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Lipid Peroxidation in Ethylene Glycol Induced Hyperoxaluria and Calcium Oxalate Nephrolithiasis
0022-5347/97/1573-1059$03.00/0
THE JOURNAL
OF UROL~CY Copyright 0 1997 by AMERICANUROLOCICAL ASS~C~ATION, INC.
Vol. 157. 1059-1063, March 1997 Pnntrd in U.S.A.
LIPID PEROXIDATION IN ETHYLENE GLYCOL INDUCED HYPEROXALURIA AND CALCIUM OXALATE NEPHROLITHIASIS SIVAGNANAM THAMILSELVAN, RAYMOND L. HACKET"*
AND
SAEED R. KHAN
From the Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Unioersity of Florida, Gainesuille, Florida
ABSTRACT
Purpose: To determine if lipid peroxidation plays a role in renal injury associated with experimental nephrolithiasis. Materials and Methods: Hyperoxaluria was produced in rats by ethylene glycol in drinking water. At 15, 30 and 60 days of treatment, urinary oxalate, lipid peroxide, calcium oxalate crystals, enzymes and tissue lipid peroxide were measured. Results: Urinary oxalate increased significantly at all time periods and was associated with crystalluria. Lipid peroxides in kidney tissue and urine increased a t all time periods. Tissue calcium oxalate crystal deposits from 0 to 1+ were present on day 15, but present in all animals on days 30 and 60. Renal tubular cell damage was confirmed by a n increase in urinary marker enzymes. Conclusions: Renal cell damage is associated with lipid peroxide production indicating cell injury due to the production of free radicals. The damage appears due primarily to hyperoxaluria a n d is augmented by crystal deposition in the renal tubules. KEY WORDS:lipid peroxide, hyperoxaluria, calcium oxalate, nephrolithiasis Hyperoxaluria is a major risk factor for calcium oxalate (CaOx) urolithiasis' and is augmented and promoted when combined with cellular degradation products derived from renal tubular injury. Oxalate (Ox) plays no known vital function in mammals and most of the Ox found in urine is endogenously produced with only a small proportion derived from ingested foodstuffs.2 Oxalate is produced primarily from glycolate or glyoxylate in the liver and it has been demonstrated that glycolic acid oxidase, xanthine oxidase, and lactate dehydrogenase can catalyze the oxidation of glyoxylate to oxalate in mammalian systems. Lactate dehydrogenase is also involved in the reduction of glyoxylate to glycolate. In addition, NAD' augments the production of Ox because in its presence not only xanthine oxidase but also lactate dehydrogenase contributes to the oxidation of glyoxylate formed from glycolate by glycolic acid oxidase.3 While the biochemistry of Ox is generally understood, the exact mechanisms involved in CaOx stone formation are not completely formulated. Under appropriate conditions, hyperoxaluria results in CaOx crystal formation,4 but it seems apparent that the levels of urinary Ox involved in stone formation require heterogenous rather than homogenous nucleation and that renal tubular cellular injury is central to this process.5 In considering the pathways for Ox production mentioned above, it seems appropriate to consider whether or not lipid peroxidation plays a role in experimental urolithiasis. Earlier studies have shown that acute and chronic production of CaOx crystal deposition induces lipid peroxidation and therefore the process may play a n important role in CaOx stone formation." Lipid peroxidation usually refers to the functional impairment of cellular components by reactive oxygen species such as superoxide radicals, hydroxyl free radicals and hydrogen peroxide. The process is initiated by the hydroxyl radical formed through the extraction of a hydrogen atom from unsaturated fatty acids of membrane phospholipids,7 the resulting chain reaction yielding several types
of secondary free radicals and a large number of reactive compounds, culminating in the destruction of cellular membranes. Previous studies from our laboratory have reported that hyperoxaluria, even without crystalluria, caused increased excretion of enzymes of renal tubular origin8 and that cellular injury potentiates CaOx crystal formation in hyperoxaluric rats.4 The present study was undertaken to examine the possible role of lipid peroxidation in stone forming rats in a chronic model for experimentally induced CaOx crystalluria and stones. MATERIALS AND METHODS
Animals. The experimental animal was the male SpragueDawley rat weighing 150-160 gm. The animals were placed in metabolic cages, three days prior to beginning the experiment, to acclimatize them to the cages. Baseline 24 hour urine collections were begun one day prior to starting the experiment. The ambient temperature was maintained at 23C ? 1 on a 12 hour lightIl2 hour dark cycle. The animals were divided into two groups. Group I: Control animals (n = 5 for the 15 day time period, and n = 6 for 30 and 60 days) received distilled drinking water. Group 11: The experimental animals (n = 5 for the 15 day time period, and n = 6 for 30 and 60 days) were given a hyperoxaluria inducing diet of 0.757~ethylene glycol in distilled drinking water. The experiment was conducted for 60 days. On day 15, five animals each from the control and experimental groups were sacrificed; on days 30 and 60, six animals from each group were sacrificed. During the experimental period all groups were provided ground regular rat chow ad libitum. Urine collection. Twenty four hour urine samples were collected on day 0, 15, 30, and 60 of the experiment. One hour urine samples were collected before the start of the 24 hour urine sample collection to determine the presence of CaOx crystalluria, which was scored on a basis of 0-4+. Twenty four hour samples were collected on ice in 50 ml. 9 p t e d for publication July 1, 1996. graduated centrifuge tubes attached to urine collection funRequests for reprints: University of Florida College of Medicine, nels. Urine samples were centrifuged at 2000 X g for 10 Department of Pathology, Immunology and Laboratory Medcine, Box minutes to remove debris. The supernatants were used to 100275, JHMHC, Gainesville, FL 32610. determine the lipid peroxides and enzymes. Supported by NIH grants Pol-DK20586 and R01-DK41434-05. 1059
1060
LIPID PEROXIDATION AND HYPEROXALURIA
Determination of lipid peroxides. Urinary lipid peroxides as malondialdehyde (MDA) coupled with thiobarbituric acid (TBA) were assayed by High-Performance Liquid Chromatography (HPLC) using a Water's p-Bondapak C18 column, eluting with phosphate-methanol (65%-35%) mobile phase. The eluted malondialdehyde complex which has a retention time of 8 minutes, was measured at 532 nm.9 1,1,3,3tetraethoxypropane was used as a standard. A portion of the kidney tissue was used to determine the products of lipid peroxidation as described by Sunderman et a1 with some modifications.10 The kidney tissues for assay were rapidly excised, decapsulated, blotted, and minced with fine scissors. Tissue samples were homogenized in 10 ml of cold KCl-EDTA solution (potassium chloride 154 mmol/L; EDTA 1 mmoVL) using tissue homogenizers. Duplicate aliquots of 0.1 ml of each homogenate were pipetted into pyrex tubes containing 3 ml of cold 1%phosphoric acid, 1ml of 0.6% thiobarbituric acid solution containing 0.01% butylated hydroxytoluene (BHT) and placed for 45 minutes in a boiling water bath. While performing the TBARS assay in control samples we included 500 pg/ml 1 pM calcium oxalate monohydrate (COM) crystals in the control sample to test whether the crystals induce TBARS during the assay. The final result was the same in controls with or without COM crystals in the assay tube after boiling with TBA and phosphoric acid; also BHT was added to the thiobarbituric acid reagent in order to prevent decomposition of lipid hydroperoxides and the formation of MDA during heating step of the assay.'' The tubes were cooled to room temperature and the pink color formed was extracted with n-butanol and measured by colorimetry at a n absorbance of 532 nm and the amount of MDA extrapolated as from the standard curve using 1,1,3,3-tetraethoxypropane standard. Enzyme determinations. Centrifuged urine supernatants were dialyzed for three hours at 4C against deionized water for determination of enzymes.'* Alanine aminopeptidase, a marker of tubular brush border, was measured a t 410 nm. and 37C by monitoring the increase of absorbance due to release of 4-nitroaniline catalyzed by alanine aminopeptidase during 3 minute intervals, according to the method of Jung and Scholz. I B N-acetyl-glucosaminidase (NAG), a lysosomal marker, was assayed by colorimetry under optimal reaction conditions a t 405 nm. using 4-nitrophenyl-glycosidesas substrate by the method of Maruhn.14 Lysozyme, after purification with a sep-pak C18 cartridge, was quantified by HPLC on a HS-5 C18 (0.4 x 12.5 cm) column. Samples for lysozyme were analyzed by elution for 25 minutes at a flow rate of 1.5 ml./min. with a linear gradient from 45% to 65'1) acetonitrile with the addition of 0.1% trifluoracetic acid. Chromatograms were recorded by monitoring absorbance at 220 nm. using a LC 85 UV detector
(Perkin Elmer) and data was processed with L-C1-100 integrator.15 Isocitrate dehydrogenase was measured by monitoring the rate of absorbance increase a t 340 nm. as NADP is reduced using the method of Goldberg.16 Lactate dehydrogenase was analyzed on the spectrophotometer by measuring the rate of decrease of absorbance of NADH at 340 nm. using pyruvate as substrate.17 Measurement of omlate. Oxalate was measured by ion chromatography as described by Singhl8 with some modification. For the determination of Ox, an aliquot of 24 hour urine samples from control and experimental subjects was acidified by the addition of 20:l dilution 0.01 M HCl and kept frozen in closed vials until analyzed. Just prior to analysis, the samples were thawed and brought to room temperature, mixed and Ox determinations were done in triplicate. Analyses were performed using Dionex DXlOO gradient ion chromatography system equipped with 0.4 x 25 cm ASlOA anion exchange analytical column containing AGlOA guard column. The 25 pl. of sample was injected using auto sampling injector and the column eluted with 40 mM NaOWDouble distilled water at a flow rate of 1.0 ml. per minute with a linear gradient from 10% to 75% NaOH, 90% to 25% deionized water after helium degassing. The column eluent was monitored using a conductivity cell set at 100 US and peak area was measured with a Dionex AI 450 interface chromatography automation soRware V 3.32. Background conductivity was minimized by using a n anion self regenerating micro membrane suppressor set at 300 mA and with recycled eluent. Sample Ox concentration was then calculated based on an Ox standard curve (range of 0.0125-0.1 mM). The Ox anion eluted at approximately 18 minutes under the chromatographic conditions used. Light microscopy. Animals were euthenized with an intraperitoneal injection of sodium pentobarbital. The kidneys were removed and fixed in 10% neutral buffered formalin, trimmed, processed, and embedded in paraffin. Two sections from each kidney were stained with hematoxylin and eosin and examined under polarized light. The presence of CaOx crystal was scored on a basis of 0-4+. Statistical evaluation. Changes were evaluated by two statistical methods, Student's t test for a comparison between mean values, and Duncan's multiple range test using a computerized program (SAS Institute, Cary, NC). The points of significance were identical and for purposes of simplicity of tables, only the Student's t test data, mean 5 SD, are provided. RESULTS
The mean urinary Ox excretion (Table 1)was 0.67 -t 0.14 mmol in 15 day control animals, and this value was found to
TABLE1. Changes in urinary aurlate, cryatalluria, crystal &position and lipid peroxide in control and experimental animals Experimental Day
Urinary oxalate (mmol)
0.63 ? 0.09
0.67t. 0.14
3.612 0.12"
0.642 0.08
-
4.02 2 0.17-
0.63 2 0.11
4.09
Crystalluria Urinary lipid peroxide
0.69 ? 0.07
0.82t 0.08
1.16 f 0.12=*
0.72 2 0.09
1.W 2 0.06"
0.73 I O . 1 1
2.06 t 0.31'"
0.48r 0.06
0.642 0.07'"
0.50? 0.05
1.19 ? 0.14'.
0.48 t 0.04
1.61 ? 0.06"'
0
5/5 rats I + + + )
-
-
++
++
tpmom hr.1 R e d tiMue lipid peroxide (nmoVmg. protein) Renal tissue crystal dew rition = p <0.001. 'p <0.01. Sigm6cmn compared to control group. EC: ethylene glycol. Values M mean z SD.
.
0
3/6ratsf+)
0
5/5rats(++)
-
?
0.14"
++++
1061
LIPID PEROXIDATION AND HYPEROXALURIA
kidney (Fig. A). The remaining two animals had 1+ deposits in the papillary tips and cortex. At 30 and 60 days 2-31 COM cortical and medullary crystal deposits were present in all kidneys (Fig. B ) . Urinary malondialdehyde (Table 1 ) were significantly elevated in the EG administered rats on day 15 when compared to values for control rats. The mean malondialdehyde content of control urine was 0.82 t 0.08 mmol per 24 hrs. At 15 days, the amounts increased significantly to 1.16 t 0.12 mmol per 24 hrs. in experimental rats. Among the animals on day 15, there was considerable individual variation as 1/5 animals excreted amounts within normal limits and formed no crystals, while one other animal, had increased lipid peroxide excretion and also formed no crystals. The mean malondialdehyde amounts in the urine were further elevated on day 30 and 60 of the experimental period and all animals exhibited significantly increased excretions a t these time periods. Lipid peroxidation in terms of TBARS production in the kidney tissue is also given in Table 1. The mean control value at 15 days was 0.48 2 0.06 nmol/mg. protein. In experimental animals this concentration increased to a mean of 0.64 -t 0.07 nmol/mg. protein on day 15. Among the animals, all except one demonstrated increased tissue concentrations of TBARS. The values increased to a mean of 1.61 -f 0.06 on day 60 of the ethylene glycol treatment. With the exception of isocitrate dehydrogenase which remained unaltered at all days tested, the urinary enzymes tended to progressively increase throughout the experiment (Table 2). This response was evident at day 15 for the lysosoma1 enzymes NAG and lysozyme, and for the brush border membrane enzyme alanine aminopeptidase. Lactate dehydrogenase also responded, but a significant increase in activity was not detected until day 30. A, renal cortex from an animal receiving 0.75% EG in drinking water for 15 days photographed under polarized light A l + deposit of COM crystals is present in the renal tubules. B , renal cortex from an animal receiving 0.75% EG in drinking water for 60 days. A 3+ deposit of COM crystals is present in the renal tubules.
DISCUSSION
Our previous studies, demonstrated that hyperoxaluria and CaOx crystalluria are accompanied by enzymuria and membranuria, a finding consistent with damage to renal tubular cells.4~8~19 Moreover, these changes were observed increase significantly to 3.51 ? 0.12 mmol in experimental even in the absence of crystalluria, suggesting that the Ox rats at 15 days reaching a maximum of greater than 4.0 induced damage was not due solely to injury produced by CaOx ~ r y s t a l s , and ~ . ~ that shedding of cell injury products mmol on days 30 and 60. Freshly voided urine was examined for CaOx crystals (Ta- contributes to heterogenous nucleation. These previous findble 1) on day 0 , 1 5 , 3 0 and 60 of the experiment. None of the ings prompted us to assess the effects of Ox on peroxidative control rats had CaOx crystalluria. Rats receiving EG dem- damage to the kidney. Considerable evidence has implicated onstrated 2+ CaOx crystalluria on days 15 and 30, and 4+ by reactive free radicals in the pathophysiology of a wide spectrum of disorders including atherosclerosis, ischemiaday 60. Light microscopic examination of H and E stained kidney reperfusion, inflammatory disorders, cancer, and aging.20 Resections from the 15 day EG treated animals revealed an cent studies have shown that urinary excretion of lipid inconsistent deposition of calcium oxalate monohydrate peroxide can be demonstrated in a variety of different disor(COM) crystals (Table 1). Two animals had no crystals in the ders including administration of adriamycin,21 diabetes melsections and third had a I+ papillary tip deposit only in each litusT2 and vitamin E defi~iency.2~ TABLE2. Effect of ethylene glycol on urinary enzyme excretion Experimental Day Baseline 'day O 1 Lactate dehydrogenase 129.4 2 4.0 lmUi24 hr.1 Lysozyme I~gm./24hr.) 20.9 2 3.0 N-acetyl 0-glucosaminidase 1.85 z 0.27 lu/24 hr.) Ismitrate dehydrogenase 99.7 2 16.5 lmUi24 hr.) Alanine aminooeotidase 195.8 1 19.9 . . (mU/24 hr.) * o <0.001. ? D <0.01. ' D <0.05. Significance compared to control group EG: ethylene glycol. Values are mean 2 SD.
15 Control cn
=
5)
132.4 f 3.5 21.9 2 4.2 1.85 2 0.20 106.3 2 22.8 202.6 2 12.6
30
E G ( n = 51 133.3 2 3.9 26.7 2 3.3'" 2.88 ? 0.24"" 112.8 ? 23.1 241.5 Z 19.7""
Control tn = 6) 132.1 z 3.7
60
E G (n
= 6)
183.5 I 4.8""
Control In 135.0
f
=
6)
6.2
E G In
=
61
325.0 I 11.4'"
21.6 Z 5.1 1.93 t 0.13
40.1 7.8"" 3.24 r 0.22'e
=
23.6 f 5.3 2.08 2 0.14
108.2 i 16.3
109.4 Z 16.9
119.4 -C 20.9
115.5 C_ 17.1
218.1
456.0
206.5
?
24.8
326.1
2
27.1'"
I 12.3
72.9 I4.9"" 7.80 I 0.53'"
f
17.6""
1062
LIPID PEROXIDATION AND HYPEROXALURIA
In the current study, &r 15 days exposure to 0.75% EG, renal cell membrane may be among the important mechahyperoxaluria, CaOx crystalluria, diuresis, and enzymuria nisms contributing to kidney stone formation. especially of cell membrane and lysosomal origin were present. These findings were expected, consistent with pubCONCLUSIONS lished resultss and established the validity of the model. At Our demonstration that TBARS production occurs in the 30 and 60 days all studied parameters showed significant changes except isocitrate dehydrogenase; these data proba- presence of enzymuria, hyperoxaluria, and crystalluria offers bly reflect progressive renal damage associated with persis- additional evidence that lipid peroxidation is a major pathtent hyperoxaluria and increasing renal parenchymal CaOx way in the mechanism involved in renal tubular cell damage during Ox induced experimental urolithiasis. The finding is deposition. The elevated concentration of malondialdehyde in important because it offers potential clues for therapeutic the urine of our study is indicative of oxidative tissue damage intervention or prevention of stones. since, a s lipid peroxides and peroxidation products accumuAcknowledgments. Technical assistance was provided by late, they may be excreted in the urine.24 The urinary data were strengthened by the observation that. TBARS produc- Mrs. Paula Scott, Ms. Karen Byer and Mrs. Patricia Glenton. tion was increased in the renal tissues. Of importance in this Mr. Ken Massey performed the Duncan's multiple range test. study was our examination of the 15 day animals. At this time, hyperoxaluria and crystalluria were present as were REFERENCES elevated concentrations of renal tissue TBARS. However, 1. Finlavson. B.: Phvsicochemical asDects of urolithiasis. Kidnev crystal deposition and increased concentration of TBARS were Int:, 13' 334, 1<78. unrelated in that the one animal with normal TBARS had 2. Menon. M. and Mahle. C. J.: Oxalate metabolism and renal papillary tip crystal deposits only, and the two animals with calculi. J. Urol., 121:.148, 1982. no crystals demonstrated increased TBARS. This suggests 3. Hodgkinson, A.: Oxalic Acid in Biology and Medicine. London, Academic Press, p. 159, 1977. that oxidative damage is more related to the hyperoxaluria 4. Khan, S. R., Shevock, P. N. and Hackett, R. L.: Acute hyperoxand crystalluria rather than to tissue crystal deposits. The aluria. renal iniurv and calcium oxalate urolithiasis. J. Urol.. latter process ultimately contributes a s TBARS production is 147: 226, 1992." greatly increased with progressive crystal deposition. 5. Hackett. R. L.. Shevock, P. N. and Khan, S. R.: Cell iniurv While tissue crystals may not be central to early damage, associated calcium oxalate crystalluria. J. Urol., 144: G35, in this study we were not able to separate the cellular effects 1990. of Ox from those due to the presence of urinary crystals since 6. Selvam, R. and Bijikurien, T.: Effect of citrate feeding on fiee both occurred together. In cell culture systems,25Ox alone radical induced changes in experimental urolithiasis. Indian J. Exp. Biol., 3 0 707, 1992. has been shown to be toxic and that CaOx crystals potentiate 7 Farber, J. L., Kyle, M. E. and Coleman, J. B.: Biology of disease. the toxicity. mechanisms of cell injury by activated oxygen species. Lab. These data give strong support to the concept that hyperInvest.. 62: 670. 1990. oxaluria and crystalluria are associated with lipid peroxida8. Khan, S. R. and Hackett, R. L.: Hyperoxaluria, enzymuria and tion in renal tubular cells. Oxalate has been shown in vitro to nephrolithiasis. Contrib. Nephol., 101: 190, 1993. generate free radicals26 and therefore can induce lipid per9. Knight, J. A., Smith, S. E., Kinder, V. E. and Pieper, R. K: oxidation. In animal experiments in which CaOx crystals and Urinary lipoperoxides quantified by liquid chromatography, hyperoxaluria are induced by calculogenic diet, renal tissue and determination of reference. Clin. Chem., 34: 1107, 1988. TBARS concentration are elevated significantly as are levels 10. Sunderman, F. W., Marzouk, A., Hopfer, S. M., Zaharia, O., and Reid, M. C.: Increased lipid peroxidation in tissues of nickel of xanthine oxidase.6 The latter is associated with decreased chloride-treated rats. Ann. Clin. Lab. Sci., 15: 229, 1985. concentrations of catalase and reduced glutathione strengthening the idea that one effect of Ox upon cell metabolism is 11. Buege, J. A. and Aust, S. D.: Microsomal lipid peroxidation. Methods Enzymol., 5 2 302, 1978. the production of lipid peroxides. Lipid peroxidation reflects 12. Hoheister, R., Bhargava, A. S. and Gunzel, P.: Value of enzyme the interaction between molecular oxygen and polyunsatudeterminations in urine for the diagnosis of nephrotoxicity in rated fatty acids, resulting in the oxidative deterioration of rats. Clin. Chim. Acta., 160:163, 1986. the latter with the breakdown products including alcohols, 13. Jung, K and Scholz, D.: An optimized assay of alanine aminopeptidase activity in urine. Clin. Chem., 2 6 1251, 1980. aldehydes, ketones and ethers.27 In order to degrade toxic reactive oxygen species, cells are 14. Maruhn, D.: Rapid colorimetric assay of P-galactosidase and N-acetyl-p glucosaminidase in human urine. Clin. Chim. equipped with various antioxidant systems including antioxiActa., 73: 453, 1976. dative enzymes.28 Therefore, it is reasonable to speculate 15. Severini, G. and Aliberti, L. M.: Diagnostic significance of urithat development of tissue injury depends upon the balance nary enzymes: Development of a high performance liquid chrobetween generation of reactive oxygen species and tissue matographic method for the measurement of urinary lyantioxidant defense mechanism.29 Selvam and BijikurienG sozyme. Clin. Chim. Acta, 163 97, 1987. have shown increased formation of hydroxyl radicals and 16. Goldberg, D. M. and Ellis, G.: Isocitrate dehydrogenase. In: Methods of Enzymatic Analysis. Edited by H. U. Bergmeyer. hydroperoxides in stone forming rats. Decreased antioxidant New York, Academic Press, pp. 183-189, 1983. enzymes with increased lipid peroxidation reactions have been reported in kidney stone formers.30 In calculi producing 17. Vassault, A.: Lactate dehydrogenase. In: Methods of Enzymatic Analysis. Edited by H. U. Bergmeyer. New York, Academic diet fed rats the activities of antioxidant enzymes decrease Press, pp. 118-126, 1983. along with a diminished vitamin E, ascorbic acid and tissue 18. Singh, R. P. and Nancollas, G. H.: Determination of phosphate, glutathione content.6 sulphate and oxalate in urine by ion chromatography. J. ChroReactive oxygen molecules have been shown to be involved matography, 433 373, 1988. in the pathogenesis of renal cellular damage.3l Oxalate is 19. Khan, S. R., Shevock, P. N. and Hackett, R. L.: Urinary enzymes and calcium oxalate urolithiasis. J. Urol., 142:846, 1989. known to induce lipid peroxidation,26 which causes disruption of the structural integrity of the membranes and Scheid 20. Halliwell, B. and Grootveld, M.: The measurement of free radical reactions in humans: Some thoughts for future experimentaet a132 have recently shown that Ox produced a significant tion. FEB Lett., 213 9, 1987. increase in both free radical production and cell death in D., Bagchi, E. A,, Hassoun, J. K. and Stohs, S. J.: LLC-PK, cell monolayer. Evidence from previous studies has 21. Bagchi, Adriamycin-induced hepatic and myocardial lipid peroxidation shown that lipid peroxidation is involved in the increased and DNA damage, and enhanced excretion of urinary lipid excretion of tubular marker enzymes into the urine.33 The metabolites in rats. Toxicology, 95: 1, 1995. results of the current study indicate that peroxidation of the 22. Gallaher, D. D., Csallany, A. S., Shoeman, D. W. and Olson,
LIPID PEROXIDATION AND HYPEROXALURIA
J. M.: Diabetes increases excretion of urinary malonaldehyde conjugates in rats. Lipids, 2 8 663, 1993. 23. Lee, H., Shoeman, D. W. and Csallany. A. S.: Urinary response to in vivo lipid peroxidation induced by vitamin E deficiency. Lipids, 27: 124, 1992. 24. Yagi, K.: Lipid peroxides and human diseases. Chem. Phy. Lipids, 45: 337, 1987. 25. Hackett, R. L., Shevock, P. N. and Khan, S. R.: Madin-Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals. Urol. Res., 2 2 197, 1994. 26. Ernster, L. and Nordenbrand, K.: Microsomal lipid peroxidation. In Methods in Enzymology, Edited by R. W. Estabrook, and M. E. Pullman, New York, Academic Press, 10: pp. 574-580, 1967. 27. Slater, T. F.: Overview of methods used for detecting lipid peroxidation. In Methods in Enzymology, Edited by F. Wold and K. Moldave, New York, Academic Press, 105: pp. 283-293, 1984. 28. Grisham, M. B. and McCord, J. M.: Chemistry and cytotoxicity of reactive oxygen metabolites in physiology of oxygen radicals.
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In American Physiological Society, Edited by A. E. Taylor, S. Matalon and P. Ward. Bethesda, MD, pp. 1-18, 1986. 29. Beckman, J. S. and Freeman, B. A.: Antioxidant enzymes as mechanistic probes of oxygen dependent toxicity in physiology of oxygen radicals. In American Physiological Society, Edited by A. E. Taylor, S. Matalon, and P. Ward. Bethesda, MD. pp. 450-453, 1986. 30. Anuradha, C. V. and Selvam, R.: Increased lipid peroxidation in RBC of kidney stone formers. Indian J. Exp. Biochem. Biophys., 2 6 39, 1989. 31. Andreoli, S. P., McAteer, J. A,, Seifert, S. A. and Kempson, S. A.: Oxidant-induced alterations in glucose and phosphate transport in LLC-PK1 cells: mechanisms of injury. Am. J. Physiol., 265: F377, 1993. 32. Scheid, C., Koul, H., Hill, W. A., Narod, J . L., Kennington, L., Honeyman, T., Jonassen, J. and Menon, M.: Oxalate toxicity in LLC-PK1 cells: role of free radicals. Kidney Int., 4 9 413, 1996. 33. Suzuki, M.: Experimental studies on the elevation of urinary enzyme activities and its pathogenesis in acute renal failure. (Abstract Only) Nippon Jinzo Gakkai Shi., 33: 387, 1991.
THE JOURNAL
OF UROL~CY Copyright 0 1997 by AMERICANUROLOCICAL ASS~C~ATION, INC.
Vol. 157. 1059-1063, March 1997 Pnntrd in U.S.A.
