Renal calcium oxalate binding protein: Studies on its properties

Kidney International, Vol. 53 (1998), pp. 125–129

Renal calcium oxalate binding protein: Studies on its properties MARIMUTHU ADHIRAI and RAMASAMY SELVAM Department of Medical Biochemistry, Dr. ALM. PGIBMS, University of Madras, Taramani, Chennai, India

Renal calcium oxalate binding protein: Studies on its properties. To understand the mechanism of calcium oxalate (CaOx) crystal retention within the kidneys, calcium oxalate binding protein was isolated and characterized. The specific activity of calcium oxalate binding protein in the homogenate was 4.54 nmol/mg protein. The renal medulla showed higher CaOx binding activity than that of papilla or cortex, and among the cellular fractions the nucleus exhibited highest specific activity. Several tissues showed CaOx binding activity suggesting its ubiquitous nature. After being subjected to acetone precipitation, ethanol precipitation and HPLC chromatography, the renal protein revealed a 57-fold purity with a specific activity of 260 nmol/mg protein and a molecular weight of 45 kDa. The CaOx binding protein had the kinetic properties of concentration and time dependency, optimum temperature and substrate saturability. Scatchard plot analysis showed a single affinity site with a kDa of 41 nM and Bmax of 6.7 nmol/mg protein. The binding activity was inhibited by the anion transport inhibitor DIDS and substrate analogs like succinate and oxamide, while EGTA or ruthenium red had no effect on binding, suggesting that the protein binding was oxalate site specific. The molecular weight of the CaOx binding protein of different tissues was similar to that of renal cells. In conclusion, the presence of CaOx binding protein is demonstrated in rat and human kidneys, as well as other rat tissues.

The interaction between renal epithelial cells and calcium oxalate (CaOx) crystals/oxalate ions has been suggested to play a critical role for the formation as well as retention of calcium oxalate stone [1]. Several studies have shown that cellular membranes [2], damaged proximal tubules and loop of Henle, and injured urothelium after treatment with gentamycin [3] induce nucleation of CaOx crystals at a lower supersaturation. Membrane damage due to lipid peroxidation or depletion of cellular antioxidants has been suggested as a predisposing factor for CaOx crystal deposition [4, 5], and administration of vitamin E or methionine prevents CaOx retention [6 – 8]. Oxalate binding proteins have been identified and characterized in human and rat kidney mitochondria [9] and nucleus [10], intestinal cytosol [11], vitamin B6-deficient rat intestinal brush border membranes [12] and bacterial system [13]. Several CaOx crystal associated proteins have been isolated from matrix of stones [14] or urine crystals [15]. All these proteins are found to be derived from different tissues and the existence of any specific protein involved in stone retention is not known. Some of the basement membrane com-

Key words: calcium oxalate binding, tissue distribution, protein kinetics, stones. Received for publication May 13, 1997 and in revised form August 20, 1997 Accepted for publication August 21, 1997

© 1998 by the International Society of Nephrology

ponents have a high affinity for CaOx crystals and promote crystal adhesion [16]. To understand the mechanism of CaOx retention within the kidneys, it is necessary to identify the CaOx binding factor. This communication presents the data for the presence of a CaOx binding protein in renal cells as well as other tissues. METHODS Adult male albino rats of the Wistar strain weighing 180 to 200 g, obtained from Tamil Nadu Veterinary University, Chennai were used for the study. The animals were fed with standard rat pellet diet supplied by M/s. Hindustan Lever Ltd. (Bombay, India) and water was provided ad libitum. [14C]-oxalate was purchased from Babha Atomic Research Centre (BARC; Bombay, India), which has a specific activity of 5.86 mCi/mmol/ml. 2-(N-morpholino) ethane sulphonic acid (MES) and 3-(N-morpholino)-propanesulphonic acid (MOPS) were obtained from Sigma Chemical Company (St. Louis, MO, USA). Millipore membrane filters 0.45 mm were obtained from Millipore India Ltd. (Bangalore, India). All other chemicals, reagents and solvents used were of the analytical grade from Glaxo Laboratories (BDH Chemicals, Bombay, India). Subcellular fractionation Rat tissues were excised from the animals following cervical decapitation and quickly dropped into ice-cold saline. Twenty percent rat tissue homogenate was made from 0.01 M TRIS-HCL CONTAINING 0.25 M sucrose pH 7.4, and then the mixture was subjected to differential centrifugation. Pellets obtained after 800 3 g for 15 minutes and 8000 3 g for 20 minutes were the nuclear and mitochondrial fractions, respectively. The remaining supernatant was for the post-mitochondrial fraction. Preparation of calcium [14C]-oxalate monohydrate crystals Calcium [14C]-oxalate monohydrate (CaOx*) crystals were synthesized essentially as described by Meyer and Smith [17]. Assay of calcium oxalate binding protein Calcium oxalate binding activity was estimated by the method described by Laxmanan et al [18] for oxalate binding with a slight modification. A 20% homogenate was prepared in MES buffer (0.1 M, pH 5.5). An aliquot of the homogenate or the subcellular fractions suspended in MES buffer (0.1 M pH 5.5) was incubated in 1 ml of 0.1 M MES buffer (pH 5.5) containing 60 nmol CaOx* (3120 cpm; 52 cpm/nmol) at room temperature for 45 minutes. The incubation mixture was filtered through a 0.45 mm Millipore membrane filter with the aid of a constant vacuum. Nonspecific binding was

