JOURNAL OF BONE AND MINERAL RESEARCH Volume 7, Number 9, 1992 Mary Ann Liebert, Inc., Publishers

The Calcium Oxalate Crystal Growth Inhibitor Protein Produced by Mouse Kidney Cortical Cells in Culture Is Osteopontin ELAINE M. WORCESTER, SAMUEL S. BLUMENTHAL, ANN M. BESHENSKY, and DONNA L. LEWAND

ABSTRACT Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 kg/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 pg/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.

INTRODUCTION

We recently purified a crystal growth inhibitory protein from mouse kidney cortical cell tissue culture media and determined the N-terminal sequence of the protein. The seRINE CONTAINS PROTEINS that inhibit the growth of calcium oxalate (CaOx) crystals. l ’ ) These proteins may quence of the first 21 amino acid residues matches the protect against the growth of CaOx crystals in vivo and be previously identified amino terminus of the bone phosphoimportant in preventing the formation of urinary calculi. protein, osteopontin (OP), also known as secreted phosOne of these crystal growth inhibitory proteins, nephrocal- phoprotein l.I6,’) The cDNA clone for this protein, obcin (NC), has been partially characterized; it is an acid gly- tained from mouse epidermal cells stimulated with tumor coprotein containing y-carboxyglutamic acid (gla). 1 2 ) promoters, has been called 2ar.l’’ This protein, which does The site of NC synthesis is thought to be the kidney.(3’ not contain gla, also appears in urine and may be a second Several crystal growth inhibitory proteins have also been urinary crystal growth inhibitor, distinct from NC. isolated from conditioned media of mouse kidney cortical tubule cells growth in defined medium. These proteins MATERIALS AND METHODS have amino acid compositions similar to that of urinary Culture technique NC, but only one of the four peaks eluted from an anionexchange column contained gla.1‘) Primary cultures of renal cortical tubule cells were pre-

U

Nephrology Section, Medical Service, Zablocki VA Medical Center and Department of Medicine, Medical College of Wisconsin, Milwaukee.

1029

1030

pared from the kidneys of 4-week-old C57b16 mice, as previously described.(8)Mice were lightly anesthetized and the kidneys removed and placed in sterile Hank’s balanced salt solution (HBSS). Under a laminar flow hood the capsule was removed, the cortex separated from the medulla, and the tissue digested for 20-40 minutes at 37°C with collagenase (0.25 mg/ml) and soybean trypsin inhibitor (0.5 mg/ ml). Digested tissue was mixed with equal volumes of 10% horse serum in HBSS and centrifuged at 700 rpm for 7 minutes at room temperature. The cell pellet was resuspended in 20 ml serum-free medium composed of equal volumes of Dulbecco’s modified medium and Ham’s F12 medium containing 10 mM HEPES, penicillin (50 units/ ml), streptomycin (50 pg/ml), 2 mM L-glutamine, transferrin ( 5 pg/ml), insulin ( 5 pg/ml), and prostaglandin E, (PGE, 25 ng/ml). The medium osmolality was adjusted to 360 mosmol/liter with choline ~ h l o r i d e . ‘ ~ ) Cultures were initiated by adding a volume of cell suspension containing 2-4 mg protein per ml to 100 mm tissue cultures dishes with 15-20 vol more of serum-free media. Cultures were incubated at 37°C under 95% air and 5 % CO, and the medium changed at 24 h and every 2-3 days thereafter. These primary cultures retain many biochemical features of proximal tubule cells, such as Na’-dependent substrate cotransport activity and 25-hydroxyvitamin D, (25-ODH) 1 ,a-hydroxylase activity.(*.’’

