Department of Medicine, The University of Chicago, Chicago, Illinois 60637
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 · min1 · mg
1.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
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.
|
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).
|
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.
|
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).
|
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Burns, JR,
and
Finlayson B.
A proposal for a standard reference artificial urine in in vitro urolithiasis experiments.
Invest Urol
18:
167-169,
1980[ISI][Medline].
2.
Burt, HM,
and
Jackson JK.
Role of membrane proteins in monosodium urate crystal-membrane interactions. II. Effect of pretreatments with membrane permeable and impermeable protein crosslinking agents.
J Rheumatol
17:
1359-1363,
1990[ISI][Medline].
3.
Burt, HM,
Jackson JK,
and
Kim KJK
Role of membrane proteins in monosodium urate crystal-membrane interactions. I. Effect of pretreatment of erythrocyte membranes with glutaraldehyde and neuraminidase.
J Rheumatol
17:
1353-1358,
1990[ISI][Medline].
4.
Cheung, HS,
Sallis JD,
Mitchell PG,
and
Struve JA.
Inhibition of basic calcium phosphate crystal-induced mitogenesis by phosphocitrate.
Biochem Biophys Res Commun
171:
20-25,
1990[ISI][Medline].
5.
Coe, FL.
Uric acid and calcium oxalate nephrolithiasis.
Kidney Int
24:
392-403,
1983[ISI][Medline].
6.
De Bruijn, WC,
Boevé ER,
van Run PRWA,
Van Miert PPMC,
de Water R,
Romijn JC,
Verkoelen CF,
Cao LC,
van `t Noordende JM,
and
Schröder FH.
Etiology of calcium oxalate nephrolithiasis in rats. II. The role of the papilla in stone formation.
Scanning Microsc
9:
115-125,
1995[ISI][Medline].
7.
Emmerson, BT,
Cross M,
Osborne JM,
and
Axelsen RA.
Ultrastructural studies of the reaction of urate crystals with a cultured renal tubular cell line.
Nephron
59:
403-408,
1991[ISI][Medline].
8.
Emmerson, BT,
Cross M,
Osborne JM,
and
Axelson RA.
Reaction of MDCK cells to crystals of monosodium urate monohydrate and uric acid.
Kidney Int
37:
36-43,
1990[ISI][Medline].
9.
Ettinger, B,
Tang A,
Citron JT,
Livermore B,
and
Williams T.
Randomized trial of allopurinol in the prevention of calcium oxalate calculi.
N Engl J Med
315:
1386-1389,
1986[Abstract].
10.
Finlayson, B,
Newman RC,
and
Hunter PC.
The role of urate and allopurinol in stone disease: a review.
In: Urolithiasis and Related Research, edited by Schwille PO,
Smith LH,
Robertson WG,
and Vahlensieck W.. New York: Plenum, 1985, p. 499-503.
11.
Grases, F,
Costa-Bauza A,
March JG,
and
Masarova L.
Glycosaminoglycans, uric acid and calcium oxalate urolithiasis.
Urol Res
19:
375-380,
1991[ISI][Medline].
13.
Hess, B,
Nakagawa Y,
and
Coe FL.
Inhibition of calcium oxalate monohydrate crystal aggregation by urine proteins.
Am J Physiol Renal Fluid Electrolyte Physiol
257:
F99-F106,
1989
14.
Hess, B,
Nakagawa Y,
Parks JH,
and
Coe FL.
Molecular abnormality of Tamm-Horsfall glycoprotein in calcium oxalate nephrolithiasis.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F569-F578,
1991
15.
Lieske, JC,
Leonard R,
Swift HS,
and
Toback FG.
Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F192-F199,
1996
16.
Lieske, JC,
Leonard R,
and
Toback FG.
Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F604-F612,
1995
17.
Lieske, JC,
Norris R,
Swift H,
and
Toback FG.
Adhesion, internalization and metabolism of calcium oxalate monohydrate crystals by renal epithelial cells.
Kidney Int
52:
1291-1301,
1997[ISI][Medline].
18.
Lieske, JC,
Norris R,
and
Toback FG.
Adhesion of hydroxyapatite crystals to anionic sites on the surface of renal epithelial cells.
Am J Physiol Renal Physiol
273:
F224-F233,
1997
19.
Lieske, JC,
Swift HS,
Martin T,
Patterson B,
and
Toback FG.
Renal epithelial cells rapidly bind and internalize calcium oxalate monohydrate crystals.
Proc Natl Acad Sci USA
91:
6987-6991,
1994[Abstract].
