Upregulated expression of human membrane type-5
matrix metalloproteinase in kidneys from diabetic patients
Anne M.
Romanic,
Cynthia
L.
Burns-Kurtis,
Zhaohui
Ao,
Anthony J.
Arleth, and
Eliot H.
Ohlstein
Department of Cardiovascular Pharmacology, GlaxoSmithKline
Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
Matrix
metalloproteinases (MMPs) are a family of proteolytic enzymes that
degrade the extracellular matrix (ECM). The membrane-type matrix
metalloproteinases (MT-MMPs) are a new family of MMPs that differ from
other MMPs in that they have a transmembrane domain that anchors them
to the cell surface. MT-MMPs have been shown to function as receptors
and activators for other MMPs and to localize extracellular matrix
proteolysis at the pericellular region. Here we report on mRNA and
protein expression of the fifth human MT-MMP (MT5-MMP), a 64-kDa
protein that is capable of converting pro-MMP-2 to its active form, in
human kidney as well as its upregulation in diabetes. We also
demonstrate upregulation of the active form of MMP-2 in kidney samples
from patients with diabetes. Through immunohistochemistry, MT5-MMP
expression was localized to the epithelial cells of the proximal and
distal tubules, the collecting duct, and the loop of Henle.
Furthermore, the tubular epithelial cells that expressed MT5-MMP were
associated with tubular atrophy. Because renal tubular atrophy is a
significant factor in the pathogenesis of diabetic nephropathy and
renal failure and the molecular mechanisms regulating this process
remain unknown, it is hypothesized that the elevated expression of
MT5-MMP contributes to the activation of pro-MMP-2, which participates
in the remodeling of the proximal and distal tubules as well as in the
collecting duct. These results provide the first evidence of the
expression of a MT-MMP in diabetes and suggest a novel role for MT5-MMP
in the pathogenesis of renal tubular atrophy and end-stage renal disease.
atrophy; extracellular matrix; protease; remodeling
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INTRODUCTION |
THICKENING OF THE
GLOMERULAR and tubular basement membranes, expanded mesangial
matrix, and tubulointerstitial fibrosis are hallmarks of diabetic
nephropathy (9). There is also a degree of tubular atrophy
that takes place as diabetes proceeds into end-stage renal disease
(13). This atrophy is associated with high proliferative
activity of tubular epithelial cells and modulation of the basement
membrane (13). Also, it has been suggested that interstitial, rather than glomerular, mechanisms may be important in
progressive loss of renal function (3, 13, 14). Changes in
the tubulointerstitial compartment include expansion of the tubular
basement membrane, alterations of extracellular matrix (ECM) in the
interstitium, interstitial cell proliferation, inflammatory cell
influx, and epithelial cell apoptosis (3, 13, 14, 16,
23). Furthermore, changes in the ECM have been shown to modulate
tubular epithelial cell function such as cell proliferation (11) and atrophy (13).
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes
that degrade the ECM and are implicated in numerous pathological conditions, including atherosclerosis, inflammation, tumor growth, and
metastasis (20, 26). The membrane-type matrix
metalloproteinases (MT-MMPs) are a new family of MMPs. The MT-MMPs
differ from other MMPs in that they have a transmembrane domain that
anchors them to the cell surface. Like most of the MMPs, however, the
MT-MMPs consist of four characteristic domains including a signal
peptide for transport from the cell; a propeptide that renders the
enzyme inactive until processed by a cysteine-switch mechanism; a
catalytic domain containing a zinc binding site; and a hemopexin-like
domain that serves as an inhibitor binding site (12, 15, 17, 18, 21, 22, 25). MT-MMPs have been demonstrated to function as
receptors and as activators for other MMPs and serve to localize extracellular matrix proteolysis at the pericellular region (1, 21, 22). Some of the MT-MMPs have also been shown to cleave ECM
molecules directly (8, 24). They have been demonstrated to
play a role in metastasis and have been identified in numerous carcinomas (5, 10, 12, 15, 18, 21). Furthermore, it has
been suggested that MT-MMPs contribute to the infiltration of
inflammatory cells such as T cells into tissues, and it can be
speculated therefore that MT-MMPs are likely to be involved in a host
of diseases in which an inflammatory response is evoked (6).
