Studies on Urothelium. III. Experimental Vesical Stone Formation in the Dog

THE JOURNAL OF UROLOGY

Vol. 89, No. 2 :February 1963 Copyright © 1963 by The Williams & Wilkins Co.

Printed in U.S.A.

STUDIES ON UROTHELIUM. III. EXPERIMENTAL VESICAL STONE FORMATION IN THE DOG CLIFFORD N. EDWARDS, FRED K. GARVEY

AND

WILLIAM H. BOYCE

From the Department of Urology, Division of Surgery, The Bowman Gray School of JJ1edicine, Winston-Salem, N. C.

The mucoprotein matrix is considered an important structural component of urinary calculi and may be essential to stone formation. 1 Its presence has been demonstrated in calculi from both humans and animals. 2 - 4 This mucoprotein matrix contains neutral polysaccharides and thus gives a positive reaction1 to the periodicacid-Schiff procedure of Mcl\!Ianus. 5 Large complex molecules are present in urine 6 and these may be increased or altered under conditions of active stone formation in the urinary tract. 7 l\!Iatrices of animal and human calculi have been shown to contain a number of proteins and mucosubstances which are also present in normal blood and urine. However, at least one mucoprotein is present in human calculous matrix and in the urine of patients forming calculi which has not been demonstrable in normal urine. 8 This has been designated matrix substance A. The site of origin in the blood, renal substance, or urinary collecting system of this Accepted for publication July 10, 1962. Supported by the Damon Runyon Cancer Foundation, The Forsyth County Cancer Society and National Institutes of Health Allocation R.G. 7189.

1 Boyce, W. H. and Garvey, F. K.: The amount and nature of the organic matrix in urinary calculi; a review. J. Urol., 76: 213, 1956. 2 Boyce, W. H., Pool, C. S., Meschan, I. and King, J. S., Jr.: Organic matrix of urinary calculi. Acta Radio!., 50: 543, 1958. 3 Keeler, R. F.: The internal structure and composition of silceous urinary calculi of bovine origin. Am. J. Vet. Research, 21: 428, 1960. 4 Cornelius, C. E. and Moulton, J.E.: Ruminant urolithiasis: II. Histochemical studies in experimental ovine calculosis. J. Urol., 84: 223, 1960. .1McManus, J. F. A.: Histological and histochemical uses of periodic acid. Stain Techn., 23:

99, 1948. 6 King, J. S., Jr., Boyce, W. H., Little, J.M. and Artom, C.: Total nondialyzable solids (TNDS) in human urine. I. The amount and composition of TNDS from normal subjects. J. Clin. Invest., 37: 315, 1958. 7 Boyce, W. H. and King, J. S., Jr.: Effects of high calcium intake5 on urine in human beings. Fed. Proc., 18: 1102, 1959. 8 King, J. S., Jr. and Boyce, W. H.: Immunological studies on serum and urinary protein in urolith matrix in man. Ann. N. Y. Acad. Sciences, in press.

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substance and its precursors is a matter of conjecture. Transitional epithelium lining the urinary tract of man, the dog and other animals contains mucopolysaccharide granules in the cytoplasm of the cells of the most superficial layer 9 - 11 as demonstrated by the periodic-acid-Schiff reaction (fig. 1). In smne cases of human stone formation, there has been observed a marked increase in mucopolysaccharide content of transitional epithelium adjacent to urinary calculi and also in the epithelium at some distance from the stones. Transitional epithelium has therefore been suggested as a possible source of the mucopolysaccharides of urine and of stone matrix. 2 Previous studies11-12 in this department have indicated that the periodic-acid-Schiff positive components of transitional epithelium are not absorbed constituents of urine and are not quantitatively increased by administration of parathormone, vitamin D or clihydrotachysterol, drugs which are known to encourage urinary stone formation. The present study was undertaken to further investigate changes in the vesical urothelium under conditions of experimental calculus formation in the clog. MATERIALS AND METHODS

Sixteen adult female mongrel dogs were utilized in the study. In each instance a tripartite 9 Mende, T. J. and Chambers, E. L.: Distribution of mucopolysaccharide and alkaline phosphatase in transitional epithelia. J. Histochem. & Cytochem., 5: 99, 1957. 10 Martin, B. F.: Histological and histochemical studies on the bladder and ureter. J. Anat., 92:

