Inhibition of p21 modifies the response of cortical proximal tubules to cisplati - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:40 |只看该作者 |倒序浏览 |打印

作者：Hua Zhou, Yoshihide Fujigaki, Akihiko Kato,, Takehiko Miyaji, Hideo Yasuda, Takayuki Tsuji, Tatsuo Yamamoto, Katsuhiko Yonemura, and Akira Hishida作者单位：1 First Department of Medicine and 3 Hemodialysis Unit, Hamamatsu University School of Medicine, Hamamatsu; and 2 Division of Nephrology, Endocrinology and Metabolism, Shizuoka Cancer Center Hospital, Shizuoka, Japan / G1 Z3 K7 E5 Y: j
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      【摘要】
   The purpose of this study was to evaluate whether upregulated p21, a cell cycle-inhibitory protein, contributes to cisplatin (CDDP)-induced acute renal failure (ARF) and to acquired resistance to rechallenge injury with CDDP in rats. ARF was induced in rats by injection of CDDP (5 mg/kg) and rechallenge injury to CDDP by the same dose of CDDP 14 days after the first CDDP injection. Rats were treated with p21 antisense oligodeoxynucleotide (ODN) or its vehicle, p21 sense ODN, every 36 h from days 0 to 5 for single CDDP and from days 13 to 19 for rechallenge injury and killed at day 3, 5, 16, or 19. The uptake of FITC-labeled p21 antisense ODNs by cortical proximal tubule (PT) cells was much greater than by PT cells in the outer stripe of outer medulla (OSOM). Administration of antisense induced partial downregulation of p21 mRNA and protein levels in whole kidneys with single CDDP treatment and its rechallenge injury. Antisense significantly aggravated PT necrosis and decreased the number of p21-positive PT cells in the cortex but not in the OSOM in both CDDP-induced ARF and its rechallenge injury. However, antisense did not alter serum creatinine (S cr ) and blood urea nitrogen (BUN) levels. Our findings suggested that p21 plays, at least in part, a cytoprotective role in cortical PTs exposed to CDDP, although this does not contribute to renal dysfunction when judged by S cr and BUN levels. Because antisense may not adequately be taken up and/or function in PTs in the OSOM, the role of p21 in PTs in the OSOM in CDDP-induced ARF remains to be clarified. $ b& |+ c5 u8 R: A3 v& G
      【关键词】 acute renal failure cisplatin renal cortex
   THE INDUCTION OF P 21, a cyclin-dependent kinase inhibitor known to play a role in stopping the cell cycle at G1/S and G2/M phase checkpoints ( 23, 24 ), is reported to protect tubular cells against ischemia- ( 13 ) and cisplatin (CDDP)-induced acute renal failure (ARF) ( 12, 14, 15 ) by preventing DNA damage from progressing without resulting in death from either apoptosis or necrosis. In tumor cells, overexpression of p21 provides resistance to CDDP ( 19, 27 ), whereas deletion of the p21 gene results in preferential sensitivity to CDDP ( 6, 22 ).

We previously reported that animals recovering from ARF are resistant to a subsequent insult with a nephrotoxic agent ( 9, 14, 21, 29 ). This phenomenon, called acquired resistance, is associated with less tubular damage and less apoptotic cell death ( 21, 29 ). In CDDP-induced ARF, we found that the number of p21-positive nuclei was increased with two peaks at days 3 and 9 after CDDP injection in rats and that the number remained high until day 14. Subsequent injection of CDDP after a 14-day interval increased p21 expression further at day 16. Therefore, we suggested that the precedence of overexpression of p21 in rechallenge injury of CDDP might contribute to repair in tumor cells, to acquired resistance to CDDP-induced ARF in rats ( 14 ). In addition to p21, we recently reported that sodium arsenite-induced attenuation of CDDP nephrotoxicity in rats was associated with augmented expression of p27, another cyclin-dependent kinase inhibitor that also induces cell cycle arrest at G1/S and G2/M, and DNA repair-related proteins, suggesting that the induction of cell cycle-regulatory and DNA repair proteins provided resistance to CDDP injury ( 30 ). Because CDDP is repeatedly used to treat carcinoma patients under a clinical situation, analyzing mechanisms of an acquired resistance to CDDP rechallenge injuries should be important for prevention of CDDP renotoxicity, which is known as subclinically chronic tubulointerstitial injury ( 4, 8 ), as well as acute tubular injury in humans.
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The initial purpose of the present study was to verify whether the induction of p21 plays a protective role in tubular cells in both CDDP-induced ARF and its resistance to CDDP. For this purpose, we used oligodeoxynucleotide (ODN) to inhibit the expression of p21 in the kidney in rats treated with CDDP. However, the p21 antisense that we used was unexpectedly taken up mainly by cortical proximal tubules (PTs) and strengthened the renal damage in the cortex without affecting the renal damage in the outer stripe of the outer medulla (OSOM) in CDDP-induced ARF and CDDP rechallenge injury. Therefore, in this study, we discuss a cytoprotective role of p21 in the PT, which has not been highlighted so far as a major damage site in CDDP-induced ARF ( 5, 14, 29 ).* \7 j' w. e* F7 K4 p2 b. _

