A possible role for metalloproteinases in renal
cyst development
Nicholas
Obermüller1,
Natividad
Morente1,
Bettina
Kränzlin1,
Norbert
Gretz1, and
Ralph
Witzgall2
1 Medical Research Center, Klinikum Mannheim, University of
Heidelberg, 68167 Mannheim; and 2 Institute for Anatomy and Cell
Biology I, University of Heidelberg, 69120 Heidelberg,
Germany
 |
ABSTRACT |
The expansion of cysts in polycystic kidneys bears several
similarities to the invasion of the extracellular matrix by benign tumors. We therefore hypothesized that cyst-lining epithelial cells
produce extracellular matrix-degrading metalloproteinases and that the
inhibition of these enzymes may represent a potential target for
therapeutic intervention. Using in situ hybridization, we first
analyzed the expression of membrane-type metalloproteinase 1 (MMP-14),
an essential matrix metalloproteinase, of its inhibitor TIMP-2, and of the cytokine transforming growth factor (TGF)-
2 in
the (cy/+) rat model of autosomal-dominant
polycystic kidney disease. Upregulated MMP-14 mRNA was predominantly
located in cyst-lining epithelia and distal tubules, whereas TIMP-2
mRNA was confined almost exclusively to fibroblasts. TGF-
2, a
cytokine known to regulate the expression of matrix metalloproteinases and their inhibitors, was also expressed by cyst wall epithelia. We
then treated (cy/+) rats with the
metalloproteinase inhibitor batimastat for a period of 8 wk. The
treatment with the metalloproteinase inhibitor batimastat resulted in a
significant reduction of cyst number and kidney weight. Our study
suggests that metalloproteinase inhibitors represent a new therapeutic
tool against polycystic kidney disease, which should be applicable
independently of the background of the disease.
autosomal-dominant polycystic kidney disease; therapy; extracellular matrix; matrix metalloproteinase-14; tissue inhibitor of
metalloproteinases-2; transforming growth factor-
2
 |
INTRODUCTION |
AUTOSOMAL-DOMINANT
POLYCYSTIC kidney disease (ADPKD) represents the most
frequently inherited nephropathy. Approximately 50% of all
affected individuals reach end-stage renal disease at the age of 60, ultimately requiring expensive renal replacement therapy such as
dialysis and transplantation (14, 22, 52). Genetically, this disease is most often caused by mutations in the PKD1
(72) and PKD2 (38) genes,
which together are mutated in far more than 90% of all ADPKD patients.
So far, however, it is only poorly understood how these mutations
contribute to cyst formation in the kidney and in other organs.
Several studies have investigated the role of abnormal cell
proliferation, fluid accumulation within cysts, and alterations of the
extracellular matrix in cystogenesis (11, 12, 78), but no
conclusive concept has surfaced so far.
It is likely, however, that the basement membrane plays an important
role in the formation of cysts, because it represents the interface
between the cyst-lining epithelium and its environment. The balance
between the increased degradation of collagens, glycoproteins, and
proteoglycans on the one hand and the enhanced synthesis and deposition
of extracellular matrix components on the other should influence the
extent of cyst enlargement and interstitial fibrosis, the latter being
typical at advanced stages of the disease (44, 50). Matrix
metalloproteinases (MMPs) are a large family of secreted and
membrane-bound zinc-dependent endopeptidases, which degrade a wide
spectrum of substrates and therefore represent key enzymes in the
turnover of the extracellular matrix (8, 37, 40). Most
MMPs are released as zymogens before being activated in the
extracellular environment; MMP-14, however, a membrane-bound metalloproteinase, is processed before its insertion into specific plasma membrane domains. In addition to its matrix-degrading
properties, MMP-14 mediates the activation of pro-MMP-2 together with
the tissue inhibitor of metalloproteinases (TIMP)-2, which acts as a
cofactor. Activation of pro-MMP-2 from this ternary complex is finally
accomplished by a second, non-TIMP-2-bound, MMP-14 molecule (20,
40). Thus net activation of MMP-2 depends on local MMP-14 and
TIMP-2 levels, and it therefore follows that the interaction of a cell
with the extracellular matrix is critically determined by the
concentrations of metalloproteinases and their natural inhibitors.
Furthermore, components of the extracellular matrix have long been
recognized as transforming growth factor (TGF)-
- and fibroblast
growth factor (FGF)-2-binding proteins. Degradation of the
extracellular matrix leads to the release of these growth factors
(7, 21, 69) and, in turn, may regulate cell division and
cyst growth.
