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A New Approach to the Study of Urinary Macromolecules as a Participant in Calcium Oxalate Crystallization
0022-534 7/88/1394-0869$02.00/0
Vol. 139, April Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1988 by The Williams & Wilkins Co.
A NEW APPROACH TO THE STUDY OF URINARY MACROMOLECULES AS A PARTICIPANT IN CALCIUM OXALATE CRYSTALLIZATION REID M. MORSE
AND
MARTIN I. RESNICK*
From the Division of Urology, Case Western Reserve University School of Medicine, Cleveland, Ohio ABSTRACT
Despite intense investigation, the relationship of urinary proteins to urinary stone matrix formation remains poorly understood. In an attempt to gain more information regarding this interaction, the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro was studied. 0.1 M calcium chloride and 0.1 M sodium oxalate were added to an aliquot of urine collected from five non-infected, non-stone forming males. The resulting calcium oxalate crystals were centrifuged and the pellet demineralized with 5% EDTA. Gel filtration chromatography was used to isolate the protein fraction from the urine samples before and after crystallization of calcium oxalate. The proteins recovered from the crystals and urine were separated by two-dimensional polyacrylamide gel electrophoresis. Many, but not all of the urinary proteins were bound by the crystals. Albumin, seen to be the most abundant urinary protein, was absent or markedly diminished in all instances, and the second most abundant urinary protein, PC-30, became the predominant protein component of the crystals. An unidentified protein with approximate molecular weight of 22,000 daltons and isoelectric point of 6.4 was highly concentrated by the crystals even when undetected in the urine. The study suggests that the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro is not a random event but rather a selective phenomenon. (J. Ural., 139:869-873, 1988) More than three hundred years have passed since Anton von Heyde demineralized a urinary calculus and observed that the gross structure of the stone remained intact.' Studies by numerous investigators continued regarding the structure of urinary stones and by the early 1820's the crystalline content was understood, although the composition of the "framework", or matrix was largely ignored. Significant effort has since been directed towards the investigation of this material and though much insight has been gained, many questions remain unanswered. On a dry weight/weight basis, matrix comprises approximately 2.5% of all calcigerous calculi. Uric acid calculi are comprised of between 0.5 and 2.0% of this material, whereas struvite and cystine stones contain 0.5% and 9.0%, respectively.2 Matrix itself is a heterogenous material composed of approximately 64% protein, 9% nonamino sugars, 5% glucosamine, 10% bound water and 12% organic ash. 3 Many distinct proteins are present in stone matrix, derived from both serum (alpha globulin, beta glubulin, albumin, etc.) and kidney (Tamm-Horsfall Mucroprotein and proteoglycans). The major controversy concerning the role of matrix in stone formation and growth is largely related to the selectivity in which the material is formed. Boyce believes that the basic composition of matrix is constant regardless of the crystalline component of a stone,3 and that the material forms in a very selective manner. It is not the random adsorption of macromolecules onto the surface of growing crystals but rather a highly selective process that is required for stone formation to occur. Accordingly, a differentiating factor between stone formers and non-stone formers relates to the ability of the stone former to produce or deposit this required material and if this cannot be done (as in the non-stone former) stone formation will not occur. Leal and Finlayson have performed experiments Accepted for publication December 14, 1987. * Requests for reprints: Division of Urology, Case Western Reserve University, School of Medicine, 2065 Adelbert Rd., Cleveland, OH44106. 869
showing that physical adsorption could not account for all of the deposition of mucoproteins in calcium oxalate stones and that some specific mechanisms are operating to account for their presence. 4 Others have suggested that because the protein content of matrix varies between stones of different crystalline composition, the process is a non-specific event and follows stone formation rather than being either required or specific. 5 • 6 Vermulen and Lyon supported this concept and concluded that the origin of stone matrix is secondary to the non-specific adsorption of urinary mucoproteins. 7 In an attempt to gain a better understanding of this subject, we have studied the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro and have extracted the urinary proteins to determine the selectivity of the process. The model utilizes a system of in vitro crystallization as well as gel filtration chromatography and two-dimensional electrophoresis. MATERIALS AND METHODS
Sample collection and initial sample preparation. Urine samples were obtained from five non-infected, non-stone forming males between 21 and 29 years of age. All participants, healthy individuals on no therapy of any type, provided a random morning sample of at least 200 ml. The urine was collected in sterile glass containers and the urinary pH immediately brought to 6.5 with either 0.1 M sodium hydroxide or 0.1 M hydrogen chloride to prevent spontaneous crystallization. After centrifugation at 1500 RPM for ten minutes, the supernatant was filtered through qualitative grade #613 filter paper and the filtrate stored at 4C for several hours until it was used for gel filtration chromatography and calcium oxalate crystallization. Gel filtration chromatography. The previously treated urine was desalted using the protocol of Tollaksen and Anderson. 8 100 ml. was applied to a 5 X 35 cm. gel filtration column containing Bio-Gel P-6 which has an exclusion of 6000 daltons. The column was eluted at 100 ml./hr. with water purified by
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filtering house-demineralized water through one Barnstead Organic Removal Cartridge and two Barnstead Mixed-Bed Ca:rtridges (Fisher Scientific Co., Pittsburgh, Pa.) The eluant was monitored for protein content at a wavelength of 280 nm. (ISCO Absorbance Monitor Model UA-5, ISCO Recorder Model 613, Lincoln Nebraska), the corresponding protein peak collected utilizing an ISCO Fraction Collector (Model 328), and this fraction lyophilized with a Labconco Freeze-Dryer (Labconco Corporation, Kansas City, Mo.) The resulting golden-brown powder was resuspended in 5.0 ml. purified water and applied to a 2.5 x 15 cm. column also containing Bio-Gel P-6. Five milliliters of 1.0 M sodium chloride was applied just prior to sample loading to free any adsorbed particles. The protein peak was collected and lyophilized in a similar manner as previously described, producing a white, electrostatic material. All columns, solutions, and glassware were maintained in a cold-room at 4C throughout the studies. Calcium oxalate crystallization. To initiate calcium oxalate crystal formation in