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THE EFFECT OF CALPROTECTIN ON THE NUCLEATION AND GROWTH OF STRUVITE CRYSTALS AS ASSAYED BY LIGHT MICROSCOPY IN REAL-TIME
0022-5347/98/1594-1384$03.00/0
Vol. 159, 1384-1389,April 1998 Printed in U . S A
THE JOURNAL OF UROLOGY
Copyright 8 1998 by A ~ R I C AUROLOGICAL N ASSOCIATION, INC.
THE EFFECT OF CALPROTECTIN ON THE NUCLEATION AND GROWTH OF STRUVITE CRYSTALS AS ASSAYED BY LIGHT MICROSCOPY IN REAL-TIME HIROTAKA ASAKLTRA, JEREMY D. SELENGUT, WILLIAM H. ORMEJOHNSON STEPHEN P. DRETLER*
AND
From the Department of Urology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts and the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
ABSTRACT
Purpose: To use light microscopy to observe the urease-induced growth of struvite crystals in real-time, and to compare the effects of various proteins on that growth. Materials and Methods: Artificial urine, with and without citrate, and a minimal urine solution containing only urea and the components of struvite and apatite were incubated with urease and test proteins in the depressions of culture slides. The number and size of rectangular and X-shaped struvite crystals were recorded using a low-power phase contrast microscope. Results: The formation of crystalline struvite appears to occur after the formation of an amorphous calcium- and magnesium-containing phase. The extent of this amorphous phase is dependent on the presence of calcium and citrate, both of which strongly promote its formation over the crystalline phase. a-globulin, y-globulin and chymotrypsin inhibitor all result in the same amount of crystalline struvite as bovine serum albumin which is used as a control. Calprotectin, on the other hand, causes consistent and significant reductions in the number and size of struvite crystals under a wide range of conditions. No changes in the morphology of the struvite crystals were observed. Conclusions: Calprotectin, the dominant protein of infection stone matrix, has distinctive properties which affect the formation and growth of struvite crystals. The presence of citrate in synthetic urine dramatically reduces the number of struvite crystals observed. The present method for observing the effects of putative infection stone inhibitors appears to have merit. KEY WORDS:urinary calculi, urease, urinary tract infections, urine
Renal calculi composed of struvite and apatite are known as infection stones due to their association with urinary tract infections, a relationship which is believed to be causal.' These account for 10%of all urinary stones, grow more rapidly than other types, and often come to fill the renal pelvis ("staghorn" morphology). Since the treatment of these calculi often requires multiple procedures, prevention is a preferable goal. To this end it is important to elucidate the mechanisms of infection stone formation, the tactics the body has evolved to prevent it and the ways in which these protective systems may be impaired. In our investigations of infection stones, we have reported the identification of calprotectin as the primary protein constituent of the stone matrix.2 Calprotectin is found in human granulocytes in high concentration: has been reported to be released into the extracellular medium upon activation and cell-death4 and exhibits inhibition of bacterial and fungal growth in vitro; an effect believed to be related to sequestration of zinc.5 Normal serum levels are -0.3 pgJml. while UTI patients exhibited -2.5 pg./ml.6 A similar study of urine calprotectin levels in children found -0.024 pg./ml. and -1 pg./ml. in pyelonephritis patient^.^ It is to be expected then, that calprotectin should be present at the site of a urinary tract infection and the subsequent growth of infectioninduced calculi. Considering these facts, the finding of calprotectin in in-
fection stones should not be surprising. Our goal is to determine whether the binding of calprotectin to growing stone material has an inhibitory or promoting effect on stone formation. To achieve this end we have developed a urine model system in which individual struvite crystals can be counted, characterized and measured in the presence of various additives. Previous experimental work on the crystallization of struvite have generally utilized glass-rod encrustation in continuous-flow11 and statics-10 reactors. Studies of this type have illustrated many properties of urine constituents and inhibitory compounds, but are unable to separately discern their effects on crystal growth properties such as nucleation, growth and morphology. The present work extends these methodologies (particularly that of McLean et al)" to allow quantitative determinations. MATERIALS AND METHODS
Calprotectin preparation. Granulocytes were prepared from human BufTy Coat (Blood Transfusion Service, Massachusetts General Hospital) as follows: granulocytes and erythrocytes were separated by centrifugation through a Ficoll-Paque density gradient. Erythrocytes were lysed by addition of isotonic NH,C1.12 This procedure yielded -95% granulocytes. Crude granulocyte lysate (CGL) was prepared by the Accepted for publication November 14, 1997. method of Ausubel.13 Calprotectin was isolated by ion* Re uests for re rints. Department of Urology, Massachusetts exchange chromatography and was eluted with 60 mM barGenerA Hospital, &ACC 4, #486, 15 Parkman St., Boston, MA bital containing 5 mM CaC1,.14 This fraction is -95% pure by 02114. Supported by ants from Add Venture, Inc. and the American SDS-PAGE (fig. 1). Sequencing of the 8-kilodalton subunit Foundation for 8ologic Disease. confirmed its assignment as calprotectin (data not shown). 1384
EFFECT OF CALPROTECTIN ON STRWITE CRYSTAL GROWTH
FIG. 1. SDS-PAGE of chromatography results using Pharmacia Phast Gel system. All samples contain 5% 2-mercaptoethanol as disulfide reductant. 20% homogeneous polyacrylamidegel. Proteins stained with silver nitrate. Each lane contains 3 pg. total protein as assayed by Bradford assay versus BSA. Lane 1, molecular weight markers; Lane 2, CGL prior to column; Lane 4, column loading fraction-unbound proteins; Lane 5, calprotectin eluted with 5 mM CaCl, in barbital buffer; Lane 6, proteins eluted with 5 mM CaCl, and 125 mM NaCl in barbital buffer.
