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Effect of Orchiectomy and Ovariectomy on Oxalate Production, Transport and Excretion in Rats
0022-534 7/84/1326-1244$02.00/0 Vol. 132, December Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Williams & Wilkins Co.
EFFECT OF ORCHIECTOMY AND OVARIECTOMY ON OXALATE PRODUCTION, TRANSPORT AND EXCRETION IN RATS LAXMANAN SEETHLAKSHMI,* CARL J. MAHLE AND MANI MENON From the Division of Urology, University of Massachusetts Medical School, Worcester, Massachusetts and the Division of Urology, Washington University, St. Louis, Missouri
ABSTRACT
The role of sex hormones on oxalate synthesis by liver, transport by renal cortical mitochondria and urinary excretion was studied in adult male and female Sprague-Dawley rats. Orchiectomy decreased the hepatic synthesis of oxalate whereas ovariectomy increased it by 10 per cent. Castration inhibited oxalate transport by the renal mitochondria uncompetitively in both sexes. Urinary levels of oxalate were unaffected after orchiectomy whereas ovariectomy resulted in an initial elevation in the urinary oxalate levels which returned to control values by 21 days. The results are discussed in light of recent data. The incidence of calcium oxalate kidney stone formation is far less in women than in men. This dissimilarity was first attributed to anatomical differences between males and females but it is now clear that such is not the case. Detailed studies on the reason for this difference have not been performed but some studies indicate that sex hormones may modulate oxalate metabolism. It has been reported that the enzymes glycolate oxidase, glycolate dehydrogenase and lactate dehydrogenase involved in the hepatic synthesis of oxalate are testosterone dependent. 1 • 2 Richardson 1 • 3·4 also reported that deposition of oxalate in the kidney is regulated by testosterone and that castrated female rats given testosterone are more prone to develop urolithiasis. In a previous study we demonstrated that oxalate binds specifically to the mitochondrial inner membranes of kidney and liver and is transported into the mitochondrial matrix space. 5 It was, therefore, thought worthwhile to investigate the role of sex hormones on the synthesis, binding, transport and excretion of oxalate. MATERIALS AND METHODS
Adult male and female Sprague-Dawley rats weighing 300 to 350 gm. were used for the experiments. The animals were divided into 4 groups: 1) sham ovariectomized, 2) ovariectomized, 3) sham orchiectomized, 4) orchiectomized. Each group had a total of 12 rats of which 4 each were sacrificed by decapitation 7, 14, and 21 days after operation. The experiment was repeated once with the same number of animals in each group. All animals were maintained under standard conditions (14 hr. light, 10 hr. darkness) and provided water and food (Purina rat chow containing 0.1 per cent oxalate) ad libitum. The average intake of food by an adult rat ranged between 15 to 20 gm./day. There was no significant change in the food intake in the postoperative state. Surgical techniques. Orchiectomy and ovariectomy were carried out under semisterile conditions. Ovariectomy was performed through an abdominal incision. The uterine artery was tied before removal of the ovary. The abdominal muscle was sutured with silk thread and the incision on the skin was closed with autoclips. Orchiectomy was performed through a scrotal incision. The testis was removed after the ligation of the epididymal artery and the incision was closed with autoclips. Accepted for publication August 2, 1984. * Requests for reprints: Division of Urology, University of Massachusetts Medical School, Worcester, MA 01605. Supported by grants from the NIADDK and the Veterans Administration.
Sham operated animals had the skin incisions alone. Oxalate synthesis by liver. On the day of sacrifice, the liver was removed from each animal, blotted free of blood and weighed. The tissue was homogenized (20 per cent w/v) in 50 mM potassium phosphate buffer, pH 7.4 using a motorized glass-teflon homogenizer. Three hundred µl. of liver homogenate from each animal were incubated in the presence of 10 mM glyoxalate. 8 The total volume of incubation mixture was made up to 1.5 ml. Incubations were carried out at 37C for 60 min. By 60 min. the oxalate production was maximum. At the end of incubation, the samples were kept in a boiling water bath for 5 min. and centrifuged at 13,000 g for 3 min. in a microcentrifuge. All samples were frozen at -4C until measurement of oxalate by ion chromatography. 6 • 7 In order to find out the level of oxalate production from the endogenous pool of glyoxalate, if any, 1 control was run by incubating only the homogenate in potassium phosphate buffer at 37C for 60 minutes. Another control was run by incubating the substrate alone under the same conditions in order to find out whether glyoxalate gets converted into oxalate non-enzymatically. In the absence of either liver homogenate or substrate, there was no detectable level of oxalate synthesis. The data are expressed as µg./hr./gm. tissue weight and as a percentage of control. Renal mitochondrial oxalate transport. Kidney cortex was pooled from all 4 rats at each time interval. Renal cortical mitochondria were isolated in 0.25 M sucrose using conventional centrifugation techniques. 