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NDT Advance Access originally published online on December 29, 2005
Nephrology Dialysis Transplantation 2006 21(3):616-623; doi:10.1093/ndt/gfi314
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Urinary marker for oxidative stress in kidneys in cisplatin-induced acute renal failure in rats

Hua Zhou1, Akihiko Kato1, Takehiko Miyaji1, Hideo Yasuda1, Yoshihide Fujigaki1, Tatsuo Yamamoto1, Katsuhiko Yonemura1, Satoru Takebayashi2, Hiroyuki Mineta2 and Akira Hishida1

1 First Department of Medicine and 2 Department of Otolaryngology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan

Correspondence and offprint requests to: Prof. Akira Hishida, MD, First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka, 431-3192, Japan. Email: ahishida{at}hama-med.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Establishment of non-invasive urinary biomarkers for the prediction of acute renal failure (ARF) is important. We evaluated whether urinary oxidative stress markers reflect intrarenal oxidative stress in cisplatin (CDDP)-induced ARF, and whether these markers can be used for the prediction of future ARF.

Methods. Urinary malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured up to day 14 post-CDDP (6 mg/kg) injection in rats. MDA and 8-OHdG expressions were examined in kidneys.

Results. CDDP induced an increase in serum creatinine (Scr), blood urea nitrogen (BUN), and tubular damage at day 5, increased urinary MDA excretion and MDA expression in kidneys at day 1 (but returned to basal values by day 3), increased urinary excretion of 8-OHdG at day 5 till day 14 (though the number of 8-OHdG-positive tubular cells increased at day 5 and then gradually decreased). Urinary MDA levels at day 1 correlated significantly with Scr ({rho} = 0.721, P<0.01) and tubular damage score ({rho} = 0.840, P<0.01) at day 5.

Conclusion. Our findings demonstrated divergent changes of urinary oxidative stress markers in CDDP-induced ARF, and suggested that urinary MDA may be a useful marker for the prediction of the development of CDDP-induced ARF.

Keywords: acute renal failure; cisplatin; malondialdehyde; 8-OHdG; urinary oxidative stress biomarker



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The mortality rate of patients with acute renal failure (ARF) has remained 25 to 70% despite the use of various pharmacological agents [1]. Earlier diagnosis and effective therapeutic strategies are important for improvement of the high morbidity and mortality rate of ARF. For this purpose, the development of non-invasive early biomarkers for the prediction of ARF is essential.

Several studies have demonstrated that oxidative stress is involved in the development of cisplatin (CDDP)-induced renal tubule injury [2–4]. In CDDP-treated kidney, we previously found that renal malondialdehyde (MDA) was increased at 6 h after the injection of CDDP [4]. Other investigators reported that CDDP induced an increase in tubular cells positive for 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat distal tubules [5]. Increased production of prostaglandin-F (2{alpha}) isomer (8-isoprostane) following CDDP exposure was also observed in cultured renal tubular epithelial cells [6].

Recently, several novel biomarkers for the induction of ARF have been identified, such as kidney injury molecule-1 (KIM-1) [7,8], interleukin-18 (IL-18) [9], cysteine-rich protein 61 (CYR61) [10], neutrophil gelatinase-associated lipocalin (NGAL) [11,12] and hepatocyte growth factor (HGF) [13]. These proteins are detectable in either urine or renal tissue and had been advocated as new markers for the diagnosis of ischaemic and nephrotoxic ARF. However, there is little study whether oxidative stress markers, which are known to be related to CDDP nephrotoxicity, increase in urine and can predict the development of CDDP-induced ARF.

