1 Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington 98195; and 2 Department of Pathology, University of Aberdeen, Aberdeen, AB25 2ZD Scotland, United Kingdom
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ABSTRACT |
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Tubulointerstitial renal injury induced by unilateral ureteric
obstruction (UUO) is characterized by marked cell proliferation and
apoptosis. Proliferation requires cell cycle transit that is positively regulated by cyclins and cyclin-dependent
kinases (CDKs) and inhibited by the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs: p21, p27, and p57). We have shown that the
absence of p27 results in markedly increased tubular epithelial cell
proliferation and apoptosis following UUO (V. Ophascharoensuk, M. L. Fero, J. Hughes, J. M. Roberts, and S. J. Shankland. Nat. Med.
4: 575-580, 1998). Since p21 mRNA is upregulated following UUO, we hypothesized that p21 would also serve to limit cell
proliferation and apoptosis. We performed UUO in p21 +/+ and p21
/
mice. Cell proliferation [bromodeoxyuridine
(BrdU), proliferating cell nuclear antigen (PCNA)], apoptosis
[terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL) method], interstitial myofibroblast accumulation (actin), macrophage infiltration (F4/80), and collagen I
expression were quantified at days 3, 7, and
14. In contrast to p27
/
mice, there was no
difference in tubular epithelial cell proliferation or apoptosis
between p21
/
and p21 +/+ mice at any time point.
However, interstitial cell proliferation at day 3 was
significantly increased in p21
/
mice [BrdU, 40.7 ± 1.9 cells/high-power field (cells/hpf) vs. 28.8 ± 2, P < 0.005], although, interestingly, no difference was seen in
interstitial cell apoptosis. Actin/BrdU double staining demonstrated
increased interstitial myofibroblast proliferation at day 3 in
p21
/
animals (10 ± 0.12 vs. 5.8 ± 0.11 cells/hpf,
P < 0.05), which was followed by increased myofibroblast
accumulation at day 7 in p21
/
mice. No
differences were detected in interstitial macrophage infiltration, collagen I deposition or transforming growth factor-
1 mRNA (in situ
hybridization) expression. In conclusion p21, unlike p27, is not
essential for the regulation of tubular epithelial cell proliferation
and apoptosis following UUO, but p21 levels do serve to limit the
magnitude of the early myofibroblast proliferation. This study
demonstrates a differential role for the CKI p21 and p27 in this model.
cell cycle; myofibroblast; apoptosis; kidney
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INTRODUCTION |
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MANY STUDIES INDICATE THAT tubulointerstitial renal disease is associated with both cell proliferation and apoptosis. However, over time, an imbalance between these processes leads to unchecked apoptosis resulting in progressive cell loss, renal tubular atrophy, and interstitial fibrosis (15). Indeed, the importance of the renal interstitium in both human nephropathies and animal models of renal disease is strongly underscored by the well-documented correlation between the severity of tubulointerstitial changes and the subsequent development and progression of chronic renal failure (4).
We now have significant insights into the basic biological mechanisms governing the proliferation of cells, which involve transit through the cell cycle (31). Cells pass from quiescence (G0) through G1 into S phase, where DNA synthesis occurs, followed by G2 and mitosis (M phase), resulting in two daughter cells. The cell cycle is tightly controlled by both positive and negative regulators. Positive regulators include the cyclins and their catalytic partners cyclin-dependent kinases (CDK) which are essential for cells to progress through each phase of the cell cycle and to overcome the various cell cycle checkpoints (34, 35). Negative regulators include the cyclin kinase inhibitors (CKI) which inhibit the cell cycle at multiple checkpoints through inactivation of cyclin-CDK complexes (36).
There are two families of CKI, which are defined on the basis of their sequence homology and the cyclin-CDK complexes that they inhibit. Individual CKI are named according to their molecular weight. The INK4 family of CKI includes p15, p16, p18, and p19 and inhibit the action of the G1 cyclin-CDK complexes (36). The CIP/KIP family of CKI includes p21WAF1/CIP1 (p21) (14, 19, 23), p27KIP1 (p27) (32, 33, 37), and p57KIP2 (p57) (25, 22), and these inhibit cyclin-CDK complexes during both G1 and S phases of the cell cycle.
