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First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
Correspondence to Dr. Akira Hishida, First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka, 431-3192, Japan. Phone: +81-53-435-2261; Fax: +81-53-434-9447; E-mail: ahishida{at}hama-med.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An increase in proliferating nuclear cell antigen (PCNA)-positive nuclei was recognized in ischemic (5) and nephrotoxic ARF including CDDP (2). The expression of PCNA initially was considered as a marker of proliferating cells (6). However, studies have demonstrated that PCNA is expressed not only in proliferating cells but also in nonproliferating cells that are in G1-phase arrest and are repairing their DNA (7,8,9,10). Thus, the increased PCNA-positive nuclei in the damaged kidney may, in part, reflect increased cells arrested in G1-phase. G1-phase arrest may be a critical step that determines the fate of a damaged cell (11). In the case of unsuccessful DNA repair, the cells will undergo apoptosis (11), which is reported to be closely associated with tubular damage in ischemic (5) and nephrotoxic (2,3) ARF.
p21, a cyclin-dependent kinase inhibitor, is known to play a key role in stopping the cycle at the G1 phase. Megyesi et al. (12) demonstrated upregulation of p21 mRNA in ARF-induced ischemia, CDDP, or ureteral obstruction. They also reported that in p21 knockout mice, CDDP caused a more rapid onset of ARF and induced more severe morphologic damage and higher mortality than in wild-type litter mates (13). These findings suggested that the induction of p21 plays a protective role in kidney cells by preventing DNA-damaged cells from progressing in the cell cycle without repair, which eventually would result in death from either apoptosis or necrosis (13). However, the exact roles of the increase in p21 in ARF remain unclear. If the p21-induced G1 arrest and DNA repair shown by PCNA expression play an important role in CDDP-induced ARF, then the degree of p21 and PCNA expression would be modulated in rechallenge injury.
In the present study, we evaluated the expression of p21 and PCNA and the number of apoptotic cells in the development of CDDP-induced ARF and in acquired resistance to CDDP. We also examined the expression of p53, which acts as a transcriptional activator to enhance expression of the p21 gene. A significant expression of p21 was observed in the development of ARF. The reduction in tubular damage after the second CDDP was associated with higher expression of p21. The expression of p21 during the development of ARF was associated with the overexpression of p53 and PCNA but not with 5-bromo 2'-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) incorporation. These data suggested that p21 may play a protective role to prevent the development of CDDP-induced ARF.
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Materials and Methods |
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Histologic Examination
For histologic examination, the excised left kidneys were fixed in 20%
neutral-buffered formalin solution. The kidney tissue block was dehydrated in
graded alcohol, embedded in paraffin, cut at 3 µm, and then stained with
periodic acid-Schiff reagent. Because tubular damage was most evident in the
outer medulla, we carried out a semi-quantitative analysis of the histologic
damage on this area. Thirty tubules were randomly selected at x400
magnification and scored for each rat. Each tubular profile was assigned one
of four categories according to the following criteria
(4): 1, areas of tubular
epithelial cell swelling, vacuolar degeneration, necrosis, and desquamation
involving <25% of tubular profile; 2, similar changes involving 
25% but
<50% of tubular profile; 3, similar changes involving 50% but <75%
of tubular profile, and 4, similar changes involving 75% of tubular
profile. To minimize observer bias, the morphometric examination was performed
in a blinded manner 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 calculated.
Immunohistochemical Detection of the Expression of p53, p21, and Cell
Cycle Markers (PCNA and BrdU Incorporation)
For immunohistochemical detection of p53, p21, and cell cycle markers,
3-µm-thick 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 polyclonal
antibody against p53 (Sant Cruz Biotechnology, Inc., Santa Cruz, CA) overnight
at 4°C, a monoclonal antibody (mAb) against p21 (Sant Cruz Biotechnology)
overnight at 4°C, a mAb against PCNA (Oncogene Science, Uniondale, NY) for
30 min at 37°C, or a mAb against BrdU (Amersham International plc,
Buckinghamshire, UK) for 1 h at 37°C. Then, samples were incubated with
peroxidase-conjugated goat anti-mouse IgG (Nichirei, Tokyo, Japan) for 10 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 or p53 were incubated overnight using
phosphate buffer solution for the primary antibody.