Reagents. “Minimal” urine solution was 3.2 mM MgCl,,
20.5 mM KH,PO,, 19 mM NH,Cl, and 416 mM urea. The pH in initial experiments were adjusted to 6.6, in subsequent experiments the pH was adjusted to 6.1 (see below). Artificial urine was prepared which additionally contained 118 mM NaC1, 16 mM Na,SO,, 2.3 mM sodium citrate, 0.149 mM sodium oxalate, 42 mM KC1, and 7 mM creatinine. In some experiments citrate was omitted (see below). CaCl, was added as at the beginning of each experiment so that variable calcium concentrations could be explored. Urease (Sigma, #U-0251), was dissolved in distilled water at 100 unitdml. Other proteins used as controls and additives were dissolved in the above minimal solution at 10 mg/ml. The following proteins were purchased from Sigma: bovine serum albumin (BSA, #A-3803),a-globulin (#G-8512), y-globulin (#G-5009) and mucin (#M-3895). Chymotrypsin inhibitor was purchased from CALBIOCHEM (#230906). Crystallization assay. A portion (-30 pl.) of test protein and suficient 1 M CaC1, to achieve the desired final calcium concentration were added to a 3 ml. centrifuge tube. Artificial urine or minimal solution was added to bring the total volume to 2.5 ml. Since calprotectin was dissolved in a barbital solution, barbital was added to all trials not containing calprotectin to a concentration of 3.8 pM. This mixture is then passed through a 0.22 pm. filter. The individual proteins were tested for losses during filtration by the Bradford assay. The amounts of protein added before filtration were adjusted to yield the desired amount after filtration (typically <20% losses, data not shown). A measured volume of filtrate (typically 1.7 ml.) was transferred to a centrifuge tube, mucin and urease were added, a timer started and the solution mixed. Mucin, a mucoprotein found on the surface of urinary tract epithelium, was added to provide a site for heterogeneous crystal nucleation. In its h e n c e , crystallization tended to initiate at sites around the periphery of the culture slide wells. This is undesirable bemuse such crystals are distorted and highly variable in number. Mucin forms a suspension which promotes heterogeRwus crystal nucleation as has been demonstrated with calcium oxalate.15 Initial experiments used a urease concen-
1385
tration of -0.75 unitdml. while later experiments used -1 univml. (fig. 2, pH profiles A and B). Variations in the experimental components and room temperature resulted in significant variations in the pH profile of the experiments. The initially determined pH profiles were used as a standard and was checked before every experiment. If this varied from the standard by more than 0.1 pH unit at any time point, the amount of urease added was altered to compensate. The amounts of urease added never varied by more than 5%from the concentrations mentioned above. Fifty pl. portions of these solutions were placed in the depressions of triple-well culture slides. A pipette tip was used to spread the droplets to within 1 mm. of the edge of the wells and each was covered with a cover slip. Portions of the remaining solution were placed in small tubes. Each time the microscope slides were evaluated microscopically one of these samples was uncovered and the pH determined. Every half-hour after the addition of urease the microscope slide wells were evaluated using a phase-contrast microscope (Olympus BHA) at 25X magnification. The number of X-shaped crystals was counted and the maximum length of each was determined using an in-line scale bar. The smallest crystals measurable were -40 pm. Photomicrographs were taken using an Olympus C-35 camera and Ilford FP4Plus film. In order to ensure reproducible results, the culture slides were soaked overnight in 5 N nitric acid, rinsed with distilled water, soaked in RBS-35 detergent (PIERCE, #27952) for 2 hours, rinsed again and then dried under a stream of filtered air inside a laminar flow hood. Data processing. Due to the fact that the nucleation of crystals is an ongoing process and that, under a certain size, crystals are not counted or measured, the observations made in this study do not conform to normal population distributions. All significance tests were carried out using the non-parametric Wilcoxon rank-sum test a t 95%and 996 confidence. Infrared analysis. X-shaped and amorphous crystals were grown from the minimal urine solution using urease in 1.5 *____..-.. --. ._..-.-0-
-
6.5ei
ureasdml., avera k of 6 trials, ised for experiments with minimal solution. pH proale B (solid diamonds, dashed line): -1.0 units u r e d m l . , average of 14 trials, used for experiments with artificial urine (with and without citrate). Bars indicate standard deviation.
