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Animal Model |
From the Departments of Internal Medicine D,*
Physiological Chemistry and Pathobiochemistry,
and Arteriosclerosis Research,¶
University ofMünster, Münster, Germany; the Department of Cellular andMolecular Pathology,
German Cancer ResearchCenter, Heidelberg, Germany; the Department of Pathology, Anatomy, andCell Biology,||
Thomas Jefferson University, Philadelphia,Pennsylvania; and the Institute of MolecularBiology,
Slovak Academy of Sciences,Bratislava, Slovak Republic
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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In light of this complexity, the experimental model of unilateral ureteral obstruction (UUO) has been widely used to study the pathogenesis of tubulointerstitial fibrosis5,6 because the evolution of fibrosis after ureteral obstruction is highly reproducible and reflects the sequence of pathogenetic events in an accelerated manner. As in human interstitial fibrosis there is rapid infiltration by mononuclear cells that finally leads to hypocellular fibrotic scarring and tubular atrophy without primary glomerular damage.7 Although important insights into the mechanisms of interstitial fibrosis have been obtained in these investigations, long-term studies are scarce, and some aspects of tissue remodeling have not yet been addressed at all. In this investigation we were interested in the sequence of pathogenetic events and in the mechanisms leading to fibrosis in the absence of the small leucine-rich proteoglycan (SLRP)8,9 decorin in obstructive nephropathy for the following reasons. First, decorin, and other SLRPs (eg, biglycan and fibromodulin) have been shown to form complexes with TGF-ß10 leading to inhibition and/or sequestration of the cytokine within the extracellular matrix.11-14 Second, decorin, biglycan, and fibromodulin as well as a fourth member of this family, lumican, interact with fibrillar collagens,15-17 thereby modulating fibril formation and stability. Ablation of each of the four SLRP genes results in grossly disturbed type I collagen-containing fibrils18-21 indicating the need for an orchestrated action of all four proteoglycans during fibril assembly. The presence of all four SLRPs in the kidney has been reported previously.22 Third, biglycan has been shown to stimulate growth and differentiation of monocytic lineage cells from various lymphatic organs23,24 and may, hence, support acute inflammation in obstructive nephropathy. Fourth, decorin expression enables endothelial cells to escape apoptosis.25,26 Furthermore, decorin expression is followed by an up-regulation of cyclin-dependent kinase inhibitors,27 probably via interaction with an epidermal growth factor-receptor family member,28 and is therefore also involved in cell cycle control. Here we show that the course of obstructive nephropathy becomes greatly altered in mice with an inactivated decorin gene because of a decrease in p27KIP1 expression, caspase induction, and tissue remodeling affecting primarily tubular epithelia. Differences between normal and decorin-/- mice in the extent of apoptosis, in TGF-ß levels, and the number of biglycan-expressing macrophages appear as major determinants for this type of end-stage kidney disease.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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All animal work was done in accordance with the German Animal Protection Law. Decorin-deficient mice were generated as described previously.18 The kidneys studied were from wild-type (WT) (n = 43), decorin-/- (Dcn-/-) (n = 78), and decorin+/- (Dcn+/-) (n = 4) mice of the same inbred strain background.18 Obstruction of the left ureter was performed in 2-month-old male mice. The contralateral and sham-operated kidney served as control. Kidneys (at least n = 4 per group) were analyzed up to 70 days after ligation.
Northern Blot Analysis and in Situ Hybridization
Total RNA was extracted from whole kidneys as
described.29
The cDNA probes for decorin and biglycan were
those used previously in the rat.29
cDNA probes for rat
fibromodulin and lumican, which are also homologous to the
respective murine sequences, were generated by reverse
transcriptase-polymerase chain reaction (PCR) from total rat tail RNA
with Super Script II reverse transcriptase (Life Technologies,
Eggenstein, Germany) and random hexanucleotide cDNA primers.