urine, 100 ml. of 0.1 M calcium chloride and 100 ml. of 0.1 M sodium oxalate was added to a 100 ml. aliquot of previously treated urine. This was performed over a period of five hours at 4C with a Buchler Multistatic 4-channel pump (Fisher Scientific Co.). A non-crushing magnetic stir bar maintained the resulting crystals in suspension during addition of the two solutions and stirring was continued for an additional four hours. Crystal composition was confirmed to be calcium oxalate by x-ray diffraction analysis performed at a commercial stone analysis laboratory (Calculab, Richmond, Virginia). After centrifugation at 2000 RPM for five minutes, the supernatant was filtered through grade #613 filter paper and the filtrate subjected to the same desalting protocol as the original urine sample. The pellet produced during centrifugation of the suspended crystals was washed to prevent contamination from the remaining urinary proteins. This was performed by resuspending the pellet in a 50 ml. aliquot of purified water and centrifuging the crystals at 2000 RPM for five minutes. The process was repeated for a total of six cycles. The crystals were then suspended in 10 ml. of purified water, placed in dialysis tubing with a 6000 dalton cut-off, (Spectropore Membrane Tubing, #132655, Spectrumedical Industries, Inc.) and demineralized against 5% ethylenedinitrilotetraacetic acid (EDTA) at pH 7.9 at 4C. The dialysate was stirred with a magnetic stir bar and changed every twelve hours until the crystals had dissolved (approximately seven days). Dialysis was then carried out against purified water at 4C, changing the dialysate every twelve hours for four days in order to remove the residuai EDTA. The remaining liquid was lyophilized to recover the non -dialyzable material. Two-dimensional electrophoresis. Two-dimensional polyacrylamide gel electrophoresis, a method of protein resolution based on varying isoelectric points and molecular weights, was used to analyze the three prepared samples (urine before and after crystallization, and crystals). This was performed on the Bio-Rad Protean-II system (Bio-Rad, Richmond, Cal.) with several modifications in the described protocol. 9 • 10 The isoelectric focusing, or first dimension, was begun by casting 15.5 cm. tube gels in 18.0 cm. glass tubes with an inside diameter of 1.5 mm. A 5.5 ml. aliquot of gel solution (48.6 gm. urea, 11.8 ml. 30% acrylamide/bis-acrylamide stock, 28.8 ml. purified water, 20.3 ml. triton X-100, 4.5 ml. Bio-Rad ampholyte 5/7, 0.5 ml. Bio-Rad ampholyte 3/10) was degassed for fifteen minutes, polymerized with 5.5 microliters of tetramethylethylenediamine (TEMED) and 7.25 microliters of ammonium persulfate, and poured into the glass tubes using a fine needle. After polymerization, (approximately 1.5 hours), 5.0 microliters of 2% sodium dodecyl sulfate (SDS) was added to the top of each tube gel, which ultimately produced a bulge at the acidic end of the gel for easy reference. The gels were prefocused for one hour at 200 volts with 0.1 N sodium hydroxide and 0.06%
phosphoric acid used as the cathode and anode solution, respectively. Samples to be run were prepared in a 9.0 M urea mix (27 gm. urea, 2.5 ml. ampholyte 3/10, 2.5 ml. 2-mercaptoethanol, purified water to 50 ml.) to denature the proteins. A Bio-Rad dye binding protein assay11 was used to monitor protein load, with 40 micrograms applied to each gel. After focusing for 14,000 volt-hours (17.5 hours at 800 volts), the tube gels were extruded from the glass tubes using a tuberculin syringe adapted with a pipet tip. Each gel was equilibrated for twenty minutes in four ml. of equilibration buffer (11.23 gm. trishydroxymethyl aminomethane (Tris), 15.75 gm. SDS, 75 ml. glycerol, 50 mg. bromphenol blue, to 750 ml. with purified water; pH 6.8) while the solution was agitated. The second dimension was run through 1.5 x 16 x 16 cm. gels cast between glass plates. Identical gels were cast simultaneously in a casting chamber with a Bio-Rad gradient former and a Manostat varistaltic pump (Manostat, N.Y.) used to produce a 10 to 20% acrylamide gradient which facilitates protein resolution. The 10% acrylamide solution (49.5 ml. 30% acrylamide/bis-acrylamide stock solution, 1.65 ml. 10% SDS, 41.25 ml. 1.5 M Tris at pH 8.8, 72.6 ml. purified water) and 20% acrylamide solution (99.0 ml. 30% acrylamide/bis-acrylamide stock solution, 1.65 ml. 10% SDS, 41.25 ml. 1.5 M Tris at pH 8.8, 23.1 ml. purified water) was degassed for fifteen minutes and polymerized with TEMED and ammonium persulfate (0.825 ml. 10% ammonium persulfate, 82.5 microliters TEMED added to 10% solution; 0.825 ml. 10% ammonium persulfate, 41.25 microliters TEMED added to 20% solution). Each gel was overlayed with 0.5 ml. of tert-amyl alcohol and allowed to polymerize for 1.5 hours. The equilibrated tube gels were loaded onto the top of the slab gels with the acidic end on the left. The slab gels were then placed into an electrophoresis tank filled with electrolyte buffer (15 gm. Tris, 72 gm. glycine, 5 gm. SDS, to 5.0 L with purified water; pH 8.3), and the system run at 30 milliamps/gel until the dye front reached the edge of the gels (approximately 7.5 hours). A circulating cooler maintained the gels at 4C, preventing thermal distortion. Gel processing and analysis. The gels were fixed and stained with silver nitrate according to the protocol by Guevara. 12 Identification of protein spots was carried out utilizing twodimensional electrophoretic maps and comparing prior reports of other researchers. 13- 17 Protein patterns were compared by directly superimposing the gels. The gels were stored in polyethylene tubing (Birnberg Machinery Inc, Skokey, 11.) with 10 ml. 0.5% acetic acid added as a preservative. RESULTS
Figures 1 through 3 are representative gels produced during these experiments. Figure 1 shows the two-dimensional pattern of the original urinary proteins, figure 2 the pattern of proteins recovered from the calcium oxalate crystals formed in the urine, and figure 3 the pattern of proteins obtained from urine following calcium oxalate crystallization. The gels are oriented so that the left side is acidic and the right side basic, with the higher molecular weight proteins located at the top. The limits of resolution range from isoelectric points (pl) of approximately 4.5 to 8.6 and molecular weights from 10,000 to 150,000 daltons. Many of the proteins exist in only one form yielding one discrete spot on a gel but others have undergone post-translational charge modifications including deamidation, phosphorylation, or sialization. These modified peptides appear as a row of horizontal spots. Proteins receiving carbohydrate groups as well as charge modifications after peptide synthesis most often appear slanted upward and to the left. 13 An example of this phenomenon can be seen in figure 1. The individual protein (B), to be discussed below, appears as four separate spots grouped around 30,000 daltons and spans pl's of 5.1 to 5.8. Comparison of protein patterns from urine prior to crystal-
URINARY MACROMOLECULES IN CALCIUM OXALATE CRYSTALLIZATION
FIG. 1. Two-dimensional electrophoretic pattern of proteins from original urine sample.