9 Briefly, the kidney cortex was homogenized in 20 mM Tris-HCl (pH 7.2) buffer containing 1 mM EDT A and 0.25 M sucrose. The homogenate was centrifuged at 750 g for 10 min. at 4C in a Beckman J2-20 preparative centrifuge. The supernatant 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 followed by another wash with 0.25 M sucrose and centrifugation under the same conditions. The final pellet was suspended in 0.25 M sucrose. The viability of mitochondria was tested by demonstrating respiration using the oxygen electrode as described by Chan et al.1° Oxalate uptake was measured by the inhibitor stop technique.11-13 Briefly, a 100 µl. aliquot from the stock mitochondrial suspension (1 to 2 mg. mitochondrial protein) was incubated in a total volume of 0.9 ml. containing 20 mM HEPES (pH 6.9), 1 mM EGTA, 100 mM KCl, 2 µg. rotenone and 2 µg. antimycin A for 30 seconds at room temperature (23C). Varying concentrations of oxalate (0.2 mM to 5 mM final concentration) containing 200 cpm/nmole 14 C-oxalate were added to the incubation medium and the volume was made up to 1 ml. Incu-
1244
1245
EFFECT OF CASTRATION ON OXALATE METABOLISM
bations were carried out in triplicate at 23C for 30 seconds and terminated by the addition of mersalyl to a final concentration of 0.5 mM. Mersalyl, which is a sulfhydryl group inhibitor, was used because it was found to inhibit oxalate uptake better than phenyl succinate and N-ethyl maleimide which are dicarboxylic acid inhibitors (data not shown). Nonspecific uptake (defined as uptake detected in the presence of mersalyl added prior to the addition of 14C oxalate) was determined in duplicate aliquots. Since the inhibitor prevents the entry of anions into the mitochondrial matrix space, the difference between total and nonspecific uptake represents the specific uptake into matrix space which is expressed as nmoles/mg. mitochondrial protein. At the end of incubation, all the tubes (for total and nonspecific uptake) were centrifuged at 13,500 g for 1 min. in a Fisher 235 A microcentrifuge. The supernatant was aspirated and the pellet was washed once with the incubation buffer and centrifuged. The sediment was suspended in 1 ml. distilled water and transferred to a glass scintillation vial containing 10 ml. PCS and radioactivity was counted. Protein was measured by the method of Lowry et al. 14 Kinetic parameters were determined from a double reciprocal plot. Oxalate determination in urine. The rats were maintained in metabolic cages for the collection of urine. Twenty-four hour urine was collected 7, 14 and 21 days after operation. The pH was adjusted to 1 with concentrated HCI and stored at -4C until used. Oxalate in the urine was determined by ion chromatography .6· 7 Statistical analysis of the data was done by a professional statistician using 2 way factorial analysis of variance on a computer.
illMa!e 0Female
120
ec "' 0"
120
100
100
80
80
60
60
40
40
20
20
"'~
~ ~
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castrated
control
castra!<1d
FIG. 1. In vitro conversion of glyoxylate (10 mM) to oxalate at 37C. As there was no significant difference among each group after 7, 14 and 21 days of treatment, values were pooled. Data represent mean (SE) from 6 pooled values for each group. p < 0.01 - male vs. female. p < 0.08 - control vs. castration. TABLE 1.
Effect of orchiectomy and ovariectory on mitochondrial transport of oxalate
Concentration of Oxalate (mM)
Control Female Male
0.2
RESULTS
0.5
Biosynthesis of oxalate from glyoxalate. The hepatic synthesis of oxalate in control and experimental animals is shown in fig. 1. Oxalate content in the liver was measured by ion chromatography6 • 7 originally developed for urinary oxalate. The data shown in fig. 1 clearly indicate that the capacity of the liver to synthesize oxalate from glyoxalate is greater (p < 0.01) in male rats than in female rats. Orchiectomy resulted in a 20 per cent decline in oxalate synthesis (p < 0.08). On the contrary the conversion of glyoxalate to oxalate by the female liver increased by 10 per cent after ovariectomy (p < 0.08). These results corroborate the previously demonstrated sexual dimorphism in oxalate production and its dependence on testosterone. Mitochondrial transport of oxalate. Mitochondrial transport by renal cortical mitochondria was measured at 23C and pH 6.9. Transport by both male and female kidney mitochondria increased with increasing concentrations of substrate in the range 0.2 to 5 mM (table 1). As there was no significant difference in the transport of oxalate by mitochondria from 7, 14 and 21 day groups, the data were pooled. Kinetic analysis revealed Km value of 1.47 + 0.07 mM (range 1.37 to 1.54) and a Vmax of 4.3 + 0.50 nmoles/mg. protein (range 3.9 to 5.1) for the control male groups. In the female control groups the mean Km value was 1.32 + 0.26 mM with a range of 1.0 to 1.64 mM and the Vmax was 3.56 ± 0.37 from a range of 3.1 to 4.0 nmoles/ mg. protein. This suggests that the kinetics of mitochondrial oxalate transport are similar in both male and female rat kidneys. Castration induced a significant decrease (p < 0.001) in both the apparent Km and Vmax of oxalate transport in either sex, indicating uncompetitive inhibition of transport that was not sex-dependent (table 1). Urinary excretion of oxalate. From the data on the urinary excretion of oxalate after 7, 14 and 21 days of orchiectomy and ovariectomy it is clear that oxalate excretion was not significantly affected after orchiectomy. On the other hand, ovariectomy resulted in an initial elevation of urinary levels of oxalate which returned to control values by 21 days (table 2).
1.0 5.0 Kinetic parameters Km(mM) Vmax (nmoles/mg. protein/30 sec.)
