Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate Chiaki Nakajima,* Koji Suzuki, and Ryuzo Tsugawa Department of Urology, Kanazawa M e d i c a l University, Kanazawa, Japan -
The presence of macromolecular substances is among the multiple factors that may influence the complex process of urinary stone formation. The aim of this study was to evaluate the inhibitory potency of crystal matrix protein (CMP). Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis and high-performance liquid chromatography (HPLC). The inhibitory potency of crystal aggregation was examined by the seed crystal method, the undiluted urine method, and the use of scanning electron microscopy (SEM). CMP showed the protein band of 31 kDa in SDS-PAGE. Anti-CMP polyclonal antibody and antihuman prothrombin antibody crossreacted well with human prothrombin and CMP in Western blotting. CMP and human prothrombin had high inhibitory potency by the seed crystal method and undiluted urine method. Using SEM, we were able to observe the high inhibitory potency of human prothrombin and undiluted CMP on the aggregation of calcium oxalate crystals. Int j Urol 1996;3(suppl 1):576-579 Key words: calcium oxalate crystal, crystal matrix protein, crystal growth and aggregation, growth and aggregation inhibitor, scanning electron microscopy
Recently, macromolecular substances have attracted the attention of investigators as inhibitors of calcium oxalate stones." The aim of this study was to examine the inhibitory potency of crystal matrix protein (CMP) extracted from newly formed calcium oxalate crystals.i We have already reported that the N-terminal amino acid sequence of CMP is completely identical to that of human prothrombin.' In the present study, therefore, we compared the inhibitory potency of CMP with that of human prothrombin. MATERIALS A N D METHODS Collection and Preparation of Urine Samples Fresh urine samples collected from 4 healthy males aged 28 to 32 years old, were centrifuged and filtered through Millipore filters to prepare spun and filtered urine (SF urine). Sodium oxalate was added to this SF urine, 30 pmol/L in excess of the metastable limit,7 in order to produce calcium oxalate crystals. Freeze-dried calcium oxalate crystals were then obtained from this urine sample. To extract CMP, the crystals were completely dissolved in 0.25 mol/L EDTA and subjected to electrodialysis using membrane cut-off substances of molecular weights under 10,000. Electrodialysis was conducted using Tdglycine solution at 60 volts for 6 hours, at 80 volts for 6 hours, then, using double distilled water, at 100 volts for 16 hours. The intracrystalline macromolecular substance thus obtained was freeze-dried. The substance was then purified using HPLC to CMP. The molecular weight of C M P was determined by SDSPAGE8 and was assessed by Western b l ~ t t i n g . ~
*Correspondence and requests for reprintz to: Department of Urology, Kanarawa Medical University, Uchinada, Kanazawa, lshikawa 920-02, ldpan.
S76
Seed Crystal Methodlo A metastable solution of calcium oxalate was prepared immediately before use. Then, calcium oxalate crystal suspension was added to the metastable solution, and the number and size of the crystals of each channel were counted immediately after the addition, then again after a 60-minute interval, using Coulter Counter TA-11. The inhibitory activity of aggregation (Ia) was calculated by the changes in the number of crystals with the addition of human prothrombin or CMP. The inhibitory activity of growth (Ig) was calculated from the change in crystal volume with prothrombin or C MP. These steps were repeated 6 times. Undiluted Urine Method" Using the ultrafiltration module, any macromolecules of more than 10,000 molecular weight were excluded to prepare ultrafiltered urine (UF urine). Excess sodium oxalate was added to U F urine, and the number and volume of crystals were counted over 90 minutes at 15-minute intervals. This experiment was repeated 6 times. Scanning Electron Microscopy (SEMI The 4 samples were: SF urine, UF urine, U F with 1 mg/L of human prothrombin added, and UF with 5 mg/L of CMP added. RESULTS SDS-PAGE and Western Blotting The molecular weight of C M P determined by SDS-PAGE was 31-kDa, differing from that of human prothrombin which has a molecular weight of 71-kDa. In Western blotting, C M P was highly reactive with
0919-8172/1996/03Sl-S76/US$3.00
0 )UA/CLJ
C. Nakajima et al.
Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate
a
human prothrombin polyclonal antibodies and CMP polyclonal antibodies, but it did not react with the monoclonal antibody of human prothrombin. These findings further suggest that C M P is an activation peptide of human prothrombin (Fig. 1).