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FIG. 3. Two-dimensional electrophoretic pattern of proteins from urine sample after calcium oxalate crystals were formed and removed.
lization and the calcium oxalate crystals reveals that many of the protein spots are included in both gels. It is also apparent that some spots are absent from the calcium oxalate gel and that there are several spots in this gel that are not present in the gel from the urinary sample. For example, albumin (A) 14 is the most abundant urinary protein present. However, of the proteins bound to the crystals, albumin is conspicuously dimin ished in quantity in this preparation and was absent in others. The second most abundant urinary protein was identified as PC-30, or, alpha-2 microglubulin. 15 This is the most prominent protein associated with the crystals (B). An unidentified protein with approximate molecular weight of 22,000 daltons and pl of 6.4 (C) was highly concentrated by the crystals even when undetected in the original urine sample. This protein was considered to be the much discussed matrix substance A (MSA) but does not meet the criteria, in that MSA has a reported pl near 4.5 and a molecular weight in the range of 30,000 to 40,000 daltons. 18• 19 Another striking example of differences between these two preparations is that of the lgG light chains which are located in a horizontal row at 25,000 daltons and range from a pl of 6.0 to 7.9 (D). 17 These appear quite prominently among the urinary proteins and are absent from the calcium oxalate crystal preparation. The gels from the urine post-crystallization are virtually identical to those of the original urine sample. It is believed that only a small portion of the proteins were extracted during the crystallization process and therefore no differences could be discerned between the two urine samples. It had been postulated that the growing crystals might completely clear one or perhaps several proteins from the urine, but this did not occur. DISCUSSION
FIG. 2. Two-dimensional electrophoretic pattern of proteins recovered from calcium oxalate crystals.
Urinary stone matrix is a complex heterogeneous mixture of organic molecules originating from either serum, kidney, urine
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or some combination of these. Its composition includes a combination of proteins, carbohydrates, nucleic acids and glycosaminoglycans. The present experiments have focused on the protein fraction and not the other materials which also have been noted to be specifically incorporated into urinary calculi. Studies have shown that the glycosaminoglycans heparan sulfate and hyaluronic acid are present in matrix in fractions disproportionate to their urinary concentrations, and that chondroitin sulfate, the most abundant glycosaminoglycan in urine, is notably absent. 2 • 20 The goals of our experimentation included the development and use of a technique to study the selectivity in which urinary macromolecules are incorporated into calcium oxalate crystals formed in urine in vitro. It was learned that urinary proteins with molecular weights between 10,000 and 150,000 daltons and isoelectric points of 4.5 to 8.6 could be recovered from calcium oxalate crystals and qualitatively analyzed with good reproducibility. The differences observed between the crystalbound proteins and the proteins present in the original urine samples suggests that the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro is a selective process. Conversely, a finding of similar protein patterns among these preparations would have favored a random, or non-selective process. It is interesting to speculate about the nature of the various unknown proteins present on the gels, for once a hypothesis regarding their identity is made, studies using specific antibodies or electrophoretic standards can be performed to establish their true identity. For example, there is an area between 35,000 and 68,000 daltons containing very prominent protein spots which may represent the vitamin K-dependant blood clotting factors. These are proteins which have molecular weights ranging from 55,000 to 65,000 daltons or containing several polypeptide chains ranging between 12,000 and 40,000 daltons. 21 • 22 They are all comprised of large amounts of amino acid gammacarboxyglutamic acid which is responsible, in part, for the binding of calcium. 22 Lian has reported that calcium containing kidney stones contain gamma-carboxyglutamic acid within their matrix 23 whereas uric acid, cystine and magnesium ammonium phosphate stones do not. Recently, this amino acid was noted to be a component in pediatric bladder stones containing calcium salts, 24 and it has also been found that 30 to 50% more free and protein-bound gamma-carboxyglutamic acid was excreted in the urine from active stone formers than from normal individuals. 25 The role of this substance in urinary stone formation has not been established, although one can speculate that it may promote nucleation by allowing the local concentration of calcium oxalate to exceed its formation product. In conclusion, the data produced in this study suggest that the incorporation of urinary proteins into the supporting matrix of calcium oxalate crystals formed in urine in vitro as a selective process. Electron microscopic studies of human stones also indicates the process is an orderly one. 26 Our goal was not to produce "artificial kidney stones", for it is well recognized that differences in temperature, mineral concentration, and physical environment exist between any in vitro system and the human kidney. We are presently conducting studies in our laboratory to investigate the protein content of calcium oxalate renal calculi using two-dimensional electrophoresis. Preliminary data reveals similarities between the protein pattern seen from these stones and the pattern observed from the calcium oxalate crystals produced in our present study. The similarities are most prevalent among the proteins which are considered to have calcium binding activity. As expected, differences are also apparent, suggesting that the native stones contain more high molecular weight proteins than the laboratory counterpart. In the future, this technique will also be used to study the matrix of different urinary calculi including uric acid, calcium phosphate, magnesium ammonium phosphate and cystine.