-

Protein purification Media containing 200 pg/ml of leupeptin in addition to the previous additives was incubated with confluent cultures for up to 72 h. Conditioned media (-3OOO ml) were diluted with water to bring the NaCl concentration to 0.05 M and protease inhibitors added to achieve the following concentrations: 1 pM leupeptin, 200 pM PMSF, 1 pM pepstatin, and 100 pM EDTA. Unless otherwise stated, protease inhibitors were present in all subsequent steps. Conditioned cell media were stirred at 25°C with DEAE-cellulose (400 ml) in 0.05 M Tris-HCI and 0.05 M NaCl buffer, pH 7.3, for 30 minutes. The cake of DEAE-cellulose was filtered on a Buchner funnel and washed with 800 ml of the same buffer and the bound protein eluted with 500 ml of 0.05 M Tris-HCI and 0.4 M NaCI, pH 7.3, and dialyzed against 5 liters of 0.05 M Tris-HCI and 0.05 M NaCl overnight at 4°C. The dialyzed eluate was applied to a DEAEcellulose column (30 x 1.5 cm) and the bound protein eluted with a linear gradient of 0.1-0.4 M NaCl in 0.05 M Tris-HCI, pH 7.3 (800 ml). Column fractions were monitored for protein by absorbance at 280 nm, for NaCl concentration with a conductivity meter (Cole-Palmer, IL), and for inhibitory activity using a seeded crystal growth assay (see later). The active peak was dialyzed against water overnight at 4”C, lyophilized, and loaded on a (3-75 gel filtration column (80 x 1.5 cm), which was calibrated with bovine serum albumin (BSA), carbonic anhydrase, cytochrome c, and aprotinin. After elution with 0.2 M NaCl and 0.05 M Tris-HCI, pH 7.3, fractions were monitored for protein and inhibitory activity as before. Protein samples were electrophoresed on 10 or 12.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels by the method of Laemmli.(’O’

WORCESTER ET AL. Proteins were transferred to 0.2 pm nitrocellulose by electroblotting at 0.8 A for 1 h by the method of Towbin et aI.(l1) (Transphor Unit; LKB, Piscataway, NJ). After blotting, protein bands were visualized with colloidal gold (Bio-Rad, Richmond, CA), or in western blots (see later). Molecular weights were determined by running protein standards (Sigma, St. Louis, MO) in parallel lanes, which were gold stained. For sequencing, protein was electroblotted to polyvinylidene (PVDF) membrane (Immobilon; Millipore, Burlington, MA) in 0.022 M N-ethylmorpholine and 10% methanol adjusted to pH 8.3 with formic acid. Proteins were visualized with amido black. Protein sequencing was carried out using the Applied Biosystems pulsed liquid-phase sequencer Model 477A. Both protein sequencing and amino acid analysis were performed at the Protein/Nucleic Acid Shared Facility at the Medical College of Wisconsin. Amino acid analysis was performed following acid hydrolysis by separation on a cation-exchange column with a Beckman 6300 amino acid analyzer.

Crystal growth inhibition assays CaOx crystal growth inhibition was measured in two ways. Chromatography fractions and conditioned media were assayed for CaOx growth inhibition using a modification of a seeded crystal growth system.‘”) This method permits rapid assay of multiple specimens for the presence of growth inhibitory activity. The assay was performed in 2 ml microfuge tubes containing 0.67 ml of 2 mM CaCI, in 5 mM barbituric acid, 133 mM NaCI, and 0.03% acetic acid, pH 5.7. An aliquot of 0.134 ml of a CaOx crystal slurry (1.5 mg/ml) and 50 pI of the protein solution to be assayed was added to the tube. Tubes with no added protein were used as controls. After equilibrating the tubes at 37°C with constant stirring for 30 minutes, the assay was started by adding 0.67 ml of 0.4 mM sodium oxalate containing 0.1 pCi/ml of [L4C]oxalateas tracer in barbituric acid and acetic acid buffer. After 40 minutes of incubation, the tubes were removed from the water bath and centrifuged for 30 s to pellet the crystals and a 100 pI aliquot of supernatant withdrawn. Aliquots were dissolved in 4 ml scintillant (Opti-fluor; Packard) and counted in a liquid scintillation counter (Packard Model 3255). All assays were performed in duplicate. One set of duplicate tubes was used to obtain initial cpm. Inhibitory activity was determined according to the equation

where [I] is proportional to the inhibitor concentration, Cexprepresents the cpm at 40 minutes in the sample with inhibitor, C, represents the cpm present initially in the sample, and C,,,,,,, represents the cpm at 40 minutes in samples without inhibitor.(13’Polyaspartic acid (PA, molecular weight around 800) was used as a standard inhibitor, and inhibitory activity was expressed as P A units when comparing conditioned media samples. The inhibitory activity of purified protein was estimated using the constant composition assay of Sheehan and Nan~ o l l a s . ( ’This ~ ’ assay permits estimates of inhibitory activ-