Thus far, five novel human MT-MMPs have been identified in the
literature (12, 15, 18, 21, 22, 25). Here we report on
mRNA and protein expression of the fifth human MT-MMP (MT5-MMP) in
human kidney. MT5-MMP has recently been identified in the literature as
a 64-kDa protein that is capable of converting pro-MMP-2 to its active
form (12, 17). MT5-MMP was identified in a brain cDNA
library and, by Northern blot, has been localized in normal brain and
in brain tumors including astrocytomas and glioblastomas (12). The results of the present study demonstrate MT5-MMP
mRNA and protein expression in kidney and its upregulation in diabetes. Results also show elevated MMP-2 protein and enzyme activity in kidney
samples from patients with diabetes. These results provide the first
evidence of the expression of an MT-MMP in diabetes and suggest a novel
role for MT5-MMP in the remodeling of the proximal and distal tubules
as well as in the collecting duct and the loop of Henle. As MMPs are
capable of modulating the tubular basement membrane and the
tubulointerstitium, it is hypothesized that remodeling of the
epithelial cell basement membrane, via MT5-MMP and MMP-2, contributes
to the tubular atrophy that occurs in diabetic nephropathy and
end-stage renal failure.
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METHODS |
TaqMan Quantitative PCR Analysis
Oligonucleotides for TaqMan PCR assay.
Table 1 shows the nucleotide sequences of
the oligonucleotide hybridization probes and primers used. These were
obtained from PE Applied Biosystems, a division of PerkinElmer (Foster City, CA). The primers of MT5-MMP were designed from the 3'-UTR region
of the gene using Primer Express Software (PE Applied Biosystems).
TaqMan quantitative PCR assay.
Multiple tissue cDNAs were obtained from Clontech Laboratories (Palo
Alto, CA). These cDNAs were pooled from multiple donors for each
tissue. The number of samples represented for each tissue ranged from 1 to 550 (heart, n = 8; brain, n = 2;
placenta, n = 7; lung, n = 2; liver,
n = 1; skeletal muscle, n = 27; kidney, n = 8; pancreas, n = 20; spleen,
n = 6; thymus, n = 9; prostate, n = 20; testis, n = 19; ovary,
n = 7; small intestine, n = 32; colon,
n = 20; peripheral blood leukocyte, n = 550). TaqMan PCR was performed according to the manufacturer's
specifications (PE Applied Biosystems). Briefly, a master mix was made
containing TaqMan buffer A, MgCl2, dNTPs,
AmpErase UNG, and AmpliTaq Gold polymerase. To this mixture was added 5 µl (1 ng) of cDNA and either MT5-MMP primers (400 nM) and probe (200 nM) or
-actin primers (200 nM) and probe (100 nM). The PCR reaction
was carried out in duplicate tubes in a TaqMan LS-50B PCR detection
system (PE Applied Biosystems) for 40 cycles.
TaqMan results are based on Ct (cycle threshold) values.
That is, when the probe fluorescent signal exceeds the background noise
level, a Ct value is generated. The Ct value
for each tissue was normalized on the basis of
-actin housekeeping
gene expression, yielding a value referred to as the "delta threshold
count." The tissue with the highest delta threshold count was
designated as having a relative copy number of one. The difference
between the tissue with the highest delta threshold count and any other
tissue was calculated and called "n." This n
value was used to calculate the relative copy number of a given tissue
(that is, relative copy number is equal to 2n).
The relative copy numbers for the tissues analyzed were utilized to
compare expression differences among tissues.
Statistical comparisons of relative copy numbers for each tissue were
based on the multiple cDNA samples per tissue (n = 1-550) run in quadruplicate. A Kruskal-Wallis one-way analysis of
variance was performed, followed by individual Mann-Whiney
U-test comparisons among tissues. Data were considered
significant if P < 0.05.
To confirm that the identity of the PCR products generated for TaqMan
analysis was in fact MT5-MMP, Southern analysis was conducted. The PCR
product (5 µl) from each tissue sample was electrophoresed on a 2.5%
agarose gel and then transferred to a nylon membrane (GeneScreen plus;
NEN Life Science Products). The membrane was prehybridized for 2 h
at 42°C with denatured ssDNA in standard buffer (50% deionized
formamide, 1.5 M NaCl, 1% SDS, 10% dextran sulfate) and then
hybridized with a MT5-MMP (1,100-bp fragment generated from an ApaI
restriction digest) cDNA probe labeled with [32P]dCTP
using random-priming synthesis (T7 QuickPrime; Pharmacia Biotech).
