Effect of Oxalate on Function of Kidney Mitochondria

0022-5347 /89/ 1412-04,23$02.00/0 °THE .]QURNAL OF UROLOGY

Vcl. 141, February Prio,ted in U.S.A.

Copyl'ight C0 1989 by The Wiiliams & Wiikins Co.

EFFECT OF OXALATE ON FUNCTION OF KIDNEY MITOCHONDRIA TADEUSZ STRZELECKI, BONNIER. McGRAW, CHERYL R. SCHEID

AND

MANI MENON*

From the University of Massachusetts Medical School, Worcester, i\1assachusetts

ABSTRACT

The effects of oxalate on kidney mitochondria were evaluated in vitro to test whether oxalate exposure leads to derangement(s) in mitochondrial function that could in turn promote the formation of kidney stones. Our previous studies demonstrated that oxalate is transported across the mitochondrial membrane via the dicarboxylate carrier. The present studies indicated that oxalate competitively inhibits the uptake and oxidation of exogenous malate and succinate in isolated mitochondria but has no effect on mitochondrial respiration in the presence of a mixture of glutamate plus malate or glutamate plus pyruvate. Oxalate attenuates the increase in mitochondrial respiration produced by the uncoupler CCCP or by the Ca2 + ionophore A23187, and the latter effect is more pronounced in kidney than in liver mitochondria. The apparent K; of oxalate for the response to Ca 2 + ionophore is 1.9 ± 0.3 mM in kidney and 6.1 ± 0.2 mM in liver mitochondria. Similarly, the ability of oxalate to attenuate calcium-induced swelling of mitochondria is more dramatic in kidney than in liver mitochondria (apparent Ks of 1.7 ± 0.1 and 18.2 ± 0.7 mM, respectively). Oxalate has no effect on the rate of calcium uptake by energized mitochondria or on the rate of ruthenium red-insensitive calcium efflux from mitochondria in either tissue. The above findings indicate that oxalate interacts with the inner mitochondrial :membrane or with processes controlling membrane integrity to a greater extent in kidney than liver mitochondria. The effects of oxalate on membrane permeability or integrity may be more important than its effects on mitochondrial energy production or calcium sequestration in the pathogenesis of calcium oxalate microlith formation in the kidney. Ural., 141: --x"''"'-~'"" 1989) The precise sequence of events involved in the formation of calcium oxalate kidney stones is unclear. Oxalate is an endproduct of metabolism that is excreted by the kidneys and under certain conditions this metabolite can interact with calcium within the tubular lumen to form calcium oxalate crystals. 1 ~3 Normally these crystals pass rapidly through the kidney and do not attain sufficient size to block a duct and form a urinary microlith. 4 The formation of calcium oxalate kidney stones appears to require the retention of crystals within the kidney. There are a number of conditions that might promote crystal retention and hence stone formation within the kidney. Tubular stasis is one such condition, although there is no evidence that this is a primary event in idiopathic nephrolithiasis.' Changes in the properties of tubular membranes that promote the adherence of these crystalline particles to the membrane could also lead to crystal retention within the Marked alterations in epithelial membranes in the vicinity of calcium oxalate crystals have been demonstrated in rat experimental models of nephrolithiasis.'- 8 The cause of the observed alterations remains unclear, however, in that the morphological changes may be a consequence of crystal adhesion or they may result from an underlying defect(s) in the renal tubular cells. Although the link between renal stone formation and derangements in renal tubular function remains controversial,7· 9-12 there is evidence that mitochondrial calcium transport is altered in many forms of nephrocalcinosis."3 Thus, over the last three years, we have been assessing the possibility that derangements in mitochondrial metabolism induced by oxalate and calcium alter renal tubular cell function and contribute to the retention and growth of calcium oxalate crystals in the kidney. Our previous studies 14 demonstrated that oxalate is transported into renal cortical mitochondria via the dicarboxylate carrier. In the present studies we compared the effects of

oxalate exposure in vitro on various aspects of mitochondrial function in mitochondria derived from the kidney and in mitochondria derived from the liver. We hypothesized that oxalate may differentially affect kidney mitochondrial and liver mitochondrial functions, because calcium oxalate stones are known to occur in kidney but not liver tissue. The results from these studies suggest that oxalate exposure selectively affects kidney mitochondrial membrane permeability. MATERIALS AND METHODS