1031

MOUSE KIDNEY CORTICAL CELLS MAKE OSTEOPONTIN ity in a system more comparable to that in vivo, in which calcium and oxalate concentrations remain stable over time; the growth rate of crystals can be more directly measured with this method. The assay was performed in a water-jacketed 5 ml glass vessel maintained at 37"C, bubbled with nitrogen, and stirred continuously. All solutions were filtered through a 0.2 pm filter before use. The reaction solution contained 0.01 HEPES, p H 7.5, and 0.15 M NaCl. The solution was brought to 0.45 mM Ca by the addition of 0.225 ml 0.01 M CaCl, in HEPES; C a activity was monitored continuously with a Ca-selective electrode (Radiometer, F2112 Ca, Ref K401) and the solution allowed to equilibrate for 10 minutes. An equal volume of 0.01 M sodium Ox was added and the solution allowed to equilibrate for another 5 minutes. The protein sample to be tested was added and the solution equilibrated for an additional 5 minutes until the baseline was stable. The reaction was initiated by the addition of 100 p1 of CaOx crystal slurry (1.5 mg/ml) prepared in HEPES. Ca and Ox activities in the reaction solution were maintained constant by potentiostatic addition of concentrated equimolar solutions of Ca and Ox in 0.01 M HEPES with automatic burettes (Radiometer, ABU 80). The volume of added titrant and the Ca activity were monitored continuously and plotted with a chart recorder. The rate at which Ca was added to the solution by the automatic titrator (Radiometer TTT8O) was taken as the rate of crystal formation. Experiments in which no protein was added were performed as controls; the rate of Ca addition to the solution was linear, with a slope of 0.254 pmol/minute. If the interaction between crystals and inhibitor protein follows a simple Langmuir adsorption isotherm, in which crystal growth rate is proportional to the number of free or unbound growth sites on the crystal surface, then the crystal growth rate at different inhibitor concentrations should reflect the affinity of the inhibitor protein for crystal surface, according to the equation k,/k,,

-

k, =

K/[4

where k, represents the rate of crystal growth at a given inhibitor concentration, k , represents the rate of crystal growth in the absence of inhibitor, K represents the dissociation constant for the crystal-inhibitor complex, and [A is the inhibitor concentration. Under conditions in which the assumptions of the Langmuir adsorption isotherm are valid, a plot of l / [ A versus k,/k, - k , yields a straight line with slope K . ('')

Immunochemical methods Antibodies (Ab) were generated against purified culture media inhibitor protein in rabbits using Freund's or Titermax (CytRx, Norcross, GA) adjuvants. Rabbits were injected intramuscularly with 50-75 pg protein, followed by repeat injections on days 30 and 60, and immune serum collected 10 days following the last boost. Ab raised to fusion proteins produced by plasmids containing N-terminal or C-terminal coding sequences of 2ar cDNA were a kind gift of Dr. David Denhardt."' Western blots were performed on protein blotted to ni-

8o

1

T

0

6

12

24

72

48

Time (hours) FIG. 1. Appearance of CaOx crystal growth inhibitory activity in serum-free media incubated with cultured mouse kidney cortical cells. After the cells reached confluence, fresh medium was placed on the cells. At the times shown, medium was removed from three to four plates, dialyzed, lyophilized, and assayed in the seeded crystal growth system. Values are mean f standard error of the mean (SEM).

0b

2

,

Elution volume ( m l )

FIG. 2. Isolation of the crystal growth inhibitor protein from conditioned media using DEAE-cellulose column chromatography (1.5 x 30 cm). Protein was eluted using a linear NaCl gradient from 0.1 to 0.4 M in 0.05 M TrisHCI, pH 7.3. Protein elution was monitored at 280 nm, and CaOx crystal growth inhibition was assayed in a seeded crystal growth system. The main peaks of inhibitory activity eluted between 23 and 27 mS. The bracket indicates the fractions pooled for further purification.

trocellulose; blots were rinsed in 0.020 M Tris-HCI, pH 7.5, and 0.5 M NaCl (TBS) for 30 minutes and then immersed in 1% gelatin-TBS overnight. This and all subsequent incubations were carried out at 25°C with gentle shaking. After blocking, the blot was placed in a 45-well Mini-blotter apparatus (Immunetics, Cambridge, MA) so that each lane of protein could be probed with three different Ab solutions. Ab were diluted in 2% BSA-TBS. The blot was incubated with the first A b for 4 h, rinsed in 0.05% Tween 20-TBS (TTBS), and incubated in alkaline phosphatase-labeled goat antirabbit IgG (1 :3000; Bio-Rad, Richmond, CA) for 1 h. After rinsing in TTBS followed by TBS, the blots were developed in a solution of 5-bromo4-chloro-3-indolyl phosphate and nitroblue tetrazoliurn.

