Renal Failure, 13(4), 227-232 (11!991)
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Protective Effect of Zinc-Induced :Metallothionein Synthesis on Gentamicin Nephrotioxicity in Rats Chao-Ling Yang,* MD, Xue-Hai IDu,* ME), Wan-Zhong Zou,t MD, and Wen Chen,* MD Department of Nephrology China-Japan Friendship Hospital, and tDepettment of Pathology Seijing Medical University Beijing, China
ABSTRACT
Wistar rats were used to study the proteciive effect t7 f zinc-induced metallothionein (MQsynthesis on gentamicin nephrotoxicity. Wejnund that s. c. pre-injection of &SO, (Zn I0 mg/kg/day)for 5 days could czmeliorafeproximal tubuhzr necrosis and acute renal failure caused by an 8-da-y S.C. iriyection of gentainicin (100 mg/kg/day),while preinjection of saline instead of zinc ox .zinc and gentamicin together could not. In the zinc-pretreated rats (n = ti), renal mrtical metallothitoneinlevel 0.OOI) and the saline conwas significantlyhigher than that of normal (n = 8, p trols (n = 6, p < 0.OOI). Since MT is a scavenger of hydroxyl radicar', it is proposed that hydroxyl radical plays a role in the pathogentpsis of gentamicin nephrotoxicity and that preinjection of zinc could ameliorate geritamicin nephroi'oxicity via the induction of renal cortical MT synthesi3. :a
INTRODUCTION
could prevent or ameliorate gentamicin nephrotoxicity in rats, and to confirm the role of hydroxyl radical in genlamicin nephrotoxicity.
The mechanism of gentamicin nephrotoxicity is unclear. Mitochondrial injury (1, 2), lysosomal injury (3, 4), inhibition of Na+ K+-ATP-ase (5) and hydroxyl radical (6) were found to play a role in the pathogenesis of gentamicin nephrotoxicity. Metallothionein (MT) can scavenge hydroxyl radical (7). This study was designed to investigate whether MT, as a hydroxyl radical scavenger,
MATERIALS AND METHODS Wistar 111iilerats weighing 250-300 g were used to study the protective effect of zinc-induced MT synthesis on 227
Copyright 0 1991 by Marcel Dekker, Inc.
Yang et al.
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228
gentamicin (GM) nephrotoxicity. Fifty-four rats were divided into 5 groups: Group I (n = 8), BUN, renal cortical metallothionein (RCMT), renal cortical superoxide dismutase (RCSOD) activities, and blood malondialdehyde (MDA) were measured as normal control. Group II (n = 8) rats were injected S.C.with GM 100 mg/kg/day for 8 days and then were sacrificed. BUN, RCSOD, and MDA were measured on the same day of sacrifice. Group III (n = 14) rats were injected S.C. with ZnS04 (Zn 10 mg/kg/day) for 5 days. On the 6th day, 6 rats were sacrificed for RCMT measurement; the other 8 rats received S.C. injection of GM 100 mglkglday for another 8 days and then were sacrificed. BUN, RCSOD, and MDA were measured on the same day of sacrifice. Group IV (n = 14) rats were treated the same way as Group IU except n o d saline was used instead of ZnS04. Parameters were measured the same way as in Group HI;except that MDA was not measured. Group V (n = 8) rats were injected S.C.with ZnS04 and GM (doses as above) simultaneously for 8 days, and then were sacrificed. BUN was measured on the day of sacrifice (Table 1). Renal specimens were taken from normals (n = 8), from Group II rats after sacrifice (n = 8), from Group III rats after 5 days of ZnS04 injection followed by an 8-day GM injection (n = 8), and from Group IV rats after 8 days of normal saline injection followed by an %day GM injection (n = 8) and from Group V rats after 8 days of ZnS04 and GM injection simultaneously (n = 8). These specimenswere examined under a light and an electron microscope. At least 3 sections were evaluated from each kidney.
Determination of Renal Cortical Superoxide Dismutase Activities The principle described by Hodgson and Fridovich (8) that SOD could inhibit light produced by 0 2 with luminal reaction, was adopted by Li et al. (9) to assay renal cortical SOD activities. Prior to the measurement of renal cortical SOD activities, an inhibition curve was drawn. The procedure was as follows: 2 , 4 , 6 and 8 pl of SOD (10 mg/ml, Sigma Chemical Company, U.S.A.) were put into 4 different glass tubes (55 X 10 mm), 5 pL of 0.5 M NaCO6 buffer @H 10.2) was used as control. Into all tubes, 10 pL of xanthine oxidase was added (0.1 mg/mL, Sigma Chemical Company, U.S.A.), and then 980 pL of a freshly prepared mixture of 0.05 M NaC06 @H 10.2) with 0.1 mM xanthion and luminal (Aldrich, U.S.A.). One minute later the luminescence was measured in a luminometer (LKB
1250 Sweden). Since SOD inhibit light produced by 0 2 with luminal, the light strength of control was considered as 100%.Then the extent of inhibition of light could be calculated from the different amounts of SOD added. According to the amount of SOD and the extent of inhibition of light, an inhibition curve could be drawn. The concentration of SOD (nanograms/milliliter)causing a 50 % inhibition of light was defined as one enzyme activity unit. Renal cortex was homogenized ( 5 % , w/v) in 0.01 M Tris-HC1 buffer @H 7.8). Supernatant fraction was obtained by centrifuging homogenate at 18,000 rpm at 4°C for 1 h. Five and 10 FL of supernatant were put into 2 tubes; then the same method was used to measure renal cortical SOD activities as described above.