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Table 1. Calcium oxalate (CaOx) binding activity in cortex and medulla of rat and human kidney and in papilla of rat kidney

Table 2. Calcium oxalate (CaOx) binding in subcellular fractions of different tissues of rat and human kidney CaOx binding nmol/mg protein

CaOx binding Rat

Cortex Medulla Papilla

Human

nmol/mg protein

nmol/g tissue

nmol/mg protein

nmol/g tissue

2.96 6 0.15 6.30 6 0.45 5.65 6 0.4

420 6 30 794 6 49 583 6 37

2.77 6 0.14 5.92 6 0.44

401 6 25 753 6 5

Homogenate of cortex, medulla or papilla (500 mg) was incubated with CaOx, and the radioactivity retained with the membrane filter discs was measured as described in the Methods section. Values are expressed as mean 6 SD of six experiments.

determined as the radioactivity retained by the filter in the presence of 13 mmol cold oxalate. The filters were dried and placed in mini vials to which 4.0 ml scintillation fluid was added, and the CaOx* was measured in a Betamatic IV liquid scintillation counter. Specific activity of oxalate binding was expressed as nmol/mg protein. Purification of calcium oxalate binding protein from human kidney, rat kidney and other rat tissues One milliliter of 20% tissue homogenate prepared in MES buffer (0.1 M, pH 5.5), and CaOx* (50,000 cpm), HCl (0.05 N, 300 ml) and MOPS buffer (0.1 M, pH 7.4, 1.5 ml) were added. The results were mixed well and incubated for one hour at room temperature. Step I: Delipidation by organic solvents This step was based on the method of Findlay [19] with slight modifications. (1.) To 1.75 ml of the above incubation mixture 7.5 ml of cold chloroform methanol (2:1 vol/vol) were added. (2.) This was shaken vigorously and centrifuged at 3000 3 g for five minutes. The clear upper part of the intermediate layer was aspirated out and used for the acetone precipitation. Step II: Acetone precipitation The aspirated layer of step I was added to an equal amount of ice cold acetone, stirred and left at 4°C for six hours. Then the mixture was centrifuged at 10,000 3 g for 20 minutes. Step III: Ethanol precipitation Acetone precipitate obtained from 500 mg of rat kidney alone was dissolved in 1 ml MES buffer (0.1 M, pH 5.5) and added to two volumes of ice cold ethanol. The mixture was left at 4°C overnight before centrifugation at 10,000 3 g for 15 minutes. High pressure liquid chromatography A modified method of Brewer and Sassenfeld [20] was used. The protein precipitates of steps II and III were suspended in suitable amount of MES buffer (0.1 M, pH 5.5) and 25 ml was loaded onto a high pressure liquid chromatography (HPLC) TSK 3000 Supelco gel permeation column that was fitted to LC-8A Schimadzu liquid chromatograph with a SPD 6A UV spectrophotometer detector. The CaOx* bound protein peak was monitored at 204 nm and radioactivity was measured. Marker proteins were also run under similar conditions to determine the molecular weight of the desired protein.