Castrated Male Female
0.74+ (0.14) 1.31+ (0.10) 1.95+ (0.31) 4.26+ (0.93)
0.70+ (0.18) 0.85+ (0.09) 1.45+ (0.14) 4.55+ (0.11)
0.70+ (0.14) 1.08+ (0.09) 1.38+ (0.36) 3.23+ (0.55)
0.47+ (0.11) 0.75+ (0.20) 1.08 (0.07) 3.64+ (0.99)
1.47 (0.07) 4.3 (0.50)
1.32 (0.26) 3.56 (0.37)
0.47* (0.01) 2.65* (0.11)
0.70* (0.01) 1.82* (0.51)
Details of the procedure for transport are given in METHODS. Upper panel shows effect of increasing concentration of oxalate on its transport. Data are expressed as nmole/mg. protein/30 sec. Six replicates were carried out for each concentration and time interval (7, 14 and 21 days). As there was no significant difference among the 3 intervals, data were pooled within the group. Kinetic parameters of oxalate were determined from double reciprocal plot using concentration range shown above. Km and Vma: were calculated for each time interval and pooled within the group. The data represent mean (SE) from all the pooled values. * p < 0.001. TABLE
2. Effect of orchiectomy and ovariectomy on urinary excretion of oxalate
Sex Male Female
7
Control 14 (Days)
21
7
Castrated 14 (Days)
21
2.09 (0.19) 1.73 (0.08)
1.54 (0.16) 1.25 (0.31)
1.32 (0.12) 1.20 (0.13)
2.44 (0.17) 2.56 (0.47)*
1.66 (0.24) 1.84 (0.04)**
1.51 (0.16) 1.39 (0.26)
Values are mean (SE) from 4 urine samples. Data expressed as mg./24 hr. urine sample. * p < 0.02, ** p < 0.01.
DISCUSSION
It is well established that idiopathic calcium oxalate renal stones are 3 to 4 times more prevalent in men than in women. 15- 17 Even when diseases with distinct female predilection such as hyperparathyroidism 18• 19 or absorptive hyperoxaluria15 are considered, proportionately more men than women develop renal calculi. The reasons for this are unclear;
1246
SEETHLAKSHMI, MAHLE AND MENON
however, experimental findings suggest that the activities of some of the liver enzymes involved in oxalate synthesis are greater in males than in females. 1 The lower activity found among females may indicate a possible inhibition by estrogens.1 Except for this information, the importance of sex differences in kidney stone formation has not been fully explored. The results of the present study corroborate Richardson's findings that the synthesis of oxalate from its precursor glyoxylate is significantly decreased by orchiectomy.1·3 In contrast, ovariectomy resulted in a slight increase in oxalate production by female liver. The major enzyme involved in the conversion of glyoxylate to oxalate is probably glycolate oxidase, the concentrations of which are decreased by orchiectomy. This explains why orchiectomy decreased hepatic oxalate synthesis. On the other hand, the increase in oxalate production following ovariectomy has not been previously demonstrated. It is known that glycolate oxidase is located in the peroxisomes of the liver 20 and that estradiol decreases the number of liver peroxisomes.21 The increase in oxalate production following ovariectomy may be related to an increase in hepatic peroxisome content or to an increase in the activity of other enzymes involved in oxalate synthesis. In addition, Sharma et al. 22 have shown that estradiol inhibits hepatic glycolic acid oxidase activity in normal rats. Ovariectomy may be expected to remove this inhibition and thus increase the production of oxalate from glyoxylate. In contrast to the increase in hepatic oxalate production induced by castration the mitochondrial transport of oxalate was inhibited by orchiectomy or ovariectomy. Also, there was no sexual difference in mitochondrial oxalate uptake in the intact rat. Although at first confusing, these findings are interesting in light of data recently reported by Koening and colleagues.23 They demonstrated that testosterone induced a rapid increase in calcium fluxes across renal cortical mitochondria. These fluxes were seen within minutes of incubating mitochondria in medium containing testosterone. The increases in calcium transport were preceded by increase in ornithine decarboxylase production and polyamine synthesis. Koening et al. postulated that testosterone increased ornithine decarboxylase production through a receptor-mediated phenomenon and that the resultant increase in protein synthesis affected mitochondrial structure and function. It is conceivable that testosterone may similarly increase mitochondrial oxalate transport and that this may explain the decrease in transport seen with orchiectomy. It should be noted, however, that we have examined the effect of depriving the intact animals of androgen whereas Koening et al. studied these effects on isolated mitochondria. In future studies we plan to examine the effects of androgens and estrogens added to the incubation medium on the transport. The reason for the decrease in oxalate transport by kidney mitochondria in the female rats remains to be elucidated. It is conceivable that the structure and function of kidney mitochondria in the female may depend upon estrogens. In the present study, the 24 hr. urinary excretion of oxalate did not change significantly after orchiectomy although the synthetic capacity of the liver was low. The reason why this occurred is unclear, although similar results have been reported by Richardson. These results also agree with the clinical data reported by Tiselius et al. 22 who found that urinary oxalate excretion did not decrease following orchiectomy or estrogen treatment in patients with prostatic carcinoma. In conclusion, the results of the present study suggest that in addition to altering hepatic oxalate production, sex hormones modulate the mitochondrial transport of oxalate. We have shown that in 2 separate models of renal stone formation a decrease in the rate of mitochondrial oxalate uptake precedes mitochondrial swelling, cellular destruction and intratubular calcium oxalate crystallization. 25 By decreasing oxalate transport, sex hormones may affect the pathogenesis of calcium oxalate renal stones, at least in these models.