MATERIALS AND METHODS6 o  X  Z- d# H& x$ d2 ?

Induction of CDDP-induced ARF and acquired resistance to CDDP nephrotoxicity. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the Hamamatsu University School of Medicine. One hundred male Sprague-Dawley rats, weighing 230-250 g (SLC, Shizuoka, Japan), were provided with standard rat chow and drinking water ad libitum. For CDDP-induced ARF, rats ( n = 48) received a single intravenous injection of 5 mg/kg body wt of CDDP (a gift from Nippon Kayaku, Tokyo, Japan) and were killed under pentobarbital sodium anesthesia (50 mg/kg) before, or at day 3 or day 5 after CDDP injection.; \. y+ z6 V2 r8 u5 I8 p& B

Acquired resistance to CDDP nephrotoxicity was induced in rats ( n = 48) by a second intravenous injection with the same dose of CDDP 14 days after the first intravenous injection of 5 mg/kg body wt of CDDP, when our previous experiments demonstrated that serum creatinine (S cr ) levels had returned to the basal value ( 14 ). Rats were killed at day 16 or 19 after the first injection of CDDP.
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Blood was obtained from the abdominal aorta for measurements of the concentration of S cr using an enzymatic assay (Mizuho Medy, Saga, Japan) and the concentration of blood urea nitrogen (BUN) using an enzymatic assay (Kanto Kagaku, Tokyo, Japan) on a autoanalyzer (U-240 Plus, Japan Tectron Instruments, Tokyo, Japan). The kidneys were removed after being flushing with PBS for immunohistochemistry, Western blot analysis, and RT-PCR. The kidney samples for Western blot analysis and RT-PCR were frozen rapidly in liquid nitrogen and stored at -80°C until analysis.

Application of p21 antisense ODN and its distribution in vivo. We used p21 antisense phosphorothioated oligodeoxynucleotide (ODN) (5'-TGTCATGCTGGTCTGCCGCC-3') ( 18 ) and a random scrambled control as a sense to p21 phosphorothioated ODN (5'-CCGGTGAACGAGCGAGCACA-3') ( 25 ). For CDDP-induced ARF, p21 antisense or sense ODNs were injected intravenously in rats at a concentration of 2 mg/kg every 36 h at 1 h and at days 1.5, 3, and 4.5 after CDDP injection and killed at days 3 and 5. For acquired resistance to CDDP nephrotoxicity, rats were administered intravenously p21 antisense or sense at a concentration of 2 mg/kg every 36 h between days 13 and 17.5 and killed at days 16 and 19. To demonstrate the delivery of ODNs into renal tubular epithelium, fluorescein isothiocyanate (FITC)-conjugated p21 antisense ODNs at a concentration of 2 mg/kg were injected intravenously into normal rats ( n = 1) and rats 1 h ( n = 2) or 13.5 days ( n = 1) after CDDP injection, and the kidneys were harvested 12 h after ODN injection and processed for fluorescence study.

Histological examination. For histological examination, the excised left kidneys were fixed in 20% neutral-buffered formalin solution. The kidney tissue blocks were dehydrated in graded alcohol, embedded in paraffin, cut at 3-µm thickness, and then stained with periodic acid-Schiff reagent. We carried out a semiquantitative analysis of the number of PT cells with necrosis in both the cortex and the OSOM. Cross-sectional PTs without wide dilation, which is often seen in the OSOM in CDDP-induced ARF, were randomly selected in 25 fields at x 400 magnification in each rat, and each PT was considered necrotic when it included any tubular cell profiles, such as vacuolar degeneration, necrosis, and desquamation. The mean number of necrotic PTs relative to the number of PTs examined in each rat and the mean number of necrotic PTs in each group were calculated." Y+ R, \9 f( i: Y
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Immunohistochemistry for p21, proliferating-cell nuclear antigen, and Ki67. For immunohistochemical detection of p21, proliferating-cell nuclear antigen (PCNA), and Ki67, 3-µm-thick, 20% neutral-buffered formalin-fixed sections were treated with 3% hydrogen peroxide for 30 min at room temperature. After incubation of the sections with 20% normal goat serum for 15 min at room temperature, samples were incubated with a monoclonal antibody (mAb) against p21 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, a mAb against PCNA (Oncogene Science, Uniondale, NY) as a marker for G1/S phases for 30 min at 37°C, and a mAb against Ki67 (Novocastra, Newcastle upon Tyne, UK) as a marker for S phase for overnight at 4°C. Then, samples were incubated with biotin-conjugated donkey anti-mouse IgG (Chemicon International, Temecula, CA) for 30 min at room temperature, washed in PBS, and then incubated with streptavidin-conjugated peroxidase (Nichirei, Tokyo, Japan) for 30 min at room temperature. Finally, the reaction products were visualized using hydrogen peroxide containing 3,3'-diaminobenzidine in 0.05 M Tris buffer. Control sections against p21, PCNA, and Ki67 were incubated with phosphate-buffered solution instead of the primary antibodies, showing negative or negligible staining in PTs.
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The numbers of p21-, PCNA-, and Ki67-positive nuclei in the cortex and the OSOM were counted under a light microscope at x 400 magnification. The number of positive nuclei at each time point was expressed as the average value based on the examination of 25 fields in each experimental animal.
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Western blot analysis of p21. Isolation of proteins from the whole kidneys was conducted as described previously ( 30 ). 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). 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 mAb against p21 (F-5, Santa Cruz) overnight at 4°C. As an internal standard, blots were re-probed with mAb against -actin (1:50,000, Sigma) for 30 min at room temperature. Binding to primary antibodies was visualized using anti-mouse HRP at 1:10,000 dilution (Amersham Biosciences, NJ) for 1 h at room temperature, followed by the ECL chemiluminescence detection system (Amersham Biosciences). Developed films were analyzed semiquantitatively by JADE2 scanning densitometry and National Institutes of Health image-analysis software. The area under the scanning curve in each blot and the relative abundance of p21 were determined by dividing by each -actin area.

As an additional study, Western blot analysis of p21 was performed in the cortex and in the outer medulla, separately in normal rat kidney ( n = 2), antisense-treated kidney at day 3 ( n = 4), and sense-treated kidney at day 3 ( n = 2) after CDDP single injection.$ B: x/ N" I, N# r7 ^1 W

p21 mRNA by real-time RT-PCR. The methods for total RNA extraction and PCR were described elsewhere ( 26 ). The primers for p21 were 5'-ACGTGGCCTTGTCGCTGTCTT-3' (sense) and 5'-TAAGGCAGAAGATGGGGAAGAG -3' (antisense) ( 28 ), giving an amplified real-time (RT)-PCR product of 236 bp. To quantitative PCR of RNAs, a housekeeping gene, 18S ribosomal RNA, was coamplified using the primers for 18S, 5'-GGAGGTTCGAAGACGATCAG-3' (sense), 5'-CCCTTCCGTCAATTCCTTTA-3' (antisense), yielding a 173-bp product ( 26 ). Cycling conditions were 15-min preincubation at 95°C, 15-s denaturation at 95°C, 20-s annealing at 57°C, and 15-s extension at 72°C, using a LightCycler (Roche Diagnostics, Tokyo, Japan). The fluorescent product was detected at the end of each cycle, and PCR data were analyzed according to instructions provided by the manufacturer.- G0 \4 u3 d* K( j# Q9 M

Statistical analyses. All data are presented as means ± SE. Significant differences among data were determined using ANOVA followed by Fisher?s test (StatView, version 5.0, SAS Institute, Cary, NC). P 5 O' ^" L; `6 w1 O9 a6 ?/ }' Z; @- ?

RESULTS
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Distribution of FITC-labeled p21 antisense ODN in vivo. FITC-labeled ODN injected into normal rats or experimental rats was taken up by PT cells in the cortex ( Fig. 1 ), with less uptake by PT cells in the OSOM 12 h after injection.+ f; O5 p% j2 y9 j4 u
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Fig. 1. Accumulation of FITC-labeled p21 antisense oligodeoxynucleotides (ODNs) in the kidney at 12 h after ODN injection in normal rats ( A ) and in rats at 1 h ( B and D ) or 13.5 days ( C ) after cisplatin (CDDP) injection. Labeled ODNs were observed exclusively in the proximal tubular cells in the cortex. G, glomerulus; PT, proximal tubules; DT, distal tubule. Dashed lines, border between the cortex and the outer stripe of outer medulla. Original magnification: x 100 (A and C); x 200 ( B ); x 400 ( D ).
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Effect of p21 antisense ODN on p21 protein and mRNA. In our previous study ( 4 ), the number of p21-positive cells in the OSOM reached a peak level at days 3 and 16 after the first CDDP injection; thus p21 protein was examined at these time points. Western blot analysis showed that p21 protein was not detected in normal whole kidneys (data not shown). Increased expression of p21 proteins was significantly reduced in antisense-treated rats at days 3 and 16 compared with that in sense-treated rats, respectively ( Fig. 2 ).# r0 Q( g1 o& {# d) X
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Fig. 2. Western blots of protein and from whole kidney incubated with antibody for p21 and -actin ( top ) and relative abundance of p21 to -actin ( bottom ). Lanes 1 and 2, sense-treated kidney at day 3; lanes 3 and 4, antisense-treated kidney at day 3; lanes 5 and 6, sense-treated kidney at day 16; lanes 7 and 8, antisense-treated kidney at day 16 after CDDP first injection; P-S, single CDDP injection with p21 sense ODN; P-AS, single CDDP injection with p21 antisense ODN; PP-S, rechallenge of CDDP with p21 sense ODN; PP-AS, rechallenge of CDDP with p21 antisense ODN. Values are means ± SE for each group ( n = 6). * P
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The expression of p21 mRNA in whole kidneys was significantly higher in single CDDP-treated rats at day 5 and CDDP-rechallenged rats at day 19 than that in normal rats, and in CDDP-rechallenged rats at day 19 than that in single CDDP-treated rats at day 5 ( Fig. 3 ). Antisense-treated rats at days 5 and 19 showed a significant but incomplete reduction in p21 mRNA expression compared with that of control rats (CDDP alone or CDDP with p21 sense ODN), respectively ( Fig. 3 ), suggesting that p21 antisense ODN partially inhibited mRNA expression in whole kidneys during the experimental periods.# h  N* f6 g0 g- u
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Fig. 3. Changes in p21 mRNA abundance in the kidneys of CDDP-induced acute renal failure (ARF) and rechallenge of CDDP. P21 expression is normalized to 18S mRNA expression detected in each sample and is expressed as a ratio to 18S. C, normal control rats; P, single CDDP injection alone; P-AS, single CDDP injection with p21 antisense ODN treatment; P-S, single CDDP injection with p21 sense ODN; PP, rechallenge of CDDP alone; PP-AS, rechallenge of CDDP with p21 antisense ODN; PP-S, rechallenge of CDDP with p21 sense ODN; NS, not significant. Values are means ± SE of each group ( n = 6). * P
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Additional Western blot analysis showed that p21 protein was almost negative in the cortex and in the outer medulla ( Fig. 4 ) and that increased expression of p21 proteins in the cortex but not in the outer medulla was reduced in antisense-treated rats at day 3 compared with that in sense-treated rats at day 3 after a CDDP single injection ( Fig. 4 ).
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Fig. 4. Western blots of protein from the cortex ( A ) and the outer medulla ( B ) incubated for p21 and -actin. Abbreviations are the same as in Fig. 3.4 \, |$ R4 p  d: R

