Adhesion of uric acid crystals to the surface of renal epithelial cells

Rima M. Koka, Erick Huang, and John C. Lieske

Department of Medicine, The University of Chicago, Chicago, Illinois 60637


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 12% of which contain uric acid (UA) either alone or admixed with calcium oxalates or calcium phosphates. UA crystals bind rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. The urinary glycoproteins osteopontin, nephrocalcin, and Tamm-Horsfall glycoprotein had no effect on binding of UA crystals to the cell surface, whereas other polyanions including specific glycosaminoglycans blocked UA crystal adhesion. Specific polycations also inhibited adhesion of UA crystals and appeared to exert their inhibitory effect by coating cells. However, removal of anionic cell surface molecules with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal binding, and sialic acid-binding lectins had no effect. These observations suggest that hydrogen bonding and hydrophobic interactions play a major role in adhesion of electrostatically neutral UA crystals to renal cells, unlike the interaction of calcium-containing crystals with negatively charged molecules on the apical cell surface via ionic forces. After adhesion to the plasma membrane, subsequent cellular events could contribute to UA crystal retention in the kidney and the development of UA or mixed calcium and UA calculi.

BSC-1 cells; glycosaminoglycans; Madin-Darby canine kidney cells; nephrolithiasis; 3T3 fibroblasts


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEPHROLITHIASIS, the formation of kidney stones, is a common condition that develops in 12% of men and 5% of women in industrialized countries (34). However, the mechanisms by which renal calculi arise are poorly understood. Although the majority of renal stones contain calcium oxalate, uric acid (UA) is also a common crystalline component of calculi, either alone or admixed with calcium salts (24). Hyperuricosuria is the sole metabolic abnormality in 15-20% of patients with recurrent calcium oxalate stones (9), and treatment of this subpopulation of hyperuricosuric calcium oxalate stone formers with allopurinol to reduce urinary UA excretion reduces subsequent formation of stones (9). Therefore, mechanisms that favor nucleation, aggregation and retention of UA crystals in the kidney could promote formation of UA as well as calcium oxalate stones. We have previously characterized the binding of calcium oxalate monohydrate (COM) and hydroxyapatite (HA) crystals to the surface of monkey (BSC-1 line) and canine [Madin-Darby canine kidney cell (MDCK) line] renal epithelial cells (15, 16, 19, 20), because adhesion of microcrystals that nucleate in metastable tubular fluid to tubular cells could mediate crystal retention within the kidney and subsequent formation of a calculus. Ionic bonding forces appear to mediate binding of COM and HA crystals to cells, and soluble anions including citrate, glycosaminoglycans, and glycoproteins such as osteopontin and nephrocalcin (16) that are found in tubular fluid can coat the crystals and diminish their adhesion. Binding sites for COM and HA crystals on the apical surface of renal epithelial cells include anionic, sialic acid-containing glycoproteins (15, 18).

UA crystals are often present in urine, especially during periods of reduced urinary pH (5), even though urine may contain molecules that inhibit their nucleation and/or growth (11). Once formed, UA crystals can adhere to renal cells (29), and the cells appear to respond to them in a specific manner (7, 8). Because of their pathogenic nature in gout, adhesion of monosodium urate (MSU) crystals to red blood cell and other membranes has also been studied (3), and it has been proposed that both hydrogen- and electrostatic bonding forces mediate this interaction (36). We hypothesized that nucleation of UA crystals in metastable tubular fluid, followed by their adhesion to the apical surface of kidney epithelial cells, could be an important event in the formation of both UA and calcium oxalate stones. Our results suggest that renal cell adhesion of electrostatically neutral UA crystals, which are held together by an elaborate hydrogen-bonding network (22), is mediated by hydrogen bonding and hydrophobic interactions.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture. Renal epithelial cells of the nontransformed African green monkey line, BSC-1, were used for study. Cells were grown in Dulbecco-Vogt DMEM containing 25 mM glucose, 1% calf serum, and 1.6 µM biotin at 38°C in a 13% CO2 incubator, as previously described (20). Under these conditions, BSC-1 cells achieved confluence at 4 × 105 cells/ 35-mm plastic plate (9.65 cm2; Nunc, Naperville, IL). High-density, quiescent cultures were prepared by plating 8 × 105 cells in a 35-mm dish. The spent medium was changed after 3 days so that there were 106 cells/dish 6 days later. Medium was then aspirated and replaced with fresh medium containing 0.01% calf serum and 16 µM biotin; 3 days later the quiescent cells were used for study.