Determination of Renal Cortical Metallothionein A modified Cd/hemoglobin affinity assay described by Eaton et al. (10) was used for the detefinination of renal cortical MT. Briefly, renal cortex was prepared for MT analysis (1:4, v/v) in 0.01 M Tris-HC1 buffer @H 7.4). The homogenate was centrifuged at 18,OOO rprn at 4°C for 45 min. Then 0.2 mL of the supernatant fraction was diluted 10-fold with 0.01 M Tris-HC1 buffer @H 7.4), added with 0.2 mL of l15Cd (2.0 pg l15Cd/mL, 0.2 pCi/mL, China Institute of Atomic Energy, Beijing), mixed, and incubated for 10 min. Next, 0.2 mL of 2% rabbit hemoglobin solution was added, mixed, and incubated for 10 min, and heated in a 100°C boiling water bath for 2 min. After centrifuging at 4,000 rpm for 10 min, another 0.2 mL of 2% rabbit hemoglobin solution was added, mixed, heated, and centrifuged repeatedly. The amount of radioactivity in the supernatant fraction was then measured on a gamma-counter (LKB 1272, Sweden). The amount of MT (Cd binding potential) in each sample was calculated according to 6 g-atom of Cd binding per mole of MT.
Serum Malondialdehyde Assay A modified assay for measurement of serum malondialdehyde (MDA) described by Yagi (11) was used. Then 0.2 mL of serum was added to 4 mL of 0.5 N H2S04and 0.5 mL of 10% phosphotungstic acid, and mixed. After 5 min at room temperature, the mixture was centrifuged at 4,000 rpm for 10 min. The supernatantwas discarded, the sediment was suspended in 4.0 mL of distilled water, and 0.5 mL of 0.67% thiobarbituric acid (TBA, Sigma Chemical Company, U.S.A.) was added. The mixture was
7.1 f l . V f
4.2 f 0.2
MDA
“p < 0.001 value vs n o d control. h,p < 0 M I ..vzh vs GFXp !I ‘p < 0.001 value vs Group IV dp < 0.001 value vs Group V . ‘p < 0.01 value vs normmaJ control. 6 < 0.01 value vs Group III. NM = not measured.
167.1 f 31.3e
275.0 f 52.1
SCSOD
NM
3.9 f 0.1
RCMT
* 30.1a
17.4 f 3.2
130.4
GM (n = 8)
II:
BUN
I: normal (n = 8)
NM
NM
50.5 f 4.3a3C
NM
ZnS04 (n = 6)
m: ~~~
5.2 f 0.1
163.2 f 49.6e
NM
35.2 f 17.4b-e
~
ZnS04-GM (n = 8)
NM
NM
4.1 f 0.2
NM
Saline (n = 6)
Saline-GM (n = 8)
NM
165.2 f 4 5 . F
NM
104.5 f 22.3
IV:
Mean f SD of B W , Renal Cortical Metallothiomein (RCMT), Renal Cortical Superoxide Dismutase Activities (RCSOD), and Serum Malondialdehyde (MDA) in Direrent Groups
Table 1
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NM
NM
NM
115.2 f 35.4
+
V: Zn GM (n =8)
nmol/mL
pg/g tissue
pg/g tissue
mgldL
units
Yang et al.
230
heated at 100"C for 60 min in a boiling water bath. After cooling, 3.0 mL of butyl alcohol was added and the mixture was shaken vigorously for 1 min. After centrifugation at 4,000 rpm for 15 min the butyl alcohol layer was taken for spectophotometer(HP 8451 A, U.S.A.) measurement, which was made at 533 nm emission. MDA of the sample was calculated and compared with standard tube. In the standard tube, 0.2 mL standard solution (tetraethoxypropane 25 nmol) was used to substitute the sediment of serum, and other procedures are the same as described above. Statistical analysis was made by paired I test, and a P value less than 0.05 was taken as statistically significant.
in the normal controls (275.0 f 52.1 pg/g tissue, n = 8, p c 0.01) (Table 1).
Serum Malondialdehyde Serum MDA levels in Group I (n = 8), Group II (n = 8), and Group III (n =8) were 4.2 f 0.2, 7.1 f 1.0, and 5.2 f 0.1 nmol/mL respectively. This showed that MDA of rats treated with gentamicin alone was significantly higher than that in normal controls (Group I, p c 0.01), and that in rats pretreated with Zn followed by gentamicin injection for 8 days (Group III,p < 0.01) (Table 2).
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Histological Changes RESULTS Renal Cortical Metallothionein Concentration Renal cortical MT in the normal rats (Group I) was 3.9 f 0.1 pg/g tissue (n = 6). After daily S.C.injection of zinc (10 mg/kg/day) for 5 days, renal cortical MT in Group III was 50.5 f 4.3 pglg tissue (n = 6), significantly higher than that of the normal @ < 0.001) and that of the saline-treated rats (Group IV, n = 6, 4.1 f 0.2 pg/g tissue, p < 0.001).
Blood Urea Nitrogen Concentration BUN was 17.4 f 3.2 mg/dL in normal control, 130.4 f 30.1 mg/dL (n = 8) in rats treated with gentamicin alone (100 mg/kg/day) for 8 days in Group I1 (p < 0.001). In the rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection, BUN was 35.2 f 17.4 mg/dL (Group 111, n = 8), significantly lower than in the rats treated with gentamicin alone (Group 11,p < 0.001), and in the saline-pretreated rats (5 days of saline pretreatment followed by an 8-day gentamicin injection) in Group IV (104.5 22.3 mg/dL, n = 8, p < O.OOl), and in rats receiving zinc and gentamicin simultaneously in Group V (115.2 f 35.4 mg/dL, n = 8, p c 0.001). There was no significant difference of BUN among Groups 11, IV and V @ > 0.05), the BUN of Group I11 was higher than the normal controls @ < 0.01) (Table 1).