Hybridization was conducted overnight at 42°C with 1 × 109 cpm/µg denatured radiolabled probe in standard
buffer, where cpm is counts per minute. The membrane was washed under
low-stringency conditions in 1× standard sodium citrate (SSC), 0.1%
SDS at 25°C, followed by a high-stringency wash in 0.1× SSC, 0.1%
SDS for 30 min at 55°C. Hybridization signals were detected by
conventional X-ray autoradiography (Hyper film; Amersham Life Science)
and phosphorimaging (Storm 860, Molecular Dynamics). The resultant predicted size for the MT5-MMP PCR product was 85 bp.
Multiple-Tissue Western Blot Analysis
Human tissue extracts prepared from the liver, lung, kidney,
skeletal muscle, heart, brain, spleen, testis, ovary, and placenta were
purchased from Clontech Laboratories. The samples were provided in
Laemmli buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, and 5%
glycerol and were reduced with
-mercaptoethanol and heat denatured at 100°C for 3 min. The samples were evaluated for MT5-MMP expression by Western blot analysis. Briefly, samples (50 µg each) were resolved by electrophoresis through a 10% polyacrylamide gel and then
transferred to a nitrocellulose membrane with a Bio-Rad semidry
transfer apparatus (Bio-Rad, Hercules, CA). Unoccupied binding sites
were blocked overnight at 4°C with 5% nonfat powdered milk in a 0.1 M Tris · HCl buffer, pH 8.0, containing 1.5 M NaCl and 0.5%
Tween-20 (TBST). A rabbit polyclonal primary antibody directed against MT5-MMP (generated against amino acid sequence CNQKEVERRRKERRL located
in the coding region of MT5-MMP), diluted 1:5,000 in TBST, was then
added to the membrane and allowed to incubate for 1 h at 25°C.
The membrane was washed three times, 15 min each, with TBST, incubated
for 30 min with a goat anti-rabbit IgG secondary antibody conjugated to
horseradish peroxidase (Bio-Rad), and diluted 1:5,000 in TBST. The
membrane was washed as above, and the blot was developed by using the
enhanced chemiluminescence method (Amersham, Arlington Heights, IL)
according to the manufacturer's instructions.
Preparation of Kidney Tissue Extracts
Human kidney tissues from diabetic and nondiabetic
individuals were obtained from the Anatomic Gift Foundation (Phoenix,
AZ). Gender, age, and serum creatinine levels of the patients are
described in Table 2. To analyze protein
expression in these kidney tissues, protein extracts of the
tissues were prepared. To prepare the tissues for extraction, they were
first weighed and then minced into 1-mm3 pieces. The minced
tissues were incubated in an extraction buffer consisting of 0.5%
Triton X-100 (Sigma, St. Louis, MO) in PBS containing 0.01% sodium
azide while being gently rotated at 4°C for 18 h. The
concentration of the initial extraction mixture for each tissue sample
was normalized to 400 mg/ml. After the extraction was complete, the
samples were centrifuged at 3,500 rpm for 15 min at 4°C, and the
supernatants were collected. The supernatants were then centrifuged at
14,000 rpm for 15 min at 4°C to clear the lysates further. Aprotinin
(Sigma) was added to each extract to a final concentration of 3 U/ml.
To check the quality of the extraction, samples of each extract
prepared were analyzed by SDS-PAGE (10% polyacrylamide) in which the
gel was stained with 0.25% Coomassie brilliant blue 250 (Sigma).
Western Blot Analysis
To investigate MT5-MMP and MMP-2 protein expression in kidney
tissue extracts (n = 6/condition), samples were
normalized for protein concentration by using a DC Protein Assay
(Bio-Rad) and prepared for Western analysis as described above, except
that only 30 µg of total protein were used. As control standards,
recombinant pro-MT5-MMP and pro and active MMP-2 were included on each
respective blot. Also, for Western analysis of MMP-2 expression, a
mouse monoclonal anti-MMP-2 primary antibody (2 µg/ml, clone 42-5D11, Oncogene Research Products, Cambridge, MA) and a goat anti-mouse IgG
secondary antibody (1:5,000, GIBCO-BRL, Bethesda, MD) were used. The
levels of intensity of each band relative to background were determined
and quantitated with a Molecular Dynamics densitometer (Molecular
Dynamics, Sunnyvale, CA). Data were expressed as means ± SE. For
statistical analysis of MT5-MMP and MMP-2 expression, Student's
t-test for unpaired data was used. Statistical significance was accepted when P < 0.05.
