Influence of vitamin E treatment on glutathione system after renal ischemia in immature and adult rats

Exp Toxic Patho11997; 49: 81-86 Gustav Fischer Verlag

Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Germany

Influence of vitamin E treatment on glutathione system after renal ischemia in immature and adult rats CH. FLECK, D. HAUBOLD, T. HILLMANN, and H. BRAuNLICH With 3 figures and 1 table Received: November 1,1995; Accepted: November 8,1995

Address for correspondence: Prof. Dr. med. habil. CH. FLECK, Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Lobder StraBe 1, D-07743 Jena, Germany. Key words: Renal ischemia; Reperfusion; Vitamin E; Glutathione; Lipid peroxidation; Kidney, age; Maturation, rat; Ischemia, renal; Peroxidation, lipid.

Summary Survival rates were not significantly different 5 days after 20-min unilateral ischemia followed by contralateral nephrectomy: 58 % in 20-day-old vs. 77 % in 55-day-old rats. This experimental approach was used to characterize age dependent differences in the susceptibility of the glutathione system to ischemia and protective effects of treatment with vitamin E (10 mg/IOO g b.wt. once daily s.c.) on the outcome after renal ischemia. The degree of postischemic changes (GSH, 'Y-GT, TBARS) was the highest on days 1 and 2 after ischemia; at this time, survival rates were similar in young and adult rats. In adult animals, both glutathione content and the activity of 'Y-GT were significantly reduced after ischemia whereas in immature rats only the glutathione content was distinctly diminished. At the 5th day after ischemia the parameters were almost normalized in the two age groups. Repeated administration of vitamin E improved the survival rate in adult rats up to 100 %; in young animals, lethality was not influenced by vitamin E treatment. This reflects the beneficial effects of vitamin E on the glutathione system in adults whereas the vitamin was without effect on the immature rats' glutathione system.

Abbreviations GSH - reduced glutathione; GSSG - oxidized glutathione;

'Y-GT - gamma-glutamy1transpeptidase; TBARS - thiobar-

bituric acid reagib1e substances; LPO - lipid peroxidation; uNX - unilateral nephrectomy

Introduction Consequences of ischemic renal injury have been investigated frequently in the last two decades (for review

see WEINBERG 1991). However, little is known about the degree of susceptibility of the glutathione system to ischemia during postnatal maturation of the kidney function. Furthermore, it is of interest to find out suitable methods to prevent postischemic kidney damage. A couple of methods is described in the literature (RANGAN and BULKLEY 1993). Deferoxamine binds Fe and reduces the amount of free radicals (AUGUSTIN et al. 1992). Allopurinol inhibits xanthine oxidase and, therefore, it diminishes the formation of radicals (JOANNIDIS et al. 1990). Calcium antagonists are described to act protectively against renal ischemic damage (BONVENTRE 1993, POMER et al. 1994). Dimethyl sulfoxide (DMSO) improves survival rates after kidney ischemia in rats (LIN AS et al. 1987, Paddock and PADDOCK 1987, FLECK and HELLER 1993). The radical scavenging effect of N-acetylcysteine has been known for many years (SCHOENBERG 1991). Methimazole, an inhibitor of thyroid hormone synthesis, decreases metabolic activity in the organism and acts as a sulfur-containing nucleophilic substance and, therefore, it protects against lipid peroxidation (SAUSEN et al. 1992, V AlL et al. 1993). Mannitol reduces the extent of ischemic injury in the kidney, especially in adult rats (ZAGER et al. 1985) and acts as a radical scavenger (RANGAN and BULKLEY 1993). A promising approach to prevent postischemic renal failure seems to be the administration of vitamin E (PINCEMAIL 1989, CHOW 1991, HAGIWARA et al. 1991). This vitamin acts as a scavenger, protective against free oxygen species occurring during the reperfusion phase after renal ischemia (SIES et al. 1992). In this study, the glutathione system was characterized after warm renal ischemia in immature and adult rats. Furthermore, the effect of treatment with vitamin E on the outcome after kidney ischemization was investigated in rats of both age groups. Exp Toxic Pathol49 (1997) 1-2