Renal Cortical Superoxide Dismutase Activities Renal cortical SOD activities were not significantly different among Group I1 (167.1 f 3 1.3 pg/g tissue, n = 8), Group I11 (163.2 f 49.6 pglg tissue, n = 8), and Group IV (165.2 f 45.8 pg/g tissue, n = 8, p > 0.05), but were significantly lower in all these three groups than
Light microscopy showed that daily injection of gentamicin alone for 8 days (Group 11) caused diffuse proximal tubular necrosis (n = S), (Fig. 1). Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed normal proximal tubules in 3 (Fig. 2), amyloidosis of proximal tubules in 1, focal proximal tubular necrosis in 3, and diffuse necrosis in 1. There was diffuse proximal tubular necrosis in Group IV (pretreated with saline for 5 days followed by an 8-day gentamicin injection, n = 8), and to a lesser degree in Group V (zinc and gentamicin injected simultaneously, n = 8) (Table 2). Proximal tubular cell injuries caused by gentamicin observed under electron microscopy were cytoplasmic vacuous, mitochondria1 swelling, membrane folds, and amorphous debris as described by Hougito et al. (12). Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed slighter changes compared to
Table 2 Histological Change of Kidney in Different Groups Grad&
Groupa
0
1
2
~~
I
(n = 8)
I1
(n
=
8)
I11 (n
=
8)
IV (n
=
8)
V
(n = 8)
3 ~
8 8 3
1
3
1 8
8
n' = the number of animals studied. 'Grade: 0 = normal; 1 = degeneration of proximal tubular epithelial cells; 2 = focal necrosis of proximal tubular epithelial cells; 3 = diffuse necrosis of proximal tubular epithelial cells.
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Zinc-Induced Metallothionein Synthesis
Figure 1. Diffuse proximal tubular necrosis after an 8-day injection of gentamicin. 100 X ,
rats with gentamicin treatment alone and rats pretreated with saline for 5 days followed by an 8-day gentamicin injection.
DISCUSSION Metallothionein, a kind of protein rich in cysteine with a molecular weight of 6,000-7,000, can be combined with metals such as Zn, Cd, and Cu (13). It distributes mainly in animals’ liver and kidney; its concentration is very low; and it can be increased by induction of metals, starvation, and toxins (13, 14). It has been shown that this protein
Figure 2. Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed normal proximal tubules. lOOX.
23 1
can protect cells against heavy metal toxicity (15,, 16), ]preventnephrotoxicity of antitumor drugs (17, 181, and ]providecells with radioresistance (19). These functions suggest thal MT could scavenge free radicals, particularly lhydroxyl radical, thus protecting tissues from injury (20, :2 1). HydroxyL radical is a strong mediator of tissue injury. [t can react with metal chelators via the Fenton reaction, iuld it can oxidize a wide variety of organic compounds iincluding ~mlyunsaturatedfatty acids (22, 23), leading to cell membrane injury and protein degeneration (23,24). Walker c:t al. (6, 25) demonstrated that gentamicin t:nhances the production of hydrogen peroxide by rat renal cortical mntochondria, and that hydroxyl scavengers and iiron chelators could prevent gentamicin-induced ARF. ‘Thus they believed that hydroxyl radical plays an irniportant irole in gentamicin-induced nephrotoxicity . Ransamm!, et al. (26) postulated that gentamicin in some imanner depresses catalase activity and that this results in the accumulation of hydrogen peroxide, which exceeds rhe metabolic capacity of cells’ residual peroxidase system including glutathione peroxidase and leads to increased generation of hydroxyl radical and peroxidation of lipids. So, it is rei*sonable to consider that if hydroxyl radical ji s eliminated or scavenged, gentamicin-induced iic~hrotoxiic:itywill be reduced. In this study, daily S.C. injection of gentamiciri 100 mg/kg/day for 8 days indued severe proximal tubular necrosis and ARF in adult Wistar rats, as described by Humes et id. (27). Rats preinjected S.C.with ZnS04 (Zn LO mg/kg/tlay) for 5 days followed by an 8-day S.C. regimen of gentamicin (100 rng/kg/day) resulted in normal OI slight changes of proximal tubular cells, and BUN was significantly lower than rats receiving gentamicin alone. Neither d i n e used instead of ZnS04 nor ZnSO,, and gentamiciri given simultaneously could ameliorate proximal tubular necrosis and/or ARF. Five days after injection of ZnS04, rat renal cortical MT level was significantly higher than that of the normal and saline control rats. These data suggest that preinjection of ZnS04 could ameliorate gentamicin-induced nephrotoxicity via the induction of renal cortical IMT synthesis, while ZnSO, and gentanwin injection used simultaneously could not, indicating that zinc itself does not possess the ability to clirectly ameliorate gentamicin-induced nephrotoxrcity . This further confirmed Walker et ale’s finding that hydroxyl radical plays a role in the pathogenesis of gentamicin-induced nephrotoxicity (25). MDA is the product of lipid peroxide (28), Ransammy et al. (26) teported that lipid peroxide increased in the
Yang et al.
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232
renal cortex of rats with gentamicin injection. We also observed an increase of serum MDA in rats treated with gentamicin alone, but M D A was significantly lower in rats pretreated with Zn. We consider that this change was the consequenceof increased MT eliminatinggentamicininduced hydroxyl radial which otherwise could attack cell membranes to accelerate lipid peroxidation. Ransammy et al. (29) showed that vitamin E, a lipid peroxide inhibitor, could neither prevent nor reduce the severity of gentamicin-induced proximal tubular cell lesions and necrosis. They concluded that lipid peroxidation was a consequence and not a cause of gentamicin nephrotoxicity. SOD is a superoxide scavenger, in this study, in rats either treated with gentamicin alone or pretreated with zinc or saline followed by gentamicin injection, the SOD activities were all significantly lower than normal controls, suggesting that amelioration of gentamicin nephrotoxicity by injection of Zn was not mediated via the scavenging of superoxide radical by increased SOD. Why SOD activity was low in gentamicin-induced nephrotoxicity remains unknown. In conclusion, gentamicin nephrotoxicity was probably caused by hydroxyl radical injury. It is proposed that preinjection of zinc could ameliorate gentamicin nephrotoxicity via the induction of renal cortical MT synthesis. Zinc itself could not prevent or reduce the gentamicin-induced nephrotoxicity.