MDCK cells were grown in DMEM containing 2% calf serum and 1.6 µM biotin as previously described (27). To prepare high-density, quiescent cultures 7 × 105 cells/35-mm dish were plated in DMEM containing 2% calf serum and 16 µM biotin. Two days later, when they were confluent at a density of 1.4 × 106 cells/dish, the medium was aspirated and replaced with fresh medium containing 0.5% calf serum and 1.6 µM biotin. The next day cultures were used for study. BALB/c 3T3 fibroblasts were grown in DMEM containing 10% calf serum, as previously described (27). To prepare high-density, quiescent cultures, 2 × 105 cells/dish were plated. Two days later the medium was aspirated and replaced with fresh medium containing 1% calf serum and 1.6 µM biotin. The cells were used for study when they were confluent at a density of 6 × 106 cells/dish.

Materials. UA crystals were prepared by Y. Nakagawa (Univ. of Chicago) by dissolving 100 mg of UA in 250 ml of hot (60°C) distilled water (29). A 100-µl aliquot of 8-[14C]UA was added (50-60 µCi/ml; 10.5 mCi/mmol; American Radiolabeled Chemicals, St. Louis, MO), followed by ethanol (250 ml), after which the solution was allowed to cool to room temperature with stirring overnight. The resulting crystal suspension was filtered, washed twice with ethanol, twice with acetone, and finally air dried. The crystals that formed were planar and 2-10 µm in size, similar to crystals that can be seen in urine, and had a specific activity of 465,000 counts · min-1 · mg-1.

Nephrocalcin (fraction B) was purified from embryonic kidney cells (25), and Tamm-Horsfall glycoprotein was isolated from the urine of two normal humans (14). Both proteins were prepared by Y. Nakagawa by using well-established methods, as was osteopontin purified from mouse renal cortical cell culture medium (37) kindly provided by E. Worcester and A. Beshensky (Univ. of Wisconsin, Milwaukee). Neuraminidase (type X), trypsin, proteinase K, dyes, lectins, and other compounds were purchased from Sigma Chemical (St. Louis, MO), unless otherwise indicated.

Adhesion of crystals to cells. High-density, quiescent cultures of BSC-1 cells, MDCK cells, or BALB/c 3T3 fibroblasts were prepared as above. At the time of a crystal adhesion assay, the culture medium was aspirated and replaced with 2 ml of artificial urine [in mM: 105.5 NaCl, 32.3 NaH2PO4, 3.21 Na3 citrate, 3.85 MgSO4, 16.95 Na2SO4, 63.7 KCl, 5.75 CaCl2, 0.318 Na2 oxalate, and 0.0276 NH4Cl, pH 5.5 (1)], to which the compound under study was added. [14C]UA crystals (125 µg/ml) were added to artificial urine overlying the cells from a freshly prepared slurry that was agitated to ensure homogeneity. Each culture dish was gently swirled by hand for 5 s so that the crystals uniformly distributed on the surface of the monolayer under the force of gravity. After 2 min, the buffer was aspirated and the cells were washed three times with artificial urine (2 ml) by using a wide-mouth tissue culture pipette. Exposure of cells to the reduced pH buffer did not appear to damage them, as confirmed by a retained ability to retain trypan blue. The cell monolayer and adherent crystals were then scraped directly into a scintillation vial containing 6 N HCl (0.5 ml) to completely dissolve the crystals, 4.5 ml of Ecoscint (National Diagnostics, E. Palmetto, FL) were added, and the amount of radioactivity was measured in a scintillation counter (Tri Carb 4640, Packard Instrument, Downers Grove, IL).

Cell coating. In experiments to assess the effect of coating cells with alcian blue and other compounds, the medium of 3-day quiescent cells was replaced with a physiological buffer [Hanks' buffered salt solution (HBSS; in mM): 137 NaCl, 5.4 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, and 5.6 glucose, pH 7.4], to which the compound under study was added. Fifteen minutes later, the buffer was aspirated from the culture dish and replaced with artificial urine, pH 5.5. Uncoated [14C]UA crystals were directly added to the artificial urine overlying the cells to achieve a final concentration of 125 µg/ml, and radioactivity associated with the cells was measured 2 min later as described above. Cells in three different cultures were coated in each of four separate experiments.

