle (1959). In their experiments with rats fed BEA, necrosis was prevented or minimized by diuresis whereas it was enhanced by antidiuresis. It is obvious from various conclusions of different investigators that a number of factors acting together or independently may cause lesions in the renal papilla. This is further complicated by the fact that in the present series of experiments the same compound produced different lesions in the papilla of two different strains of rats and, furthermore, that the renal papillary changes produced in one of these strains was more profound in males than females. REFERENCES BESTWICK, I. P., and SCHATZKI, P. F. (1960). Experimental renal papillary necrosis. Arch. Pathol. 69, 733-739. BORLAND, V. G., and JACKSON, C. M. (1931). Effects of fat-free diet on the structure of the kidney in rats. Arch. Path& 11, 687-708. FORDHAM,C. C., III, HUFFINES, W. D., and WELT,L. G. (1965).Phenacetin-inducedrenal diseasein rats. Ann. Intern. Med. 62, 738-743. FUWA, M., and WAUGH, D. (1967). Experimental renal papillary necrosis.Production by venousstasisand by toxic amines.Fed.Proc. Fed. Amer. Sot. Exp. Biol. 26, 517. FUWA, M., and WAUGH,D. (1968).Experimentalrenal papillary necrosis.Effects of diuresis and antidiuresis.Arch. Pathol. 85, 404-409. GOTTSCHALK, C. W., and MYLLE, M. (1959).Micropuncture study of the mammalianurinary concentrating mechanism.Amer. J. Physiol. 196, 927-936. GULLBRTNG, B., LJUNGQVIST, A., and RICHARDSON, J. (1966).Studieson the pathogenesisof serum-inducedrenal papillary necrosisin the rat. 11.The effect of heatinghumanserumand the localization of humanprotein in the affectedkidneys.Lab. Zmest.15, 1486-1489.
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J. V., PATRICK, R. L., and KROE, D. J. (1963). Experimental renal papillary necrosis. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 22, 549. KROE, D. J., and KLAVINS, J. V. (1965). Renal and extra-renal lesions induced by heterologous serum. Arch. Pathol. 80, 345-350. LEVADITI, C. (1901). Experimentelle Untersuchungen iiber die Nekrose der Nierenpapille. Arch. Int. Pharmacodyn. Ther. 8, 45-65. LJUNGQVIST, A., and RICHARDSON, J. (1966). Studies on the pathogenesis of serum-induced renal papillary necrosis in the rat. I. Stereomicroangiographic and histologic examination of the renal lesion in non-heparinized and heparinized animals and in animals given diuretic drugs. Lab. Znuest. 15, 1395-1401. MALLORY, G. K., CRANE, A. R., and EDWARDS, J. E. (1940).Pathology of acute and of healed experimentalpyelonephritis. Arch. Pathol. 30, 330-347. MANDEL, E. E., and POPPER, H. (1951).Experimentalmedullary necrosisof kidney; a morphologic and functional study. Arch. Pathol, 52, 1-17. MUIRHEAD, E. E., VANATTA, J., and GROLLMAN,A. (1950).Papillary necrosisof the kidney; clinical and experimentalcorrelations.J. Amer. Med. Ass. 142, 627-63 1. PATRICK,R. L., KROE,D. J., and KLAVINS,J. V. (1964).Renal papillary necrosisinducedby heterologousserum.Arch. Pathol. 78, 108-l 13. Physician’sDesk Reference(1970).PONSTEL (mefenamicacid). 1020-1021. REHNS,J. (1901).D’une n&rose typique de la papilla renale determineepar la tetrahydroquinoleine et certainsde sesderives.Arch. Zrzt.Pharmacodyn.Ther. 8, 199-205. SHEEHAN, H. L., and DAVIS, J. C. (1959). Experimental hydronephrosis.Arch. PathoE.68, 185-225. WHITE, J., and MORI-CHAVEZ,P. (1952). Acute necrotizing renal papillitis experimentally produced in rats fed mono-N-methylaniline.J. Nat. CancerInst. 12, 777-787. WIZGIRD, J., FRENCH, S. W., and COULSON, W. (1965).Pathogenesis of serum-inducedpapillary necrosis.Arch. Pathol. 79, 57-64. KLAVINS,
AND APPLIED PHARMACOLCKiY19,
Strain-Dependent
D. L. BOKELMAN,
ill-124(1971)
Renal Toxicity of a Nonsteroid Antiinflammatory Agent W. J. BAGDON, P. A. MATTIS,
AND P. F. STONIER
Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486 Received October 29. 1970
