Binding of Oxalate to Mitochondrial Inner Membranes of Rat and Human Kidney

0022-534 7/86/1354-0862$02.00/0 Vol. 135, April

THE JOURNAL OF UROLOGY

Copyright© 1986 by The Williams & Wilkins Co.

Printed in U.S.A.

BINDING OF OXALATE TO MITOCHONDRIAL INNER MEMBRANES OF RAT AND HUMAN KIDNEY SEETHALAKSHMI LAXMANAN,* RAMASAMY SELVAM,t CARL J. MAHLE

AND

MANI MENON

From the Division of Urology, University of Massachusetts Medical Center, Worcester, Massachusetts

ABSTRACT

Oxalate bound specifically to homogenates of rat kidney and liver but not to homogenates prepared from. heart, lung, skeletal muscle, spleen, stomach, small or large intestine. In the rerial cortex, binding was localized to the inner mitochondrial membrane where it was enriched fourfold when compared to homogenate. Binding of the oxalate reached equilibrium in two minutes at 23C. Analysis of the binding sites by Scatchard plot ihdicated that the maximum binding capacity was 49 pmol./:mg. protein and the apparent dissociation constant (K.i) was 43 nM. The IC50 of oxalate was 0.25 µM. Among the inhibitors studied the IC50 was in the following order: oxalate < oxamate < parabanate < glyoxalate < oxaloacetate < malate < citrate = glycollate. Heat and treatment with lubrol abolished the binding completely. Binding was not enhanced by the presence of calcium in the incubation medium; neither was it inhibited by the presence of calcium together with its transport inhibitors. A binding substance with some characteristics similar to the rat mitochondrial binding factor was also found in the human renal cortex. The exact mechanism of the initiation of calcium oxalate stone form~tion is not completely understood. One theory maintains that at certain critical periods, renal tubular concentrations of .calcium and oxalate exceed the thermodynamic solubility product of these ions and this causes spontaneous crystallization of calcium oxalate within the renal tubule. 1 In some unexplained fashion, these crystals become kidney stones. An alternate theory states that the crystals form first within the tubular cells. 2· 3 For example, calcium phosphate crystals can be seen in the mitochondria and the cytoplasm of renal cells uncler conditions of cellular ischemia or hyperparathyroidism.2-4 Excessive crystallization leads to cell death. The agglometate of crystals and cell debris is extruded from the cell, blocks the tubule and acts as the nidus for renal stone formation. If intracellular and intramitochondrial crystallization is important in renal stone formation, a clearer understanding of the uptake of calcium, phosphate and oxalate by renal cells or cellular components may provide an insight into early events in the pathogenesis of the disease. Although considerable data are available concerning calcium and phosphate uptake by the renal cells, very little is known about oxalate uptake. In the present investigation, we have examined the uptake of oxalate by subcellular fractions of renal cortical tissue in adult male Sprague Dawley tats. Preliminary findings about oxalate uptake by human kidney are also given. MATERIALS AND METHODS

Preparation of homogenates. Adult male Sprague Dawley rats weighing 400 to 450 grams were anesthetized with diethyl ether and the internal organs (liver, kidney, brain, heart, skeletal muscle, stomach, spleen, lung, small and large intestine) were removed. All tissues except skeletal muscle were homogenized in 5 volumes (W/V) of 50-mM phosphate buffer, pH 7.4 using a Teflon glass homogenizer (8 to 10 strokes at 500 rpm). Accepted for publication October 29, 1985. . * Requests for reprints: Division of Urology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01605. Supported by Biomedical Research Grant 3375 (51054B from the Washington University School of Medicine, USPHS Award IR23AM 29831-01 from the NIADDKD and a Veterans Administration Research Grant. t Current address: Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Madras-60041, India. 862