Crystal coating. In experiments to assess the effect of coating crystals, [14C]UA crystals were incubated in artificial urine containing the compound of interest in an Eppendorf tube that was rotated end to end at 4°C for 15 min. Each tube was then centrifuged at 3,000 g for 15 s, and the supernatant was gently aspirated and replaced with artificial urine to resuspend the coated crystals. The crystals were washed and resuspended three times in succession to ensure removal of nonadsorbed compounds. Control crystals were identically rinsed in artificial urine. Coated crystals were then added to cell monolayers in which the medium had been replaced with artificial urine as described above, and cell-associated radioactivity was measured 2 min later. Crystals were added to three separate cultures in each of four different experiments.

Enzymatic treatment of cells. To assess the role of cell surface molecules during adhesion of UA crystals, monolayer cultures of BSC-1 cells were pretreated with neuraminidase (type X, E.C. 3.2.1.18, 1 U/ml, HBSS, pH 5, 1 h, 37°C); trypsin (E.C. 232 650 8, 50 µg/ml, HBSS, pH 7.4, 15 min, 37°C); proteinase K (E.C. 4.2.2.8, 10 µg/ml, HBSS, pH 7.4, 15 min, 37°C); heparitinase I (E.C. 4.2.2.8, 2 unit/ml, HBSS, pH 7.4, 50 min, 43°C); or chondroitinase ABC (E.C. 4.2.2.4, 2 U/ml, 4.75 mM Tris, 9.5 mM NaCl, 0.1 mg/ml BSA, pH 7.4, 30 min, 37°C). The enzyme-containing buffer was then aspirated and replaced with 2 ml of artificial urine to which [14C]UA crystals were added as described above. For each enzyme, three cultures were treated in each of two separate experiments.

Surface charge of UA crystals [Zeta potential (ZP)]. Net crystalline surface charge was measured as previously described (13). To prevent settling, UA crystals (0.3 mg/ml) were suspended in a 50% sucrose solution containing 2 mM NaCl, and the pH was adjusted to 5.0, 5.5, or 6.0. The electrophoretic mobility of the crystals was measured at 25°C by assessing their rate of movement in an electrical field [electrophoretic mobility (EM)] by using a Zeta Meter ZM-77 (Zeta Meter, New York, NY). At least five different crystals in every suspension were tracked for 20 min with an applied voltage of 100 V (direct current). The ZP was calculated as ZP = Ct · EM, where Ct was taken as 12.9 poises at 25°C (13).

Statistics. Data were compared by Student's t-test; P < 0.05 was accepted as significant. Values are expressed as means ± SE. When no measures of variance appear on a figure, it is because they are smaller than symbols used for the means.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rapid binding of UA crystals to renal epithelial cells. Binding of UA crystals (125 µg/ml) to high-density, quiescent monolayer cultures of BSC-1 cells was detected within 15 s and approached a plateau by 2 min (Fig. 1A). Addition of crystal quantities from 100 to 500 µg/ml (200-1,000 µg/dish) was associated with a linear increase in crystals bound when measured 2 min later (Fig. 1B). Inspection of 20 fields in each of 3 separate cultures revealed that the percentage of cells associated with a crystal also increased progressively, from 2.3 to 9.1% (Fig. 1C), confirming that cell-associated radioactivity correlated with bound crystals. Therefore, UA crystals rapidly adhere to BSC-1 cells in a concentration-dependent manner.


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Fig. 1.   Time and concentration dependence of uric acid (UA) crystal binding to BSC-1 cells. High-density, quiescent cultures were prepared as described in MATERIALS AND METHODS, and the medium was replaced with artificial urine. A: at specific times after addition of [14C]UA crystals (125 µg/ml), the cell layer was rinsed 3 times, and cell-associated radioactivity was measured. Association of crystals with cells was detectable after 15 s and approached a plateau by 2 min. B: as the amount of [14C]UA crystals added to cells was raised, cell-associated radioactivity 2 min later increased progressively. C: monolayers to which an increasing amount of UA crystals had been added were rinsed 3 times and inspected under a polarizing microscope 2 min later. A greater percentage of cells were associated with a crystal as the quantity added was increased. The specific activity of crystals used in this and subsequent figures was 465,000 counts · min-1 (CPM) · mg-1, so that 10,000 CPM/dish is the equivalent of 3.5 µg of crystal bound/cm2 of cell surface area. Values are means ± SE for at least 6 cultures.