Acknowledgment. The authors are indebted to Chung Lee, Ph.D., Department of Urology, Northwestern University School of Medicine, who has been a diligent advisor during the course of this study. REFERENCES L Butt, A. S.: History of treatment of urinary lithiasis. In: Treatment of Urinary Lithiasis. Edited by Butt, A. S. Thomas, Springfield, pp 3-89, 1960. 2. Roberts, S. D. and Resnick, M. I.: Glycosaminoglycans content of stone matrix. J. Urol., 135: 1078, 1986. 3. Boyce, W. H.: Organic matrix of human urinary concretions. Am. J. Med., 45: 673, 1968. 4. Leal, J. J. and Finlayson, B.: Adsorption of naturally occuring polymers onto calcium oxalate crystal surfaces. Invest. Urol., 14: 278, 1977. 5. Spector, A. R., Gray, A. and Prien Jr., E. L.: Kidney stone matrix. Differences in acidic protein composition. Invest. Urol., 13: 387, 1976. 6. Foye, W. 0., Hong, S. H., Kim, M. C. and Prien Sr., E. L.: Degree of sulfation in mucopolysaccharide sulfates in normal and stoneforming urines. Invest. Urol., 14: 33, 1976. 7. Vermulen, C. W. and Lyon, E. S.: Mechanisms of genesis and growth of calculi. Am. J. Med., 45: 684, 1968. 8. Edwards, J. J., Tollaksen, S. L. and Anderson, N. G.: Proteins of human urine. III. Identification and two-dimensional Electrophoretic map positions of some major urinary proteins. Clin. Chem., 28: 941, 1982. 9. Two-dimensional polyacrylamide gel electrophoresis. Bio-Rad. Bulletin #1144, 1985. 10. Acrylamide polymerization-A practical approach. Bio-Rad. Bulletin #1156, 1984. 11. Bio-Rad Protein Assay. Technical Bulletin. #1051, 1977. 12. Guevara Jr., J., Johnston, A., Ramagalli, L. S., Martin, B. A., Capetillo, S. and Rodriguez, L. V.: Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gels. Electrophoresis, 3: 197, 1082. 13. Anderson, N. G., Anderson, N. L. and Tollaksen, S. L.: Proteins of human urine. I. Concentration and analysis by two-dimensional electrophoresis. Clin. Chem. 25: 1199, 1979. 14. Frearson, N., Taylor, R. D. and Perry, S. V.: Proteins in the urine associated with Duchenne muscular dystrophy and other neuromuscular disease. Clin. Science, 61: 141, 1981. 15. Lee, C.: Personal Communication. 16. Tsai, Y. C., Harrison, H. H., Lee, C., Daufeldt, 0. L. and Grayhack, J. T.: Systematic characterization of human prostatic fluid proteins with two-dimensional electrophoresis, Clin. Chem., 30: 2026, 1984. 17. Tracy, R. P. and Young, D, S.: Clinical applications of two-dimensional gel electrophoresis. In: Two-Dimensional Gel Electrophoresis of Proteins. Methods and applications. Edited by Celis, J. A. and Bravo, R. Academic Press, Orlando, pp. 194-240, 1984. 18. Boyce, W. H., King Jr., J. S. and Fielden, M. L.: Total nondialyzable solids (TNDS) in human urine. XIII. Immunological detection of a component peculiar to renal calculous matrix and to urine of calculous patients. J. Clin. Invest., 41: 1180, 1962. 19. King, J. S. and Boyce, W. H.: Immunological studies on serum and urinary proteins in uroltith matrix in man. Ann. NY Acad. Sci., 104: 579, 1963, 20. Nishio, S., Abe, Y., Wakatuki, A., Iwata, H., Ochi, K., Takeuchi, M. and Matsumoto, A.: Matrix glycosaminoglycans in urinary stones. J. Urol., 134: 503, 1985. 21. Rosenberg, R. D.: Hemorrhagic disorders I. Protein interactions in the clotting mechanisms. In: Hematology. Edited by Beck, W. S. MIT Press, Cambridge, pp. 373-400, 1981. 22. Nelsestuen, G. L.: Calcium function in vitamin K-dependent proteins. In: Calcium-Binding Proteins and Calcium Function. Edited by Wasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R.H., MacLennan, D. H. and Siegel, F. L. North-Holland, New York, pp. 323-332, 1977. 23. Lian J. B., Prien, E. L., Glimcher Jr., M. J. and Gallop, P. M.: The presence of protein bound gamma-carboxyglutamic acid in calcium containing renal calculi. J. Clin. Invest., 59: 1151, 1977. 24. Ogasawara, K., Reen, R. V. and Ako H.: Gamma-carboxyglutamic acid, a component in human pediatric bladder stones containing
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URINARY MACROMOLECULES IN CALCIUM OXALATE CRYSTALLIZATION calcium salts. J. Urol., 137: 349, 1987. 25. Resnick, M. I., Gammon, C. W., Sorrell, M. B. and Boyce, W. H.: Calcium-binding proteins and renal lithiasis. Surgery, 88: 239, 1980.
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26. Boyce, W. H.: Some observations on the ultrastructure of "idiopathic" human renal calculi. In: Urolithiasis. Physical Aspects. Edited by Finlayson, B., Hench, L. L. and Smith, L. H. National Academy of Sciences, Washington, D.C., pp. 97-114, 1972.