Strain-DependentRenal Toxicity of a Nonsteroid Antiinflammatory Agent. BOKELMAN, D.L., BAGDON, W.J., MATTIS, P.A.,and STONIER, P.F. (1971), Toxicol. Appl. Pharmacol. 19, 111-124. Manor Farms SPF and CharlesRiver CD rats were given a nonsteroid antiinflammatory agent (1-p-chlorobenzylidine-5-methoxy-2-methyl-3-indenea~ticacid) po, at doselevelsof 3, 6, or 12mg,/kg/day,for periodsup to 29 wk. Hematuria wasobservedin all groups of male Manor Farms SPF rats, both control and drug-treated. The incidenceof hematuria was dose-dependent,and occurred 6, 12, or 24 timesmore often in male rats receiving 3, 6, or 12 mg/kg/day than in the controls. In contrast, hematuriawasnot observed in any control femalesand was infrequent in drug-treated females. The hematuriaobservedin the Manor FarmsSPFrats wasdue to lesions in the renal papilla. The initial lesionconsistedof focal hyperplasiaof the epitheliumcovering the renal papilla. Singlesmallfoci progressedto large, multiple, polypoid hyperplastic lesions. Areas of necrosis frequently observed within hyperplastic epithelium led to hemorrhage from the underlying vasarectae. Basically the lesionswere identical in the control and treated rats, but were more severein the latter. In contrast, renal lesionswere not observedin untreated CharlesRiver CD rats of either sex. When this strain of rat was treated with the same compoundat the samedoses,hematuriawasrarely seen,and no lesionsof the renal papillary epithelium were found. On the other hand, very slight interstitial edemaand necrosiswere observedin the interior of the papilla of a few drug-treatedrats. Theseobservationsdemonstratea type of problemthat can arisein the safety evaluation of a compounddue to strain-specificlesions. Lesions of the renal papilla have been produced experimentally by a variety of compounds, such as vinylamine (Levaditi, 1901; Mandel and Popper, 1951), tetrahydroquinoleine (Rehns, 1901), bromethylamine hydrobromide (Fuwa and Waugh, 1967), mono-N-methylaniline (White and Mori-Chavez, 1952), phenacetin (Fordham et al., 1965), mefenamic acid (Parke Davis Co., January, 1967), and human serum (Klavins et al., 1963; Patrick et al., 1964; Wizgird et al., 1965; Kroe and Klavins, 1965; Ljungqvist and Richardson, 1966; Gullbring et al., 1966). Other methods of producing lesions of the renal papilla have included fat-free diet (Borland and Jackson, 1931), ureteral ligation with or without bacterial infection (Mallory et al., 1940; Muirhead et al., 1950; Sheehan and Davis, 1959), and obstruction of the renal vein (Bestwick and Schatzki, 1960). 111
112
BOKELMAN
ET
AL.
The lesion most commonly produced by these various experimental procedures was necrosis of the renal papilla or associated changes, such as interstitial edema of the papilla. The incidence and severity of lesions have been variable, giving rise to numerous hypotheses to explain the pathogenesis of these lesions. Routine safety evaluation studies of a nonsteroid antiinflammatory agent in Holtzman rats for 4 wk and in Charles River CD strain rats for 16 wk showed no signs of renal toxicity. In a longer-term study, begun later in Manor Farms SPF rats, a low incidence of hematuria was noted 3 wk after the start of the study. The incidence of the hematuria was drug-related, but was noted also in a few control rats. The hematuria was traced to a series of lesions in the renal papilla. The present study was initiated to confirm these findings in another group of Manor Farms SPF rats, and also to investigate the responseof another strain of rats (Charles River CD) to this compound after prolonged treatment. This study points out the differences in responseof two different strains of rats to the samecompound and how the differences may influence the interpretation of an experiment. The study also illustrates a unique spontaneouslesion of the renal papilla of the Manor Farms SPF rat and how this condition was aggravated by the administration of the test compound. METHODS
Albino rats of two different strains, Manor Farms SPF’ and Charles River CD2, approximately 5-6 wk old, were given 1-p-chlorobenzylidine-5-methoxy-2-methyl3-indeneacetic acid, a nonsteroid antiinflammatory compound, the structure of which is shown in Fig. 1. CH,CO,H
C”3
CH
I-P-CHLOROBENZYLIDINE-5METHYL - 3- INDENEACETIC FIG.