Isolation of mitochondria. Sprague-Dawley rats weighing 220 to 300 gm. were used. Animals were anesthesized with nembutal (50 mg./kg. i.p.). The kidney and a portion of the liver were excised and placed in ice-cold homogenization solution. The renal cortex was from the medulla with a scalpel. The renal cortex liver were then homogenized in a solution of 225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA (ethylenebis( oxyethylenenitrilo )tetraacetic 0.1 % bovine sErum albumin (fraction acid free) and five mM MOPS (3morpholinopropanesulfonic pH 7.2. Mitochondria were isolated using standard differential centrifugation procedures. 15 Briefly, unbroken cells and nuclei were sedimented by centrifugation at 600 g for five min. and mitochondria were sedimented at 15,000 g for 10 min. Respiration of mitochondria. The respiration rate of mitochondria was determined by measuring 0 2 consumption polarographically with a Clark-type electrode in a water-jacketed incubation cell at 28C. The incubation medium was composed of 135 mM KCl, five mM K2HP0 4 , 0.1 mM EGTA, 20 mM MOPS, pH 7.2, and either one mM or OmM MgCl 2 as indicated in the figure legends. In some experiments five mM glutamate or five mM succinate plus two µg./ml. rotenone were used; others employed a mixture of five mM glutamate plus one mM malate or pyruvate. The amount of mitochondrial protein Accepted for publication August 3, 1988. ranged from 0.85 to 1.1 mg./ml. Some experiments also em* Requests for reprints: Div. of Urology, University of Massachusetts ployed the uncoupling agent CCCP at a concentration of 0.25 Medical School, 55 Lake Ave. North, Worcester MA 01655. Supported by grant DK36696 from the NIH, and a grant from the µM or the Ca 2+ ionophore A23187 at a concentration of 2.5 Eleanor Naylor Dana Foundation. nmol./mg. 16 ' 17 423

424

STRZELECKI AND ASSOCIATES

In experiments in which we monitored the rate of uptake and oxidation of exogenous substrates, mitochondria were first depleted of endogenous substrates by preincubating with one mM ADP for 30 to 40 min. at 25C. 18 Oxidation was then initiated by addition of the desired amount of malate or succinate, as indicated in the figure legends, and respiration was then followed in the presence or absence of oxalate. In all other experiments, oxalate was present in the incubation medium prior to the addition of mitochondria. Protein content was determined by the Biuret method. 14 Swelling of mitochondria. Calcium-induced swelling of mitochondria was detected by monitoring the change in optical density at 540 nm. produced by the addition of CaClz (110 nmol/mg. of mitochondrial protein) to a mitochondrial suspension. Mitochondria were suspended at a concentration 0.55 to 0.65 mg./ml. in a medium consisting of 200 mM mannitol, 75 mM sucrose, two mM K 2HP04, five mM succinate, two µg./ml. rotenone, and 20 mM MOPS, pH 7.2, 28C. The process was initiated by the addition of 110 nmol CaClz/mg. of mitochondrial protein, and optical density was recorded continuously using a Gilford spectrophotometer. 19 Calcium flux in mitochondria. Assessment of mitochondrial calcium uptake was performed using Arsenazo III as described previously. 2° For these experiments, mitochondria (0.85 to 1.1 mg./ml.) were suspended in buffers similar to those employed for the respiration experiments except for the addition of 75 µM Arsenazo III. The concentration of the extramitochondrial Ca-Arsenazo III complex was monitored spectrophotometrically by alternately assessing the optical density of the suspension at 665 and 685 nm in a Gilford spectrophotometer. Recordings were carried out 12 times per minute in a temperature controlled cuvette chamber at 28C. The rate of change in the level of extramitochondrial calcium was calculated using linear regression analysis of each range of calcium (six to nine measurement points in each range) with the median calcium level indicated in the table. The concentration of free calcium ions in the presence ofCa-EGTA buffers was calculated as described by Bers. 21 Materials. The organic acids, nucleotides, uncoupling agent CCCP (carbonyl cyanide p-chlorophenylhydrazone), and calcium ionophore A23187 (calmycin) were obtained from Sigma Chemical Co. All other reagents were of high purity and were obtained from commercial sources. The sucrose was depleted of contaminating calcium by chromatographing the sucrose solutions on CHELEX resin obtained from Sigma Chemical Co. Statistical analysis. Statistical analysis of the data presented was performed using the Student's unpaired test in the RSl statistics package for the IBM PC. Differences were considered statistically significant at p <0.05. The Wilkinson's procedure was used for calculating kinetic parameters. 22 For responses that were transient or nonlinear we presented the results from a single representative experiment out of the three or more experiments that were performed. RESULTS