Double-stranded cDNAs were generated by using primers that contained
additionally appropriate sequences with restriction sites for
SmaI and HindIII, respectively. For fibromodulin
the primer pair was 5'-TCCCCCGGGGATGCAGTGGGCCTCCATC-3', and
5'-GCTCCAGATCTCGATGAGGCT-3', thus yielding a rat fibromodulin cDNA
encompassing bp 54 to 1184 (GenBank X82152). For lumican, the
respective primer pair was 5'-TCCCCCGGGGATGAATGTATGTACGTTCAC-3' and
5'-CCCAAGCTTTAATTAACCGTGATTTCA-3' yielding lumican cDNA between bp 46
to 1062 (GenBank X84039). Endonuclease-treated PCR products were cloned
into pGEM 3Z (Promega, Madison, WI) and verified by DNA sequencing.
Mouse caspase-3 and caspase-8 were generated with the primer pairs
5'-TGTCATCTCGCTCTGGTACG-3', 5'-AGCATGGACACAATACACGG-3' (245 to 869 bp,
GenBank NM009810) and 5'-CCGAGAGGAGATGGTGAGAG-3',
5'-TCGGTAGGAAACGCAGTTCT-3' (292 to 1291 bp, GenBank AJ007749),
respectively, and cloned into pCRII (Invitrogen, Groningen, The
Netherlands). cDNAs for 
1(I) collagen, TGF-ß1, and GAPDH
were from ATCC (Rockville, MD). Northern blots were performed and
analyzed as described previously.13
In situ
hybridization of renal sections from WT and Dcn-/- mice
was performed with the sense and antisense probes in parallel and under
the same conditions.13
Real-Time Semiquantitative PCR
cDNA synthesis from total RNA extracted from whole kidneys and amplification were performed as described previously13 using the primers for the sequences of p21CIP1/WAF1: 5'-CTGCCCAAGCTCTACCTTCC-3' and 5'-CAGGTCCACATGGTCTTCCT-3' (123 bp); p27KIP1: 5'-CCGGCTAACTCTGAGGACAC-3' and 5'-AGAAGAATCGTCGGTTGCAG-3' (120 bp); GAPDH: 5'-GTCAGTGGTGGACCTGACCT-3' and 5'-ACCTGGTGCTCAGTGTGCC-3' (123 bp). Melting curves and agarose gel electrophoresis were used to verify the quality of the PCR products. The data obtained were referred to standard curves where plasmids with authentic cDNAs were analyzed analogously. Finally, the values were normalized to the results of GAPDH-reverse transcriptase-PCR. Statistical analysis was performed with Prism, version 3 (GraphPad Software, San Diego, CA).
Morphological and Immunohistochemical Studies
Serial sections (2 to 6 µm) of paraffin-embedded samples were stained with periodic acid-Schiff reaction (PAS) and processed for immunohistochemical studies or in situ hybridization.
Morphometric evaluation of tubulointerstitial damage (defined as the occurrence of dilated or collapsed tubules with flattened and vacuolated epithelia), glomerular volumes, interstitial fibrosis, and infiltration of mononuclear cells was performed by a computer-aided image analysis system.30
Paraffin sections were stained by immunoperoxidase or APAAP techniques.29 Primary antibodies included LF-113 (a rabbit anti-murine decorin antiserum31 kindly provided by Dr. L.W. Fisher, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD), a chicken antibody against the Ser67 to Gln78 peptide of rat biglycan,29 rabbit anti-rat type I collagen (Biogenesis, Berlin, Germany) and the marker for murine monocytes/macrophages: rat anti-mouse F4/80 (Serotec, Eching, Germany). After blocking endogenous peroxidase and incubation with respective antibodies, sections were developed with the Diaminobenzidine Substrate Kit (Vector Laboratories, Burlingame, CA). Antibodies against proliferating cell nuclear antigen were visualized with the EPOS-horseradish peroxidase-coupled system (DAKO, Hamburg, Germany). Counterstaining was with methyl green. Mouse anti-fibronectin extradomain A (FN-EDA+), clone OST-9 (kindly provided by Dr. L. Zardi, Genova, Italy), was conjugated with digoxigenin. The active form of caspase-3, the proform of caspase-8, and its active p20 subunit were visualized with digoxigenin-conjugated affinity-purified antibodies (R&D Systems, Wiesbaden, and Santa Cruz, Heidelberg, Germany, respectively) in paraffin and frozen sections. Early apoptosis was evaluated in frozen sections by a digoxigenin-conjugated monoclonal antibody to single-stranded DNA (F7-26: Alexis, Grünberg, Germany). Antibody-treated tissue sections were incubated with alkaline phosphatase-conjugated Fab fragments of anti-digoxigenin antibodies.29 The specificity of immunostaining was tested by omitting the primary antibody, by using nonimmune serum/unspecific IgG, and by preabsorption of antisera with antigens (decorin, biglycan).