Correspondence to: Dr. Xue-Hai Du, Departmnt of Nephrology, China-Japan Friendship Hospital, Beijing 1OOO29,China.
REFERENCES 1. Klune WM, Hook JB: Functional nephrotoxicityof gentamicin in the rat. ToxicolAppl Phurmacol45:163-175,1978. 2. Simmons CF, Ronald TB, Humes H D Inhibitory effects of gentamicin on renal mitochondrialoxidative phosphorylation.J Phurmacol Erg 7her 214:709-719,1980. 3. Powell JH, Reidenberg MM: In vitro response of rat and human kidney lysonases of aminoglycosides. Bochem Phurmacol 31~3447-3453,1982. 4. Powell JH,Reidenberg MM: Further studies of the response of kidney lysosomes to aminoglycosidesand other cations. Biochem Phurma~0132:3213-3220,1983. 5. Williams PD, Holohan PD, Ross CR: Gentamicin nephrotoxicity . 1. acute biochemicalcorrelatesin rats. T'cof Appl Phuimacol 61~234-242,1981. 6. Walker PD, Sham SV: Evidence suggesting a role for hydroxyl radical in gentamicin-induced acute renal failure in rats. J Clin Invest 81(2):334341,1988. 7. Thornalley PJ, Vasar M: Possible role for metallothioneinin protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Actu 827:36-44,1985.
8. Hodgson EK, Fridovich I: The d t i o n of superroxide radical during the aerobic action of -thine oxidase. Biochim Biophys act^ 430:182-188, 1976. 9. Li YX, Fang YZ, Lui ZF: A micro w a y for superoxide dismutase in blood and tissues.A m A d Milit Med Sci 31(3):359-362,1984. 10. Eaton DL, Toal B F Evaluation of the Cdmrmoglobinaffinity assay for the rapid determinationof metalbh'onein in biological tissues. Toxicul Appl Pharmacol66:134-142, 1982. 11. Yagi K: A simple fluommetric assay for lipoperoxide in blood plasma. Biochem Med 15:212-216, 1976. 12. Houghton DC,Plamp CE, Defehr JM, Bennett WM, Porter G, Gilbt D: Gentamicin and tobramycin nephctoxicity. Am J Puthol 931137-152,1978. 13. Kigi JHR, Nordbcrg M: Report from the first international meeting on metallothionein and other proteins, Ziirich, 1978.fipen'entio Sypl. 34:51-55,1979. 14. Squebb KS, Cousins RI: Control of cadmiunbinding protein synthesis in rat liver. Environ Physiol Biochem 4:24-30, 1974. 15. Cherian MG, Nordberg M: Cellular adaption in metal toxicology and metallothionein. Toxicology 28:1-15, 1983. 16. Jin T, Nordberg GF, Nordberg M: Resistance to acute nephrotoxicity induced by cadmium-mdothionein dependence on pretreatment with cadmium chloride. Phunmcol Toxicol61:89-93,1987. 17. Bakka AB, Endresen L, Johnsen ABS, Edminson PD, Rugstad HE: Resistance against cis-dichlorodiammineplatinumin cultured cells with a high content of metallothionein. Toxicol Appl PhurIMCOI 61~215-226,1981. 18. Naganuma A, Satoh M, Imura N: Prevention of lethal and renal toxicity of cis-didnedichloroplatinum(II) by induction of metallothionein synthesis without compromisingits antitumor activity in mice. Cancer Res 47:983-987,1987. 19. Bakka A, Johnsen AS, Endresen L,Rugstad HE: Radioresistance in cell with high content of metallothionein. Experientiu 38~381-383,1982. 20. Abel J, Ruiter DN: Inhibition of hydroxyl-radical-generatedDNA degradation by metalldionein. Toxicol Lert 47:191-196,1989. 21. Mimura T, Tsujikawa K, Yasuda N, Nakajima H, Haruyama M, Obmura T, Okabe M: Suppressionof gastric ulcer induced by stress and HC1-ethanol by intravenously administered metallothioneinII. Biochem Bwphys Res Commun 151:725-729, 1987. 22. Maestro RFD: An approach to fxee radicals in medicine and physiology. Aau Physiol Scud Suppl492:153-168, 1980. 23. Freeman BA, Crapo J: Biology of disease: free radicals and tissue injury. Lab Invest 47:412-426,1982. 24. Pryor WA: Oxy-radialsand related species: their formation, lifetimes, and reactions. Annu Rev Physiol48:657-667,1986. 25. Walker PD, Sham S V Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria. Am J Physiol 253zC495-499, 1987. 26. Ramsammy L, Ling KY, Josepovitz C, k i n e R, Kaloyanides GJ: Effect of gentamicinon lipid peroxidation in rat renal cortex. Biochem Phurmucol 343895-3900, 1985. 21 Hum= HD,Sastrasinh M, Weinberg Jhf: Calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions and dietary calcium supplementation protects against gentamicin nephrotoxicity. J Clin Invest 73:134-147,1984. 28. Blaker DR, Allen RE, Lunec J: Free radicals in biologicalsystems: a review orientated to inflammatory processes. Br Med Bull 43 :371-385, 1987. 29, Ramsammy LS,Josepovitzc, Ling KY, Lane BP, Kaloyanides GJ: Failure of inhibition of lipid peroxidation by vitamin E to protect against gentamkin nephrotoxicity in the rat. Biochem Pharmacol 36~2125-2132,1987. I
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Protective Effect of Zinc-Induced :Metallothionein Synthesis on Gentamicin Nephrotioxicity in Rats Chao-Ling Yang,* MD, Xue-Hai IDu,* ME), Wan-Zhong Zou,t MD, and Wen Chen,* MD Department of Nephrology China-Japan Friendship Hospital, and tDepettment of Pathology Seijing Medical University Beijing, China
ABSTRACT
Wistar rats were used to study the proteciive effect t7 f zinc-induced metallothionein (MQsynthesis on gentamicin nephrotoxicity. Wejnund that s. c. pre-injection of &SO, (Zn I0 mg/kg/day)for 5 days could czmeliorafeproximal tubuhzr necrosis and acute renal failure caused by an 8-da-y S.C. iriyection of gentainicin (100 mg/kg/day),while preinjection of saline instead of zinc ox .zinc and gentamicin together could not. In the zinc-pretreated rats (n = ti), renal mrtical metallothitoneinlevel 0.OOI) and the saline conwas significantlyhigher than that of normal (n = 8, p trols (n = 6, p < 0.OOI). Since MT is a scavenger of hydroxyl radicar', it is proposed that hydroxyl radical plays a role in the pathogentpsis of gentamicin nephrotoxicity and that preinjection of zinc could ameliorate geritamicin nephroi'oxicity via the induction of renal cortical MT synthesi3. :a
INTRODUCTION
could prevent or ameliorate gentamicin nephrotoxicity in rats, and to confirm the role of hydroxyl radical in genlamicin nephrotoxicity.
