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Expression of aldehydic lipid peroxidation products in rat kidneys during warm ischemia
ELSEVIER
Expression of Aldehydic Lipid Peroxidation Products in Rat Kidneys During Warm Ischemia P. Eschwege, M. Conti, V. Paradis, M. Pudliszewski, E. Prieur, A. Bendavid, P. Bedossa, A. Jardin, and G. Benoit U R I N G warm ischemia (WI), liberation of metabo• lites such as superoxide anions and hydroxyl radicals induce severe tissue damage. 1 Stress resulting from oxygenderived free radical is highly involved in WI damage to kidney. The main targets of these molecules are membrane lipids.2 The two prominent lipid degradation products are malondialdehyde (MDA) and 4-hydroxynonenal (HNE). They can be detected with biochemical assays or immunostaining using antibodies against protein-linked MDA and HNE. This article describes the study to determine qualitatively and quantitatively the liberation of MDA and HNE. These molecules measured in kidney biopsies reflect kidney damage and could be considered as significant markers especially after WI.
25
D
MATERIALS AND METHODS
Thirty Sprague-Dawley male rats were used for the experiments. The rats were anesthetized with 1 mg/100 g of rat intramuscular ketamine. The animals were divided into five groups (n = 6 rats/groups) that represented different time of WI: 0, 15, 30, 45, and 60 minutes. Warm ischemia was performed by clamping left and right renal pedicle, except for the t = 0 minutes group. A left-sided nephrectomy was performed at the end of the WI period. Part of the left kidneys were placed in Bouin liquid and the other part in liquid nitrogen. The animals were studied for 10 days following WI. Blood samples were collected before and after the clamping period at 1, 2, 4, 6, 8, and 10 days. Biochemical Study of MDA
Biopsies were homogenized in HCIO4, 0.3 N, After centrifugation, supernatant was neutralized at pH = 6 and was submitted to the assay. After reaction with diethylthiobarbituric acid, MDA was measured using synchronous fluorescence as previously described.3 MDA and HNE Immunostaining
MDA and HNE immunostaining was performed on paraffin embedded kidney samples using rabbit antisera against protein-linked MDA and HNE. RESULTS
The curve of MDA amounts (pmol/mg of biopsies) as a function of different WI times (0 to 60 minutes) was plotted © 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2437-2438 (1997)
D.
20
.9 ..Q 15
~
10
<
5
-t
I
0
i
0
I
,
20
I
40 Time
,
I
60
(min)
Fig 1. Curve of MDA amounts (pmol/mg of biopsies) as a function of different Wl times (0 to 60 minutes).
in Fig 1. In Fig 2, we have exhibited specific immunostaining of MDA in the rat kidney biopsies after 45 minutes of WI (cortex and kidney medullar immunostaining). We have measured in the control group (t = 0), 12.9 --- 5 pmol/mg of MDA. At 15 minutes, we observed an increased MDA value up to 15.7 - 6 pmol/mg. Then a regular and small decrease of MDA value to 11.9 +_ 3 pmol/mg was observed. Controls were not positive after MDA and HNE immunostaining. In contrast, at 15 minutes superficial cortex were stained, then deep cortex at 30 minutes, and medullar at 45 and 60 minutes. These data correlated with an increase in animal mortality at 45 minutes (30%) and at 60 minutes (60%) compared with 0% at 0, 15, and 30 minutes. Creat-
From the Laboratoire de Chirurgie Experimentale (P.E., M.P., E.P., A.B., A.J., G.B.), Faculte de Medecine Paris Sud; Laboratoire de Biochimie (M.C.), and Laboratoire d'AnatomiePathologique (V.P., P.B.), H6pital de BicC)tre, Le Kremlin Bic#}tre, France. Supported by grants from Fondation de I'Avenir, Etude ET5118, and Etablissement Fran(;ais des Greffes. Address reprint requests to P. Eschwege, Laboratoire de Chirurgie Experimentale, Facult6 de M~decine Paris Sud, 63 rue Gabriel P6ri, 94270 Le Kremlin Bic6tre, France. 0041-1345/97/$17.00 PII S0041-1345(97)00440-5 2437
2438
ESCHW#GE, CONTI, PARADIS ET AL
Table 1. Creatininemia of Animals Before and After Different Wl Periods (t~mol/L) 0 DO D1 D2 D4 D6 D8 D10
15
63 + 16 55_+ 16 95-+38 98_+2 130+2 104_+68 73_+3 68_+ 10 67 +_ 4 68 _+ 8 67_+5 63_+5 71 _+11 79_+24
30
45
60
5 6 ± 16 75___5 135± 106 57___21 58 ± 11 75±26 54±14
51 _+5 382_+ t00 412_+470 278_+213 202 _+ 20 107_+ 19 68_+10
51 _+8 489_+ 169 605_+268 388_+ 135 184 _+ 141 92_+34 89_+4
DISCUSSION
Fig 2. Immunostaining of MDA in the rat kidney biopsies after 45 minutes of WI. (A) Cortex. (B) Medullar kidney immunostaining. (Magnification ×40).
