Ruminant Urolithiasis: II. Histochemical Studies in Experimental Ovine Calculosis

Tt--IE JOURNAL OF' UROLOGY

Vol. 84, '\To. 2, August 10611 Pn'uled in c~.S.A.

RTl\HNANT "CROLITHIASIS: II. IIISTOCHl:DMICAL STCDIES Ii\'. EXPERil\IE;\;TAI, OVI1\E CALCTLOSIS c. K ccm:-,ELIU8

AND

.r.

E. JIOULTON

Frnm. the Departments of Clinical Palhology, and Pathology, School of Velerinary ?iler/ieine, Un11'eis1Ju o/

Davis, Cal.

Information concerning tlw origin and chemical natnrC' of the prntein-honnd carbohydrate ('.omplexes isolated from the· matrix of urinary calculi is incomplete. The cxtensivC' biochemical stmlies of Boyce and coworkern 1 · 2 concerning the frac·tiorn1tion and analysis for the elemental/ amino acicl,4 and carbohyclrate 4 composition of the) urinary colloids and organic matrices of human renal calculi have contributed greatly in the darification of the rnlc of matrix in urolith fommtion in man. A. specific: urinary glycoprotein, uromucoid, which presumably originates in the secretions of the transitional epithelial cells, 5 waR found to have a dosc· but not exact analytical relationship to stone nrntrix. Histoc:hemical stuclic's on the matrices of calculi in man 6 have; revealed the presence of a protein-bound carbohydrate material by the periodic acid-Schiff (P),.S) rraction. N C'utrnl Accepted for publication January 29, 1960. This study was supported in part by a grant (USPH-A-3296) from the National Institute of Arthritis and Metabolic Diseases, and Regional Research Funds from the United States Department of Agriculture for co-operative research on Western Regional Frnject W-41. 1 Boyce, vV. H., Garvey, F. K. and ~orfieet, C. ~1.: Proteins and other biocolloids of urine in health and in calculmrn disease. I. Electrophoretic studies at pH 4.5 and 8.6 of these components soluble in molar sodium chloride. J. Clin. Invest .. 33: 1287, 1954. 2 Boyce, W. H. and Swanson, NI.: Biocolloids of mine in health and in calculous disease. II. Electrophoretic and biochemical studies of a mucoprotein insolnble in molar sodium chloride. J. Clin. Invest., 34: 1581, HJ55. 3 Boyce, W. H. and Snlkin, N. M.: Biocolloids in urine in health and in calculous disease. III. The mncoprotein matrix of nrinary calculi. ,J. Cliu. Investig., 35: 106, 1956. 4 King, J. S., Jr. and Boyce, W. H.: Amino acid and carbohydrnte composition of the mucopro1.ein matrix in various calculi. Proc. Soc. Exp. Biol. & Med., 95: 183, 1957. 'Boyce, W. H. and GarveY, F. K.: The mnount and nature of the organic matrix in urinary calculi: A review. J. Urol., 76: 21:3, 1956. u Boyce, W. H., Pool, C. S., Meschan, I. and IGng, J. S., Jr.: Organic matrix of nrinar:v calculi. l\licroradiographie comparison of crystnllinc strnctm·c with microscopic and histochemical studies. Acb1 radiol., 50: 54:J, HJ58. 223

carbohydrate moieties \H)l'C obc:C'rved in tlw matrices of all moliths as .iuclged by the orthochromatic reaction of toluicli1w blue, except for stones containing apatite, whose matrices stain:·d eonsistently metachromatic.
Histochemical studies corn·erning tlw systems were made on 2 groups of shcrp: 1) 2:1 seven-month old normal castrated mules (wethC'rs) receiving alfalfa bay; and 2) S castrated males on an cxperinwntal diet with complete or partial urethral occlusion from impac-trcl c·alculi. The urirrnry hladdcrn of fom wethers in group 2 hacl ruptured. Tlw mental diet employee! in this <,xperimcnt wa8 pelleted and composed of wheat straw pe1 cent), dried beet pulp (:30 per cent), linsecci meal (17 per cent), oats per cent), wheat. (1.5 per cent), :\TaCl (0.5 per cent), and K,HP01 (2.5 per cent). Tlw chemical and calculi-provoking nature of this dil't in shr:ep has been pn'viously reported. 8 7 De Albuquerque, P. F. and Paola, n. Experiments in urolithiasis: l. Histodwmicnl studies. J. Urol., 81: 345, 1959. 8 Cornelius, C. E .. :\lonhon, J. F and J\,foCowan, B.: Ruminant urolithirrnis: I. Prelimi uary observations in experimental ovinc cakulosis. Arn. ,J. Vet. Res., 20: 86:3, 19.5G.