The numbers of p53-, p21-, PCNA-, and BrdU-positive nuclei in the outer stripe of the outer medulla were counted under a light microscope at x400 magnification. The number of positive nuclei for each time point was expressed as the average value based on the examination of 50 fields in each experimental animal by an investigator who was blinded to the treatment of each animal.
Assessment of Apoptosis
The degree of apoptosis was assessed using the in situ DNA nick
end labeling (TUNEL). We previously confirmed that the same dosage of CDDP
induced nuclear condensation on electron microscopy and a ladder pattern on
DNA electrophoresis in rats
(3). The tissue was
deparaffinized and rehydrated, followed by incubation of 3-µm-thick
sections with 20 µg/ml proteinase K for 15 min and immersion in distilled
water containing 3% hydrogen peroxide for 30 min at room temperature.
Detection of DNA fragmentation was performed using ApoTag plus in
situ Apoptosis Detection Kit (Oncor, Gaithersburg, MD). A
semiquantitative analysis was performed by counting TUNEL-positive cells per
field at x400 magnification in the outer stripe of the outer medulla.
The mean number of stained cells in 50 randomly selected fields in each kidney
was expressed as the number of TUNEL-positive cells.
mRNA Isolation and Semiquantitative Analysis of p21 mRNA by
RT-PCR
For isolation of p21 mRNA, the kidneys were perfused with icecold
diethyl pyrocarbonate-treated phosphate buffer solution. Total RNA was
extracted from whole kidneys in each rat (n = 3) using ISOGEN (Nippon
Gene, Inc., Tokyo, Japan) according to the manufacturer's protocol. After
treatment with DNase, we obtained poly-A+ enriched RNA by binding to oligo
(dT) cellulose beads according to Megyesi's protocol
(11). mRNA samples were
quantified by spectrophotometry at 260 nm.
Oligo-dT-primed cDNA was used to amplify for specific p21 mRNA species by RT-PCR. RT-PCR was performed using Super-Script ONE-STEP RT-PCR System (Life Technologies BRL, Gaithersburg, MD) according to the manufacturer's protocol. Each PCR cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 63°C (p21) or at 51°C (glyceraldehyde phosphate dehydrogenase [GAPDH]) for 1 min, and an extension step at 72°C for 1 min. The primers for p21 were 5'-CGGGCAGTCCCTTCTAGTTCC-3' (sense; 451 to 470 bp) and 5'-AATGCTTGAGCACACACGAG-3' (antisense; 654 to 635 bp), respectively. An intron was not included between two primers. These primers yielded a 204-bp product by amplifying at 30 cycles. RT-PCR products were run on 2% agarose gels and were visualized by ethidium bromide staining. All ethidium bromide—stained bands were quantified using NIH image version 1.61. We confirmed that mRNA samples without RT did not yield any band on agarose gel, indicating that PCR products were derived specifically from p21 mRNA but not from contaminated genomic DNA. The semiquantitative analysis of this RT-PCR product was conducted by factoring GAPDH-PCR products similarly determined by RT-PCR as the internal standard. At least three different samples were investigated at each time point.
Statistical Analyses
All data were presented as mean ± SEM. Significant differences among
data were determined using one-way ANOVA with standard post hoc
testing (Statview, version 5.0, Abacus Concepts, Berkeley, CA). P
< 0.05 denoted the presence of a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No staining of nuclei for p53 and p21 was found in normal kidneys. CDDP significantly increased the number of p53- and p21-positive nuclei in the outer stripe of the outer medulla (Figure 2, C and D). The number of p53-positive nuclei reached a peak at day 1 (6.16 ± 0.38/HPF, P<0.01 versus day 0) and disappeared by day 5 (Figure 3B). In contrast, the number of p21-positive nuclei had two peaks, at day 3 (2.17 ± 0.23/HPF, P<0.01 versus day 0) and day 9 (4.14 ± 0.35/HPF, P<0.01 versus day 0) (Figure 2E). A significant increase in p21-positive nuclei was sustained until 14 d after the injection of CDDP (Figure 3C). p21-positive nuclei were located mainly in the damaged cells at day 3 but in flat epithelial cells in regenerating tubules at day 9 (Figure 2E). No specific signal was noticed in the negative control sections (Figure 2G). p21 mRNA extracted from the CDDP-treated kidneys was detected as a 204-bp product by RT-PCR (Figure 4A). An increase in relative density of p21 mRNA divided by GAPDH mRNA became significant by day 2 and remained high until day 14 after the CDDP injection (Figure 4B).
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CDDP also induced two peaks of the increment of the number of PCNA-positive nuclei, at day 3 (11.01 ± 0.38/HPF, P < 0.01 versus day 0) and day 12 (10.25 ± 0.86/HPF, P < 0.01 versus day 0). The significant increase in PCNA expression was continued through day 14 (Figure 5A). In contrast, a significant increase in BrdU incorporation into the nuclei was not observed until day 5. A significant increase in BrdU incorporation began at day 5 and peaked at day 7 (8.42 ± 1.71/HPF, P < 0.01 versus day 0) and then gradually decreased through day 14 (Figure 5B).
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Rechallenge with CDDP Treatment
In group 2, the subsequent CDDP was given 14 d after the first CDDP
injection at the time when Scr returned to normal values. The second CDDP
injection also induced a significant increase in Scr, but the increase in Scr
levels 5 d after the second injection of CDDP (group 2) was significantly less
than those after the first injection (group 1, 1.70 ± 0.34 mg/dl; group
2, 0.98 ± 0.07 mg/dl; P < 0.05;
Figure 1). Pretreatment with
CDDP also significantly attenuated the CDDP-induced tubular damage score (3.91
± 0.04 versus 2.16 ± 0.14, n = 8; P
< 0.001). The number of TUNEL-positive cells at the time of CDDP injection
was slightly increased in group 2 when compared with group 1 (group 1, 0.01
± 0.01/HPF; group 2, 0.52 ± 0.28/HPF; P = NS). The
second injection of CDDP did not induce further increases in the number of
TUNEL-positive cells in the outer stripe of the outer medulla
(Figure 2B,
Figure 3A). The number of
TUNEL-positive cells in group 2 was significantly lower than in group 1 at day
5 (P < 0.01; group 1, 6.24 ± 0.91; group 2, 0.30 ±
0.07/HPF; Figure 3A).
The number of p53-positive nuclei in group 2 was identical with those in normal rats at the time of injection. The second injection of CDDP significantly increased the number of p53-positive nuclei in the outer stripe of the outer medulla at day 1 (2.17 ± 0.37/HPF, P < 0.01, versus day 0 in group 2). This significant increase in p53 expression was also found at day 2 (2.62 ± 0.25/HPF, P < 0.01, versus day 0 in group 2) but disappeared by day 3. The increase in p53 expression in group 2 was significantly less than that in group 1 at days 1 and 2 (Figure 3B).
The increased number of p21-positive nuclei in the outer stripe of the outer medulla was still observed on the day of the second CDDP injection (Figure 3C). The second injection of CDDP increased further the p21 expression at day 2 (Figure 2F). Thus, the number of p21-positive nuclei in group 2 was significantly higher than that in group 1 at days 0 through 2 (Figure 3C). The expression of p21 mRNA in kidneys on the day of the second CDDP injection was also significantly increased compared with normal kidneys (Figure 6). However, the second injection of CDDP did not induce any change in the expression of p21 mRNA. The degree of p21 mRNA expression at days 2 through 5 was similar between both groups (Figure 6).
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The numbers of PCNA- and BrdU-positive nuclei in the outer medulla before the second CDDP injection were higher than those in control rats. The number of PCNA-positive nuclei was also significantly increased by day 2 after the second CDDP injection (Figure 5A). The PCNA expression in group 2 was significantly higher than those in group 1 on days 0 through 2 (Figure 5A). In contrast, the number of BrdU-positive nuclei was significantly decreased at 5 d after the second CDDP injection in group 2 compared with those in group 1 (Figure 5B).