METHOXY-PACID
1. I-p-Chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic
acid.
The compound was given at the following approximate dose levels: 0 (control), 3, 6, or 12mg/kg/day. Twenty malesand 20 femaleswere included in each dosegroup in the Manor Farms SPF series,and 10 malesand 10 femalesmade up each dosegroup in the Charles River CD series.The drug was mixed in the food (milled Wayne Lab Rat Blox), and its concentration was adjusted periodically according to the results of food consumption and body weight measurements.Food consumption was determined weekly on 5 male and 5 female rats from each group. All surviving Manor Farms SPF animals at a doseof 3 or 6 mg/kg/day were treated for 17wk. Other rats in this experiment, given * Manor Farms Research Inc., Stattsburg, New York. 2 The Charles River Breeding Laboratories, Inc., North Wilmington,
Massachusetts.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
113
RATS
12 mg/kg/day, were treated for 20 wk, and then treatment was suspended for 4 wk followed by an additional 5 wk treatment period. Control rats of this strain were continued on test with the high dose group animals. All Charles River CD rats were treated continuously for 23 wk. Each rat was individually caged in air-conditioned quarters with food and water available at all times. All rats were selected for physical soundness and normal behavior and placed into groups so that average weights of groups by sex were similar. Physical signs were noted daily and weights were recorded weekly. Urine samples were obtained periodically by placing the animals in metabolism cages and the urine sediment was examined microscopically after centrifugation. Urine samples were collected from the Manor Farms SPF rats as follows : from 10 males and 10 females of each group in the 8th wk, from all surviving rats in the 12th wk, and from all surviving control and high dosage group animals weekly from the 19th through the 28th wk. Urine was examined from all Charles River CD rats three times prior to the experiment and during weeks 2,3,4,6,8,10,13,15, and 23 of the study. Urine of selected rats from both series showing hematuria was also examined at various additional times during the experiments. Hematologic, biochemical, and ophthalmic examinations were made at various intervals. All rats were necropsied, and sections of the various organs were fixed in 10 % aqueous formalin. Both kidneys of all animals were cut sagittally, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Semiserial sections were made of the kidneys of selected animals. Selected tissue sections were also stained with Masson (Goldner-Foot) Trichrome and Alcian Blue-periodic acid Schiff. RESULTS
Manor Farms SPF Rats Urinalysis. Grossly visible hematuria was first seenin the 3rd wk in 2 male rats receiving 12mg/kg/day. From this time on, the incidence of hematuria in the treated rats increased and the results of the urine examinations in the 8th and 12th wk are shown in Table 1. Blood was present in the urine of 2 control malesat 8 wk and in 1 control male TABLE 1 INCIDENCE OF HEMATURIAIN MANOR FARMS SPF RATSDURINGTHE 8TH AND 12TH WKS
Female0
Male”
Treatment bdWday)
8 wk
12 wk
8 wk
12 wk o/20 o/20 2120 o/20
Control
2/10
l/18
o/10
3 6 12
3110 4/10 5/10
8/20*b 8/20* 10/18**
O/l0 l/l0 o/10
a Number of animals with 10 or more erythrocytes per high power field/total animals examined. b Values marked with asterisks differ significantly from control values: *Pio.o5;**P
114
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ET AL.