Some studies have indeed provided circumstantial evidence for the
importance of certain matrix metalloproteinases and their natural
counterparts, the tissue inhibitors of metalloproteinases (TIMPs), in
polycystic kidney disease (23, 42, 55, 56, 61, 64). More
direct experimental support for the role of MMPs and TIMPs in the
formation of tubular and cystic structures has come from organ culture
experiments and from the analysis of the invasive behavior of cells in
collagen gels. Murine embryonic kidneys have not only been shown to
produce MMPs in vitro, but moreover renal organogenesis is impaired by
the inhibition of MMP-9 (35), MMP-14 (31),
and possibly of MMP-2 (31, 35). Similar results have
arisen from the three-dimensional culture of certain cell lines. When
renal epithelial cell lines such as Madin-Darby canine kidney (MDCK)
and mIMCD3 cells are grown in collagen gels, they form arborized
tubular structures, a phenomenon called branching morphogenesis. In
that process they synthesize MMPs such as MMP-1 (59),
MMP-2 (33), MMP-9 (33), and MMP-14 (30). The addition of natural (TIMP-1, TIMP-2) and
synthetic (batimastat, see below) MMP inhibitors drastically reduces
branching morphogenesis (30, 33), thus emphasizing the
importance of MMPs.
We therefore performed an in situ hybridization study to localize
MMP-14, TIMP-2, and TGF-
2 in polycystic kidneys of the (cy/+) rat, a model that closely resembles human
ADPKD (16, 17, 26, 34, 44, 62). These experiments
demonstrated the distinct expression of all three mRNAs in polycystic
kidneys, with MMP-14 mRNA being expressed by cyst-lining epithelia in particular.
Because a local degradation of extracellular matrix components appears
necessary for cyst expansion, inhibition of metalloproteinases could be
a useful strategy to influence the clinical course of polycystic kidney
disease. Many synthetic inhibitors of metalloproteinases presently
being studied have been developed as anticancer agents to prevent tumor
cell invasion through matrix barriers (10). They
essentially are collagen peptide mimetics and in addition contain
zinc-binding groups such as hydroxamates to block the active site of
the MMPs (6, 79). Batimastat, also known as BB-94, was one
of the first synthetic inhibitors of metalloproteinases; it
specifically and potently inhibits MMPs without showing major toxicity
in animals (79). As a test of principle, we used
batimastat as a therapeutic agent against polycystic kidney disease and
were able to reduce renal cyst number in the
(cy/+) rat.
 |
MATERIALS AND METHODS |
Animals.
Our colony of (cy/+) rats is derived from the
Han:SPRD rat strain. It has been inbred for over 20 generations in
Mannheim and has therefore been registered as PKD/Mhm (for polycystic
kidney disease, Mannheim; inbred strains of rats,
http://www.informatics.jax.org/external/festing/rat/docs/PKD.shtml). Animals were maintained under the control of N. Gretz at the Animal Care Facility in Mannheim. Only male wild-type and heterozygous rats
were used in this study (homozygously affected progeny do not live
longer than 3-4 wk). A total of 53 animals originating from 9 different litters were enrolled randomly over a period of 16 days in
the treatment protocol. Starting on postnatal day 14, rats
either received daily intraperitoneal injections of batimastat (British
Biotech, Oxford, UK) at a dosage of 25 mg/kg body wt or vehicle (0.9%
NaCl containing 0.01% Tween-20) for 6 wk, followed by 3 injections/wk
for another 2 wk. The applied daily dosage of batimastat used in this
trial was based on previous studies in human subjects. Body weight was
monitored weekly, and blood samples were collected 2 wk after the
beginning and at the end of the treatment period. The rats received
standard rat chow (containing 19% protein) and tap water. Two to three
days before the conclusion of the study period, animals were placed
into metabolic cages to analyze their urine parameters. All experiments
were conducted in accordance with the German Animal Protection Law and
were approved by the local government (Regierungspräsidium
Karlsruhe, Germany).
Tissue preparation.
For histomorphological analysis and histochemical experiments, all
animals in the study were subjected to perfusion-fixation. Rats were
anaesthetized with pentobarbital sodium (40 mg/kg body wt) and perfused
through the distal abdominal aorta with 2.5% freshly depolymerized
paraformaldehyde in PBS, pH 7.4, at a pressure level of 180-200
mmHg for 3 min. Subsequently, kidneys were carefully removed and
weighed. A complete slice from the midportion was cut from the right
kidneys, immersion-fixed overnight in the same fixative, and embedded
in paraffin. Four- to five-micrometer-thick paraffin sections were
stained with hemotoxylin and eosin and examined morphometrically. For
subsequent in situ hybridization experiments, the remaining portions of
the right and left kidneys were cut into pieces and incubated in a 18%
sucrose solution in PBS for 4 h on a shaking platform at room
temperature before being snap-frozen in liqid nitrogen-cooled
isopentane. Twelve-week-old, male heterozygous rats, which were used
for initial in situ hybridization studies, were perfused in a similar
way, and both kidneys were processed as described above.
