EXPERIMENTAL UROLITHIASIS: CALCIUM OXALATE STONE*

Transcription

1 EXPERIMENTAL UROLITHIASIS: CALCIUM OXALATE STONE* THADDEUS J. DOMANSKI, MAJOR, MSC, USAF From the Department of Biology, Washington Square College, Neio York University, Neiv York This paper discusses the influence upon the formation of urinary stone of an increased ingestion of ammonium chloride and ammonium oxalate, administered separately and together. The work was undertaken to determine the effectiveness for the formation of calcium oxalate stone of an increased urinary excretion of calcium and oxalate. MATERIALS AND METHODS Male albino rats (Carworth Farms Wistar Strain), six to eight weeks old at the start of the study, were used. The Control Group and Groups I, II and III were maintained on a diet (basic diet A) known to be adequate for the rat; in addition ammonium chloride was fed orally to Group I, ammonium oxalate to Group II and ammonium chloride plus ammonium oxalate to Group III. Neither salt was added to the diet of the Control Group. Group IV was maintained on a diet (basic diet B) from which were omitted the vitamin A and D concentrates and the meat and bone scraps used in the preparation of basic diet A. Group IV. received the same amounts of ammonium chloride and ammonium oxalate as did Group III. The duration of the feeding experiment was 115 days. The composition of basic diets A and B is shown in Table 1. Basic diet A was based upon the stock diet developed and used for the maintenance, growth and reproduction of rats at the Department of Biology, Washington Square College, New York University. Calf mealf contained yellow corn meal (417 parts by weight); 34 per cent protein O.P. linseed meal (300); ground malt barley (00); wheat red dog (440); oat flour (300); dried skimmed milk (60); soluble blood flour (40); irradiated yeast (0.50); dicalcium phosphate (15); ground limestone (5); iodized salt (0); vitamin A feeding-oil (.5). The vitamin A feeding-oil (1,816,000 U.S.P. units vitamin A per pound) present in calf meal OAf was replaced by an equal weight of yellow corn meal. The meat and bone scraps (50 per cent Armour's meat and bone scraps) contained: protein, 50 per cent; bone phosphates of lime, 0 per cent; fat, 6 per cent; crude fiber, not over 6 per cent. The foregoing are manufacturer's assays. For Groups I, III and IV, ammonium chloride constituted 0.6 percent of the * Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, New York University, A r ew York, New York, while a member of the United States Army. The author's present address is United States Air Force School of Aviation Medicine, Randolph Air Force Base, Texas. Received for publication, March 13, t Cooperative G. L. F. Mills, Inc., Buffalo, New York. J Prepared upon request by courtesy of Cooperative C. L. F. Mills, Inc. Ammonium Chloride (NH 4 C1) Merck Reagent, A.C.S. 707

2 708 DOMANSKI basic diet during the initial 15 days of the study, 1 per cent during the sixteenth through the twenty-third day (eight days), per cent during the twenty-fourth through the fifty-eighth day (35 days) and 3 per cent during the remainder of the study (57 days). For Groups II, III and IV ammonium oxalate* constituted 0.4 per cent of the basic diet during the initial two days of the study, 0.45 per cent during the third through the seventeenth day (15 days) and 0.6 per cent during the eighteenth through the forty-second day (5 days); thereafter the ammonium oxalate was removed from the basic diet and placed in the animals' drinking water, the concentration of ammonium oxalate in water being 0.6 per cent. Except during the collection of 4-hour urine specimens, an excess of food and water (or oxalate solution) was available to the animals at all times. Both kidneys of every animal which died or was sacrificed (ethyl ether) were dissected and examined with the aid of a dissecting microscope. Representative TABLE 1 THE COMPOSITION OF BASIC DIETS A AND B COMPONENT Meat and bone scraps Brewer's yeast* Calf meal Calf meal-oa Cod liver oil, U.S.P. BASIC DIET A 360 Cm. 10 Cm. To make a total of 4000 Gm. of food mixture 80 ml. per 4000 Gm. food mixture 10 Gm. BASIC DIET B To make a total of 4000 Gm. of food mixture * Pure, dry Brewer's Yeast, type 019, Standard Brands, Inc., New York, New York. kidney specimens were submitted for histologic study.j The maximum experimental period (Table ) was: Group I, 115 days; Group II, 114 days; Group III, 11 days; Group IV, 109 days; Control Group, 115 days. Urinary calculi were analyzed by the method previously described. At least one of the renal, ureteral and bladder stones found in each of the stone-forming rats was analyzed. Observations concerning the acid-insoluble fraction found in papillary plaques were made at the time of addition of 10 per cent hydrochloric acid to the specimen. Beginning five weeks after the start of the study, 4-hour urine specimens! collected at intervals of one or two weeks were examined for calcium and oxalate. Sulkowitch's reagent 7 was used for the calcium determination. For the determination of oxalate 1 ml. CaCl (5 per cent) was added to ml. urine, followed by the addition of drops CH 3 COOTI (5 per cent). The solution was mixed by inversion * Ammonium Oxalate [(NH 4 ) C 0rH 0l Merck Reagent, A.C.S., ph t Histologic preparations and interpretations were made by the Columbia University Research Division, Welfare Island, New York City. t Actual duration of urine collection was? hours. In addition there were 3 feeding periods of one-half hour each. Each specimen examined consisted of the pooled urine of C animals.

