Experimentally Induced Uracil Urolithiasis in Rats

THE JOURNAL OF UROLOGY

VoL 95, Jan Printed in U.S.A.

Copyright © 1966 by The Williams & Wilkins Co.

EXPERIMENTALLY INDUCED URACIL UROLITHIASIS IN RATS JOSEPH J. LALICH From the Department of Pathology, University of Wisconsin Medical School, Madison, Wisconsin

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During studies of the effect of uracil upon the tissue response in /3-aminopropionitrile-fed rats nmnerous calculi were observed in the urinary tract. Control studies indicated that uracil calculus formation was not related to the presence of odoratogenic drugs in the diet. To our knowledge there have been no previous reports of uracil calculi. These observations are presented in the hope that the conditions under which such uracil calculi are produced may contribute to the understanding of the general problem of calculus formation.

noted (table 1). Three control rats fed 25 per cent calcium caseinate grew and gained weight satisfactorily. Gross and microscopic inspection revealed normal tissues. vVhen 25 gm. uracil per kg. of diet was fed there was appreciable suppression of weight gain. Gross inspection revealed calculi in the bladder or ureters in 4 of 6 rats. Such calculi invariably predisposed to dilatation of the renal pelvis. Because both the protein and carbohydrate concentrations in the diet are known to exert a profound influence on urolithiasis 2 - 5 the next series of assays were made with three different concentrations of uracil in a commercial diet. It is evident that both weight gain and growth are related to the concentration of uracil which was added to the diet. One of the 7 rats fed 30 gm. per kg. of diet died with ascites and hydronephrosis after 11 days" Calculi were most frequently observed in the bladder and less often in the ureters in the other 6 rats. Hydronephrosis was the next most common alteration encountered. Some degree of hydronephrosis was always associated with hydrometer (fig. 1, A). Wedgeshaped white infarcts were observed in the kidneys of 5 of 31 rats fed uracil (fig. 1, B). Minimal to moderate swelling of the renal cortex was an associated complication of hydronephrosis. Examination of tissues in rats fed barbituric acid, hydantoin and cytosine revealed no tissue alterations on gross examination. Microscopic inspection revealed atrophy of tubular epithelium, dilatation of both proximal

METHOD

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Uracil and related chemicals were fed to weanling male Sprague-Dawley rats in a semisynthetic or a commercial diet for 29 to 54 days. Uracil was fed at concentrations of 20, 25 and 30 gm per kg. of diet. Barbituric acid, hydantoin and cytosine were all fed at 20 gm. per kg. of diet. Granulated uracil was uniformly mixed in a semisynthetic 25 per cent casein1 or a finely ground commercial diet.* The other chemicals were mixed in the commercial diet and fed ad libitum. After periods of 29 to 54 days the experiments were terminated, the rats were killed and the tissues were examined. All but one of the test rats fed 30 gm. of uracil per kg. of diet survived, as did those fed the various other chemicals previously indicated. Representative samples of muscle parenchymatous organs and endocrine glands were fixed in acetic ethanol formal solution. After fixation, the tissues were dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin.

2 Sager, R. H. and Spargo, B.: The effects of a low phosphorus ration on calcium metabolism in the rat with the production of calcium citrate urinary calculi. Metabolism, 4: 519-530, 1955" 3 Van Reen, R., Lyon, H. W. and Losee, F. L.: Urolithiasis in the rat. I. The influence of diet on the formation and prevention of calcium citrate calculi. J. Nutr., 69: 392-396, 1959. 4 Van Reen, R., Indacochea, N. and Hess, W. C.: Urolithiasis in the rat. II. Studies on the effect of diet on the excretion of calcium, citric acid and phosphate. J. Nutr., 69: 397-402, 1959. 5 Van Reen, R.: Urolithiasis in the rat. III. Effects of proteins, carbohydrate and phosphate on the occurrence of calcium citrate stones. J. Nutr., 77: 137-141, 1962.

RESULTS

The nurn.ber of assays performed, the chemicals fed and the alterations encountered have been Accepted for publication March 4, 1965. Supported by grant AM-06444-02 from the Division of Research Grants, Public Health Services. 1 Merkow, L. P. and Lalich, J. J.: Influence of diet on the incidence of angiorrhexis in b-aminopropionitrile-fed rats. AMA Arch. Path., 70: 752756, 1960. * Rockland Rat Diet, :Monmouth, Illinois. 83

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LALICH TABLE

Diet

Casein Casein Commercial Commercial Commercial Commercial Commercial Commercial Commercial