Albumin ELISA.
The concentration of albumin in rat urine was determined by using a
competitive two-step ELISA, which was developed in the Medical Research
Center in Mannheim. During the first step, a chicken anti-rat albumin
antibody (catalog no. 55727, Cappel, Eppelheim, Germany) was incubated
with the sample. After complexes between albumin and the anti-rat
albumin antibody had formed, this mixture was transferred to a 96-well
plate coated with rat albumin (catalog no. A-4538, Sigma, Deisenhofen,
Germany). The amount of antibody bound to the albumin in the 96-well
plates was determined with a peroxidase-coupled antibody directed
against chicken IgG (catalog no. A-9792, Sigma) and subsequent
photometric measurement at 450 nm. Each measurement included rat
albumin samples with known concentrations, so that the albumin
concentration in the urine could be determined from a standard curve.
Morphometry.
For morphometric measurements hemotoxylin- and eosin-stained paraffin
sections from all animals were analyzed by using the image-analysis
system Quantimet 600 (LEICA Q600, Leica Cambridge, Cambridge, UK) and
QWin software (v 1.05). To differentiate between normal tubular
profiles and cysts, the roundedness and the open diameter of the
profiles were taken into account. Thus only profiles with approximately
the size of a glomerulus (~9,000 µm2) and larger were
counted as cysts.
Preparation of riboprobes for in situ hybridization.
Recombinant plasmids containing a 2.5-kbp cDNA fragment of rat TIMP-2,
a 2.4-kbp cDNA fragment of rat MMP-14 (both kindly provided by Michael
T. Crow, National Institutes of Health, Baltimore, MD), and a 1.4-kbp
cDNA fragment of mouse TGF-
2 (a kind gift of Clemens
Suter-Crazzolara, Institute for Anatomy and Cell Biology III,
Heidelberg, Germany) were digested appropriately. Antisense and sense
RNA probes were synthesized and labeled by in vitro transcription using
digoxigenin-11-UTP according to the protocol supplied by the
manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). The
transcripts were finally subjected to partial alkaline hydrolysis to
obtain fragments of a calculated average length of 250 nucleotides.
In situ hybridization.
This procedure was essentially carried out as described in detail
previously (43). In brief, 6-µm-thick cryostat sections were hybridized with a solution containing 50% formamide and 5-8 ng/µl of hydrolyzed TIMP-2, MMP-14, or TGF-
2 RNA probes.
Hybridization was performed overnight at 45°C, followed by several
stringent washes [the most stringent wash was in 0.2× standard sodium
citrate (SSC) containing 50% formamide at 52°C for 1 h]. The
specificity of the obtained in situ hybridization signal was verified
by parallel incubation with antisense and sense riboprobes on alternate
sections. Throughout all experiments, sense probes did not produce any
detectable signals. As further negative controls, some sections were
processed without anti-digoxigenin antibody, which also yielded
completely negative results.
Preparation of total RNA.
The kidneys from 10-wk-old male (cy/+) and
(+/+) rats were rapidly removed, and total RNA
was extracted according to the acid-guanidinium-phenol-chloroform protocol of Chomczynski and Sacchi (13). The resulting RNA
pellets were dissolved in diethylpyrocarbonate-treated water, and the yield was measured by spectrometry at 260 nm. Samples were stored at
80°C until further use. The integrity of the extracted RNA was
checked by agarose gel electrophoresis.
RNase protection assay.
RNAse protection analysis was performed according to standard protocols
(3). An ~300-bp rat MMP-14 cDNA fragment, a 361-bp rat
TIMP-2 cDNA fragment (both kindly provided by Michael T. Crow), a
290-bp rat TGF-
2 cDNA fragment (kindly provided by Ian McLennan and
Kyoko Koishi, University of Otago, Dunedin, NZ), and a 80-bp 18S cDNA
fragment (Ambion, Austin, TX) were used for in vitro transcription.
Plasmids were digested appropriately, and radiolabeled antisense cRNA
probes were synthesized in vitro with viral RNA polymerases (Roche
Molecular Biochemicals) in the presence of [
-32P]UTP.
