YAMATE, Takanori; UMEKAWA, Tohru; I Masanori; KURITA, Takashi; KOHRI, K. Citation 泌尿器科紀要 (1997), 43(9):

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1 itle Detetion of osteopontin as ontaining urinary stones matrix Author(s) YAMAE, akanori; UMEKAWA, ohru; I Masanori; KURIA, akashi; KOHRI, K Citation 泌尿器科紀要 (1997), 43(9): Issue Date URL Right ype Departmental Bulletin Paper extversion publisher Kyoto University

2 Ata Urol. Jpn. 43: , DEECION OF OSEOPONIN AS MARIX PROEIN IN CALCIUM-CONAINING URINARY SONES akanori YAMAE, ohru UMEKAWA, Masanori IGUCHI and akashi KURIA From the Department of Urology, Kinki University Shool of Mediine Kenjiro KOHRI From the Department of Urology, Medial Shool, Nagoya City University Osteopontin (OPN) has been identified as a matrix protein of alium oxalate urinary stones by sequening -DNA for urinary stone protein. OPN, a phosphoprotein, is suseptible to thrombin digestion and speifially stainable with Stains-All. We examined the matrix in 4 kinds of urinary stones, alium oxalate dihydrate, alium phosphate, magnesium ammonium phosphate and uri aid, for the presene of OPN by staining thrombin-digestion and undigested matrix with Stains-All. Matrix was extrated with a 0.1 M ethylenediamine tetraaeti aid (ED A) solution. Furthermore, the amino aid sequene was determined for the NH2-terminal 20 amino aid residues. OPN was identified in alium oxalate dihydrate and alium phosphate stones, but was absent in magnesium ammonium phosphate and uri aid stones. Our findings suggest that OPN, whih binds tightly to hydroxyapatite and is related to bone formation as bone matrix, also partiipates in the formation of alium-ontaining usinary stones. (Ata Urol. Jpn. 43: , 1997) Key words: Calium-ontaining stone, Matrix protein, Osteopontin, Stains-All INRODUCION Although many reports have suggested the importane of the matrix in urinary stone formation, its role has remained unidentified. Boye et al. l ) and King et al. 2 ) reported that the matrix was omposed of 64% protein, 5% proteoglyans and 10% bound wa ter. U meka wa et al. reported the presene of alprotetin 3 ) and alpha l-antitripsin 4 ) in the matrix of alium oxalate urinary stones. Aording to the amino aid analysis onduted by Spetor et al. 5 ) aidi amino aids suh as asparti aid and glutami aid are the major onstituent amino aids of the matrix. Lian et al. 6 ) reported the presene of gamma arboxyglutami aid, and Melik et al. 7) identified siali aid. wo dimensional polyarylamide gel eletrophoresis by Jones et al. 8 ) revealed that most of the proteins in the matrix of the urinary stones are low moleular weight proteins. Our reent study revealed that the -DNA sequene of osteopontin (OPN) enodes the urinary alium oxalate stone protein, suggesting that OPN is involved in stone formation as the stone matrix 9 ) he expression of OPN mrna and protein was observed in stone forming kidneyslo) OPN was isolated from the mineralized matrix of bovine bone. his protein, a sereted phosphoprotein-i, binds tightly to hydroxyapatite l1 ) Sine previous studies on the stone matrix protein primarily used Coomassie brilliant blue stain, highly phosphorylated OPN protein may not have been identified. In this study, we demonstrated the presene of OPN in the stone proteins by using Stains-All staining, a new moleular biologial tehnique that an stain highly phosphorylated protein. he purpose of this study is to examine the existene of OPN in urinary stone omponents as a matrix. MAERIALS AND MEHODS Extration proedures of osteopontin protein he urinary stones obtained after extraorporeal shok wave lithotripsy (ESWL) were analyzed by infrared spetrosopy. Four stones with over 96% of any of the 4 kinds of stone omponents of alium oxalate dihydrate, alium phosphate, magnesium