286, 1958. 11 Edwards, C. N., Boyce, W. H. and King, J. S., Jr.: Studies of urothelium: I. Characteristics of canine transitional epithelium following isolation from the urinary stream. J. Urol., 85: 802, 1961. 12 Edwards, C. N., Boyce, W. H. and Drummond, C. S., Jr.: Studies on urothelium. II. Effect of parathyroid extract, vitamin D, and dihydrotachysterol on some histochemical characteristics of canine transitional epithelium. J. Urol., 86: 364,.

1961.

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Fm. 1. Normal transitional epithelium (canine). Fine, periodic-acid-Schiff positive granular stippling of cytoplasm of superficial layer cells only. PAS-hematoxylin, diastase-digested. X400. bladder pouch preparation11 • 13 was constructed (fig. 2). In 4 animals dried, autoclaved fragments of human urinary calculi were inserted into the isolated bladder cavities. In two of these a similar additional fragment was placed within the functioning area of the bladder in contact with the urinary stream (fig. 3, A). All fragments were weighed prior to insertion. In 12 similar preparations, a silk suture (fig. 3, B) anchored through the epithelial layers was placed in both pouches and in the functioning bladder cavity as a foreign body. Biopsies of the transitional epithelium of the bladder were obtained from each animal at the time of construction of the bladder pouches. The dogs were sacrificed at varying intervals of 2 to 17 months. Biopsies of the transitional epithelium of the bladder, right and left isolated pouch cavities, renal pelves, renal parenchyma and ureters were preserved in each case. Clarkson, T. B., Boyce, W. H. and King, J. S., Jr.: A surgical technique for the formation of isolated pouches of the canine bladder. Am. J. Vet. Research, 19: 661, 1958. 13

Fm. 2. Drawing shows completed (healed) tripartite bladder preparation. All sections, after appropriate fi.'l:ation, were examined by hematoxylin-eosin, aqueous toluidin blue, the periodic-acid-Schiff reaction (McManus) with and without prior diastase digestion, Best's

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Fm. 3. A, human stone fragments implanted surgically in each of 3 compartments of tri-partite bladder preparation. B, silk sutures placed surgically through mothelium of isolated areas and functioning bladder area of tri-partite bladder preparation.

Fm. 4. Experimentally produced canine bladder stone formed about human renal calculus-fragment, used as foreign body in functioning bladder. carmine, and Gomori technique for alkaline phosphatase. The histochemical techniques were the standard methods used in similar studies previously reported from this laboratory.11· 12 Decrystallization of calculi ,rns accomplished by dialysis in cello11hane bags against buffered, aqueous EDTA solution, 5 per cent, at pH 7.8.

RESULTS

The human stone fragments used as foreign bodies in isolated pouches of the bladder failed to increase in size in seven of the 8 pouches. There was a consistent minimal reduction in weight of these 7 pieces which was attributed to loss of small fragments by handling. The surfaces of

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increase (compared to original control biopsies from the same animals) of PAS-positive, diastaseresistant intracytoplasmic granulation, orthochromatic to toluidin blue, adjacent to the calculous fragments. This varied from experiment to eiqJeriment and from area to area in the same animal. Moderate to marked inflammatory changes were noted; squamous metaplasia was observed in one area of an isolated pouch. There appeared to be no consistent histological or quantitative histochemical difference in over-all pattern between the epithelium adjacent to the human calculous fragments in the pouches where no matrix or crystalline deposit was observed and that adjacent to the rapidly growing fragments within the functioning bladder cavity, except that, in the latter, reduplication of the juxtaluminal layer containing the PAS-positive substance was more frequent. Alkaline phosphatase content in the deeper layers of the epithelium was unchanged in the amount and FIG. 5. Tripartite bladder preparation in which human stone fragment was placed in each compartment 15 months previously. Communication has developed (indicated by probe) between right isolated pouch and bladder allowing urine to ,mter. Fragment A in this area and B in functioning bladder have increased markedly in size; fragment C which remained isolated in left pouch shows no growth. the calculi remained irregular and granular and no matrix deposition was noted. In the 2 animals in which a stone fragment was in1planted in the bladder cavity, in contact with the urinary stream, an ovoid crystalline calculus was deposited about the original foreign body (fig. 4). Decrystallization of the newly deposited calculus revealed a fine latticework of matrix substance containing but few indistinct and incomplete lamina. Histochemical studies of this matrix revealed it to be PAS-positive and diastaseresistant with only minimal, irregularly situated traces of metachromasia on treatment with toluidin blue. This matrix was thus similar to human stone matrix in respect to these reactions. 1 In one instance a communication developed between the bladder cavity and an isolated pouch; deposition of canine calculus then occurred in contact with the urinary stream and the fragment doubled in size and weight (fig. 5). Histochemical examination of the transitional -epithelium lining the isolated pouch cavities and the functioning bladder areas showed some