The CKI p21 is different from other members of the Cip/Kip
family. Unlike p27 or p57, p21 has the additional function
of binding and inhibiting the proliferating cell nuclear antigen
(PCNA), which plays an important role in both DNA replication and DNA repair (5, 6, 24). Furthermore, p21 also inhibits G2/M kinases, and limits mitosis (38). p21 levels are transcriptionally regulated through p53-dependent (14) and p53-independent pathways (8,
26), particularly by the action of the cytokine transforming growth
factor- (TGF-
) (8, 23). In addition, p21 has been shown to
regulate apoptosis in a variety of cell types in vitro (3, 12, 16, 17).
The model of unilateral ureteric obstruction (UUO) induces severe tubulointerstitial injury and is characterized by tubulointerstitial inflammation comprising an interstitial mononuclear cell infiltrate with both interstitial myofibroblast and tubular epithelial cell proliferation early in the course of the disease (10, 11). However, over time, continued apoptosis of all cell populations results in significant loss of renal tissue and scarring (11, 15).
Although p21 levels are increased in experimental UUO, the exact role
of this cell cycle regulatory protein in this disease is not known
(26). To test the hypothesis that p21 acts to limit tubulointerstitial
inflammation, we utilized mice with a targeted disruption of the p21
gene (p21 /
) (9). The current study demonstrates that, in
contrast to p27 knockout mice (30), overall tubular epithelial cell
proliferation and apoptosis are unchanged in p21
/
mice
compared with control p21 +/+ mice, indicating that the function of p21
is redundant in the tubular compartment of the kidney in this model.
Similarly, an absence of p21 had no effect upon interstitial cell
apoptosis or interstitial macrophage infiltration. However, p21
/
mice develop augmented proliferation of interstitial
myofibroblasts early in the course of the disease without increased
apoptosis, resulting in an increased accumulation of
myofibroblasts. This study demonstrates that p21 plays a
role in limiting the early proliferation of interstitial myofibroblasts in this model of tubulointerstitial renal injury. In addition, these
data suggest that the effect of p21 may vary according to the
particular cell type and also the nature of the injurious stimulus.
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METHODS |
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Experimental animals. Deletion of the murine p21 gene was
performed by targeted disruption. Briefly, the p21 gene was mutated by
deletion of exon 2 with creation of a null mutation through homologous
recombination (9). Homozygous null mutant mice (p21 /
)
were viable and fertile, and breeding pairs of a C57 BL6 genetic
background were established to produce the animals used in this study.
Wild-type (p21 +/+) breeding pairs of the same background were used as
controls. The genotype of animals was confirmed by PCR analysis.
Description of the model of UUO. Experimental unilateral
ureteric ligation resulting in UUO was performed in 10- to 14-wk-old female p21 /
and p21 +/+ mice by ligation of the left
ureter of each animal at the ureteropelvic junction under anesthesia (intraperitoneal injection of ketamine and xylazine), as previously described (30). Mice were killed on days 3, 7, and
14 (n = 5-6 at each time point), and renal
biopsies were performed. To accurately measure DNA synthesis, each
animal was injected with bromodeoxyuridine (BrdU) intraperitoneally 4 h
prior to death (1 ml/100g body wt, Cell proliferation kit; Amersham
Pharmacia Biotech, Bucks, UK). Renal biopsies were fixed in Formalin or
methyl Carnoy's solution (60% methanol, 30% chloroform, and 10%
acetic acid) and embedded in paraffin. These studies were performed in
an accredited animal care facility in accordance with the National
Institutes of Health "Guide for the Care and Use of Laboratory Animals."
Renal morphology and immunohistochemistry. To examine renal histology, 4-µm sections were stained with periodic acid-Schiff reagent and counterstained with hematoxylin. To perform single immunoperoxidase staining, methyl Carnoy's fixed tissue sections were incubated with the following primary and secondary antibodies as indicated.