LIPID PEROXIDATION IN ETHYLENE GLYCOL INDUCED HYPEROXALURIA AND CALCIUM OXALATE NEPHROLITHIASIS SIVAGNANAM THAMILSELVAN, RAYMOND L. HACKET"*
AND
SAEED R. KHAN
From the Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, Unioersity of Florida, Gainesuille, Florida
ABSTRACT
Purpose: To determine if lipid peroxidation plays a role in renal injury associated with experimental nephrolithiasis. Materials and Methods: Hyperoxaluria was produced in rats by ethylene glycol in drinking water. At 15, 30 and 60 days of treatment, urinary oxalate, lipid peroxide, calcium oxalate crystals, enzymes and tissue lipid peroxide were measured. Results: Urinary oxalate increased significantly at all time periods and was associated with crystalluria. Lipid peroxides in kidney tissue and urine increased a t all time periods. Tissue calcium oxalate crystal deposits from 0 to 1+ were present on day 15, but present in all animals on days 30 and 60. Renal tubular cell damage was confirmed by a n increase in urinary marker enzymes. Conclusions: Renal cell damage is associated with lipid peroxide production indicating cell injury due to the production of free radicals. The damage appears due primarily to hyperoxaluria a n d is augmented by crystal deposition in the renal tubules. KEY WORDS:lipid peroxide, hyperoxaluria, calcium oxalate, nephrolithiasis Hyperoxaluria is a major risk factor for calcium oxalate (CaOx) urolithiasis' and is augmented and promoted when combined with cellular degradation products derived from renal tubular injury. Oxalate (Ox) plays no known vital function in mammals and most of the Ox found in urine is endogenously produced with only a small proportion derived from ingested foodstuffs.2 Oxalate is produced primarily from glycolate or glyoxylate in the liver and it has been demonstrated that glycolic acid oxidase, xanthine oxidase, and lactate dehydrogenase can catalyze the oxidation of glyoxylate to oxalate in mammalian systems. Lactate dehydrogenase is also involved in the reduction of glyoxylate to glycolate. In addition, NAD' augments the production of Ox because in its presence not only xanthine oxidase but also lactate dehydrogenase contributes to the oxidation of glyoxylate formed from glycolate by glycolic acid oxidase.3 While the biochemistry of Ox is generally understood, the exact mechanisms involved in CaOx stone formation are not completely formulated. Under appropriate conditions, hyperoxaluria results in CaOx crystal formation,4 but it seems apparent that the levels of urinary Ox involved in stone formation require heterogenous rather than homogenous nucleation and that renal tubular cellular injury is central to this process.5 In considering the pathways for Ox production mentioned above, it seems appropriate to consider whether or not lipid peroxidation plays a role in experimental urolithiasis. Earlier studies have shown that acute and chronic production of CaOx crystal deposition induces lipid peroxidation and therefore the process may play a n important role in CaOx stone formation." Lipid peroxidation usually refers to the functional impairment of cellular components by reactive oxygen species such as superoxide radicals, hydroxyl free radicals and hydrogen peroxide. The process is initiated by the hydroxyl radical formed through the extraction of a hydrogen atom from unsaturated fatty acids of membrane phospholipids,7 the resulting chain reaction yielding several types
of secondary free radicals and a large number of reactive compounds, culminating in the destruction of cellular membranes. Previous studies from our laboratory have reported that hyperoxaluria, even without crystalluria, caused increased excretion of enzymes of renal tubular origin8 and that cellular injury potentiates CaOx crystal formation in hyperoxaluric rats.4 The present study was undertaken to examine the possible role of lipid peroxidation in stone forming rats in a chronic model for experimentally induced CaOx crystalluria and stones. MATERIALS AND METHODS
Animals. The experimental animal was the male SpragueDawley rat weighing 150-160 gm. The animals were placed in metabolic cages, three days prior to beginning the experiment, to acclimatize them to the cages. Baseline 24 hour urine collections were begun one day prior to starting the experiment. The ambient temperature was maintained at 23C ? 1 on a 12 hour lightIl2 hour dark cycle. The animals were divided into two groups. Group I: Control animals (n = 5 for the 15 day time period, and n = 6 for 30 and 60 days) received distilled drinking water. Group 11: The experimental animals (n = 5 for the 15 day time period, and n = 6 for 30 and 60 days) were given a hyperoxaluria inducing diet of 0.757~ethylene glycol in distilled drinking water. The experiment was conducted for 60 days. On day 15, five animals each from the control and experimental groups were sacrificed; on days 30 and 60, six animals from each group were sacrificed. During the experimental period all groups were provided ground regular rat chow ad libitum. Urine collection. Twenty four hour urine samples were collected on day 0, 15, 30, and 60 of the experiment. One hour urine samples were collected before the start of the 24 hour urine sample collection to determine the presence of CaOx crystalluria, which was scored on a basis of 0-4+. Twenty four hour samples were collected on ice in 50 ml. 9 p t e d for publication July 1, 1996. graduated centrifuge tubes attached to urine collection funRequests for reprints: University of Florida College of Medicine, nels. Urine samples were centrifuged at 2000 X g for 10 Department of Pathology, Immunology and Laboratory Medcine, Box minutes to remove debris. The supernatants were used to 100275, JHMHC, Gainesville, FL 32610. determine the lipid peroxides and enzymes. Supported by NIH grants Pol-DK20586 and R01-DK41434-05. 1059
1060
LIPID PEROXIDATION AND HYPEROXALURIA
Determination of lipid peroxides. Urinary lipid peroxides as malondialdehyde (MDA) coupled with thiobarbituric acid (TBA) were assayed by High-Performance Liquid Chromatography (HPLC) using a Water's p-Bondapak C18 column, eluting with phosphate-methanol (65%-35%) mobile phase. The eluted malondialdehyde complex which has a retention time of 8 minutes, was measured at 532 nm.9 1,1,3,3tetraethoxypropane was used as a standard. A portion of the kidney tissue was used to determine the products of lipid peroxidation as described by Sunderman et a1 with some modifications.10 The kidney tissues for assay were rapidly excised, decapsulated, blotted, and minced with fine scissors. Tissue samples were homogenized in 10 ml of cold KCl-EDTA solution (potassium chloride 154 mmol/L; EDTA 1 mmoVL) using tissue homogenizers. Duplicate aliquots of 0.1 ml of each homogenate were pipetted into pyrex tubes containing 3 ml of cold 1%phosphoric acid, 1ml of 0.6% thiobarbituric acid solution containing 0.01% butylated hydroxytoluene (BHT) and placed for 45 minutes in a boiling water bath. While performing the TBARS assay in control samples we included 500 pg/ml 1 pM calcium oxalate monohydrate (COM) crystals in the control sample to test whether the crystals induce TBARS during the assay. The final result was the same in controls with or without COM crystals in the assay tube after boiling with TBA and phosphoric acid; also BHT was added to the thiobarbituric acid reagent in order to prevent decomposition of lipid hydroperoxides and the formation of MDA during heating step of the assay.'' The tubes were cooled to room temperature and the pink color formed was extracted with n-butanol and measured by colorimetry at a n absorbance of 532 nm and the amount of MDA extrapolated as from the standard curve using 1,1,3,3-tetraethoxypropane standard. Enzyme determinations. Centrifuged urine supernatants were dialyzed for three hours at 4C against deionized water for determination of enzymes.'