Oxalate inhibited mitochondrial respiration in the presence of malate. After preincubating mitochondria for 30 to 40 min with ADP, all endogenous substrates were depleted, and respiration was negligible. The addition of malate enhanced the respiration, and subsequent addition of oxalate instantly diminished oxygen consumption by mitochondria. Fig. 1 illustrates the relationship between malate concentration and the rate of oxygen consumption in rat and liver mitochondria. Data are plotted as the double reciprocal, 1/[SJ vs 1/[VJ. In the absence of oxalate, the apparent Km for malate was 0.31 ± 0.10 and 0.067 ± 0.06 mM in kidney and liver mitochondria respectively, values similar to those reported previously. 18' 23 In the presence of oxalate, malate uptake and oxidation was inhibited and this effect was more dramatic in

kidney than in liver mitochondria. The apparent K for oxalateinduced inhibition of malate oxidation, determined from Dixon plots (not shown) was 1.3 ± 0.45 and 8.1 ± 2.0 mM for the kidney and liver respectively (p <0.05). The rate of succinate oxidation was determined under the same experimental conditions as described for malate oxidation. The apparent Km for succinate oxidation was 1.1 ± 0.05 mM in kidney and 1.6 ± 0.05 mM in liver mitochondria. Oxalate competitively inhibited succinate uptake and oxidation in both kidney and liver mitochondria with apparent K,s of 4. 7 ± 0. 7 and 3.3 ± 0.5 mM, respectively (p >0.05). Table 1 shows the effect of oxalate on kidney mitochondrial respiration with five mM succinate or a mixture of glutamate plus malate or glutamate plus pyruvate as substrates. Under resting (State 4) as well as ADP-stimulated conditions (State 3), the presence of three mM oxalate had no significant effect on kidney mitochondrial respiration with the various substrate combinations tested. The ADP /0 ratios and the acceptor control indices (ACI) were unchanged by oxalate. In liver mitochondria, the respiration rates and phosphorylation of ADP were also unchanged by three mM oxalate (data not shown). Fig. 2 shows recording traces of oxygen consumption by kidney and liver mitochondria uncoupled by a proton ionoa) Kidney mit.

b) Liver mit.

N

'o

t,

16

X

'Een X

'c,E

Oxalate

·~: X

12

X

8

0 E

B

"'enC

2,5mM t,

,,I'

4

•

/.

/4M

......

0~

0

4

4

8

8

1/S, malate (1/mM)

FIG. 1. Oxygen consumption by kidney and liver mitochondria in presence of malate and oxalate. Mitochondria were preincubated at 28C in medium containing 135 mM KCl, 5 mM K 2HPO., 1 mM MgC[z, 1 mM ADP and 20 mM MOPS, pH 7.2 for 35 min. to deplete endogenous substrates. Additions of malate and oxalate were made at concentrations indicated. Data are means from 3 separate experiments and are represented in form of double reciprocal plot (1/V vs 1/[S]) of rate of oxygen consumption as function of malate concentration in presence of varying concentrations of oxalate. 1. Acceptor-control index (AC!) and ADP/0 ratio of rat kidney cortical mitochondria in presence of 3 mM oxalate in vitro

TABLE

Experimental Conditions

State 4

State 3

ngatom 0 2 Glutamate, malate control oxalate Glutamate, pyruvate control oxalate Succinate control oxalate

X

mg.- 1

ACI X

ADP/0

min.- 1

24.4 ± 2.0 23.3 ± 1.1

237 ± 11 233 ± 15

4.55 ± 0.34 4.76 ± 0.27

2.46 ± 0.08 2.47 ± 0.08

24.1 ± 2.0 23.3 ± 1.3

181 ± 14 162 ± 9

3.45 ± 0.33 2.98 ± 0.51

2.38 ± 0.11 2.36± 0.07

77.0± 4.2 82.5 ± 1.5

372 ± 25 361 ± 9

3.70 ± 0.39 3.55 ± 0.10

1.48 ± 0.03 1.41 ± 0.01

Data represent the means ± SEM from 4 experiments. Abbreviations are ACI, the acceptor control index, which is the ratio of the respiration ratio of mitochondria in the presence of ADP to the respiration rate when all added ADP has been phosphorylated: ADP /0, which is the ratio of the number of nmol of ADP added to the number of ngatom 0 2 consumed. There are no statistically significant differences (p > 0.05).