Inhibition of UA crystal binding to renal cells by anionic molecules. We next sought to identify molecules that alter the rapid adhesion of UA crystals to the surface of BSC-1 cells. Because it has been proposed that UA crystals can adsorb anionic urinary crystallization inhibitors (39), we evaluated the effect of diverse anions on UA crystal adhesion. Polyvinyl sulfate (100,000 Da), a potent inhibitor of COM (16) and HA (18) crystal adhesion to BSC-1 cells, served as a prototypical polyanion. At a concentration as low as 0.01 µM, polyvinyl sulfate reduced adherence of UA crystals (125 µg/ml) by 36% (P < 0.001; Fig. 2). Maximal inhibition (83%) was observed at concentrations >= 0.1 µM. Other polyanions were also effective. Heparin (15,000 Da) and polyaspartate (10,000 Da) inhibited adhesion to cells when present at concentrations >= 0.1 and 0.001 µM, respectively, whereas polyglutamate (13,600 Da) reduced adhesion by 40% at a concentration of 1 µM. Polyvinyl sulfate, polyaspartic acid, and heparin are not found in human urine, but certain glycosaminoglycans are. Chondroitin sulfate A (45,000 Da) decreased adhesion 40% at a concentration of 0.5 µM, with maximal inhibition of 73% at concentrations >= 10 µM (Fig. 2). Conversely, heparan sulfate (12,000 Da; up to 10 µM) and hyaluronic acid (4,500,000 Da; up to 1 µM) each had only a minimal effect on UA crystal adhesion (not shown).


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Fig. 2.   Effect of anionic molecules on adhesion of UA crystals to BSC-1 cells. Heparin (), polyglutamate (open circle ), chondroitin sulfate A (black-diamond ), polyvinyl sulfate () ,or polyaspartate (black-down-triangle ) was added to high-density, quiescent cultures of BSC-1 cells in which the medium had been replaced by artificial urine. [14C]UA crystals (125 µg/ml) were then added, and 2 min later the buffer was removed, the cell layer was rinsed, and cell-associated radioactivity was measured. UA crystal adhesion was significantly decreased by polyvinyl sulfate, heparan sulfate, heparin, and polyaspartate, but only minimally by polyglutamate. * P < 0.001.

Urine contains multiple proteins that inhibit nucleation, growth, or aggregation of urinary crystals (13, 32, 38) as well as their adhesion to renal epithelial cells (16, 18). We evaluated the effect on UA crystal binding to BSC-1 cells of three glycoproteins that have been studied in stone disease: nephrocalcin (14,000 Da; up to 1 µM), osteopontin (45,000 Da; up to 0.25 µM), and Tamm-Horsfall glycoprotein (80,000 Da; up to 0.5 µM). However, neither of the three glycoproteins significantly altered UA crystal adhesion. Urine also contains abundant citrate (28). However, neither citrate nor phosphocitrate (4), each of which blocked COM and HA crystal adhesion to cells, had a significant effect on UA crystal adhesion.

Inhibition of UA crystal binding to renal cells by cationic molecules and lectins. COM and HA crystals adhere to polyanionic molecules anchored on the surface of renal epithelial cells (15, 18). To evaluate the potential role of cell surface anions during UA crystal adhesion, cationized ferritin (440,000 Da) was added to the artificial urine overlying cells immediately before the crystals. Cationized ferritin decreased adherence of UA crystals by 33% at a concentration of 0.5 µM (P < 0.001), with maximal inhibition (77%) at concentrations >= 5 µM (Fig. 3). Cetylpyridinium chloride (340 Da), a cationic compound used to precipitate polyanions such as glycosaminoglycans from solution, inhibited UA crystal adhesion by 73% at a concentration of 200 µM (Fig. 3). The effect of diverse polycationic dyes on UA crystal adhesion was also evaluated. Polyethylenamine (750,000 Da) inhibited crystal adhesion by 50% at a concentration of 1 µM, with maximal inhibition of 80% at concentrations >= 5 µM (Fig. 3). Alcian blue (1,298 Da) and brilliant blue R-250 (826 Da) also decreased binding by 86 (at 500 µM) and 53% (at 200 µM), respectively (Fig. 3). Therefore, numerous polycations can block adhesion of UA crystals, possibly by interacting with polyanions on the cell surface.