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Discussion |
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The expression of p21 mRNA is usually limited in untreated adult animal kidney (11, 14) but upregulated after ischemia-and CDDP-induced renal injury (11). In this study, we also confirmed by RT-PCR analysis a faint expression of p21 mRNA in the kidney before CDDP injection. CDDP increased the amount of PCR products by day 2 and lasted until day 14 after the CDDP injection. The number of p21-positive nuclei was also increased in the outer stripe of the outer medulla 3 d after CDDP injection, and this significant increase was observed until day 14. These data suggest that CDDP upregulates the long-term expression of p21.
p21, which forms a quaternary complex with cyclins, cyclin-dependent kinases (cdk), and PCNA (15), is a multifunctional protein that is involved in coordination of the cellular response to stress (11). p21 has been shown to inhibit one or more of the cyclin-cdk activities (16, 17), and the induction of p21 is associated with cell-cycle interruption and G1 arrest. PCNA plays a role in nucleotide excision DNA repair (18) in addition to its putative role in replicative DNA synthesis (7,8,9,10). p21 is reported to bind PCNA and inhibit the action of PCNA (15, 19, 20), especially the action in DNA replication (18). p21 itself does not inhibit the DNA repair action of PCNA (21). In this study, we demonstrated that CDDP injection induced a significant expression of p21 and PCNA in the outer stripe of the outer medulla by day 3. However, we could not find a significant increase in BrdU-positive nuclei, an S-phase marker (22), at this early phase, suggesting that the expression of PCNA observed at 3 d after CDDP injection may reflect a facilitation of DNA repair rather than cell proliferation. These observations also suggest that the increase in p21 in the early phase of CDDP-induced ARF may stop the cell cycle at the G1 phase and could inhibit the damaged tubular cells from entering to S phase, providing the time for DNA repair.
In the current study, we found a significant increase in p21-positive nuclei mainly in the flat epithelium of dilated tubules at the recovery phase of CDDP-induced ARF. Differentiating cells are known to express p21 and to manifest a prolongation of the G1 phase of the cell cycle and an inhibition of G1-S transition (23,24,25,26). The induction of p21 in cellular differentiation is coupled with the expression of early differentiation markers (26), suggesting that p21 protein may function as an inducible growth inhibitor that contributes to stopping the cell cycle and facilitating differentiation (24). The expression of p21 in differentiating cells is reported to be p53 independent (25, 26). In accordance with these findings, the expression of p21 during the recovery phase was not associated with the increase in p53-positive nuclei in the outer stripe of the outer medulla. Taken together, these findings suggest that p21 expression in the recovery phase of CDDP-induced ARF could play a role in the redifferentiation of the proliferating tubular cells in the damaged tubules via a p53-independent pathway. To our knowledge, this is the first study that showed the induction of p21 expression in the recovery phase of CDDP-induced ARF and the possible involvement of p21 in the differentiation of the damaged tubules.
It is known that prior ARF provides resistance to a subsequent rechallenge with renal insults (1, 2, 27). We recently found that rats that are recovering from ARF are functionally and morphologically resistant to CDDP (3, 4). Pretreatment with 2 mg/kg CDDP also affords a protective effect for subsequent 5 mg/kg CDDP nephrotoxicity (4). In accordance with these previous reports, the second injection of CDDP in group 2 was associated with the attenuated increase in Scr and less tubular damage. The numbers of p21- and PCNA-positive nuclei were increased in the outer stripe of the outer medulla after the second injection of CDDP in group 2, and the number of p21- and PCNA-positive nuclei were significantly higher than those in group 1 at days 1 through 3. The higher expression of PCNA after the second CDDP injection was associated with a significant suppression of BrdU incorporation, suggesting that this higher expression of PCNA did not indicate an increase in cell proliferation. These observations suggest the possibility that the increases in p21 and PCNA in group 2 may indicate enhanced DNA repair in damaged tubules and that p21 may contribute to the reduced amount of tubular damage in acquired resistance by stopping the cell cycle in the G1 phase and providing enough time for DNA repair.