81

Material and methods Experimental design The experiments were carried out on female Wi star rats (Uje: WIST) of our institute's own out-breed stock. Young animals were nursed by their dams. Adult rats were fed a standard diet (Rezeptur VTD I, Versuchstierproduktion Schonwalde, Germany) and tap water ad libitum. At the ages of 20 ± 2 or 55 ± 5 days the rats were anaesthetized by intraperitoneal administration of hexobarbitone (Hexobarbital-Natriumo, Arzneimittelwerk Dresden, 10 mg/lOO g b.wt.). For ischemization of the kidney the following type of surgical procedure was used: Unilateral ligation of the renal artery (20-min-ischemia) and contralateral nephrectomy after 24 hours: From median incision, the left kidney was laid bare and the renal artery was ligated for 20 minutes; for sham operation, the kidney was freed only without ligating the renal artery. After ischemia, the abdominal wound was carefully sewn up. In general, the rats woke up 30 to 60 min after the end of the surgical procedure. One day later the right kidney was removed via a dorsal incision under ether anaesthesia (uNX) to exclude compensatory effects of the contralateral kidney. In sham operated rats (see above), uNX was performed, too. Animals woke up 2-5 min after ether withdrawal. Day one of the experiments was the first day after uNX.

Treatment with vitamin E 14 days before ischemization or sham operation, treatment with vitamin E was started. Vitamin E (Jenapharm GmbH, Jena) was given in a dose of 10 mg/IOO g b.wt., dissolved in 0.1 ml oil, once daily subcutaneously. As reported by ANDERSEN and ANDERSEN (1993), this dose regimen is optimally effective and well tolerated. Administration of vitamin E was continued after ischemization or sham operation up to the day of experimentation. Control rats received an aliquot of the solvent (01. Arachid.).

Determination methods Protein concentrations in urine were estimated by a modified Biuret method (KLINGER and MULLER 1974). Glutathione: Reduced (GSH) and oxidized glutathione (GSSG) were determined according to ELLMAN and LYSKO (1979) and HISSIN and HILF (1976), respectively. Adaptation of both methods to kidney tissue was performed as described previously (ApPENROTH and SEIDEL 1989). The activity of renal y-glutamyl-transpeptidase (y-GT) was estimated with L-gamma-glutamyl-p-nitroanilide as substrate and glycylglycine as acceptor (KRETZSCHMAR and KLINGER 1989). Lipid peroxidation (LPO) was characterized by measurement of thiobarbituric acid reagible substances (TBARS) in the kidney tissue according to Y AGI (1987).

Statistical analysis Arithmetic means ± S.E.M. are given; n = 3-6 for each determination. Differences between means were statistically 82

Exp Toxic Pathol49 (1997) 1-2

analyzed using STUDENT'S t-test. Although the number of animals per group was relatively low, it was possible to use the t-test, because a bulk of previous experimental data indicates that the data of our laboratory animals are normally distributed. Lethality rates were compared with the X2-test.

Results For better comparison of the parameters measured, relative values are given in the figures. The absolute values of the corresponding control groups are shown in table 1 for the first day after ischemization or sham operation. The GSH content is similar in 20- and 55-day-old sham operated rats. On the first day after ischemia, however, this parameter is significantly reduced in young rats whereas it is decreased in adults at the second day (see fig. 1). Interestingly, the activity ofy-GT is significantly lower in 20-day-old animals, but it is significantly reduced after ischemia in adults only. Ischemization for 20 minutes resulted in small changes in the course of the glutathione status in 55-day-old rats (fig. 1): GSH and GSSG (not shown) contents in kidney tissue were diminished only on day 2 after ischemia, the activity of y-GT was decreased only at days 2 and 3 after ligation of the renal artery and moderate signs of enhanced lipid peroxidation occured in kidney tissue. Quite comparable results have been obtained in 20-day-old rats (fig. 1): Only the GSH and GSSG (not shown) contents were significantly reduced in the kidney whereas the activity ofy-GT was slightly diminished. The content of TBARS in renal tissue from 20-day-old rats was distinctly, but not significantly, enhanced compared with adult animals. Under these experimental con-