Effect of p21 antisense ODN on kidneys with single CDDP treatment. The S cr level at day 5 and BUN level at days 3 and 5 in rats with a single CDDP injection were significantly increased compared with that in normal control rats ( Fig. 5, A and C ). S cr and BUN levels were significantly higher at day 5 than at day 3 in rats with a single CDDP injection ( Fig. 5, A and C ). p21 antisense ODN treatment did not affect S cr and BUN levels at days 3 and 5 ( Fig. 5, A and C ). A single CDDP injection induced PT necrosis mainly in the OSOM at days 3 and 5. Rats with antisense treatment showed a significant increase in the number of necrotic PTs in the cortex but not in the OSOM at days 3 and 5 compared with those in control rats, respectively ( Figs. 6, A - F, and 7, A and B ). A single CDDP injection induced p21-positive PT cells in both the cortex and the OSOM at days 3 and 5, and the number of p21-positive PT cells was significantly lower in the cortex but not in the OSOM in antisense-treated rats at days 3 and 5 than that in control rats, respectively ( Figs. 8, A - D, and 9, A and B ). In contrast, the numbers of PCNA- and Ki67-positive PT cells were significantly higher in the cortex but not in the OSOM in antisense-treated rats at days 3 and 5 than those in control rats, respectively ( Figs. 10, A - D, 11, A and B, and 12, A and B ). Most PCNA-positive PT cells were also positive for Ki67 in consecutive sections of the cortex in rats at day 5 after a single CDDP injection ( Fig. 10, I and J ).
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Fig. 5. Serum creatinine (S cr; A and B ) and blood urea nitrogen (BUN; C and D ) concentrations in CDDP-injected rats at days 3 and 5 ( A and C ) and its rechallenge injury of CDDP at days 16 and 19 ( B and D ). Abbreviations are the same as in Fig. 3. Values are means ± SE. * P
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Fig. 6. Photomicrographs of the cortex ( A, C, E, G, and I ) and outer stripe of the outer medulla (OSOM; B, D, F, H, and J ) stained with periodic acid-Schiff from normal control rats ( A and B ), rats 3 days after single CDDP injection with sense ( C and D ) and antisense treatment ( E and F ), and rats 5 days after CDDP rechallenge with sense ( G and H ) and antisense treatment ( I and J ). *Proximal tubules with necrosis. Original magnification, x 200.
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Fig. 7. PT necrosis score per field in the cortex (CO; A and C ) and OSOM ( B and D ). Abbreviations are the same as in Fig. 3. Values are means ± SE. * P

Fig. 8. Photomicrographs of immunostaining for p21 (brown nuclei) in the cortex ( A, C, E, and G ) and OSOM ( B, D, F, and H ) in rats 3 days after single CDDP injection with sense ( A and B ) and antisense treatment ( C and D ) and rats 5 days after CDDP rechallenge with sense ( E and F ) and antisense treatment ( G and H ). Original magnification, x 400.3 Z" ]$ h: B. m% G( p5 k! b& G
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Fig. 9. Number of p21-positive proximal tubular cells per field in the cortex ( A and C ) and the OSOM ( B and D ). Abbreviations are the same as in Fig. 3. Values are means ± SE. * P

Fig. 10. Photomicrographs of immunostaining for proliferating-cell nuclear antigen (PCNA; brown nuclei) in the cortex ( A, C, E, and G ) and OSOM ( B, D, F, and H ) in rats 3 days after single CDDP injection with sense ( A and B ) and antisense treatment ( C and D ) and rats 5 days after CDDP rechallenge with sense ( E and F ) and antisense treatment ( G and H ). Immunostaining of PCNA ( I )- and Ki67 ( J )-positive proximal tubular cells (arrows) in cortex in consecutive sections at day 5 after CDDP injection is shown. The same numbers indicate the identical PT cells. Original magnification, x 400.