1101 100-
90 -
Seed Crystal Method
Human prothrombin exhibited 50% of Ia at the concentration of 0.5 n m o l L and 50% of Ig at 5 nmoliL. CMP exhibited 50% of Ia and Ig at a concentration of 0.5 nmoliL. These data showed that crystal growth and aggregation were highly inhibited by an extremely low concentration of human prothrombin or CMP (Fig. 2a, b).
80 n
70-
v
60 -
s
0)
PTh la
a
_.....*
PTh lg
Undiluted Urine Method
The crystal volume tended to decrease following the addition of human prothrombin or CMP in the undiluted urine method. Furthermore, both human prothrombin and C M P inhibited the increase of crystal volume at low concentrations from 0.01 mg/L to 0.1 mg/L (Fig. 3a, b). SEM SEM showed the severe aggregation of crystals in U F urine without macromolecular substances in urine. In contrast, crystals were seen scattered in S F urine containing macromolecular substances. These findings proved that macromolecular substances had a high inhibitory activity of aggregation of calcium oxalate crystals. One milligram per liter of human prothrombin and 5 mg/ L of CMP were added to U F urine and observed by scanning electron microscopy. Both substances highly inhibited the aggregation of calcium oxalate crystals (Fig. 4).
0.5
0.1
1
5
10
50
100
Concentration of PTh (nmol/L)
b 1103
10090 80 -
70 60
-
50 40 -
CMP la CMP lg
30 20 -
1
10-
Concentration of CMP (nrnol/L) Fig. l a . Inhibitory activity of aggregation (la) and growth (Ig) of human prothrombin (PTh). b. Inhibitory activity of aggregation (la) and growth (lg) of CMP.
DISCUSSION
Fig. 1. SDS-PAGE. Lane 1, protein standard; lane 2, peak fraction of HPLC eluted at 12.06 min, a 31-kDa protein band (CMP) can be seen; lane 3, human prothrombin (PTh) (71 kDa). Western blotting of CMP (lane l ) , and human prothrombin (lane 2). Anti-CMP antibody reacted strongly with 31-kDa protein as well as prothrombin. Antiprothrombin monoclonal antibody nonreacted with 31 -kDa protein. Antimothrombin Dolvclonal antibodv reacted well to both PTh and 31-k‘Da protein.
Through these experiments, we have shown the inhibitory activities of human prothrombin and CMP on calcium oxalate crystal aggregation. With regard to inhibitors of crystal growth and aggregation, Nakagawa extracted nephrocalcin from macromolecular substances of human urine in 1983.’ Shiraga extracted uropontin from human urine in 1992.4 For inhibitory effects on growth, these substances were examined only by the seed crystal method, not by the undiluted urin’e method.
s77
Int J Urol 1996;3(suppl 1):576-579
a
b 1000 1
-
1
2500
3
!
\
-5
!
I .7
2000-
v
al
:
-
1500-
>
1
3
1000
-
-
O.Olrng/L O.OSrng/L O.lrng/L O.Srng/L lmg/L
-O-
_t
---t
5mg/L
-O-
..... .A.
V
.... ~
O.Olrng/L O.OSrng/L O.lrng/L O.Smg/L Irng/L Srng/L
SF UF
500
L
01
__1
10
100
Diameter (urn)
1
100
10
Diameter
(urn)
Fig. 3a. Crystal volume distribution at 90 minutes after the load of human prothrombin (PTh) in UF. b. Crystal volume distribution at 90 minutes after the load of CMP in UF.
Fig. 4. Scanning electron microscopic findings of calcium oxalate crystals at 90 minutes after addition of an overload of sodium oxalate to the undiluted urine method. a . Spun and filtered urine
578
(S&F). b. Ultrafiltered urine (UF). c. U F 1mdL. d. UF + CMP 5rndL.
+
human prothrombin
Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate
T h e seed crystal method is a n easy and reproducible method, a n d there are only 2 solution components used, oxalate a n d calcium. However, this does not offer a natural model for evaluation of inhibitory potency; the undiluted urine method should be used, instead. Our present s t u d y confirmed that CMP had an apparent inhibitory activity o f aggregation not only b y the seed crystal method but also b y the undiluted urine method. We extracted CMP, which is a crystal aggregation inhibitor, from calcium oxalate crystals. We then examined its inhibitory activity on crystal aggregation using the seed crystal method, undiluted u r i n e method and scanning electron microscopy. As the control, human p r o t h r o m b i n was examined using the same methods. CMP a n d human prothrombin showed the high inhibitory activity against crystal growth and aggregation. In conclusion, CMP is a crystal aggregation inhibitor. In the future, it will b e necessary to further s t u d y the relationship between CMP a n d other inhibitors such as nephrocalcin, osteopontin or glycosaminoglycans. REFERENCES
1. Nakagawa Y, Abram V, Kezdy EJ, Kaiser ET, and Coe FL. Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. J Biol Chem 1983;258:12594-12600. 2. Robertson WG, Knowles F, Peacock M. Urinary acid mucopolysaccharide inhibitors of calcium oxalate crystallization. In: Fleish H, Robertson WG, Smith LH, Vahlensieck W (eds). Urolithiasis research. New York: Plenum Press, 1976:331-334.