1. Calculi observed in urinary tract after feeding of various chemicals Chemical Added to Diet Gm/Kg

None Uracil None Uracil Uracil Uracil Barb. acid Hydantoin Cytosine

25 20 25 30 20 20 20

No. Rats Fed

Diet Fed Days

3 6 9 12 6

49 49 49 49 54 11-55 50 50 29

7

6 36 3

Gross Observations Urinary Tract Weight Calculus HydroGained Ureters nephrosis Gm/day Bladder L R L R

4.6 2.2 5.2 4.1 3.3 2.2 4.5 4.9 4.2

0/3 4/6 0/9 8/12 6/6 5/7 0/6 0/36 0/3

0/3 1/6 0/9 1/12 2/6

0/3 1/6 0/9 2/12 4/6 2/7 0/6 0/6 0/36 0/36 0/3 0/3

0/3 4/6 0/9 4/12 6/6 5/7 0/6 0/36 0/3

0/3 4/6 0/9 6/12 G/6 4/7 0/6 0/36 0/3

Fm. 1. A, rat fed 25 gm. uracil/kg. commercial diet for 49 days. During this interval, rat gained from 44 to 238 gm. At autopsy calculi, hydroureter and hydronephrosis were seen on left side. Only minimal precipitation occurred in right ureter. Acute renal infarcts were also seen in both kidneys in this rat. B, rat fed 25 gm. of uracil in calcium caseinate diet for 42 days. Infarct 3 by 7 mm. across was seen on

anterior midportion of right kidney. In section through infarcted area rather extensive dystrophic calcification was observed. H & E, X 37.

and distal convoluted tubules, focal hyalinization and fibrosis of glomeruli. Eventually a reduction in the numbers of tubules with interstitial lymphocytic infiltration and fibrosis occurred. These changes first occurred in the renal cortex

adjacent to the renal pelves. Another common observation was hyperplasia of transitional epithelium in the renal pelvis, the ureters and bladder. Intratubular renal obstruction was uncommon, the chemical aggregating into calculi

85

URACIL UROLITHIASIS IN RATS

Fm. 2. A, rat fed 25 gm. uracil/kg. of calcium caseinate diet for 48 days. At autopsy infarct was seen in right kidney. In section through renal pelvis of right kidney area of papillary necrosis was also found near calculus. Note extensive loss of tubules and glomeruli occurred adjacent to area of papillary necrosis. H & E, X 28. B, rnt fed 20 gm. of uracil in commercial diet for 49 days. At autopsy calculi were found in left ureter and bladder. This section of bladder illustrates hyperplasia of mucosa, ulceration and early submucosal inflammation. H & E, X 4L

only in the pelves, the ureters or bladder. Calculi were common in the ureters and bladder and infrequent in the renal pelvis where they were occasionally associated with papillary necrosis (fig. 2, A). Ulceration of the mucosa of both the renal pelvis and bladder presumably due to the calculi commonly occurred. Following ulceration there was evidence of either acute pyelitis or cystitis (fig. 2, B). Focal white infarcts with subsequent dystrophic calcification were infrequently observed. Hyaline thrombosis in the interlobular arteries was occasionally encountered. Inspection of other organs of the uracil-fed rats revealed nothing unusual. The tissues of rats fed barbituric acid and hydantoin were normal on microscopic inspection. The tissues of rats fed cytosine for 29 days were normal except for the livers in which a moderate vacuolar degeneration of liver cells was observed. The fact that chemicals somewhat similar to uracil did not produce urinary calculi may be of some theoretical interest.

CHEMICAL STUDIES

Urinary calculi recovered from 5 test rats were pulverized and washed twice with 25 mL of 50 and 95 per cent ethanol. After drying, from 20 to 75 mg. of dry white amorphous powder was recovered in each case. The white residue was dissolved in 0.05 N HCL Absorption spectra of this residue in the range from 245 to 289 mµ, corresponded to that of a known sample of uracil. This material was spotted on "\Vhatman's No. l paper and chromatogramed in butanol-urea or a dibasic sodium phosphate-isoamyl alcohol solvent. 6 In these 2 solvent systems the R1 was identical to uracil. These preliminary findings indicate that the material in the urinary calculi is uracil. 6 Block, R. J., Durrum, E. L. and Zweig, G.: A Manual of Paper Chromatography and Paper Electrophoresis. New York: Academic Press, Inc., 2nd edit., chapt. 9, 1958.