3 EXPERIMENTAL UROLITHIASIS 709 and read immediately and, again, 30 minutes later. Both calcium and oxalate results were recorded in terms of a graded response (0 to +4). Prior to the performance of the above two tests, the urine was cleared of protein and sediment (heat and acetic acid followed by centrifugation). TABLE OCCURRENCE OF STONE IN ANIMALS TllAT DlED OB WERE SACRIFICED AFTER SlXTY OF EXPERIMENTAL FEEDING DAYS DUMBER OF ANIMALS Group I Group II Group III Group IV Control Group Days S 3S 6S IS S S* 3 S 5 S 1 S* ID* D* 7S 15 S* 1 D* 1 S S* 7D* S* 1 S 9S* 13 S 14 S 6S D indicates animal died; S, animal was sacrificed. * Indicates stone was present. TABLE 3 INCIDENCE OF STONE AFTER THE SIXTIETH DAY OF EXPERIMENTAL FEEDING GROUP TOTAL NUMBER OF ANIMALS NUMBER OF ANIMALS WITH STONE PER CENT WITH STONE Control I II III IV IS S EXPERIMENTAL RESULTS A. Occurrence of Stones Urinary stones were not found until the sixty-first clay of experimental feeding. At that time there were 33 animals remaining in the Control Group, 5 in Group I, 9 in each of Groups II and III and 19 in Group IV. The incidence of stones in these animals is shown in Table 3. Table 4 shows the relative incidence of renal, ureteral and bladder calculi and the relative incidence of unilateral and bilateral involvement. In stone-forming rats, kidney stone was always present. Bladder stone was seen in only 3 rats; one of these was a Group IV animals. Stones were seen in the renal cortex and medulla in 7 Group IV members and in 4 members of Group III. In each instance such stones were bilateral and mul-

4 1710 DOMANSKr tiple in both kidneys. They were always associated with larger renal calculi. It is possible that the actual incidence of stone in the renal cortex and medulla in 'Groups III and IV was greater than herein reported, since identification was based upon the ability to isolate these pinpoint concretions. B. Nature of Stones llenal calculi were found either attached to the tip of the papilla or loose in the kidney pelvis. The diameter of these stones was 0.8 to.5 mm. Representative renal calculi are shown in Figure 1. Very often, renal stone was associated, with.the occurrence of one or more papillary plaques (plate-like deposits on or embedded in the surface of the papilla). The smallest of these plaques had a diameter of 0. mm. while the largest covered an area of.5 x 1.5 TABLE 4 THE RELATIVE INCIDENCE OF RENAL, URETERAL AND BLADDER STONES AND OF UNILATERALITY AND BLLATERALITY CROUP ii GROUP III GROUP IV Number o( Rats Incidence of Stone Number of Rats Incidence of Stone Number of Rats Incidence of Stone per cent per cent 100 per cent 100 Animals with stone Location of stone Kidney only Kidney and ureter Kidney, ureter and bladder... Unilateral stones Renal Ureteral : Bilateral stones Renal Ureteral : S9 6 mm. Plaques had a thickness of 0.1 to 0. mm. A plaque which had been dislodged from the lateral border of the papilla is shown in Figure. Ureteral calculi (Fig. 3) had a diameter of 0.7 to 1. mm. and bladder stones varied from 1.0 to 1. mm. in diameter. All calculi had a depression or facet which was most pronounced in stones removed from the renal papilla. In the case of papillary stones it was clear that the facet always occurred on the attachment surface of the stone. Figure 4 indicates the common presence of a facet in renal, ureteral and bladder stones. A similar depression or facet was clearly visible on the attachment surface of the larger of the papillary plaques. In each instance of papillary stone the papilla presented a flattened eroded appearance (Fig. 6). The calculi and the plaques analyzed were composed of calcium oxalate; phosphates were either absent or present only in traces. In the course of the chemical analysis of papillary plaques it was possible to demonstrate the presence of an acid-insoluble fiber-like network. The discrete tiny masses of salt