REFERENCES
1. Richardson, K. E.: Effect of testosterone on the glycolic acid oxidase levels in male and female rat liver. Endocrinology, 74: 128, 1964. 2. Sharma, V., Murtry, M. S. R., Thind, S. K. and Nath, R.: Effect of sex hormone on oxalate synthesizing enzymes in weanling rats. Biochemistry International, 3: 507, 1981. 3. Richardson, K. E.: Effects of vitamin B6, glycolic acid, testosterone, and castration on the synthesis, deposition and excretion of oxalic acid in rats. Toxicol. Appl. Pharmacol., 10: 40, 1967. 4. Richardson, K. E.: The effects of partial hepatectomy on the toxicity of ethylene glycolic acid, glyoxylic acid and glycine. Toxicol. Appl. Pharmacol., 24: 530, 1973. 5. Selvam, R. and Menon, M.: Oxalate binding to subcellular fractions of rat and human kidney: evidence for the existence of a specific binding protein. Proc. 2nd Int. Urolithiasis Conference, Singapore. In Press, 1983. 6. Mahle, C. J. and Menon, M.: Determination of urinary oxalate by ion chromatography: preliminary observations. J. Urol., 127: 159, 1982. 7. Menon, M. and Mahle, C. J.: Ion chromatographic measurement of oxalate in unprocessed urine. Clin. Chem., 29: 369, 1983. 8. Fry, D. W. and Richardson, K. E.: Isolation and characterization of glycolic acid oxidase from human liver. Biochem. Biophys. Acta, 568: 135, 1979. 9. Sottacosa, G. L., Bokuylenstierna, L. E. and Bergstrom, A.: Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria. Methods Enzymol., 10: 448, 1967. 10. Chan, T. L., Greenwalt, J. W. and Pederson, P. L.: Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matric activities. J. Cell. Biol., 45: 291, 1970. 11. Palmieri, F., Prezioso, G., Quagliariello, E. and Klingenberg, M.: Kinetic study of the dicarboxylate carrier in rat liver mitochondria. Eur. J. Biochem., 22: 66, 1971. 12. Robinson, B. H. and Williams, G. R.: The sensitivity of dicarboxylate anion exchange reactions to transport inhibitors in rat liver mitochondria. Biochem. Biophys. Acta, 216: 63, 1970. 13. Quagliariello, E., Palmieri, F., Prezioso, G. and Klingenberg, M.: Kinetics of succinate uptake by rat liver mitochondria. FEBS Lett., 4: 251, 1969. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R.: Protein measurement with the follin phenol reagent. J. Biol. Chem., 193: 265, 1951. 15. Pak, C. Y. C., Britton, F., Peterson, R., Ward, D., Northcutt, C., Breshlau, N. A., McGuire, J., Sakhee, K., Bush, S., Nicar, M., Norman, D. A. and Peters, P.: Ambulatory evaluation of nephrolithiasis: classification, clinical presentation and diagnostic criteria. Am. J. Med., 69: 19, 1980. 16. Yendt, E. R. and Cohlanim, M.: Prevention of calcium stones with thiazides. Kidney Int., 13: 397, 1978. 17. Menon, M.: Calcium oxalate renal lithiasis: endocrinology and metabolism. In: Urologic Endocrinology. Edited by E. Rajfer. W. B. Saunders, Philadelphia, 1984. 18. Nicar, M. J., Skurla, C., Sakhee, K. and Pak, C. Y. C.: Low urinary citrate excretion in nephrolithiasis. Urology, 21: 8, 1983. 19. Wikstrom, B., Backman, U., Danielson, B. G., Fellstrom, B., Johannsson, G. and Ljunghall, S.: Ambulatory diagnostic evaluation of 389 recurrent renal stone formers. Klin. Worchenschr., 61: 85, 1983. 20. Masters, C. J. and Homes, R. S.: Peroxisomes: new aspects of cell physiology and biochemistry. Physiol. Rev., 57: 816, 1977. 21. Svoboda, D., Azarnoff, D. and Reddy, J.: Microbodies in experimentally altered cells. II. The relationship of microbody proliferation to endocrine glands. J. Cell. Biol., 40: 734, 1969. 22. Sharma, V., Murthy, M. S. R., Thind, S. K. and Nath, R.: Regulation of oxalate synthesizing enzymes by sex hormones in weanling rats. Urol. Res., 12: 75, 1984. 23. Koening, H., Goldstone, A. and Chung, Y. L.: Polyamines regulate calcium fluxes in a rapid plasma membrane response. Nature, 305: 530, 1983. 24. Tiselilus, H. G., Varenhorst, E., Carlstrom, K. and Larsson, L.: Urinary oxalate excretion during anti-androgenic therapy. Invest. Urol., 18: 110, 1980. 25. Menon, M., Pak, C. Y. C., Gregory, J. G. and Seethalakshi, L.: Mitochondrial oxalate binding and transport in experimental renal stone formation. J. Urol., 131: 258A, 1984.