OXALATE AND KIDNEY MITOCHONDRIA

Liver mit.

Kidney mit.

~~ ? cc{

ADP

I'\"-.(p

a)

-------

0.8

/'

CCCP

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~ ,1,

0.6

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; I

c:i 0.4 ci

CCCP


ngatom 0 2 /ml

/,-o•

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b)

200

:'/·

~

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0.2

1

425

><

3 3~ ><

100

3

(C

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100 Oxalate

-

0 Oxalate, mM

1 min Control

0.5

2.5mM 0.5mM

Control

FIG. 2. Recording traces of oxygen consumption by kidney and liver mitochondria uncoupled by CCCP. Mitochondria (0.9-1.1 mg./ml.) were maintained in medium consisting of 135 mM KCl, 5 mM K 2HP04 , 1 mM MgCl2 , 5 mM glutamate, 1 mM malate, 20 mM MOPS, pH 7.2, and oxalate as indicated, 28C. Additions of 0.35 mM ADP or 0.3 µM CCCP are indicated by arrows. In kidney mitochondria in absence but not presence of ADP, oxalate reduced effects of CCCP on respiration. This effect of oxalate was not apparent in liver mitochondria. Data are from 1 experiment; similar data were obtained in 3 additional experiments.

phore, CCCP. Addition of CCCP to the mitochondrial suspension dissipated the mitochondrial proton gradient and produced a marked linear increase in 02 consumption. Oxalate addition had no effect on this response to CCCP in the presence of ADP. In kidney mitochondria in the absence of added ADP, however, oxalate attenuated the effects of CCCP such that the rate of oxygen consumption was reduced and nonlinear with time. In liver mitochondria, oxalate had no effect on CCCP-induced uncoupling with or without ADP. The data presented in fig. 2 are representative of data obtained in four separate experiments. The effect of oxalate on Ca-stimulated mitochondrial respiration is shown in fig. 3. Mitochondria were maintained in our standard incubation medium containing either zero or one mM MgCl2 • Extramitochondrial calcium concentrations were varied by the addition of Ca-EGTA buffers and the resultant extramitochondrial Ca2 + concentrations were determined spectrophotometrically using Arsenazo III. Note that the addition of 2.5 rr:.M oxalate had no effect on extramitochondrial free Ca2 + concentrations (fig. 3A). Calcium cycling across the mitochondrial membrane was induced by the addition of a calcium ionophore, A23187 in the presence of varying levels of extramitochondrial Ca 2+, and mitochondrial respiration was monitored.13 Note that in the absence of MgCb, oxalate markedly attenuated the increase in mitochondrial respiration seen at ratios of Ca/EGTA of 1.6 or greater (corresponding to free Ca2 + levels of 52 µMand above). In the medium containing one mM magnesium, however, oxalate had no inhibitory effect on calcium-stimulated respiration (fig. 3B). Oxalate also attenuated mitochondrial respiration induced by Ca2 + cycling in liver mitochondria, although the inhibitory effect of oxalate was less pronounced than in kidney mitochondria (fig. 4). At a level of free calcium that produced maximal stimulation of mitochondrial respiration (350 µM), oxalate inhibited respiration in a dose-dependent manner with an apparent K of 1.93 ± 0.03 mM in kidney and 6.1 ± 0.2 mM in liver mitochondria, p <0.05. Table 2 shows the effect of oxalate on the rates of calcium flux across the membrane of intact, energized kidney mitochondria in the absence of magnesium ions. The rate of calcium

1.0

1.5

2.0 0.5

1.0

1.5

2.0

Ca/EGTA

FIG. 3. Effect of oxalate on extramitochondrial free calcium levels and on oxygen consumption by kidney mitochondria stimulated by calcium cycling across mitochondrial membrane. Mitochondria (0.81.1 mg./ml.) were m8:intained in standard incubation medium (28C, pH 7.2)_ supplemented with 75 µM Arsenazo III, 5 mM succinate, 0.10 mM EGTA, 2 µg./ml. rotenone and 2 µM A23187. A, optical density of CaArsenazo III complex recorded at 665 and 685 nm. Note that the addition of 2.5 mM oxalate (solid symbols) has little effect in the presence (circles) or absence (triangles) of 1 mM MgCI,. B, respiration rates stimulated by A23187.

200

'eEn X

-__f t""-t'f---1 f-........__

X

E

100

.9 IC

C) C: <'<

Kidney mit~

----+-~-----..J!

0 <3

Liver mit.