M D A and HNE detection methods will be developed in order to appreciate the ischemic status of kidneys after various WI periods. Peroxydation during ischemia is major in the first 15 minutes and then slowly decreases to reach a value below that of the control. M D A and HNE were first observed at 15 minutes in proximal tubular cells (cortex), and then observed in the kidney medullar at 45 minutes. Immunostaining of M D A and HNE showed a progression during WI periods in the kidney that correlates with a decrease of the renal function and an increase in animal mortality. The medullar lesions seem to indicate an important degradation of the kidney tissue. M D A and HNE detection methods will be developed in order to obtain an objective evaluation of warm ischemic lesions.
REFERENCES
ininemia increased the first day for all animals and decreased thereafter, returning toward normal at the end of follow-up except for the rats with 45 and 60 minutes of WI (Table 1).
1. Grune T, Sommerburg O, et al: Free Rad Biol Med 18:21, 1995 2. Conti M, Morand PC, et al: Clin Chem 37:1273, 1991 3. Parks DA, Bukley GB, et al: Surgery:428, 1983
Expression of Aldehydic Lipid Peroxidation Products in Rat Kidneys During Warm Ischemia P. Eschwege, M. Conti, V. Paradis, M. Pudliszewski, E. Prieur, A. Bendavid, P. Bedossa, A. Jardin, and G. Benoit U R I N G warm ischemia (WI), liberation of metabo• lites such as superoxide anions and hydroxyl radicals induce severe tissue damage. 1 Stress resulting from oxygenderived free radical is highly involved in WI damage to kidney. The main targets of these molecules are membrane lipids.2 The two prominent lipid degradation products are malondialdehyde (MDA) and 4-hydroxynonenal (HNE). They can be detected with biochemical assays or immunostaining using antibodies against protein-linked MDA and HNE. This article describes the study to determine qualitatively and quantitatively the liberation of MDA and HNE. These molecules measured in kidney biopsies reflect kidney damage and could be considered as significant markers especially after WI.
25
D
MATERIALS AND METHODS
Thirty Sprague-Dawley male rats were used for the experiments. The rats were anesthetized with 1 mg/100 g of rat intramuscular ketamine. The animals were divided into five groups (n = 6 rats/groups) that represented different time of WI: 0, 15, 30, 45, and 60 minutes. Warm ischemia was performed by clamping left and right renal pedicle, except for the t = 0 minutes group. A left-sided nephrectomy was performed at the end of the WI period. Part of the left kidneys were placed in Bouin liquid and the other part in liquid nitrogen. The animals were studied for 10 days following WI. Blood samples were collected before and after the clamping period at 1, 2, 4, 6, 8, and 10 days. Biochemical Study of MDA
Biopsies were homogenized in HCIO4, 0.3 N, After centrifugation, supernatant was neutralized at pH = 6 and was submitted to the assay. After reaction with diethylthiobarbituric acid, MDA was measured using synchronous fluorescence as previously described.3 MDA and HNE Immunostaining
MDA and HNE immunostaining was performed on paraffin embedded kidney samples using rabbit antisera against protein-linked MDA and HNE. RESULTS
The curve of MDA amounts (pmol/mg of biopsies) as a function of different WI times (0 to 60 minutes) was plotted © 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2437-2438 (1997)
D.