20.
Lieske, JC,
Walsh-Reitz MM,
and
Toback FG.
Calcium oxalate monohydrate crystals are endocytosed by renal epithelial cells and induce proliferation.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F622-F630,
1992
21.
Lonsdale, K.
Epitaxy as a growth factor in urinary calculi and gallstones.
Nature
217:
56-58,
1968[ISI][Medline].
22.
Mandel, G,
and
Mandel N.
Crystal-crystal interactions.
In: Kidney Stones: Medical and Surgical Management, edited by Coe FL,
Favus MJ,
Pak CYC,
Parks JH,
and Preminger GM.. New York: Lippincott-Raven, 1996, p. 115-127.
23.
Mandel, NS.
The structural basis of crystal-induced membranolysis.
Arthritis Rheum
19:
439-445,
1976[ISI][Medline].
24.
Mandel, NS,
and
Mandel GS.
Urinary tract stone disease in the United States veteran population. II. Geographical analysis of variations in composition.
J Urol
142:
1516-1521,
1989[ISI][Medline].
25.
Nakagawa, Y,
Sirivongs D,
Novy MB,
Netzer MF,
Michaels E,
Vogelzang NJ,
and
Coe FL.
Nephrocalcin: biosynthesis by human renal carcinoma cells in vitro and in vivo.
Cancer Res
52:
1573-1579,
1992[Abstract].
26.
Pak, CY,
and
Arnold LH.
Heterogeneous nucleation of calcium oxalate by seeds of monosodium urate.
Proc Soc Exp Biol Med
149:
930-932,
1975[Abstract].
27.
Pak, CY,
Koenig K,
Khan R,
Haynes S,
and
Padalino P.
Physicochemical action of potassium-magnesium citrate in nephrolithiasis.
J Bone Miner Res
7:
281-285,
1992[ISI][Medline].
28.
Parks, JH,
and
Coe FL.
Urine citrate and calcium in calcium nephrolithiasis.
Adv Exp Med Biol
208:
445-449,
1986[Medline].
29.
Riese, RJ,
Kleinman JG,
Wiessner JH,
Mandel GS,
and
Mandel NS.
Uric acid crystal binding to renal inner medullary collecting duct cells in primary culture.
J Am Soc Nephrol
1:
187-192,
1990[Abstract].
30.
Ringertz, H.
The molecular and crystal structure of uric acid.
Acta Cryst
20:
397-403,
1966[ISI].
31.
Ryall, RL,
Hibberd CM,
and
Marshall VR.
The effect of crystalline monosodium urate on the crystallisation of calcium oxalate in whole human urine.
Urol Res
14:
63-65,
1986[ISI][Medline].
32.
Shiraga, H,
Min W,
VanDusen WJ,
Clayman MD,
Miner D,
Terrell CH,
Sherbotie JR,
Foreman JW,
Przysiecki C,
Neilson EG,
and
Hoyer JR.
Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.
Proc Natl Acad Sci USA
89:
426-430,
1992[Abstract].
33.
Tiselius, HG,
and
Larsson L.
Urinary excretion of urate in patients with calcium oxalate stone disease.
Urol Res
11:
279-283,
1983[ISI][Medline].
34.
Unwin, R,
Wrong O,
Cohen E,
Tanner M,
and
Thakker R.
Unraveling of the molecular mechanisms of kidney stones.
Lancet
348:
1561-1565,
1996[ISI][Medline].
35.
Verkoelen CF, van der Boom BG, and Romijn JC. Cell density
dependent calcium oxalate crystal binding to sulphated proteins at the
surface of MDCK cells (Abstract). Urolithiasis: 208-211, 1996.
36.
Weissmann, G,
and
Rita GA.
Molecular basis of gouty inflammation: interaction of monosodium urate monohydrate crystals with lysosomes and liposomes.
Nature
240:
167-172,
1972.
37.
Worcester, EM,
Blumenthal SS,
Beshensky AM,
and
Lewand DL.
The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.
J Bone Miner Res
7:
1029-1036,
1992[ISI][Medline].
38.
Worcester, EM,
Nakagawa Y,
Wabner CL,
Kumar S,
and
Coe FL.
Crystal adsorption and growth slowing by nephrocalcin, albumin, and Tamm-Horsfall protein.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F1197-F1205,
1988
39.
Zerwekh, JE,
Holt K,
and
Pak CY.
Natural urinary macromolecular inhibitors: attenuation of inhibitory activity by urate salts.
Kidney Int
23:
838-841,
1983[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |