Induction of lipid peroxidation by oxalate in experimental rat urolithiasis

J. Biosci., Vol. 12, Number 4, December 1987, pp. 367 373. Printed in India. Induction of lipid peroxidation by oxalate in experimental rat urolithiasis R. SELVAM and T. BIJI KURIEN Department of Medical Biochemistry, P. G. Institute of Basic Medical Sciences, University of Madras, Taramani, Madras 600 113, India MS received 31 March 1987; revised 14 September 1987 Abstract. The function of lipid peroxidation and the antiperoxidative enzymes of rat liver and kidney were studied in stone formation induced by intraperitoneal administration of sodium oxalate (7 mg/100 g body weight). The animals sacrificed 3 and 12 h after administration of sodium oxalate had higher level of malondialdehyde in liver and kidney than control animals. A significantly pronounced release of malondialdehyde was observed in treated liver and kidney homogenates when incubated with either ferrous sulphate or hydrogen peroxide compared to control liver and kidney. Superoxide dismutase activity was increased only in liver and not in kidney in treated animals compared to the control. A highly significant decrease in catalase activity was observed in both liver and kidney of treated animals. Keywords. Induction; lipid peroxidation; oxalate; urolithiasis; catalase; superoxide dismutase. Introduction Renal lithiasis is a condition in which concretions form within the pelvis and calyces. A majority of renal stones are composed of calcium oxalate. More than 90% of the urinary oxalate is of endogenous origin since exogenous oxalate is poorly absorbed in the intestine (Barilla et al., 1978). Oxalate, a non-essential end product of metabolism, is excreted in the urine unchanged. Among many mechanisms proposed for the formation of kidney stones, supersaturation of precipitating ions, matrix nucleation and absence of inhibitors are the most popular. The current view is that the basic problem in the stone formers is a cellular defect in the handling of oxalate and calcium. Lipid peroxidation, a type of oxidative degeneration of polyunsaturated lipids, has been implicated in a variety of pathogenic processes (Dianzani and Ugazio, 1978; Slater, 1972; Plaa and Witschi, 1976). Ascorbic acid, a precursor of oxalate biosynthesis has been shown to increase peroxide formation in tissues non-enzymatically. This ascorbic acid linked lipid peroxidation has been found to be activated by iron complexes and oxalate (Ernster and Nordenbrand, 1967). Oxalate was shown to bind to membrane proteins in our earlier studies (Selvam and Menon, 1982, 1983; Seethalakshmi et al., 1986). In this communication we report for the first time that oxalate or calcium oxalate induces lipid peroxidation in tissues during stone formaion and the induction is through oxalate-enzyme interaction. Abbreviations used: MDA, Malondialdehyde; TCA, trichloroacetic acid; SOD, superoxide dismutase. 367

368 Selvam and Biji Kurien Materials and methods Animals Male Wistar strain rats weighing about 120 g were used for this study. The animals were divided on their weight basis into two groups. Group I served as control and Group II as the test group. All animals received the regular rat chow supplied by Hindustan Lever Ltd., Bombay. The feed contained protein (21%), lipids (5%), crude fibre (4%), ash (8%), calcium (1%), phosphorus (0 6%), nitrogen free extract (55%), stabilized water and fat soluble vitamins, minerals and trace elements. It provided metabolizable energy of 3600 Kcal kg. All the animals were fed with food and water ad libitum. Methods Group I rats (n = 6) received only 0 9% saline intraperitoneally. Group II rats (n=12) were injected intraperitoneally (Bhaskar and Selvam, 1985) with 0 22 Μ sodium oxalate (7 mg/100 g body weight) dissolved in 0 9% saline. Six animals were sacrificed 3 h after the injection and 6 were killed after 12 h. These time points were chosen for the present study as our earlier studies (Bhaskar and Selvam, 1985) had shown maximal deposition of calcium oxalate crystals in the kidney 12 h after the injection. The animals were killed by decapitation and liver and kidney were quickly dissected into ice cold saline. The tissues were trimmed free of connective tissue and blotted with filter paper to remove moisture. They were then homogenized in Tris- HCl buffer (0 01 M, ph 7 4) using a Potter-Elvehjem homogenizer to give a 10% homogenate. Lipid peroxidation Lipid peroxidation was assayed by the method of Brogan et al. (1981) in which malondialdehyde (MDA) released served as the index of lipid peroxidation. 1, 1, 3, 3 tetraethoxypropane malonaldehyde bis(diethyl acetal) was used as the standard. Three sets of tubes were used for the assay. The first set had 2 2 ml of buffer and 0 2 ml of FeSO 4 (0 7 mm final concentration); another set had 2 2 ml of buffer and 0 2 ml of H 2 O 2 (0 75 mm final concentration); the third set contained 2 2 ml of buffer and 0 2 ml of distilled water. Each set was divided into two batches. To each tube of one batch 0 6 ml of the tissue homogenate was added. The tubes were incubated on a mechanical shaker at 100 120 oscillation/min (for aeration) for 60 min. After the incubation 0 5 ml of 40% trichloroacetic acid (TCA) was added. To each tube of the second batch 0 5 ml of 40 % TCA was added at zero time immediately after the addition of 0 6 ml of the homogenate. To all the tubes 0 25 ml of 5 Ν HCl was added and the contents mixed thoroughly. This was followed by the addition of 0 5 ml of 2% thiobarbituric acid. The tubes were shaken and incubated in a water bath at 90 C for 20 min. They were then cooled and 3 ml of chloroform was added. After thorough mixing they were centrifuged for 15 min at 3000 g. The supernatant was aspirated and its absorbance at