0

3

5

Oxalate, mM FIG. 4. Effect of oxalate on respiration of kidney and liver mitochondria stimulated by calcium cycling. Respiration rates were determined as described in fig. 5 before and after addition of 350 µM CaCl2 • Data represents means ± SEM from 4 experiments.

TABLE 2.

Calcium uptake by kidney mitochondria in the presence and absence of 2.5 mM oxalate

Extramitochondrial Calcium Level

6µM

11 µ

17 µM

ngatom Ca 2+ x mg. 1 X min.-,

Ca2 + influx control oxalate

7.0 ± 0.6 5.8 ± 0.4

20.5 ± 2.1 23.2 ± 2.3

63.8 ± 3.5 58.7 ± 1.6

Calcium uptake was measured after additions of various amounts of CaCl, to mitochondria suspended in a standard incubation medium without MgCl2 ~nd with 50 µM Arsenazo III (see Methods section). The rate was calculated from the linear regression approximated at the level of extramitochondrial calcium as indicated. Data represents means ± SEM from 3 experiments. There are no significant differences between groups (p > 0.05).

426

STRZELECKI AND ASSOCIATES

influx was not altered by 2.5 mM oxalate when extramitochondrial calcium ranged between six and 17 µM. We did not assess influx rates at higher extramitochondrial calcium levels because of the possibility that higher levels would evoke calcium release from the mitochondria (see below). Oxalate also had no effect on the steady state extramitochondrial calcium levels established after accumulation of 20 ngatom Ca2 + /mg. protein by kidney mitochondria. The subsequently determined rate of ruthenium red-insensitive calcium efflux was also unchanged by oxalate (2.6 ± 0.5 vs. 2.1 ± 0.8 ngatom Ca2 + /mg./min. in kidney and liver, respectively). When 110 ngatom of calcium/mg. of protein was added to the mitochondrial suspension in a medium containing respiratory substrate and phosphate, mitochondria quickly accumulated the calcium and, after about a 30 sec. lag phase, started to swell (fig. 5). This phenomenon has been attributed to a nonspecific increase in membrane permeability that accompanies the collapse in mitochondrial membrane potential and the release of calcium and other ions from the mitochondrial matrix. 19 Oxalate markedly inhibited the rate and amplitude of calcium-dependent swelling in kidney, but not in liver mitochondria, despite the fact that liver mitochondria exhibited greater swelling per mg. of protein (fig. 5). The apparent K of oxalate for the maximal rate of mitochondrial swelling, calculated assuming Michaelis-Menten kinetics for this phenomenon, was 1.7 ± 0.1 mM in kidney and 18.2 ± 0.1 mM in liver mitochondria. Parallel studies of calcium influx were also carried out under the experimental conditions employed for mitochondrial swelling experiments (fig. 6). These studies indicated that oxalate did not affect the rate or amount of calcium accumulation by mitochondria.

0. D. A665-685

0.10 ca++

i

~=--=========--

0 2.5 5.0

Oxalate, mM

1 min FIG. 6. Effect of oxalate on Ca2 + accumulation by kidney mitochondria. 50 µM Arsenazo III was included. Extramitochondrial Ca-Arsenazo complex was monitored at 665 and 685 nm.

DISCUSSION

The present studies formed a part of our long range investigation examining the hypothesis that calcium oxalate kidney stone formation may be attributable to derangement(s) in renal cortical mitochondrial function. In the studies reported in this communication, we have assessed the p0t,sibility that oxalate exposure could compromise kidney mitochondrial metabolism by effects on one of several processes, including 1) mitochondrial oxidation since oxalate is structurally similar to pyruvate and oxaloacetate 24 ' 25 and since oxalate competes with dicarboxylates for transport into mitochondria 14 or 2) calcium handling in mitochondria since oxalate serves as a calcium trapping agent in other cellular organelles. 26 We have also examined if the effects of oxalate are unique to kidney mitochondria by performing parallel studies in mitochondria derived from the liver. al Kidney mit.

bl Liver mit.

O.D.r-540 0.20

Control

3mM 1mM Control

1 min

FIG. 5. Recording traces of calcium-induced swelling of kidney and liver mitochondria. Mitochondria (0.6-0.65 mg./ml.) were maintained in medium of 200 mM mannitol, 75 mM sucrose, 2 mM K 2 P0 4 , 5 mM succinate, 2 µg./ml. rotenone, and 20 mM MOPS, pH 7.2, 28C. Aliquots of CaCl, (70 nmole/ml.) were introduced to portions of mitochondrial suspension (arrows) and optical density was monitored at 540 mM against remaining portions of mitochondrial suspension. Data are from 1 experiment; similar data were obtained in 3 additional experiments.