Fifty micrograms each of total RNA were hybridized with the
radiolabeled MMP-14, TIMP-2 and TGF-
2 riboprobes, whereas 50 ng of
total RNA were hybridized with the radiolabeled 18S riboprobe; tRNA
from Escherichia coli served as a negative control.
Hybridization was conducted overnight at 42°C. After digestion with
RNase A and T1, the protected fragments were separated on a 4%
polyacrylamide/6 M urea gel. Thereafter, the gel was dried for 2 h
and analyzed in Fujifilm BAS-2500 and Bio-Rad GS-525 Phosphorimagers.
Processing of figures.
Black-and-white photographs were scanned with a Nikon Coolscan LS-2000
by using Silverfast 4.1 software (LaserSoft, Kiel, Germany) and then
processed with Photoshop 5.0 (Adobe Systems, San Jose, CA).
Statistics.
Statistical evaluations were performed by using the statistical
analysis system (SAS) from SAS Institute (Cary, NC) The following procedures were applied: medians and quartiles were calculated by using
the PROC UNIVARIATE software, Wilcoxon tests were carried out with PROC NPAR1WAY, and
2-tests were
done with PROC FREQ. Statistical significance was considered at a P value
0.05.
 |
RESULTS |
Expression pattern of the MMP-14,
TIMP-2 and TGF-
2 mRNAs.
By using polycystic kidneys from 12-wk-old male
(cy/+) rats, the expression patterns of the mRNAs
encoding the metalloproteinase MMP-14, the metalloproteinase inhibitor
TIMP-2, and the cytokine TGF-
2 were determined by in situ
hybridization. MMP-14 mRNA was expressed in tubular profiles of the
cortex and outer medulla (Fig. 1,
A and B). Whereas in the renal cortex MMP-14 was
prominently expressed in cyst-lining epithelia (Fig.
2A), in the inner stripe a
number of thick ascending limb profiles were stained for MMP-14 mRNA
(Fig. 2B). In addition, MMP-14 mRNA expression could also be
localized to few fibroblasts in the vicinity of cystic and noncystic
tubules (Fig. 2C). By contrast, in situ hybridization for
TIMP-2 mRNA in polycystic kidneys demonstrated an almost exclusive expression in fibroblasts (Fig. 3). In
rare cases, specific hybridization signals for MMP-14 and TIMP-2 were
also observed in the renal capsule, in parietal cells of Bowman's
capsule, and in the papillary epithelium (data not shown). In situ
hybridization for TGF-
2 in polycystic kidneys resulted in the
labeling of a subset of cysts with a mosaic expression pattern (Fig.
4). When kidneys from age-matched
wild-type rats were analyzed by in situ hybridization, virtually no
specific expression was seen in the case of MMP-14 and TGF-
2, and
only scarce expression of TIMP-2 mRNA in fibroblasts (data not shown).
To corroborate these findings, RNase protection assays with RNAs
isolated from total kidneys were carried out. We were able to detect
all three mRNAs in both (cy/+) and
(+/+) rat kidneys, but there were no pronounced
differences between (cy/+) and
(+/+) kidneys (Fig.
5).

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Fig. 1.
Overview of membrane-type metalloproteinase 1 (MMP-14) mRNA
expression on a kidney section of a 3-mo-old
(cy/+) rat. In situ hybridization for MMP-14 mRNA
was carried out by using digoxigenin-labeled antisense and sense RNA
probes. An overview of the cortex (A) and the inner stripe
(B) demonstrates the expression of MMP-14 mRNA in cystic
(A) and noncystic (B) tubular profiles. Because
no signal was obtained in the inner medulla, the positive tubules
extending into the inner stripe most likely correspond to thick
ascending limbs. Hybridization with MMP-14 sense RNA yielded no
specific signals in the cortex (C) and the inner stripe
(D). Bars, 200 µm.
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Fig. 2.
Detailed analysis of MMP-14 mRNA expression on a kidney
section of a 3-mo-old (cy/+) rat. Strong
expression of MMP-14 mRNA can be clearly seen in cyst-lining epithelial
cells in the cortex (A) and in thick ascending limb cells in
the inner stripe (B). Inset: expression of MMP-14
in cyst-lining epithelial cells at a higher magnification. In some
cases, MMP-14 mRNA was also detected in fibroblasts surrounding tubular
profiles in the cortex (C). Bars, 100 µm (A),
50 µm (B, C).
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Fig. 3.