C. Nakajima et al.
3 . Nakagawa Y, Ahmed M, Hall SL, Deganello S , Coe FL. Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in ycarboxyglutamic acid. J Clin Invest 1987;79:1782-1787. 4. Shiraga H, Min W, VanDusen WJ, Clayman MD, Miner D, Przysiecki C, Neilson Terrel CH, Sherbotie JR, Foreman JW, EG, Hoyer JR. Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of aspartic acid rich protein superfamily. Proc Natl Acad Sci USA 1992;89:426-430. 5. Doyle IR, Ryall RL, Marshall VR. Inclusion of proteins into calcium oxalate crystal precipitated from human urine: a highly selective phenomenon. Clin Chem 1991;37: 15891594. 6. Suzuki K, Moriyama M, Nakajima C, Kawamura K, Miyazawa K,Tsugawa R, Kikuchi N , Nagata K. Isolation and characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation: Evidence of activation peptide of human prothrombin. Urol Res 1994;22:45-50. 7. Suzuki K, Miyazawa K, Tsugawa R. A simple method for determining the metastable limit of calcium oxalate. In: Walker VR, Sutton RAL, Cameron ECB, Pak CYC, Robertson WG (eds) Urolithiasis. New York: Plenum Press, 1989:65-66. 8 . Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacterio-phage T4. Nature 1970;227: 680-685. 9. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;7 6 :43 5 0-43 54. 10 Suzuki K, Miyazawa K,Tsugawa R. Inhibitory effect of sodium pentosan polysulfate on the formation, growth and aggregation of calcium oxalate in vitro. Jpn J Urol 1989;80:526-531. 11 Ryall RL, Hibberd CM, Marshall VR. A method for studying inhibitory activity in whole urine. Urol Res 1985;13:285289.
s79
The presence of macromolecular substances is among the multiple factors that may influence the complex process of urinary stone formation. The aim of this study was to evaluate the inhibitory potency of crystal matrix protein (CMP). Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis and high-performance liquid chromatography (HPLC). The inhibitory potency of crystal aggregation was examined by the seed crystal method, the undiluted urine method, and the use of scanning electron microscopy (SEM). CMP showed the protein band of 31 kDa in SDS-PAGE. Anti-CMP polyclonal antibody and antihuman prothrombin antibody crossreacted well with human prothrombin and CMP in Western blotting. CMP and human prothrombin had high inhibitory potency by the seed crystal method and undiluted urine method. Using SEM, we were able to observe the high inhibitory potency of human prothrombin and undiluted CMP on the aggregation of calcium oxalate crystals. Int j Urol 1996;3(suppl 1):576-579 Key words: calcium oxalate crystal, crystal matrix protein, crystal growth and aggregation, growth and aggregation inhibitor, scanning electron microscopy
Recently, macromolecular substances have attracted the attention of investigators as inhibitors of calcium oxalate stones." The aim of this study was to examine the inhibitory potency of crystal matrix protein (CMP) extracted from newly formed calcium oxalate crystals.i We have already reported that the N-terminal amino acid sequence of CMP is completely identical to that of human prothrombin.' In the present study, therefore, we compared the inhibitory potency of CMP with that of human prothrombin. MATERIALS A N D METHODS Collection and Preparation of Urine Samples Fresh urine samples collected from 4 healthy males aged 28 to 32 years old, were centrifuged and filtered through Millipore filters to prepare spun and filtered urine (SF urine). Sodium oxalate was added to this SF urine, 30 pmol/L in excess of the metastable limit,7 in order to produce calcium oxalate crystals. Freeze-dried calcium oxalate crystals were then obtained from this urine sample. To extract CMP, the crystals were completely dissolved in 0.25 mol/L EDTA and subjected to electrodialysis using membrane cut-off substances of molecular weights under 10,000. Electrodialysis was conducted using Tdglycine solution at 60 volts for 6 hours, at 80 volts for 6 hours, then, using double distilled water, at 100 volts for 16 hours. The intracrystalline macromolecular substance thus obtained was freeze-dried. The substance was then purified using HPLC to CMP. The molecular weight of C M P was determined by SDSPAGE8 and was assessed by Western b l ~ t t i n g . ~
*Correspondence and requests for reprintz to: Department of Urology, Kanarawa Medical University, Uchinada, Kanazawa, lshikawa 920-02, ldpan.