Skeletal muscle was homogenized in a polytron blender (setting at 2 for 20 seconds). The tissue homogenate was used immediately. Aliquots of homogenates (lOQ µl. containing 2 to 3 mg. protein) were incubated with 100 nM 14C oxalate in 0.9 ml. of potassium phosphate buffer pH 7.4 for 20 min. at 23C. To differentiate specific from non-specific binding, parallel incubations were performed in the presence of 100 µM unlabeled oxalate. At the end of incubation, the samples were centrifuged at 37,500 g for 20 min. The pellets were washed three times with 2 ml. of buffer and the radioactivity co1,1nted ("pellet" or "membrane"). The supernatant was decanted and treated with oxalate adsorbant (1 gm./ml. of supernatant, Sigma Chemical Co.) for 30 min. at 23C in order to remove the unbound 14C oxalate. After the sedimentation of the absorbant, the supernatant was decanted and centrifuged at 37,500 g for 20 minutes. The radioactivity bound in the clear supernatant represented cytosolic binding. Under these conditions all the radioactivity was removed by oxalate adsorbant incubated with free 14C oxalate. Fresh human kidney and muscle were obtained at the time of nephrectomy for renal cell carcinoma and were verified to be normal by histologic examination. All assays were performed in duplicate. The numbers of experiments performed are indicated in the tables and figure legends. Subcellular fractionation. Rat renal cortex was homogenized in 20 per cent (W/V) buffer containing 2 mM potassium phosphate, 1 mM EDTA and 0.25 M sucrose. The crude nuclear fraction was sedimented at 800 g for 15 min. at 4C in a Beckman J2-20 preparative centrifuge. The pellet was washed once and used for the nuclear binding studies. The supernatant was used to prepare the mitochondria and microsomes. The intact mitochondria and microsomes were prepared by the conventional centrifugation techniques. 5 The supernatant from 800 g spin was centrifuged at 6500 g for 20 min. at 4C. The pellet containing the mitochondria was washed twice with buffer and centrifuged at 6500 g for 20 min. at 4C and the final pellet containing the intact mitochondria was suspended in 50 mM potassium phosphate buffer. The supernatant from the mitochondriai preparation was spun for 1 hr. at 100,000 g at 4C in a Beckman ultracentrifuge (model L8-55M) in order to prepare the microsomes. The pellet was suspended in 50 mM potassium phosphate buffer. The

863

RENAL OXALATE BINDING

purity of the preparation was tested by determining glucose -6- phosphatase activity. 6 Inner and outer mitochondrial membranes were prepared according to the sucrose density gradient method of Sottacosa et al. 5 Briefly, 50 mg. of mitochondria were swollen at OC for 5 minutes in 7.5 ml. of hypotonic medium containing 10 mM Tris-phosphate, pH 7.5. To the mitochondria 2.5 ml. of a solution containing 1.8 M sucrose, 2 mM ATP and 2 mM MgS0 4 was added. After 5 min. at OC the mitochondria were sonicated and layered on a discontinuous sucrose gradient of 0.45 M, 0.76 M and 1.32 M sucrose. The mitochondria were spun in a SW 27 rotor at 25,000 rpm for 3 hours in a Beckman ultracentrifuge (model L8-55M). The pellet contained the inner mitochondrial membrane. The fluffy layer was outer mitochondrial membrane. The purity of mitochondrial membranes was examined by determining cytochrome oxidase7 and monamine oxidase8 in inner and outer mitochondrial membranes respectively. Basolateral membranes and brush border membranes were prepared according to the methods of Proverbio and Castillo9 and Biber et al. 10 respectively. The purity of the preparations was checked by determining the activities of alkaline phosphatase in the brush border membranes 11 and Na+ K+ ATPase in the basolateral membranes. 9 Aliquots of each fraction containing 2 to 3 mg. protein in 100 µl. were incubated for 20 min. at 23C in a 0.9 ml. volume of buffer containing 100 nM 14C oxalate. Non-specific binding was determined as explained above. All experiments were repeated at least twice. Statistical analysis was done using Student's t test. RESULTS

Oxalate binding to different organs. Table 1 indicates that oxalate specifically binds to homogenates of rat kidney and liver and not to homogenates prepared from heart, lung, spleen, stomach, skeletal muscle, small and large intestine. Binding TABLE 1.

Oxalate binding to rat tissues

Tissues

Oxalate bound

(pmol./mg. protein)

Liver Kidney Brain Heart Spleen Lung Small intestine Lg. Intestine Stomach Skeletal Muscle

Supernatant 0.25 ± 0.01 ND ND ND 0.26 ± 0.02 0.05 ± 0.004 0.28 ± 0.02 0.25 ± 0.03 ND ND

Pellet 4.75 ± 0.44 4.50 ± 0.39 1.05 ± 0.51 0.20 ± 0.01 0.12 ± 0.02 0.25 ± 0.03 ND ND 0.10 ± 0.01 0.10 ± 0.005

N 17 19 2 4 2 2

3

2 2 2

The values are mean ± S.E. ND= Not detectable. N = Number of experiments.