1032

Competitive enzyme-linked immunosorbent assay (ELISA) was performed in 96-well microtiter plates (Costar) coated with purified inhibitor protein (0.5 pg/ml) in 0.02 M borate buffer, pH 8.4, overnight. The coating solution was removed, and nonspecific protein binding blocked by addition of 0.25% gelatin in 0.01 M sodium phosphate buffer containing 0.15 M NaCl (PBS), pH 7.3, to each well for 1 h; this and all subsequent incubations were done at 37°C. Blocking solution was removed, 50 p1 fresh blocking solution was placed in each well, and 50 pl of either purified inhibitor protein (1 pg/ml) or pooled mouse urine (diluted with blocking solution) was added to the first row of wells. Serial 1:2 dilutions were done in the plate, and then 50 pl antisera against inhibitor protein, diluted 1:1O,OOOin blocking solution, was added to each well and incubated for 3 h. Plates were washed with PBS three times and 100 p1 biotinylated goat antirabbit IgG (1:3OOO; Vector Laboratories) added to each well for 1 h. Plates were washed with PBS three times and 100 p1 avidin-biotinylated horseradish peroxidase complex added for 30 minutes. The plates were washed with PBS a final time, and 100 pl substrate solution containing 0.1 To orthophenylenediamine and 0.01% H,O, in 0.05 M citrate buffer (pH 4) was added to each well. After 30 minutes the color reaction was stopped by addition of 50 pl 4 N HISO, and the absorbance at 490 nm read (Dynatech MicroElisa Reader; Dynatech Laboratories, Alexandria, VA). The standard curve was constructed using a logit-log regression, and the concentration of crystal growth inhibitor in the urine sample calculated.

Other methods The gla assay was performed by the method of Nishimoto.'lsb Proteins (20 pg) were spotted onto nylon 66' membrane (Hoefer Scientific Instruments, San Francisco, CA) and incubated overnight in a solution of 4.8 x lo-' M 4-diazobenzenesulfonic acid (Fluka), 3.6 x lo-' M NaNO,, and 0.113 M acetate buffer, pH 4.6, at 25°C. Color was developed by bringing the solution to 6.6% NaOH and 0.066 M EDTA. Prothrombin was the positive control; the detection limit was 250 ng of this protein. Urine was aspirated from the bladders of several anesthetized mice. Urine was stored at 4°C with 0.01% a i d e and protease inhibitors before immunoassay. Protein concentration was determined by alkaline hydrolysis, followed by reaction with ninhydrin, using BSA as the standard.(16) We prepared seed crystals of CaOx monohydrate using standard methods(131;their structure was verified by x-ray crystallography. Statistical analysis was perfomred using Student's unpaired I-test.

WORCESTER ET AL. mond, CA). U[l5C]oxalic acid, 112 mCi/mmol, was purchased from Amersham. All other reagents were the highest grade available. Water was passed through a reverse osmosis apparatus and then purified with a Nanopure I1 system (Barnstead Thermolyne, Dubuque, LA).

RESULTS Production of crystal growth inhibitory activity by mouse kidney cell cultures over time is shown in Fig. 1. Media was collected from confluent cultures at 0, 6, 12, 24, 48, and 72 h and assayed for crystal growth inhibitory activity. As shown, activity increases rapidly over the first 12 h and continues to rise more slowly thereafter. At 24 h, the inhibitory activity is 15 times that in control media, p = 0.003. The inhibitory protein was isolated from media incubated on confluent cell cultures for 24 h using anion-exchange chromatography. Two major peaks of inhibitory activity eluted between 23 and 27 mS (Fig. 2). Both peaks were electrophoresed on 12.5% SDS-PAGE, blotted to nitrocellulose, and gold-stained, and each contained one major band of protein at 63 kD. The first peak, which appeared to contain the majority of the protein, was purified further by rechromatographing on a gel-filtration column. A single peak of inhibitory activity eluted at -78,OOO daltons (Fig. 3). Active fractions were pooled, separated by SDS-PAGE, and blotted t o nitrocellulose (Fig. 4A). The protein was assessed by gold-staining electroblots rather than by silver staining the gel because the protein does not visualize well with the latter technique. The apparent molecular weight of the protein varied depending on the percentage cross-linkage of the gel. On 12.5% gels, the major band was seen at a molecular weight of 63 kD, but on 10% gels, the band was consistently seen at a molecular weight of 80 kD. The apparent molecular weight did not change after incubation of the protein with 50 mM EDTA for 4 days (not shown).