S76
Seed Crystal Methodlo A metastable solution of calcium oxalate was prepared immediately before use. Then, calcium oxalate crystal suspension was added to the metastable solution, and the number and size of the crystals of each channel were counted immediately after the addition, then again after a 60-minute interval, using Coulter Counter TA-11. The inhibitory activity of aggregation (Ia) was calculated by the changes in the number of crystals with the addition of human prothrombin or CMP. The inhibitory activity of growth (Ig) was calculated from the change in crystal volume with prothrombin or C MP. These steps were repeated 6 times. Undiluted Urine Method" Using the ultrafiltration module, any macromolecules of more than 10,000 molecular weight were excluded to prepare ultrafiltered urine (UF urine). Excess sodium oxalate was added to U F urine, and the number and volume of crystals were counted over 90 minutes at 15-minute intervals. This experiment was repeated 6 times. Scanning Electron Microscopy (SEMI The 4 samples were: SF urine, UF urine, U F with 1 mg/L of human prothrombin added, and UF with 5 mg/L of CMP added. RESULTS SDS-PAGE and Western Blotting The molecular weight of C M P determined by SDS-PAGE was 31-kDa, differing from that of human prothrombin which has a molecular weight of 71-kDa. In Western blotting, C M P was highly reactive with
0919-8172/1996/03Sl-S76/US$3.00
0 )UA/CLJ
C. Nakajima et al.
Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate
a
human prothrombin polyclonal antibodies and CMP polyclonal antibodies, but it did not react with the monoclonal antibody of human prothrombin. These findings further suggest that C M P is an activation peptide of human prothrombin (Fig. 1).
1101 100-
90 -
Seed Crystal Method
Human prothrombin exhibited 50% of Ia at the concentration of 0.5 n m o l L and 50% of Ig at 5 nmoliL. CMP exhibited 50% of Ia and Ig at a concentration of 0.5 nmoliL. These data showed that crystal growth and aggregation were highly inhibited by an extremely low concentration of human prothrombin or CMP (Fig. 2a, b).
80 n
70-
v
60 -
s
0)
PTh la
a
_.....*
PTh lg
Undiluted Urine Method
The crystal volume tended to decrease following the addition of human prothrombin or CMP in the undiluted urine method. Furthermore, both human prothrombin and C M P inhibited the increase of crystal volume at low concentrations from 0.01 mg/L to 0.1 mg/L (Fig. 3a, b). SEM SEM showed the severe aggregation of crystals in U F urine without macromolecular substances in urine. In contrast, crystals were seen scattered in S F urine containing macromolecular substances. These findings proved that macromolecular substances had a high inhibitory activity of aggregation of calcium oxalate crystals. One milligram per liter of human prothrombin and 5 mg/ L of CMP were added to U F urine and observed by scanning electron microscopy. Both substances highly inhibited the aggregation of calcium oxalate crystals (Fig. 4).
0.5
0.1
1
5
10
50
100
Concentration of PTh (nmol/L)
b 1103
10090 80 -
70 60
-
50 40 -
CMP la CMP lg
30 20 -
1
10-
Concentration of CMP (nrnol/L) Fig. l a . Inhibitory activity of aggregation (la) and growth (Ig) of human prothrombin (PTh). b. Inhibitory activity of aggregation (la) and growth (lg) of CMP.
DISCUSSION
Fig. 1. SDS-PAGE. Lane 1, protein standard; lane 2, peak fraction of HPLC eluted at 12.06 min, a 31-kDa protein band (CMP) can be seen; lane 3, human prothrombin (PTh) (71 kDa). Western blotting of CMP (lane l ) , and human prothrombin (lane 2). Anti-CMP antibody reacted strongly with 31-kDa protein as well as prothrombin. Antiprothrombin monoclonal antibody nonreacted with 31 -kDa protein. Antimothrombin Dolvclonal antibodv reacted well to both PTh and 31-k‘Da protein.