To evaluate individual kidneys, 20 randomly selected nonoverlapping fields of renal cortex were examined under higher magnification (x400) and the number of biglycan-positive cells, proliferating cell nuclear antigen-labeled nuclei, and apoptotic nuclei in tubular and interstitial cells were evaluated. Mean values of at least four kidneys per group were averaged. Morphometrical evaluation of FN-EDA+ was performed as described previously.32 In brief, a grid containing 117 (13 x 9) sampling points was superimposed on images of cortical high-power fields (x400) and FN-EDA+ deposits were counted and expressed as percentage of all sampling points. All morphometric evaluations were performed by a blinded observer.
Confocal Microscopy
For the characterization of biglycan-positive cells double-labeling of 6-µm frozen sections was performed with antibodies against biglycan (1:500) and F4/80 (1:10) for identification of macrophages. Unspecific staining was blocked with phosphate-buffered saline/1% bovine serum albumin/20% goat serum. For visualization 1:200 dilutions of Texas Red-conjugated donkey anti-chicken IgG and fluorescein-conjugated goat anti-rat IgG (Dianova, Hamburg, Germany) were used. Laser scan microscopy was performed with a Nikon confocal microscope PCM 2000 (Nikon, Duesseldorf, Germany). Nonspecific staining was determined by the use of secondary antibodies alone.
Assay of Caspase-3 Activity
Caspase-3 activity was determined in homogenates of sham-operated, obstructed, and contralateral kidneys by the Caspase-3/CPP32 Colorimetric Assay Kit (BioCat, Heidelberg, Germany). In this assay, active caspase-3 cleaves the chromophore p-nitroanilide (pNA) from the labeled substrate DEVD-pNA. Enzyme activity was measured as described previously.33 Briefly, whole kidneys were homogenized in 2 ml of cell lysis buffer at 4°C for 15 minutes. After centrifugation at 10,000 x g, proteins were measured in the supernatants (cytosolic extractions) and adjusted to a concentration of 200 µg/50 µl. After incubation with substrate for 2 hours at 37°C, the optical density was determined using a microplate reader at 405 nm. Data were calculated as the average of duplicates for each sample and per whole kidney. Mean values of three kidneys per group were averaged.
Other Procedures
Thin section electron microscopy was performed as
described.15
Serum urea and creatinine levels were
measured using a Hitachi 747 autoanalyzer and urinary and
tissue protein by the BCA Protein Assay Reagent (Pierce, Rockford,
IL). Western blots were performed and quantified as described
previously13
using rabbit
anti-p21CIP1/WAF1,
anti-p27KIP1, and anti-ß-tubulin as primary
antibodies (all from Santa Cruz). Type I collagen was determined after
exhaustive pepsin digestion of whole minced kidneys followed by 4 to
12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under
reducing and nonreducing conditions and quantification (IQ Solutions
Image Quant Software: Molecular Dynamics, Uppsala, Sweden) of Coomassie
Blue-stained bands of 1(I)- and 2(I)-chains of collagen
I.34
A high- and low-molecular weight mixture of proteins
(Life Technologies) and pepsin-digested purified type I, III, and IV
collagens were used as standards. For Western blotting and type I
collagen quantification the results from three kidneys per group were
averaged.
Statistics
Results are expressed as means ± SEM. Statistical analysis was performed by the unpaired Student’s t-test. Significance was accepted at the 5% level.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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UUO was performed in WT and Dcn-/- animals, and the
development of hydronephrosis was followed throughout a period of 70
days (Figure 1A)
. No macroscopical
differences in the development of hydronephrosis between WT and
Dcn-/- mice were evident for up to 35 days after UUO.
However, thereafter (42, 53, and 70 days) obstructed kidneys from
Dcn-/- mice became progressively smaller in size and
weight. After aspiration of fluid from the dilated pyelon the weight of
Dcn-/- kidneys was 123 ± 11 mg versus
201 ± 17 mg in WT animals at day 53 (n =
4, P < 0.05) and 87 ± 9 mg versus
152 ± 14 mg at day 70 (n = 6,
P < 0.05). These differences have to be interpreted in
light of the finding that weights of normal kidneys were comparable in
age- and sex-matched WT and Dcn-/- mice. No differences
were observed in body weight or in the weight of contralateral kidneys
between both groups at any time point.
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Histomorphological Changes in Obstructed Kidneys
Already at day 7 quantitative histomorphometry of PAS-stained
sections (Figure 1, B and C)
from obstructed kidneys revealed a
significantly higher percentage (P < 0.05) of
damaged tubules in Dcn-/- (58 ± 10%,
n = 6) than in WT mice (20 ± 6%,
n = 4). During the subsequent course of the disorder
this difference increased further, and at day 53 tubules with
segment-specific differentiation were no longer recognizable in
decorin-deficient mice whereas in obstructed kidneys from WT animals
such tubules could still be occasionally detected (Figure 1, D and E)
.
Another striking difference in end-stage fibrosis concerned the extent
of infiltration by mononuclear cells. At day 53 an area covering
34 ± 3% of the tissue sections was occupied by infiltrating
mononuclear cells, frequently in the form of lymphoid follicles, in
Dcn-/- animals (Figure 1, D and E)
whereas the
corresponding value in WT animals was 2.3 ± 0.4%
(n = 4 for each group, P <
0.05). Similar data were obtained from kidneys on day 70. The majority
of these cells were positive for monocyte/macrophage markers. At days
53 and 70, the volumes of glomeruli in obstructed kidneys from WT were
1.8-fold to 2.9-fold higher as compared to Dcn-/- animals.
Early tubular damage and enhanced infiltration of macrophages were the
two most prominent histomorphological differences observed in
obstructed kidneys from Dcn-/- as compared to WT mice. In
contrast, semiquantitative evaluation of interstitial fibrosis showed
no difference in the extent of fibrosis in obstructed kidneys from WT
and Dcn-/- animals. In addition, there were no differences
in PAS-stained sections from contralateral and sham-operated kidneys in
either genotype.
Enhanced Early Tubular Apoptosis in Obstructed Kidneys from Dcn-/- Mice
The earliest detectable difference between WT and
Dcn-/- kidneys concerned the expression of
p27KIP1. Overexpression of
p27KIP1 in obstructed kidneys from WT both on the
mRNA (Figure 2A)
and protein level
(Figure 2, C and D)
was detected at 12 hours, whereas in
Dcn-/- kidneys p27KIP1 started to
increase at 36 hours after UUO. Lower levels of
p27KIP1 were detected in obstructed
Dcn-/- than in WT kidneys until day 4. In later stages of
UUO levels of p27KIP1 did not differ between WT
and Dcn-/- kidneys. In agreement with earlier
reports,6
p21CIP1/WAF1 mRNA (Figure 2B)
was up-regulated in obstructed kidneys starting at 36 hours.
However, no genotype-specific differences were observed (Figure 2
; B,
C, and E) at any experimental time point (12 hours to 70 days). Because
a lack of p27KIP1 has been shown to enhance
tubular epithelial cell apoptosis in UUO35
and because
decorin exhibits an anti-apoptotic effect in
vitro,25
we wondered, whether the extensive tubular
damage in Dcn-/- mice described above might have been the
consequence of an earlier and/or more pronounced induction of apoptotic
pathways in these animals. Hence, we quantified the mRNAs for caspase-8
and caspase-3, performed immunostainings for the active forms of these
enzymes, measured caspase-3 activity, and stained single-stranded DNA.
|
. Besides the overexpression of
caspase-8, there was also an up-regulation of caspase-3. After 36
hours, enhanced mRNA levels for caspase-3 were observed only in
obstructed kidneys from Dcn-/- mice (Figure 3B)
. At day 4,
induction of caspase-3 mRNA was observed also in obstructed kidneys
from WT animals. However, the expression of caspase-3 was 1.4-fold
higher in Dcn-/- mice. The active form of caspase-3 as
well as the proform of caspase-8 and its active subunit were found at
days 4 and 7 mainly in tubular epithelial cells (Figure 3, C and D)
.