I

"

u

Absorbance

+ lnhibdwn 08

05

0.2

00 0

50

100

150

Elution volume (ml)

Materials DEAE-cellulose was obtained from Pierce (Rock ford, IL) and recycled using the manufacturer's procedure. Sephadex G-75 was purchased from Pharmacia. Electrophoresis reagents were purchased from Bio-Rad (Rich-

FIG. 3. Gel-filtration chromatography on a Sephadex G-75 column (1.5 x 80 cm) of the pooled fractions from the DEAE-cellulose column. The crystal growth inhibitor is eluted with 0.2 M NaCl and 0.05 M Tris-HCI, pH 7.3, and a single peak of protein and crystal growth inhibition is seen.

1033

MOUSE KIDNEY CORTICAL CELLS MAKE OSTEOPONTIN

The amino-terminal sequence of this protein was deter- 4B). Both sets of antisera stained the same bands. Higher mined: Leu-Pro-Val-Lys-Val-Thr-Asp-Ser-Gly-Ser-X-Glumolecular weight bands may represent aggregates of the Glu-Lys-Leu-Tyr-Ser-X-His-Pro-Asp. A search of the protein; the lower molecular weight band may be a proteoGenbank and EMBL data bases using the program Word- lytic fragment. search (Genetics Computer Group, Inc., Madison, WI) The effect of the purified protein on the rate of CaOx showed that this sequence matched the N terminus of crystal growth in the constant composition assay is shown mouse o ~ t e o p o n t i n / 2 a r . ( ~The . ~ ~amino ) acid (aa) composi- in Fig. 5 . Protein concentrations of 0.5-1.5 pg/ml were tion of the purified protein is shown in Table 1, along with used in the assay. Increasing concentrations of protein propreviously reported aa compositions of the crystal growth gressively slowed the rate of crystal growth. The conceninhibitor NC isolated from rat kidney'18) and human tration of protein predicted to slow the growth of crystals urine(*) and the predicted amino acid composition of by 50% is 0.85 pg/ml. The dissociation constant of the mouse osteopontin/2ar. (7) This protein was assayed for protein for crystal, estimated from the Langmuir isotherm, M; in making this estimate a molecular the presence of gla after spotting on nylon membrane. No was 3.7 x gla was detected in 20 pg inhibitor, but 250 ng prothrom- weight of 44,OOO daltons was assumed (see later). The concentration of O P in pooled mouse urine, meabin gave a positive signal. Antisera raised to the purified protein in two different sured using antisera raised to the purified inhibitor from rabbits were compared in a western blot to antisera raised culture media, was 8 pg/ml. This is well above the concento fusion proteins containing either the N-terminal (2arN) tration prodicted to slow the growth of crystals by 50% in o r C-terminal (2arC) portions of osteopontin/2ar (Fig. our assay.

DISCUSSION

A

116 97.4

66

B

1

2

205 97.4 66 -

45 45

36 -

29 29

FIG. 4. Protein from the active peak of G75 column separated by 10% SDS-PAGE, under reducing conditions, and blotted to nitrocellulose. (A) Colloidal gold stain of 1 pg protein; estimated molecular weight of band is 80 kD. (B) Western blot using 3 pg protein per lane, showing the immunoreactivity toward the inhibitor protein of antisera raised to the purified protein and antisera raised to fusion proteins produced by plasmids containing the N-terminal (2arN) or C-terminal (2arC) coding sequences of 2ar cDNA. 2ar is the mouse homolog of osteopontin. Lane 1, antisera t o purified inhibitor: a , nonimmune rabbit serum 1:1000; b, serum H7 1:1000; c, serum H6 1:1000. Lane 2, antisera to 2ar fusion proteins: a, nonimmune serum 1:40; b, 2arN antiserum 1:40; c, 2arC antisera 1:40.