The mechanism of gentamicin nephrotoxicity is unclear. Mitochondrial injury (1, 2), lysosomal injury (3, 4), inhibition of Na+ K+-ATP-ase (5) and hydroxyl radical (6) were found to play a role in the pathogenesis of gentamicin nephrotoxicity. Metallothionein (MT) can scavenge hydroxyl radical (7). This study was designed to investigate whether MT, as a hydroxyl radical scavenger,
MATERIALS AND METHODS Wistar 111iilerats weighing 250-300 g were used to study the protective effect of zinc-induced MT synthesis on 227
Copyright 0 1991 by Marcel Dekker, Inc.
Yang et al.
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228
gentamicin (GM) nephrotoxicity. Fifty-four rats were divided into 5 groups: Group I (n = 8), BUN, renal cortical metallothionein (RCMT), renal cortical superoxide dismutase (RCSOD) activities, and blood malondialdehyde (MDA) were measured as normal control. Group II (n = 8) rats were injected S.C.with GM 100 mg/kg/day for 8 days and then were sacrificed. BUN, RCSOD, and MDA were measured on the same day of sacrifice. Group III (n = 14) rats were injected S.C. with ZnS04 (Zn 10 mg/kg/day) for 5 days. On the 6th day, 6 rats were sacrificed for RCMT measurement; the other 8 rats received S.C. injection of GM 100 mglkglday for another 8 days and then were sacrificed. BUN, RCSOD, and MDA were measured on the same day of sacrifice. Group IV (n = 14) rats were treated the same way as Group IU except n o d saline was used instead of ZnS04. Parameters were measured the same way as in Group HI;except that MDA was not measured. Group V (n = 8) rats were injected S.C.with ZnS04 and GM (doses as above) simultaneously for 8 days, and then were sacrificed. BUN was measured on the day of sacrifice (Table 1). Renal specimens were taken from normals (n = 8), from Group II rats after sacrifice (n = 8), from Group III rats after 5 days of ZnS04 injection followed by an 8-day GM injection (n = 8), and from Group IV rats after 8 days of normal saline injection followed by an %day GM injection (n = 8) and from Group V rats after 8 days of ZnS04 and GM injection simultaneously (n = 8). These specimenswere examined under a light and an electron microscope. At least 3 sections were evaluated from each kidney.
Determination of Renal Cortical Superoxide Dismutase Activities The principle described by Hodgson and Fridovich (8) that SOD could inhibit light produced by 0 2 with luminal reaction, was adopted by Li et al. (9) to assay renal cortical SOD activities. Prior to the measurement of renal cortical SOD activities, an inhibition curve was drawn. The procedure was as follows: 2 , 4 , 6 and 8 pl of SOD (10 mg/ml, Sigma Chemical Company, U.S.A.) were put into 4 different glass tubes (55 X 10 mm), 5 pL of 0.5 M NaCO6 buffer @H 10.2) was used as control. Into all tubes, 10 pL of xanthine oxidase was added (0.1 mg/mL, Sigma Chemical Company, U.S.A.), and then 980 pL of a freshly prepared mixture of 0.05 M NaC06 @H 10.2) with 0.1 mM xanthion and luminal (Aldrich, U.S.A.). One minute later the luminescence was measured in a luminometer (LKB
1250 Sweden). Since SOD inhibit light produced by 0 2 with luminal, the light strength of control was considered as 100%.Then the extent of inhibition of light could be calculated from the different amounts of SOD added. According to the amount of SOD and the extent of inhibition of light, an inhibition curve could be drawn. The concentration of SOD (nanograms/milliliter)causing a 50 % inhibition of light was defined as one enzyme activity unit. Renal cortex was homogenized ( 5 % , w/v) in 0.01 M Tris-HC1 buffer @H 7.8). Supernatant fraction was obtained by centrifuging homogenate at 18,000 rpm at 4°C for 1 h. Five and 10 FL of supernatant were put into 2 tubes; then the same method was used to measure renal cortical SOD activities as described above.