Table 1. Influence of 20-minute-ischemia on glutathione status (GSH, GSSG, y-GT) and lipid peroxidation in 20and 55-day-old rats on the first day after ischemization. Comparison with sham operated animals (SOP). Arithmetic means ± S.E.M.; n = 6. * - significant difference between ischemia and sham operation (p ~ 0.05) + - significant difference between both age groups (p ~ 0.05) Parameter

age (days) 20

55

GSH (Ilg/g kidney)

SOP ischemia

1252 ± 122 683 ± 243*

1389 ± 83 1205 ± 289

GSSG (Ilg/g kidney)

SOP ischemia

319 ± 24 141 ± 54

393 ± 14 347 ± 55

y-GT (nmol/mg protein x min)

SOP ischemia

56 ± 30 47 ± 16

279 ± 10+ 186 ± 14*

SOP TBARS (nmol/g wet weight) ischemia

145 ± 23 200 ± 20

129 ± 23 159 ± 20

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100

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80

Fig.2. Survival rates (%) after 20-minute-ischemia and influence of vitamin E treatment (10 mg/lOO g b.wt.; cf. methods) on postischemic lethality in 20- and 55-day-old rats (n = 12-14).

~120

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ditions (20-min unilateral renal ischemia followed by contralateral uNX 24 hours later), lethality was not signifij cantly different in young and adult rats 2 days after ische~ 40 mization (18 vs. 22 %). Figure 2 shows the survival rates 20 . . . . . . . . . . . . . . . . . . . . • . . . . of 20- and 55-day-old rats after unilateral renal ischemia. At day 5 the survival rate in 55-day-old rats (77 %) is, disoL--------------------------------------J tinctly but not significantly, higher than in young animals 11 2 8 o 5 (58 %). As also shown in this figure, repeated treatment TBARS with vitamin E prevented lethality in adult rats undergoing renal ischemia. The same treatment is not effective in 20160,--------------------------------------, day-old rats with immature kidney function (fig. 2). After treatment with vitamin E, the glutathione system 140 of adult rats seems to be well protected against ischemic injury (fig. 3b). The content of GSH in the kidney remained normal, enhanced lipid peroxidation did not occur and ~100r-~------------~~=======t~~--~t-~ the activity of y-GT was depressed only at the 1st day c: ~ 80 . . . . . . • . . . . . . after ischemia. On the contrary, in 20-day-old rats treatment with vitamin E had nearly no influence on the glutathione system compared to rats which received the solvent only (fig. 3a). ()

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Fig. 1. Time course of glutathione status (GSH, y-GT) and lipid peroxidation (TBARS) in kidney tissue of 20- and 55day-old rats after 20-minute-ischemia. 100 % - mean value of the corresponding sham operations; arithmetic means ± S.E.M., n = 4-6 * - significant differences between ischemia and sham operation (p ~ 0.05)

Discussion After 20-minute-ischemia of the left kidney and subsequent removal of the contralateral kidney, in both immature and adult rats distinct signs of anoxic impairment and reoxygenation injury occured. In accordance with the literature, the maximal production of free oxygen radicals can be measured in the initial phase of acute ischemic renal failure (BA YATI et al. 1990; RABL et al. 1993). Interestingly, in the two age groups investigated in this study, the severity of ischemic damage is quite comparable, and Exp Toxic Pathol49 (1997) 1-2

83

GSH

GSH 160

160

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• vitamin E

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time after ischemia (days)

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a

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Fig.3. Influence of treatment with vitamin E (10 mg/lOO g b.wt.; cf. methods) on glutathione status (GSH, y-GT) and lipid peroxidation (TBARS) after 20-minute-ischemia in 20-day-old rats (figure 3a) and 55-day-old rats (figure 3b); comparison with rats receiving the solvent (01. Arachid.) only. 100 % - mean value of the corresponding sham operations; arithmetic means ± S.E.M., n = 3-4

* - significant differences between ischemia and sham operation (p ~ 0.05)

+ - significant differences between rats treated with vitamin E or the solvent (p ~ 0.05) 84