at 12 wk, but not in female controls at either period. The incidence of hematuria was significantly increased in the treated males at all dose levels, especially during the 12th wk, when 40% or more of all treated animals were affected. Hematuria was evident in only 1 female at 8 wk and 2 females at 12 wk. In each case, the animals were in the 6 mg/kg/day dose group. The incidence of grossly visible hematuria for the first 17 wk of the study was dosedependent (Table 2) in the male rats, occurring 6, 12, and 24 times more often in rats given 3,6, or 12 mg/kg/day than in the controls. No hematuria was evident in the control females and the incidence was low in the treated females. TABLE INCIDENCE
2
OF MACROSCOPIC HEMATURIA IN MANOR FARMS
DURING
No. of rats with hematuria Treatment b-WWday) Control 3 6 12
THE FIRST
17 WIG
SPF RATS
Male
Female
Incidence (days) of hematuria -___ Male Female
6
0
11 13
1 5
13 78 152
19
2
318
0 3 16 8
During the 21st wk, the surviving rats in the high dose and control groups were removed from drug and no drug was given again until the 25th wk. Urine samplesof these rats were examined weekly from the 19th through the 28th wk. The results of thesestudies (Table 3) indicated that the incidence of hematuria decreasedin the drugtreated male rats during the period of drug withdrawal, and did not significantly increase again when drug administration was reinstituted. TABLE RESULTS
OF URINE
STUDIES
3
PERFORMED FROM (MANOR FARMS
THE 19~~ WK SPF RATS)
THROUGH
THE 28TH
WK
Number of animalswith 10 or more erythrocytes/HPF/total animals No drug Treatment (mg/kg/day) Weeks: 19 Control $ 126 Control 9 12?
20
21
22
23
24
25
26
27
28
l/18 O/l8 O/18 2/18 l/l8 l/l8 O/l8 2/18 O/l7 O/18 S/16 7/16 2/16 3/16 3/16 4/16 4/16 6/16 5/16 2/11 o/20 o/20
o/20 l/20
o/20 o/20
o/20 o/20
o/20 o/20
o/20 l/20
o/20 l/20
o/20 l/20
o/20 2120
o/20 l/20
The duration of hematuria in individual rats varied from 1 day to several weeksbut was usually intermittent. Hematologic changes,body weight differences or changesin food consumption were not evident.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
115
Histologic observations. Focal hyperplasia of the epithelium of the renal papilla was first observed in 1 of 2 control male rats killed after 8 wk because of hematuria. This lesion consisted of a few well-localized foci of rounded polyhedral cells, 3-8 cells thick (Fig. 2). Two male rats in the 12 mg/kg/day dosage group were also killed for the same reason at this time and focal hyperplasia of the epithelium of the papilla was also evident. The lesion in these treated rats was identical to that found in the controls, except that areas of necrosis were evident in the hyperplastic epithelium (Fig. 3). Necrosis extended from the surface of the epithelium into the underlying vasa rectae causing hemorrhage into the pelvis. More advanced hyperplasia was evident in a high dose male rat sacrificed after 12 wk. The hyperplasia in this instance was polypoid, consisting principally of a central core of connective tissue covered by hyperplastic epithelium projecting into the renal pelvis (Figs. 4 and 5). Lesions occurred singly, but more often were multiple, forming clusters. Dilated collecting ducts and blood vessels were also frequently present below the hyperplastic epithelium. Lesions occurred most often near the junction (fornix) of the papillary and pelvic epithelium. The changes found in the kidneys of animals receiving 3 and 6 mg/kg/day and killed after 16 wk of drug administration are summarized in Table 4. Varying degrees of focal hyperplasia of the renal papillary epithelium occurred in approximately half the males in both groups, and foci of necrosis and hemorrhage accompanied the hyperplasia in about half of those affected. Lesions again were most prevalent near the fornix of the pelvis. Grading of