ammonium phosphate, and uri aid were used as speimens. Stones were destroyed by extraorporeal shok wave lithotripsy, allowed to spontaneously disharge, washed with water, dried, and stored. One hundred fifty mg of eah powdered stone was put into a 1.5 ml Safe-Lok (Eppendorf) mirofuge tube and extrated with 1 ml of 0.5 M ethylenediamine tetraaeti aid (ED A), prepared in 50 mm ris/ hydrohlori aid (HCl) buffer, ph 7.4, ontaining 1 mm phenylmethyl sulfonyl fluoride (PMSF). he extration was performed for 24 hours at 4 C with gentle shaking. he speimen was washed three times In phosphate buffered solution (PBS) ontaining PMSF for 30 minutes eah, between extrations. he suspension was entrifuged at 8,000

3 624 Ata Urol. Jpn. Vol. 43, No.9, 1997 rpm on an Eppendorf mirofuge tube for 10 minutes, and the supernatant was dialyzed against three hanges of 10 mm ammonium biarbonate ontaining 0.05% Bolyxyethleme Lauryl ether (BRIJ 35) lyophilized and kept at -80 C. hrombin digestion Digestion was performed at 3rC for 30 minutes in 1O,u1 of 10 mm ris/hci buffer ph 8.0 ontaining 10 mm alium hloride (CaCI 2 ) and I unit of thrombin (Sigma Chern. Co., St Louis). Digestion was terminated by the addition of 0.25 volume of a 4-fold onentrated sodium dodeyl sulfate-polyarylamide gel eletrophoresis (SDS-PAGE) sample buffer ontaining 150,ug dithiothreitol (D) and heating to 56 C for 25 minutes. Poylarylamide gel eletrophoresis Proteins were analyzed by SDS-PAGE eletrophoresis using the disontinuous ris/glyine buffer system with 10% ross-linked linear gels as desribed by Goldberg et a1. 12 ) and Nagata et a1. 13 ) Speimens were dissolved in 1O,u1 of sample buffer ontaining 1% SDS, 2M urea, and bromophenol blue marker. For analysis of proteins under reduing onditions, 150,ug D was indued. he speimens were heated to 56 C for 30 minutes and ooled immediately before appliation to individual wells. A 10% gel plate was put into an eletrophoresis tank filled with eletrolyte buffer (15.1 g/i, ris, 72 g/i (kda) glyine). Eah lane was applied with thrombin undigested sample, thrombin digested sample, and prestained SDS-.PAGE standard markers. Eletrophoresis was arried out for I hour at 150 V Following separation, the gels were stained in the dark for at least 48 hours with 0.025% Stains-All (3, 3' -diethyl-9-methyl-4, 5, 4', 5' -dibenzothiaarboyanine bromide) (Nakalai esque Co., Kyoto), 25% isopropyl alohol, 7.5% formamide and 30 mm ris base, ph 8.8. Amino aid sequene analysis NH2-terminal analysis of 20 amino aids using pmol samples of protein was arried out by a gas phase protein sequener 477A (Applied Biosystems). M(kDa) RESULS Figures 1-3 and 4 show the eletrophoreti patterns for the matrix protein extrated from 4 kinds of stone omponents and stained with Stains-All. he alium oxalate dihydrate stones showed 3 bands of 49.5,32.5, and 25 kda (Fig. I, lane C). he 49.5 kda band disappeared after thrombin digestion, and the 32.5 band was intensified (Fig. I, lane ). he alium phosphate stones showed 3 bands of 45, 32.5, and 25 kda (Fig. 2, lane C). After thrombin digestion, the 45 kda band disappeared and the 32.5 kda band was intensified (Fig. 2, lane ). Magne Fig. 1. Eletrophoreti patterns by Stains-All staining of matrix protein extrated from alium oxalate dihydrate (weddelite) he 3 bands of 49.5, 32.5 and 25 kda appeared in the thrombin undigested lane (lani C). he 49.5 kda band disappeared after thrombin digestion, and the 32.5 kda band was in tensified (lane ). Fig. 2. Eletrophoreti patterns by Stains-All staining of matrix protein extrated from alium phosphate (brushite) he 3 bands of 45, 32.5 and 25 KDa appeared in the thrombin undigested lane (lane ) he 45 kda band disappeared after thrombin digestion, and the 32.5 kda band was intensified (lane ).