1386
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
ml. tubes. The resulting precipitates were centrifuged, the supernatants decanted and the precipitates air dried. "he precipitate was mixed with KBr and compressed into a wafer. Spectra of these materials were obtained using an infrared spectrophotometer and compared to spectra of MAP and calcium phosphate. RESULTS
Two types of precipitates were observed in these expenments. In the presence of calcium, apparently amorphous crystals appeared first at -pH 7.2 and then X-shaped struvite crystals appeared at -pH 8.2-8.5 (fig. 3). Struvite crystals exhibited a roughly X-shaped morphology most of the time. Sometimes this morphology was imperfect and only 2 or 3 arms were observed. Occasionally trapezoidal, rectangular or octahedral morphologies were found. These observations are consistent with earlier reports by McLean.11 Infrared spectroscopy demonstrated that the X-shaped crystals consisted of struvite and that the amorphous-appearing materials consisted of -98% calcium phosphate. This analysis could not determine the more detailed mineralogy of the calcium phosphate phase and the low-power objective used in this study had insufficient magnification to discriminate between a truly amorphous phase and microcrystalline material. Initial experiments were carried out in minimal solution using pH profile A (fig. 2), and the variation of calcium concentration was evaluated for its effect on the formation of struvite crystals. These trials included 100 pgJml. BSA. In the absence of calcium the number of struvite crystals formed per well was large, exceeding 50 by 2 hours. The length of
1
this many crystals could not be quickly and conveniently measured. By comparison, addition of 0.4, 1 or 2 mM Ca+2 resulted in -10 struvite crystals per well by 5 hours. We estimate that, by the time pH 9 is reached, nearly all of the magnesium must have precipitated from solution (due to the excess of and NH4+ at this pH, a solution with 3.2 mM Mg would be -1000-fold supersaturated with respect to struvite since I& =, [Mg][POJ[NHJ = 2.5 X In the presence of calcium, therefore, the majority of the magnesium must be found in the X-shaped or amorphous precipitates. Several other authors have also reported the presence of amorphous magnesium-containing precipitates in experiments using synthetic ~ r i n e . ~ . l ' "he addition of 100 pg./ml. calprotectin in place of BSA resulted in significant decreases in the total length (sum of individual crystal lengths per well) of the crystals at calcium concentrations of 0.4 and 1 mM (fig. 4,A, B ) . Similar, although not statistically significant, differences in total length were found at 2 mM calcium, although significant decreases in the number of crystals are found under these conditions (fig. 4,C). At 0.4 mM calcium, the initiation of crystallization was delayed 1 to 1.5 hours; after this period the rate of nucleation was roughly the same for the BSA and calprotectin trials. The crystal growth rate appeared to be higher with calprotectin, averaging 0.57mm./hour between 2 and 5 hours compared with 0.32 mm./hour for BSA over the same period, resulting in roughly the same crystal size by the end of the experiment. Three other control proteins were tested in the minimal solution at 0.4 mM calcium: a-globulin, y-globulin and chy-
Frc. 3. Growth o f t ical X shaped struvite crystal. Minimal solution, [Cal = 0.4 mM, [Ureasel = 1.56 unitdml., [BSA] = 100 mg./ml. A, 2.5 hours, pH = 8.49,cngth 18 mm. B, 3 hours, pH = 8.72, length = 40 mm. C, 3.5 hours, pH = 8.87, length = 48 mm. D , 5.5 hours, pH = 9.1, length = 84 mm. Bar = 10 mm.
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
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Incubation time (hours) FIG. 5. Comparison of struvite p w t h with BSA (solid circles, solid line) and cal rotectin (open circles, dashed line) in arti!icial urine (pH profile A, [Ca] = 0.4 mhf, N = 9 for BSA, 8 for calprotectin. B, [Ca] = 2.0 mM, N = 8 for each. C, [CaI = 4.0 mM, N = 6 for each.
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Incubation time (hours) FIG.4. Corn arison of struvite p w t h with BSA (solid circles, solid line) ancfcalprotectin (open circle, dash+ line) in minimal solution. Total len h of crystals per well versus tune ( H profile A); bar indicates s t a n g d deviation of the mean (standaderror), numbers indicate average number of crystals per well. * p c0.05, cO.01 (in parenthesis, indicate for number of c stals only)*> [Cal = 0.4 mM, N = 6 for each. B , [Ca] = 1.0 m M 7 for BSA, 8 for calprotectin. C, [Ca] = 2.0 mM, N = 6 for each.
dramatically increased the number of struvite crystals formed (but not their size). It seems then, that the other differences between the minimal solution and artificial urine (the presence of sulfate, oxalate and creatinine and increased ionic strength) had only minimal effects on the formation of these crystals. In artificial urine containing citrate, calprotectin had a dramatic effect on struvite crystallization, delaying nucleation and reducing the overall size of the crystals at all motrypsin inhibitor. The globulins have been reported to be calcium concentrations (fig. 5). Without citrate a less drafound in elevated levels in urine from stone patients and matic effect was observed at 0.4 mM calcium. In 1 mM urinary tract infection patients, respectively.17 Chymotryp- calcium a small but reproducible effect could be seen, while sin inhibitor has been included because, unlike BSA and the globulins, it has a molecular weight of 38 kDa which is no effect was observed at 4 mM calcium (fig. 6). comparable to that of calprotectin (MW = 35 None of DISCUSSION these proteins could be distinguished from BSA in terms The model presented in this paper is related to the work of q s t a l number, average size, total length or morphology McLean11 in that the crystals are evaluated by light micros(data not shown). In order to test the effects of calprotectin in the more copy. In their work, crystals are grown in an agitated, clinically relevant artificial urine solution, we used pH pro- continuous-flow system where the processes of aggregation file B (fig. 2). The use of artificial urine resulted in far fewer and crystal fracturing cannot be discounted. Here, the soluidentifiable struvite crystals than the corresponding minimal tion is static and individual crystals can be obsemed repeat solution experiments, and the crystals that did form were edly to determine growth rates and crystal morphology. The somewhat smaller (fig. 5). This effect was not due to the present methodology uses the diagonal length of the change in pH profile-use of pH profile B with the minimal X-shapedstruvite crystals as a measure of size. This crude solution system resulted in a greater number of crystals but rapid measurement is sufficient for the conclusions (data not shown). The absence of citrate, however (fig. 61, drawn in this work and as a demonetration of the atwaJr's
g=
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
1388
B
6+3
0
2 3 4 Incubation time (hours) 1
5
FIG. 6. Comparison of struvite growth with BSA (solid circles, solid line) and calprotectin ( 0 n circles, dashed line) in artificial urine without citrate (pH p r o g e B). A, [Cal = 0.4 mM, N = 5 for BSA, 4 for calprotectin. B, [Cal = 1.0 mM, N = 5 for BSA, 4 for calprotectin. C, [Cal = 4.0 mM, N = 3 for each.