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Fig. 3.   Effect of cationic molecules on adhesion of UA crystals to BSC-1 cells. High-density, quiescent cultures of BSC-1 cells were prepared, and the medium was replaced with artificial urine. Brilliant blue R-250 (), alcian blue (black-diamond ), cetylpyridinium chloride (down-triangle), polyethylenamine (black-triangle), or cationized ferritin () was added, followed by [14C]UA crystals (125 µg/ml). Two minutes later, the buffer was removed, the cell layer was rinsed 3 times with artificial urine, and cell-associated radioactivity was measured. Each polycation significantly decreased UA crystal adhesion. * P < 0.001.

One family of anions on the cell surface are glycoproteins, some of which contain sialic acid residues. Therefore, we evaluated the capacity of specific lectins to modify the crystal-cell interaction, because these molecules specifically bind glycoconjugates. Neither lectin from Triticum vulgaris (wheat germ agglutinin) nor hemocyanins from keyhole limpets or Limulus polyphemus, each of which has an affinity for sialic acid-containing glycoconjugates, had any effect on the adhesion of UA crystals to the cell membrane (not shown). These results suggest that, unlike COM and HA crystals, UA crystals do not interact with sialic acid-containing glycoconjugates during adhesion to the cell surface (15, 18).

Enzymatic treatment of cells. Another method of investigating the role of specific cell surface molecules during crystal attachment is pretreatment of cells with enzymes that remove candidate binding molecules. Although pretreatment of BSC-1 cells with the proteases trypsin (50 µg/ml) or proteinase K (10 µg/ml) markedly diminished COM and HA crystal adhesion to cells, pretreatment with neither enzyme altered UA crystal adhesion (Fig. 4). Surprisingly, neuraminidase (1 unit/ml), which cleaves terminal sialic acid residues of glycoconjugates, enhanced adhesion by 313% (Fig. 4). Treatment of cells with heparitinase I (2 U/ml) or chondroitinase ABC (2 U/ml) also increased UA adhesion by 315 and 445%, respectively. Together, these observations suggest that negatively charged cell surface molecules containing sialic acid or sulfate groups play a different role during adhesion of UA crystals, because their removal markedly increased binding (Fig. 4) than during binding of HA and COM crystals, where their removal decreased binding (15, 18, 35).


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Fig. 4.   Effect of enzymatic treatment of BSC-1 cells on adhesion of UA crystals. High-density, quiescent cultures of BSC-1 cells were prepared, and the medium was replaced with either Hanks'/ balanced salt solution (HBSS) containing neuraminidase (NA), heparitinase I (H), chondroitinase ABC (CSABC), trypsin, or proteinase K (PK). Control cultures (Cont) were incubated in enzyme-free HBSS. After incubation with the enzyme for the times indicated in the MATERIALS AND METHODS, HBSS was replaced with artificial urine (pH 5.5), and adhesion of [14C]UA crystals was measured 2 min later. Treatment with NA, H, and CSABC each significantly increased UA crystal adhesion, whereas treatment with the 2 proteases had no effect. * P < 0.001.

Coating of crystals or cells by molecules that block adhesion of crystals. Next, studies were performed to determine whether inhibition of UA crystal adhesion by the ions identified above was mediated by an interaction at the crystal and/or cell surface. Binding of uncoated UA crystals to cell monolayers that had first been exposed to the polycations ferritin (5 µM) or alcian blue (500 µM) was significantly reduced compared with untreated cells, whereas precoating cells with the polyanions polyvinyl sulfate (1 µM), heparin (5 µM), or chondroitin sulfate A (1 µM) had no effect (Fig. 5A). In the complementary experiment, preexposing crystals to the polyanions polyvinyl sulfate, heparin, or chondroitin sulfate A had no effect on adhesion to uncoated monolayers, whereas preexposing UA crystals to the polycations alcian blue and cationized ferritin increased adhesion by 333 and 267% respectively, compared with uncoated crystals (Fig. 5B). Therefore, our results suggest these polycations can act on the cell surface to block adhesion of UA crystals. However, precoating UA crystals with these two representative polycations markedly increased UA crystal adhesion, even though precoating cells with the same cations decreased adhesion. One possibility is that under these experimental conditions the cations may coat crystals, perhaps via hydrophobic interactions, thereby potentiating their subsequent adhesion to cell surface anions that were never exposed to the cations. Even though addition of certain polyanions to artificial urine immediately before UA crystals deceased crystal adhesion to cells (Fig. 1), precoating crystals or cells with each of three representative anions had no effect. Therefore, our experiments cannot discern whether these polyanions decreased UA crystal adherence by binding to the cell surface and/or crystal surface.