Lipid peroxidation in urolithiasis 369 532 nm determined using a water blank. Standard malonaldehyde was treated in a similar fashion and the colour developed was measured. Enzymes Superoxide dismutase (SOD) activity was determined by the method of Marklund and Marklund (1974). The activity was expressed as units/mg protein; 1 unit corresponds to the amount of enzyme needed to inhibit the autoxidation of pyrogallol by 50%. Catalase activity was determined by the method of Takahara et al (1960). It was expressed as μmol of H 2 O 2 consumed/mg protein/min. Protein was estimated by the method of Lowry et al (1951) using bovine serum albumin as standard. Results The function of lipid peroxidation and the antiperoxidative enzymes of rat liver and kidney were studied under stone forming conditions. Calcium oxalate stones were induced by intraperitoneal administration of sodium oxalate solution which produced deposition immediately and maximally by 12 h after administration. Lipid peroxidation in liver Table 1 presents MDA release in the liver of control and treated rats. MDA in control liver homogenate (61 pmol/mg protein) increased significantly (165 pmol/mg protein) when incubated for 60 min. The rats administered sodium oxalate solution showed higher lipid peroxidation than control rats at 3 and 12 h after treatment. Incubation of the homogenate with FeSO 4 (0 7 mm) or H 2 O 2 (0 75 mm) stimulated a further release of MDA. It is very interesting to note that a significant increase in MDA was observed in the liver homogenate of treated rats in the presence of FeSO 4. MDA levels in the presence of FeSO 4 (354 and 390 pmol/mg protein at 3 and 12 h, respectively) were 75 90% higher than those in the absence of FeSO 4. H 2 O 2 stimulated the release of MDA to a smaller extent. Table 1. Effect of sodium oxalate injection on rat liver lipid peroxidation. Statistical comparisons were between test value and corresponding control value. Significant difference at a P<0 05 and b P< 0 001.

370 Selvam and Biji Kurien Lipid peroxidation in kidney Table 2 presents the data for kidney of control and test groups. MDA release in control kidney homogenate (75 pmol/mg protein) increased (111 pmol/mg protein) after 60 min incubation. It was even higher (155 pmol/mg protein) in the presence of FeSO 4 (0 7 mm). In contrast, MDA release in the presence of FeSO 4 in treated kidney homogenate was quite striking (230 and 296 pmol/mg protein at 3 and 12 h, respectively). H 2 O 2 stimulated MDA release only slightly, as in liver and the degree of stimulation was more in the treated kidney homogenate. Table 2. Effect of sodium oxalate injection on rat kidney lipid peroxidation. Statistical comparisons were between test value and corresponding control value. Significant difference at a P < 0 05, b P < 0 01 and c P < 0 001. SOD and catalase activities in liver and kidney SOD and catalase activities in liver and kidney of control and treated rats are presented in table 3. SOD activity in liver of treated rats (5 9 and 7 2 units/mg protein at 3 and 12 h, respectively) was significantly higher (P <0 01) at 12 h than that in liver of control rats (5 4 units/mg protein). However, there was no significant change in SOD activity in kidney of treated rats compared to that of control rats. Catalase activity in liver of treated rats (138 and 97 μmol H 2 O 2 consumed/mg protein/min at 3 and 12 h, respectively) was significantly lower (Ρ <0 001) than that in liver of control rats (168 μmol H 2 O 2 consumed/mg protein/min). A similar significant decrease in catalase activity was found in kidney of treated rats. Table 3. Effect of sodium oxalate injection on SOD and catalase activities in rat liver and kidney. Numbers are activities expressed in materials and methods. Statistical comparisons as in tables 1 and 2.