Determination of Renal Cortical Metallothionein A modified Cd/hemoglobin affinity assay described by Eaton et al. (10) was used for the detefinination of renal cortical MT. Briefly, renal cortex was prepared for MT analysis (1:4, v/v) in 0.01 M Tris-HC1 buffer @H 7.4). The homogenate was centrifuged at 18,OOO rprn at 4°C for 45 min. Then 0.2 mL of the supernatant fraction was diluted 10-fold with 0.01 M Tris-HC1 buffer @H 7.4), added with 0.2 mL of l15Cd (2.0 pg l15Cd/mL, 0.2 pCi/mL, China Institute of Atomic Energy, Beijing), mixed, and incubated for 10 min. Next, 0.2 mL of 2% rabbit hemoglobin solution was added, mixed, and incubated for 10 min, and heated in a 100°C boiling water bath for 2 min. After centrifuging at 4,000 rpm for 10 min, another 0.2 mL of 2% rabbit hemoglobin solution was added, mixed, heated, and centrifuged repeatedly. The amount of radioactivity in the supernatant fraction was then measured on a gamma-counter (LKB 1272, Sweden). The amount of MT (Cd binding potential) in each sample was calculated according to 6 g-atom of Cd binding per mole of MT.
Serum Malondialdehyde Assay A modified assay for measurement of serum malondialdehyde (MDA) described by Yagi (11) was used. Then 0.2 mL of serum was added to 4 mL of 0.5 N H2S04and 0.5 mL of 10% phosphotungstic acid, and mixed. After 5 min at room temperature, the mixture was centrifuged at 4,000 rpm for 10 min. The supernatantwas discarded, the sediment was suspended in 4.0 mL of distilled water, and 0.5 mL of 0.67% thiobarbituric acid (TBA, Sigma Chemical Company, U.S.A.) was added. The mixture was
7.1 f l . V f
4.2 f 0.2
MDA
“p < 0.001 value vs n o d control. h,p < 0 M I ..vzh vs GFXp !I ‘p < 0.001 value vs Group IV dp < 0.001 value vs Group V . ‘p < 0.01 value vs normmaJ control. 6 < 0.01 value vs Group III. NM = not measured.
167.1 f 31.3e
275.0 f 52.1
SCSOD
NM
3.9 f 0.1
RCMT
* 30.1a
17.4 f 3.2
130.4
GM (n = 8)
II:
BUN
I: normal (n = 8)
NM
NM
50.5 f 4.3a3C
NM
ZnS04 (n = 6)
m: ~~~
5.2 f 0.1
163.2 f 49.6e
NM
35.2 f 17.4b-e
~
ZnS04-GM (n = 8)
NM
NM
4.1 f 0.2
NM
Saline (n = 6)
Saline-GM (n = 8)
NM
165.2 f 4 5 . F
NM
104.5 f 22.3
IV:
Mean f SD of B W , Renal Cortical Metallothiomein (RCMT), Renal Cortical Superoxide Dismutase Activities (RCSOD), and Serum Malondialdehyde (MDA) in Direrent Groups
Table 1
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NM
NM
NM
115.2 f 35.4
+
V: Zn GM (n =8)
nmol/mL
pg/g tissue
pg/g tissue
mgldL
units
Yang et al.
230
heated at 100"C for 60 min in a boiling water bath. After cooling, 3.0 mL of butyl alcohol was added and the mixture was shaken vigorously for 1 min. After centrifugation at 4,000 rpm for 15 min the butyl alcohol layer was taken for spectophotometer(HP 8451 A, U.S.A.) measurement, which was made at 533 nm emission. MDA of the sample was calculated and compared with standard tube. In the standard tube, 0.2 mL standard solution (tetraethoxypropane 25 nmol) was used to substitute the sediment of serum, and other procedures are the same as described above. Statistical analysis was made by paired I test, and a P value less than 0.05 was taken as statistically significant.
in the normal controls (275.0 f 52.1 pg/g tissue, n = 8, p c 0.01) (Table 1).
Serum Malondialdehyde Serum MDA levels in Group I (n = 8), Group II (n = 8), and Group III (n =8) were 4.2 f 0.2, 7.1 f 1.0, and 5.2 f 0.1 nmol/mL respectively. This showed that MDA of rats treated with gentamicin alone was significantly higher than that in normal controls (Group I, p c 0.01), and that in rats pretreated with Zn followed by gentamicin injection for 8 days (Group III,p < 0.01) (Table 2).
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Histological Changes RESULTS Renal Cortical Metallothionein Concentration Renal cortical MT in the normal rats (Group I) was 3.9 f 0.1 pg/g tissue (n = 6). After daily S.C.injection of zinc (10 mg/kg/day) for 5 days, renal cortical MT in Group III was 50.5 f 4.3 pglg tissue (n = 6), significantly higher than that of the normal @ < 0.001) and that of the saline-treated rats (Group IV, n = 6, 4.1 f 0.2 pg/g tissue, p < 0.001).
Blood Urea Nitrogen Concentration BUN was 17.4 f 3.2 mg/dL in normal control, 130.4 f 30.1 mg/dL (n = 8) in rats treated with gentamicin alone (100 mg/kg/day) for 8 days in Group I1 (p < 0.001). In the rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection, BUN was 35.2 f 17.4 mg/dL (Group 111, n = 8), significantly lower than in the rats treated with gentamicin alone (Group 11,p < 0.001), and in the saline-pretreated rats (5 days of saline pretreatment followed by an 8-day gentamicin injection) in Group IV (104.5 22.3 mg/dL, n = 8, p < O.OOl), and in rats receiving zinc and gentamicin simultaneously in Group V (115.2 f 35.4 mg/dL, n = 8, p c 0.001). There was no significant difference of BUN among Groups 11, IV and V @ > 0.05), the BUN of Group I11 was higher than the normal controls @ < 0.01) (Table 1).