D affect the timing andor extent of crystalline struvite prezipitation or whether this apparently amorphous phase can lffect the nucleation of crystalline struvite is presently un:lear. However, it seems reasonable that similar relationships between amorphous and crystalline phases may be important in the process of infection stone formation. In the present study we have found that a-globulin and y-globulin, two proteins associated with urinary tract disease, have effects indistinguishable from BSA and chymotrypsin inhibitor, two proteins not expected to have a n inhibitory effect. Although statistical comparison of calprotectin data with control data containing BSA (at matched time points) were only intermittently found to represent significantly different populations, the totality of the data is unambiguous-with rare exception, calprotectin results in the production of fewer crystals of smaller size than any of the other proteins observed in this study. Although the binding of calprotectin to the crystals observed in this study was not evaluated, we speculate that calprotectin may induce its effects on MAP by either binding to MAP and slowing its growth, binding to MAP nuclei and capping them off before they can grow t o visible X-shaped crystals or by binding to the calcium phosphate crystals, allowing incorporation of an increased amount of magnesium. Considering this information and the recent report of a calprotectin-like protein extracted from calcium oxalate stones and its inhibitory effect towards calcium oxalate crystallization,la it appears that further study of this protein and its effect on stone formation is warranted. Separate from these observations of the effects of proteins, we have observed substantial differences between struvite crystallization from a minimal solution and that from a more complete artificial urine formulation. The primary effector of these differences appears to be citrate. Citrate will form a complex with either calcium or magnesium, reducing the degree of saturation of these two ions. After calcium has precipitated from solution in these experiments, all of the citrate becomes available t o bind magnesium. Considering the stability constant of magnesium citrate, the free Mg concentration will be reduced by more than 70%.'9 Most likely this shifts the precipitation of the bulk of the MAP to a pH at which X-shaped crystal formation is not favored. Additionally, citrate has been reported to inhibit the growth and aggregation of calcium oxalate and calcium phosphate. 16-20 Acknowledgments. The authors wish to thank Ms. Mary Chung and Mitsubishi Kagaku, Bio-Clinical Laboratories.
REFERENCES
utility. Further applications of this technique might warrant a more sophisticated approach such as computer-based image analysis to better approximate the crystal volume, or to assess subtle changes in crystal habit. It is clear from our observations and those of others that the precipitation of struvite is often preceded by the formation of an amorphous or microcrystalline phase.3." This phase probably does contain microcrystalline material, but the low power objective used in this study does not allow further morphological characterization. This material is most likely comprised of amorphous or microcrystalline calcium phosphate, as is found in the absence of magnesium. Although a separated magnesium phosphate phase is unlikely to be present in large amounts, it is possible that significant amounts of magnesium may be present as impurities within the calcium phosphate phase. When the initial amorphous precipitate is analyzed for magnesium content by a colorimetric method, an incorporation of up to 20% magnesium relative to calcium has been observed (data not shown). Whether this sink of magnesium is of sufficient magnitude
1. Griffith, D. P., Musher, D. M. and Itin, C.: Urease: the primary cause of infection-induced urinary stones. Invest. Urol., 1: 346, 1976. 2. Bennett, J. M., Dretler, S. P., Selengut, J . D. and Orme-Johnson, W. H.: Identification of the calcium-binding protein calgranulin in the matrix of struvite stones. J . Endourol., 8: 95, 1994. 3. Edgeworth, J. M., Gorman, R., Bennet, P., Freemont, P. and Hogg, N.: Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J. Biol. Chem., 266. 7706, 1991. 4. Fagerhol, M. K.,Dale, I. and Anderson, T.: Release and quantitation of a leukocyte derived protein. Scand. J. Haematol., 24:393,1980. 5. Sohnle, P.G., Collins-Lech, C. and Wiessner, J . H.: The zincreversible, antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J . Infect. Dis., 164: 137, 1991. 6. Sander, J., Fagerhol, M. K., Bakken, J. S. and Dale, I.: Plasma levels of the leukocyte L1 protein in febrile conditions: relation to aetiology, number of leukocytes in blood, blood sedimentation reaction and C-reactive protein. Scand. J. Clin. Lab. Invest., 44:357, 1984. 7. Holt, J., Fagerhol, M. K. and Dale, I.: Quantitation of a leukocyte protein (Ll)in urine. Acta Pediatr. Scand., 72: 615,1983.
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH 8. Hugosson, J., Grenabo, L., Hedelin, H. and Pettersson, S.: Effects of serum, albumin and immunoglobulins on ureaseinduced crystallization in urine. Urol. Res., 18: 407,1990. 9. Grenabo, L.,Hedelin, H. and Petterson, S.: The inhibitory effect of human urine on urease-induced crystallization in vitro. J. Urol., 136: 416,1986. 10.Hedelin, H.,Grenabo, J., Hugosson, J. and Petterson, S.: The influence of zinc and citrate on urease-induced urine crystallization. Urol. Res., 17: 177,1989. 11. McLean, R. J., Downey, J., Clapham, L. and Nickel, J. C.: A simple technique for studying struvite crystal growth in vitro. Urol. Res., 18: 39,1990. 12. Eggleton, P.,Gargan, R. and Fisher, D.: Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers. J. Immun. Methods, l2k 105,1989. 13. Ausubel, F. M. et al.: Preparation of nuclear and cytoplasmic extracts from mammalian cells. In: Current Protocols in Molecular Biology. New York John Wiley & Sons, vol. 2, pp. 12.1.1.-12.1.9,1990. 14. Fagerhol, M. K., Dale, I. and Naesgaard, I.: Purified human
1389
granulocyte L1 proteins methods for their preparation, monospecific antibodies and test kits. United States Patent #4833074,1989. 15. Grases, F.,MasBmvl, L., S h n e l , 0. and Coeta-Baud, A:Agglomeration of calcium oxalate monohydrate in synthetic urine. Br. J. Urol., 70: 240, 1992. 16. Berland, Y. and Dussol, B.: New insights into renal stone formation. Curr. Opin. Nephrol. Hypertens., 3 417,1994. 17. Boyce, W. H.: Proteinuria in kidney calculua dieease. In:Proteins in normal and pathological urine. New York Karger, pp. 235243,1970. 18. Umekawa, T. and Kurita, T.: Calproktin-lihe protein is related to soluble orgauic matrix in calcium oxalate urinary stone. Bioch. Mol. Biol. Int., 34:309, 1994. 19. Dawson, R. M. C., Elliott, D. C., Elliott, W.H. and Jones,K M.: Data for BiochemicalResearch,3rd Edition,Oxford University Press, Oxford, UK,p. 411,1986. 20. Tieselius, H.G.,Fornander, A. M.and Nileson, M.A:Effecta of citrate on the M e r e n t phases of calcium oxalate erpstallization. Scanning Microsc., ?: 381,1993.