Both caspases were not detectable by immunohistochemistry in
contralateral kidneys. In agreement with earlier
reports,33
a quantitative assay for caspase-3 activity in
homogenates of whole kidneys showed increased activity of this enzyme
in obstructed kidneys between days 4 and 35 after ligation (Table 1)
. However, similar to the mRNA results
for caspase-3, the caspase-3 activity was higher in obstructed kidneys
from Dcn-/- versus WT mice only at days 4 and 7
(Table 1)
. From day 14 onward, no genotype-specific differences were
observed in the expression of caspase-3 and caspase-8 mRNAs and in the
caspase-3 activity.
|
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; Table 1
). Enhanced apoptosis in Dcn-/- mice was
exclusively observed in tubular epithelial cells. In contrast, the
degree of apoptosis in interstitial cells was not different in
obstructed kidneys from WT and Dcn-/- mice throughout the
whole experimental period (Table 1)
. The rather moderate increases of
caspase-3 mRNA and activity in Dcn-/- compared to WT
animals were probably because of the fact that both parameters were
measured in homogenates of whole kidneys. In contrast, the differences
in the degree of apoptosis were limited to tubular epithelial cells and
quantified there in situ. In obstructed kidneys from Dcn-/- animals there was not only an augmentation in induced cell death but also a moderate increase in cell proliferation. This was particularly evident between days 4 and 14, when the number of proliferating cell nuclear antigen-positive cells, which were mostly of epithelial origin, was 1.2-fold to 1.9-fold higher.
Increased TGF-ß1 Expression in Decorin-Deficient Kidneys
Based on the finding that decorin forms complexes with TGF-ß and
thereby modulates the activity of this cytokine, we investigated the
expression of TGF-ß1 during the course of UUO. Although UUO led to
increased TGF-ß1 mRNA expression in both WT and Dcn-/-
kidneys, this up-regulation was up to twofold higher in
decorin-deficient tissue (Figure 4A)
.
|
. The localization of TGF-ß1 mRNA did
not differ in obstructed kidneys from WT and Dcn-/- mice,
but the number of TGF-ß1-positive cells was higher in ligated kidneys
from Dcn-/- mice. In sham-operated kidneys TGF-ß1 mRNA
was expressed only sporadically in some peritubular mesenchymal cells
without genotype-specific differences.
Staining for fibronectin extradomain A (FN-EDA+),
an indicator of active TGF-ß, became evident at day 7 in obstructed
kidneys from Dcn-/- mice, whereas in WT animals
FN-EDA+ positivity was first detectable at day
14. FN-EDA+ was immunolocalized predominantly in
the peritubular space and in atrophic tubuli (Figure 4C)
. When
quantified morphometrically, the staining was significantly more
enhanced in obstructed kidneys from Dcn-/- mice (day 21:
Dcn-/-: 22 ± 3%, n = 6
versus WT: 13 ± 2%, n = 8; day 70:
Dcn-/-: 27 ± 3%, n = 6
versus WT: 19 ± 2%, n = 8,
P < 0.05).
Expression and Immunolocalization of SLRPs
In mice with ablated individual SLRP genes the possibility of a partial compensation by related SLRP members has been described.18-20 We compared, therefore, the expression of decorin, biglycan, lumican, and fibromodulin in whole kidneys before and after ligation.
Renal RNA from WT animals contained a decorin transcript of 1.8 kb. The
expected second decorin transcript of 1.6 kb was detectable only in
heavily overloaded gels. In Dcn+/- mice the normalized
decorin mRNA levels were reduced by only 40%, and they were absent in
Dcn-/- mice. In obstructed kidneys from WT animals
enhanced expression of decorin became evident 36 hours after ligation,
and decorin mRNA remained up-regulated throughout the experiment.
Overexpression of decorin was especially pronounced at earlier stages
with a more than sevenfold up-regulation of decorin mRNA between days 4
and 7 (Figure 5, A and D)
. Differences
between sham-operated and contralateral kidneys were not observed.
