Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy
Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy
Sang Soo Kim 0 1 2
Nari Shin 0 1
Sun Sik Bae 0 1
Min Young Lee 0 1 2
Harin Rhee 0 1 2
Il Young Kim 0 1
Eun Young Seong 0 1 2
Dong Won Lee 0 1
Soo Bong Lee 0 1
Ihm Soo Kwak 0 1 2
David H. Lovett 0 1 3
Sang Heon Song 0 1 2
0 Funding: This study was supported by Biomedical Research Institute Grant (2015-3), Pusan National University Hospital (
1 Editor: Utpal Sen, University of Louisville , UNITED STATES
2 Biomedical Research Institute and Department of Internal Medicine, Pusan National University Hospital , Busan , Republic of Korea, 2 Department of Pathology, Pusan National University Yangsan Hospital , Yangsan, Gyeongnam , Republic of Korea, 3 MRC for Ischemic Tissue Regeneration , Medical Research Institute, and Department of Phamacology, Pusan National University School of Medicine, Yangsan, Republic of Korea, 4 Research Institute for Convergence of Biomedical Science and Technology and Department of Internal Medicine, Pusan National University Yangsan Hospital , Yangsan, Gyeongnam , Republic of Korea
3 The Department of Medicine, San Francisco Department of Veterans Affairs Medical Center/University of California San Francisco , San Francisco, California , United States of America
Data availability statement; All relevant data are within the paper
We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in
HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE) and tested the effects of
the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). The streptozotocin (STZ) murine
model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were
Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with
HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but
potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression
of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold).
Isoform-specific immunohistochemical staining revealed low, but detectable levels of the
used in this study.
Diabetes, Digestive and Kidney Disease grant
RO1DK39776. DHL's contributions are the result of
work supported with the resources and the use of
facilities at the San Francisco VA Medical Center.
Competing interests: The authors have declared
that no competing interests exist.
MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest
increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2
was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform
expression as quantified by qPCR were both significantly elevated in renal biopsies of
human diabetic nephropathy (12-fold and 3-fold, respectively).
The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for the treatment of diabetic renal disease.
Diabetes mellitus (DM) has an increasing prevalence worldwide and is associated with major
morbidity and mortality. According to the 2014 National Diabetes Statistics Report, over 9%
of the US population (or 29.1 million people) have diabetes mellitus [
]. Diabetic nephropathy
is an increasingly frequent microvascular complication of diabetes mellitus and the leading
cause of end-stage renal disease (ESRD) worldwide, including Korea. Multiple pathologic
processes contribute to the development and progression of diabetic nephropathy, including
acute and chronic hyperglycemia, hypertension, dyslipidemia, oxidative stress, inflammation
and genetics [
]. At present, it is not possible to predict in which patients diabetic nephropathy
will develop, presumably due to the complexity of the individual contributing factors.
Matrix metalloproteinase-2 plays a major role in the pathogenesis of cardiovascular and
renal disease [3±7]. In terms of the kidney, enhanced expression of MMP-2 has been described
in several experimental models of renal injury, including ischemia/reperfusion injury [8±10].
In addition, transgenic renal proximal tubule-specific expression of the secreted, full length
MMP-2 (FL-MMP-2) isoform is sufficient to induce all of the common features of progressive
human renal disease, including glomerulosclerosis, tubular atrophy, interstitial fibrosis and
]. These observations suggest that MMP-2 may represent a common pathway
for the development of progressive renal disease.
We recently reported on the identification and characterization of a novel intracellular
isoform of MMP-2 generated by oxidative stress-mediated activation of an alternate promoter
located in the first intron of the MMP-2 gene [
]. This event leads to the synthesis of an
NTerminal Truncated MMP-2 isoform (NTT-MMP-2) that localizes to mitochondria and
triggers a primary innate immune response resulting in inflammation and cell death [11). Both
the acute and chronic hyperglycemia of diabetes mellitus is associated with increased oxidative
]. We hypothesized, in the setting of experimental and clinical diabetic
nephropathy, that hyperglycemia-mediated oxidative stress enhances tubular epithelial expression of
both MMP-2 isoforms, leading to cellular injury. The experiments outlined in this report
confirm our hypothesis as detailed below.
Materials and methods
HK2 cell culture experiments
The immortalized human proximal tubule cell line HK2 was obtained from ATCC (Manassas,
VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5.5 mM
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D-glucose, 10% heat-inactivated fetal bovine serum and antibiotics-antimycotics (Gibco1/Life
Technologies, Paisley, UK). Cells were incubated at 37ÊC in a humidified incubator with 5%
CO2. For experimental studies, HK2 cells were cultured with different concentrations of
Dglucose (5mM, 30mM) for 2, 24 and 48h, respectively. Additionally, 4-hydroxy-2-hexenal
(HHE) was obtained from Cayman Chemical, Inc. (Ann Arbor, MI, USA). HHE induces lipid
peroxidation and is a stimulant of oxidative stress. HK2 cells were cultured with 10μM HHE
for 2, 24 and 48h and compared with a non-treated control group. Control cells were harvest
at 2hr because the expression of MMP-2 was not significantly different according to varying
time point (data not shown). RNA was extracted from cells using TRIzol1 reagent (Invitrogen;
Life Technologies Korea, Seoul, Korea) according to the manufacturer's protocol and
processed to measure the expression of two MMP-2 isoforms using a quantitative real-time
polymerase chain reaction (qPCR) detailed below. An antibody to p65-NFkB (ser536) was
obtained from Cell Signaling Technology (Danvers, MA, USA). The antioxidant and NF-kB
inhibitor pyrrolidine dithiocarbamate (PDTC) was obtained from Sigma-Aldrich, Inc.