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Williams & Wilkins Co.
EFFECT OF ORCHIECTOMY AND OVARIECTOMY ON OXALATE PRODUCTION, TRANSPORT AND EXCRETION IN RATS LAXMANAN SEETHLAKSHMI,* CARL J. MAHLE AND MANI MENON From the Division of Urology, University of Massachusetts Medical School, Worcester, Massachusetts and the Division of Urology, Washington University, St. Louis, Missouri
ABSTRACT
The role of sex hormones on oxalate synthesis by liver, transport by renal cortical mitochondria and urinary excretion was studied in adult male and female Sprague-Dawley rats. Orchiectomy decreased the hepatic synthesis of oxalate whereas ovariectomy increased it by 10 per cent. Castration inhibited oxalate transport by the renal mitochondria uncompetitively in both sexes. Urinary levels of oxalate were unaffected after orchiectomy whereas ovariectomy resulted in an initial elevation in the urinary oxalate levels which returned to control values by 21 days. The results are discussed in light of recent data. The incidence of calcium oxalate kidney stone formation is far less in women than in men. This dissimilarity was first attributed to anatomical differences between males and females but it is now clear that such is not the case. Detailed studies on the reason for this difference have not been performed but some studies indicate that sex hormones may modulate oxalate metabolism. It has been reported that the enzymes glycolate oxidase, glycolate dehydrogenase and lactate dehydrogenase involved in the hepatic synthesis of oxalate are testosterone dependent. 1 • 2 Richardson 1 • 3·4 also reported that deposition of oxalate in the kidney is regulated by testosterone and that castrated female rats given testosterone are more prone to develop urolithiasis. In a previous study we demonstrated that oxalate binds specifically to the mitochondrial inner membranes of kidney and liver and is transported into the mitochondrial matrix space. 5 It was, therefore, thought worthwhile to investigate the role of sex hormones on the synthesis, binding, transport and excretion of oxalate. MATERIALS AND METHODS
Adult male and female Sprague-Dawley rats weighing 300 to 350 gm. were used for the experiments. The animals were divided into 4 groups: 1) sham ovariectomized, 2) ovariectomized, 3) sham orchiectomized, 4) orchiectomized. Each group had a total of 12 rats of which 4 each were sacrificed by decapitation 7, 14, and 21 days after operation. The experiment was repeated once with the same number of animals in each group. All animals were maintained under standard conditions (14 hr. light, 10 hr. darkness) and provided water and food (Purina rat chow containing 0.1 per cent oxalate) ad libitum. The average intake of food by an adult rat ranged between 15 to 20 gm./day. There was no significant change in the food intake in the postoperative state. Surgical techniques. Orchiectomy and ovariectomy were carried out under semisterile conditions. Ovariectomy was performed through an abdominal incision. The uterine artery was tied before removal of the ovary. The abdominal muscle was sutured with silk thread and the incision on the skin was closed with autoclips. Orchiectomy was performed through a scrotal incision. The testis was removed after the ligation of the epididymal artery and the incision was closed with autoclips. Accepted for publication August 2, 1984. * Requests for reprints: Division of Urology, University of Massachusetts Medical School, Worcester, MA 01605. Supported by grants from the NIADDK and the Veterans Administration.
Sham operated animals had the skin incisions alone. Oxalate synthesis by liver. On the day of sacrifice, the liver was removed from each animal, blotted free of blood and weighed. The tissue was homogenized (20 per cent w/v) in 50 mM potassium phosphate buffer, pH 7.4 using a motorized glass-teflon homogenizer. Three hundred µl. of liver homogenate from each animal were incubated in the presence of 10 mM glyoxalate. 8 The total volume of incubation mixture was made up to 1.5 ml. Incubations were carried out at 37C for 60 min. By 60 min. the oxalate production was maximum. At the end of incubation, the samples were kept in a boiling water bath for 5 min. and centrifuged at 13,000 g for 3 min. in a microcentrifuge. All samples were frozen at -4C until measurement of oxalate by ion chromatography. 6 • 7 In order to find out the level of oxalate production from the endogenous pool of glyoxalate, if any, 1 control was run by incubating only the homogenate in potassium phosphate buffer at 37C for 60 minutes. Another control was run by incubating the substrate alone under the same conditions in order to find out whether glyoxalate gets converted into oxalate non-enzymatically. In the absence of either liver homogenate or substrate, there was no detectable level of oxalate synthesis. The data are expressed as µg./hr./gm. tissue weight and as a percentage of control. Renal mitochondrial oxalate transport. Kidney cortex was pooled from all 4 rats at each time interval. Renal cortical mitochondria were isolated in 0.25 M sucrose using conventional centrifugation techniques. 