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. However, in Dcn-/- mice this
up-regulation was much more pronounced, resulting in a 15-fold increase
at day 7. The greater degree of overexpression of biglycan in
Dcn-/- mice persisted throughout the whole experimental
period (Figure 5E)
. Differences between sham-operated and contralateral
kidneys or age-dependent changes were not observed.
Other SLRPs were not up-regulated similarly during the course of UUO.
The expression of fibromodulin remained unchanged for 21 days (Figure 5, C and F)
. Thereafter, a slight overexpression (up to twofold) was
found by Northern blot analysis in the obstructed kidneys from both WT
and Dcn-/- mice. Genotype-specific differences did
not become evident. The renal expression of lumican was very weak
and remained unchanged in obstructed kidneys from WT and
Dcn-/- mice throughout the experiment.
In situ hybridization and immunohistochemical stainings
indicated that in unaffected kidneys decorin mRNA and core protein were
detectable predominantly in the adventitia of blood vessels. Beginning
at day 7 after UUO, decorin mRNA could be found in peritubular
mesenchymal cells where it remained visible until the end of the
experimental period. Immunostainings for decorin also showed an
accumulation of decorin protein in the peritubular space (Figure 6)
.
|
. Genotype-specific differences were
not evident. At day 4 after ureteral ligation, the expression of
biglycan was detected in some epithelial cells of dilated tubules. At
day 7, however, tubular epithelial expression of biglycan became
already weaker, whereas infiltrating and interstitial cells strongly
expressed biglycan. At later stages, the expression of biglycan in
obstructed kidneys was almost exclusively because of infiltrating and
interstitial cells. A similar switch in the cellular source of biglycan
was observed at the protein level. Initially, staining for biglycan
protein was detected only in some tubular epithelial cells (day 4),
whereas at day 7 infiltrating and interstitial cells became
positive (Figure 7A)
. At day 70, the majority of infiltrating and
interstitial cells stained for biglycan (Figure 7A)
. The localization
of biglycan mRNA and protein did not differ in the obstructed kidneys
from WT and Dcn-/- mice, but the number of
biglycan-positive cells was higher in ligated kidneys from
Dcn-/- mice.
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The nature of the biglycan-positive infiltrating cells was evaluated by
double-immunofluorescence staining for biglycan and F4/80 (a marker of
murine macrophages). Confocal microscopy indicated that a subset of
macrophages expressed both the F4/80 antigen and biglycan (Figure 7B)
.
Discrepancy Between Increased mRNA Expression of 1(I) Collagen
and Collagen Accumulation in Decorin-Deficient Kidneys
In light of the enhanced TGF-ß1 activity and the differences in
the long-term development of kidney fibrosis some aspects of type I
collagen metabolism were studied. An overexpression of 1(I) collagen
was noted in obstructed kidneys from both WT and Dcn-/-
animals beginning at day 4 after ligation. In WT and
Dcn-/- animals maximal induction of 1(I) collagen,
resulting in a 12-fold and 21-fold increase, respectively, was observed
at day 7 (Table 2)
. In agreement with
these data deposition of type I collagen, assessed by quantification of
extractable collagens by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and densitometry (sum of 1(I)- and 2(I)-chains),
was higher (180%) at day 7 in obstructed kidneys from
Dcn-/- mice (Figure 8
and
Table 2
). However, although 1(I) collagen mRNA approached similar
levels in WT and Dcn-/- kidneys after prolonged periods of
time, the content of type I collagen became remarkably reduced in
decorin-deficient organs, achieving only 20% of the quantity present
in WT organs at day 70 (Figure 8
and Table 2
). Immunostaining for type
I collagen confirmed these differences in renal accumulation of
collagen between WT and Dcn-/- animals (data not shown).
