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*
Renal Pathology Laboratory, Department of Pathology The Methodist Hospital
Houston, Texas.
Department of Medicine, The Methodist Hospital Houston, Texas.
Department of Pediatrics, Texas Children's Hospital and Baylor College of
Medicine, Houston, Texas.
Correspondence to Dr. Luan D. Truong, Department of Pathology, M.S. 205, The Methodist Hospital, Houston, TX 77030. Phone: 713-394-6481; Fax: 713-793-1473; E-mail: ltruong{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular control of apoptosis is only partially understood. It likely involves two distinct pathways that share a common downstream arm. The first pathway involves the activation of death receptors (Fas, tumor necrosis factor-1 [TNFR-1], TRAIL) by their respective ligands (9,10,11,12,13). The generated signal activates cytosolic adapter molecules (e.g., TRADD, RIP, FADD, FAF) and results in the conversion of pro-caspase 8 to caspase 8. Caspase 8 converts pro-caspases 3, 6, and 7 into their respective active caspases, which are responsible for the cellular changes that are characteristic of apoptosis (10,11,14). The second pathway involves the bcl-2 family and is initiated by a large variety of environmental stresses, such as radiation, heat, or growth factor deficiency (15,16,17,18). These signals are transmitted to the mitochondria by an unknown mechanism and influence the balance between apoptosis promoters (bax, bcl-xS, bad, and bak) and inhibitors (bcl-2, bcl-xL, bcl-w, and bfl-1) among the bcl-2 family. These proteins reside in the mitochondrial membrane and share structural homology (15,16). Apoptotic signals that originate from this pathway result in the activation of pro-caspase 9 by cytochrome c and apoptosis protease activating factor-1 (15,19). Caspase 9, in turn, activates pro-caspases 3,6, and 7, a step that represents the onset of the common arm of both death receptor and bcl-2 apoptotic pathways (15). Other caspases, including caspases 1, 11, and 12, have been recognized recently (20) and reported to have a regulatory role in apoptosis and inflammatory response (20,21,22). However, their location in the apoptotic pathways has not been firmly established.
Although it is well established that p53 promotes apoptosis (6,23,24,25), the underlying molecular mechanism for its action remains unclear. Although interaction between p53 protein and the death receptor pathway has not been established, p53 can regulate the cellular levels of members of the bcl-2 family, including bax and bcl-2 (23,26,27). In addition, p53 has been suggested recently to activate a number of caspases in vitro (28,29). Last, p53 can induce cell-cycle arrest through upregulation of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (23,30,31).
In this study, we report that p53 gene deficiency attenuates renal cell apoptosis after ureteric obstruction. This effect may be attributable to downregulation of caspase activity, including caspases 1, 11, and 12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA Extraction and Southern Blotting
To determine p53 genotypes of the bred mice, DNA was extracted from tail
fragments, as previously described
(32). Ten µg of genomic DNA
from each animal were cleaved with BamHI, subjected to 0.9% agarose
gel electrophoresis, blotted onto nylon membrane, hybridized to a probe
specific for exons 2 through 6 of the p53 gene
(32), washed, and
autoradiographed. This probe detects both the wild-type and mutant
p53 genes. However, Because of the deletion of exon 5 in p53 null
mice, the DNA product detected by the probe in p53 null mice is smaller than
that seen in wild-type samples and, thus, easily distinguishable.
Creation of COU
Under inhalation anesthesia with methoxyflurane, COU was created in
12-wk-old, p53+/+, p53+/-, and p53-/- mice by complete ligation of the left
ureter at the ureteropelvic junction with the use of double silk sutures.
Animals were placed on regular diets, allowed free access to tap water, and
killed at day 4, 7, 15, 20, or 30. Three to six mice were used for each time
point. These time points were chosen because, in a pilot study, they were
shown to span the entire quantitative spectrum of tubular and interstitial
cell apoptosis. Kidneys of p53+/+, p53+/-, and p53-/- sham-operated mice were
harvested at days 0, 15, and 30 to confirm that mice without urinary
obstruction of these genotypes have the same pattern of apoptosis.