86

LALICH DISCUSSION

Phosphate calculi were first observed in rats in 1917, by Osbourne and associates who attributed their production to vitamin A deficiency.7 Schneider and Steenbock produced urinary calculi in rats by feeding a low phosphorus diet8 and found that diets low in phosphorus but complete in other respects predisposed to the formation of calcium citrate calculi. 9 Sager and Spargo enhanced the tendency of low phosphate diets to produce calculi by reducing the protein concentration in the diet. 2 Van Reen and associates later extended the observations on the influence of protein concentration on the formation of urinary calculi in rats fed low phosphate diets. They observed that urolithiasis occurred when a Hubbell salt mixture was fed at 15 per cent casein level but not when 20 per cent casein was fed to NMRI-D rats. 3 Elevated concentrations of dietary casein reduced the urinary excretion of calcium and citric acid which in turn prevented the formation of calculi. 4 In addition to protein the concentration of dietary carbohydrate apparently also exerts some influence on urolithiasis. Van Reen demonstrated that starch-containing diets produced fewer calculi than did dextrose or sucrose. 5 Other types of diet have also predisposed to renal calculi. Magnesium-deficient diets may cause calcific deposits within the renal tubules of rats. 10 Pyridoxine-deficient diets with proper concentrations of calcium and glycine supplements promote the formation of calcium oxalate calculi in rats. 11 Review of the literature does not indicate that uracil is related to any of the previously used dietary forms of urolithiasis. However, the possibility exists that uracil derivatives may be present in some diets and can predispose to urolithiasis. While studying the carcinogenic effects of 4dimethylaminoazobenzene, supplements of thiou7 OsbornE:, '!'· B., Mendel, L. B. and Ferry, ~. L.: The rncidence of phosphatic urinary calculi m rats fed on experimental rations. J.A.M.A. 69· 32-33, 1917. ' . 8 Schn~ider, H. and Steenbock, H.: A low phosphorus diet and the response of rats to vitamin D2. J. Biol. Chem., 128: 159-171 1939 . • 9 Schneid:r, H. and Steenb~ck, H.: Calcium citrate urohths on a low phosphorus diet. J. Urol. 43: 339-344, 1940. ' 10 Whang, R., Oliver, J., McDowell, M. and Welt, L. G.: The renal lesion of magnesium depletion. Clin. Res., 10: 257 1962. 11 Andrus, .s. B., Gershoff, S ..N., Faragalla, F. F. and Pnen, E. L.: Production of calcium oxalate renal calculi in vitamin B, deficient rats. Lab. Invest., 9: 7-27, 1960.

racil were added to test for its protective activity.12 Feeding 0.4 gm. thiouracil per kg. of food up to 150 days resulted in thiouracil calculi in 14 of 72 rats. It would appear that suppression of thyroid activity by thiouracil is not necessary for calculus formation because the feeding of metabolically inert uracil will also cause calculi. Spontaneous renal calculi have also been observed in breeder rats of the Sprague-Dawley strain.1s Male rats are apparently more susceptible than female rats to spontaneous calculus formation. Our results probably have no bearing on this type of stone formation since much younger rats were used in our studies and nothing suggestive of renal or bladder calculi was found in the controls, or the rats fed barbituric acid or hydantoin. Vermeulen and associates analyzed the factors responsible for calculus formation and considered the following to be important: 1) urinary colloids may increase the solubility of solutes, 2) organic solubilizers or hydrotropic agents may prevent calculus formation, 3) inorganic salts in urine can exert a stabilizing influence on urinary solutes, and 4) guttulate precipitation will prevent the formation of larger aggregates. 14 Which of these factors may be involved in uracil calculus formation is not apparent from our studies. However, it is our opinion that uracil lithiasis in rats is partly dependent upon the intestinal absorption of uracil, its poor solubility in water and the concentration which it attains in the urine. Calculi once formed act as local irritants which may ulcerate the mucosa in the renal pelvis or bladder and thus predispose to infection. SUMMARY

Diets that contained 20 to 30 gm. of uracil per kg. of diet were fed to weanling Sprague-Dawley rats up to 54 days. Minimal to moderate suppression in weight gain occurred in the uracil-fed rats. Calculi in the bladder, the ureters or the renal pelves were encountered in a majority of the uracil fed rats. Chemicals resembling uracil such as barbituric acid, hydantoin and cytosine when fed at 20 gm. per kg. diet did not produce calculi. . 12 Harris, P. N. and Clowes, G. H. A.: Observations on carcinogenesis by 4-dimethylaminoazobenzene. Cancer Res., 12: 471-479 1952. 13 W E:xler_, B. C.: Histopathology of kidney stone format10n m repeatedly bred rats. J. Urol. 90· 527-534, 1963. ' . 14 Vermeulen, C .. W., Miller,. G . :a;. and Chapman, W. H.: Experimental urohthiasis: X. On the state of calcium in the urine. J. Urol. 75: 592-601 1956. ' '