We have shown that a CaOx crystal growth inhibitory protein is secreted by primary cultures of mouse cortical tubule cells and that the N-terminal amino acid sequence of this protein is identical to that of 0steopontin/2ar.(*.~) Two amino acid residues could not be identified during sequencing. The amino acid at position l l predicted from both the genomic and cDNA sequences is Ser, and the residue at position 18 is Leu. During sequence analysis, it may be difficult to identify amino acids with posttranslational modifications, such as phosphorylation or glycosylation; this may account for the inability to identify the Ser residue. In addition to sequence homology, the inhibitor protein and OP share immunochemical similarity since Ab raised to both NH,-terminal and COOH-terminal 2ar fusion proteins recognize the crystal growth inhibitor on western blots. The molecular weight predicted from the aa sequence and the analysis of monosaccharides and phosphate bound to the protein isolated from rat bone matrix is 41,285 daltons."9' This agrees well with the molecular weight of 44,OOO obtained by sedimentation equilibrium analysis. (I9) However, the protein is known to migrate anomalously in SDS gels, and apparent molecular weight of 45-75 kD have been The apparent molecular weight of the inhibitor protein in SDS gels of 63,000 and 80,000 daltons reported here is consistent with these observations. The tendency of purified OP t o aggregate during electrophoresis, as seen on the western blots, has been noted by other investigators as well. In calculating molar concentrations, a molecular weight of 44 kD was assumed. OP, a phosphorylated glycoprotein, was isolated originally from rat bone matrix.(6' The protein is secreted by rat osteosarcoma cells in culture (ROS 17/2.8)(20)and by normal bone cells isolated from fetal calvaria. ( 2 0 ) Subsequently, mRNA for the mouse homolog, named 2ar, was found in cultures of mouse epidermal cells stimulated with phorbol Studies have shown that induction of OP occurs in response to several agents in many cultured

1034

WORCESTER ET AL. TABLE1. AMINOACIDCOMPOSITION OF INHIBITORISOLATEDFROM MOUSEKIDNEY CORTICAL CELLTISSUE CULTURE MEDIA(WEIGHT070)” Nephrocalcin Amino acid

Lysine Histidine Arginine Asp/Asn Threonine Serine GluGln Proline GI ycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Cysteine Gla

Mouse culture medium

2ar (Predicted)

Human urineb

Rat kidneyc

3.8 4.3 1.5 22.0 5.5 12.4 14.6 5.3 3.8 6.5 6.2 0.9 1.3 5 .O 1 .O 2.4 NM NM 0

6.1 5.4 2.2 17.6 4.3 17.6 14.4 5.4 3.2 4.3

3.6 2.0 4.1 10.6 8.8 9.7 12.2 5.9 10.8 1.6

2.8 1.8 2.8 11.0 8.3 11.0 14.7 6.4 8.3 5.5 6.4 0.9 3.7 7.3 1.8 3.7 NR 1.8 1.8

5 ‘4

6.4

1.1 1.8 7.6 1.4 1.8 0.4 0 0

0.6 2.4 6.3 1 .o 3.1 0.9 1.7 2.0

acornparison with composition of previously isolated crystal growth inhibitor proteins. NM, not measured; NR, not reported. bFrom Nakagawa et al. 1983 J Biol Chem 258:12594-12600. ‘From Nakagawa et al. 1984 Am J Physiol 247:F765-772.

Q)

Y

k -5 Y Y

0

0.02

0.04

0 06

0 08

1/[OP] (nM/L) FIG. 5. The effect of the inhibitor purified from mouse kidney cortical cell culture media on the growth of CaOx crystals. Growth rate data plotted in the form of a Langmuir-type isotherm. k, and k , refer to crystal growth in the presence and absence of purified inhibitor, respectively. The slope of the regression line, 3.7 x M , is the dissociation constant for the protein with respect to CaOx crystal. cell lines, but expression of OP mRNA in vivo is limited to a few tissues. In addition to bone-forming cells, mRNA for O P has been found in embryonic and postnatal kidney, placenta, and sensory epithelium of the ear.(zzI J 1 The protein has also been localized to these sites by immunohisto-