* Alanine aminopeptidase, a marker of tubular brush border, was measured a t 410 nm. and 37C by monitoring the increase of absorbance due to release of 4-nitroaniline catalyzed by alanine aminopeptidase during 3 minute intervals, according to the method of Jung and Scholz. I B N-acetyl-glucosaminidase (NAG), a lysosomal marker, was assayed by colorimetry under optimal reaction conditions a t 405 nm. using 4-nitrophenyl-glycosidesas substrate by the method of Maruhn.14 Lysozyme, after purification with a sep-pak C18 cartridge, was quantified by HPLC on a HS-5 C18 (0.4 x 12.5 cm) column. Samples for lysozyme were analyzed by elution for 25 minutes at a flow rate of 1.5 ml./min. with a linear gradient from 45% to 65'1) acetonitrile with the addition of 0.1% trifluoracetic acid. Chromatograms were recorded by monitoring absorbance at 220 nm. using a LC 85 UV detector
(Perkin Elmer) and data was processed with L-C1-100 integrator.15 Isocitrate dehydrogenase was measured by monitoring the rate of absorbance increase a t 340 nm. as NADP is reduced using the method of Goldberg.16 Lactate dehydrogenase was analyzed on the spectrophotometer by measuring the rate of decrease of absorbance of NADH at 340 nm. using pyruvate as substrate.17 Measurement of omlate. Oxalate was measured by ion chromatography as described by Singhl8 with some modification. For the determination of Ox, an aliquot of 24 hour urine samples from control and experimental subjects was acidified by the addition of 20:l dilution 0.01 M HCl and kept frozen in closed vials until analyzed. Just prior to analysis, the samples were thawed and brought to room temperature, mixed and Ox determinations were done in triplicate. Analyses were performed using Dionex DXlOO gradient ion chromatography system equipped with 0.4 x 25 cm ASlOA anion exchange analytical column containing AGlOA guard column. The 25 pl. of sample was injected using auto sampling injector and the column eluted with 40 mM NaOWDouble distilled water at a flow rate of 1.0 ml. per minute with a linear gradient from 10% to 75% NaOH, 90% to 25% deionized water after helium degassing. The column eluent was monitored using a conductivity cell set at 100 US and peak area was measured with a Dionex AI 450 interface chromatography automation soRware V 3.32. Background conductivity was minimized by using a n anion self regenerating micro membrane suppressor set at 300 mA and with recycled eluent. Sample Ox concentration was then calculated based on an Ox standard curve (range of 0.0125-0.1 mM). The Ox anion eluted at approximately 18 minutes under the chromatographic conditions used. Light microscopy. Animals were euthenized with an intraperitoneal injection of sodium pentobarbital. The kidneys were removed and fixed in 10% neutral buffered formalin, trimmed, processed, and embedded in paraffin. Two sections from each kidney were stained with hematoxylin and eosin and examined under polarized light. The presence of CaOx crystal was scored on a basis of 0-4+. Statistical evaluation. Changes were evaluated by two statistical methods, Student's t test for a comparison between mean values, and Duncan's multiple range test using a computerized program (SAS Institute, Cary, NC). The points of significance were identical and for purposes of simplicity of tables, only the Student's t test data, mean 5 SD, are provided. RESULTS
The mean urinary Ox excretion (Table 1)was 0.67 -t 0.14 mmol in 15 day control animals, and this value was found to
TABLE1. Changes in urinary aurlate, cryatalluria, crystal &position and lipid peroxide in control and experimental animals Experimental Day
Urinary oxalate (mmol)
0.63 ? 0.09
0.67t. 0.14
3.612 0.12"
0.642 0.08
-
4.02 2 0.17-
0.63 2 0.11
4.09
Crystalluria Urinary lipid peroxide
0.69 ? 0.07
0.82t 0.08
1.16 f 0.12=*
0.72 2 0.09
1.W 2 0.06"
0.73 I O . 1 1
2.06 t 0.31'"
0.48r 0.06
0.642 0.07'"
0.50? 0.05
1.19 ? 0.14'.
0.48 t 0.04
1.61 ? 0.06"'
0
5/5 rats I + + + )
-
-
++
++
tpmom hr.1 R e d tiMue lipid peroxide (nmoVmg. protein) Renal tissue crystal dew rition = p <0.001. 'p <0.01. Sigm6cmn compared to control group. EC: ethylene glycol. Values M mean z SD.
.
0
3/6ratsf+)
0
5/5rats(++)
-
?
0.14"
++++
1061
LIPID PEROXIDATION AND HYPEROXALURIA
kidney (Fig. A). The remaining two animals had 1+ deposits in the papillary tips and cortex. At 30 and 60 days 2-31 COM cortical and medullary crystal deposits were present in all kidneys (Fig. B ) . Urinary malondialdehyde (Table 1 ) were significantly elevated in the EG administered rats on day 15 when compared to values for control rats. The mean malondialdehyde content of control urine was 0.82 t 0.08 mmol per 24 hrs. At 15 days, the amounts increased significantly to 1.16 t 0.12 mmol per 24 hrs. in experimental rats. Among the animals on day 15, there was considerable individual variation as 1/5 animals excreted amounts within normal limits and formed no crystals, while one other animal, had increased lipid peroxide excretion and also formed no crystals. The mean malondialdehyde amounts in the urine were further elevated on day 30 and 60 of the experimental period and all animals exhibited significantly increased excretions a t these time periods. Lipid peroxidation in terms of TBARS production in the kidney tissue is also given in Table 1. The mean control value at 15 days was 0.48 2 0.06 nmol/mg. protein. In experimental animals this concentration increased to a mean of 0.64 -t 0.07 nmol/mg. protein on day 15. Among the animals, all except one demonstrated increased tissue concentrations of TBARS. The values increased to a mean of 1.61 -f 0.06 on day 60 of the ethylene glycol treatment. With the exception of isocitrate dehydrogenase which remained unaltered at all days tested, the urinary enzymes tended to progressively increase throughout the experiment (Table 2). This response was evident at day 15 for the lysosoma1 enzymes NAG and lysozyme, and for the brush border membrane enzyme alanine aminopeptidase. Lactate dehydrogenase also responded, but a significant increase in activity was not detected until day 30. A, renal cortex from an animal receiving 0.75% EG in drinking water for 15 days photographed under polarized light A l + deposit of COM crystals is present in the renal tubules. B , renal cortex from an animal receiving 0.75% EG in drinking water for 60 days. A 3+ deposit of COM crystals is present in the renal tubules.