Immunohistochemistry
Human kidney tissue (cortex and medulla) from four
insulin-dependent diabetic and four nondiabetic patients was analyzed
for MT5-MMP protein expression by immunohistochemistry. Formalin-fixed, paraffin-embedded, 4-µm-thick serial sections of cortex and medulla were obtained from Clinomics Laboratories (Pittsfield, MA). Staining was conducted by Clinomics using the Ventana Medical Systems ES System.
Briefly, sections were deparaffinized in xylenes and rehydrated in
graded alcohols and distilled water. Endogenous peroxidase was quenched
with 0.3% H2O2. Nonspecific immunoglobulin
binding sites were blocked with normal goat serum for 16 min, and the sections were then incubated with a rabbit polyclonal primary antibody
directed against MT5-MMP for 32 min at room temperature. As a negative
control, serial sections were treated with primary antibody that was
preincubated with MT5-MMP protein (5-fold molar excess, overnight at
4°C). The sections were then stained by using the
avidin-biotin-peroxidase method. The reaction was visualized with
3,3'-diaminobenzidine (DAB) as substrate. Sections were counterstained with Gill's hematoxylin solution, dehydrated in graded alcohols and
xylenes, mounted, and then examined by light microscopy by using an
Olympus IX70 microscope. Alternating sections from each kidney were
also stained with hematoxylin and eosin (H&E).
MMP-2 Activity ELISA
MMP-2 activity was quantitated with a human MMP-2 Activity ELISA
System (Amersham Pharmacia Biotech, Piscataway, NJ) according to the
manufacturer's instructions. Briefly, protein extracts, normalized in
assay buffer [0.03 M phosphate buffer, pH 7.0, containing 0.1 M NaCl2,
1% (wt/vol) BSA, and 0.01 M EDTA], were incubated for 2 h at
room temperature in microtitre wells coated with anti-MMP-2 antibody.
Components of the extract other than MMP-2 were removed with four
rinses in wash buffer (0.01 M phosphate buffer, pH 7.5, containing
0.05% Tween 20). Peroxidase-labeled Fab' antibody to MMP-2 was added
to the wells for 1 h at room temperature. After excess peroxidase
conjugate was removed with four rinses in wash buffer, the amount of
peroxidase bound to each well was determined by the addition of 3, 3',
5, 5'-tetramethylbenzidine (TMB)/hydrogen peroxide in dimethylformamide
(20%, vol/vol). The reactions were stopped with the addition of 1 M
sulphuric acid, and the plate was read at 450 nm in a SPECTRAmax 250 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA). The
concentration of active MMP-2 in each sample was determined by
interpolation from a standard curve using SOFTmax PRO software
(Molecular Devices). Data were expressed as means ± SE. For
statistical analysis of MMP-2 activity, Student's t-test
for unpaired data was used. Statistical significance was accepted when
P < 0.05.
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RESULTS |
MT5-MMP mRNA Expression in Human Brain, Kidney, and Pancreas
The expression of MT5-MMP mRNA was analyzed by TaqMan
quantitative PCR analysis (Fig. 1). cDNA
samples from human heart, brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary,
small intestine, colon, and peripheral blood leukocytes were evaluated
for MT5-MMP copy number relative to
-actin. The results showed that
significant MT5-MMP mRNA expression was detected in brain, kidney, and
pancreas (Fig. 1A). Visualization of the MT5-MMP PCR
products on an agarose gel as well as Southern analysis of the PCR
products using a probe specific for MT5-MMP revealed a single band of
85 bp in all tissue samples determined to express MT5-MMP by TaqMan
analysis (Fig. 1B). Characteristic of the high sensitivity
of Southern blot analysis, relative to PCR analysis, MT5-MMP mRNA
expression was also detected in additional tissues, albeit at very low
levels (Fig. 1B).

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Fig. 1.
TaqMan quantitative PCR analysis of human membrane type-5 matrix
metalloproteinase (MT5-MMP) mRNA expression. A: TaqMan PCR
analysis, conducted to quantitate MT5-MMP mRNA expression in multiple
human tissue cDNA samples. As a control, TaqMan PCR analysis was
conducted for mRNA expression of -actin in these samples.