Fig. 11. Number of PCNA-positive PT cells in the cortex ( A and C ) and the OSOM ( B and D ). Abbreviations are the same as in Fig. 3. Values are means ± SE. * P 7 y; l# U6 W& g
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Fig. 12. Number of Ki67-positive PT cells per field in the cortex ( A and C ) and OSOM ( B and D ). Abbreviations are the same as in Fig. 3. Values are means ± SE. * P
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Effect of p21 antisense ODN on kidneys with CDDP rechallenge injury. CDDP rechallenge did not result in a significant increase in the S cr level at days 16 and 19 compared with that in normal control rats ( Fig. 5 B ). However, the BUN level at days 16 and 19 in CDDP-rechallenged rats was significantly increased compared with that in normal control rats ( Fig. 5 D ), and the BUN level was significantly higher at day 19 than at day 16 ( Fig. 5 D ). CDDP-rechallenged rats showed a minimal number of necrotic PTs in both the cortex and the OSOM ( Fig. 6, G and H ). Although dilated tubules were detected in the OSOM at days 16 and 19 ( Fig. 6, G and H ), such dilated tubules were found in the cortex at days 16 and 19 after the first CDDP injection (not shown). The effect of p21 antisense ODN on the kidneys in the CDDP-rechallenged rats was similar to that in rats with a single CDDP injury. Treatment with p21 antisense did not affect S cr and BUN levels ( Fig. 5, B and D ) but significantly increased the number of necrotic PTs in the cortex only at days 16 and 19 compared with control rats ( Figs. 6, G - J, and 7, C and D ). The number of p21-positive PT cells was significantly lower only in the cortex in antisense-treated rats at days 16 and 19 than that in control rats ( Figs. 8, E - H, and 9, C and D ). In contrast, the numbers of PCNA- and Ki67-positive PT cells were significantly higher in the cortex but not in the OSOM in antisense-treated rats at days 16 and 19 than those in control rats ( Figs. 10, E - H, 11, C and D, and 12, C and D ) and both showed similar distribution in the cortex in CDDP-rechallenged rats (not shown).8 a* ]7 @0 Y9 O+ E; E/ W, W. i
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DISCUSSION

In our previous study ( 14 ), our data suggested that induction of p21 plays a protective role in PT cells not only in CDDP-induced ARF as reported by others ( 12, 15 ) but also in its acquired resistance to CDDP. The present study was an extension of that study and was designed to evaluate whether upregulated p21 contributes to resistance to CDDP-induced ARF and to acquired resistance to CDDP rechallenge injury using antisense ODN. Because after CDDP administration, the PT cells in the OSOM (S3 segment) become especially sensitive and undergo extensive necrosis in vivo ( 5, 14, 29 ), we paid attention mainly to this area and hypothesized that any alterations should appear in the S3 segment in the OSOM if resistance to CDDP was negated by the inhibition of p21 induction. In the present study, we confirmed our previous report ( 14 ) that the number of p21-positive PT cells significantly increased in the OSOM at days 3 and 5 after CDDP injection ( Fig. 9 ). We also found that the number of p21-positive PT cells significantly increased in the cortex in rats with CDDP-induced ARF and CDDP-rechallenged injury, although this was not given attention in our previous study. Unexpectedly, we found that p21 antisense could significantly aggravate the number of PTs with necrosis only in the cortex but not in the OSOM of rats with CDDP-induced ARF and CDDP-rechallenged injury. p21 antisense could significantly reduce the number of p21-positive PT cells in the cortex but not in the OSOM. However, the number of p21-positive PT cells in the OSOM showed a tendency to decrease. This indicates that p21 mRNA was inhibited mainly in the cortex and may explain the partial downregulation of p21 mRNA and protein in the whole kidneys of single-CDDP and CDDP-rechallenged injuries. This was supported by the additional data that antisense reduced p21 protein levels in the cortex but not in the outer medulla of rats with a single CDDP treatment.

The exact cause of the lack of p21 antisense ODN effect in PT cells in the OSOM is not clear. The uptake of FITC-labeled p21 antisense ODNs by cortical PT cells was much greater than by PT cells in the OSOM. Like other 18- or 20-mer phosphorothioate ODNs injected into mice or rats ( 1, 16, 20 ), it is conceivable that the administered antisense could inhibit p21 induction mainly in the cortical PT cells, leading to increased sensitivity of cortical PT cells only to CDDP and leading them to undergo tubular damage. The other possible cause of the lack of effect is severe injury of PT cells in the OSOM following a single CDDP injection, rendering p21 antisense ODN nonfunctional or the fact that PT injury was already maximal after the single CDDP injection. Furthermore, antisense might not function properly in the regenerated PT cells that have acquired resistance, an unknown feature of the rechallenge injury of CDDP. Nevertheless, cortical PT cells protected by p21 induction do not seem to contribute largely to resistance to CDDP because renal function judged by S cr and BUN levels did not change significantly with antisense administration." Y6 h8 H4 I( B, t; K  l