Renal Cortical Superoxide Dismutase Activities Renal cortical SOD activities were not significantly different among Group I1 (167.1 f 3 1.3 pg/g tissue, n = 8), Group I11 (163.2 f 49.6 pglg tissue, n = 8), and Group IV (165.2 f 45.8 pg/g tissue, n = 8, p > 0.05), but were significantly lower in all these three groups than
Light microscopy showed that daily injection of gentamicin alone for 8 days (Group 11) caused diffuse proximal tubular necrosis (n = S), (Fig. 1). Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed normal proximal tubules in 3 (Fig. 2), amyloidosis of proximal tubules in 1, focal proximal tubular necrosis in 3, and diffuse necrosis in 1. There was diffuse proximal tubular necrosis in Group IV (pretreated with saline for 5 days followed by an 8-day gentamicin injection, n = 8), and to a lesser degree in Group V (zinc and gentamicin injected simultaneously, n = 8) (Table 2). Proximal tubular cell injuries caused by gentamicin observed under electron microscopy were cytoplasmic vacuous, mitochondria1 swelling, membrane folds, and amorphous debris as described by Hougito et al. (12). Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed slighter changes compared to
Table 2 Histological Change of Kidney in Different Groups Grad&
Groupa
0
1
2
~~
I
(n = 8)
I1
(n
=
8)
I11 (n
=
8)
IV (n
=
8)
V
(n = 8)
3 ~
8 8 3
1
3
1 8
8
n' = the number of animals studied. 'Grade: 0 = normal; 1 = degeneration of proximal tubular epithelial cells; 2 = focal necrosis of proximal tubular epithelial cells; 3 = diffuse necrosis of proximal tubular epithelial cells.
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Zinc-Induced Metallothionein Synthesis
Figure 1. Diffuse proximal tubular necrosis after an 8-day injection of gentamicin. 100 X ,
rats with gentamicin treatment alone and rats pretreated with saline for 5 days followed by an 8-day gentamicin injection.
DISCUSSION Metallothionein, a kind of protein rich in cysteine with a molecular weight of 6,000-7,000, can be combined with metals such as Zn, Cd, and Cu (13). It distributes mainly in animals’ liver and kidney; its concentration is very low; and it can be increased by induction of metals, starvation, and toxins (13, 14). It has been shown that this protein
Figure 2. Rats pretreated with zinc for 5 days followed by an 8-day gentamicin injection showed normal proximal tubules. lOOX.
23 1
can protect cells against heavy metal toxicity (15,, 16), ]preventnephrotoxicity of antitumor drugs (17, 181, and ]providecells with radioresistance (19). These functions suggest thal MT could scavenge free radicals, particularly lhydroxyl radical, thus protecting tissues from injury (20, :2 1). HydroxyL radical is a strong mediator of tissue injury. [t can react with metal chelators via the Fenton reaction, iuld it can oxidize a wide variety of organic compounds iincluding ~mlyunsaturatedfatty acids (22, 23), leading to cell membrane injury and protein degeneration (23,24). Walker c:t al. (6, 25) demonstrated that gentamicin t:nhances the production of hydrogen peroxide by rat renal cortical mntochondria, and that hydroxyl scavengers and iiron chelators could prevent gentamicin-induced ARF. ‘Thus they believed that hydroxyl radical plays an irniportant irole in gentamicin-induced nephrotoxicity . Ransamm!, et al. (26) postulated that gentamicin in some imanner depresses catalase activity and that this results in the accumulation of hydrogen peroxide, which exceeds rhe metabolic capacity of cells’ residual peroxidase system including glutathione peroxidase and leads to increased generation of hydroxyl radical and peroxidation of lipids. So, it is rei*sonable to consider that if hydroxyl radical ji s eliminated or scavenged, gentamicin-induced iic~hrotoxiic:itywill be reduced. In this study, daily S.C. injection of gentamiciri 100 mg/kg/day for 8 days indued severe proximal tubular necrosis and ARF in adult Wistar rats, as described by Humes et id. (27). Rats preinjected S.C.with ZnS04 (Zn LO mg/kg/tlay) for 5 days followed by an 8-day S.C. regimen of gentamicin (100 rng/kg/day) resulted in normal OI slight changes of proximal tubular cells, and BUN was significantly lower than rats receiving gentamicin alone. Neither d i n e used instead of ZnS04 nor ZnSO,, and gentamiciri given simultaneously could ameliorate proximal tubular necrosis and/or ARF. Five days after injection of ZnS04, rat renal cortical MT level was significantly higher than that of the normal and saline control rats. These data suggest that preinjection of ZnS04 could ameliorate gentamicin-induced nephrotoxicity via the induction of renal cortical IMT synthesis, while ZnSO, and gentanwin injection used simultaneously could not, indicating that zinc itself does not possess the ability to clirectly ameliorate gentamicin-induced nephrotoxrcity . This further confirmed Walker et ale’s finding that hydroxyl radical plays a role in the pathogenesis of gentamicin-induced nephrotoxicity (25). MDA is the product of lipid peroxide (28), Ransammy et al. (26) teported that lipid peroxide increased in the
Yang et al.
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232
renal cortex of rats with gentamicin injection. We also observed an increase of serum MDA in rats treated with gentamicin alone, but M D A was significantly lower in rats pretreated with Zn. We consider that this change was the consequenceof increased MT eliminatinggentamicininduced hydroxyl radial which otherwise could attack cell membranes to accelerate lipid peroxidation. Ransammy et al. (29) showed that vitamin E, a lipid peroxide inhibitor, could neither prevent nor reduce the severity of gentamicin-induced proximal tubular cell lesions and necrosis. They concluded that lipid peroxidation was a consequence and not a cause of gentamicin nephrotoxicity. SOD is a superoxide scavenger, in this study, in rats either treated with gentamicin alone or pretreated with zinc or saline followed by gentamicin injection, the SOD activities were all significantly lower than normal controls, suggesting that amelioration of gentamicin nephrotoxicity by injection of Zn was not mediated via the scavenging of superoxide radical by increased SOD. Why SOD activity was low in gentamicin-induced nephrotoxicity remains unknown. In conclusion, gentamicin nephrotoxicity was probably caused by hydroxyl radical injury. It is proposed that preinjection of zinc could ameliorate gentamicin nephrotoxicity via the induction of renal cortical MT synthesis. Zinc itself could not prevent or reduce the gentamicin-induced nephrotoxicity.