Tissue sections were incubated with a murine IgG2a monoclonal antibody against BrdU (Cell proliferation kit; Amersham) at room temperature for 60 min, followed by a peroxidase-conjugated goat anti-mouse IgG2a (Cell proliferation kit, Amersham) at room temperature for 30 min. Prior to incubation with these antibodies, the tissue sections underwent an antigen retrieval step comprising boiling in 0.01 M sodium citrate buffer for a total of 10 min.
Tissue sections were incubated in murine IgM monoclonal antibody against PCNA (Coulter Immunology, Hialeah, FL) at 4°C overnight, followed by a peroxidase-conjugated rat anti-mouse IgM monoclonal antibody (Zymed Laboratories, San Francisco, CA) at room temperature for 60 min.
Tissue sections were incubated in a mouse IgG2a monoclonal antibody against smooth muscle actin (Sigma Chemical, St. Louis, MO) at 4°C overnight, followed by a biotinylated rat anti-mouse IgG2a IgG (Zymed) at room temperature for 60 min.
Tissue sections were incubated in a rat IgG2b monoclonal antibody (F4/80) against mouse macrophages (Caltag Laboratories, Burlingame, CA) at 4°C overnight, followed by a mouse-adsorbed biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) at room temperature for 30 min.
Tissue sections were incubated with a guinea pig anti-rat type I collagen antibody that also detects with murine type I collagen (gift of Dr H. Sage, University of Washington) at 4°C overnight, followed by a biotinylated goat anti-guinea pig IgG (Vector Laboratories) at room temperature for 30 min.
Horseradish peroxidase-conjugated avidin D (Vector Laboratories) was used after all biotinylated secondary antibodies at room temperature for 20 min. Black nuclear (BrdU, PCNA) or brown cytoplasmic (actin, F4/80) or interstitial (collagen I) staining was developed using diaminobenzidine (DAB, Sigma Chemical) with or without nickel as the chromogen, respectively, and counterstained with methyl green and eosin. An irrelevant primary antibody of the same isotype was used as negative control. Positive control tissue included sections from diseased mice that expressed these antigens.
The number of PCNA- or BrdU-positive cells in each biopsy was calculated in a blinded fashion by counting the number of positive tubular and interstitial cells in 20 sequentially selected fields of renal cortex at ×250 magnification and expressed as the mean number ± SE per high-power field (hpf). Smooth muscle actin expression, macrophage infiltration, and collagen I expression in each biopsy was calculated in a blinded fashion by scoring 20 sequentially selected fields of renal cortex at ×250 magnification using a semiquantitative scale of 0 to 4 as follows: grade 0, no expression; grade 1, minimal expression (1-9% of interstitium); grade 2, mild expression (10-49%); grade 3, moderate expression (50-89%); grade 4, marked expression (>90%). Data were expressed as the mean score ± SE per hpf.
Detection of apoptosis. Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay as previously described (2). Briefly, 4-µm Formalin-fixed tissue sections were deparaffinized and rehydrated in ethanol followed by an antigen retrieval step consisting of boiling in 0.01 M sodium citrate buffer for 2 min. Sections were then incubated with proteinase K (6.2 µg/ml; Boehringer Mannheim, Indianapolis, IN) followed by TdT (300 enzyme units/ml; Pharmacia Biotech, Piscataway, NJ) and Bio-14-dATP (0.94 nM; GIBCO-BRL, Life Technologies). Biotinylated ATP was detected using the ABC staining method (Vector Laboratories; following the manufacturer's protocol). As a positive control, slides were pretreated with DNase (20 Kunitz units/ml; Sigma Biosciences, St. Louis, MO). Cells were regarded as TUNEL positive if their nuclei were stained black and displayed a typical apoptotic morphology with chromatin condensation. The number of TUNEL-positive cells in each biopsy was calculated in a blinded fashion by counting the number of TUNEL-positive tubular and interstitial cells in 20 sequentially selected fields of renal cortex at ×250 magnification and expressed as the mean number ± SE per hpf.