Tissue Preparation
Control, ligated, and contralateral kidneys were harvested. Portions of the
kidney tissue were fixed in 10% formalin for in situ end labeling of
fragmented DNA and routine histology (hematoxylin and eosin, periodic
acid-Schiff, and trichrome stains). Tissue portions were also snap-frozen in
liquid nitrogen and stored at -70°C for subsequent RNA extraction. Because
the obstructed kidneys of the same duration did not show any significant
difference among mice of the same genotype (see Results section), kidney
tissues from mice of the same genotype, obtained at the same experimental time
points, were pooled for RNA extraction.
Light Microscopy and Morphometry
Tissue sections were observed independently by two renal pathologists
(Y.-J.C. and L.D.T.) who were unfamiliar with the experimental conditions.
Morphometric evaluation of renal tubular diameters and interstitial volume was
assessed as described previously
(3). Briefly, a graded 1-cm
micrometer, viewed under the 40x objective of a Nikon microscope
(Melville, NY), was used to measure tubular diameters in periodic
acid-Schiff-stained sections. The diameter (in micrometers) was measured for
20 random tubular cross sections in both the cortex and the medulla in each
kidney section. Interstitial volume was determined with the use of a
point-counting technique on trichrome-stained sections. The interstitial
volume was expressed as the percentage of grid points of a 1-cm2
graded ocular grid, which is situated within the interstitial area, viewed at
20x magnification. Five to 10 random fields were used for
morphometry.
Detection and Quantification of Renal Cell Apoptosis by In Situ End
Labeling of Fragmented DNA
In situ end labeling of fragmented DNA was performed as detailed
previously (3). The following
morphologic criteria were used to ensure accurate identification of apoptotic
cells: Only tissue sections without background staining were used for
evaluation. Most positive cells also should have displayed light microscopic
features of apoptosis (shrunken or fragmented and condensed nuclei).
Unequivocally positive cells without obvious light microscopic features of
apoptosis were also counted because it has been shown that they may represent
cells in which DNA fragmentation already occurs but precedes other apoptotic
changes that are recognizable by light microscopy
(3). Necrotic cells, which were
stained positive, were not counted. These cells occurred in small patches
involving several contiguous cells at the papillary tip and were distinguished
easily from apoptotic cells, which occurred in isolation and were scattered
throughout the renal parenchyma. Necrosis occurred in three obstructed kidneys
only and was not a significant lesion after ureteric obstruction. Apoptotic
cells were counted under the 40x objective lens of a Nikon microscope.
All apoptotic cells within 5 to 10 random fields were counted, each delineated
by a 1-cm2 ocular grid attached to the eyepiece. Differentiation
between cortex and medulla regions in obstructed kidneys was possible until 20
d postobstruction, and the random fields chosen equally represented cortex and
medulla. Beyond this time point, however, advanced parenchymal injury
precluded identification of specific structures in obstructed kidneys, and the
fields that were chosen to count apoptosis in these kidneys were truly random.
The number of apoptotic tubular cells, cortical plus medullary, was expressed
as a percentage of total number of tubular cells in the fields that were
chosen initially for counting. Quantification of interstitial cell apoptosis
was performed in the same manner. It should be noted that interstitial and
inflammatory cells appear similar as they develop apoptosis; hence, precise
quantification of interstitial and inflammatory cells that were undergoing
apoptosis was not carried out.
Northern Hybridization for p53 mRNA
Northern hybridization was used to determine p53 gene expression
in control, contralateral, and ligated kidneys of mice that belonged to each
genotype (p53+/+, p53+/-, and p53-/-). Pooled kidney tissues, at each time
point, were used to extract total cellular RNA, with the use of the RNAzol-B
method (Tel Test B, Friendswood, TX). Ten µg of the total RNA were loaded
per lane onto 1% denaturing agarose gel and separated by electrophoresis. RNA
was transferred to a nylon membrane and hybridized simultaneously with
32P-labeled cDNA probes for p53 mRNA
(32) and 28S rRNA,
respectively.