Tissues

Homogenate

Nucleus

Mitochondria

Post-mitochondrial fraction

Brain Lungs Spleen Heart Small intestine Liver Kidney rat Kidney human

3.20 6 0.21 1.36 6 0.07 4.23 6 0.31 2.98 6 0.22

4.17 6 0.30 2.52 6 0.17 6.08 6 0.45 5.48 6 0.39

1.62 6 0.12 1.10 6 0.07 2.85 6 0.17 1.02 6 0.04

1.00 6 0.05 0.78 6 0.03 1.18 6 0.09 1.00 6 0.05

0.98 6 0.06 0.82 6 0.05 3.81 6 0.27 5.60 6 0.42

0.91 6 0.06 2.15 6 0.19

0.61 6 0.04 0.97 6 0.07

4.54 6 0.25 6.54 6 0.5

2.74 6 0.21

1.01 6 0.06

3.10 6 0.27

1.02 6 0.06

4.20 6 0.2

5.93 6 0.48

Values are expressed as mean 6

SD

of six experiments.

RESULTS Calcium oxalate binding was studied in different regions of rat and human kidneys (Table 1). Maximal CaOx binding was observed in the medullary region of both rat and human kidney, followed by the papilla and cortex regions. The ubiquitous nature of CaOx binding protein was found by its binding with various subcellular fractions of different tissues (Table 2). Kidney, liver, spleen and brain showed good binding activity, while the lungs and intestine showed poor activity. Among the different subcellular fractions in all of the tissues, the nucleus showed the highest CaOx binding activity. Kinetic studies on calcium oxalate binding protein Calcium oxalate binding activity increased linearly up to a protein concentration of 900 mg (specific activity 5 4.54 nmol), and above that concentration there was no further increase in binding for both rat and human kidney homogenates. Hence, further studies were carried out at the protein concentration of 500 mg. Calcium oxalate binding was linear up to 45 minutes, after which there was no further increase in binding activity in both rat and human kidney. Experiments were carried out with a 60minute incubation time. Maximal CaOx binding was seen at pH 5.5 and further studies were carried out at that pH. The optimum temperature for CaOx binding was 30°C. Calcium oxalate binding activities were studied with increasing CaOx concentrations from 5 to 150 mM. The CaOx binding activity was dependent on the concentration of calcium oxalate in the medium. The saturation concentration of CaOx binding was observed above 80 mM (Fig. 1). Scatchard plot analysis was carried out by plotting bound CaOx/free CaOx ratio versus bound CaOx. As shown in Figure 2, the dissociation constant (Kd) of rat and human kidney CaOx binding proteins were computed to be 39.7 and 39.49 nM, and the maximum binding (Bmax) to be 6.5 nmol and 6.2 nmol/mg protein, respectively. To determine the specificity of CaOx binding of rat and human kidney protein,studies were carried out in the presence of oxamide, citrate, glycolate and glyoxylate (Table 3). Of all the analogs, succinate showed the maximum inhibition and glyoxylate showed the minimum inhibition for both rat and human kidney proteins.

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Table 3. Effect of substrate analogs, thiol modifiers, Ca21 chelators and interferers on (CaOx) binding in kidney homogenate CaOx binding nmol/mg protein Additives 2 mM

Fig. 1. Effect of substrate concentration on calcium oxalate (CaOx) binding in rat (F) and human (1) kidney homogenates.

None Oxamide Succinate Malate Trisodium citrate Glycolate Glyoxylate Phenyl succinate N-ethyl maleimide p-chloromercury benzoate GSSG GSH b-mercaptoethanol Dithiothrietol EGTA Ruthenium red CaCl2 z 2H2O MgCl2 z 6H2O Na2SO4 NaHCO3

Rat

% of control

Human

% of control

4.54 6 0.25 1.31 6 0.06b 0.60 6 0.02b 1.98 6 0.12b 1.89 6 0.11b 2.64 6 0.17b 3.10 6 0.21b 1.40 6 0.1 2.66 6 0.19b 3.46 6 0.24b

100 29 13 44 42 58 68 31 59 76

4.20 6 0.24 1.80 6 0.1b 1.91 6 0.12 2.50 6 0.15b 2.10 6 0.14b 2.70 6 0.19b 3.20 6 0.25b 1.62 6 0.11b 2.16 6 0.17b 2.5 6 0.19b

100 43 46 60 50 64 76 39 51 60

4.37 6 0.35 1.62 6 0.11b 3.1 6 0.22b 1.65 6 0.13b 4.20 6 0.23 5.20 6 0.25a 3.09 6 0.19b 4.38 6 0.24 1.05 6 0.1b 2.19 6 0.17b

96 36 68 36 93 115 86 97 23 48

4.1 6 0.31 2.35 6 0.17b 3.2 6 0.2b 1.92 6 0.16b 4.19 6 0.2 4.30 6 0.23 3.60 6 0.17b 4.21 6 0.23 0.90 6 0.03b 2.10 6 0.15b

98 56 76 46 100 102 86 100 21 50

Values are expressed as mean 6 SD of six experiments. a P , 0.01; b P , 0.001 vs. control.