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Fig. 5.   Effect on adhesion of UA crystals of coating crystals or cells with specific anions or cations. A: to coat cells, the medium of quiescent cultures was aspirated and replaced with HBSS to which alcian blue (AB), cationized ferritin (CF), polyvinyl sulfate (PVS), heparin (H), or chondroitin sulfate A (CSA) were added. Fifteen minutes later, buffer was aspirated, and the monolayer was rinsed 3 times with artificial urine after which [14C]UA crystals were added to the monolayer. Precoating cells with the cations alcian blue and cationized ferritin each decreased UA crystal adhesion, whereas the anions heparin, PVS, and CSA had no effect. B: [14C]UA crystals were uncoated (Cont) or incubated with the same concentrations of the compounds cited in A. They were then washed and added to high-density, quiescent BSC-1 cultures in which the medium had been replaced with artificial urine. Two minutes later, the buffer was removed, the cell monolayer was washed, and cell-associated radioactivity was measured. Precoating crystals with the cations alcian blue and cationized ferritin each increased their adhesion to cells, whereas the anions heparin, PVS, and CSA had no effect. * P < 0.001.

UA crystal binding to other types of cells. To determine whether specific polyanions or polycations that inhibit adhesion of UA crystals to the surface of BSC-1 cells act similarly on other types of cells, another renal epithelial cell line, canine MDCK cells, and mouse 3T3 fibroblasts were studied. The polyanions heparin (5 µM) and polyvinyl sulfate (1 µM) inhibited adhesion of UA crystals to MDCK cells by 86 and 80%, respectively (Fig. 6), and to 3T3 fibroblasts by 80 and 90%, respectively (Fig. 7). Therefore, the effect of these two anions on adhesion of UA crystals was similar in all three cell types (Fig. 2). The polycations alcian blue and polyethylenamine inhibited adhesion of UA crystals to MDCK cells by 30 and 66%, respectively, and to 3T3 fibroblasts by 92 and 95%, respectively (Fig. 7). Interestingly, at low concentrations these two cations modestly increased adhesion of UA crystals, whereas at higher concentrations they were inhibitory (Fig. 7). A similar but less pronounced dual action on UA crystal adhesion to BSC-1 cells was observed for certain polycations (Fig. 3).


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Fig. 6.   Effect of the anions heparin [Madin-Darby canine kidney cells (MDCK; ) 3T3 ()] and polyvinyl sulfate [MDCK (), 3T3(open circle )] on binding of UA crystals to MDCK cell (solid lines) or 3T3 fibroblasts (dashed lines). High-density, quiescent cultures of MDCK cells or 3T3 fibroblasts were prepared as described in MATERIALS AND METHODS, and the medium of each was replaced with artificial urine to which the anions under study was added followed by [14C]UA crystals (125 µg/ml). Two minutes later, the buffer was removed, the cell layer was rinsed 3 times with artificial urine, and cell-associated radioactivity was measured. Both anions decreased UA crystal adhesion to both cell types over a similar concentration range.



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Fig. 7.   Effect of the cations alcian blue [MDCK (black-triangle); 3T3 (triangle )] and polyethylenamine [MDCK (), 3T3 (open circle )] on binding of UA crystals to MDCK cells (solid lines) or 3T3 fibroblasts (dashed lines). High-density, quiescent cultures of MDCK cells or 3T3 fibroblasts were prepared as described in MATERIALS AND METHODS, and the medium of each was replaced with artificial urine to which the cations under study was added followed by [14C]UA crystals (125 µg/ml). Two minutes later, the buffer was removed, the cell layer was rinsed 3 times with artificial urine, and cell-associated radioactivity was measured. Both cations increased adhesion of UA crystals to each cell type at low concentrations but blocked crystal adhesion at higher concentrations. * P < 0.001.