The purpose of the present study was to test whether the changes of urinary excretion of oxidative stress markers, such as MDA and 8-OHdG reflect the oxidative stress in kidneys, and can be used as early biomarkers for the prediction of development of CDDP-induced ARF. For this purpose, the urinary excretion of MDA and 8-OHdG and renal expression of MDA and 8-OHdG were measured serially over a period of 14 days after injection of CDDP. Since the increases in urinary excretion and renal expression of MDA occurred before the increase in serum creatinine (Scr), we prospectively evaluated whether the early increase in urinary MDA predicts the future increase in Scr in CDDP-treated rats.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Induction of ARF
One hundred and five male Sprague–Dawley rats, weighing 230–250 g, were provided with standard rat chow and drinking water ad libitum. Animal care in this study was based on the criteria of the Ethics Review Committee for Animal Experimentation of Hamamatsu University School of Medicine and NIH Guidelines for the Care and Use of Laboratory Animals. ARF was provoked by a single intravenous injection of CDDP (a kind gift from Nippon Kayaku Inc., Tokyo, Japan). We conducted two experimental protocols in this study. In the first protocol (n = 75), we administered 6 mg/kg body weight (BW) of CDDP to rats and collected 24 h urine in metabolic cages for the measurement of MDA and 8-OHdG and we obtained blood samples and kidneys before (day 0) and at days 1, 2, 3, 5, 7, 10 and 14 following CDDP injection in each of 6 rats (n = 48). The kidneys of these rats were used for histological and immunohistological studies and western blot analysis. Using an additional 6 rats, we serially collected urine and examined urinary MDA excretion before and at days 1, 2, 3, 5, 7, 10 and 14 following CDDP injection. We also measured urinary MDA without CDDP treatment to evaluate the urinary MDA excretion in normal rats (n = 21). In the second protocol (n = 30), we injected with 3 mg/kg BW (n = 4), 4.5 mg/kg BW (n = 16) or 6 mg/kg BW (n = 10) of CDDP and collected 24 h urine samples at day 1 and blood samples from the abdominal aorta and kidneys under pentobarbital anesthesia (50 mg/kg) at day 5 after CDDP. In this study, urine was used for measurement of MDA and creatinine, blood for Scr and blood urea nitrogen (BUN), while the kidneys served for evaluation of tubular damage. All urine samples were centrifuged at 1500 rpm for 10 min at 4°C, and then stored at –80°C until the examination. The concentrations of creatinine in serum and urine were measured by an enzymatic method (CreE test, Mizuho Medy. Inc., Saga, Japan). The concentration of BUN was determined by an enzymatic assay.

Histological examination
For histological examination, the harvested left kidney from each animal was immediately fixed in 20% neutral buffered formalin solution. The kidney tissue block was dehydrated through a graded alcohol series, embedded in paraffin, and cut into 3 µm sections and then stained with periodic acid Schiff (PAS) reagent. Since tubular damage was most evident in the outer stripe of the outer medulla in CDDP-treated kidney, we carried out semi-quantitative analysis of histological damage on this area. For this purpose, 100 tubules were selected in 25 random fields (four tubules in the centre of each field) at 400x magnification. Each tubular profile was assigned to one of five categories according to the following criteria: 0, normal; 1, areas of tubular epithelial cell swelling, vacuolar degeneration, necrosis and desquamation involving <25% of the tubular profile; 2, similar changes involving ≥25% but <50% of the tubular profile; 3, similar changes involving ≥50% but <75% of the tubular profile; and 4, similar changes involving ≥75% of the tubular profile. To minimize observer bias, the morphometric examination was performed in a blinded fashion without knowledge of the treatment group from which the tissue originated. The mean score for each rat and the mean score for each group were then calculated.

Assessment of apoptosis in renal tissues
The extent of apoptosis was assessed using the terminal deoxynucleotidyl transferase (TUNEL) technique. The tissue was deparaffinized and rehydrated, followed by incubation of 3 µm thick kidney sections with 20 µg/ml proteinase K; for 15 min at room temperature, and immersion in distilled water containing 3% H2O2 to inactivate endogenous peroxidase. Detection of DNA fragmentation was performed using an ApoTag plus in situ Apoptosis Detection Kit (Oncor, Gaithersburg, MD). A semi-quantitative analysis was performed by counting the number of TUNEL-positive cells per field at 400x magnification. The mean number of stained cells in 50 fields in each kidney was expressed as the number of TUNEL-positive cells.

Immunohistochemical examination of MDA and 8-OHdG
The paraffin samples were cut at 3 µm thickness, deparaffinized, and rehydrated. After consuming endogenous peroxidase with 3% H2O2, slides were pre-incubated with 10% normal donkey serum to block non-specific reaction. The samples were incubated respectively with polyclonal rabbit anti-MDA (1:100, code MDA 11-S, Alpha Diagnostic Intl, San Antonio, TX) and monoclonal antibody against 8-OHdG (2 µg/ml, Japan Institute for Control of Aging, Shizuoka, Japan) overnight at 4°C. After washing in phosphate-buffered saline (PBS, pH 7.4), the samples were incubated with biotin-conjugated donkey anti-rabbit IgG or anti-mouse IgG (Chemicon International Inc., Temecula, CA) for 30 min at room temperature, washed in PBS, and then incubated with streptavidin-conjugated peroxidase for 30 min at room temperature. The reaction products were visualized using H2O2 containing 3,3'-diaminobenzidine in 0.05 M Tris buffer. The number of 8-OHdG -positive nuclei in the outer stripe of the outer medulla was counted under a light microscope at 400x magnification in random 50 fields in each experimental animal by an investigator who was blinded to the treatment, and calculated its average value at each time point.