Fig. 3. Effect of DIDS on calcium oxalate (CaOx) binding in rat (F) and human (1) kidney homogenates.

Fig. 2. Scatchard plot of calcium oxalate (CaOx) binding activity in kidney homogenates of rat (E) and human (F).

DIDS concentration up to 0.5 mM, above which there was no further inhibition of binding (Fig. 3). Purification of calcium oxalate binding protein

However, none were able to inhibit CaOx binding completely, thus demonstrating high specificity of CaOx binding to rat and human kidney proteins. Thiol reagents were found to inhibit CaOx binding activity of both rat and human kidney protein by 30% to 60%, but oxidized glutathione had no effect. Sulphate and carbonates inhibited CaOx binding very effectively whereas EGTA, ruthenium red and MgCl2 did not affect CaOx binding. Calcium oxalate binding was inhibited linearly with increasing

Calcium oxalate binding protein was purified from 4.25 ml of 20% rat kidney homogenate with a total protein concentration of 112.6 mg, and the total bound CaOx* was 511.32 nmol. The specific activity of CaOx binding protein of rat kidney homogenate was found to be 4.54 nmol CaOx/mg protein, and this was increased to 25.2 nmol when the homogenate was extracted with chloroform and methanol, as described in the Methods section, with a yield of 50%. Acetone precipitate of this extract showed 29.7% yield and 22-fold purity. Further precipitate with ethanol gave 10% yield with 45-fold purity, and the specific activity was

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101 nmol/mg protein. The precipitate was loaded onto HPLC column and the elution of CaOx binding protein was monitored by measuring radioactivity of CaOx bound protein peak. The CaOx binding protein had a specific activity of 260 nmol/mg protein and showed 57-fold purity with a final yield of 6.2%. Calf-thymus histone To determine whether histone protein was responsible for CaOx binding (Selvam and Prasannalakshmi [10], Selvam and Kannabiran [21]), calf-thymus histone (H1) was incubated with CaOx, and then extracted with chloroform-methanol followed by precipitation with acetone, as described for the purification of CaOx binding protein in the Methods section. There was little CaOx binding activity in the acetone precipitate, suggesting that proteins other than histones were responsible for the CaOx binding. Molecular weight of calcium oxalate binding protein The acetone precipitate of different tissues of rat and human kidney and the ethanol precipitate of rat kidney were loaded onto a HPLC column. From the calibration curve of standard molecular weight markers the molecular weight of CaOx binding protein of these tissues was computed to be around 45 to 47 kDa. The specific activity of CaOx binding protein in the acetone precipitate of each tissue was between 190 and 207 nmol/mg protein, whereas the specific activity in ethanol precipitate was 260 nmol of CaOx*/mg protein. DISCUSSION Calcium oxalate binding activity is enriched in the nuclear fraction of renal cells of both human and rat tissue. The same is true of the other tissues examined in this study. Liver and spleen are the other tissues having good calcium oxalate binding activity. The physiological significance of nuclear binding in renal cells as well as other tissues is not well understood, however, there is evidence showing that the binding may be physiologically important for certain nuclear function. Lieske et al [22] demonstrated that monkey kidney epithelial cells (BSC-1) in culture can also internalize CaOx crystals and undergo proliferation in the presence of the crystals. Internalized crystals are found to be distributed to daughter cells during cell division. It has recently been shown by Hammes et al [23] that CaOx crystals induce expression of immediate early genes C-myc, C-jun, EGR-1 and NVR-77 and genes encoding plasminogen activator inhibitor (PAI-1) and platelet-derived growth factor (PDGF)-A in BAC-1 kidney epithelial cells in primary cultures of rat proximal tubular epithelium exposed to oxidative stress [24]. Calcium oxalate crystals have been observed in normal thyroid tissue [25] and mammary gland [26]. The presence of these crystals is attributed to the normal function of thyroid gland, since these crystals are absent in abnormal function. The calcium oxalate binding protein has the following characteristic properties of saturation of binding: optimum pH at 5.5, time and temperature dependency, substrate specificity, inhibition by oxalate analogs but not by calcium chelators, and inhibition of binding by thiol modifiers and the anion transport inhibitor, DIDS. Saturation studies reveal the maximal CaOx binding at about 5 nmol of CaOx/mg protein. This suggests that about 100 to 120 mg CaOx can bind per gram of tissue. Binding is maximal at pH 5.5.