9 Briefly, the kidney cortex was homogenized in 20 mM Tris-HCl (pH 7.2) buffer containing 1 mM EDT A and 0.25 M sucrose. The homogenate was centrifuged at 750 g for 10 min. at 4C in a Beckman J2-20 preparative centrifuge. The supernatant 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 followed by another wash with 0.25 M sucrose and centrifugation under the same conditions. The final pellet was suspended in 0.25 M sucrose. The viability of mitochondria was tested by demonstrating respiration using the oxygen electrode as described by Chan et al.1° Oxalate uptake was measured by the inhibitor stop technique.11-13 Briefly, a 100 µl. aliquot from the stock mitochondrial suspension (1 to 2 mg. mitochondrial protein) was incubated in a total volume of 0.9 ml. containing 20 mM HEPES (pH 6.9), 1 mM EGTA, 100 mM KCl, 2 µg. rotenone and 2 µg. antimycin A for 30 seconds at room temperature (23C). Varying concentrations of oxalate (0.2 mM to 5 mM final concentration) containing 200 cpm/nmole 14 C-oxalate were added to the incubation medium and the volume was made up to 1 ml. Incu-
1244
1245
EFFECT OF CASTRATION ON OXALATE METABOLISM
bations were carried out in triplicate at 23C for 30 seconds and terminated by the addition of mersalyl to a final concentration of 0.5 mM. Mersalyl, which is a sulfhydryl group inhibitor, was used because it was found to inhibit oxalate uptake better than phenyl succinate and N-ethyl maleimide which are dicarboxylic acid inhibitors (data not shown). Nonspecific uptake (defined as uptake detected in the presence of mersalyl added prior to the addition of 14C oxalate) was determined in duplicate aliquots. Since the inhibitor prevents the entry of anions into the mitochondrial matrix space, the difference between total and nonspecific uptake represents the specific uptake into matrix space which is expressed as nmoles/mg. mitochondrial protein. At the end of incubation, all the tubes (for total and nonspecific uptake) were centrifuged at 13,500 g for 1 min. in a Fisher 235 A microcentrifuge. The supernatant was aspirated and the pellet was washed once with the incubation buffer and centrifuged. The sediment was suspended in 1 ml. distilled water and transferred to a glass scintillation vial containing 10 ml. PCS and radioactivity was counted. Protein was measured by the method of Lowry et al. 14 Kinetic parameters were determined from a double reciprocal plot. Oxalate determination in urine. The rats were maintained in metabolic cages for the collection of urine. Twenty-four hour urine was collected 7, 14 and 21 days after operation. The pH was adjusted to 1 with concentrated HCI and stored at -4C until used. Oxalate in the urine was determined by ion chromatography .6· 7 Statistical analysis of the data was done by a professional statistician using 2 way factorial analysis of variance on a computer.
illMa!e 0Female
120
ec "' 0"
120
100
100
80
80
60
60
40
40
20
20
"'~
~ ~
"if'.
castrated
control
castra!<1d
FIG. 1. In vitro conversion of glyoxylate (10 mM) to oxalate at 37C. As there was no significant difference among each group after 7, 14 and 21 days of treatment, values were pooled. Data represent mean (SE) from 6 pooled values for each group. p < 0.01 - male vs. female. p < 0.08 - control vs. castration. TABLE 1.
Effect of orchiectomy and ovariectory on mitochondrial transport of oxalate
Concentration of Oxalate (mM)
Control Female Male
0.2
RESULTS
0.5
Biosynthesis of oxalate from glyoxalate. The hepatic synthesis of oxalate in control and experimental animals is shown in fig. 1. Oxalate content in the liver was measured by ion chromatography6 • 7 originally developed for urinary oxalate. The data shown in fig. 1 clearly indicate that the capacity of the liver to synthesize oxalate from glyoxalate is greater (p < 0.01) in male rats than in female rats. Orchiectomy resulted in a 20 per cent decline in oxalate synthesis (p < 0.08). On the contrary the conversion of glyoxalate to oxalate by the female liver increased by 10 per cent after ovariectomy (p < 0.08). These results corroborate the previously demonstrated sexual dimorphism in oxalate production and its dependence on testosterone. Mitochondrial transport of oxalate. Mitochondrial transport by renal cortical mitochondria was measured at 23C and pH 6.9. Transport by both male and female kidney mitochondria increased with increasing concentrations of substrate in the range 0.2 to 5 mM (table 1). As there was no significant difference in the transport of oxalate by mitochondria from 7, 14 and 21 day groups, the data were pooled. Kinetic analysis revealed Km value of 1.47 + 0.07 mM (range 1.37 to 1.54) and a Vmax of 4.3 + 0.50 nmoles/mg. protein (range 3.9 to 5.1) for the control male groups. In the female control groups the mean Km value was 1.32 + 0.26 mM with a range of 1.0 to 1.64 mM and the Vmax was 3.56 ± 0.37 from a range of 3.1 to 4.0 nmoles/ mg. protein. This suggests that the kinetics of mitochondrial oxalate transport are similar in both male and female rat kidneys. Castration induced a significant decrease (p < 0.001) in both the apparent Km and Vmax of oxalate transport in either sex, indicating uncompetitive inhibition of transport that was not sex-dependent (table 1). Urinary excretion of oxalate. From the data on the urinary excretion of oxalate after 7, 14 and 21 days of orchiectomy and ovariectomy it is clear that oxalate excretion was not significantly affected after orchiectomy. On the other hand, ovariectomy resulted in an initial elevation of urinary levels of oxalate which returned to control values by 21 days (table 2).
1.0 5.0 Kinetic parameters Km(mM) Vmax (nmoles/mg. protein/30 sec.)