Double immunostaining. BrdU/actin double immunostaining was performed to determine which interstitial cell-type underwent increased DNA synthesis. Sections were first stained for actin as described above using DAB without nickel as the chromogen. Sections were then washed and boiled in 0.01 M sodium citrate buffer (pH 6.0) for a total of 10 min before being stained for BrdU using DAB with nickel as the chromogen. The number of BrdU+/actin+ cells in each biopsy was calculated in a blinded fashion by counting the number of BrdU+/actin+ interstitial cells in 30 sequentially selected fields of renal cortex at ×630 magnification and expressed as the mean number ± SE per hpf.
To identify and accurately quantify proximal versus distal tubular
epithelial cell proliferation and apoptosis, Fx1A/BrdU and Fx1A/TUNEL
double labeling was performed. The Fx1A antibody was raised against rat
proximal tubular brush border antigen as described previously and
specifically stains the brush border of proximal tubular epithelial
cells (13). Sections were incubated with biotinylated sheep Fx1A
antibody at 4°C overnight, followed by horseradish
peroxidase-conjugated avidin D (Vector Laboratories) for 20 min at room
temperature. Staining was developed using DAB without nickel to produce
a brown color. BrdU or TUNEL staining was then performed as indicated
previously using DAB with nickel as the chromogen. The number of
Fx1A+/BrdU+,
Fx1A/BrdU+,
Fx1A+/TUNEL+, and
Fx1A
/TUNEL+ cells in each biopsy was
calculated in a blinded fashion by counting 20 sequentially selected
fields of renal cortex at ×250 magnification and expressed as the
mean number ± SE per hpf.
In situ hybridization for TGF-1. A mouse
TGF-
1 cDNA clone (gift of Dr H. L. Moses, Vanderbilt University,
Nashville, TN) was transcribed into 35S-labeled antisense
and sense cRNA probes. The probes correspond to 975 bp
(421-1395) of mouse TGF-
1. In situ hybridization
was then performed as previously described (29). Briefly,
Formalin-fixed sections were digested with 10 µg/ml proteinase K and
hybridized with 5 × 105 cpm of
35S-labeled cRNA probe. Overnight incubation was followed
by 2× SSC washes, then RNase A digestion (20 µg/ml) for 30 min
at 37°C. Three stringency washes comprising 0.1× SSC and
0.5% Tween at 50°C were followed by dehydration and air drying.
The slides were dipped in photographic emulsion (Kodak NTB2), exposed
at 4°C for 4 wk and developed with D-19 developer (Eastman Kodak,
Rochester, NY), and counter-stained with hematoxylin and eosin. In situ
hybridization was quantified in a blinded fashion as previously
described (18). Briefly, sequentially selected fields of renal cortex
at ×400 magnification were examined using a grid until at least
200 positive cells per biopsy had been counted. Data was expressed as
the mean number of positive cells ± SE per square millimeter.
Statistical methods. All values are expressed as means ± SE. Statistical significance (defined as P < 0.05) was evaluated using the Student's t-test.
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RESULTS |
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Renal tubular epithelial cell proliferation is unchanged in p21
/
mice with UUO. Tubular epithelial cell
proliferation and DNA synthesis was assessed by the incorporation of
BrdU. There was no difference in the basal level of renal tubular
epithelial cell proliferation between p21
/
and p21 +/+
mice, since p21
/
mice have normal kidneys (results not
shown). There was a 4.3-fold increase in tubular epithelial cell
proliferation at day 3, the peak of proliferation, in p21 +/+
mice (Fig. 1). At days 7 and
14 proliferation was increased two- to threefold in p21 +/+
mice compared with controls. However, there was no significant difference in tubular epithelial cell proliferation between p21
/
and p21 +/+ mice at any of the time points studied.
