Abstract
Our aim was to examine the attachment to, and incorporation of intact, highly phosphorylated osteopontin (OPN) into inorganic (i) and urinary (u) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. uCOM and uCOD crystals were precipitated from ultrafiltered (UF) urine containing human milk OPN (mOPN) labelled with Alexa Fluor 647 fluorescent dye at concentrations of 0.1–5.0 mg/L. iCOM and iCOD crystals were generated in aqueous solutions at concentrations of 0.01–0.5 mg/L. Crystals were demineralised with EDTA and the resulting extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting, or examined by fluorescent confocal microscopy and field emission scanning electron microscopy before and after washing to remove proteins bound reversibly to the crystal surfaces. Binding of mOPN to pre-formed iCOM crystals was also studied in phosphate-buffered saline (PBS) and ultrafiltered (UF) urine. mOPN attached to the {100} faces and to the {010} sides of the {100}/{010} edges of iCOM crystals was removed by washing, indicating that it was not incorporated into the mineral bulk. In both PBS and urine, mOPN was attached to the {021} faces of pre-formed iCOM crystals as well as to the {100}/{010} edges, but was concentrated at the intersection points of the {100} and {121} faces at the crystal tips. Attachment in UF urine appeared to be greater than in PBS and stronger at higher calcium concentrations than lower calcium concentrations. In uCOM crystals, the distribution of fluorescence and patterns of erosion after washing suggested attachment of mOPN to the four end faces, followed by interment within the mineral phase. Fluorescence distributions of mOPN associated with both iCOD and uCOD crystals were consistent with uniform binding of the protein to all equivalent {101} faces and concentration along the intersections between them. Persistence of fluorescence after washing indicated that most mOPN was incarcerated within the mineral phase. We concluded that attachment of mOPN to calcium oxalate crystals is face-specific and depends upon the anatomical and genetic source of the protein and whether the crystals are (1) COM or COD; (2) pre-formed or precipitated from solution, and (3) precipitated from urine or aqueous solutions. Our findings emphasise the need for caution when drawing conclusions about possible roles of OPN or other proteins in urolithiasis from experimental data obtained under inorganic conditions.
Similar content being viewed by others
References
Addadi L, Weiner S (1985) Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc Natl Acad Sci USA 82:4110–4114
Addadi L, Weiner S, Geva M (2001) On how proteins interact with crystals and their effect on crystal formation. Z Kardiol 90:92–98
Weiner S, Addadi L (1991) Acidic macromolecules of mineralized tissues: the controllers of crystal formation. Trends Biochem Sci 16:252–256
Fleming DE, van Riessen A, Chauvet MC, Grover PK, Hunter B, van Bronswijk W, Ryall RL (2003) Intracrystalline proteins and urolithiasis: a synchrotron X-ray diffraction study of calcium oxalate monohydrate. J Bone Min Res 18:1282–1291
Ryall RL, Chauvet MC, Grover PK (2005) Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals. BJU Int 96:654–663
Doyle IR, Ryall RL, Marshall VR (1991) Inclusion of proteins into calcium oxalate crystals precipitated from human urine: a highly selective phenomenon. Clin Chem 37:1589–1594
Merchant M, Cummins T, Wilkey D, Salyer S, Powell D, Klein J, Lederer E (2008) Proteomic analysis of renal calculi indicates an important role for inflammatory processes in calcium stone formation. Am J Physiol Renal Physiol 295:F1254–F1258
McKee MD, Nanci A, Khan SR (1995) Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi. J Bone Min Res 10:1913–1929