Nonradioactive in situ hybridization for tissue inhibitor
of metalloproteinases (TIMP)-2 mRNA in a 3 mo-old
(cy/+) rat kidney. An overview through the renal
cortex (A) shows numerous cells expressing TIMP-2; on closer
examination, these cells were identified as fibroblasts located in the
close vicinity of cortical cysts (B). Hybridization with
TIMP-2 sense RNA yielded no specific signals (C). Bars, 200 µm (A, C), 50 µm (B).
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Fig. 4.
Nonradioactive in situ hybridization for transforming
growth factor (TGF)- 2 mRNA in a 3-mo-old
(cy/+) rat kidney. TGF- 2 was strongly
expressed by portions of the cyst wall epithelium, whereas no signal
could be demonstrated in epithelial cells of noncystic tubular profiles
nor in the interstitium [overview (A), higher magnification
(B)]. Hybridization with TGF- 2 sense RNA yielded no
specific signals (C). Bars, 100 µm (A,
C), 50 µm (B).
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Fig. 5.
RNase protection assay for the detection of MMP-14, TIMP-2, and
TGF- 2 mRNA. Two different (+/+) and
(cy/+) rats, each 10 wk old, were used to isolate
RNA from whole kidneys. Fifty micrograms of total RNA were hybridized
with MMP-14, TIMP-2, and TGF- 2 antisense RNA, respectively, whereas
50 ng of total RNA were hybridized with antisense RNA directed against
the 18S subunit of rRNA. The expression levels were determined by
quantitating the protected bands (indicated by an arrow in all 4 cases)
with a Phosphorimager and normalizing the expression of MMP-14, TIMP-2,
and TGF- 2 to that of 18S rRNA. Bar graphs below the gels represent
the mean values obtained from (+/+) and
(cy/+) rats [the level in
(+/+) rats was arbitrarily set as 100%].
Although in the case of TGF- 2 a >2-fold change could be detected,
no drastic changes were observed with MMP-14 and TIMP-2. *, In the
protection assay with MMP-14 antisense RNA, 2 nonspecific bands were
observed, which survived the RNase digest probably due to a particular
intrinsic property of the probe.
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Inhibition of MMP activity leads to reduced cyst
number.
These results prompted us to study the effect of the metalloproteinase
inhibitor batimastat on the progression of cystic disease. Starting at
postnatal day 14 and lasting until postnatal day
70, a total of 53 animals were treated with batimastat or vehicle; no mortality was observed during the study. Treatment with batimastat was associated with a statistically significant reduction in absolute kidney weights (Table 1) in both
(cy/+) rats (median, 4.98 vs. 6.05 g,
P = 0.007) and wild-type (+/+)
animals (median, 2.38 vs. 3.13 g, P = 0.001). The
reduction in kidney weight was still statistically significant when
corrected for body weight. Body weights were only moderately lowered in
the case of batimastat-treated (cy/+) rats,
whereas in (+/+) animals treatment with
batimastat led to a statistically significant reduction in body weights
(Table 1). Morphometric analysis revealed that treatment with the
metalloproteinase inhibitor was associated with a marked and
statistically significant reduction in cyst number in
(cy/+) animals (375 vs. 474, P = 0.026; Table 1). When the distribution of different cyst sizes in
batimastat- and placebo-treated (cy/+) rats was
analyzed, it could be seen that batimastat treatment resulted in a
decreased number of cysts of all sizes (Table
2), although it had a more pronounced
inhibitory effect on the occurrence of smaller cysts (Table 2).
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Table 1.
Median values for body weights, kidney weights, and morphometric data
obtained from batimastat- and placebo-treated
(cy/+) and
(+/+) rats
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Renal function as assessed from serum urea and creatinine values was
unaltered in batimastat compared with placebo groups, and treatment
with the metalloproteinase inhibitor also had no effect on the absolute
and relative 24-h urine volume production in
(cy/+) and wild-type animals (Table
3). However, sodium excretion per 24 h was significantly increased in batimastat-treated vs. placebo-treated
(cy/+) rats (0.44 vs. 0.29 mmol
Na+ · 24 h
1 · 100 g body
wt
1, P = 0.009; Table 3). This effect was
not found in the wild-type animals. No statistically significant
differences were encountered in batimastat vs. placebo groups with
regard to the excreted amounts of other electrolytes; moreover, total
urinary excretion of osmolytes was also unchanged (Table 3).
One side effect of the batimastat treatment was a higher urinary
protein excretion, although this difference was statistically significant only in wild-type rats (4.87 vs. 4.09 mg
protein · 24 h
1 · 100 g body
wt
1, P = 0.003; Table 3), while reaching
borderline significance in (cy/+) rats (4.95 vs.