RNase Protection Assay
RNase protection assay was carried out per the manufacturer's instructions
(RiboQuant kit; PharMingen, San Diego, CA) and used to detect and quantify the
expression of mRNA of apoptosis-related molecules
(33,34).
Three sets of template cDNA were used, each containing multiple cDNA probes,
which allowed simultaneous detection of mRNA corresponding to the death
receptor family (Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAP, and
FAF), bcl-2 family (bax, bad, bak, bcl-xL/xS, bcl-2, bcl-w, and bfl-1), and
effector family (caspases 1, 2, 3, 6, 7, 8, 11, and 12). cDNA probes for
ribosomal protein L32 and glyceraldehyde-3-phosphate dehydrogenase were
included as internal controls.
Labeled antisense RNA probes were synthesized from cDNA multiprobe
templates with the use of [
-32P] UTP in an in vitro
transcription reaction performed according to the manufacturer's instructions.
The labeled RNA probes were hybridized for 16 h at 56°C with samples of 10
µg of total RNA extracted from control, ligated, and contralateral kidneys.
The hybridized products were treated with a digestion mixture, including RNase
and proteinase. During this procedure, the unhybridized RNA and the free RNA
probes were digested but the hybridized RNA were protected from digestion
(RNase protection assay). The hybridized RNA were heat-denatured and
electrophoresed on polyacrylamide gel for 2 h, 50 W at 45 to 50°C. Gels
were dried and exposed to x-ray film at -70°C. The resultant bands were
scanned and quantified using PhotoShop and the University of Texas Health
Science Center, San Antonio, Image Tool program. Band intensity was normalized
to that of L32 in the same reaction.
Statistical Analyses
The morphometric data and the frequency of apoptosis were expressed as mean
± SD. Statistical differences between p53+/+ and p53-deficient mice
groups were evaluated with the use of Kruskal-Wallis analysis. P <
0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathologic Changes of COU
The control and contralateral kidneys from p53+/+, p53+/-, and p53-/- mice
were histologically normal (Figure
2A). Regardless of the underlying genotype, obstructed kidneys
uniformly developed progressive tubulointerstitial changes characteristic of
COU (Figure 2, B through D).
The tubular changes included atrophy, dilation, simplification of the tubular
epithelium, and tubular cell apoptosis. The interstitial changes included
fibroblast proliferation, fibrosis, inflammatory cell infiltration, and
apoptosis of interstitial cells. The glomeruli and blood vessels remained
intact throughout the experimental period. Although the spectrum of changes
was the same for p53+/+, p53+/-, and p53-/- kidneys, the severity of these
changes was different among these genotypes. There was no difference between
p53+/- and p53-/- mice in the severity of tubular atrophy (P = 0.20)
and interstitial widening (P = 0.83;
Figure 3). Compared with p53+/+
mice, obstructed kidneys from p53-deficient mice showed significantly less
tubular atrophy (P 
0.05) and less interstitial widening
(P 0.05) between days 7 and 15, but no significant differences
were noted after day 20 (P 
0.34).
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Apoptosis of Tubular and Interstitial Cells
The control and contralateral kidneys showed rare apoptotic cells, which
did not differ among genotypes and experimental duration.
(Figure 2, E and I). Obstructed
kidneys of each genotype showed increased tubular
(Figure 2, F through H) and
interstitial (Figure 2, J through
L) cell apoptosis, but the frequency of apoptotic cells was
influenced by the p53 genotype. Kidneys from p53-/- mice, compared with those
from p53+/- mice, showed, at various time points, a similar or mildly
decreased frequency of apoptotic cells (up to 8 and 9% for tubular and
interstitial cell apoptosis, respectively). However, compared with p53+/+
mice, kidneys from both groups showed a significant attenuation of tubular
(44% at day 7 [P = 0.02] and 70% at day 15 [P = 0.02]) and
interstitial cell apoptosis (50% at day 15 [P = 0.009]). In addition,
p53-deficient kidneys showed an earlier peak of tubular cell apoptosis (day 4
for p53-/- and p53+/- versus day 15 for p53+/+) and a later peak of
interstitial cell apoptosis (day 20 for p53-/- and p53+/- versus day
15 for p53+/+; Figure 4).