Vol. 139, April Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1988 by The Williams & Wilkins Co.
A NEW APPROACH TO THE STUDY OF URINARY MACROMOLECULES AS A PARTICIPANT IN CALCIUM OXALATE CRYSTALLIZATION REID M. MORSE
AND
MARTIN I. RESNICK*
From the Division of Urology, Case Western Reserve University School of Medicine, Cleveland, Ohio ABSTRACT
Despite intense investigation, the relationship of urinary proteins to urinary stone matrix formation remains poorly understood. In an attempt to gain more information regarding this interaction, the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro was studied. 0.1 M calcium chloride and 0.1 M sodium oxalate were added to an aliquot of urine collected from five non-infected, non-stone forming males. The resulting calcium oxalate crystals were centrifuged and the pellet demineralized with 5% EDTA. Gel filtration chromatography was used to isolate the protein fraction from the urine samples before and after crystallization of calcium oxalate. The proteins recovered from the crystals and urine were separated by two-dimensional polyacrylamide gel electrophoresis. Many, but not all of the urinary proteins were bound by the crystals. Albumin, seen to be the most abundant urinary protein, was absent or markedly diminished in all instances, and the second most abundant urinary protein, PC-30, became the predominant protein component of the crystals. An unidentified protein with approximate molecular weight of 22,000 daltons and isoelectric point of 6.4 was highly concentrated by the crystals even when undetected in the urine. The study suggests that the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro is not a random event but rather a selective phenomenon. (J. Ural., 139:869-873, 1988) More than three hundred years have passed since Anton von Heyde demineralized a urinary calculus and observed that the gross structure of the stone remained intact.' Studies by numerous investigators continued regarding the structure of urinary stones and by the early 1820's the crystalline content was understood, although the composition of the "framework", or matrix was largely ignored. Significant effort has since been directed towards the investigation of this material and though much insight has been gained, many questions remain unanswered. On a dry weight/weight basis, matrix comprises approximately 2.5% of all calcigerous calculi. Uric acid calculi are comprised of between 0.5 and 2.0% of this material, whereas struvite and cystine stones contain 0.5% and 9.0%, respectively.2 Matrix itself is a heterogenous material composed of approximately 64% protein, 9% nonamino sugars, 5% glucosamine, 10% bound water and 12% organic ash. 3 Many distinct proteins are present in stone matrix, derived from both serum (alpha globulin, beta glubulin, albumin, etc.) and kidney (Tamm-Horsfall Mucroprotein and proteoglycans). The major controversy concerning the role of matrix in stone formation and growth is largely related to the selectivity in which the material is formed. Boyce believes that the basic composition of matrix is constant regardless of the crystalline component of a stone,3 and that the material forms in a very selective manner. It is not the random adsorption of macromolecules onto the surface of growing crystals but rather a highly selective process that is required for stone formation to occur. Accordingly, a differentiating factor between stone formers and non-stone formers relates to the ability of the stone former to produce or deposit this required material and if this cannot be done (as in the non-stone former) stone formation will not occur. Leal and Finlayson have performed experiments Accepted for publication December 14, 1987. * Requests for reprints: Division of Urology, Case Western Reserve University, School of Medicine, 2065 Adelbert Rd., Cleveland, OH44106. 869
showing that physical adsorption could not account for all of the deposition of mucoproteins in calcium oxalate stones and that some specific mechanisms are operating to account for their presence. 4 Others have suggested that because the protein content of matrix varies between stones of different crystalline composition, the process is a non-specific event and follows stone formation rather than being either required or specific. 5 • 6 Vermulen and Lyon supported this concept and concluded that the origin of stone matrix is secondary to the non-specific adsorption of urinary mucoproteins. 7 In an attempt to gain a better understanding of this subject, we have studied the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro and have extracted the urinary proteins to determine the selectivity of the process. The model utilizes a system of in vitro crystallization as well as gel filtration chromatography and two-dimensional electrophoresis. MATERIALS AND METHODS
Sample collection and initial sample preparation. Urine samples were obtained from five non-infected, non-stone forming males between 21 and 29 years of age. All participants, healthy individuals on no therapy of any type, provided a random morning sample of at least 200 ml. The urine was collected in sterile glass containers and the urinary pH immediately brought to 6.5 with either 0.1 M sodium hydroxide or 0.1 M hydrogen chloride to prevent spontaneous crystallization. After centrifugation at 1500 RPM for ten minutes, the supernatant was filtered through qualitative grade #613 filter paper and the filtrate stored at 4C for several hours until it was used for gel filtration chromatography and calcium oxalate crystallization. Gel filtration chromatography. The previously treated urine was desalted using the protocol of Tollaksen and Anderson. 8 100 ml. was applied to a 5 X 35 cm. gel filtration column containing Bio-Gel P-6 which has an exclusion of 6000 daltons. The column was eluted at 100 ml./hr. with water purified by
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filtering house-demineralized water through one Barnstead Organic Removal Cartridge and two Barnstead Mixed-Bed Ca:rtridges (Fisher Scientific Co., Pittsburgh, Pa.) The eluant was monitored for protein content at a wavelength of 280 nm. (ISCO Absorbance Monitor Model UA-5, ISCO Recorder Model 613, Lincoln Nebraska), the corresponding protein peak collected utilizing an ISCO Fraction Collector (Model 328), and this fraction lyophilized with a Labconco Freeze-Dryer (Labconco Corporation, Kansas City, Mo.) The resulting golden-brown powder was resuspended in 5.0 ml. purified water and applied to a 2.5 x 15 cm. column also containing Bio-Gel P-6. Five milliliters of 1.0 M sodium chloride was applied just prior to sample loading to free any adsorbed particles. The protein peak was collected and lyophilized in a similar manner as previously described, producing a white, electrostatic material. All columns, solutions, and glassware were maintained in a cold-room at 4C throughout the studies. Calcium oxalate crystallization. To initiate calcium oxalate crystal formation in urine, 100 ml. of 0.1 M calcium chloride and 100 ml. of 0.1 M sodium oxalate was added to a 100 