Similar results were also obtained with PCNA immunostaining, a marker of DNA synthesis (data not shown). In addition, there was no increase of proximal (Fx1A+/BrdU+) or distal
(Fx1A
/BrdU+) tubular epithelial cell
proliferation demonstrated in p21
/
mice (data not
shown).
|
Interstitial cell proliferation is increased at day 3 in p21
/
mice with UUO. Interstitial cell proliferation as
measured by BrdU, and PCNA staining was markedly increased in p21 +/+
mice at day 3 following UUO (Figure
2). However, a significant further increase
in proliferation was evident in the interstitium of p21
/
mice at the peak of proliferation at day 3 by BrdU
immunostaining (Figs. 2 and 3). Formal quantification demonstrated a
41% increase in BrdU-positive interstitial cells at day 3 in
p21
/
mice compared with p21 +/+ mice (P < 0.005), and although there was also a trend at day 7, this was
not significant. The significant increase in interstitial cell DNA
synthesis in the obstructed kidneys of p21
/
mice was
confirmed with PCNA staining. p21
/
mice demonstrated a
significant 32% increase in interstitial cell PCNA expression at
day 3 compared with their wild-type controls (Fig. 2).
|
Interstitial myofibroblast proliferation is increased at day 3 in
p21 /
mice with UUO. The interstitial population of
cells following UUO is composed mainly of myofibroblasts, fibroblasts, and infiltrating macrophages (10, 11). To determine which interstitial
cells were undergoing increased proliferation, we performed double
labeling for BrdU, a marker of DNA synthesis, and smooth muscle actin,
a marker of myofibroblasts (Figure 3), since previous work indicated that this cell type plays a prominent role in this model (26). Quantification demonstrated a 72% increase in
actively proliferating myofibroblasts
(BrdU+/actin+) in p21
/
mice at
day 3 following UUO (Figure 4).
These results show that the myofibroblast was the predominant cell type
proliferating in p21
/
mice following renal injury
induced by obstruction.
|
|
Interstitial myofibroblast accumulation is increased at day 7 in
p21 /
mice with UUO. Interstitial myofibroblast
accumulation was identified by immunostaining for smooth muscle actin.
There was a significant 45% increase in interstitial actin expression at day 7 in p21
/
mice compared with p21 +/+ mice
(P = 0.005), indicating increased myofibroblast accumulation in
p21
/
mice at day 7 (Figs. 4 and 5).
Renal tubular epithelial cell and interstitial cell apoptosis is
unchanged in p21 /
mice with UUO. Apoptosis of
tubular and interstitial cells was quantified by the TUNEL assay (Fig. 5). Apoptosis of both cell populations
increased inexorably throughout the study at the time points examined.
However, there was no detectable difference in tubular epithelial cell
apoptosis between p21
/
and p21 +/+ mice at any time
point studied (Fig. 6). In addition, there
was no increase of proximal (Fx1A+/TUNEL+) or
distal (Fx1A
/TUNEL+) tubular epithelial
cell apoptosis demonstrated in p21
/
mice (data not
shown). Similarly, there was no difference in interstitial cell
apoptosis between p21
/
and +/+ mice (Fig. 6). These
results suggest that the increased interstitial accumulation of
myofibroblasts evident at day 7 in p21
/
mice is
unlikely to be due to differences in the rates of cell death between
p21 knockout and wild-type mice.
|
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Macrophage recruitment, collagen I deposition and
TGF-1 mRNA (day 3) are unchanged in p21
/
mice with UUO. There was no detectable difference in interstitial
macrophage infiltration between p21
/
and p21 +/+ mice at
the time points studied (Fig. 7). Despite
the increased myofibroblast accumulation seen in p21
/
mice at day 7 there was no difference in interstitial
deposition of collagen I between p21
/
and +/+ mice at
days 7 and 14 (Fig. 7). Last, there was no detectable
difference in TGF-
1 mRNA expression (assessed by in situ
hybridization) at day 3 between p21
/
(39 ± 12 positive cells/mm2) and p21 +/+ mice (36 ± 11 positive cells/mm2).
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DISCUSSION |
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This study addresses the role of the CKI p21 in tubulointerstitial renal disease induced by unilateral ureteral obstruction (UUO). We show that the absence of p21 was not associated with increased tubular epithelial cell proliferation and apoptosis as expected, but rather was associated with an early increase in interstitial cell (myofibroblast) proliferation. These results show a differential effect of p21 in tubulointerstitial injury.