4 (kda) YAMAE, et al.: Osteopontin, Calium ontaining stones 625 (kda) Fig. 3. Eletrophoreti patterns by Stains-All staining of matrix protein extrated from magnesium ammonium phosphate (struvite) he 2 bands of35 and 25 kda appeared in the thrombin undigested lane (lane C). hey were unhanged after thrombin digestion (lane ). sium ammonium phosphate stones showed 2 bands of 35 and 25 kda (Fig. 3, lane C). he bands were unhanged after thrombin digestion (Fig. 3, lane ). No bands appeared after Stains-All staining for the uri aid stones (Fig. 4, lane C, lane ). he NH2- terminal 20 amino aids in the band were the same as those of 0 PN 9 ) DISCUSSION Although many reports have suggested the importane of the matrix in urinary stone formation, little is known about the omposition of the stone matrix l - S ) Our previous moleular study showed that the -DNA sequene of OPN enoded the alium oxalate urinary stone protein 9 ). he present study using Stains-All showed that the alium ontaining urinary stones (alium oxalate, and alium phosphate) ontained OPN protein. OPN is a major omponent of mineralizing onnetive tissues suh as bone and dentine l4 ) Nagata et a1. 15 ) reported that OPN ould be extrated from bone and identified with Stains-All staining. Stains-All is a ationi arboyanine dye and is suitable for staining sialoglyoproteins and phosphoproteins l6 ) OPN is a sialated phosphorylated glyoprotein. Sine OPN has a thrombin-suseptible site (Arg-Gly-Asp tripeptides) in the middle of the moleule, thrombin digestion produes 23 kda and 33 kda bands from a 52 kda band l7 ) he 49.5 and 45 kda bands (in lane C of Fig. I and 2) from Fig. 4. Eletrophoreti patterns by Stains-All staining of matrix protein extrated from uri aid. No bands appeared in the thrombin undigested lane (lane C) and after thrombin digestion (lane ). the alium oxalate and alium phosphate stones, respetively were attributed to OPN. he 2 low moleular bands of 32.5 and 25 kda were obtained from both the alium oxalate and alium phosphate stones after thrombin digestion. Only 2 low moleular bands of 35 and 25 kda were observed in the proteins extrated from the struvite stones (Fig. 3). he absene of OPN in struvite stones may be beause alium ions are not involved in the formation of struvite stones as with uri aid stones, and therefore, OPN with affinity for alium ions was not neessary. Another possibility is degradation of OPN in the formation proess of struvite stones. Further studies are neessary. On the other hand, the lak of a definite band in Fig. 4 suggested that the uri aid stones did not ontain OPN. his study revealed that OPN was extrated from only aliumontaining stones among 4 kinds of stones. OPN binds Ca 2 +, and has a negative harge, in addition to having a high affinity for hydroxyapatite. It has a potential to at as both a nuleator of hydroxyapatite rystal formation as well as a regulator of rystal growth and dissolution, these ativities being dependent upon the physial state of the protein l8 ) Shiraga et a1. 19 ) disovered In human unne uropontin, whih is a protein with homology at 40 sites of amino aids for rat, mouse and porine OPN. hey reported that this uropontin inhibited the rystal growth of alium oxalate in vivo, although they had expeted it to promote the rystal growth in vivo. We previously identified OPN by a moleular