ml. aliquot of previously treated urine. This was performed over a period of five hours at 4C with a Buchler Multistatic 4-channel pump (Fisher Scientific Co.). A non-crushing magnetic stir bar maintained the resulting crystals in suspension during addition of the two solutions and stirring was continued for an additional four hours. Crystal composition was confirmed to be calcium oxalate by x-ray diffraction analysis performed at a commercial stone analysis laboratory (Calculab, Richmond, Virginia). After centrifugation at 2000 RPM for five minutes, the supernatant was filtered through grade #613 filter paper and the filtrate subjected to the same desalting protocol as the original urine sample. The pellet produced during centrifugation of the suspended crystals was washed to prevent contamination from the remaining urinary proteins. This was performed by resuspending the pellet in a 50 ml. aliquot of purified water and centrifuging the crystals at 2000 RPM for five minutes. The process was repeated for a total of six cycles. The crystals were then suspended in 10 ml. of purified water, placed in dialysis tubing with a 6000 dalton cut-off, (Spectropore Membrane Tubing, #132655, Spectrumedical Industries, Inc.) and demineralized against 5% ethylenedinitrilotetraacetic acid (EDTA) at pH 7.9 at 4C. The dialysate was stirred with a magnetic stir bar and changed every twelve hours until the crystals had dissolved (approximately seven days). Dialysis was then carried out against purified water at 4C, changing the dialysate every twelve hours for four days in order to remove the residuai EDTA. The remaining liquid was lyophilized to recover the non -dialyzable material. Two-dimensional electrophoresis. Two-dimensional polyacrylamide gel electrophoresis, a method of protein resolution based on varying isoelectric points and molecular weights, was used to analyze the three prepared samples (urine before and after crystallization, and crystals). This was performed on the Bio-Rad Protean-II system (Bio-Rad, Richmond, Cal.) with several modifications in the described protocol. 9 • 10 The isoelectric focusing, or first dimension, was begun by casting 15.5 cm. tube gels in 18.0 cm. glass tubes with an inside diameter of 1.5 mm. A 5.5 ml. aliquot of gel solution (48.6 gm. urea, 11.8 ml. 30% acrylamide/bis-acrylamide stock, 28.8 ml. purified water, 20.3 ml. triton X-100, 4.5 ml. Bio-Rad ampholyte 5/7, 0.5 ml. Bio-Rad ampholyte 3/10) was degassed for fifteen minutes, polymerized with 5.5 microliters of tetramethylethylenediamine (TEMED) and 7.25 microliters of ammonium persulfate, and poured into the glass tubes using a fine needle. After polymerization, (approximately 1.5 hours), 5.0 microliters of 2% sodium dodecyl sulfate (SDS) was added to the top of each tube gel, which ultimately produced a bulge at the acidic end of the gel for easy reference. The gels were prefocused for one hour at 200 volts with 0.1 N sodium hydroxide and 0.06%
phosphoric acid used as the cathode and anode solution, respectively. Samples to be run were prepared in a 9.0 M urea mix (27 gm. urea, 2.5 ml. ampholyte 3/10, 2.5 ml. 2-mercaptoethanol, purified water to 50 ml.) to denature the proteins. A Bio-Rad dye binding protein assay11 was used to monitor protein load, with 40 micrograms applied to each gel. After focusing for 14,000 volt-hours (17.5 hours at 800 volts), the tube gels were extruded from the glass tubes using a tuberculin syringe adapted with a pipet tip. Each gel was equilibrated for twenty minutes in four ml. of equilibration buffer (11.23 gm. trishydroxymethyl aminomethane (Tris), 15.75 gm. SDS, 75 ml. glycerol, 50 mg. bromphenol blue, to 750 ml. with purified water; pH 6.8) while the solution was agitated. The second dimension was run through 1.5 x 16 x 16 cm. gels cast between glass plates. Identical gels were cast simultaneously in a casting chamber with a Bio-Rad gradient former and a Manostat varistaltic pump (Manostat, N.Y.) used to produce a 10 to 20% acrylamide gradient which facilitates protein resolution. The 10% acrylamide solution (49.5 ml. 30% acrylamide/bis-acrylamide stock solution, 1.65 ml. 10% SDS, 41.25 ml. 1.5 M Tris at pH 8.8, 72.6 ml. purified water) and 20% acrylamide solution (99.0 ml. 30% acrylamide/bis-acrylamide stock solution, 1.65 ml. 10% SDS, 41.25 ml. 1.5 M Tris at pH 8.8, 23.1 ml. purified water) was degassed for fifteen minutes and polymerized with TEMED and ammonium persulfate (0.825 ml. 10% ammonium persulfate, 82.5 microliters TEMED added to 10% solution; 0.825 ml. 10% ammonium persulfate, 41.25 microliters TEMED added to 20% solution). Each gel was overlayed with 0.5 ml. of tert-amyl alcohol and allowed to polymerize for 1.5 hours. The equilibrated tube gels were loaded onto the top of the slab gels with the acidic end on the left. The slab gels were then placed into an electrophoresis tank filled with electrolyte buffer (15 gm. Tris, 72 gm. glycine, 5 gm. SDS, to 5.0 L with purified water; pH 8.3), and the system run at 30 milliamps/gel until the dye front reached the edge of the gels (approximately 7.5 hours). A circulating cooler maintained the gels at 4C, preventing thermal distortion. Gel processing and analysis. The gels were fixed and stained with silver nitrate according to the protocol by Guevara. 12 Identification of protein spots was carried out utilizing twodimensional electrophoretic maps and comparing prior reports of other researchers. 13- 17 Protein patterns were compared by directly superimposing the gels. The gels were stored in polyethylene tubing (Birnberg Machinery Inc, Skokey, 11.) with 10 ml. 0.5% acetic acid added as a preservative. RESULTS
Figures 1 through 3 are representative gels produced during these experiments. Figure 1 shows the two-dimensional pattern of the original urinary proteins, figure 2 the pattern of proteins recovered from the calcium oxalate crystals formed in the urine, and figure 3 the pattern of proteins obtained from urine following calcium oxalate crystallization. The gels are oriented so that the left side is acidic and the right side basic, with the higher molecular weight proteins located at the top. The limits of resolution range from isoelectric points (pl) of approximately 4.5 to 8.6 and molecular weights from 10,000 to 150,000 daltons. Many of the proteins exist in only one form yielding one discrete spot on a gel but others have undergone post-translational charge modifications including deamidation, phosphorylation, or sialization. These modified peptides appear as a row of horizontal spots. Proteins receiving carbohydrate groups as well as charge modifications after peptide synthesis most often appear slanted upward and to the left. 13 An example of this phenomenon can be seen in figure 1. The individual protein (B), to be discussed below, appears as four separate spots grouped around 30,000 daltons and spans pl's of 5.1 to 5.8. Comparison of protein patterns from urine prior to crystal-
URINARY MACROMOLECULES IN CALCIUM OXALATE CRYSTALLIZATION
FIG. 1. Two-dimensional electrophoretic pattern of proteins from original urine sample.