Vol. 159, 1384-1389,April 1998 Printed in U . S A
THE JOURNAL OF UROLOGY
Copyright 8 1998 by A ~ R I C AUROLOGICAL N ASSOCIATION, INC.
THE EFFECT OF CALPROTECTIN ON THE NUCLEATION AND GROWTH OF STRUVITE CRYSTALS AS ASSAYED BY LIGHT MICROSCOPY IN REAL-TIME HIROTAKA ASAKLTRA, JEREMY D. SELENGUT, WILLIAM H. ORMEJOHNSON STEPHEN P. DRETLER*
AND
From the Department of Urology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts and the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
ABSTRACT
Purpose: To use light microscopy to observe the urease-induced growth of struvite crystals in real-time, and to compare the effects of various proteins on that growth. Materials and Methods: Artificial urine, with and without citrate, and a minimal urine solution containing only urea and the components of struvite and apatite were incubated with urease and test proteins in the depressions of culture slides. The number and size of rectangular and X-shaped struvite crystals were recorded using a low-power phase contrast microscope. Results: The formation of crystalline struvite appears to occur after the formation of an amorphous calcium- and magnesium-containing phase. The extent of this amorphous phase is dependent on the presence of calcium and citrate, both of which strongly promote its formation over the crystalline phase. a-globulin, y-globulin and chymotrypsin inhibitor all result in the same amount of crystalline struvite as bovine serum albumin which is used as a control. Calprotectin, on the other hand, causes consistent and significant reductions in the number and size of struvite crystals under a wide range of conditions. No changes in the morphology of the struvite crystals were observed. Conclusions: Calprotectin, the dominant protein of infection stone matrix, has distinctive properties which affect the formation and growth of struvite crystals. The presence of citrate in synthetic urine dramatically reduces the number of struvite crystals observed. The present method for observing the effects of putative infection stone inhibitors appears to have merit. KEY WORDS:urinary calculi, urease, urinary tract infections, urine
Renal calculi composed of struvite and apatite are known as infection stones due to their association with urinary tract infections, a relationship which is believed to be causal.' These account for 10%of all urinary stones, grow more rapidly than other types, and often come to fill the renal pelvis ("staghorn" morphology). Since the treatment of these calculi often requires multiple procedures, prevention is a preferable goal. To this end it is important to elucidate the mechanisms of infection stone formation, the tactics the body has evolved to prevent it and the ways in which these protective systems may be impaired. In our investigations of infection stones, we have reported the identification of calprotectin as the primary protein constituent of the stone matrix.2 Calprotectin is found in human granulocytes in high concentration: has been reported to be released into the extracellular medium upon activation and cell-death4 and exhibits inhibition of bacterial and fungal growth in vitro; an effect believed to be related to sequestration of zinc.5 Normal serum levels are -0.3 pgJml. while UTI patients exhibited -2.5 pg./ml.6 A similar study of urine calprotectin levels in children found -0.024 pg./ml. and -1 pg./ml. in pyelonephritis patient^.^ It is to be expected then, that calprotectin should be present at the site of a urinary tract infection and the subsequent growth of infectioninduced calculi. Considering these facts, the finding of calprotectin in in-
fection stones should not be surprising. Our goal is to determine whether the binding of calprotectin to growing stone material has an inhibitory or promoting effect on stone formation. To achieve this end we have developed a urine model system in which individual struvite crystals can be counted, characterized and measured in the presence of various additives. Previous experimental work on the crystallization of struvite have generally utilized glass-rod encrustation in continuous-flow11 and statics-10 reactors. Studies of this type have illustrated many properties of urine constituents and inhibitory compounds, but are unable to separately discern their effects on crystal growth properties such as nucleation, growth and morphology. The present work extends these methodologies (particularly that of McLean et al)" to allow quantitative determinations. MATERIALS AND METHODS
Calprotectin preparation. Granulocytes were prepared from human BufTy Coat (Blood Transfusion Service, Massachusetts General Hospital) as follows: granulocytes and erythrocytes were separated by centrifugation through a Ficoll-Paque density gradient. Erythrocytes were lysed by addition of isotonic NH,C1.12 This procedure yielded -95% granulocytes. Crude granulocyte lysate (CGL) was prepared by the Accepted for publication November 14, 1997. method of Ausubel.13 Calprotectin was isolated by ion* Re uests for re rints. Department of Urology, Massachusetts exchange chromatography and was eluted with 60 mM barGenerA Hospital, &ACC 4, #486, 15 Parkman St., Boston, MA bital containing 5 mM CaC1,.14 This fraction is -95% pure by 02114. Supported by ants from Add Venture, Inc. and the American SDS-PAGE (fig. 1). Sequencing of the 8-kilodalton subunit Foundation for 8ologic Disease. confirmed its assignment as calprotectin (data not shown). 1384
EFFECT OF CALPROTECTIN ON STRWITE CRYSTAL GROWTH
FIG. 1. SDS-PAGE of chromatography results using Pharmacia Phast Gel system. All samples contain 5% 2-mercaptoethanol as disulfide reductant. 20% homogeneous polyacrylamidegel. Proteins stained with silver nitrate. Each lane contains 3 pg. total protein as assayed by Bradford assay versus BSA. Lane 1, molecular weight markers; Lane 2, CGL prior to column; Lane 4, column loading fraction-unbound proteins; Lane 5, calprotectin eluted with 5 mM CaCl, in barbital buffer; Lane 6, proteins eluted with 5 mM CaCl, and 125 mM NaCl in barbital buffer.