Laxmanan et al [18] have demonstrated maximal oxalate binding in rat kidney mitochondria at pH 6.0. Oxalate binding histone protein has exhibited maximal binding at pH 4.2 [10]. The CaOx binding data by Scatchard plot analysis reveals the presence of a single type of binding site. This is similar to the oxalate binding protein of rat and human mitochondria [9], which exhibits a single affinity site. However, the presence of two types of affinity sites, one with a high affinity and the other with a low affinity for oxalate, has been reported for histone-oxalate binding protein [10, 21] and for intestinal brush border membrane vesicle (BBMV) oxalate binding protein of vitamin B6-deficient rats [27]. Effect of substrate analogs Calcium oxalate binding proteins of rat and human kidney homogenate seem to be highly specific for oxalate and are only partially inhibited by substrate analogs. The structurally related compounds to oxalate are better inhibitors than dicarboxylates or tricarboxylates, showing high specificity of oxalate binding. Similar findings have been reported by Laxmanan et al [18]. Further inhibition of CaOx binding by bicarbonate and sulphate suggests that these anions are much more effective inhibitors than substrate analogs. Similar observations have been reported in cell culture studies [28]. The Ca21 chelators and interferers such as EGTA, ruthenium red, CaCl2 and MgCl2, do not affect CaOx binding activity. This proves that it is only the oxalate moiety that is responsible for specific binding of CaOx and not calcium. Our observations are in contrast to the cytosolic oxalate binding protein of human intestine [11], which is Ca21 dependent. Characterization of calcium oxalate binding protein The purified calcium oxalate binding protein shows a specific activity of 260 nmol/mg protein. Rat protein has a molecular weight of 45 kDa as determined by HPLC. Various oxalate transport/binding proteins with different molecular weights from different sources have been isolated, purified and characterized [10 –12, 29, 30]. The presence of CaOx binding protein in different tissues suggests its ubiquitous nature. Further CaOx binding is not confined to one particular subcellular fraction. It is present in the nucleus, mitochondria and microsomal fractions of all the tissues tested. The nuclear CaOx binding is not due to histone-oxalate binding protein, since histone H1 does not show CaOx binding activity when subjected to a similar treatment of protein extraction as that of CaOx binding protein. The approximate molecular weight of the CaOx binding protein of different tissues determined by HPLC is found in the range of 45 to 47 kDa. The tissues, kidney and liver that are handling oxalate show a higher CaOx binding activity than most other tissues except spleen. Though CaOx binding protein is present in a number of tissues studied, only kidney is known to form calcium oxalate stones; other tissues do not form stones. In pathological conditions such as systematic oxalosis, many tissues including dermis [31], testis [32] and joint fluid [33] are deposited with CaOx. This implies that sufficient saturation may not exist in other tissues for the initiation of nucleation even in the presence of CaOx binding protein, whereas a saturation of CaOx is possible in the kidney that results in hyperoxaluria, and initiation of nucleation can be aided by CaOx binding proteins. Sloughed cellular membranes are shown to induce nucleation

Adhirai and Selvam: Renal calcium oxalate binding protein

of CaOx crystals at a lower supersaturation. and can also be involved in crystal aggregation and retention. Further, it is shown that some of the basement membrane components having a high affinity for CaOx may help to promote crystal adhesion [34]. The significance of the occurrence of CaOx binding protein in the subcellular fractions is not well understood. However, studies by Khan [35] suggest that crystal retention occurs by adherence to other cells, and they move from lumen to cellular and then to the interstitium. Tubular epithelial cells endocytose the crystals on the luminal side and exocytose them on the basolateral side. Consequently, crystals move from the lumen to the interstitial location in the kidney. In the process, crystals come into contact with the basement membrane, to which they bind and are thus anchored in the kidneys. These observations suggest that CaOx crystals move from one side of the cell to other side for permanent attachment. This process may be facilitated by the presence of CaOx binding protein in different subcellular fractions. ACKNOWLEDGMENTS M.A. was recipient of Junior Research Fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi. This work was partly supported by Lady Tata Memorial Trust, Bombay, India. Reprint requests to Dr. R. Selvam, Department of Medical Biochemistry, Dr. A.L.M.P.G. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, India.

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