the lesions was arbitrary (trace to marked), depending upon the number of lesions and the amount of the papillary epithelium affected. Both isolated low-grade (trace to very slight) foci of hyperplastic epithelium and polypoid hyperplasia (usually very slight or greater) were evident. In all kidneys where hyperplasia was severe it was polypoid. Scars were evident in the subepithelial areas of hyperplastic foci in many instances. These were undoubtedly sites of previous necrosis and hemorrhage which had healed. Mineralization of scar tissue was also commonly found. Inflammatory cell infiltration was not commonly associated with the lesions and was never pronounced. The severity of these changes was similar in males of both treatment groups, but females of both groups were markedly less affected. Trace amounts of epithelial hyperplasia were evident in the papilla of one low dose female animal, and similarly, traces of hyperplasia were found in the papilla of 6 females in the 6 mg/kg/day dose group. The incidence of necrosis and hemorrhage in the hyperplastic areas was also considerably less in females; only traces of each were found in 1 rat in the 6 mg/kg/day dose group. Scarring of the renal papilla was not evident in female rats. Trace amounts of focal hyperplasia of the renal papillary epithelium were evident in 2 of 18 male and 3 of 20 female control rats after 29 wk (Table 5). A tiny scar was also evident in the subepithelial area of the epithelium of one of the affected males. A higher incidence of hyperplasia was evident in the treated animals, especially the males. Hyperplasia of the renal papillary epithelium was evident in 6 of 12 high dose males and in 7 of 20 high dose females. The severity of this change in high dose males was also much greater than in the control males and also significantly greater than that seen in the high dose females. Marked hyperplasia of the renal papillary epithelium was found in 1 high dose female, and a large calculus (magnesium, ammonium, and phosphate ions) and pyelonephritis were also present.
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BOKELMAN
ETAL.
FIG. 2. Focus of hyperplasia in epithelium covering the renal papilla of an untreated Manor Farms SPF rat. Sacrificed after 8 wks because of hematuria. Hematoxylin and eosin. :<100. FIG. 3. Focus of hyperplasia in epithelium covering the renal papilla with necrosis and hemorrhage. From a Manor Farms SPF rat sacrificed because of hematuria after 8 wks treatment, 12 mg/kg/day. Hematoxylin and eosin. x40.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
FIG. 4. Polypoid hyperplasia of epithelium covering the renal papilla of a Manor Farms SPF male rat treated for 12 wks with 12 mg/kg/day. Hematoxylin and eosin. ~40. FIG. 5. Pronounced polypoid hyperplasia of epithelium covering the renal papilla of a Manor Farms SPF rat treated with 12 mg/kg/day for 12 wks. Note dilated ducts and necrosis and debris in stroma below epithelium. Hematoxylin and eosin. x40.
14
10
9 19
8 9
12 12
16 17 6 13
18
20 12 20
0
1 (0-I)
0
1 (01.)
0
0
6 (O+-++)
4 (0-l -+)
0 5 (O+-++) 2 (+-++>
1 CO+)
Scarring
OF TREATMENT
Hemorrhage
3 CO+) 6 CO+-++ ) 7 (O+-i--t-ti-)b
Necrosis
2 CO+)
Hyperplasia
PAPILLARY EPITHELIUM OF RATS SACRIFICED (MANOR FARMS SPF)
Normal
IN THE RENAL
No. rats examined
OF LESIONS
’ Symbols: Of = trace; + = very slight; +-+ = slight; +++ = moderate; ++++ = marked. ’ Calculus in pelvis and pyelonephritis in animal with ++++, others 0+ or +.
8 ?
Control Control
SEVERITY
Sex
AND
Treatment (mdkdday)
INCIDENCE
TABLE 5
= marked.
AFTER 29 WKS
6 (Oi--++) 1 co+)
1 @+I
6 CO+-++)
6 CO+)
5 (0+---t+)
5 (O+-++)
Scarring
16 WKS OF TREATMENT
Hemorrhage
AFTER
11 (O+-++) 1 (Oi-) 10 (0+-i-+)
SACRIFICED
Necrosis
OF RATS
Hyperplasia
’ Symbols: Of = trace; + = very slight; ++ = slight; +++ = moderate; ++++
20 20 20 20
8 ? d ?