5 626 Ata Urol. Jpn. Vol. 43, No.9, 1997 biologial method. he separation and identifiation of OPN in the stone protein was diffiult by the onventional method. OPN IS a highly phosphorylated protein and was not stained with Coomassie brilliant blue. All onventional staining methods of the stone protein used Coomassie brilliant blue. In this study, OPN in the stone protein ould be stained with Stains-All, but further studies are needed on the role, of OPN in stones and its involvement in in vitro rystallization. he finding obtained in the present study and the strong binding properties of OPN to Ca 2 + suggest that OPN is related to stone formation in the aliumontaing stones. ACKNOWLEDGMENS he authors wish to thank Drs. K. Kaho, N. Amasaki, A. Suzuki, K. Yoshida for their valuable tehnial advie. his work was supported by Grants in Aid from the Ministry of Siene, Eduation, Sports, and Culture of the Japan, Suzuki Urologial Promotion Fund, and Grant-in-Aid of he Japan Medial Assoiation. REFERENCES 1) Boye WH and Garvey FK: he amount and nature of the organi matrix in urinary aluli. J Urol 76: , ) King JS and Boye WH: Amino aid and arbohydrate_ omposition of the muoprotein matrix in various aluli. Pro So Exp Bioi Med 95: , ) Umekawa and Kurita : Calprotetin-like protein is related to soluble organi matrix in alium oxalate urinary stone. Biohem Mole Bio Int 34: , ) Umekawa, Yamate, Kurita, et al.: Sequening of a urinary stone proteins, idential to alpha-one antitripsin, whih laks 22 amino aids. Biohem Biophys Res Commun 193: , ) Speter AR, Gray A and Prien WLJr: Kidney stone matrix. Differenes in aidi protein omposition. Invest Urol 13: , ) LianJB, PrienJr, Glimher EL, et al. : he presene of protein-bound y-arboxyglutami aid In alium-ontaining renal aluli. Clin Si 59: , ) Meli RA, Quelh KJ and Rhodes M: he demonstration of siali aid in kidney stone matrix. Clin Soi 59: , ) Jones W and Resnik MI : he haraterization of soluble matrix proteins in seleted human renal aluli using two-dimensional polyarylamide gel eletrophoresis. J Urol 144: , ) Kohri K, Yam ate, Kurita, et al.: Moleular loning and sequening of edna enoding urinary stone protein, whih is idential to osteopontin. Biohem Biophys Res Commun 184: , ) Kohri K, Umekawa, Kurita, et al. : Struture and express of the mrna enoding urinary stone protein (osteopontin). J Bioi Ch"em 268: , ) Sodek J, Chen J, Nagata, et al. : Sialoproteins in bone remodeling. Bio Meha ooth Cranio 38: , ) Goldberg HA, Domeniui C, Sodek J, et al.: Mineral-binding proteoglyans of fetal porine alvarial bone. J Biolumin Chemilumin 263: , ) Nagata, Domeniui C, Sodek J, et al.: Biosynthesis of bone proteins by fetal porine alvariae in vitro. Rapid assoiation of sulfated sialoproteins and hondroitin sulfate proteoglyan with bone mineral. Matrix 11: , ) Mark MP, Butler W, Ruh JV, et al.: Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone y-arboxyglutami aid-ontaining alifying tissues of rat. 136, 1988 protein (osteoalin) In Differentiation 37: ) Nagata, odesan R, Goldberg HA, et al.: Sulphation of sereted phosphoprotein I (SPPI, osteopontin) is assoiated with mineralized tissue formation. Biohem Biophys Res Commun 165: , ) Campbell KP, MaLennan DH and Jorgensen AO: Staining of the Ca 2 + -binding proteins, Calsequestrin, Calmodulin, roponin C, and S-100, with the ationi arboyanine dye 'Stains-All'. J Biolumin Chemilumin 18: , ) Nagata, Kasugai S, Sodek J, et al. : Biosynthesis of bone proteins (seretad phosphoprotein-i, osteopontin), BSP (bone sialoprotein) and SPARC (osteonetin) in assoiation with mineralized-tissue formation by fetal-rat arvarial ells in ulture. Biohem J 274: , ) Linde A and Lussi A: Mineral indution by polyanioni dentin and bone proteins at physiologial ioni onditions. Connet issue Res 21: , ) Shiraga H, Min W, Hoyer JR, et al. : Inhibition of alium oxalate rystal growth In vitro by uropontin: another protein superfamily , 1992 member of asparti aid-rih Pro Natl Aad Si USA 82 : ( Reeived on January 7, 1997) Aepted on June 12, 1997

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