4.40 mg protein · 24 h
1 · 100 g body
wt
1, P = 0.065; Table 3). A similar, but
more pronounced effect could be found, when the urinary albumin
excretion was analyzed; in both the (cy/+) (1.11 vs. 0.79 mg albumin · 24 h
1 · 100 g body
wt
1, P = 0.013; Table 3) and the
wild-type (0.55 vs. 0.28 mg albumin · 24 h
1 · 100 g body wt
1,
P < 0.001; Table 3) rats, the albumin excretion in the
urine was significantly higher in the batimastat groups compared with the respective placebo groups.
During the removal of the renal capsules after the perfusion procedure,
it was noticed that the renal capsular tissue of most batimastat-treated (cy/+) and wild-type rats had
lost its typical transparency and appeared as an opaque capsule with a
rigid consistency. The expression of MMP-14, TIMP-2, and TGF-
2 mRNA
was also studied after treatment. There was no obvious difference
between the batimastat-treated and placebo-treated animals (data
not shown).
 |
DISCUSSION |
Expression pattern of the MMP-14,
TIMP-2, and TGF-
2
mRNAs.
Our results show that the mRNA for a crucial membrane-bound
metalloproteinase, MMP-14, is expressed in cyst-lining cells of polycystic kidneys, whereas in normal kidneys expression levels were
below the detection limit of the in situ hybridization experiments. Previous analysis of patients (23, 41, 42) and
(cpk/cpk) mice (55, 56) already has
provided evidence for the upregulation of secreted MMPs, whereas one
publication found a downregulation of MMP expression in tubular
cultures from polycystic kidneys (61). Furthermore, there
also is direct evidence for an increased degradation of the
extracellular matrix in patients with polycystic kidney disease
(41, 64). We believe that our study makes an important
contribution to a better understanding of cystogenesis, because we are
the first to identify the MMP-synthesizing cells in polycystic kidneys
in situ. MMP-14, a membrane-bound metalloproteinase, plays a key role
in the turnover of the extracellular matrix (20, 40),
which is demonstrated by the severe phenotype of the MMP14- knockout mice (27, 83) and the rather mild phenotype of
the MMP2 (29)- and MMP9
(76)- knockout mice. The importance of MMP-14 is further
emphasized by the striking invasive phenotype of MDCK cells
overexpressing MMP-14 compared with other MMPs (28). Nevertheless, we cannot rule out the possibility that other
matrix-degrading enzymes also play an important role for the expansion
of cysts.
In addition to the detection of MMP-14 mRNA in cyst-lining epithelial
cells, MMP-14 mRNA expression was seen in a portion of thick ascending
limb profiles, which, however, was not or only moderately dilated.
Interestingly, clusterin, a molecule upregulated in states of cell
injury, is also strongly expressed by noncystic distal tubules in
heterozygous rat kidneys (44). The expression of MMP-14 in
noncystic distal tubules is another indication that the underlying
defect in the (cy/+) kidneys also affects the
distal portion of the nephron, but at this point it is not understood why in this model cysts almost exclusively develop in the proximal tubule. It is possible that additional defects, e.g., in cell-matrix or
cell-cell contacts, are present in the proximal tubule. Alternatively, because MMP-14 activates other metalloproteinases, it is conceivable that proximal tubules but not thick ascending limbs express additional metalloproteinases, which, on activation, contribute to cyst expansion.
Our study also shows the increased cell-specific expression of TIMP-2
mRNA in kidneys of the (cy/+) rat, again a
finding in agreement with previous reports analyzing human
(42), mouse (56), and rat (61)
polycystic kidneys. Whereas MMP-14 mRNA was expressed predominantly by
epithelial cells, TIMP-2 mRNA was almost exclusively located in
fibroblasts. The membrane-bound protease MMP-14 directly influences
pericellular proteolysis, and it therefore is conceivable that an
imbalance of MMP-14 over TIMP-2 contributes to cell migration and cyst
enlargement in the early stages of cyst development. In contrast, the
observed upregulation of endogenous MMP inhibitors such as TIMP-2 may
represent a defense mechanism of the surrounding interstitium to
diminish the effects of MMPs, thus neutralizing soluble MMPs and
leading to fibrosis at advanced stages of the disease
(44). This hypothesis is also supported by the recently
generated MMP-14-knockout mouse, hallmarks of which are impaired
connective tissue growth and fibrosis of soft tissues (27,
83).