Although the main site damaged by CDDP is the S3 segment of PTs, the less severe damage with apoptosis in PTs in the cortex was reported in mice and rabbits ( 10, 11 ). Renal functional and morphological damage in PTs in the cortex was also reported in rats ( 3, 18 ). However, the role of cortical PT damage in renal function in CDDP-induced ARF is unclear. Our findings suggest that cortical PT damage does not significantly affect renal function and that PT cells in the cortex are partially protected by induction or precedence of p21 in both CDDP-induced ARF and its acquired resistance. Megyesi et al. ( 12 ) reported that the p21 -/- mice showed aggravated PT damage as early as day 1 after CDDP injection compared with wild-type mice. They also showed that p21 -/- mice exhibited PT necrosis extending throughout S1 and S2 in the cortex and S3 segments in the OSOM, although wild-type mice exhibited PT necrosis restricted to the S3 segment. They concluded that induction of p21 after CDDP administration is a protective event for kidney cells ( 12 ). However, they did not specifically mention the role of p21 in cortical PT in CDDP-induced ARF. Because knockout of certain molecules can be sometimes compensated for by other molecules in vivo, the effect of knockout of p21 might differ from that of p21 antisense ODN in vivo. Our data support the findings using p21 knockout mouse by Megyesi et al. ( 12 ) that p21 induction may protect PT cells from death at least in the cortex in the CDDP-induced ARF model.
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We reported that overexpression of both p21 and PCNA in PT cells with a rechallenge injury may contribute to acquired resistance in CDDP-induced ARF via enhanced DNA repair ( 14 ). PCNA is expressed not only in proliferating S-phase cells but also in nonproliferating cells that are in G1-phase arrest and are in the DNA-repair phase ( 2, 7 ). Thus the increased number of PCNA-positive nuclei in the damaged kidney may, in part, reflect increased cells arrested in the G1 phase. In the present study, the number of PCNA-positive cortical PTs was increased in p21 antisense-treated kidneys in contrast to the decreased number of p21-positive PTs. This increased number of PCNA-positive PT cells may simply reflect increased proliferation activity after PT injury because most PCNA-positive PT cells were also positive for Ki 67 as a specific S-phase marker in the cortex.& x6 l* w8 P( H" _
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Our previous findings suggested that antiapoptotic mechanisms may be operational in acquired resistance of ARF ( 14, 21, 29 ). Recently, Price et al. ( 17 ) reported complete protection of a mouse kidney PT cell culture from CDDP-induced apoptosis by various cell cycle inhibitors, including a p21 adenovirus and the drugs roscovitine and olomoucine. They concluded that the protection by p21 was independent of the effect on the cell cycle and was likely due to selective inhibition of caspase-dependent and -independent cell death pathways. With regard to the link between p21 and apoptosis, it is worth examining the exact role of p21 in PT cells in the OSOM in CDDP-rechallenge injury by using other approaches (e.g., RNA interference) to inhibit p21.. ^- @3 z9 m0 P2 _' d
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In conclusion, we demonstrated in the present study that p21 in cortical PTs may, at least in part, play a cytoprotective role in both CDDP-induced ARF and its acquired resistance. However, the protection of PT cells in the cortex was not associated with protection of renal function; thus it is necessary to evaluate the exact role of p21 in PTs of the OSOM in CDDP-induced ARF and its acquired resistance.

GRANTS

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 15590846) and a Grant-in-Aid for the Center of Excellence from the Ministry of Education, Culture, Sports, Science and Technology (Japan).

ACKNOWLEDGMENTS9 k+ y3 X. ~1 C& m

We thank Nippon Kayaku Co., Ltd. (Tokyo, Japan), for kindly providing the CDDP for this study.
      【参考文献】
Butler M, Stecker K, and Bennett CF. Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues. Lab Invest 77: 379-388, 1997.

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Celis JE and Madsen P. Increased nuclear cyclin/P.CNA antigen staining of non-S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett 209: 277-283, 1986.
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