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p53 mRNA Expression and Correlation with Apoptosis
The ligated kidneys of the p53+/+ mice showed a marked increase of p53 mRNA
compared with control or unligated contralateral kidneys. This increase was
observed day 4, peaked between days 7 and 15, and decreased and returned to
the control level at day 30. The pattern of p53 mRNA expression paralleled
that of renal cell apoptosis. A slight increase of p53 mRNA was detected in
ligated kidneys of p53+/- mice, in a pattern similar to that of p53+/+ mice.
p53 mRNA was not detected in kidneys of p53-/- mice, irrespective of
experimental protocol (Figure
5).
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RNase Protection Assay for Apoptosis-Related Molecules
Death Receptor Family. The Fas ligand mRNA showed a pattern of
expression similar for all three genotypes. It was not seen in control or
contralateral kidneys but was increased mildly after ureteric ligation. A
baseline level of mRNA of other molecules of the death receptor family was
noted in control or contralateral kidneys; this level was less in p53+/- and
p53-/- mice than in p53+/+ mice. Ureteric ligation induced increased
expression of these molecules; however, this expression was more pronounced in
p53+/- and p53-/- mice than in p53+/+ mice. This difference was most evident
for TRAIL, TNFR-1, and FAP mRNA (Figure
6).
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Bcl-2 Family. The difference between contralateral or control kidneys on the one hand and ligated kidneys on the other was similar among the three genotypes. Within each genotype, the ligated kidneys, compared with the control or contralateral kidneys, expressed the following pattern of mRNA expression: markedly increased bfl-1 (antiapoptotic molecule), increased bax, bak, and bcl-xS (proapoptotic molecules), decreased bcl-w (antiapoptotic molecule), and unchanged bcl-2, bcl-xL, and bad (Figure 7).
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Caspase Family. The baseline expression of caspases 2, 3, 6, 7, and 8, in control and unobstructed contralateral kidneys, was very low in p53+/- and p53-/- mice but mild in p53+/+ mice. These caspases were increased in ligated kidneys in a pattern similar for all three genotypes. The expression of caspases 1, 11, and 12 at baseline (control and unobstructed contralateral kidneys) was virtually undetectable in all three genotypes. However, these caspases (especially caspase 11) displayed in ligated kidneys distinctive patterns of expression that were significantly different among the various genotypes. Caspase 11 mRNA was markedly increased in ligated kidneys from p53+/+ mice and was virtually undetectable in p53+/- and p53-/- mice at any experimental time point. Caspases 1 and 12 mRNA were increased in the ligated kidneys of all genotypes. However, the increase was much less in p53+/- and p53-/- mice than in p53+/+ mice, especially between days 4 and 7.
The time frame of caspase expression in the ligated kidneys was also different among the various genotypes. In p53+/+ mice, they were increased at day 4 after ureteric ligation reached a plateau at day 15, decreased at day 20, and increased again at day 30. However, in p53+/- and p53-/- mice, these caspases peaked between days 7 and 15, decreased at day 20, and increased again at day 30 (Figure 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p53 protein is a transcription factor that regulates both apoptosis and growth arrest and is one of the best characterized tumor suppressor genes (24,25,28). A marked increase in p53 expression is observed in cells that are treated with DNA-damaging agents (35,36), in hepatocytes (5), and in neurons (6) that are undergoing apoptosis. Although increased p53 expression has been described briefly in kidneys after ureteric ligation (5,31), our study demonstrates unequivocally a marked increase in p53 mRNA in kidneys of p53+/+ mice after ureteric ligation, in a manner that parallels the incidence of renal cell apoptosis. This observation establishes a clear correlation between the p53 gene and apoptosis in ureteric obstruction. The mechanism by which COU causes increased renal expression of p53 remains unclear, although ischemia, increased renin production, and elevated intratubular pressure may be relevant (31).