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FIG. 3. Two-dimensional electrophoretic pattern of proteins from urine sample after calcium oxalate crystals were formed and removed.
lization and the calcium oxalate crystals reveals that many of the protein spots are included in both gels. It is also apparent that some spots are absent from the calcium oxalate gel and that there are several spots in this gel that are not present in the gel from the urinary sample. For example, albumin (A) 14 is the most abundant urinary protein present. However, of the proteins bound to the crystals, albumin is conspicuously dimin ished in quantity in this preparation and was absent in others. The second most abundant urinary protein was identified as PC-30, or, alpha-2 microglubulin. 15 This is the most prominent protein associated with the crystals (B). An unidentified protein with approximate molecular weight of 22,000 daltons and pl of 6.4 (C) was highly concentrated by the crystals even when undetected in the original urine sample. This protein was considered to be the much discussed matrix substance A (MSA) but does not meet the criteria, in that MSA has a reported pl near 4.5 and a molecular weight in the range of 30,000 to 40,000 daltons. 18• 19 Another striking example of differences between these two preparations is that of the lgG light chains which are located in a horizontal row at 25,000 daltons and range from a pl of 6.0 to 7.9 (D). 17 These appear quite prominently among the urinary proteins and are absent from the calcium oxalate crystal preparation. The gels from the urine post-crystallization are virtually identical to those of the original urine sample. It is believed that only a small portion of the proteins were extracted during the crystallization process and therefore no differences could be discerned between the two urine samples. It had been postulated that the growing crystals might completely clear one or perhaps several proteins from the urine, but this did not occur. DISCUSSION
FIG. 2. Two-dimensional electrophoretic pattern of proteins recovered from calcium oxalate crystals.
Urinary stone matrix is a complex heterogeneous mixture of organic molecules originating from either serum, kidney, urine
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or some combination of these. Its composition includes a combination of proteins, carbohydrates, nucleic acids and glycosaminoglycans. The present experiments have focused on the protein fraction and not the other materials which also have been noted to be specifically incorporated into urinary calculi. Studies have shown that the glycosaminoglycans heparan sulfate and hyaluronic acid are present in matrix in fractions disproportionate to their urinary concentrations, and that chondroitin sulfate, the most abundant glycosaminoglycan in urine, is notably absent. 2 • 20 The goals of our experimentation included the development and use of a technique to study the selectivity in which urinary macromolecules are incorporated into calcium oxalate crystals formed in urine in vitro. It was learned that urinary proteins with molecular weights between 10,000 and 150,000 daltons and isoelectric points of 4.5 to 8.6 could be recovered from calcium oxalate crystals and qualitatively analyzed with good reproducibility. The differences observed between the crystalbound proteins and the proteins present in the original urine samples suggests that the binding of urinary proteins to calcium oxalate crystals formed in urine in vitro is a selective process. Conversely, a finding of similar protein patterns among these preparations would have favored a random, or non-selective process. It is interesting to speculate about the nature of the various unknown proteins present on the gels, for once a hypothesis regarding their identity is made, studies using specific antibodies or electrophoretic standards can be performed to establish their true identity. For example, there is an area between 35,000 and 68,000 daltons containing very prominent protein spots which may represent the vitamin K-dependant blood clotting factors. These are proteins which have molecular weights ranging from 55,000 to 65,000 daltons or containing several polypeptide chains ranging between 12,000 and 40,000 daltons. 21 • 22 They are all comprised of large amounts of amino acid gammacarboxyglutamic acid which is responsible, in part, for the binding of calcium. 22 Lian has reported that calcium containing kidney stones contain gamma-carboxyglutamic acid within their matrix 23 whereas uric acid, cystine and magnesium ammonium phosphate stones do not. Recently, this amino acid was noted to be a component in pediatric bladder stones containing calcium salts, 24 and it has also been found that 30 to 50% more free and protein-bound gamma-carboxyglutamic acid was excreted in the urine from active stone formers than from normal individuals. 25 The role of this substance in urinary stone formation has not been established, although one can speculate that it may promote nucleation by allowing the local concentration of calcium oxalate to exceed its formation product. In conclusion, the data produced in this study suggest that the incorporation of urinary proteins into the supporting matrix of calcium oxalate crystals formed in urine in vitro as a selective process. Electron microscopic studies of human stones also indicates the process is an orderly one. 26 Our goal was not to produce "artificial kidney stones", for it is well recognized that differences in temperature, mineral concentration, and physical environment exist between any in vitro system and the human kidney. We are presently conducting studies in our laboratory to investigate the protein content of calcium oxalate renal calculi using two-dimensional electrophoresis. Preliminary data reveals similarities between the protein pattern seen from these stones and the pattern observed from the calcium oxalate crystals produced in our present study. The similarities are most prevalent among the proteins which are considered to have calcium binding activity. As expected, differences are also apparent, suggesting that the native stones contain more high molecular weight proteins than the laboratory counterpart. In the future, this technique will also be used to study the matrix of different urinary calculi including uric acid, calcium phosphate, magnesium ammonium phosphate and cystine.