Tawada T, Fujita K, Sakakura T, Shibutani T, Nagata T, Iguchi M, Kohri K (1999) Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone. Urol Res 27:238–242
Hoyer JR, Otvos L, Urge L (1995) Osteopontin in urinary stone formation. Ann N Y Acad Sci 760:257–265
Kleinman JG, Wesson JA, Hughes J (2004) Osteopontin and calcium stone formation. Nephron Physiol 98:43–47
Denhardt DT, Guo X (1993) Osteopontin: a protein with diverse functions. FASEB J 7:1475–1482
Wang L, Qiu SR, Zachowicz W, Guan X, De Yoreo JJ, Nancollas GH, Hoyer JR (2006) Modulation of calcium oxalate crystallization by linear aspartic acid-rich peptides. Langmuir 22:7279–7285
Christensen B, Nielsen MS, Haselmann KF, Petersen TE, Sørensen ES (2005) Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five o-glycosylation sites and their biological implications. Biochem J 390:285–292
Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS (2001) Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochem Biophys Res Commun 280:460–465
Shiraga H, Min W, VanDusen WJ, Clayman MD, Miner D, Terrell CH, Sherbotie JR, Foreman JW, Przysiecki C, Neilson EG, Hoyer JR (1992) Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily. Proc Natl Acad Sci USA 89:426–430
Worcester EM, Blumenthal SS, Beshensky AM, Lewand DL (1992) The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin. J Bone Min Res 7:1029–1036
Boskey AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW (1993) Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatine-gel. Bone Miner 22:147–159
Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA (1996) Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 317:59–64
Saavedra RA (1994) The roles of autophosphorylation and phosphorylation in the life of osteopontin. Bioessays 16:913–918
Langdon A, Wignall GR, Rogers K, Sørensen ES, Denstedt J, Grohe B, Goldberg HA, Hunter GK (2009) Kinetics of calcium oxalate crystal growth in the presence of osteopontin isoforms: an analysis by scanning confocal interference microscopy. Calcif Tissue Int 84:240–248
Hunter GK, Grohe B, Jeffrey S, O’Young J, Sørensen ES, Goldberg HA (2009) Role of phosphate groups in inhibition of calcium oxalate crystal growth by osteopontin. Cells Tissue Org 189:44–50
Gericke A, Qin C, Spevak L, Fujimoto Y, Butler WT, Sørensen ES, Boskey AL (2005) Importance of phosphorylation for osteopontin regulation of biomineralization. Calcif Tissue Int 77:45–54
de Bruijn WC, de Water R, van Run PR, Boevé ER, Kok DJ, Cao LC, Romijn HC, Verkoelen CF, Schröder FH (1997) Ultrastructural osteopontin localization in papillary stones induced in rats. Eur Urol 32:360–367
de Water R, Noordermeer C, van der Kwast TH, Nizze H, Boevé ER, Kok DJ, Schröder FH (1999) Calcium oxalate nephrolithiasis: effect of renal crystal deposition on the cellular composition of the renal interstitium. Am J Kidney Dis 33:761–771
de Water R, Noordermeer C, Houtsmuller AB, Nigg AL, Stijnen T, Schröder FH, Kok DJ (2000) Role of macrophages in nephrolithiasis in rats: an analysis of the renal interstitium. Am J Kidney Dis 36:615–625
de Water R, Leenen PJ, Noordermeer C, Nigg AL, Houtsmuller AB, Kok DJ, Schröder FH (2001) Cytokine production induced by binding and processing of calcium oxalate crystals. Am J Kidney Dis 38:331–338
Mandel NS, Mandel GS (1989) Urinary tract stone incidence in the US veteran population: II. Geographical analysis of variations in composition. J Urol 142:1516–1521
Dyer R, Nordin BE (1967) Urinary crystals and their relation to stone formation. Nature 215:751–752
Elliot JS, Rabinowitz IN (1980) Calcium oxalate crystalluria: crystal size in urine. J Urol 123:324–327
Wesson JA, Worcester E (1996) Formation of hydrated calcium oxalates in the presence of poly-l-aspartic acid. Scanning Microsc Int 10:415–424
Wesson JA, Worcester EM, Wiessner JH, Mandel NS, Kleinmann JG (1998) Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules. Kidney Int 53:952–957