TABLE

2. Oxalate binding to subcellular fractions of rat kidney

Fraction Homogenate Crude nuclei Intact mitochondria Mitochondrial inner membrane Mitochondrial outer membrane Brush border membrane Basolateral membrane Microsomes

Total Oxalate Binding (pmol./gm. Tissue) 1080 ± 712 ± 356 ± 324 ±

80.2 31.l 16.16 6.8

Oxalate Bound (pmol./mg. Protein) 6.4 8.5 20.9 23.5*

± ± ± ±

0.04 0.51 1.92* 1.52

% Distribution

100 66 33 30

10± 1.0

6.0 ± 1.0

0.9

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2.7 ± 0.30

0.8

6 ± 0.5 9 ± 0.1

0.6 ± 0.05 0.11 ± 0.01

0.5 0.8

The values are mean ± S.E. * P <0.001 (compared with homogenate).

14C-Oxalate Bound ( pmol mg" 1 Protein)

32

8

0

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4 Time in Minutes

6

8

FIG. 1. Effect of time on oxalate binding. Rat kidney inner mitochondrial membranes (1-2 mg. protein/100 µl.) were incubated at 23C in 1 ml. volume of 50 mM phosphate buffer (pH 7.4) containing 100 nM 14C oxalate. At indicated times, tubes were centrifuged at 37500 g for 10 min., sedimented membranes washed gently with buffer and radioactivity determined. Non-specific binding was subtracted at each point.

was also observed in brain homogenates, but averaged 22 to 23 per cent of binding present in liver and kidney homogenates. The results also indicate that the binding predominantly occurred in the membrane fraction as there was very negligible or no binding at all in the supernatant. Subcellular localization. To localize the oxalate binding in the kidney, subcellular fractionation of renal tissues was performed according to standard techniques and verified by determining the enhancement of appropriate marker enzymes. As seen in table 2, two thirds of the total binding was localized to the crude nuclear pellet and one third to the mitochondrial fractions. When related to protein content, oxalate binding was enriched primarily in the inner mitochondrial membrane (p < 0.001) where it was approximately four times that detected in the homogenate. Very little binding was detected in the brush border membranes or basolateral membranes or microsomes. Kinetic studies. All kinetic studies were performed on inner mitochondrial membranes. Effect of time. Binding of oxalate at 23C increased with time and reached equilibrium in 2 min. (fig. 1). No further increase in uptake was detected up to 2 hr. Therefore in all subsequent studies incubations were performed up to 20 min., which far exceeds the time required for equilibrium. Effect of temperature on oxalate binding was studied by altering the incubation temperature from OC to 50C (fig. 2). Maximum binding was observed at 23C. In the insert of the figure is shown a plot of the same data. The energy of activation for binding was calculated to be 53.5 KJ/mol. Effect of concentration on oxalate binding. Fig. 3 illustrates the effect of increasing substrate concentration on oxalate binding by the inner mitochondrial membrane. Specific binding of oxalate increased with increasing substrate concentration and was saturated at an external oxalate concentration of 240 nM. Analysis of the data by Scatchard plot indicated that the maximal binding capacity was 49 pmol./mg. protein and the apparent disassociation constant Kd was 43 nM. At a concentration of 64 nM oxalate in the incubation medium, half the available binding sites for oxalate were saturated (table 3, fig. 3).

Specificity of oxalate binding to mitochondrial membranes. The specificity of binding was studied by determining the ability of other anions to compete with oxalate (fig. 4). The concentration of competing anion that displaces 50 per cent of specifically bound oxalate is considered to be the half saturation concentration (lCso) of the inhibitor. The IC 50 for oxalate was

864

LAXMANAN AND ASSOCIATES

0.25 µM. Among the inhibitors studied ICso was in the following order: oxalate < oxamate < parabanate < glyoxalate < oxalo acetate < malate < citrate = glycollate. Thus the compounds that are structurally related to oxalate such an oxamate, parabanate and glyoxalate had lower IC 50, as compared to other dicarboxylics, citrate and glycollate. The IC5o ratio of oxalate to glyoxalate was 1:300 while even 10,000-fold excess of dicarboxylates was ineffective in competition demonstrating the high specificity of oxalate binding to the inner mitochondrial membrane. Effect of calcium and calcium inhibitors on oxalate binding. In order to find out whether calcium enhances the binding of oxalate, the binding studies were carried out in the presence of 1 mM calcium, and calcium chelaters like EDTA and EGTA, competitive inhibitors of calcium binding like lanthanum chloride or inhibitors of calcium transport such as ruthenium red