Castrated Male Female
0.74+ (0.14) 1.31+ (0.10) 1.95+ (0.31) 4.26+ (0.93)
0.70+ (0.18) 0.85+ (0.09) 1.45+ (0.14) 4.55+ (0.11)
0.70+ (0.14) 1.08+ (0.09) 1.38+ (0.36) 3.23+ (0.55)
0.47+ (0.11) 0.75+ (0.20) 1.08 (0.07) 3.64+ (0.99)
1.47 (0.07) 4.3 (0.50)
1.32 (0.26) 3.56 (0.37)
0.47* (0.01) 2.65* (0.11)
0.70* (0.01) 1.82* (0.51)
Details of the procedure for transport are given in METHODS. Upper panel shows effect of increasing concentration of oxalate on its transport. Data are expressed as nmole/mg. protein/30 sec. Six replicates were carried out for each concentration and time interval (7, 14 and 21 days). As there was no significant difference among the 3 intervals, data were pooled within the group. Kinetic parameters of oxalate were determined from double reciprocal plot using concentration range shown above. Km and Vma: were calculated for each time interval and pooled within the group. The data represent mean (SE) from all the pooled values. * p < 0.001. TABLE
2. Effect of orchiectomy and ovariectomy on urinary excretion of oxalate
Sex Male Female
7
Control 14 (Days)
21
7
Castrated 14 (Days)
21
2.09 (0.19) 1.73 (0.08)
1.54 (0.16) 1.25 (0.31)
1.32 (0.12) 1.20 (0.13)
2.44 (0.17) 2.56 (0.47)*
1.66 (0.24) 1.84 (0.04)**
1.51 (0.16) 1.39 (0.26)
Values are mean (SE) from 4 urine samples. Data expressed as mg./24 hr. urine sample. * p < 0.02, ** p < 0.01.
DISCUSSION
It is well established that idiopathic calcium oxalate renal stones are 3 to 4 times more prevalent in men than in women. 15- 17 Even when diseases with distinct female predilection such as hyperparathyroidism 18• 19 or absorptive hyperoxaluria15 are considered, proportionately more men than women develop renal calculi. The reasons for this are unclear;
1246
SEETHLAKSHMI, MAHLE AND MENON
however, experimental findings suggest that the activities of some of the liver enzymes involved in oxalate synthesis are greater in males than in females. 1 The lower activity found among females may indicate a possible inhibition by estrogens.1 Except for this information, the importance of sex differences in kidney stone formation has not been fully explored. The results of the present study corroborate Richardson's findings that the synthesis of oxalate from its precursor glyoxylate is significantly decreased by orchiectomy.1·3 In contrast, ovariectomy resulted in a slight increase in oxalate production by female liver. The major enzyme involved in the conversion of glyoxylate to oxalate is probably glycolate oxidase, the concentrations of which are decreased by orchiectomy. This explains why orchiectomy decreased hepatic oxalate synthesis. On the other hand, the increase in oxalate production following ovariectomy has not been previously demonstrated. It is known that glycolate oxidase is located in the peroxisomes of the liver 20 and that estradiol decreases the number of liver peroxisomes.21 The increase in oxalate production following ovariectomy may be related to an increase in hepatic peroxisome content or to an increase in the activity of other enzymes involved in oxalate synthesis. In addition, Sharma et al. 22 have shown that estradiol inhibits hepatic glycolic acid oxidase activity in normal rats. Ovariectomy may be expected to remove this inhibition and thus increase the production of oxalate from glyoxylate. In contrast to the increase in hepatic oxalate production induced by castration the mitochondrial transport of oxalate was inhibited by orchiectomy or ovariectomy. Also, there was no sexual difference in mitochondrial oxalate uptake in the intact rat. Although at first confusing, these findings are interesting in light of data recently reported by Koening and colleagues.23 They demonstrated that testosterone induced a rapid increase in calcium fluxes across renal cortical mitochondria. These fluxes were seen within minutes of incubating mitochondria in medium containing testosterone. The increases in calcium transport were preceded by increase in ornithine decarboxylase production and polyamine synthesis. Koening et al. postulated that testosterone increased ornithine decarboxylase production through a receptor-mediated phenomenon and that the resultant increase in protein synthesis affected mitochondrial structure and function. It is conceivable that testosterone may similarly increase mitochondrial oxalate transport and that this may explain the decrease in transport seen with orchiectomy. It should be noted, however, that we have examined the effect of depriving the intact animals of androgen whereas Koening et al. studied these effects on isolated mitochondria. In future studies we plan to examine the effects of androgens and estrogens added to the incubation medium on the transport. The reason for the decrease in oxalate transport by kidney mitochondria in the female rats remains to be elucidated. It is conceivable that the structure and function of kidney mitochondria in the female may depend upon estrogens. In the present study, the 24 hr. urinary excretion of oxalate did not change significantly after orchiectomy although the synthetic capacity of the liver was low. The reason why this occurred is unclear, although similar results have been reported by Richardson. These results also agree with the clinical data reported by Tiselius et al. 22 who found that urinary oxalate excretion did not decrease following orchiectomy or estrogen treatment in patients with prostatic carcinoma. In conclusion, the results of the present study suggest that in addition to altering hepatic oxalate production, sex hormones modulate the mitochondrial transport of oxalate. We have shown that in 2 separate models of renal stone formation a decrease in the rate of mitochondrial oxalate uptake precedes mitochondrial swelling, cellular destruction and intratubular calcium oxalate crystallization. 25 By decreasing oxalate transport, sex hormones may affect the pathogenesis of calcium oxalate renal stones, at least in these models.