Lieske JC, Leonard R, Toback FG (1995) Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions. Am J Physiol 268:F604–F612
Kumar V, Farell G, Lieske JC (2003) Whole urinary proteins coat calcium oxalate monohydrate crystals to greatly decrease their adhesion to renal cells. J Urol 170:221–225
Grover PK, Thurgood LA, Ryall RL (2007) Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis. Am J Physiol Renal Physiol 292:F1396–F1403
Grover PK, Wang T, Thurgood LA, Ryall RL (2009) The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine. BJU Int (epub)
Chien YC, Masica DL, Gray JJ, Nguyen S, Vali H, McKee MD (2009) Modulation of calcium oxalate dihydrate crystal growth by selective crystal face binding of phosphorylated osteopontin and poly-aspartate peptide showing occlusion by sectoral (compositional) zoning. J Biol Chem 284:23491–23501
Qiu SR, Wierzbicki A, Orme CA, Cody AM, Hoyer JR, Nancollas GH, Zepeda S, De Yoreo JJ (2004) Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. Proc Natl Acad Sci 101:1811–1815
Taller A, Grohe B, Rogers KA, Goldberg HA, Hunter GK (2007) Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohydrate crystals. Biophys J 93:1768–1777
Grohe B, O’Young J, Ionescu DA, Lajoie G, Rogers KA, Karttunen M, Goldberg HA, Hunter GK (2007) Control of calcium oxalate crystal growth by face-specific adsorption of an osteopontin phosphopeptide. J Am Chem Soc 129:14946–14951
O’Young J, Chirico S, Al Tarhuni N, Grohe B, Karttunen M, Goldberg HA, Hunter GK (2009) Phosphorylation of osteopontin peptides mediates adsorption to and incorporation into calcium oxalate crystals. Cells Tissues Organs 189:51–55
Senger DR, Perruzzi CA, Papadopoulos A, Tenen DG (1989) Purification of a human milk protein closely similar to tumor-secreted phosphoproteins and osteopontin. Biochim Biophys Acta 996:43–48
Ryall RL, Grover PK, Thurgood LA, Chauvet MC, Fleming DE, van Bronswijk W (2007) The importance of a clean face: the effect of different washing procedures on the association of Tamm-Horsfall glycoprotein and other urinary proteins with calcium oxalate crystals. Urol Res 35:1–14
Bautista DS, Denstedt J, Chambers AF, Harris JF (1996) Low-molecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones. J Cell Biochem 61:402–409
Hoyer JR, Pietrzyk RA, Liu H, Whitson PA (1999) Effects of microgravity on urinary osteopontin. J Am Soc Nephrol 10:S389–S393
Thurgood LA, Grover PK, Ryall RL (2008) High calcium concentration and calcium oxalate crystals cause significant inaccuracies in the measurement of urinary osteopontin by enzyme linked immunosorbent assay. Urol Res 36:103–110
Min W, Shiraga H, Chalko C, Goldfarb S, Krishna GG, Hoyer JR (1998) Quantitative studies of human urinary excretion of uropontin. Kidney Int 53:189–193
Cook AF, Grover PK, Ryall RL (2008) Face-specific binding of prothrombin fragment 1 and human serum albumin to inorganic and urinary calcium oxalate monohydrate crystals. BJU Int 103:826–835
Hess B, Ryall RL, Kavanagh JP, Khan SR, Kok D-J, Rodgers AL, Tiselius H-G (2001) Methods for measuring crystallization in urolithiasis research—why, how and when? Eur Urol 40:220–230
Brown LF, Berse B, Van de Water L, Papadopoulos-Sergiou A, Perruzzi CA, Manseau EJ, Dvorak HF, Senger DR (1992) Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Mol Biol Cell 3:1169–1180
Jung T, Sheng X, Choi CK, Kim WS, Wesson JA, Ward MD (2004) Probing crystallization of calcium oxalate monohydrate and the role of macromolecule additives with in situ atomic force microscopy. Langmuir 20:8587–8596
Millan A (2001) Crystal growth shape of whewellite polymorphs: influence of structure distortions on crystal shape. Cryst Growth Des 1:245–254
Ryall RL, Fleming DE, Doyle IR, Evans NA, Dean CJ, Marshall VR (2001) Intracrystalline proteins and the hidden ultrastructure of calcium oxalate urinary crystals: implications for kidney stone formation. J Struct Biol 134:5–14