Western blot analysis of renal tissue MDA
A portion of the outer renal medulla from each rat was homogenized with PBS containing 1% Igepal CA-630 (Sigma Chemical Co., St Louis, MO), 0.5% sodium deoxycholate (Sigma), 0.1% sodium dodecyl sulfate (SDS), 10 mg/ml phenylmethylsulfonyl fluoride (PMSF, Sigma), 100 mM sodium orthovanadate (Na3VO4, Sigma) and 0.5 mg/ml aprotinin at 4°C. The homogenate was centrifuged at 15000 g for 20 min at 4°C. A volume equivalent to 60 µg of protein was separated on a NuPAGE Bis-Tris Gel (Invitrogen Life Technologies, San Diego, CA), and then electroblotted onto Hybond ECL nitrocellulose (Amersham Pharmacia Biotech UK, Buckinghamshire, UK). The membranes were blocked with 5% (wt/vol) skim milk powder in 0.1% (vol/vol) Tween-20 Tris-buffered saline for 1 h at room temperature. Blots were probed with polyclonal rabbit anti-MDA (1:800, code MDA 11-S, Alpha Diagnostic Intl) overnight at 4°C. As an internal standard, blots were re-probed with a monoclonal anti-ß-actin (1:50000, Sigma) for 30 min at room temperature. Binding to primary MDA antibodies were visualized using anti-rabbit HRP at 1:10000 dilution (for binding to primary ß-actin antibody, anti-mouse HRP at 1:10000 dilution) (Amersham Biosciences, NJ) for 1 h at room temperature, followed by the ECL chemiluminescence detection system (Amersham Biosciences). Developed films were analysed semi-quantitatively by volume densitometry using JADE2 scanning densitometry and NIH Image software version 1.60. We determined the area under the scanning curve in each blot, and the relative abundance of MDA was determined, by dividing by each ß-actin area.

Measurement of urinary MDA and 8-OHdG
Urinary MDA was determined by measurement of thiobarbituric acid reactive substances (TBARS) [14]. We selected the TBA test in this study since this method is popular and clinically used to measure MDA [15]. Aliquots of 500 µl of urine or MDA standards were mixed with 500 µl thiobarbituric acid (1%, pH 1.5) and boiled for 30 min. After cooling at room temperature, its absorbance was measured at 540 nm with a microplate reader. We also measured the absorbance of reactive solution of every urine sample and vehicle of TBA as the blank of TBARS. The final concentration of MDA was expressed as the difference between TBARS and blank in order to diminish the interference of urine chromogens.

Before measuring urinary MDA in our experimental samples, we evaluated the accuracy of our method for measuring MDA in urine. In the first study, we added known amounts of standard MDA (10, 20 and 40 nmol/ml) to urine obtained from normal rats and measured MDA concentration before and after the addition of standard MDA. The difference between these two values was 94.4±3.5% (mean±SEM) (n = 6) of added amounts of MDA. In the second study, we measured MDA twice on different days using the same urine samples obtained from normal rats (n = 9) 1 day post-CDDP-injection (n = 12). MDA in normal rat urine were 15.72±1.93 on the first day and 14.94±1.60 nmol/ml on the second day respectively. The mean±SEM of the difference between two measurements was 0.78±0.33 nmol/ml. The results of two measurements on MDA in urine obtained from CDDP-treated rats were 41.46±5.11 and 42.79±5.67 nmol/ml, respectively, and the mean±SEM in the difference was 1.33±0.54 nmol/ml.

Urinary 8-OHdG concentrations were measured by enzyme-linked immunosorbent assay using commercially available competitive 8-OHdG ELISA kit (Japan Institute for Control of Aging, Shizuoka, Japan). The kit can measure 8-OHdG values ranging from 0.125 to 10 ng/ml using a monoclonal specific antibody, N45.1. This antibody does not cross-react with the original four deoxyribonucleosides, 2'-deoxyinosine, 8-hydroxy-27-deoxyadenine or O6-methyl-2'-deoxyguanosine) [16]. Urinary concentrations of MDA and 8-OHdG were expressed per milligram of urinary creatinine.