Surface charge of UA crystals (ZP). None of the UA crystals that were tracked moved along the electrical field. Therefore, the surface charge of the UA crystals at pH 5.0, 5.5, and 6.0 is neutral. In contrast, COM crystals had a ZP of +13.8 ± 0.3 mV in a previous study (13). Because our experiments were conducted at pH 5.5, charge is probably not a major determinant of UA crystal-cell interactions.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study indicate that UA crystals bind rapidly to the surface of renal epithelial cells in a concentration-dependent manner. Although in the present and previous studies (6, 16, 18), certain polyanions and polycations inhibited UA, COM and HA crystal adhesion to cells (Table 1), the mechanisms that mediate cellular binding of UA and these two calcium-containing crystals differ substantially, and hydrogen bonding and hydrophobic interactions appear to play a major role in mediating adhesion of electrostatically neutral UA crystals.

                              
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Table 1.   Comparison of the effect of anions and cations on adhesion of COM, HA, or UA crystals to cells

The interaction between a COM or HA crystal and the renal epithelial cell surface appears to be regulated, at least in part, by the presentation of electrical charge on the surface of each. The crystal behaves as if it has a net positive charge through which it binds to negatively charged molecules that protrude from the apical surface of the plasma membrane (15, 16, 18). Polyanions can act to coat COM or HA crystals whereas polycations can act to coat cells, so that both effectively block adhesion.

This model cannot explain the interaction of UA crystals and cultured renal epithelial cells. Under our experimental conditions that employed artificial urine (pH 5.5), UA crystals appear to be electrostatically neutral, suggesting that charge does not play a major role in interactions between UA crystals and cells. Although certain anions in solution blocked UA crystal adhesion, many that blocked COM and HA adhesion had no effect on UA binding (Table 1). Other features differentiate UA from COM and HA crystal adhesion to cells. Specific polycations can coat BSC-1 cells and block UA crystal adhesion (Fig. 5A), but sialic acid-binding lectins had no effect on UA crystal adhesion, and removal of cell surface anions with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal adhesion (Table 2 and Fig. 4). Perhaps polycations that bind to cell surface anions mask nearby nonanionic hydrophobic sites that mediate UA crystal adhesion. Polycations that blocked UA crystal adhesion to cells when present in solution or used to coat cells instead increased adhesion to cells when used to coat crystals, suggesting that these cations can coat UA crystals and these cation-coated crystals can bind to anions on the cell surface, a property uncoated UA crystals do not appear to possess. In addition, coating of UA crystals by cationized ferritin or alcian blue could be mediated by hydrogen rather than ionic bonding. The dual effect of those polycations that increased binding at low concentrations but deceased adhesion at higher concentrations, most prominently observed in experiments that employed MDCK and 3T3 cells (Fig. 7), could be explained by a similar paradigm. At lower concentrations cations may preferentially coat UA crystals, perhaps by hydrophobic interactions, thereby enabling their adhesion to cell surface anions. At higher concentrations these cations may also coat cells and block UA crystal binding sites, both anionic (for cation-coated crystals) and neutral (for uncoated crystals).

                              
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Table 2.   Comparison of agents that modify adhesion of COM, HA, and UA crystals to cells

The results of these studies do not clarify the effect of polyanions on UA crystal adhesion to renal cells. When certain polyanions were added to artificial urine before crystals, they blocked UA adhesion. However, precoating crystals or cells with the same anions had no effect on UA crystal binding. It is possible that the polyanions form a weak interaction with either the crystal or cell surface and prevent cell-crystal interactions when present in solution, but are removed with washing. In any event, our experiments do suggest that certain anions such as chondroitin sulfate A could act in tubular fluid in vivo to decrease UA crystal adhesion to cells. Our results also suggest that urine contains numerous molecules that block COM or HA crystal adhesion to cells but have no effect on UA crystal binding (e.g., osteopontin, nephrocalcin, heparan sulfate, citrate; Table 1). Therefore, tubular cells appear relatively undefended against UA crystal binding compared with COM or HA crystals.

Differences among COM, HA, and UA crystal adhesion to renal cells may be contributed to by differences in the three crystal structures. UA crystals have a perfect hydrogen bonding system that makes use of all the available hydrogen atoms (30). The purine molecules pack so as to form rows of coplanar molecules connected by two N-O hydrogen bonds, whereas two additional N-O hydrogen bonds connect rows of adjacent sheets (22, 30). Under the present experimental conditions UA crystals had no net surface charge compared with the highly positive Zeta potential of COM crystals (13). Therefore, the apical cell surface most likely has organic molecules organized according to a motif that satisfies a demand of the UA crystalline lattice for hydrogen bonding interactions, and perhaps it is not surprising that hydrogen rather than ionic bonding appears to play a much greater role in UA crystal-cell surface interactions in contrast to COM or HA crystal-cell binding.