Acknowledgment. The authors are indebted to Chung Lee, Ph.D., Department of Urology, Northwestern University School of Medicine, who has been a diligent advisor during the course of this study. REFERENCES L Butt, A. S.: History of treatment of urinary lithiasis. In: Treatment of Urinary Lithiasis. Edited by Butt, A. S. Thomas, Springfield, pp 3-89, 1960. 2. Roberts, S. D. and Resnick, M. I.: Glycosaminoglycans content of stone matrix. J. Urol., 135: 1078, 1986. 3. Boyce, W. H.: Organic matrix of human urinary concretions. Am. J. Med., 45: 673, 1968. 4. Leal, J. J. and Finlayson, B.: Adsorption of naturally occuring polymers onto calcium oxalate crystal surfaces. Invest. Urol., 14: 278, 1977. 5. Spector, A. R., Gray, A. and Prien Jr., E. L.: Kidney stone matrix. Differences in acidic protein composition. Invest. Urol., 13: 387, 1976. 6. Foye, W. 0., Hong, S. H., Kim, M. C. and Prien Sr., E. L.: Degree of sulfation in mucopolysaccharide sulfates in normal and stoneforming urines. Invest. Urol., 14: 33, 1976. 7. Vermulen, C. W. and Lyon, E. S.: Mechanisms of genesis and growth of calculi. Am. J. Med., 45: 684, 1968. 8. Edwards, J. J., Tollaksen, S. L. and Anderson, N. G.: Proteins of human urine. III. Identification and two-dimensional Electrophoretic map positions of some major urinary proteins. Clin. Chem., 28: 941, 1982. 9. Two-dimensional polyacrylamide gel electrophoresis. Bio-Rad. Bulletin #1144, 1985. 10. Acrylamide polymerization-A practical approach. Bio-Rad. Bulletin #1156, 1984. 11. Bio-Rad Protein Assay. Technical Bulletin. #1051, 1977. 12. Guevara Jr., J., Johnston, A., Ramagalli, L. S., Martin, B. A., Capetillo, S. and Rodriguez, L. V.: Quantitative aspects of silver deposition in proteins resolved in complex polyacrylamide gels. Electrophoresis, 3: 197, 1082. 13. Anderson, N. G., Anderson, N. L. and Tollaksen, S. L.: Proteins of human urine. I. Concentration and analysis by two-dimensional electrophoresis. Clin. Chem. 25: 1199, 1979. 14. Frearson, N., Taylor, R. D. and Perry, S. V.: Proteins in the urine associated with Duchenne muscular dystrophy and other neuromuscular disease. Clin. Science, 61: 141, 1981. 15. Lee, C.: Personal Communication. 16. Tsai, Y. C., Harrison, H. H., Lee, C., Daufeldt, 0. L. and Grayhack, J. T.: Systematic characterization of human prostatic fluid proteins with two-dimensional electrophoresis, Clin. Chem., 30: 2026, 1984. 17. Tracy, R. P. and Young, D, S.: Clinical applications of two-dimensional gel electrophoresis. In: Two-Dimensional Gel Electrophoresis of Proteins. Methods and applications. Edited by Celis, J. A. and Bravo, R. Academic Press, Orlando, pp. 194-240, 1984. 18. Boyce, W. H., King Jr., J. S. and Fielden, M. L.: Total nondialyzable solids (TNDS) in human urine. XIII. Immunological detection of a component peculiar to renal calculous matrix and to urine of calculous patients. J. Clin. Invest., 41: 1180, 1962. 19. King, J. S. and Boyce, W. H.: Immunological studies on serum and urinary proteins in uroltith matrix in man. Ann. NY Acad. Sci., 104: 579, 1963, 20. Nishio, S., Abe, Y., Wakatuki, A., Iwata, H., Ochi, K., Takeuchi, M. and Matsumoto, A.: Matrix glycosaminoglycans in urinary stones. J. Urol., 134: 503, 1985. 21. Rosenberg, R. D.: Hemorrhagic disorders I. Protein interactions in the clotting mechanisms. In: Hematology. Edited by Beck, W. S. MIT Press, Cambridge, pp. 373-400, 1981. 22. Nelsestuen, G. L.: Calcium function in vitamin K-dependent proteins. In: Calcium-Binding Proteins and Calcium Function. Edited by Wasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R.H., MacLennan, D. H. and Siegel, F. L. North-Holland, New York, pp. 323-332, 1977. 23. Lian J. B., Prien, E. L., Glimcher Jr., M. J. and Gallop, P. M.: The presence of protein bound gamma-carboxyglutamic acid in calcium containing renal calculi. J. Clin. Invest., 59: 1151, 1977. 24. Ogasawara, K., Reen, R. V. and Ako H.: Gamma-carboxyglutamic acid, a component in human pediatric bladder stones containing
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URINARY MACROMOLECULES IN CALCIUM OXALATE CRYSTALLIZATION calcium salts. J. Urol., 137: 349, 1987. 25. Resnick, M. I., Gammon, C. W., Sorrell, M. B. and Boyce, W. H.: Calcium-binding proteins and renal lithiasis. Surgery, 88: 239, 1980.
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26. Boyce, W. H.: Some observations on the ultrastructure of "idiopathic" human renal calculi. In: Urolithiasis. Physical Aspects. Edited by Finlayson, B., Hench, L. L. and Smith, L. H. National Academy of Sciences, Washington, D.C., pp. 97-114, 1972.