Statistical analysis
All data were presented as mean±SEM. Differences among data were determined using one-way ANOVA with standard post hoc testing (StatView, version 5.0, Abacus Concepts, Berkeley, CA). Pearson Parametric correlation was used in relationship between urninary MDA and Scr; Spearman Rank correlation coefficient was used in the relationship between urinary MDA and tubular damage score. A P<0.05 denoted the presence of a statistically significant difference.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Induction of ARF by CDDP
CDDP injection (6 mg/kg BW) induced a significant increase in Scr and renal morphological damage including extensive tubular damage and intratubular cast formation in the outer stripe of the outer medulla. TUNEL-positive cells were detected in the outer stripe of the outer medulla. Scr and BUN reached a peak level at day 5, and then gradually decreased to the basal value by day 14 (Figure 1A and B). Tubular damage and TUNEL-positive cells were observed at day 3, reached a peak at day 5 and then gradually decreased to the basal level by day 14 (Figure 1C). In contrast, 4.5 mg/kg BW of CDDP (n = 16) only provoked mild renal injury until 5 days after CDDP injection (Scr at day 5, ranging from 0.84 to 1.89 mg/dl). There was no significant increase in Scr at 3 mg/kg BW (n = 4, ranging from 0.55 to 0.81 mg/dl).


Figure 1
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  		Fig. 1. Scr, BUN and TUNEL-positive tubular cells in CDDP-treated rats. CDDP significantly increased Scr (A) and BUN (B) at day 5. CDDP also significantly increased the number of TUNEL-positive nuclei from day 3 to day 7, reaching a peak level at day 5 (C). Data are mean±SEM. *P<0.01, #P<0.05 vs day 0.

 
Expression of MDA and 8-OHdG in CDDP-treated kidney
Immunohistochemical examination revealed that MDA stained faintly in the cytoplasm of tubular cells in the normal kidneys (Figure 2A). CDDP injection increased the intensity of MDA staining in the cytoplasm of the proximal tubular cells at day 1 (Figure 2C). The positive staining in the cytoplasm gradually diminished thereafter (Figure 2E and G). A band positively probed with anti-MDA was found in the position between molecular weight markers 51 and 64 kDa in kidneys obtained at day 1 after CDDP injection (Figure 3A), but not in normal kidneys. We evaluated this band till day 14 after CDDP injection as the MDA-modified proteins in CDDP-induced ARF. Western blot analysis showed no other CDDP-induced MDA-modified protein in kidneys. CDDP significantly increased the abundance of MDA-modified protein at day 1, but it decreased at day 2, and finally disappeared at day 3 and thereafter (Figure 3B and C).


Figure 2
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  		Fig. 2. Photomicrographs of immunostained sections for MDA (A, C, E and G) and 8-OHdG (B, D, F and H) in the outer stripe of the outer medulla after CDDP injection. A very faint staining of MDA was found in normal kidney tissue (A). MDA-positive immunostaining was found in the cytoplasm of tubular cells at day 1 (C), then decreased at day 5 (E), and disappeared at day 14 (G). Magnification 200x. There was no staining for 8-OHdG in the normal kidney (B) and CDDP-treated kidney at day 1 (D). An increase in 8-OHdG-positive nuclei was observed at day 5 (F) but disappeared at day 14 (H) after CDDP injection. Magnification 400x.

 

Figure 3
Figure 3
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  		Fig. 3. Changes in MDA level (A, B and C) and the number of 8-OHdG-positive nuclei (D) in CDDP-treated kidney. Western blotting analysis showed that a band positively probed with anti-MDA was found in the position between 51 and 64 kDa at day 1 after CDDP injection, but not in normal kidneys (arrow indicated band); lane 1, molecular weight marker; lanes 2, 4 and 6, dose of 50, 20, and 10 µg protein from normal kidney; lanes 3, 5 and 7, CDDP-treated kidneys with 50, 20, and 10 µg of protein (A). The band between 51 and 64 kDa was considered as CDDP-induced MDA-modified proteins, and evaluated in (B) and (C), CDDP significantly and transiently increased MDA level in the outer medulla at day 1 (B and C). The number of 8-OHdG-positive nuclei in the outer stripe of the outer medulla was significantly increased at day 5, and then gradually decreased until day 14 (D). Data are expressed as mean±SEM at each time point (n = 6). *P<0.01, #P<0.05.