FIG. 6. Bladder calculi deposited on silk sutures within functioning bladder area of dog. Note silk sutures in isolated pouches of bladder preparation show no encrustation when they are not in contact with urinary stream.

STUDIES ON UROTHELIUM

distribution. Glycogen content of the superficial cells showed the usual wide variation observed in normal and pathological canine urothelium. In the 12 animals in which silk was used as the foreign body, no crystal or matrix deposition was noted where the silk remained isolated from the urinary stream (22 pouch compartments in 12 dogs). Within the functional bladder, the silk suture formed a nidus for stone formation in nine of the 12 dogs (fig. 6). In 2 cases, one of the surgically isolated compartments developed a small communication with the bladder cavity allowing urine to come into contact with the silk and stone formation occurred within the pouch. Chemical analysis of the calculi formed in contact with the urinary stream showed the principal crystalline component to be ammonium magnesium phosphate with occasional small amounts of calcium oxalate. Decrystallization revealed the presence of a PAS-positive matrix, histochemically indistinguishable from that

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deposited about the human stone fragments in the earlier experiments. Histochemical examination of the epithelium lining the isolated compartments and the functioning bladder area was carried out in all cases and compared with control biopsies from the same animals obtained prior to the experiments. Alkaline phosphatase and glycogen content and distribution were again not remarkable. The PAS-positive, diastase-resistant granulation within the juxtaluminal transitional epithelial layer showed considerable variation in amount, In six of the 12 animals no appreciable increase over primary control sections was observed; these included 4 cases in which stones formed within the bladder. In three of the 12 animals a moderate increase involving both isolated areas and the functioning bladder was noted; these included 1 case in which no vesical stone was formed. In one of the 12 animals, the bladder adjacent to a large calculus showed a marked mcrease as compared to both the initial biopsy

Frn. 7. Extreme PAS-positive, diastase-resistant, mucopolysaccharide deposition in cells of superficial several layers of transitional epithelium. In some areas large accumulations (arrows) have replaced cells. This tissue was removed from isolated bladder compartment, in which no matrix or crystalline calculus was deposited on silk thread; same animal formed a large foreign-body calculus about similar thread placed in functioning bladder in contact with urine, although epithelium adjacent to growing calculus showed much less polysaccharide deposition. PAS-hematoxylin. X600.

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Fm. 8. Changes in canine urothelium adjacent to foreign body stone in functioning bladder. Moderate increase of PAS-positive, diastase resistant stippling is seen. Note marked increase in number of layers (hyperplasia) of epithelium and subepithelial inflammatory edema and infiltrate. P AS-hematoxylin. X400.