20
.9 ..Q 15
~
10
<
5
-t
I
0
i
0
I
,
20
I
40 Time
,
I
60
(min)
Fig 1. Curve of MDA amounts (pmol/mg of biopsies) as a function of different Wl times (0 to 60 minutes).
in Fig 1. In Fig 2, we have exhibited specific immunostaining of MDA in the rat kidney biopsies after 45 minutes of WI (cortex and kidney medullar immunostaining). We have measured in the control group (t = 0), 12.9 --- 5 pmol/mg of MDA. At 15 minutes, we observed an increased MDA value up to 15.7 - 6 pmol/mg. Then a regular and small decrease of MDA value to 11.9 +_ 3 pmol/mg was observed. Controls were not positive after MDA and HNE immunostaining. In contrast, at 15 minutes superficial cortex were stained, then deep cortex at 30 minutes, and medullar at 45 and 60 minutes. These data correlated with an increase in animal mortality at 45 minutes (30%) and at 60 minutes (60%) compared with 0% at 0, 15, and 30 minutes. Creat-
From the Laboratoire de Chirurgie Experimentale (P.E., M.P., E.P., A.B., A.J., G.B.), Faculte de Medecine Paris Sud; Laboratoire de Biochimie (M.C.), and Laboratoire d'AnatomiePathologique (V.P., P.B.), H6pital de BicC)tre, Le Kremlin Bic#}tre, France. Supported by grants from Fondation de I'Avenir, Etude ET5118, and Etablissement Fran(;ais des Greffes. Address reprint requests to P. Eschwege, Laboratoire de Chirurgie Experimentale, Facult6 de M~decine Paris Sud, 63 rue Gabriel P6ri, 94270 Le Kremlin Bic6tre, France. 0041-1345/97/$17.00 PII S0041-1345(97)00440-5 2437
2438
ESCHW#GE, CONTI, PARADIS ET AL
Table 1. Creatininemia of Animals Before and After Different Wl Periods (t~mol/L) 0 DO D1 D2 D4 D6 D8 D10
15
63 + 16 55_+ 16 95-+38 98_+2 130+2 104_+68 73_+3 68_+ 10 67 +_ 4 68 _+ 8 67_+5 63_+5 71 _+11 79_+24
30
45
60
5 6 ± 16 75___5 135± 106 57___21 58 ± 11 75±26 54±14
51 _+5 382_+ t00 412_+470 278_+213 202 _+ 20 107_+ 19 68_+10
51 _+8 489_+ 169 605_+268 388_+ 135 184 _+ 141 92_+34 89_+4
DISCUSSION
Fig 2. Immunostaining of MDA in the rat kidney biopsies after 45 minutes of WI. (A) Cortex. (B) Medullar kidney immunostaining. (Magnification ×40).
M D A and HNE detection methods will be developed in order to appreciate the ischemic status of kidneys after various WI periods. Peroxydation during ischemia is major in the first 15 minutes and then slowly decreases to reach a value below that of the control. M D A and HNE were first observed at 15 minutes in proximal tubular cells (cortex), and then observed in the kidney medullar at 45 minutes. Immunostaining of M D A and HNE showed a progression during WI periods in the kidney that correlates with a decrease of the renal function and an increase in animal mortality. The medullar lesions seem to indicate an important degradation of the kidney tissue. M D A and HNE detection methods will be developed in order to obtain an objective evaluation of warm ischemic lesions.
REFERENCES
ininemia increased the first day for all animals and decreased thereafter, returning toward normal at the end of follow-up except for the rats with 45 and 60 minutes of WI (Table 1).
1. Grune T, Sommerburg O, et al: Free Rad Biol Med 18:21, 1995 2. Conti M, Morand PC, et al: Clin Chem 37:1273, 1991 3. Parks DA, Bukley GB, et al: Surgery:428, 1983