 
In normal kidneys, 8-OHdG was negative in tubular cells (Figure 2B). No significant staining for 8-OHdG was observed by day 3 after CDDP (Figures 2D and 3D), but it appeared at day 5 in the nuclei of damaged tubular cells in the outer stripe of the outer medulla. The number of 8-OHdG-positive nuclei decreased thereafter and disappeared by day 14 (Figures 2F, 2H and 3D).

Urinary excretion of MDA and 8-OHdG in CDDP-induced ARF
The urinary excretion of MDA measured in 33 normal rats was 31±4 nmol/mg creatinine. Urinary MDA significantly increased at day 1 after CDDP injection compared with the basal level (91±10 vs 37±8 nmol/mg creatinine, P<0.01). The increase in MDA urinary excretion was transient as in the case of renal expression of MDA. The urinary excretion of MDA at day 3 and thereafter was similar to that in normal rats. In contrast, urinary 8-OHdG did not increase until day 5 (baseline: 7.05±1.49, day 5: 17.93±4.60 ng/mg creatinine, P<0.05), and then progressively and significantly increased until day 14 (38.24±7.70 ng/mg creatinine, P<0.01, Figure 4B).


Figure 4
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  		Fig. 4. Serial changes in urinary MDA (A) and 8-OHdG (B) during 14 days post-CDDP injection. Urinary MDA transiently increased at day 1 but decreased to the basal level since day 3 (A). Urinary 8-OHdG was significantly increased at day 5, then progressively increased until day 14 (B). Data are expressed as mean ± SEM at each time point (n = 6). *P<0.01, #P<0.05.

 
Relationship between urinary excretion of MDA at day 1 and renal damage at day 5 after CDDP
To test whether urinary MDA levels at day 1 predict the Scr and tubular damage at day 5, we treated rats with three different dosages of CDDP (3.0, 4.5, and 6.0 mg/kg BW, n = 30). There was a significant and positive correlation between urinary MDA/cratinine at day 1 and Scr at day 5 ({rho} = 0.721, P<0.01, Figure 5A, by Pearson correlation) and tubular damage score at day 5 ({rho} = 0.840, P<0.01, Figure 5B, by Spearman correlation). We also found a significant and positive relationship between daily MDA excretion (ng/day) at day 1 and subsequent increases in Scr ({rho}=0.701, P<0.01) and tubular damage score at day 5 ({rho} = 0.813, P<0.01).


Figure 5
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  		Fig. 5. Correlation between urinary MDA at day 1 and renal damage at day 5 in CDDP-induced ARF. Note the significant and positive correlation between urinary MDA at day 1 and subsequently elevated Scr ({rho} = 0.721, P<0.01) calculated by Pearson correlation (A) and mean tubular damage score ({rho} = 0.840, P<0.01) by Spearman Rank correlation coefficient (B) at day 5 after CDDP injection.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
The major findings of the present study were the following: (1) urinary MDA excretion and renal MDA abundance were transiently but significantly increased at day 1 following CDDP injection, (2) urinary 8-OHdG level and the number of 8-OHdG-positive nuclei in the outer stripe of the outer medulla were significantly increased at day 5, (3) urinary 8-OHdG continued to increase until day 14, but the number of 8-OHdG-positive nuclei started to decrease after day 5, (4) a significant and positive relationship was found between urinary MDA at day 1 and renal damage (Scr and tubular damage score) at day 5.

The diagnosis of ARF is based on increases in blood urea nitrogen, Scr, or urinary excretion of enzyme in tubular cells [N-acetyl-beta-glucosaminidase (NAG)] or low molecular protein [beta-2-microglobulin (ß2MG)]. The increase in urinary NAG or ß2MG in ARF is induced by the leakage of tubular enzyme or by defective tubular reabsorption [1,17]. Recently, several new urinary markers, which are induced in kidneys with ARF, were reported as specific and early diagnostic markers of ARF. For example, Kim-1 and CYR61, which are induced in tubular cells exposed to ischaemia, are reported to be increased in the kidney and urine [7,8,10]. In addition, various markers of inflammation, such as IL-18 and NGAL, were evaluated as candidate urinary markers of ARF, since inflammatory response is known to be involved in the development of ARF [9,11,12]. In the current study, we focused on oxidative stress markers as an early marker of the development of CDDP-induced ARF, since oxidative stress is known to play important roles in CDDP-induced ARF [2–4].

Among many markers of oxidative stress, renal MDA level and 8-OHdG-positive cells were reported to be increased following injection of CDDP or mercuric chloride or ureteral obstruction in rats [4,5,18,19]. 8-OHdG, 8-isoprostane, 4-hydroxynoneal, and low-density lipoprotein (LDL) were also reported to be increased in the kidneys in ischaemic ARF and uraemia [20,21].