Reagents. “Minimal” urine solution was 3.2 mM MgCl,,
20.5 mM KH,PO,, 19 mM NH,Cl, and 416 mM urea. The pH in initial experiments were adjusted to 6.6, in subsequent experiments the pH was adjusted to 6.1 (see below). Artificial urine was prepared which additionally contained 118 mM NaC1, 16 mM Na,SO,, 2.3 mM sodium citrate, 0.149 mM sodium oxalate, 42 mM KC1, and 7 mM creatinine. In some experiments citrate was omitted (see below). CaCl, was added as at the beginning of each experiment so that variable calcium concentrations could be explored. Urease (Sigma, #U-0251), was dissolved in distilled water at 100 unitdml. Other proteins used as controls and additives were dissolved in the above minimal solution at 10 mg/ml. The following proteins were purchased from Sigma: bovine serum albumin (BSA, #A-3803),a-globulin (#G-8512), y-globulin (#G-5009) and mucin (#M-3895). Chymotrypsin inhibitor was purchased from CALBIOCHEM (#230906). Crystallization assay. A portion (-30 pl.) of test protein and suficient 1 M CaC1, to achieve the desired final calcium concentration were added to a 3 ml. centrifuge tube. Artificial urine or minimal solution was added to bring the total volume to 2.5 ml. Since calprotectin was dissolved in a barbital solution, barbital was added to all trials not containing calprotectin to a concentration of 3.8 pM. This mixture is then passed through a 0.22 pm. filter. The individual proteins were tested for losses during filtration by the Bradford assay. The amounts of protein added before filtration were adjusted to yield the desired amount after filtration (typically <20% losses, data not shown). A measured volume of filtrate (typically 1.7 ml.) was transferred to a centrifuge tube, mucin and urease were added, a timer started and the solution mixed. Mucin, a mucoprotein found on the surface of urinary tract epithelium, was added to provide a site for heterogeneous crystal nucleation. In its h e n c e , crystallization tended to initiate at sites around the periphery of the culture slide wells. This is undesirable bemuse such crystals are distorted and highly variable in number. Mucin forms a suspension which promotes heterogeRwus crystal nucleation as has been demonstrated with calcium oxalate.15 Initial experiments used a urease concen-
1385
tration of -0.75 unitdml. while later experiments used -1 univml. (fig. 2, pH profiles A and B). Variations in the experimental components and room temperature resulted in significant variations in the pH profile of the experiments. The initially determined pH profiles were used as a standard and was checked before every experiment. If this varied from the standard by more than 0.1 pH unit at any time point, the amount of urease added was altered to compensate. The amounts of urease added never varied by more than 5%from the concentrations mentioned above. Fifty pl. portions of these solutions were placed in the depressions of triple-well culture slides. A pipette tip was used to spread the droplets to within 1 mm. of the edge of the wells and each was covered with a cover slip. Portions of the remaining solution were placed in small tubes. Each time the microscope slides were evaluated microscopically one of these samples was uncovered and the pH determined. Every half-hour after the addition of urease the microscope slide wells were evaluated using a phase-contrast microscope (Olympus BHA) at 25X magnification. The number of X-shaped crystals was counted and the maximum length of each was determined using an in-line scale bar. The smallest crystals measurable were -40 pm. Photomicrographs were taken using an Olympus C-35 camera and Ilford FP4Plus film. In order to ensure reproducible results, the culture slides were soaked overnight in 5 N nitric acid, rinsed with distilled water, soaked in RBS-35 detergent (PIERCE, #27952) for 2 hours, rinsed again and then dried under a stream of filtered air inside a laminar flow hood. Data processing. Due to the fact that the nucleation of crystals is an ongoing process and that, under a certain size, crystals are not counted or measured, the observations made in this study do not conform to normal population distributions. All significance tests were carried out using the non-parametric Wilcoxon rank-sum test a t 95%and 996 confidence. Infrared analysis. X-shaped and amorphous crystals were grown from the minimal urine solution using urease in 1.5 *____..-.. --. ._..-.-0-
-
6.5ei
ureasdml., avera k of 6 trials, ised for experiments with minimal solution. pH proale B (solid diamonds, dashed line): -1.0 units u r e d m l . , average of 14 trials, used for experiments with artificial urine (with and without citrate). Bars indicate standard deviation.