Exp Toxic Pathol49 (1997) 1-2

only negligible age differences exist. For example, in 20day-old rats the maximal decrease of GSH content can be seen on day 1 after ischemization whereas in adult animals it occurred at the 2nd day. ZAGER (1988) assumed that reduced GSH concentration in the tissue is the consequence of enhanced GSH loss out of the cells and of increased glutathione consumption in the kidney cell. A loss of intracellular GSH is followed by an enhanced content of lipid peroxides (BOUDJEMA et al. 1991). Increase in lipid peroxidation is more pronounced in young rats. The lower mitochondrial acti vity of rat kidney during ontogeny (DELA VAL et al. 1990; PRIEUR et al. 1995). The lower content of energy rich substrates in the kidney cells of immature rats (LINAS et al. 1987, SCADUTO et al. 1988, SLUSSER et al. 1990) and, on the other hand, the higher compensation capacity of the growing kidney in 20-dayold rats (BONV ALET 1978) could be the reason for the age differences measured. Furthermore, GAUDIO et al. (1994) described that lower energy requirement for transport processes in young rats protects against ischemic renal damage. The absence of distinct age differences is reflected by corresponding survival rates after temporary ligation ofthe left renal artery. Summing up these results, the experimental approach used is well qualified for the investigation of age differences in protective treatment against ischemiaireperfusion injury. A reduction of free oxygen radical production during and/or after ischemization or a removal of these oxygen species could reduce or even prevent postischemic damage (RANGAN and BULKLEY 1993) as shown after administration of different antioxidants (SHANLEY et al. 1990, GREENE and PALLER 1991 ).It is necessary that high levels of the antioxidant are available within the tissue during and immediately after ischemization (SCHILLER et al. 1991). A commonly accepted view is that the presence of vitamin E in tissues is, due to its function to serve as an antioxidant, protecting unsaturated lipids from degradation by free radical chain reactions (DARGEL 1992). For this reason, in this study a long lasting administration of high doses of vitamin E was used to prevent postischemic renal damage. Following this dose regimen the vitamin E concentrations in the kidney were enhanced by more than 35 % (BRAuNLICH et al. 1997). The vitamin E content of immature rat's kidney tissue is lower than that of adult animals (SCHMIDT et al. 1990). Treatment with vitamin E was followed by enhanced survival rates after renal ischemia, especially in adult rats. This finding was correlated with beneficial effects concerning glutathione status and lipid peroxidation in 20- and, more pronounced, in 55-day-old nits. Vitamin E is reported to prevent the elevation in renal lipid peroxidation found in GSH-depleted rats (TORRES et al. 1991). However, the relatively low effects of vitamin E are discussed in the literature to be caused by competition phenomena between the glutathione system and vitamin E in the detoxification of free oxygen radicals (LEMOYNE et al. 1987). Especially a protective effect of vitamin E treatment on the activity of'Y-GT seems to be possible (PASCOE and REED 1989). Vitamin E treat-

ment reduces the consumption of GSH in the kidney and, therefore, the 'Y-GT activity could be lower (UHLIG and WENDEL 1992). A disadvantage of vitamin E treatment could be its glutathione mediated activation to an oxidant. On the other hand, this fact can be neglected in the presence of sufficient amounts of ascorbic acid (BOWRY et al. 1992; BARCLAY 1993; BOWRY and STOCKER 1993). A further reason for the low effectiveness of vitamin E to act protectively against postischemic injury could be the disturbed distribution of the vitamin within the kidney after ischemization as a consequence of the postischemic impairment of kidney perfusion (LINAS et al. 1987; HAGIwARA et al. 1991). Age differences in the effects of antioxidants have been reported by other authors, too (SABBATrNI et al. 1994). Using dimethylthiourea, protective effects in young rats were found, whereas after administration of superoxide dismutase only adult rats were protected against postischemic injury. Altogether it can be summarized from the literature that renal ischemia is followed by a multiple impairment of kidney function (WEINBERG 1991). Therefore it seems to be unlikely that a single therapeutic arrangement as vitamin E treatment is able to prevent postischemic renal injury completely. It is more probable that a complex therapeutic strategy using substances with different modes of action on the cellular level is effective in the prevention of free oxygen radical mediated cell damages (BRETSCHNEIDER et al. 1988; SCHILLER et al. 1990; BOUDJEMA et al. 1991; RANGAN and BURKLEY 1993).

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