As described for the dermis18
collagen fibrils in
obstructed kidneys of Dcn-/- mice had irregular-shaped
cross-sections on electron microscopic examination.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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First, the earliest detectable difference between WT and Dcn-/- kidneys was a lower expression of p27KIP1 followed by a more rapid and extensive activation of both the initiator caspase-8 and effector caspase-3 and the induction of programmed cell death in the absence of decorin. Virus-mediated decorin induction has previously been shown to be sufficient for allowing endothelial cells to escape apoptosis.25 In vivo differences in the extent of apoptosis that could be related to the availability of decorin have not been reported before. Signaling via decorin is a novel area of research and may differ between various cell types. They may involve binding to members of the epidermal growth factor receptor family,28 and up-regulation of p21CIP1/WAF1 27 and p27KIP1.37,38 p27KIP1 strongly represses tubular epithelial cell apoptosis and proliferation after ureteral obstruction,34 whereas p21CIP1/WAF1 limits interstitial cell proliferation in this model.39 Our findings suggest that anti-apoptotic and anti-proliferative effects of decorin are primarily focused on tubular epithelial cells and seem to be mediated by activation of p27 whereas p21 seems not to be involved. It is interesting to note that, at least in endothelial cells, decorin-mediated up-regulation of p21CIP1/WAF1 and p27KIP1 occurs by different signaling pathways,38 which may explain that in UUO the expression of only one of the two cyclin-dependent phosphatase inhibitors is specifically altered in Dcn-/- mice.
The second explanation is based on the capability of decorin to form
complexes with TGF-ß110
and on its ability to
down-regulate the cytokine’s activity in the kidney.40
Until now, this mechanism was the only one taken into account for
explaining the effects of decorin on renal fibrosis. Increased TGF-ß
levels in Dcn-/- kidneys readily account for the
up-regulation of biglycan and collagen 1(I) mRNAs. However, biglycan
also forms high-affinity complexes with TGF-ß,10
and one
wonders why it may not exhibit a beneficial effect in fibrotic
diseases.41
It might be speculated that either the role of
decorin in diminishing the degree of apoptosis is of greater importance
than complex formation with TGF-ß in preventing tissue fibrosis, or
that TGF-ß/biglycan complexes are still able to activate the
signaling receptors of the cytokine.
Third, biglycan, like decorin, may also be a signaling molecule. It has been suggested that it acts as a growth factor in certain cell types, eg, for microglial/monocytic cells.23,24 This possibility may be relevant for interpreting the difference between WT and Dcn-/- animals with respect to the larger number of biglycan-expressing macrophages in decorin-deficient obstructed kidneys. If biglycan exhibits growth factor activity for these infiltrating cells, a self-perpetuating cycle could be envisaged in which the overexpression of biglycan leads to growth stimulation of immature macrophages with induced biglycan synthesis and production of an inflammation-supporting cytokine pattern.
A fourth possible explanation for the different courses of UUO in WT and Dcn-/- animals is based on the observation that Dcn-deficient kidneys become more atrophic and contain considerably lower quantities of type I collagen despite an initially higher collagen expression, both on the mRNA and on the protein level. Greater quantities of extracellular matrix would be expected as a consequence of TGF-ß overproduction.5 It is tempting to speculate that the presence or absence of decorin may have influenced the turnover of collagen fibrils. Decorin decorates collagen in a regular manner along the c1-band of the collagen fibril D period.42 It is conceivable that the proteoglycan thereby protects fibrils from collagen-degrading enzymes. The absence of decorin would then result in a greater susceptibility of fibrils toward proteolytic degradation. Another possible explanation for the lower type I collagen content in the absence of decorin might be that the activity of collagen-degrading enzymes was increased for example because of secretion of the respective metalloproteinases by a higher number of infiltrating macrophages.
In summary, the present study provides evidence that targeted disruption of the decorin gene exerts profound effects on the course and final outcome of ureteral kidney obstruction. These differences could be explained by specific effects of decorin on apoptosis via p27KIP1 signaling, TGF-ß activity, and collagen turnover.
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Acknowledgements |
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Footnotes |
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Supported by a Lise-Meitner stipend (to L. S.); the Interdisciplinary Center for Clinical Research (IZKF-Project D18); the Medical Faculty, University of Muenster; the Deutsche Forschungsgemeinschaft (SFB 492, project A6; SFB 293, project A7, and SFB 405, project B10); and the National Institutes of Health (grant RO1 CA-39481).
Accepted for publication November 29, 2001.
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