The finding that TGF-
2 is expressed by cyst-lining epithelia points
to its possible role in the progression of polycystic disease. The
regulation of matrix metalloproteinases and their inhibitors by growth
factors has been described (19, 58). TGF-
inhibits the
synthesis of matrix metalloproteinases and increases the expression of
TIMPs, therefore promoting matrix deposition and fibrosis (9,
59), which is a prominent feature in polycystic kidneys from old
(cy/+) rats (44). Among the three TGF-
s, TGF-
2 may be of particular importance in the kidney, because renal alterations have only been described in mice lacking a
functional TGF-
2-encoding gene and not in mice, in which the genes
coding for TGF-
1 and TGF-
3 were inactivated (60).
Under physiological conditions, TGF-
2 is synthesized only during
nephrogenesis in the kidney, where its mRNA is found in tubules
(63) and the protein is detected in the tubular basement
membrane (53). Our observation that TGF-
2 mRNA is
synthesized by cyst wall epithelia reiterates the phenomenon that
cyst-lining epithelial cells dedifferentiate and reactivate genes that
have been transcribed earlier in development.
The result that by RNase protection analysis we were not able to detect
any drastic differences in the expression levels of MMP-14, TIMP-2, and
TGF-
2 mRNAs between (cy/+) and
(+/+) kidneys may not be too surprising
considering the following facts. It is estimated that only a minor
percentage of cortical proximal tubules become cystic in the
(cy/+) rat (16, 44, 62). Because there already is a baseline expression of all three mRNAs in
(+/+) rat kidneys, and because MMP-14 and
TGF-
2 are expressed only in a subset of cysts, even a pronounced
increase of expression in some cysts would only lead to a moderate
increase of expression as judged from analyzing RNA from whole kidneys.
The finding that according to the RNase protection assay the expression
of TIMP-2 even decreased to a small degree shows that data obtained by
extracting RNA from a whole organ have to be interpreted with a lot of caution.
Inhibition of matrix metalloproteinase activity leads to reduced
cyst number.
The second important result of our study is the evidence that treatment
with the synthetic metalloproteinase inhibitor batimastat leads to
decreased kidney weight and a reduced number of cysts in
(cy/+) rat kidneys. Although the 21% decline in
cyst number was statistically significant, one might have hoped for a
larger reduction. On the other hand, treatment with batimastat did not commence until 2 wk after birth, when renal cyst development is well
underway (Obermüller and Witzgall, personal observations), and
therefore a complete disappearance of cysts could not be expected. An
analysis of the serum creatinine and serum urea levels showed no change
under batimastat treatment. The creatinine values were still in the
normal range, and therefore the lack of a decrease is not surprising.
In our experience, the serum urea levels in (cy/+) rats are not a very accurate indicator for
the progression of polycystic kidney disease; in rats at such an early
age and considering the relatively modest reduction of cyst number, we therefore would not have expected a drop in serum urea levels. Our
therapeutic study was intended to provide proof of principle, that
treatment with a metalloproteinase inhibitor can have a beneficial effect on the progression of polycystic kidney disease. Clearly, dose-finding and longer-lasting studies are needed to corroborate our
initial findings.
Somewhat surprisingly, the ratio of cyst area to kidney area and the
mean cyst area increased by 9 and 3%, respectively, after batimastat
treatment (these changes were not statistically significant). The
slight increase in the ratio of cyst area to kidney area can be
explained by the fact that treatment with batimastat led not only to a
reduction of cyst number but also to a decrease in kidney weight by
18%. In addition, batimastat treatment changed the distribution of
cyst sizes, because the percentage of larger cysts grew (although the
absolute numbers of both smaller and larger cysts decreased), and this
effect also caused an increase in the mean cyst area. From these data
it can be hypothesized that batimastat exerts a more pronounced
inhibitory effect on the initial phase of cyst development, for which
there may be several reasons. First, although batimastat inhibits a
number of MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-14) with
comparable efficiency (6, 82), it is possible that in the
course of cyst development the cyst-lining epithelial cells start to
express MMPs that are not inhibited by batimastat. Second, the results
presented in this study demonstrate that epithelial cells from normal
proximal tubules and from small cysts did not show detectable amounts
of MMP-14 mRNA by in situ hybridization, whereas large cysts contained
numerous epithelial cells expressing MMP-14 mRNA. In recent
publications, a downregulation, but not a complete loss, of MMP-14 mRNA
expression was demonstrated during postnatal development of the normal
rat kidney (31, 51, 71), which is also corroborated by our
RNase protection data. We therefore speculate that normal tubules and
smaller cysts produce amounts of MMP-14 mRNA that are below the
detection limit for nonradioactive in situ hybridization. According to
this hypothesis, the dosage of batimastat used in our study would
have been more efficient in inhibiting the small levels of MMP-14
present in normal tubules and small cysts, but too low to effectively
block the higher amounts of MMP-14 being expressed in larger cysts. If
this scenario were true, the application of higher doses of batimastat
should exert a stronger inhibitory effect on the growth of larger cysts
as well.