3 3 6 6
4
PAPILLARY EPITHELIUM (MANOR FARMS SPF)
Normal
IN THE RENAL
No. rats examined
OF LESIONS
Sex
AND SEVERITY
Treatment bx/kdday)
INCIDENCE
TABLE
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
119
The degree of hyperplasia in other affected females was very slight in 1 and trace in 5 others. Foci of necrosis and hemorrhage were found in I high dose male, but were not seen in either of the control groups or females in the high dose group. Areas of scarring in the edges of the papilla adjacent to the hyperplastic epithelium were found in all but one affected high dose male and in 2 of 7 affected females in this group. All the lesions of the papilla, i.e., hyperplasia, necrosis, hemorrhage, and scarring in the most severely affected individuals were bilateral, but were very frequently unilateral in the less severely affected animals. Both kidneys were also not always affected to the same degree. The overall incidence of renal papillary lesions in male rats is summarized in Table 6. Blood-tinged urine was found in the urinary bladder of 6 high dose rats sacrificed after 8-27 wk of treatment, but was not present in any other animals. The usual incidence of chronic nephritis was observed equally in the control and treated rats and was not considered to bear any relationship to the lesions described. One rat in the high dose group died of diffuse peritonitis resulting from a perforated small bowel ulcer, but no other changes attributable to treatment were observed in any other organs in any of the animals. Charles Riaer CD Rats Urinalysis. Slight hematuria was grossly visible during the study in 7 drug-treated males: 4 receiving the low dose, 1 receiving the middle dose, and 2 receiving the high dose. Hematuria was not observed more than 4 times during the study in male rats, except in I low dose rat in which it was frequently observed from the 18th through the 20th wk. One high dose female had hematuria once in the 10th wk. The incidence of urine samples with 10 or more erythrocytes per high power field was very low. Three rats had a large number of erythrocytes in the urine; 1 high dose female had 13-40 cells per high power field during the pretest observations, 1 high dose male had 70-80 cells per high power field during the 8th wk of drug administration, and 1 low dose male had 20-30 cells per high power field. Histologic obsermtions. Microscopic changes attributable to treatment were observed in the kidneys of rats in the 3 and 12 mg/kg/day dose groups, but no changes were evident in the kidneys of animals in the 6 mg/kg/day dose group or controls. Focal interstitial edema of the renal papilla was observed in 5 ratsin the high dosegroup and 1 animal in the low dose group. This was characterized by minimal accumulation of finely granular eosinophilic material in the interstitium and was always confined to the central part of the papilla near the tip, but never bordering on the papillary epithelium. Interstitial edema of the papilla accompanied by focal necrosis of the papilla was found in 2 rats in the high dose group and papillary necrosis alone was evident in another high dose animal (Figs. 6 and 7). The location of necrosis was exactly the same as the interstitial edema described earlier. The papillary epithelium was never involved. The cells of the necrotic tubules were absent or ill-defined, and if nuclei were visible they were pyknotic. Empty spaces were found where many tubules were necrotic. In some instances, the collecting ducts were slightly dilated proximal to the lesion in the papilla. Trace amounts of hemorrhage at the site of edema or necrosis were also present in 2 of the high dose animals. A very small amount of blood was evident in the renal pelvis of 1 high dose animal, but no kidney lesions were evident in this rat.
BOKELMAN
ET AL.
STRAIN-DEPENDENT
RENAL
TOXICITY
IN
RATS
121
FIGS . 6 and 7. Focal interstitial edema with minimal necrosis of the interior of the renal papi lla lofa Charle s River CD rat treated for 24 wk with 12 mg/kg/day. Hematoxylin and eosin. Fig. 6: x40; Fi lg. 7: x100.
The:se lesions were usually unilateral and focal. Inflammatory cells were not associated with this lesion and thrombosis of regional vesselswas not evident. Other PIarts of the nephron were not affected.
122
BOKELMAN
ET
AL.