Many forms of acute tubulointerstitial disease are characterized by the proliferation of resident (tubular and interstitial) cells and an influx of infiltrating cells into the regions of injury. Moreover, tubulointerstitial injury is often accompanied by an increase in resident renal cell death due to apoptosis. These early events underlie and often predict the development of progressive tubulointerstitial disease with scarring and declining renal function.
There is a growing body of literature showing that specific cell cycle proteins regulate proliferation and apoptosis. Earlier cell culture studies showed that proliferation requires the activation of CDK by partner cyclins. Moreover, the inhibitory threshold of CKI such as p21 needs to be overcome to allow CDK activation. More recent studies have examined the role of cell cycle proteins in inflammatory renal and nonrenal diseases. In this study, we focused on the role of the CKI p21 in inflammatory tubulointerstitial disease, because p21 mRNA has been shown to be rapidly upregulated in experimental tubulointerstitial injury induced by ischemia-reperfusion and following UUO (26, 28).
This evidence suggested but did not prove that CKIs may act to limit
cell proliferation and apoptosis following an injurious insult and
thereby play an important role in the resolution of tubulointerstitial
renal injury. Accordingly, in this study, we induced acute ureteral
obstruction in p21 /
mice and controls to determine the
role of p21 in tubular and interstitial cell proliferation and
apoptosis in inflammation. The first major finding was that the absence
of p21 had no discernible effect upon tubular epithelial cell
proliferation or apoptosis in tubulointerstitial disease induced by
UUO. These results were somewhat unexpected, because the absence of p21
has been shown to be associated with increased tubular epithelial cell
proliferation and apoptosis in similar (p21
/
) mice with
tubulointerstitial disease induced by cisplatin (27).
Moreover, we have recently shown that acute UUO induced in p27 knockout
mice was associated with a markedly increased tubular epithelial cell
proliferation and apoptosis (30). The second major finding in this
study was that, in contrast to the tubular epithelial cell, the same
form of injury was associated with an early increase in interstitial
cell proliferation in obstructed p21
/
mice compared with
controls. Double labeling indicated that most of these proliferating
cells were myofibroblasts. These results showed that the absence of p21
had a differential role in regulating proliferation in the two resident
cell types of the tubulointerstitium. Interestingly, the absence of p21
had no significant effect upon the rate of myofibroblast apoptosis early in the disease, since there was a net accumulation of
myofibroblasts at day 7. However, it should be noted that the
interstitial myofibroblast population does decrease significantly
between day 7 and day 14 in p21
/
mice.
One can speculate that this might be secondary to a wave of increased
apoptosis in the knockout animals at some time point prior to 14 days,
since it is not apparent in TUNEL stained slides from day 14 biopsies.
If there was an increased rate of interstitial myofibroblast apoptosis later in the disease, then it may be secondary to factors such as a limited supply of cytokine survival factors as well as the proapoptotic effects of ischemia and hypoxia upon the expanded myofibroblast population. Unfortunately, it is extremely difficult to study the rates of apoptosis of individual cell populations within solid tissues. This is because previous studies have shown that the majority of apoptotic cells in both physiological and pathological circumstances are actually within the phagolysosomes of adjacent cells or infiltrating professional phagocytes such as the macrophage (2, 7). Therefore, in the case of myofibroblasts, performing actin/TUNEL double immunostaining would be unhelpful since the TUNEL-positive apoptotic cell may be derived from a myofibroblast, a fibroblast, or even a macrophage which has subsequently been phagocytosed by an actin-positive myofibroblast. This problem therefore mitigates against an accurate quantitative analysis of interstitial myofibroblast death in this study.
In vitro studies have shown that lowering of p21 levels is
associated with an increase in proliferation in response
to mitogenic stimuli, and that forced overexpression of p21 inhibits
proliferation. This study failed to show the predicted increase in
tubular epithelial cell proliferation following injury, thereby raising
a number of important biological lessons. First, we showed that there
is a differential role for p21 in limiting proliferation within the same organ that may be cell type specific. Here, we
showed that p21 limits interstitial myofibroblast but not tubular
epithelial cell proliferation following injurious stimuli that underlie
a fibrogenic response. Moreover, we have shown that
immune mediated injury to the glomerulus is associated with increased
glomerular cell proliferation in p21 /
mice compared with
controls (21).