Recent studies demonstrated the existence of apoptotic cell death in several ARF models induced by ischemia-reperfusion (5), uranium acetate (2), ureteral obstruction (11), and CDDP (3, 4). In our previous study, we found a close correlation between the numbers of TUNEL-positive cells and the degree of tubular damage and renal dysfunction (3), suggesting an important role of apoptotic cell death in the development of CDDP-induced ARF. However, the exact mechanism for the induction of apoptosis in ARF is not clear. In addition to several molecules, including the Bc1-2 family (28) and Fas (29), p53 is known to be a key protein of apoptotic cell death (30, 31). The expression of p53 in the kidney was shown by 24 h after CDDP injection in mice (12). In our study, the number of p53-positive nuclei was increased transiently between days 1 and 3 after CDDP injection, followed by the expression of TUNEL-positive cells. Furthermore, the reduced induction of p53-positive nuclei after the second CDDP injection in group 2 was associated with the suppression of the increase in TUNEL-positive cells. Thus, it is possible that, in part, p53 may play a role in the induction of apoptosis in CDDP-induced ARF.
It is also demonstrated that the cdk inhibitor, p21, can inhibit apoptosis in C2C12 myocyte (32) and human breast epithelial cells (33). In the current study, we found that the induction of p21 by the pretreatment with CDDP significantly attenuated the increase in TUNEL-positive cells after the second injection of CDDP in group 2, suggesting that p21 plays a role in the modulation of CDDP-induced apoptotic cell death. In agreement with this hypothesis, Megyesi et al. (13) reported that CDDP-induced renal dysfunction, tubular damage, and the number of TUNEL-positive cells were significantly greater in mice homozygous for the p21 deletion than wild-type litter mates. In rats that are recovering from previous CDDP-induced ARF, antiapoptotic protein, Bcl-2, is also overexpressed (3). However, it is not known which of these antiapoptotic factors (Bcl-2 and p21) is more important in the acquisition of resistance to the rechallenge injury, and the relationship between p21 and Bcl-2 expression also remains to be clarified.
In case of DNA damage, the p21 gene is induced both by p53-dependent (34,35,36) and p53-independent (14, 37, 38) pathways. Megyesi et al. (12) demonstrated by using p53 knockout mice that CDDP injection induced high levels of p21 mRNA in kidneys through both p53-dependent and p53-independent pathways. In our study, p21 protein and mRNA expression shortly after the first CDDP injection were provoked following the increment of p53, a compatible finding by Megyesi et al. (12). The increase in p21-positive nuclei observed 2 d after the second CDDP injection was also preceded by the increase in p53-positive nuclei. However, this increment of p21 protein after the second CDDP injection was not associated with the increase in mRNA level. The reasons for this unchanged mRNA level after the second CDDP attack remain unknown. Immunohistochemical examination revealed a similar positive staining of p21 in tubular nuclei mainly located in the outer stripe of the outer medulla as well as single CDDP injection. Interestingly, Gorospe et al. (11) reported that p21 expression in DNA injury was regulated through a posttranscriptional mechanism including stabilization of p21 mRNA and that this induction was also dependent on the presence of functional p53. Thus, it is possible that the expression of p21-positive nuclei in group 2, which was associated with the increased expression of p53 but not with the increase in p21 mRNA, may be regulated through a posttranscriptional mechanism. We, however, used whole kidney to evaluate the changes in p21 mRNA in the current study; therefore, we cannot exclude the possibility that a small increase in p21 mRNA, which might cause the increased p21 protein in rechallenge injury, was missed. The increase in p21 expression during the recovery phase was not associated with an increased expression of p53 in the kidney. This finding is in agreement with previous reports, which showed that the expression of p21 in differentiating cells was p53 independent (23, 24).
In conclusion, CDDP injection induced an increase in the number of p21-positive nuclei in the outer stripe of the outer medulla and the expression of p21 mRNA in the kidneys. The increase in p21, which was induced by p53-dependent and p53-independent pathways, might contribute to the DNA repair and redifferentiation in damaged tubules in CDDP-induced ARF. The acquired resistance to CDDP in rechallenge injury might be mediated in part by the increase in p21.
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Acknowledgments |
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References |
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holoenzyme. Proc Natl Acad Sci
USA 91:8655
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