DISCUSSION
The pathologic effects of I-p-chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic acid on the kidneys of 2 different strains of rats were manifested in 2 different ways. In both instances, lesions were confined to the papilla. In one strain, Manor Farms SPF, lesions were present in the epithelium covering the papilla; in the other strain, Charles River CD, the changes were evident only in the inner zone of the papilla near the tip. A few control rats of the Manor Farms SPF strain developed an intermittent hematuria early in the study, but hematuria of a greater incidence and severity was found in animals treated with the drug. As the experiment progressed, the incidence of hematuria in Manor Farms SPF control animals remained at a very low incidence and was extremely sporadic. On the other hand, the incidence and duration of hematuria in treated males of this strain increased steadily and was dose related. The lack of hematuria in control females and the low incidence of hematuria in treated females was also significant. The hematuria evident in the Manor Farms SPF animals was directly attributable to lesions of the epithelium of the renal papilla. These observations suggest that the Manor Farms SPF animals developed spontaneous renal papillary lesions of undetermined etiology and that the administration of l-p-chlorobenzylidine-5-methoxy-2-methyl-3-indeneacetic acid increased the incidence and severity of this change. The renal papillary changes observed in both control and treated Manor Farms SPF animals in the early part of the experiment were identical. The earliest change observed was the occurrence of focal areas of hyperplasia of the renal papillary epithelium, most often located near the fornix. Tiny foci of necrosis occurred within these hyperplastic areas and eroded into the vulnerable underlying vasa rectae causing hemorrhage into the pelvis. These lesions subsequently healed, and the necrotic tissue was replaced by fibrous scar tissue, and hematuria no longer occurred from that site. The intermittence of the hematuria may be explained by the healing of these lesions followed by necrosis and hemorrhage at another site in the hyperplastic epithelium. The incidence and severity of the hyperplasia in drug-treated animals, especially males, increased markedly during the experiment. The polypoid type of epithelial hyperplasia became more evident and in some instances the pelvis was studded with these projections. As the degree of hyperplasia increased, the incidence of necrosis and hemorrhage did also. The cause of the renal papillary lesions in the control animals is unknown. Changes of this nature have never been observed in control rats in our laboratory before. Our experience with the Manor Farms SPF strain of rats was, however, limited to experiments with this compound. Very tiny insignificant foci of hyperplasia, 2-3 cells thick, have occasionally been evident in the epithelium of the papilla of control rats of other strains used by us, but in no instances have they progressed in severity or led to necrosis and hemorrhage as was observed in this study. The reason for the increased incidence and severity of renal lesions in treated rats is not known. Metabolism experiments (unpublished data) in rats have demonstrated that the compound is well absorbed and the major route of excretion is in the feces. Tissue distribution studies (unpublished data) have likewise demonstrated that the concentration of the drug in the kidneys is “low.” The increased incidence of the condition in the treated animals may be related to the potent antiinflammatory activity of the
STRAIN-DEPENDENT RENAL TOXICITY IN RATS
123
compound, but this has not been investigated. The lesion was not due to precipitation of crystals in the urine since these were never evident in microscopic observations of urine sediment. White and Mori-Chavez (1952) described similar changes in the epithelium of the renal papilla of rats after oral administration of mono-N-methylaniline. However, in their experiments the primary renal lesion was hyaline thrombosis of smaller vessels resulting in necrosis of the entire tip of the papilla. In addition, they noted marked edema and mucoid degeneration of the interstitium of the papilla. In Manor Farms SPF rats, lesions were evident only in the surface epithelium of the papilla and its immediately adjacent tissue. Vascular lesions were not evident in these animals. Hyperplasia of the pelvic epithelium was also described in rats given a fat-free diet (Borland and Jackson, 1931). The diet