5 EXPERIMENTAL UROLITHIASIS 711 impacted in the interstices of the sponge-like structure appeared to be continuous with the main mass of the salt deposit. The lattice, when freed of calcium oxalate, was soluble in dilute aqueous alkali. o FIG. 1. Renal calculi. X 14. FIG.. Papillary plaque, showing surface of attachment. X 35. FIG. 3. Renal and ureteral calculi. The two smallest arc ureteral calculi. X 14. FIG. 4. Presence of a facet in urinary calculi. Top left, renal calculus at ureteral orifice; top right, bladder calculus (facet in profile); bottom left, ureteral calculus; bottom right, renal calculus removed from the top of papilla. X 14. C. Histologic Findings Histologic examination of the kidneys of animals which had died after less than 30 days of treatment disclosed an absence of renal disease. These findings

6 71 DOMANSKI apply to members of each of Groups I and III and to 3 members'of Group IV. Renal disease was also absent in 5 members of each of Groups I and II and the Control Group after 85 to 115 days of treatment. One stone-forming member of Group II was represented in the latter series. In a sampling of 5 members of Group III which had been treated for 85 to 104 days, histologic examination disclosed a mild deposition of calcium in the collecting tubules of animals with stone ( animals), while animals which had not formed stone were either free of calcium deposits (1 animal) or had a moderate deposition of calcium in the convoluted tubules, with trace deposits or none in the collecting tubules ( animals). In addition, stone-forming members of Group-III showed a heavy deposit of anisotropic crystals in the tubules of the cortex arid medulla and lying free in the calyces. In a sampling of 6 members of Group IV which had been treated for 90 to 109 days there was seen a deposition of calcium in the collecting tubules of stone-formers (5 animals); these deposits were less heavy than corresponding calcium deposits seen in Group III stone-formers. The solitary Group IV member which had not formed stone was free of renal calcium deposits. The distribution of anisotropic crystals (Fig. 8) in Group IV stone-formers was the same as that described for Group III but the deposits were considerably heavier in the Group IV animals. The kidneys of the one Group IV rat which had not formed stone were free of anisotropic crystals. There was no evidence of keratinization of the pelvic epithelium in any of the kidneys examined. D. Urinary Findings All groups'of animals maintained an acid urine throughout the period of observation; ph* 6.4 to 6.6 for the Control Group, ph 5.5 to 5.8 for Group I, ph 6.0-to 6.3 for Group II and ph 5.4 to 5.8 for Groups III and IV. The concentration of urinary calcium was highest (+4) in Group I, relatively high (generally, +1) in Groups III and IV and lowest (0 or trace) in the Control Group and Group II. The concentration of urinary oxalate was highest (+4) in Group II and relatively high (generally, +) in Groups III and IV. The test for oxalate was negative in the Control Group and in Group I urines. DISCUSSION The stones produced had a facet which was most pronounced in those removed from the renal papillae. In the case of the latter it was clear that the facet occurred on the attachment surface of the stone. It was also possible to discern the presence of a facet on the attachment surface of the larger of the papillary-plaques. The roof and walls of the facet in papillary stones and plaques were pitted, as though the deposit had been molded about a convex base pierced by many protruding tubules. In all other calculi the facet was smooth or nearly smooth, never pitted. It has been postulated by Randall 6 that the initiating lesion which leads to the formation of stone occurs on the papilla, and he was of the opinion * Nitrazine Paper, 13. R. Squibb & Sons, Now York, N. Y.

7 FIG. 5. Normal renal papilla (Control Group). X 14. F'IG. 6. Renal papilla, showing erosion at site of stone formation. X 14. FIG. 7. Histologic view of an eroded papilla following removal of a papillary stone; ordinary light. X 114. (Compare with Fig. 8.) FIG. 8. From same field as shown in Figure 7. The use of polarized light makes easily visible the heavy deposits of anisotropic crystals. X