Ryall RL, Fleming DE, Grover PK, Chauvet MC, Dean CJ, Marshall VR (2000) The hole truth: intracrystalline proteins and calcium oxalate kidney stones. Mol Urol 4:391–402
Ryall RL (2004) Macromolecules and urolithiasis: parallels and paradoxes. Nephron 98:37–42
Khan SR, Kok DJ (2004) Modulation of urinary stone formation. Front Biosci 9:1450–1482
Chauvet MC, Ryall RL (2005) Intracrystalline proteins and calcium oxalate crystal degradation in MDCK II cells. J Struct Biol 151:12–17
Grover PK, Thurgood LA, Fleming DE, van Bronswijk W, Wang T, Ryall RL (2008) Intracrystalline urinary proteins facilitate degradation and dissolution of calcium oxalate crystals in cultured renal cells. Am J Physiol 294:F336–F355
Wang L, Guan X, Tang R, Hoyer JR, Wierzbicki A, De Yoreo JJ, Nancollas GH (2008) Phosphorylation of osteopontin in required for inhibition of calcium oxalate crystallization. J Phys Chem B 112:9151–9157
Christensen B, Petersen TE, Sørensen ES (2008) Post-translational modification and proteolytic processing of urinary osteopontin. Biochem J 411:53–61
Kon S, Maeda M, Segawa T, Hagiwara Y, Horikoshi Y, Chikuma S, Tanaka K, Rashid MM, Inobe M, Chambers AF, Uede T (2000) Antibodies to different peptides in osteopontin reveal complexities in the various secreted forms. J Cell Biochem 77:487–498
Hunter GK, Kyle CL, Goldberg HA (1994) Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation. Biochem J 300:723–728
Giachelli CM, Steitz S (2000) Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol 19:612–622
Kasemo B, Lausmaa J (1994) Material-tissue interfaces: the role of surface properties and processes. Environ Health Perspect 102(Suppl 5):41–45
Boskey AL (1995) Osteopontin and related phosphorylated sialoproteins: effects on mineralization. Ann N Y Acad Sci 760:249–256
Beshensky AM, Wesson JA, Worcester EM, Sorokina EJ, Snyder CJ, Kleinman JG (2001) Effects of urinary macromolecules on hydroxyapatite crystal formation. J Am Soc Nephrol 12:2108–2116
Guo SW, Ward MD, Wesson JA (2002) Direct visualization of calcium oxalate monohydrate crystallization and dissolution with atomic force microscopy and the role of polymeric additives. Langmuir 18:4284–4291
Grohe B, Taller A, Vincent PL, Tieu LD, Rogers KA, Heiss A, Sørensen ES, Mittler S, Goldberg H, Hunter GK (2009) Crystallization of calcium oxalate is controlled by molecular hydrophilicity and specific polyanion-crystal interactions. Langmuir 25:11635–11646
Asplin JR, Arsenault D, Parks JH, Coe FL, Hoyer JR (1998) Contribution of human uropontin to inhibition of calcium oxalate crystallization. Kidney Int 53:194–199
Sheng X, Ward MD, Wesson JA (2005) Crystal surface adhesion explains the pathological activity of calcium oxalate hydrates in kidney stone formation. J Am Soc Nephrol 16:1904–1908
Chen Y, Bal BS, Gorski JP (1992) Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone. J Biol Chem 267:24871–24878
Tazzoli V, Domeneghetti C (1980) The crystal structures of whewellite and weddellite: re-examination and comparison. Am Mineral 65:327–334
Acknowledgments
The authors are indebted to Dr Jennifer Clarke for assistance with the confocal microscopy and to Dr Roger Qiu of the Lawrence Livermore National Laboratory, USA, for invaluable advice regarding the labelling of urinary COM crystal faces. Support from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant 1R01-DK-064050-01A1) is gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Thurgood, L.A., Cook, A.F., Sørensen, E.S. et al. Face-specific incorporation of osteopontin into urinary and inorganic calcium oxalate monohydrate and dihydrate crystals. Urol Res 38, 357–376 (2010). https://doi.org/10.1007/s00240-010-0300-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00240-010-0300-7