1386
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
ml. tubes. The resulting precipitates were centrifuged, the supernatants decanted and the precipitates air dried. "he precipitate was mixed with KBr and compressed into a wafer. Spectra of these materials were obtained using an infrared spectrophotometer and compared to spectra of MAP and calcium phosphate. RESULTS
Two types of precipitates were observed in these expenments. In the presence of calcium, apparently amorphous crystals appeared first at -pH 7.2 and then X-shaped struvite crystals appeared at -pH 8.2-8.5 (fig. 3). Struvite crystals exhibited a roughly X-shaped morphology most of the time. Sometimes this morphology was imperfect and only 2 or 3 arms were observed. Occasionally trapezoidal, rectangular or octahedral morphologies were found. These observations are consistent with earlier reports by McLean.11 Infrared spectroscopy demonstrated that the X-shaped crystals consisted of struvite and that the amorphous-appearing materials consisted of -98% calcium phosphate. This analysis could not determine the more detailed mineralogy of the calcium phosphate phase and the low-power objective used in this study had insufficient magnification to discriminate between a truly amorphous phase and microcrystalline material. Initial experiments were carried out in minimal solution using pH profile A (fig. 2), and the variation of calcium concentration was evaluated for its effect on the formation of struvite crystals. These trials included 100 pgJml. BSA. In the absence of calcium the number of struvite crystals formed per well was large, exceeding 50 by 2 hours. The length of
1
this many crystals could not be quickly and conveniently measured. By comparison, addition of 0.4, 1 or 2 mM Ca+2 resulted in -10 struvite crystals per well by 5 hours. We estimate that, by the time pH 9 is reached, nearly all of the magnesium must have precipitated from solution (due to the excess of and NH4+ at this pH, a solution with 3.2 mM Mg would be -1000-fold supersaturated with respect to struvite since I& =, [Mg][POJ[NHJ = 2.5 X In the presence of calcium, therefore, the majority of the magnesium must be found in the X-shaped or amorphous precipitates. Several other authors have also reported the presence of amorphous magnesium-containing precipitates in experiments using synthetic ~ r i n e . ~ . l ' "he addition of 100 pg./ml. calprotectin in place of BSA resulted in significant decreases in the total length (sum of individual crystal lengths per well) of the crystals at calcium concentrations of 0.4 and 1 mM (fig. 4,A, B ) . Similar, although not statistically significant, differences in total length were found at 2 mM calcium, although significant decreases in the number of crystals are found under these conditions (fig. 4,C). At 0.4 mM calcium, the initiation of crystallization was delayed 1 to 1.5 hours; after this period the rate of nucleation was roughly the same for the BSA and calprotectin trials. The crystal growth rate appeared to be higher with calprotectin, averaging 0.57mm./hour between 2 and 5 hours compared with 0.32 mm./hour for BSA over the same period, resulting in roughly the same crystal size by the end of the experiment. Three other control proteins were tested in the minimal solution at 0.4 mM calcium: a-globulin, y-globulin and chy-
Frc. 3. Growth o f t ical X shaped struvite crystal. Minimal solution, [Cal = 0.4 mM, [Ureasel = 1.56 unitdml., [BSA] = 100 mg./ml. A, 2.5 hours, pH = 8.49,cngth 18 mm. B, 3 hours, pH = 8.72, length = 40 mm. C, 3.5 hours, pH = 8.87, length = 48 mm. D , 5.5 hours, pH = 9.1, length = 84 mm. Bar = 10 mm.
EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
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Incubation time (hours) FIG. 5. Comparison of struvite p w t h with BSA (solid circles, solid line) and cal rotectin (open circles, dashed line) in arti!icial urine (pH profile A, [Ca] = 0.4 mhf, N = 9 for BSA, 8 for calprotectin. B, [Ca] = 2.0 mM, N = 8 for each. C, [CaI = 4.0 mM, N = 6 for each.
b).
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Incubation time (hours) FIG.4. Corn arison of struvite p w t h with BSA (solid circles, solid line) ancfcalprotectin (open circle, dash+ line) in minimal solution. Total len h of crystals per well versus tune ( H profile A); bar indicates s t a n g d deviation of the mean (standaderror), numbers indicate average number of crystals per well. * p c0.05, cO.01 (in parenthesis, indicate for number of c stals only)*> [Cal = 0.4 mM, N = 6 for each. B , [Ca] = 1.0 m M 7 for BSA, 8 for calprotectin. C, [Ca] = 2.0 mM, N = 6 for each.
dramatically increased the number of struvite crystals formed (but not their size). It seems then, that the other differences between the minimal solution and artificial urine (the presence of sulfate, oxalate and creatinine and increased ionic strength) had only minimal effects on the formation of these crystals. In artificial urine containing citrate, calprotectin had a dramatic effect on struvite crystallization, delaying nucleation and reducing the overall size of the crystals at all motrypsin inhibitor. The globulins have been reported to be calcium concentrations (fig. 5). Without citrate a less drafound in elevated levels in urine from stone patients and matic effect was observed at 0.4 mM calcium. In 1 mM urinary tract infection patients, respectively.17 Chymotryp- calcium a small but reproducible effect could be seen, while sin inhibitor has been included because, unlike BSA and the globulins, it has a molecular weight of 38 kDa which is no effect was observed at 4 mM calcium (fig. 6). comparable to that of calprotectin (MW = 35 None of DISCUSSION these proteins could be distinguished from BSA in terms The model presented in this paper is related to the work of q s t a l number, average size, total length or morphology McLean11 in that the crystals are evaluated by light micros(data not shown). In order to test the effects of calprotectin in the more copy. In their work, crystals are grown in an agitated, clinically relevant artificial urine solution, we used pH pro- continuous-flow system where the processes of aggregation file B (fig. 2). The use of artificial urine resulted in far fewer and crystal fracturing cannot be discounted. Here, the soluidentifiable struvite crystals than the corresponding minimal tion is static and individual crystals can be obsemed repeat solution experiments, and the crystals that did form were edly to determine growth rates and crystal morphology. The somewhat smaller (fig. 5). This effect was not due to the present methodology uses the diagonal length of the change in pH profile-use of pH profile B with the minimal X-shapedstruvite crystals as a measure of size. This crude solution system resulted in a greater number of crystals but rapid measurement is sufficient for the conclusions (data not shown). The absence of citrate, however (fig. 61, drawn in this work and as a demonetration of the atwaJr's
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EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH
1388
B
6+3
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2 3 4 Incubation time (hours) 1
5
FIG. 6. Comparison of struvite growth with BSA (solid circles, solid line) and calprotectin ( 0 n circles, dashed line) in artificial urine without citrate (pH p r o g e B). A, [Cal = 0.4 mM, N = 5 for BSA, 4 for calprotectin. B, [Cal = 1.0 mM, N = 5 for BSA, 4 for calprotectin. C, [Cal = 4.0 mM, N = 3 for each.