Lipid peroxidation in urolithiasis 371 In vitro studies with oxalate on lipid peroxidation To determine whether oxalate induced lipid peroxidation was by direct interaction with the membrane or by means of indirect interaction further studies were carried out. Table 4 presents the data on tissue lipid peroxidation upon incubation with sodium oxalate, calcium oxalate and calcium chloride in vitro. Sodium oxalate and calcium oxalate stimulated lipid peroxidation in liver homogenate by 142 and 140%, respectively. When FeSO 4 was included along with sodium oxalate a further increase in lipid peroxidation was observed. A significant increase in lipid peroxidation was observed in the presence of sodium oxalate or calcium oxalate or sodium oxalate and FeSO 4 in kidney homogenate also. It is very interesting to note that calcium chloride (0 5 mm) had no effect on lipid peroxidation in both liver and kidney homogenates. Table 4. Effect of various metabolites on rat liver and kidney lipid peroxidation. The metabolites were incubated with the homogenate for 60 min and the ΜDA released was determined. Values are average of 5 determinations. In vitro studies with oxalate on catalase activity Table 5 presents the effect of different oxalate concentration on liver lipid peroxidation and catalase activity. Lipid peroxidation was maximum at 1 mm oxalate. Catalase activity progressively decreased from 167 117 units with increasing Table 5. Effect of different concentrations of oxalate on liver lipid peroxidation and catalase activity. Oxalate was incubated with the liver homogenate for 60 min and the MDA released was determined. MDA at zero time of incubation was 60 pmol/mg protein. a Quenching effect was observed above this concentration. Values are mean of 5 determinations.

372 Selvam and Biji Kurien concentrations of oxalate. When incubated with sodium azide a significant increase in lipid peroxidation was observed in both control and treated liver homogenates showing that the lipid peroxidation was mediated by inhibition of catalase. Discussion The precise mechanism by which kidney stones originate is still not well understood. The possible relationship between cell dysfunction and renal stone formation is yet to be explored. Many pathological changes have been reported during deposition of calcium oxalate in kidney (Khan et al., 1982) after administration of sodium oxalate solution. This study revealed elevated level of lipid peroxidation in both liver and kidney, increased SOD activity in liver and decreased catalase activity in both liver and kidney of rats administered sodium oxalate. The effect was severe in the rats 12 h after administration. The induction of lipid peroxidation in the test animals may be a consequence of the direct influence of oxalate or calcium oxalate as revealed from the in vitro studies. This observation is quite interesting because of the fact that calcium oxalate is the major constituent of most of the renal stones. Since calcium chloride has no effect on lipid peroxidation it is oxalate that induces this action. The mechanism of induction of lipid peroxidation by oxalate may involve inhibition of catalase activity since in vitro studies have revealed progressive inhibition of catalase activity and increase in lipid peroxidation with increasing oxalate concentration. Our earlier studies have revealed maximal deposition of calcium oxalate crystals in kidney by 12 h after administration of sodium oxalate solution. The oxalate concentration in kidney was found to be 0 47 mg/mg protein and increased to 0 62 mg after 3 h and to 1 67 mg after 12 h (Bhaskar and Selvam, 1985). These data support the belief that the observed increased lipid peroxidation in the tissues of rats 12 h after oxalate administration may be due to the increased oxalate concentration in vivo. The increased SOD activity and decreased catalase activity in oxalate treated animals are expected to result in accumulation of H 2 O 2 and thereby increased lipid peroxidation in the presence of ferrous sulphate as a result of Fenton type reactions (Borg et al., 1978). Further iron salts have been shown to promote lipid peroxidation in the microsomal system (Wills, 1969; Brogan et al., 1981) and in the liposomal system (Agarwal et al., 1985). Several compounds such as ascorbic acid, NADPH and ADP have been shown to have significant modulating action in FeSO 4 induced lipid peroxidation (Bucher et al., 1983). The presence of ferrous or ferric iron has been found to be essential for the enzymatically induced lipid peroxidation of microsomal lipids (Wills, 1969; Brogan et al., 1981). The observed enhanced Stimulation of lipid peroxidation in the presence of Fe 2+ in the tissues of rats administered sodium oxalate may be both enzyme mediated as well as a result of inhibition of catalase by oxalate. This possibility is supported by the fact that oxalate concentration is increased in tissues of rats administered oxalate and so the increased lipid peroxidation is a cumulative effect of stimulation by ferrous ion and inhibition of catalase by oxalate. A similar mechanism for induced lipid peroxidation has been proposed for turpentine-induced inflammation in rats (Nadkarni et al., 1986). References Agarwal, S., Banerjee, S. and Chatterjee, S. N. (1985) Indian J. Biochem. Biophys., 22, 331. Barilla, D. E, Notz, C, Kennedy, D. and Pak, C. Y. C. (1978) Am. J. Med., 64, 579.

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