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(table 4). At the concentrations used, binding was not changed by the presence of calcium in the medium. Inhibitors of calcium binding or uptake in the medium did not decrease the binding of oxalate to mitochondrial inner membranes. Effect of detergent/heating on mitochondrial oxalate binding. Heating of mitochondrial membranes at 60C for 10 min. or treatment with low concentrations of Lubrol (0.16 mg./mg. protein) completely abolished the binding. Binding of oxalate to human tissues. The binding of oxalate by human tissues exhibited some qualitative similarity to oxalate binding by rat tissues (table 3). Binding was most marked in the renal inner mitochondrial membranes. Skeletal muscle contained less than 5 per cent of the binding seen in the kidney (table 5). Saturation analysis showed the number of available binding sites to be 13 pmol./mg. protein with an apparent dissociation constant (Kd) of 149 nM. The ICso of oxalate was 0.20 µM which is similar to the IC50 of oxalate in rat kidney (0.25 mM). Mitochondrial binding was inhibited by 10 µM concentrations of glyoxalate but not by citrate or glycollate.

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The results of the present study clearly indicate that oxalate bound specifically to homogenates of rat liver and kidney. The binding substance was localized to the mitochondrial inner membranes and exhibited several of the kinetic properties of a binding protein: saturability, high affinity, tissue specificity and ligand specificity. Furthermore, a binding protein with many characteristics similar to the rat oxalate binding protein was present in human kidney. The reason for the existance of such a binding protein on the mitochondrial inner membranes remains speculative at the moment. Oxalate is a metabolic end product and serves no vital Characteristics of oxalate binding to inner mitochondrial membranes of the kidney Man Rat No. of binding sites (pmol./mg. protein) 13 49 Apparent Kd (x10-• M) 149 43 Half-saturation concentration (x10-• M) 51 64 IC50 (x10-< M) Oxalate 0.20 0.25 Glyoxylate 10.00 70.00 75.00 660.00 Malate 1000.00 Citrate >1000.00 Glycollate >1000.00 >1000.00

TABLE 3.

FIG. 2. Effect of temperature on oxalate binding. Membranes (1-2 mg. protein/100 µl.) were incubated for 20 min. in 1 ml. phosphate buffer (50 mM, pH 7.4) containing 100 nM 14C oxalate at indicated temperature. Non-specific binding was determined in presence of 100 µM oxalate. Insert: Arrhenius plot of same data. Log V is expressed in nmol./mg. protein/20 min. and temperature in absolute (k). Energy of activation for binding was found to be 53.5 KJ/mol. C

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FIG. 3. Saturation analysis. A, mitochondrial membranes (2 mg. protein/100 µI.) were incubated at 23C for 20 min. in 1 ml. of 50 mM phosphate buffer (pH 7.4) containing 5-240 nM 14C oxalate. Non-specific binding at each point was defined as binding retained in presence of 100 µM non-radioactive oxalate. Experimental details are described in METHODS. 0-0-0 = specific binding;•-•-•= non-specific binding. B, Scatchard plot: data obtained in fig. 3A was used for construction of Scatchard plot. F = Free 14C oxalate. B = Specifically bound 14C-oxalate. Number of available binding sites (N) was calculated to be 49 pmol./mg. protein and apparent dissociation constant (Kd) was 43nM.

865

RENAL OXALATE B!NDING TABLE 4.

Effect of calcium, calcium chelators and inhibitors on oxalate binding

® -Glycol late 1111'-Citrote ® -Glutamate

Concentration (mM)

Additive None EDTA EGTA Ruthenium red Lanthanum chloride Calcium

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Oxalate Bound (pmol./mg. Protein)

21.0 ± 2.02 19.4 ± 0.61 19.8 ± 0.2 19.5 ± 1.5 21.0 ± 3.3 1.0 21.4 ± 1.4 The rat kidney inner mitochondrial membranes (-2 mg./100 ul.) were incubated at 23C in a 1 ml. volume of 50 mM phosphate buffer (pH 7.4). The above mentioned additives were added at the indicated concentration followed by the addition of 14C oxalate (100 nM) and incubated for 20 min. and centrifuged at 37500 g for 10 minutes, and the radioactivity was counted in the pellet. The values are mean ± S.E.