The results from these studies indicated that oxalate does selectively affect some metabolic processes in renal cortical mitochondria but does not appear to compromise mitochondrial energy production. For example, although we found that oxalate inhibited malate uptake and oxidation to a greater extent in kidney than in liver mitochondria, this difference could be explained by the different malate affinities in liver vs. kidney mitochondria (the apparent Km for malate in liver is -5-fold lower than that of kidney mitochondria). The products of the Km values for malate times the K; for oxalate were similar in both types of mitochondria, 0.4 and 0.5 for liver and kidney mitochondria, respectively. Moreover, for a substrate such as succinate for which liver and kidney mitochondria have comparable affinities, oxalate had no selective inhibitory effect on the uptake or oxidation of this substrate in kidney mitochondria (fig. 3). In addition, when higher levels of succinate were employed (-5 times the apparent Km) or when malate was employed in conjunction with glutamate to provide the constant supply of oxaloacetate required for glutamate transport and subsequent oxidation, oxalate had no effect on mitochondrial respiration. The oxalate levels employed (3 mM) were some five to 10 times higher than those normally occurring in the rat renal cortex but were comparable to maximal oxalate concentrations in the human kidney, and in the Km range of the oxalate carrier in rat renal cortex. 14 Moreover, three mM oxalate did not affect the ability of either kidney or liver mitochondria to phosphorylate ADP, measured by the ADP /0 ratio and by the acceptor control index (table 1). Therefore, it seems rather unlikely that oxalate would impair the energy supply from renal cortical mitochondria under in vivo conditions. Oxalate did have an effect on the uncoupling of mitochondria produced by proton or calcium ionophores, and the effects were more pronounced in kidney than in liver mitochondria. The increased mitochondrial respiration produced by CCCP in kidney mitochondria was progressively inhibited by oxalate despite adequate substrate and oxygen (fig. 2). Moreover, these effects

OXALATE AND KIDNEY MITOCHONDRIA

of oxalate were observed in the absence but not the presence of ADP, a finding that suggests that this compound is interacting with the inner mitochondrial membrane. Oxalate also selectively inhibited the increase in mitochondrial respiration produced by addition of A23187 plus calcium in kidney mitochondria (fig. 4). This effect of oxalate appeared to involve a direct intramitochondrial action of this metabolite (an effect on the coupling between 0 2 consumption and electron transport rather than an effect on the rate of calcium uptake by mitochondria). Separate studies of calcium uptake at low extramitochondrial calcium concentrations indicated no effects of oxalate on calcium transport in energized mitochondria (table 2). Thus, it would appear that oxalate interacts with the inner mitochondrial membrane or with the processes controlling membrane integrity and that kidney mitochondria are more vulnerable than liver mitochondria to the deleterious effects of oxalate. The recent finding 28 that oxalate binds to the inner mitochondrial membrane of kidney and liver mitochondria is consistent with this site of action of oxalate. The observed effects of oxalate on calcium-induced swelling of renal cortical mitochondria are also consistent with an effect of oxalate on mitochondrial membrane integrity. When renal cortical mitochondria were exposed to llO ngatom of calcium/ mg. of protein, oxalate markedly inhibited the rate and amplitude of mitochondrial swelling but had no effect on the rate of calcium uptake. In liver mitochondria, however, oxalate inhibited neither calcium uptake nor mitochondrial swelling. This dichotomy of the swelling response in the two types of mitochondria despite equivalent calcium uptakes strongly suggests that the effects of oxalate on the kidney mitochondrial membrane are direct, and not the result of intramitochondrial calcium oxalate precipitation. In conclusion, the above findings indicate that oxalate, at concentrations five to ten-fold higher than those normally occurring in the rat renal cortex, exerts direct effects on the integrity or permeability of the mitochondrial membrane. Moreover, these findings demonstrate that kidney mitochondria are more vulnerable than liver mitochondria to the membrane effects of oxalate. The observation (fig. 3) that magnesium depletion exacerbates some of the effects of oxalate is of interest, especially given the clinical evidence linking hypomagnesemia with an increased incidence of kidney stones." Studies are now in progress to examine the interaction between oxalate and magnesium in more detail and to obtain further insights as to the nature of the effects of oxalate on cellular membranes. These effects of oxalate may play a role in the process of calcium oxalate microlith formation in the

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