DISCUSSION
Our previous studies, demonstrated that hyperoxaluria and CaOx crystalluria are accompanied by enzymuria and membranuria, a finding consistent with damage to renal tubular cells.4~8~19 Moreover, these changes were observed increase significantly to 3.51 ? 0.12 mmol in experimental even in the absence of crystalluria, suggesting that the Ox rats at 15 days reaching a maximum of greater than 4.0 induced damage was not due solely to injury produced by CaOx ~ r y s t a l s , and ~ . ~ that shedding of cell injury products mmol on days 30 and 60. Freshly voided urine was examined for CaOx crystals (Ta- contributes to heterogenous nucleation. These previous findble 1) on day 0 , 1 5 , 3 0 and 60 of the experiment. None of the ings prompted us to assess the effects of Ox on peroxidative control rats had CaOx crystalluria. Rats receiving EG dem- damage to the kidney. Considerable evidence has implicated onstrated 2+ CaOx crystalluria on days 15 and 30, and 4+ by reactive free radicals in the pathophysiology of a wide spectrum of disorders including atherosclerosis, ischemiaday 60. Light microscopic examination of H and E stained kidney reperfusion, inflammatory disorders, cancer, and aging.20 Resections from the 15 day EG treated animals revealed an cent studies have shown that urinary excretion of lipid inconsistent deposition of calcium oxalate monohydrate peroxide can be demonstrated in a variety of different disor(COM) crystals (Table 1). Two animals had no crystals in the ders including administration of adriamycin,21 diabetes melsections and third had a I+ papillary tip deposit only in each litusT2 and vitamin E defi~iency.2~ TABLE2. Effect of ethylene glycol on urinary enzyme excretion Experimental Day Baseline 'day O 1 Lactate dehydrogenase 129.4 2 4.0 lmUi24 hr.1 Lysozyme I~gm./24hr.) 20.9 2 3.0 N-acetyl 0-glucosaminidase 1.85 z 0.27 lu/24 hr.) Ismitrate dehydrogenase 99.7 2 16.5 lmUi24 hr.) Alanine aminooeotidase 195.8 1 19.9 . . (mU/24 hr.) * o <0.001. ? D <0.01. ' D <0.05. Significance compared to control group EG: ethylene glycol. Values are mean 2 SD.
15 Control cn
=
5)
132.4 f 3.5 21.9 2 4.2 1.85 2 0.20 106.3 2 22.8 202.6 2 12.6
30
E G ( n = 51 133.3 2 3.9 26.7 2 3.3'" 2.88 ? 0.24"" 112.8 ? 23.1 241.5 Z 19.7""
Control tn = 6) 132.1 z 3.7
60
E G (n
= 6)
183.5 I 4.8""
Control In 135.0
f
=
6)
6.2
E G In
=
61
325.0 I 11.4'"
21.6 Z 5.1 1.93 t 0.13
40.1 7.8"" 3.24 r 0.22'e
=
23.6 f 5.3 2.08 2 0.14
108.2 i 16.3
109.4 Z 16.9
119.4 -C 20.9
115.5 C_ 17.1
218.1
456.0
206.5
?
24.8
326.1
2
27.1'"
I 12.3
72.9 I4.9"" 7.80 I 0.53'"
f
17.6""
1062
LIPID PEROXIDATION AND HYPEROXALURIA
In the current study, &r 15 days exposure to 0.75% EG, renal cell membrane may be among the important mechahyperoxaluria, CaOx crystalluria, diuresis, and enzymuria nisms contributing to kidney stone formation. especially of cell membrane and lysosomal origin were present. These findings were expected, consistent with pubCONCLUSIONS lished resultss and established the validity of the model. At Our demonstration that TBARS production occurs in the 30 and 60 days all studied parameters showed significant changes except isocitrate dehydrogenase; these data proba- presence of enzymuria, hyperoxaluria, and crystalluria offers bly reflect progressive renal damage associated with persis- additional evidence that lipid peroxidation is a major pathtent hyperoxaluria and increasing renal parenchymal CaOx way in the mechanism involved in renal tubular cell damage during Ox induced experimental urolithiasis. The finding is deposition. The elevated concentration of malondialdehyde in important because it offers potential clues for therapeutic the urine of our study is indicative of oxidative tissue damage intervention or prevention of stones. since, a s lipid peroxides and peroxidation products accumuAcknowledgments. Technical assistance was provided by late, they may be excreted in the urine.24 The urinary data were strengthened by the observation that. TBARS produc- Mrs. Paula Scott, Ms. Karen Byer and Mrs. Patricia Glenton. tion was increased in the renal tissues. Of importance in this Mr. Ken Massey performed the Duncan's multiple range test. study was our examination of the 15 day animals. At this time, hyperoxaluria and crystalluria were present as were REFERENCES elevated concentrations of renal tissue TBARS. However, 1. Finlavson. B.: Phvsicochemical asDects of urolithiasis. Kidnev crystal deposition and increased concentration of TBARS were Int:, 13' 334, 1<78. unrelated in that the one animal with normal TBARS had 2. Menon. M. and Mahle. C. J.: Oxalate metabolism and renal papillary tip crystal deposits only, and the two animals with calculi. J. Urol., 121:.148, 1982. no crystals demonstrated increased TBARS. This suggests 3. Hodgkinson, A.: Oxalic Acid in Biology and Medicine. London, Academic Press, p. 159, 1977. that oxidative damage is more related to the hyperoxaluria 4. Khan, S. R., Shevock, P. N. and Hackett, R. L.: Acute hyperoxand crystalluria rather than to tissue crystal deposits. The aluria. renal iniurv and calcium oxalate urolithiasis. J. Urol.. latter process ultimately contributes a s TBARS production is 147: 226, 1992." greatly increased with progressive crystal deposition. 5. Hackett. R. L.. Shevock, P. N. and Khan, S. R.: Cell iniurv While tissue crystals may not be central to early damage, associated calcium oxalate crystalluria. J. 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LIPID PEROXIDATION AND HYPEROXALURIA
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