Significant MT5-MMP gene expression was detected in the brain, kidney,
and pancreas. The gene expression is calculated as relative copy
number, using -actin as a housekeeping gene control. PBL, peripheral
blood leukocyte. * P < 0.05. B: PCR products
electrophoresed on a 2.5% agarose gel and visualized on ethidium
bromide staining and ultraviolet detection. Southern analysis of the
PCR product demonstrated the presence of a single band of 85 bp,
confirming that the PCR product was MT5-MMP.
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MT5-MMP Protein Expression in Human Kidney
MT5-MMP protein expression was evaluated in various human tissue
extracts by Western blot. The results demonstrated that MT5-MMP protein
was detected only in kidney (Fig. 2).
Notably, two bands were detected. The molecular mass of the top band
was ~64 kDa, in agreement with the predicted size of pro-MT5-MMP
(17), and the molecular mass of the bottom band was ~58
kDa, presumably the active form of MT5-MMP. It should be noted that
mRNA and protein expression are not always directly correlated, as was
the case here in which MT5-MMP mRNA expression was detected in brain
and pancreas but protein expression was not detected.

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Fig. 2.
Multiple-tissue Western blot of MT5-MMP protein expression. Protein
extracts of the following tissues were analyzed for expression with a
polyclonal antibody directed against human MT5-MMP: liver, lung,
kidney, skeletal muscle, heart, brain, spleen, testis, ovary, and
placenta. MT5-MMP protein expression was detected only in the kidney.
Two bands were detected, a 64-kDa band corresponding to pro-MT5-MMP and
a 58-kDa band corresponding to active MT5-MMP.
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Upregulated MT5-MMP Protein in Kidney Samples From Diabetic
Patients
Because MT5-MMP protein was detected in normal human kidney, the
possibility of further upregulation of MT5-MMP in kidney disease was
investigated. Kidneys from nondiabetic and diabetic patients
(n = 6/condition, Table 2) were analyzed by Western blot for MT5-MMP protein expression (Fig.
3A). The results demonstrated that MT5-MMP protein expression, in the active form, was significantly upregulated 22-fold (P < 0.02) in diabetic kidney
samples (Fig. 3B).

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Fig. 3.
Western blot of MT5-MMP protein expression in kidney
tissue samples from nondiabetic and diabetic patients. Protein extracts
were prepared from kidney tissues of nondiabetic and diabetic patients
(n = 6 each) and analyzed for MT5-MMP expression.
A: Western blot of MT5-MMP expression. Lanes
1-6, nondiabetic kidney samples; lanes 7-12,
kidney samples from patients with diabetes. A recombinant standard
(std.) of pro-MT5-MMP was included for identification and comparison.
MT5-MMP protein expression, in the active form, was markedly
upregulated in the kidney samples from patients with diabetes.
B: quantitation of MT5-MMP protein expression in kidney
samples from nondiabetic and diabetic patients measured by
densitometry. MT5-MMP expression was upregulated 22-fold in the
diabetic samples. Data are presented as optical density of the bands
and are expressed as means ± SE. *P < 0.05.
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Upregulated MT5-MMP Protein in Cells of the Proximal and Distal
Tubules and Collecting Ducts in Kidney From Diabetic Patients
Tissue samples from renal cortex (n = 4/condition)
and renal medulla (n = 4/condition) of nondiabetic and
diabetic patients were evaluated by immunohistochemistry. The results
indicated that MT5-MMP was highly expressed in the epithelial cells of
the proximal and distal convoluted tubules in the cortex (Fig.
4, A and B) as well
as in the collecting duct and loop of Henle of the medulla (Fig.
5, A and B).
Furthermore, tubular atrophy was noted in the diabetic kidney samples
(Fig. 5, A and B). There was no MT5-MMP
expression detected in the glomeruli (Fig. 4). Also, there was no
appreciable staining for MT5-MMP in the nondiabetic kidney samples
(Figs. 4 and 5, C and D). Additionally, no
significant staining for MT5-MMP was observed in serial sections of
diabetic kidney samples in which the primary antibody was preincubated and blocked with MT5-MMP protein (Figs. 4 and 5E).

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Fig. 4.
Immunohistochemical localization of MT5-MMP protein expression in
the cortex of kidney from diabetic patients. Human kidney cortex
sections from nondiabetic and diabetic patients (n = 4 each) were analyzed by immunohistochemistry for MT5-MMP expression.