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TABLE

5. Binding of 14 C-oxalate to human tissues

Molarity

Fm. 4. Specificity of 14C-oxalate binding. Aliquots of membrane (2 mg. protein/100 µI.) were incubated for 20 min. at 23C in 1 ml. phosphate buffer (50 mM, pH 7.4) containing 100 nM 14C oxalate with or without 5 to 10,000-fold excess unlabeled competing substrates as indicated. Non-specific binding, which was binding not displaced by the addition of 1000-fold excess unlabeled oxalate, was subtracted in all instances. IC 50 was concentration of inhibitor needed to displace 50% of 14C oxalate binding and was calculated to be: oxalate = 0.25 µM; glyoxalate = 70 µM; glycollate = 1 mM; oxamate = 60 µM; parabanate = 50 µM; citrate = 1 mM; malate = 0.66 mM; glutamate = 1 mM, oxaloacetate = 0.55 mM; pyruvate = 1 mM.

function in mammals. However, it is filtered by the glomerulus and secreted by the proximal tubule, and so some amount of oxalate is present in the tubular cell. In addition, W edden et al. 12 have demonstrated recently that kidney cortical slices can accumulate oxalate in vitro. These observations, together with the high specificity of the binding of oxalate to the mitochondrial inner membrane suggest to us that binding of oxalate to mitochondrial inner membranes may be related to transport of oxalate by renal cortical mitochondria. This will be the subject of a further communication. REFERENCES 1. Robertson, W. G. and Peacock, M.: Calcium oxalate crystalluria

and inhibitors of crystallization in recurrent renal stone formers. Clin. Sci., 43: 499, 1972. 2. Rasmusson, H.: Possible cellular aspects of renal stone formation. In: Urolithiasis-Physical Aspects. Edited by Finlayson, B., Hench, L. L. and Smith, L. H. National Academy of Sciences, pp. 161-168, 1972. 3. Trump, B. F., Dees, J. H., Kim, K. M. and Sahaphong, S.: Some aspects of kidney structure and function, with comments on tissue calcification in the kidney. In: Urolithiasis-Physical Aspects. Edited by Finlayson, B., Hench, L. L. and Smith, L. H. National Academy of Sciences, pp. 1-39, 1972. 4. Caulfied, J. B. and Schrag, P. E.: Electron microscopic study of

Tissue

Fraction

Skeletal muscle

homogenate inner mitochondrial membranes homogenate inner mitochondrial membranes

Kidney

Oxalate Bound (pmol./mg. Protein) 0.20 ± 0.05 0.30 ± 0.05 NS 1.92 ± 0.27 7.77 ± 0.69*

The values are mean ± S.E. * p < 0.001. NS = Nonsignificant. renal calcification. Am. J. Pathol., 44: 365, 1964. 5. Sottocosa, L., Kuylenstierna, B., Ernster, L. and Bergstrand, A.: Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria. In: Methods in Enzymology. Edited by Colowick, S. P. and Kaplan, N. 0. Academic Press, New York, vol. 10, pp. 448, 1967. 6. Bergmeyer, H. V.: Methods of Enzymatic Analysis: Academic Press, New York, vol. 3, pp. 876, 1974. 7. Yonetani, T.: Cytochrome oxidase. In: Methods in Enzymology, Edited by Esterbrook, R. and Pullman, M. Academic Press, New York, vol X, pp. 332, 1967. 8. Tabor, C. W., Tabor, H. and Rosenthal, S. M.: Purification of amine oxidase from beef plasma. J. Biol. Chem., 208: 645, 1954. 9. Proverbio, F. and Castillo, J. R.: Na+ stimulated ATPase activities in kidney basolateral plasma membranes. Biophys. Acta, 646: 99, 1981. 10. Biber, J., Stiger, B., Haase, W. and Murar, H.: A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. Biochem. Biophys. Acta, 647: 169, 1981. 11. Belfield, A. and Goldberg, D. M.: Revised assay for serum phenyl phosphatase activity using 4-amino antipyrine. Enzyme, 12: 561, 1971. 12. Wedden, R. P., Levendoglu-Tugal, 0., Batuman, V. and Cheeks, C.: Oxalate accumulation in rat renal cortical slices. Proc. Soc. Exp. Biol. Med., 177: 120, 1984.