Correspondence to: Dr. Xue-Hai Du, Departmnt of Nephrology, China-Japan Friendship Hospital, Beijing 1OOO29,China.
REFERENCES 1. Klune WM, Hook JB: Functional nephrotoxicityof gentamicin in the rat. ToxicolAppl Phurmacol45:163-175,1978. 2. Simmons CF, Ronald TB, Humes H D Inhibitory effects of gentamicin on renal mitochondrialoxidative phosphorylation.J Phurmacol Erg 7her 214:709-719,1980. 3. Powell JH, Reidenberg MM: In vitro response of rat and human kidney lysonases of aminoglycosides. Bochem Phurmacol 31~3447-3453,1982. 4. Powell JH,Reidenberg MM: Further studies of the response of kidney lysosomes to aminoglycosidesand other cations. Biochem Phurma~0132:3213-3220,1983. 5. Williams PD, Holohan PD, Ross CR: Gentamicin nephrotoxicity . 1. acute biochemicalcorrelatesin rats. T'cof Appl Phuimacol 61~234-242,1981. 6. Walker PD, Sham SV: Evidence suggesting a role for hydroxyl radical in gentamicin-induced acute renal failure in rats. J Clin Invest 81(2):334341,1988. 7. Thornalley PJ, Vasar M: Possible role for metallothioneinin protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Actu 827:36-44,1985.
8. Hodgson EK, Fridovich I: The d t i o n of superroxide radical during the aerobic action of -thine oxidase. Biochim Biophys act^ 430:182-188, 1976. 9. Li YX, Fang YZ, Lui ZF: A micro w a y for superoxide dismutase in blood and tissues.A m A d Milit Med Sci 31(3):359-362,1984. 10. Eaton DL, Toal B F Evaluation of the Cdmrmoglobinaffinity assay for the rapid determinationof metalbh'onein in biological tissues. Toxicul Appl Pharmacol66:134-142, 1982. 11. Yagi K: A simple fluommetric assay for lipoperoxide in blood plasma. Biochem Med 15:212-216, 1976. 12. Houghton DC,Plamp CE, Defehr JM, Bennett WM, Porter G, Gilbt D: Gentamicin and tobramycin nephctoxicity. Am J Puthol 931137-152,1978. 13. Kigi JHR, Nordbcrg M: Report from the first international meeting on metallothionein and other proteins, Ziirich, 1978.fipen'entio Sypl. 34:51-55,1979. 14. Squebb KS, Cousins RI: Control of cadmiunbinding protein synthesis in rat liver. Environ Physiol Biochem 4:24-30, 1974. 15. Cherian MG, Nordberg M: Cellular adaption in metal toxicology and metallothionein. Toxicology 28:1-15, 1983. 16. Jin T, Nordberg GF, Nordberg M: Resistance to acute nephrotoxicity induced by cadmium-mdothionein dependence on pretreatment with cadmium chloride. Phunmcol Toxicol61:89-93,1987. 17. Bakka AB, Endresen L, Johnsen ABS, Edminson PD, Rugstad HE: Resistance against cis-dichlorodiammineplatinumin cultured cells with a high content of metallothionein. Toxicol Appl PhurIMCOI 61~215-226,1981. 18. Naganuma A, Satoh M, Imura N: Prevention of lethal and renal toxicity of cis-didnedichloroplatinum(II) by induction of metallothionein synthesis without compromisingits antitumor activity in mice. Cancer Res 47:983-987,1987. 19. Bakka A, Johnsen AS, Endresen L,Rugstad HE: Radioresistance in cell with high content of metallothionein. Experientiu 38~381-383,1982. 20. Abel J, Ruiter DN: Inhibition of hydroxyl-radical-generatedDNA degradation by metalldionein. Toxicol Lert 47:191-196,1989. 21. Mimura T, Tsujikawa K, Yasuda N, Nakajima H, Haruyama M, Obmura T, Okabe M: Suppressionof gastric ulcer induced by stress and HC1-ethanol by intravenously administered metallothioneinII. Biochem Bwphys Res Commun 151:725-729, 1987. 22. Maestro RFD: An approach to fxee radicals in medicine and physiology. Aau Physiol Scud Suppl492:153-168, 1980. 23. Freeman BA, Crapo J: Biology of disease: free radicals and tissue injury. Lab Invest 47:412-426,1982. 24. Pryor WA: Oxy-radialsand related species: their formation, lifetimes, and reactions. Annu Rev Physiol48:657-667,1986. 25. Walker PD, Sham S V Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria. Am J Physiol 253zC495-499, 1987. 26. Ramsammy L, Ling KY, Josepovitz C, k i n e R, Kaloyanides GJ: Effect of gentamicinon lipid peroxidation in rat renal cortex. Biochem Phurmucol 343895-3900, 1985. 21 Hum= HD,Sastrasinh M, Weinberg Jhf: Calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions and dietary calcium supplementation protects against gentamicin nephrotoxicity. J Clin Invest 73:134-147,1984. 28. Blaker DR, Allen RE, Lunec J: Free radicals in biologicalsystems: a review orientated to inflammatory processes. Br Med Bull 43 :371-385, 1987. 29, Ramsammy LS,Josepovitzc, Ling KY, Lane BP, Kaloyanides GJ: Failure of inhibition of lipid peroxidation by vitamin E to protect against gentamkin nephrotoxicity in the rat. Biochem Pharmacol 36~2125-2132,1987. I