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In addition to routine hematoxylin and eosin stains of tissues, the following histochemical tests or staining methods were used: l) von Kossa's technique 9 to identify phosphatic accumulations; 2) periodic acid-Schiff (PAS) method 9 to identify carbohydrate material by the oxidation of glycols to aldehydes; 3) toluidine blue 9 stain to demonstrate ,vhcther the proteinbound carbohydrate is an acidic mucoprotein (acidic mucoproteins stain metachromatically due to the presence of chondroitin sulfates, whereas neutral carbohydrate material stains orthochromatically); and 4) the alcian blue technique 10 which stains blue to blue-green with acidic carbohydrates in contrast to the background which stains a pale light-green. The calculous material was suspended as a powder in paraffin blocks and also subjected to the previously mentioned histochemical techniques. Kidneys from the two groups of sheep were specifically examined for the number of PAS positive casts present in the collecting tubules. An arbitrarily selected triangular area of approximately 0.5 cm. 2 was examined microscopically in one kidney from each sheep and the number of PAS positive casts were reported per low power field (2.4 mm. 2). Standard statistical methods were used for the calculation of means (X), standard deviations (
Signs of partial or complete urethral blockage and uremia first appeared in the group of sheep receiving the experimental diet 2 weeks following the commencement of feeding. Impaction of the processus urethrae (fig. l, D) and/or sigmoid urethra was observed in these wethers. Clinical signs and biochemical findings have been previously reportecl.8 Urine sediments from these wethers contained packets of microcalculi (fig. 1, A) which resembled the structural units observed in crushed calculous material from the bladder (fig. 1, B). These packets of microcalculi (fig. 2, B), as well as a few rare rn.icrocalculi observed in the renal collecting tubules (fig. 2, A) 9 Lillie, R. D.: Histopathologic Technique and Practical Histochemistrv. New York: The Blakiston Co., Inc., pp. 120, 264, 285. 10 Wagner, B. M. and Shapiro, S. H.: Application of alcian blue as a histochemical method. Lab. Invest., 6: 472, 1957.

exhibited black centers upon staining with von Kossa (fig. 2, A-1 and B-1) and magenta staining PAS-positive envelopes and matrices (fig. 2, A-2 and B-2). These findings were interpreted as presumptive evidence that the microcalculi were composed of phosphatic accumulations and that they were enclosed and bound by a proteincarbohydrate complex. These packets of microcalculi in turn coalesced to form large masses of calculous material. In the majority of kidneys which were examined microscopically from sheep with urolithiasis, an obvious increase in PAS-positive material was present. l\1any large, round, PASpositive granules were observed in the cytoplasm of the cells of the proximal (fig. 2, D) and distal convoluted tubules in certain nephrons. These magenta colored granules filled the entire cells in some nephrons and measured up to 4 microns in diameter. The brush borders of the proximal convoluted tubules contained more PAS-positive material than in the kidneys of the control sheep. The basement membranes exhibited neither thickening nor an increase in the amount of PAS or von Kossa staining material. An increased number of small PAS-positive granules were alRo observed within the cells of the collecting tubules and transitional epithelium of both the renal pelvis and urinary bladder. These intracellular granules in the kidneys stained PAS-positive, after pretreatment with saliva for thirty minutes at 23°C. An increase in PASpositive constituents was also observed between the transitional epithelial cells of the renal pelvis and bladder. All 23 normal control sheep, which were necropsied and examined for PAS-positivl' accumulations in their urinary systems, exhibited this material to a lesser degree than observed in sheep with urolithiasis. A thin layer of PASpositive material ,ms observed on the brush borders of the proximal convoluted tubules and the transitional epithelial cells of the urinary bladder and renal pelvis. Rarely, a fow PASpositive intracellular granules up to 1 micron in diameter were found in a few of the cells of the renal collecting tubules, and transitional epithelial cells of the renal pelvis and bladder of control sheep. Quantitative counts for PAS-positive renal casts within the lumina of the renal collecting