REFERENCES
1. Richardson, K. E.: Effect of testosterone on the glycolic acid oxidase levels in male and female rat liver. Endocrinology, 74: 128, 1964. 2. Sharma, V., Murtry, M. S. R., Thind, S. K. and Nath, R.: Effect of sex hormone on oxalate synthesizing enzymes in weanling rats. Biochemistry International, 3: 507, 1981. 3. Richardson, K. E.: Effects of vitamin B6, glycolic acid, testosterone, and castration on the synthesis, deposition and excretion of oxalic acid in rats. Toxicol. Appl. Pharmacol., 10: 40, 1967. 4. Richardson, K. E.: The effects of partial hepatectomy on the toxicity of ethylene glycolic acid, glyoxylic acid and glycine. Toxicol. Appl. Pharmacol., 24: 530, 1973. 5. Selvam, R. and Menon, M.: Oxalate binding to subcellular fractions of rat and human kidney: evidence for the existence of a specific binding protein. Proc. 2nd Int. Urolithiasis Conference, Singapore. In Press, 1983. 6. Mahle, C. J. and Menon, M.: Determination of urinary oxalate by ion chromatography: preliminary observations. J. Urol., 127: 159, 1982. 7. Menon, M. and Mahle, C. J.: Ion chromatographic measurement of oxalate in unprocessed urine. Clin. Chem., 29: 369, 1983. 8. Fry, D. W. and Richardson, K. E.: Isolation and characterization of glycolic acid oxidase from human liver. Biochem. Biophys. Acta, 568: 135, 1979. 9. Sottacosa, G. L., Bokuylenstierna, L. E. and Bergstrom, A.: Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria. Methods Enzymol., 10: 448, 1967. 10. Chan, T. L., Greenwalt, J. W. and Pederson, P. L.: Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matric activities. J. Cell. Biol., 45: 291, 1970. 11. Palmieri, F., Prezioso, G., Quagliariello, E. and Klingenberg, M.: Kinetic study of the dicarboxylate carrier in rat liver mitochondria. Eur. J. Biochem., 22: 66, 1971. 12. Robinson, B. H. and Williams, G. R.: The sensitivity of dicarboxylate anion exchange reactions to transport inhibitors in rat liver mitochondria. Biochem. Biophys. Acta, 216: 63, 1970. 13. Quagliariello, E., Palmieri, F., Prezioso, G. and Klingenberg, M.: Kinetics of succinate uptake by rat liver mitochondria. FEBS Lett., 4: 251, 1969. 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R.: Protein measurement with the follin phenol reagent. J. Biol. Chem., 193: 265, 1951. 15. Pak, C. Y. C., Britton, F., Peterson, R., Ward, D., Northcutt, C., Breshlau, N. A., McGuire, J., Sakhee, K., Bush, S., Nicar, M., Norman, D. A. and Peters, P.: Ambulatory evaluation of nephrolithiasis: classification, clinical presentation and diagnostic criteria. Am. J. Med., 69: 19, 1980. 16. Yendt, E. R. and Cohlanim, M.: Prevention of calcium stones with thiazides. Kidney Int., 13: 397, 1978. 17. Menon, M.: Calcium oxalate renal lithiasis: endocrinology and metabolism. In: Urologic Endocrinology. Edited by E. Rajfer. W. B. Saunders, Philadelphia, 1984. 18. Nicar, M. J., Skurla, C., Sakhee, K. and Pak, C. Y. C.: Low urinary citrate excretion in nephrolithiasis. Urology, 21: 8, 1983. 19. Wikstrom, B., Backman, U., Danielson, B. G., Fellstrom, B., Johannsson, G. and Ljunghall, S.: Ambulatory diagnostic evaluation of 389 recurrent renal stone formers. Klin. Worchenschr., 61: 85, 1983. 20. Masters, C. J. and Homes, R. S.: Peroxisomes: new aspects of cell physiology and biochemistry. Physiol. Rev., 57: 816, 1977. 21. Svoboda, D., Azarnoff, D. and Reddy, J.: Microbodies in experimentally altered cells. II. The relationship of microbody proliferation to endocrine glands. J. Cell. Biol., 40: 734, 1969. 22. Sharma, V., Murthy, M. S. R., Thind, S. K. and Nath, R.: Regulation of oxalate synthesizing enzymes by sex hormones in weanling rats. Urol. Res., 12: 75, 1984. 23. Koening, H., Goldstone, A. and Chung, Y. L.: Polyamines regulate calcium fluxes in a rapid plasma membrane response. Nature, 305: 530, 1983. 24. Tiselilus, H. G., Varenhorst, E., Carlstrom, K. and Larsson, L.: Urinary oxalate excretion during anti-androgenic therapy. Invest. Urol., 18: 110, 1980. 25. Menon, M., Pak, C. Y. C., Gregory, J. G. and Seethalakshi, L.: Mitochondrial oxalate binding and transport in experimental renal stone formation. J. Urol., 131: 258A, 1984.