MT5-MMP is highly expressed in the proximal and distal convoluted
tubules in the cortex from the diabetic patients and absent in the
nondiabetic cortex. Epithelial cells lining the Bowman's capsule are
positive as well. Representative results are shown.
A: cortex section, from the kidney of a diabetic patient,
incubated with anti-MT5-MMP antibody (original magnification ×25).
B: same as in A (original magnification ×50).
C: cortex section of a kidney, from a nondiabetic
patient, incubated with anti-MT5-MMP antibody (original
magnification ×25). D: same as in C (original
magnification ×50). E: negative control (serial section of
diabetic kidney shown in B, in which the primary antibody
was preincubated and blocked with MT5-MMP protein). F:
hematoxylin-and-eosin-stained serial section of diabetic kidney shown
in B.
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Fig. 5.
Immunohistochemical localization of MT5-MMP protein expression in
the medulla of kidney from diabetic patients. The medullas from
nondiabetic and diabetic patients (n = 4 each) were
analyzed by immunohistochemistry for MT5-MMP expression. MT5-MMP was
expressed in the collecting duct and loop of Henle of the medulla from
the diabetic patients. The tubules from the diabetic patients appear to
have undergone atrophy. MT5-MMP expression was not detected in the
nondiabetic medulla. Representative results are shown. A:
medulla section of a kidney, from a diabetic patient, incubated with
anti-MT5-MMP antibody (original magnification ×25). B: same
as in A (original magnification ×50). C: medulla
section of a kidney, from a nondiabetic patient, incubated with
anti-MT5-MMP antibody (original magnification ×25). D: same
as in C (original magnification ×50). E:
negative control (serial section of diabetic kidney shown in
B, in which the primary antibody was preincubated and
blocked with MT5-MMP protein). F:
hematoxylin-and-eosin-stained serial section of diabetic kidney shown
in B.
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Elevated MMP-2 Protein and Enzyme Activity in Kidney Samples
from Diabetic Patients
MMP-2 protein and enzyme activity were evaluated in kidney samples
obtained from nondiabetic and diabetic patients (n = 6/condition) by Western blot and by an activity-based ELISA. The ELISA
is specific for measuring MMP-2 enzyme activity only. The results
generated by Western blot demonstrated significant elevation of MMP-2
protein of the active form (3.8-fold, P < 0.01) in
kidney samples from diabetic patients compared with samples from
nondiabetic patients (Fig. 6). As
demonstrated by the activity-based ELISA, MMP-2 activity was also
significantly elevated (6-fold, P < 0.05) in kidney
samples from diabetic patients compared with samples from nondiabetic patients (Fig. 7).

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Fig. 6.
Western blot of MMP-2 protein expression in kidney tissue
samples from nondiabetic and diabetic patients. Protein extracts were
prepared from kidney tissues of nondiabetic and diabetic patients
(n = 6 each) and analyzed for MMP-2 expression.
A: Western blot of MMP-2 expression. Lanes
1-6, nondiabetic kidney samples; lanes 7-12,
kidney samples from patients with diabetes. A standard of recombinant
pro- and active MMP-2 was included for identification and comparison.
MMP-2 protein expression, in the active form, was markedly upregulated
in the kidney samples from patients with diabetes. B:
quantitation of MMP-2 protein expression in kidney samples from
nondiabetic and diabetic patients was measured by densitometry. MMP-2
expression was upregulated 3.8-fold in the diabetic samples. Data are
presented as optical density of the bands and are expressed as
means ± SE. **P < 0.01.
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Fig. 7.
Expression of MMP-2 enzyme activity in kidney from
diabetic patients. MMP-2 enzyme activity was evaluated in kidney
extracts from nondiabetic and diabetic patients (n = 6, each condition) by using an activity-based ELISA. MMP-2 activity was
elevated 6-fold in the samples from diabetic patients compared with
nondiabetic patients, *P < 0.05.
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DISCUSSION |
In the present study, it is demonstrated that MT5-MMP mRNA and
protein are expressed in the kidney and that expression is increased
dramatically in patients with diabetes. Also, expression of MT5-MMP
correlates with elevated MMP-2 enzyme activity. Furthermore, the data
indicate that the induction of MT5-MMP protein expression occurs
primarily in the epithelial cells of the proximal and distal tubules,
the collecting duct, and the loop of Henle, and this supports the
growing evidence that interstitial mechanisms are involved in the
progressive renal damage often associated with diabetes.