D affect the timing andor extent of crystalline struvite prezipitation or whether this apparently amorphous phase can lffect the nucleation of crystalline struvite is presently un:lear. However, it seems reasonable that similar relationships between amorphous and crystalline phases may be important in the process of infection stone formation. In the present study we have found that a-globulin and y-globulin, two proteins associated with urinary tract disease, have effects indistinguishable from BSA and chymotrypsin inhibitor, two proteins not expected to have a n inhibitory effect. Although statistical comparison of calprotectin data with control data containing BSA (at matched time points) were only intermittently found to represent significantly different populations, the totality of the data is unambiguous-with rare exception, calprotectin results in the production of fewer crystals of smaller size than any of the other proteins observed in this study. Although the binding of calprotectin to the crystals observed in this study was not evaluated, we speculate that calprotectin may induce its effects on MAP by either binding to MAP and slowing its growth, binding to MAP nuclei and capping them off before they can grow t o visible X-shaped crystals or by binding to the calcium phosphate crystals, allowing incorporation of an increased amount of magnesium. Considering this information and the recent report of a calprotectin-like protein extracted from calcium oxalate stones and its inhibitory effect towards calcium oxalate crystallization,la it appears that further study of this protein and its effect on stone formation is warranted. Separate from these observations of the effects of proteins, we have observed substantial differences between struvite crystallization from a minimal solution and that from a more complete artificial urine formulation. The primary effector of these differences appears to be citrate. Citrate will form a complex with either calcium or magnesium, reducing the degree of saturation of these two ions. After calcium has precipitated from solution in these experiments, all of the citrate becomes available t o bind magnesium. Considering the stability constant of magnesium citrate, the free Mg concentration will be reduced by more than 70%.'9 Most likely this shifts the precipitation of the bulk of the MAP to a pH at which X-shaped crystal formation is not favored. Additionally, citrate has been reported to inhibit the growth and aggregation of calcium oxalate and calcium phosphate. 16-20 Acknowledgments. The authors wish to thank Ms. Mary Chung and Mitsubishi Kagaku, Bio-Clinical Laboratories.
REFERENCES
utility. Further applications of this technique might warrant a more sophisticated approach such as computer-based image analysis to better approximate the crystal volume, or to assess subtle changes in crystal habit. It is clear from our observations and those of others that the precipitation of struvite is often preceded by the formation of an amorphous or microcrystalline phase.3." This phase probably does contain microcrystalline material, but the low power objective used in this study does not allow further morphological characterization. This material is most likely comprised of amorphous or microcrystalline calcium phosphate, as is found in the absence of magnesium. Although a separated magnesium phosphate phase is unlikely to be present in large amounts, it is possible that significant amounts of magnesium may be present as impurities within the calcium phosphate phase. When the initial amorphous precipitate is analyzed for magnesium content by a colorimetric method, an incorporation of up to 20% magnesium relative to calcium has been observed (data not shown). Whether this sink of magnesium is of sufficient magnitude
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EFFECT OF CALPROTECTIN ON STRUVITE CRYSTAL GROWTH 8. Hugosson, J., Grenabo, L., Hedelin, H. and Pettersson, S.: Effects of serum, albumin and immunoglobulins on ureaseinduced crystallization in urine. Urol. Res., 18: 407,1990. 9. Grenabo, L.,Hedelin, H. and Petterson, S.: The inhibitory effect of human urine on urease-induced crystallization in vitro. J. Urol., 136: 416,1986. 10.Hedelin, H.,Grenabo, J., Hugosson, J. and Petterson, S.: The influence of zinc and citrate on urease-induced urine crystallization. Urol. Res., 17: 177,1989. 11. McLean, R. J., Downey, J., Clapham, L. and Nickel, J. C.: A simple technique for studying struvite crystal growth in vitro. Urol. Res., 18: 39,1990. 12. Eggleton, P.,Gargan, R. and Fisher, D.: Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers. J. Immun. Methods, l2k 105,1989. 13. Ausubel, F. M. et al.: Preparation of nuclear and cytoplasmic extracts from mammalian cells. In: Current Protocols in Molecular Biology. New York John Wiley & Sons, vol. 2, pp. 12.1.1.-12.1.9,1990. 14. Fagerhol, M. K., Dale, I. and Naesgaard, I.: Purified human
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granulocyte L1 proteins methods for their preparation, monospecific antibodies and test kits. United States Patent #4833074,1989. 15. Grases, F.,MasBmvl, L., S h n e l , 0. and Coeta-Baud, A:Agglomeration of calcium oxalate monohydrate in synthetic urine. Br. J. Urol., 70: 240, 1992. 16. Berland, Y. and Dussol, B.: New insights into renal stone formation. Curr. Opin. Nephrol. Hypertens., 3 417,1994. 17. Boyce, W. H.: Proteinuria in kidney calculua dieease. In:Proteins in normal and pathological urine. New York Karger, pp. 235243,1970. 18. Umekawa, T. and Kurita, T.: Calproktin-lihe protein is related to soluble orgauic matrix in calcium oxalate urinary stone. Bioch. Mol. Biol. Int., 34:309, 1994. 19. Dawson, R. M. C., Elliott, D. C., Elliott, W.H. and Jones,K M.: Data for BiochemicalResearch,3rd Edition,Oxford University Press, Oxford, UK,p. 411,1986. 20. Tieselius, H.G.,Fornander, A. M.and Nileson, M.A:Effecta of citrate on the M e r e n t phases of calcium oxalate erpstallization. Scanning Microsc., ?: 381,1993.