8 714 DOMANSKI that the presence of a facet suggests that the stone had possessed a mural attachment. The presence of a facet in renal, ureteral and bladder stones creates the impression of a common site of origin of all of the stones produced, viz., the renal papillae. It may be that the size of the stone at the time it became dislodged from the papilla was a main determinant of the subsequent resting-place of the stone, i.e., renal pelvis, ureter or bladder. A stone which had broken free of its papillary mooring might be expected to act as a nidus for the precipitation of urinary salts, resulting in a gradual increase in the size of the stone and an attendant levelling of the originally irregular surface of the facet. The size of papillary stones was roughly the same as the amount of gross destruction of tissue observed. The impression received was one of replacement of tissue by stone, with deposition of salt from calyceal urine contributing very little to the size and shape of the calculus. It would appear that the prime growth of stone had been directed towards the base of the pyramid. All animals maintained an acid urine throughout the period of observation. Stone, when present, would seem to have occurred without the intervention of urea-splitting organisms. Group I remained free of stones despite a much increased concentration of urinary calcium. Urinary oxalate was'absent by the test employed; this does not necessarily mean that the urine was totally devoid of oxalate but that the concentration of oxalate, if present at all, was low as compared with the findings in Groups II, III, and IV. In terms of a precipitation phenomenon it can be reasoned that calcium oxalate did not precipitate in the urinary tract of Group I animals because, the concentration of oxalate ions was so low as to negate the potential precipitability of calcium ions present in high concentration, It is similarly possible to reason that the precipitation of calcium phosphate was prevented by the high urinary acidity in Group I animals. Apparently, an increase in the concentration of urinary calcium did not, of itself, constitute a sufficient condition for the formation of stone, The high incidence of stone in Group III (about 69 per cent) suggests an effectiveness for calcium oxalate stone formation of a simultaneous increase in the concentrations of urinary calcium and oxalate. The diet of Group I animals represented an acidification of the Control Group diet, by use of ammonium chloride, Polak 5 found that a vitamin A deficiency diet containing per cent ammonium chloride resulted in kidney or bladder stone, or both, in 5 of 0 rats maintained on such a diet for six and one-half to eight weeks. In the present study Group I animals were maintained on a diet rich in vitamin A and containing to 3 per cent ammonium chloride for approximately IS- weeks, without the occurrence of stone. On the basis of available evidence it is impossible to attribute a stone-forming property to ammonium chloride since it has been demonstrated that the rat as well as other laboratory animals may develop stones when subjected to a diet deficient in vitamin A in the absence of ammonium chloride. 1 ' 3 ' 4 Moreover it is seldom convincing that the so-called vitamin A deficient diets have been limited only to a deficiency of that vitamin.

9 EXPERIMENTAL UROLITHIASIS 715 The diet of Group IV differed from that of Group III in that the cod liver oil, vitamin A concentrate and meat and bone scraps present in the diet of Group III were withheld. On the altered diet there occurred an increase of almost 6 per cent in the incidence of stone. Data are insufficient for an evaluation of the cause or causes of the increased incidence of stone. With respect to the diminished intake of vitamin A, Group IV gave no histologic evidence of a vitamin A deficiency. Weight gain was less than in Group III animals, but there were no other clinical signs which might be related to a vitamin A deficiency. It is clear only that a plentiful intake of vitamin A, plus the inclusion of meat and bone scraps in the diet, did not prevent a high incidence of stone in Group III. SUMMARY The addition of ammonium chloride plus ammonium oxalate to an otherwise nutritionally adequate diet resulted in a high incidence (about 69 per cent) of urinary stone in the rat. When each salt was added separately to the same basic diet, the feeding of ammonium oxalate resulted in a relatively low incidence of stone (about 10 per cent). Ammonium chloride administered alone was not associated with urinary stone formation. The calculi produced were composed of calcium oxalate. Acknowledgment. The author gratefully acknowledges the help and advice of Professor Milan J. Kopae, Now York University, during the course of this stud}'. REFERENCES 1. Buss, A. R. JR., LIVERMORE, G. R., AND PRATHER, E. 0., JR.: The relationship of vitamin A and vitamin D to urinary calculus formation. J. Urol., 30: , DOMANSKI, T..].: Analysis of urinary calculi. Am. J. Clin. Path., 8: (Tech. Suppl., :) , HIGOINS, C. C: Urinary lithiasis; experimental production and solution with clinical application and end results. J. Urol., 36: , OSBORNE, T. B., MENDEL, L. B., AND FERRY, E. L.: The incidence of phosphatic urinary calculi in rats fed on experimental rations. J. A. M. A., 69: 3-33, POLAK, A.: On the relationship between kidneystone- and bladderstone-formation and nutrition. Arch, nderl. de physiol., 19: , RANDALL, A.: The origin and growth of renal calculi. Ann. Surg., 105: , TODD, J. C, AND SANFORD, A. H.: Clinical Diagnosis by Laboratory Methods. Ed. 10. Philadelphia: W. B. Saunders Co., 1943, p. 13.