The application of higher doses of batimastat, however, may be limited
by the observed side effects, in particular, albuminuria. It was beyond
the scope of the present manuscript to determine the origin of
albuminuria, but it could, for example, result from an altered turnover
of the glomerular basement membrane. A solution to this problem could
be the use of more specific metalloproteinase inhibitors, which may
show fewer side effects, or slightly lower doses of the drug. Already a
slowed progression of the disease would be of great therapeutic value:
because ~50% of ADPKD patients reach end-stage renal disease by the
age of 60 (14, 22, 52), a delay of 10 yr or longer would
mean that a number of patients would not require renal replacement
therapy in their lifetimes. The decreased kidney weight also in the
wild-type rats probably results from the fact that the therapy was
already started on postnatal day 14, when the kidney is
still growing. Because it is very well possible that metalloproteinases
play a role in renal growth (31, 35), their inhibition may
result in smaller kidneys. We also want to point out that previous
studies did not report these side effects. In a human phase I trial
with batimastat, no adverse effects of batimastat on renal function
were observed, although the authors did not elaborate on how renal
function was examined (5). Because we started the
administration of batimastat already on postnatal day 14,
the kidney was not yet fully developed, which may have rendered it more
susceptible to the action of batimastat. The same probably is true for
the weight loss, which was observed in the (+/+)
rats in our study but not in other animal studies, where the treatment
with batimastat did not commence until postnatal weeks
6-8 (e.g., Ref. 77).
At this point, we tend to believe that the beneficial effect of
batimastat is due to the inhibition of matrix degradation by
metalloproteinases, which is consistent with previous studies demonstrating a strong inhibitory effect of batimastat on the invasion
of cells into collagen gels (28, 30). It is also possible,
however, that batimastat exerts a negative effect on cell
proliferation. The epidermal growth factor family of proteins has been
shown to be important for cystogenesis at least under some
circumstances (57, 66-68). Unless many other secreted
proteins, EGF family members are synthesized as membrane-bound
precursors, and the inhibition of metalloprotease activity has been
shown to reduce EGF receptor-mediated signaling in general (18,
54), and cell proliferation and migration in particular
(18) by preventing the cleavage of these membrane-bound
precursors. Further experiments are needed to determine the
role of the EGF receptor in cyst formation in this particular rat model
of polycystic kidney disease.
In summary, our present study describes a completely novel experimental
approach to positively influence the progression of polycystic kidney
disease. Previous investigations have focused on the potential benefits
of protein restriction (1, 2, 4, 15, 48, 74), other
dietary changes (45-47, 70, 75), and also
pharmaceutical intervention (24, 25, 32, 36, 39, 49, 65, 67, 80,
81). Although some of these animal studies have demonstrated
beneficial effects, they may not be easily transferred to human
patients (73), and certain therapeutic strategies only
work in some animal models but not in others. Because it is possible
that cyst expansion in general depends on the removal of extracellular
matrix by matrix-degrading enzymes, a therapeutic strategy based on
inhibitors of those enzymes should be independent of the underlying
cause of the disease and therefore would be widely applicable.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the kind gifts of rat TIMP-2 and MMP-14
cDNAs from Michael T. Crow, mouse TGF-
2 cDNA from Clemens Suter-Crazzolara, and rat TGF-
2 cDNA from Ian McLennan and Kyoko Koishi. Jutta Christophel and Bernd Schnabel skillfully performed the
albumin ELISAs and prepared the tissue sections. We are also thankful
for the expert photographic work of Ingrid Ertel and for the processing
of the figures by Rolf Nonnenmacher. The batimastat used in our study
was provided by British Biotech.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. Witzgall, Institute for Anatomy and Cell Biology I, Univ. of
Heidelberg, Im Neuenhimer Feld 307, 69120 Heidelberg, Germany (E-mail:
ralph.witzgall{at}urz.uni-heidelberg.de).
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. Section 1734 solely to indicate this fact.
Received 22 March 2000; accepted in final form 10 November 2000.
 |
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