chemical staining. (I4) Immunohistochemistry of bone and neural cells identified staining within the Golgi apparatus, however, and staining in kidney tubule cells was localized to lysosome-like bodies, suggesting that these cells were taking up OP from the circulation. This is the first report of OP synthesis by cultured renal tubular cells. Although these cultures possess many characteristics of proximal tubule epithelium, the original preparation from which the monolayers are grown is not homogeneous, and cultures may contain cells derived from other parts of the nephron. OP mRNA has been found in the proximal and distal tubules and in the loop of Henle.(I3l Fibroblastic cells derived from rat kidney (NRK cells) have been reported to make O P in culture(”’; the cultures reported here d o not contain appreciable numbers of fibroblasts. The function of O P is not known. In mineralized tissue, expression is increased by 1,2Sdihydroxyvitamin D(16’and transforming growth factor /3(20B and decreased by paraIt contains a n Arg-Gly-Asp (RGD) thyroid sequence that is found in many cell adhesion molecules that bind to integrin-type receptors, such as fibronectin,(61 and the protein promotes the attachment of cultured osteosarcoma cells to plastic culture dishes.(61 OP also binds strongly to apatite,‘”’ and this suggests that it may promote cell attachment to mineralized matrix. We have shown that O P is a potent inhibitor of CaOx crystal growth in vitro. Our data suggest that the protein exerts its effect by adsorbing to the crystal surface. One in-

1035

MOUSE KIDNEY CORTICAL CELLS MAKE OSTEOPONTIN teresting feature of the structure of the OP is a series of 10 consecutive Asp residues at positions 69-78. Polyaspartic acid is a potent inhibitor of CaOx crystal growth,(**)and it is possible that this region of the protein is responsible for crystal binding and crystal growth inhibition. Presumably OP inhibits growth of crystals in vivo as well and may help prevent the growth of CaOX crystals in tubular fluid and urine, which are supersaturated with respect to this salt.(29' The protein is present in mouse urine in concentrations sufficient to significantly reduce crystal growth, as predicted by its behavior in the constant composition assay. A CaOx crystal growth inhibitory protein was recently isolated from human urine that has sequence homology to human osteopontin and was named ~ r o p o n t i n ( ~this ~ ' pro; tein may be the mouse homolog. A CaOx crystal growth inhibitor called N C has previously been isolated from the urine and kidneys of several mammalian species. ( 2 . 1 9 1 NC contains several residues of gla per molecule. A gla-containing crystal growth inhibitor has also been found in conditioned media from cultured mouse and human kidney cortical cell~.''.'~)O P has not been reported to contain gla, and the aa sequence predicted from cDNA and genomic analysis does not contain the conserved sequences identified in all known gla-containing proteins, such as o s t e ~ c a I c i n , (which ~ ~ ) are thought to be important for interaction of these proteins with the vitamin K-dependent carboxylase. Table 1 shows the aa compositions of the protein we isolated from culture medium, OP/2ar, and NC. All contain an abundance of acid residues and few basic residues, and all are glycosylated. The Asp content of OP is higher than that of NC, however, and OP contains no Cys or gla. It appears that O p and NC are distinct and that urine contains at least two proteins that inhibit CaOx crystal growth, both of which are produced by kidney cortical cells. In summary, mouse renal cortical tubule cells produce a crystal growth inhibitory protein with sequence and immunochemical similarity to OP. Ab to this protein recognize a protein in mouse urine. We propose that OP produced in the kidney is secreted into the urine and functions as a urinary crystal growth inhibitor. It may have other functions as well. Further studies are needed to understand the regulation of OP in kidney tissue and its role in stone formation.

REFERENCES 1. Coe FL, Margolis HC, Deutsch LH, Strauss AL 1980 Uri-

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

ACKNOWLEDGMENTS This work was supported by the Department of Veterans Affairs, and by research Grants DK41725-02 and EH1R01-ES0426 from the National Institutes of Health. We thank Dr. David Denhardt for the kind gift of antisera against 2ar fusion proteins, Dr. Neil Mandel and the National VA Crystal ID Center for crystallographic assistance, and Dr. Liane Mende-Mueller and the staff of the Protein and Nucleic Acid Facility of the Medical College of Wisconsin for their assistance. Presented in part at the 22nd Annual Meeting of the American Society of Nephrology, Washington, DC, December 3-6, 1989, and the 24th Annual Meeting of the American Society of Nephrology, Baltimore, MD, November 17-20, 1991.

14.

15.

16.

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Address reprint requests to: Elaine Worcester, M .D. Nephrology Section/l I I K Zablocki VAMC 5000 W . National Avenue Milwaukee, WI 53295 Received for publication August 2, 1991; in revised form March 23, 1992; accepted April 7, 1992.