Through these experiments, we have shown the inhibitory activities of human prothrombin and CMP on calcium oxalate crystal aggregation. With regard to inhibitors of crystal growth and aggregation, Nakagawa extracted nephrocalcin from macromolecular substances of human urine in 1983.’ Shiraga extracted uropontin from human urine in 1992.4 For inhibitory effects on growth, these substances were examined only by the seed crystal method, not by the undiluted urin’e method.
s77
Int J Urol 1996;3(suppl 1):576-579
a
b 1000 1
-
1
2500
3
!
\
-5
!
I .7
2000-
v
al
:
-
1500-
>
1
3
1000
-
-
O.Olrng/L O.OSrng/L O.lrng/L O.Srng/L lmg/L
-O-
_t
---t
5mg/L
-O-
..... .A.
V
.... ~
O.Olrng/L O.OSrng/L O.lrng/L O.Smg/L Irng/L Srng/L
SF UF
500
L
01
__1
10
100
Diameter (urn)
1
100
10
Diameter
(urn)
Fig. 3a. Crystal volume distribution at 90 minutes after the load of human prothrombin (PTh) in UF. b. Crystal volume distribution at 90 minutes after the load of CMP in UF.
Fig. 4. Scanning electron microscopic findings of calcium oxalate crystals at 90 minutes after addition of an overload of sodium oxalate to the undiluted urine method. a . Spun and filtered urine
578
(S&F). b. Ultrafiltered urine (UF). c. U F 1mdL. d. UF + CMP 5rndL.
+
human prothrombin
Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate
T h e seed crystal method is a n easy and reproducible method, a n d there are only 2 solution components used, oxalate a n d calcium. However, this does not offer a natural model for evaluation of inhibitory potency; the undiluted urine method should be used, instead. Our present s t u d y confirmed that CMP had an apparent inhibitory activity o f aggregation not only b y the seed crystal method but also b y the undiluted urine method. We extracted CMP, which is a crystal aggregation inhibitor, from calcium oxalate crystals. We then examined its inhibitory activity on crystal aggregation using the seed crystal method, undiluted u r i n e method and scanning electron microscopy. As the control, human p r o t h r o m b i n was examined using the same methods. CMP a n d human prothrombin showed the high inhibitory activity against crystal growth and aggregation. In conclusion, CMP is a crystal aggregation inhibitor. In the future, it will b e necessary to further s t u d y the relationship between CMP a n d other inhibitors such as nephrocalcin, osteopontin or glycosaminoglycans. REFERENCES
1. Nakagawa Y, Abram V, Kezdy EJ, Kaiser ET, and Coe FL. Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. J Biol Chem 1983;258:12594-12600. 2. Robertson WG, Knowles F, Peacock M. Urinary acid mucopolysaccharide inhibitors of calcium oxalate crystallization. In: Fleish H, Robertson WG, Smith LH, Vahlensieck W (eds). Urolithiasis research. New York: Plenum Press, 1976:331-334.
C. Nakajima et al.
3 . Nakagawa Y, Ahmed M, Hall SL, Deganello S , Coe FL. Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in ycarboxyglutamic acid. J Clin Invest 1987;79:1782-1787. 4. Shiraga H, Min W, VanDusen WJ, Clayman MD, Miner D, Przysiecki C, Neilson Terrel CH, Sherbotie JR, Foreman JW, EG, Hoyer JR. Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of aspartic acid rich protein superfamily. Proc Natl Acad Sci USA 1992;89:426-430. 5. Doyle IR, Ryall RL, Marshall VR. Inclusion of proteins into calcium oxalate crystal precipitated from human urine: a highly selective phenomenon. Clin Chem 1991;37: 15891594. 6. Suzuki K, Moriyama M, Nakajima C, Kawamura K, Miyazawa K,Tsugawa R, Kikuchi N , Nagata K. Isolation and characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation: Evidence of activation peptide of human prothrombin. Urol Res 1994;22:45-50. 7. Suzuki K, Miyazawa K, Tsugawa R. A simple method for determining the metastable limit of calcium oxalate. In: Walker VR, Sutton RAL, Cameron ECB, Pak CYC, Robertson WG (eds) Urolithiasis. New York: Plenum Press, 1989:65-66. 8 . Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacterio-phage T4. Nature 1970;227: 680-685. 9. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;7 6 :43 5 0-43 54. 10 Suzuki K, Miyazawa K,Tsugawa R. Inhibitory effect of sodium pentosan polysulfate on the formation, growth and aggregation of calcium oxalate in vitro. Jpn J Urol 1989;80:526-531. 11 Ryall RL, Hibberd CM, Marshall VR. A method for studying inhibitory activity in whole urine. Urol Res 1985;13:285289.
s79