EXPERIMENTAL OVINE CALCULOSIS

225

Frn. 1. A, urine sediment from sheep with experimental calculosis. Note unstained aggregates of "microcalculi," X400. B, calculous material, recovered from urinary bladder, crushed, and suspended in 0.9M NaCl. Note similarity of "microcalculi" to A, X900. C, powdery calculous material (Mg, Kand P) recovered from urinary bladder of one sheep receiving experimental diet (air dried): and D, necropsy of prepuce and penis showing 1) normal processus urethrae, 2) impaction and blockage of urethra by calculous material, and 3) excised hemorrhagic urethra. tubules were made on both normal control sheep and on those with experimental urolithiasis. Twenty-three normal sheep exhibited an average of 0.83 ± 0.94 PAS-positive casts per low power field (2.4 mm. 2) as compared to 4.12 ± 3.21 for eight sheep with clinical urolithiasis. The difference was statistically significant (P = 1.75). Histochemical examination of the PAS-positive intracellular granules and casts in kidneys from both normal sheep and those with urolithiasis revealed an orthochromatic staining reaction

with toluidine blue. Alcian blue stained these granules and casts a pale light-green. Crushed P AS-pm,itive calculous material, which was recovered from the urinary bladders of two sheep, stained orthochromatically with toluidine blue with only a trace of metachromasia. The calculous material stained a very pale light blue-green with alcian blue. The toluidine blue and alcian blue stains were interpreted as having suggested that only a trace of an acidic carbohydrate component was present in the calculous material.

226

C. E. CORNELIUS AND J. E. MOULTON

Fm. 2. A, section of ovine kidney exhibiting aggregate of "microcalculi" in renal collecting tubule in experimental calculosis, X900. 1, darkly staining von Kossa-positive material and 2, periodic acidSchiff (PAS) positive envelope and matrices. B, stained urine sediment from sheep with experimental calculosis, PAS-von Kossa, X400. Observe aggregates of "microcalculi" staining as in A-1 and 2. C, section of kidney from sheep with experimental calculosis. Observe massive formation in collecting tubules of orthochromatic-staining casts with toluidine blue, X200. D, section of renal cortex of sheep \Yith experimental calculosis. K ote PAS-positive granules in cells of certain convoluted tubules, X900.

Dii:iCUSSJON

Of special interest in this study was the simultaneous appearance in the sheep with urolithiasis of an abnormal increase in PAB-positive material \\'ithin the cells of the proximal and distal convoluted tubules and collecting tubules, on the brush borders of the proximal convoluted tubules, as cast material within the lumina of the collecting tubules, and on the surface of the transitional epithelial cells of the renal pelvis and urinary bladder. A similar staining material was

also observed in the rnicrocakuli of the urine sediment and in the larger aggregates of calculous material. These observations suggest that the mucoid material ,,-hich forms the matrices of calculous material in sheep may also originate at a higher level in the urinary system than in the transitional epithelial cells as previously suggested.2, 3 A previous observation that the plasma seromucoid as well as a urinary mucoid fraction was elevated in a group of wethers on an experimental calculi-provoking diet, 8 suggests the