Riese and colleagues (29) described adhesion of UA crystals to cultured rat medullary collecting duct cells. As in our study, crystal binding was concentration dependent. Furthermore, adhesion was only partially inhibited by prebound COM crystals suggesting, as in our study, that different populations of binding sites mediated UA and COM crystal-cell interactions. The interaction of MSU crystals and membranes has been studied more widely, due to the role of this crystal in gout (2, 3). For example, pretreatment of red blood cells with neuraminidase increased MSU crystal-induced cell lysis (3), analogous to the increased binding of UA crystals to neuraminidase-pretreated BSC-1 cells that we observed (Fig. 4). However, important crystallographic differences between UA and MSU crystals (22, 23) limit comparisons between the two crystals.

Important factors that promote UA stone formation have been identified, including increased UA excretion, perhaps related to protein ingestion, and urinary pH (33). Urinary pH is particularly important because the pKa of undissociated UA is 5.5, and below this value the protonated form of UA, which is insoluble, becomes predominant. Nevertheless, additional factors that promote retention of those UA crystals that do form, such as adhesion to cells, could also be important determinants of stone formation. The mechanisms whereby hyperuricosuria promotes calcium oxalate stone formation are less clear. UA appears to promote calcium oxalate crystal growth in vitro (39). Proposed mechanisms include epitaxial (21) or heterogeneous (26) growth of calcium oxalate on UA crystals. UA crystals may also adsorb or otherwise remove calcium oxalate crystal growth inhibitors from solution, favoring growth of calcium salts (11, 39). UA in solution, without forming crystals, also appears to promote calcium oxalate crystal growth (31). Finally, stasis of tubular fluid due to collecting duct obstruction with UA crystals could favor growth of calcium oxalate crystals (10). None of these possibilities has been unequivocally demonstrated in vivo.

Previous investigations have suggested that renal cells respond to adhesion of UA crystals. In a study that employed canine renal cells (MDCK line), a specific reaction to MSU crystals was observed (8). In response to MSU crystals, cells appeared enlarged and raised above the monolayer culture in clumps termed "reaction sites." Intracellular MSU crystals were detected by electron microscopy, and release of cellular lactate dehydrogenase into the medium was demonstrated, which suggested cell injury. Because cellular uptake of COM crystals enhances adhesion of additional crystals (17), it is tempting to speculate that abnormal reactive cells that have engulfed a UA crystal might be more likely to bind additional UA crystals, or possibly COM or HA crystals. In this fashion, adhesion of a UA crystal to the surface of a renal epithelial cell could promote UA or calcium stone formation and contribute to the association between hyperuricosuria and calcium urolithiasis.

In summary, UA crystals bind rapidly to the surface of renal epithelial cells, and a subpopulation of polyanions in tubular fluid can block this event. The interaction of UA crystals with the apical surface of kidney epithelial cells appears to differ in important ways from both COM and HA crystal-cell interactions. Hydrogen bonding and hydrophobic interactions may mediate the interaction between electrostatically neutral UA crystals and structures on the apical cell surface, whereas ionic bonding is predominant during COM and HA crystal interactions with cultured renal cells. After adhesion to the apical plasma membrane, subsequent cellular events could potentiate the development of UA or mixed calcium and UA calculi.


    ACKNOWLEDGEMENTS

We thank Y. Nakagawa for preparation of valuable reagents and discussions, J. Asplin, F. Coe, S. Deganello, and F.G. Toback for valuable advice and discussions, and R. Norris for technical assistance. We also thank A. Beshensky and E. Worcester for mouse renal cortical cell culture medium used to isolate osteopontin and J. Sallis for kindly supplying phosphocitrate.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Clinical Investigator Award K08 DK-02272 and Grant R01 DK-53399 (to J. C. Lieske), the O'Brien Kidney Research Center at the University of Chicago (P50 DK-47631), and the Oxalosis and Hyperoxaluria Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. C. Lieske, Mayo Foundation, Div. of Nephrology, 200 First St. SW, Rochester, MN 55905.

Received 20 July 1999; accepted in final form 6 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 278(6):F989-F998
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