p53 can promote apoptosis by a direct effect on apoptosis-related molecules (35,36,37,38). Our study showed, for the first time, that kidneys from p53-deficient mice with COU, compared with those from wild-type mice, showed a mild but definite increase (reaching twofold) of FAP, TRAIL, and TNFR-1, whereas other molecules of this family (Fas, Fas ligand, FADD, TRADD, RIP, and FAF) were unchanged. These findings indicate that the apoptotic pathway, mediated by the death receptors, is not only intact but also enhanced in ligated kidneys from p53-deficient mice.
Cell susceptibility to apoptosis is determined partially by the ratio of bcl-2 protein, an apoptosis inhibitor, and bax protein, an apoptosis promoter (15,16,39,40,41). p53 null mice manifest an attenuated apoptotic response and show a decrease in bax protein with (26) or without (40) an increase of bcl-2 protein. It is postulated that the protection from apoptosis observed in these mice might be due to a relative predominance of bcl-2 protein (40). These mechanisms, however, do not seem to account for the protection of apoptosis induced by p53 deficiency noted in this study. mRNA levels of bax, bcl-2, and other molecules of the bcl-2 family (bad, bak, bcl-xL/xS, bcl-w, and bfl-1) were not different in the ligated kidneys among the various p53 genotypes.
The most distinctive and novel findings in the current study were a virtual loss of caspase 11 and a marked decrease of caspases 1 and 12 in ligated kidneys of p53-deficient mice. The hierarchy of caspases 1, 11, and 12 within the general apoptotic pathways is not well known, and they are poor substrates for other caspases (22). Murine caspase 11, the murine homolog of human caspase 4, is a member of the interleukin-1ß converting enzyme (ICE) subfamily of protease and shares 54% identity with murine ICE/caspase 1 (20). Pro-caspase 11 was found recently to interact physically with pro-ICE, and the expression of caspase 11 is required for activation of ICE (21). Overexpression of caspase 11 induces apoptosis, which could be prevented by bcl-2 and crmA (20). Expression of caspase 11 is highly inducible by lipopolysaccharides (20,21) and cathepsin B (22), suggesting that caspase 11 may have a regulatory role in both apoptosis and inflammatory response (20,21,22). Findings from the current study confirm the apoptotic roles of caspases 1, 11, and 12, at least for renal cell apoptosis associated with ureter ligation, and suggest, for the first time, that p53 may be essential for their activation. These findings also raise the possibility that p53 might be an upstream regulator of caspase 11 activation and that caspase 11 could be another downstream molecule of the p53-dependent apoptosis pathway. A few reports indicated that caspases are activated by p53 (28,29). Sabbatini et al. (28) demonstrated that all apoptotic pathways downstream of p53-mediated transcription converge upon the activation of caspase 3. Ding et al. (29) reported that p53-dependent activation of caspases in cell-free systems does not require the presence of bax or cytochrome c. However, they did not elucidate the specific relationship between p53 and individual caspases; they just provided indirect evidence for caspase activation, i.e., cleavage of poly (ADP-ribose) polymerase, which is a target for caspase 3 (29). The study presented here, in conjunction with previous reports, suggests that p53 protein can transduce apoptotic signals through protein—protein interactions and that a pathway that is characterized by p53-dependent caspase activation exists (29).
In summary, this study suggests that apoptosis in kidneys with COU is attenuated but not eliminated by deletion of the p53 gene and is both p53 dependent and p53 independent. The p53-dependent pathway may involve p53-mediated transduction of apoptotic signals through the activation of caspases, especially caspases 1, 11, and 12, and this activation requires p53 homozygosity.
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Acknowledgments |
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References |
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