and the isolated areas. Contrariwise, in the remaining 2 animals, somewhat more PASpositive diastase-resistant material was seen within the cytoplasm of the cells of the isolated compartments than within the bladder lumen in which active stone growth was taking place; this group included the most extreme example of increased epithelial mucopolysaccharide seen in this study (fig. 7). Review of the sections thus revealed no discernible relationship between the presence of increased mucopolysaccharide content within the transitional epithelium and the occurrence or non-occurrence of active stone formation within the bladder. However, one positive correlation could be made: areas exhibiting the phenomenon of increased intracellular mucopolysaccharide invariably showed considerable evidence of inflammatory reaction. These areas for the most part also showed an increase in the number of layers of epithelial cells as a whole, constituting a hyperplasia of the urothelium (fig. 8) and in

rare instances metaplasia was observed. The converse was not true; inflammatory change, even to the point of abscess formation, was by no means invariably associated with increase of the PAS-positive, diastase-resistant fraction. The upper urinary tracts of the animals in both series were examined by the same techniques. Pyelonephritis varying from minimal unilateral changes to severe involvement of both kidneys was observed in all cases in which a stone was formed within the urinary bladder and in some instances in which no calculus resulted. In one instance an area of marked increase of intracytoplasmic mucopolysaccharide was observed within the cells lining the pelvis of the kidney; this was associated with marked pyelonephritis and pyelitis. DISCUSSION

The presence of increased PAS-positive, diastase-resistant, intracytoplasmic granular material in the cells of transitional epithelium adjacent to

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areas of active stone formation in the human has been previously observed and described. 1 This phenomenon was confirmed in this series of experimental animals under conditions of foreign body induced vesical stone formation. However, increased mucopolysaccharide granulation was demonstrated in only a scant majority of instances and its distribution did not coincide with the areas of active stone formation in that equal or more extensive deposition of mucopolysaccharide was often observed in areas surgically isolated from the urinary stream, and in one instance in the renal pelvis. Neither stone matrix nor crystal deposition occurred within isolated pouches of the bladder, despite the presence of normal or excessive polysaccharide granulation of the epithelium, and the demonstrable presence within these pouches of fluid containing a higher concentration of calcium than blood serum.U However, in three fortuitous instances in which urine gained access to the foreign body within such an isolated pouch, rapid stone formation occurred. Histologically, increase of mucopolysaccharide, when it occurred, was invariably associated with severe inflammatory (chemical or bacterial) changes in the epithelium, which often took the form of hyperplasia or squamous metaplasia of the urothelium. It is difficult to visualize how granulation of the deep layers of a hyperplastic cell lining could contribute, in a secretory fashion, to matrix deposition within the bladder lumen. It is more possible that the phenomenon under discussion represents a secondary reaction of some areas of urothelium to the presence of a foreign body or irritant, whether in the form of a silk suture, a stone, or bacterial infection. Limitations of a study such as the present one are obvious: 1) It is only semi-quantitative in that assessment of the extent of histochemically reacting granulation of the cytoplasm must vary somewhat in the eyes of different beholders. 2) Only small areas of the total urothelium can, for practical and economic reasons, be examined in any individual case. 3) Histochemical tech-

niques such as these indicate only the amount of a substance (in this case mucopolysaccharide) present at any one time in a given cell: no measure of metabolic turnover is obtained. 4) All animals with foreign body stones in the bladder showed evidence of pyelonephritis and it is felt that, until animals can be maintained in a germfree environment, it may be impossible to evaluate the effect of bacteria on experimental calculosis. SUMMARY

The presence of urine appears necessary for the deposition of matrL\'. or crystalloids within the bladder of the dog, under conditions favorable to experimental stone production by a foreign body. The presence of increased intracellular PASpositive, diastase-resistant mucopolysaccharide substance within the cytoplasm of transitional epithelium in cases of urolithiasis is suggested to be a secondary phenomenon related to irritation by the presence of a foreign body, a stone, or bacterial infection in the urinary tract. The phenomenon, previously described, accompanies active stone formation in barely more than 50 per cent of instances and is often found in areas in which neither matrix nor crystal deposition is occurring. CONCLUSION

These histochemical studies of canine transitional epithelium, under conditions of experimental vesical stone formation, failed to show evidence to support a contention that the mucopolysaccharides of transitional epithelium lining the urinary tract take part in calculus formation as a precursor of stone matrix. The authors acknowledge the technical assistance of Mrs. Elizabeth O'Byrne Blalock, M.T. (A. S. C. P.), Mrs. Ida W. Johnson, M.T. (A. S. C. P.) and Mr. Robert Jackson; and thank Mr. George Lynch of the Department of Medical Illustrations for figures 2 and 3.