was not balanced, however, and deficiencies of one or several vitamins may have contributed to this lesion. Lesions noted in Charles River CD animals coincide more with those described in the literature as renal papillary necrosis, but the pathogenesis of this change is equally obscure. Vinylamine (Levaditi, 1901; Mandel and Popper, 1951) and tetrahydroquinoleine (Rehns, 1901) have caused papillary necrosis in animals due to thrombosis of the vasa rectae. Extensive papillary necrosis occurred in rats after injection of human serum (Patrick et al., 1964), but not bovine or rabbit serum, suggesting an antigenantibody reaction. Fuwa and Waugh (1968) pointed out that localization of the necrosis in the inner medulla and papilla (similar to our observations in Charles River CD rats) of rats fed 2-bromethylamine hydrobromide (BEA) corresponded with the area of maximal osmolality as demonstrated by micropuncture studies conducted by Gottschalk and Mylle (1959). In their experiments with rats fed BEA, necrosis was prevented or minimized by diuresis whereas it was enhanced by antidiuresis. It is obvious from various conclusions of different investigators that a number of factors acting together or independently may cause lesions in the renal papilla. This is further complicated by the fact that in the present series of experiments the same compound produced different lesions in the papilla of two different strains of rats and, furthermore, that the renal papillary changes produced in one of these strains was more profound in males than females. REFERENCES BESTWICK, I. P., and SCHATZKI, P. F. (1960). Experimental renal papillary necrosis. Arch. Pathol. 69, 733-739. BORLAND, V. G., and JACKSON, C. M. (1931). Effects of fat-free diet on the structure of the kidney in rats. Arch. Path& 11, 687-708. FORDHAM,C. C., III, HUFFINES, W. D., and WELT,L. G. (1965).Phenacetin-inducedrenal diseasein rats. Ann. Intern. Med. 62, 738-743. FUWA, M., and WAUGH, D. (1967). Experimental renal papillary necrosis.Production by venousstasisand by toxic amines.Fed.Proc. Fed. Amer. Sot. Exp. Biol. 26, 517. FUWA, M., and WAUGH,D. (1968).Experimentalrenal papillary necrosis.Effects of diuresis and antidiuresis.Arch. Pathol. 85, 404-409. GOTTSCHALK, C. W., and MYLLE, M. (1959).Micropuncture study of the mammalianurinary concentrating mechanism.Amer. J. Physiol. 196, 927-936. GULLBRTNG, B., LJUNGQVIST, A., and RICHARDSON, J. (1966).Studieson the pathogenesisof serum-inducedrenal papillary necrosisin the rat. 11.The effect of heatinghumanserumand the localization of humanprotein in the affectedkidneys.Lab. Zmest.15, 1486-1489.
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J. V., PATRICK, R. L., and KROE, D. J. (1963). Experimental renal papillary necrosis. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 22, 549. KROE, D. J., and KLAVINS, J. V. (1965). Renal and extra-renal lesions induced by heterologous serum. Arch. Pathol. 80, 345-350. LEVADITI, C. (1901). Experimentelle Untersuchungen iiber die Nekrose der Nierenpapille. Arch. Int. Pharmacodyn. Ther. 8, 45-65. LJUNGQVIST, A., and RICHARDSON, J. (1966). Studies on the pathogenesis of serum-induced renal papillary necrosis in the rat. I. Stereomicroangiographic and histologic examination of the renal lesion in non-heparinized and heparinized animals and in animals given diuretic drugs. Lab. Znuest. 15, 1395-1401. MALLORY, G. K., CRANE, A. R., and EDWARDS, J. E. (1940).Pathology of acute and of healed experimentalpyelonephritis. Arch. Pathol. 30, 330-347. MANDEL, E. E., and POPPER, H. (1951).Experimentalmedullary necrosisof kidney; a morphologic and functional study. Arch. Pathol, 52, 1-17. MUIRHEAD, E. E., VANATTA, J., and GROLLMAN,A. (1950).Papillary necrosisof the kidney; clinical and experimentalcorrelations.J. Amer. Med. Ass. 142, 627-63 1. PATRICK,R. L., KROE,D. J., and KLAVINS,J. V. (1964).Renal papillary necrosisinducedby heterologousserum.Arch. Pathol. 78, 108-l 13. Physician’sDesk Reference(1970).PONSTEL (mefenamicacid). 1020-1021. REHNS,J. (1901).D’une n&rose typique de la papilla renale determineepar la tetrahydroquinoleine et certainsde sesderives.Arch. Zrzt.Pharmacodyn.Ther. 8, 199-205. SHEEHAN, H. L., and DAVIS, J. C. (1959). Experimental hydronephrosis.Arch. PathoE.68, 185-225. WHITE, J., and MORI-CHAVEZ,P. (1952). Acute necrotizing renal papillitis experimentally produced in rats fed mono-N-methylaniline.J. Nat. CancerInst. 12, 777-787. WIZGIRD, J., FRENCH, S. W., and COULSON, W. (1965).Pathogenesis of serum-inducedpapillary necrosis.Arch. Pathol. 79, 57-64. KLAVINS,