Recent studies reported the enhanced excretion of these markers in the urine in experimental and human kidney diseases. For example, enhanced urinary excretion of MDA was reported in ß-thalassemic patients with proximal tubular damage [22]. Other studies reported increased urinary 8-OHdG concentrations in diabetic nephropathy [23,24], and increased urinary level of 8-isoprostane in diabetes [25], hypertension [26], and acute cholestasis-induced ARF [27]. To our knowledge, however, no previous studies have studied which of these markers of oxidative stress increases in kidneys and urine in CDDP-induced ARF.

We chose MDA and 8-OHdG as candidates of urinary markers for intrarenal oxidative stress by CDDP in this study, and evaluated their expression in kidneys and urinary excretion levels in rats with CDDP-induced ARF. We showed that intrarenal and urinary MDA levels were significantly and transiently increased following CDDP injection in animals. The increase of MDA level in kidneys and urine reached peak levels at day 1 and then rapidly decreased to the basal level. Since the increase in urinary MDA excretion reflects the increase in MDA in renal tissue and preceded the development of the increase in Scr and tubular damage score, we then investigated whether one can predict the development of acute renal failure by measurements of urinary excretion of MDA at day 1 after CDDP injection. These studies showed a significant correlation between urinary excretion of MDA and the levels of Scr and tubular damage score at day 5, when these changes reach a peak in our model of CDDP-induced ARF. These findings suggest that early measurement of urinary MDA after CDDP treatment may be useful for the prediction of CDDP-induced renal damage.

Myeloperoxidase activity, an index of tissue neutrophil content, was reported to be increased until day 3 in CDDP-treated kidneys [30]. In this study, however, urinary excretion of MDA was transiently increased at day 1, and returned to base since day 2. The abundance of MDA-modified protein also disappeared in kidney until day 3. The reasons for this short decline of urinary MDA remain unclear despite the progression of ARF. MDA was mainly immunostained in the cytoplasma of the proximal tubular cells. Since most of the tubular cells revealed prominent tubular damage such as vacuolation, necrosis and desquamation until day 5, reduced urinary excretion may reflect less tubular MDA production rather than the degree of lipid peroxidation.

Our preliminary observation showed that urinary MDA excretion was increased by 24 h after the treatment with CDDP in 8 human cancer patients (data are not shown). Therefore, we think the increase in urinary MDA might be a candidate for an early urinary marker for the prediction of the development of ARF in a clinical setting. Since the increase in Scr was not significant in our preliminary clinical study, and the dose of CDDP used in rats of our study was 2–3 fold higher than that used in human patients, further studies are needed to confirm the association between the increase in urinary MDA and future development of ARF in humans.

We also found increases in 8-OHdG-positive cells in kidneys and 8-OHdG in urine. The significant increase in urinary 8-OHdG was not observed until day 5 after CDDP (Figure 4B). This finding was in agreement with the results of a previous study in which no increase in urinary 8-OHdG excretion was observed at day 1 in CDDP-treated rats [28]. The increase in urinary 8-OHdG excretion after day 5 was associated with a decrease in 8-OhdG-positive cells in kidneys, suggesting that urinary 8-OHdG is not derived from tubular cells at day 7 and thereafter. Since CDDP is known to cause a slow cellular damage in neurons [29], increased urinary 8-OHdG may reflect DNA damage in non-renal tissues. The slow increase in urinary 8-OHdG and the dissociation from the number of 8-OHdG-positive tubular cells suggest that urinary 8-OHdG is not a useful marker of oxidative DNA damage in renal tubules in CDDP-induced ARF.

In summary, we found that MDA was transiently increased in the kidneys and urine at day 1 following CDDP injection. Urinary MDA level at day 1 was significantly correlated with Scr and tubular damage score at day 5, when the renal damage reached a peak in this model of ARF. In contrast, urinary 8-OHdG was not increased in the early stage of CDDP-induced ARF. These findings suggest that early measurement of urinary MDA might be a useful marker for the prediction of CDDP-induced renal damage.



   Acknowledgments
 
We thank Nippon Kayaku Co. Ltd (Tokyo, Japan) for kindly providing the CDDP for this study. This work was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS) (No. 15590846) and a Grant-in-Aid for the Center of Excellence (COE) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 8. 8.05
Accepted in revised form: 11.11.05


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