EXPER!MEXTAL OVJ:\"J>! CALCULOSJS

a,dditional possibilit)· of a pre-n,nal origin. That mucopol:rnacdrnridPs can originate from a pren:nal origin has been 8110wn in the experiments of Pipcr,1 1 m which intravenously injected heparin was found to be excreted into urim, by both glomerular filtration and tubular sPcrdion in rabbits. There werr no olJ\'ious indications of glonwrular filtration of a mucoicl in the sheep with urolithiasis smce the incrrases in PASpositive material \VC're confinrcl to the renal tubules. Bo) 0<·e ancl Garvcv'' reported a positive correlation between tlw weoperative concentration of uromuc:oid in tlw urine and the presence of PAS-positive cytoplasmic granules in the transitional epithelium of' the ureter and urinary bladder in man 1vith renal calculi. ".\.lbuquerque ,.md Paola 7 reportecl tlmt in no instance \\'ere PAS-positive granules identified intraC'dlularly in kidm'ys with ncphrolithiasis whiC'h were surgi· <·;illy n'moved or biopsied. Urnrnucoid (T & H mucoid), which i~ quantit,1tively the largest C'arbohydrnte-prokin conjugate in human urine, has lwrn postulat,,d to form thr rnatricc,s of human c:alculi b)· a molecular orientation.'' This nmcoid stains orthochromatirally witli toluidine blue and is believed to be a secretory proclnC't of the trnrn,itional epithelium, The l'AS-positin· and orthochromatic staining aC'cumulations obsen·ed in rcxcess in the tissue sections of cases of urolithiasis are citlwr a rwntrnl glycoprotein complex or an acidic mucoprotrin, which no longer stains metaduomatically due to clepolynwrization. 12 ThP origin of this PAS-positive material in sheep is not dear. A neutral glytoprotrin t'omplex ma)· originate both from the plasma seromucoicl fraction or from cells of thr: urinar.,· tract, per se. Depolymerized acidic polysac:charicle-protrin coneould con<·ci vably originate from the chondromuc:oid uf bone or from the connective tisRue of both the urinary system and/or dsr\Yherc in the bocl_1·. 12 , H The PAS-positive ma11 Piper, J.: The fate of heparin in rabbits after intnwe11ous injection. Aeta Plrnrmacol. et Toxicol., 3: :37:3, 1947. 0

12 Baker, R., Reaveu, G. and Snwyer, J.: Ground substance nnd rnlcification. J. Urol., 71: 511, HJ54. 13 Rubin, P. S. and Howard, J.E.: Histochemical studies in the role of acid mncopo1 0·saccharides in ealcifiability and cnlcifie1tion. Trans. 2nd Jos. :\foey Conf. JV[etab. Hl50. 11 ]1ich, C. aml W. P. L.: Excre1ion of acid mucopolysaccharides in the nrine of patients with malignant neoplastie disPases. J. Lab. and Clin. l\focl., 54: 22:.i, 1%0.

terial might, in addition, 1mrtly result from ,t eelluln,r reaction of the urinary system to obstruction. However, 3 dwqi rc n'i,·ing tlw experinwntal diet and not exhibiting obstrncti()n hacl similar PAS-positive aceumubt.ions 1n their urinary s_1·stcrn.s. Tlw prescnc1" in the cakulmrn rnakrial of trncr: of metachronrnsia with toluidine blue am! light blue-green staining with alcian hhw suggests that small amounts of ac-idi <· C'harides arc present. Since :1<·idic chariclcs are commonly present in urine, trn.ct' amounts could be occlucktl in or adsorbed onto the calculous sludge. Bo:-·r-t' and :1ssoc·iat(•s 15 obsl'TY<'d that the
SU:\IJVL\.HY

Histoc'.hemical examination of the systems of sht'l']) with experinwntal urolithia:,:1:, revrnlccl an incn'asc m periodic acid-Ncl1 ifi (PAS) positive accumulations within the c·vll, of the proximal aml distal com·olutr·d on the brush borders of the proximal com·uluted tubules, ancl \\'ithin the cells of the tubulrs. lm:rcased PAS-positive nrnteria,I wn,~ also observed in casts 1Yithiu the lumi1rn of thl' (·.ollPc:ting tubules and within ancl upon the surfaces of the transitional epithelial <'l'lls renal pelvis and minary hlaclclel'. In the matricrs of cakulous material isobted multaneously from the mimu:-· bbclclns of thcs(: sheep exhibitPcl similar staining c:harackri~tit's The aforc·mentionecl P,\8·positive accwuuh tions 1vithin the kidneys sfained orthochrom:1.t· ically with toluicline him' and a with alcian blue. Crushecl calculou~ nrni,<'rial n·covr·red from the urinary libdd<'r at necrops:· stained similarly c'xt·ept for :t trace of mc,ta <·hronrnsia with toluiclirw blue and a light blue green stain with alcian blue. Thr of these findings is clisc:ussecl. lt 1n1s l,vicknt from these Ptudiec; that carhohydrate-protrin compkxes, important in nrntri:,; fornmtion, may originate from sourTt's otlwi than tlw transitional epithelium.