The ECM in the kidney, as in other organs, can affect cell function in
addition to contributing to the structural integrity of the tissue. In
renal disease, for example, the modulation of the ECM contributes to
tubular epithelial cell proliferation (11) and atrophy
(13). It has been suggested that the tubular basement membrane mediates communication between epithelial cells and
fibroblasts, and it has been demonstrated that components of the
basement membrane such as laminin and collagen IV modulate signaling
events between these two cell types (11). Qualitative and
quantitative changes in the basement membrane occur in renal disease,
suggesting that modulation of the basement membrane components
contribute to pathological changes in the tubulointerstitium, in part,
by altering tubular epithelial cell function (11, 14).
MMP-2 is a proteolytic enzyme that degrades components of the basement
membrane including laminin, collagen IV, and fibronectin
(20), and immunolocalization of this enzyme has been
detected in interstitial fibroblasts and epithelial cells of rat kidney
(7). Proteolytic products of the basement membrane such as
laminin and collagen IV fragments stimulate proliferation of epithelial
cells (2, 27), and enhanced tubuloepithelial cell
proliferation has been associated with elevated epithelial cell atrophy
(13). Also, it has been demonstrated by Frisch and Francis
(4) that disruption of epithelial cell-matrix interactions
induces apoptosis. Furthermore, Pullan et al. (19)
have shown in the mammary gland that apoptosis is suppressed in
epithelial cells when they are adherent to their basement membrane.
However, during mammary gland involution, cell loss due to
apoptosis coincides with MMP expression, basement membrane
degradation, and cell detachment. Notably, Park et al. (16) have demonstrated that detachment from the ECM
induces apoptosis in kidney collecting duct cells. Furthermore,
Thomas et al. (23) have correlated epithelial cell
apoptosis, which may contribute to tubular atrophy, with the
progression of renal damage. Thus it has been suggested that
tubulointerstitial damage is a more consistent predictor of functional
impairment rather than glomerular damage (14).
MT5-MMP has recently been identified in the literature as a new member
of the growing MT-MMP family and has been demonstrated to convert
pro-MMP-2 to its active form (12, 16). The pro form of
MMP-2 is not catalytically active until its propeptide is cleaved off,
and MT-MMP-mediated cleavage of the propeptide is one means of MMP-2
activation. Our results show elevated expression of active MMP-2 enzyme
in kidney from diabetic patients. It is hypothesized that the elevated
expression of MT5-MMP contributes to the activation of pro-MMP-2, which
participates in the remodeling of the proximal and distal tubules as
well as in the collecting duct. By immunohistochemistry, the expression
of MT5-MMP protein correlated with epithelial cells in atrophied
tubules. Renal tubular atrophy is a significant factor in the
pathogenesis of diabetic nephropathy and renal failure, but the
molecular mechanisms regulating this process remain unknown. It is
hypothesized that the expression of MT5-MMP along with MMP-2 plays an
important role in the remodeling of the basement membrane associated
with the epithelial cells of the proximal and distal tubules and the
collecting duct of the diseased kidney. Furthermore, the colocalization
of MT5-MMP to sites of basement membrane remodeling suggests a
potential role for this molecule as a receptor for and/or modulator of
MMP-2 activity. We speculate that during diabetic nephropathy,
MMP-mediated remodeling of the basement membrane contributes to
epithelial cell detachment from the basement membrane, contributing to
apoptosis of tubular epithelial cells and tubular atrophy.
These events play an important role in the pathogenesis of renal
tubular atrophy and end-stage renal disease, ultimately causing
nonfunctional renal tubules in the kidney.
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ACKNOWLEDGEMENTS |
The authors are grateful for the generosity of Dr. Nicholas Laping
and Ms. Barbara Olsen in providing us with the kidney tissue samples
for ELISA and Western blot analyses and to John Martin for help in
making the antigenic peptides used for antibody production. We also
thank Dr. David Brooks for helpful discussions during the writing of
this manuscript.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. M. Romanic, GlaxoSmithKline Pharmaceuticals, Dept. of Cardiovascular Pharmacology, 709 Swedeland Rd., King of Prussia, PA 19406 (E-mail: anne_romanic-1{at}sbphrd.com).
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 7 September 2000; accepted in final form 17 April 2001.
 |
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