Second, the protective function of p21 within a specific cell type may
depend on the form of injury. Thus, in contrast to the marked increase
in tubular epithelial cell proliferation and apoptosis in tubular
epithelial cells induced by the genotoxin cisplatin,
ureteral obstruction was not associated with increased tubular
epithelial cell proliferation in p21 /
mice compared with
wild-type mice. This disparity in the effect of an absence of p21 in
tubulointerstitial disease induced by UUO compared with cisplatin may
be explained at least in part by differences in the type of injury and
the role of p53 in the model. p21 is the mediator of the cell cycle
arrest that follows activation of p53 by injury such as irradiation or
genotoxic drugs such as cisplatin, which results in genomic DNA
damage. Although p53 induction occurs in both models of
renal injury, it is likely to be much higher in the cisplatin model.
The absence of p21 in the context of widespread DNA damage may be more
critical, since the failure to induce cell cycle arrest in these cells
prevents DNA repair from occurring, allowing progression
through the cell cycle with cells dying by default as a result.
Finally, it is noteworthy that tubular epithelial cell DNA synthesis is
also increased in p21
/
mice with diabetes mellitus
compared with p21 +/+ mice with the same degree of hyperglycemia, a
result which underscores the importance of the form of injury upon
which genes are protective (1).
Third, this study shows that different CKIs may have biological effects
in vivo different from what has been reported in vitro. In contrast to
p21 /
mice in the current study, we have previously shown
that tubular epithelial cell proliferation and apoptosis are markedly
increased in p27
/
mice with ureteral obstruction compared
with controls. However, another possibility is that other CKIs are
upregulated in order to compensate for the lack of p21. However, we
found no evidence of increased expression of p27 or p57 in this model
by immunostaining (data not shown), although we are cognizant of the
fact that simple detection of CKI protein by immunostaining does not
necessarily correlate with functional activity.
The third major finding of this study is that, although
the absence of p21 is associated with significantly enhanced early proliferation of interstitial myofibroblasts, this augmented
proliferation was not sustained over time. Both interstitial and
tubular epithelial cell proliferation peak at day 3 following
UUO and subside thereafter, indicating that the tubulointerstitial
"microenvironment" is less supportive of cellular proliferation
later in the disease. For example, the antiproliferative cytokine
TGF-1 produced by interstitial macrophages may modulate intrinsic
intracellular mechanisms, such as the induction of p21, which act to
switch off myofibroblast proliferation by inducing cell cycle arrest.
It should be noted that we found no difference in either macrophage
infiltration (at any time point) or TGF-
1 mRNA expression as
assessed by in situ hybridization at day 3. This data indicates
that the increased myofibroblast proliferation seen at day 3 in
p21
/
mice is not the result of differences in either
macrophage infiltration or TGF-
1 production between wild-type and
knockout mice but specifically results from the absence of p21 from the
intrinsic cell cycle machinery of the myofibroblast.
In conclusion, this study reveals differential effects of the CKI p21 compared with p27 in UUO with the function of p21 in the tubular epithelium being redundant. However, there was clear evidence of a role for p21 in the early wave of proliferation and later accumulation of interstitial myofibroblasts in this model of tubulointerstitial injury. Lastly, this work reinforces the concept that the effects of an absence of individual cell cycle proteins will depend not only upon the particular cell type but also upon the nature of organ injury.
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ACKNOWLEDGEMENTS |
---|
J. Hughes is a Wellcome Advanced Training Fellow, and P. Brown was supported by a Peel Traveling Scholarship. S. J. Shankland is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52121, DK-51096, and DK-34198 and by George M. O'Brien Kidney Research Center Award DK-47659.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Hughes, Division of Nephrology, Box 356521, University of Washington, Seattle, WA 98195-6521 (E-mail: jeremyh{at}u.washington.edu).
Received 19 March 1999; accepted in final form 30 June 1999.
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