(St. Louis, Mo, USA). PDTC treatment was performed at 50 μM 1h prior to D-glucose
Immunofluorescence studies of HK2 cells
HK2 cells were grown on sterilized coverslips in 6-well plates, and then washed with PBS,
fixed with 4% buffered paraformaldehyde (4ÊC) for 20 min and permeabilized in 0.1% Triton
X-100/PBS 1X for 10 min. Subsequently, the cells were incubated with a FL-MMP-2-specific
monoclonal antibody (prediluted) directed against the N-terminal domain (ab50441, Abcam,
Cambridge, UK). A NTT-MMP2-specific antibody targeting the S1' substrate binding loop
was used at 20 μg/ml 0.1% BSA/PBS [
]. Both primary antibody incubations were
performed at 4Ê C overnight . The coverslips were then incubated with the FITC-conjugated
secondary antibody (Molecular Probes) for MMP-2 (1:500, Alexa fluor1 488 goat anti-mouse
IgG); for NTT-MMP-2 (1:800, Alexa fluor1 488 rabbit anti-goat IgG) at room temperature
for 30 min. Cell nuclei were stained with DAPI (Molecular Probes, Oregon, USA) 1:2000 for
10 min. The coverslips were mounted onto microscopy glass slides with mounting solution.
Images were obtained with a Leica TCS-SP8 confocal microscope (Leica, Mannheim,
Germany). Immunofluorescence intensity was measured using the Image J program (National
Institutes of Health, Bethesda, MD, USA).
Murine model of diabetic nephropathy
The animal protocol (2014±069) used in this study was reviewed and approved by Pusan
National University±Institutional Animal Care and Use Committee (PNU-IACUC) on their
ethical procedures and scientific care. Mice were randomized into control and diabetic groups.
Diabetes mellitus was induced by five daily intraperitoneal injections of streptozotocin (40mg/
kg in citrate buffer, pH4.5, Sigma-Aldrich) in 8-week-old C57/BL6 male mice. Control mice
received citrate buffer alone. After 1 week, blood glucose levels were measured using a blood
glucometer by tail vein puncture blood sampling. Body weight was measured weekly and
blood glucose concentrations were monitored every four weeks to confirm hyperglycemia.
Mice were sacrificed under isoflurane anesthesia at 12 weeks and 24 weeks, respectively. Each
group was comprised of eight mice. Kidneys were perfused with 4Ê C phosphate-buffered
saline (PBS) and then excised. The renal cortex was harvested after excision of the medullary
portion. Half of the kidney was fixed in 10% neutralized formalin for immunohistochemical
study. The remaining portions were used for Western blot, zymography and qPCR analysis as
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Quantitative real-time Reverse Transcriptase-Polymerase Chain
Reaction (PCR) analysis
The measurements of FL-MMP-2 and NTT-MMP-2 mRNA expression were conducted with
quantitative RT-PCR. Total RNA was isolated using TRIzol1 reagent (Invitrogen; Life
Technologies Korea, Seoul, Korea) according to the manufacturer's protocol. In brief, 2 μg of total
RNA was used to synthesize cDNA using oligo-dT primers and M-MLV RTase (Promega,
Madison WI, USA). The reaction was performed at 42ÊC for 1 h. The primers used for
realtime PCR are summarized in Table 1. The amplification reaction was conducted with 40 cycles
(95ÊC for 15 sec; 60ÊC for 45 sec; and 72ÊC for 1 min) using Fast Start Universal SYBR Green
Master Mix (Rox dye, Roche) on an ABI 7500 Real-time PCR System (Applied Biosystems).
For a housekeeping internal control, β-actin or ribosomal 36B4 mRNA were quantified in
parallel with the target genes and all products were verified using melting curve analysis (95ÊC
15sec, 60ÊC 15sec, 95ÊC 15sec). Normalization and fold-change for each of the genes were
calculated using 2-ΔΔCT method.
Western blot methods
Proteins were extracted from the excised renal cortex with a protein extraction solution
(PRO-PREP™; iNtRON Biotechnology, Korea). The protein concentration was measured by
the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories Inc., Hercules, CA, USA).
Proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred
onto a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech Inc.,
Piscataway, NJ, USA). Blots were blocked for 2 h at room temperature with 5% (w/v) non-fat dried
milk in Tris-buffered saline (10 mM Tris/HCl, pH 8.0, and 150 mM NaCl) solution containing
0.05% Tween-20. The membranes were immunoblotted with the following specific primary
antibodies (1∶1000 dilution): rabbit polyclonal antibodies for p-NF-κB p65 (Cell Signaling
Technology, Inc.), and GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The
blots were then incubated with the corresponding conjugated anti-rabbit immunoglobulin
Ghorseradish peroxidase (1∶2,000 dilution, Abcam, Cambridge. UK). Immunoreactive proteins
were detected with the ECL Western blotting detection system (AB Frontier, Seoul, Korea).
Zymography was performed to identify MMP-2 activity in kidney tissue. Kidney cortices were
extracted using lysis buffer (25mM Tris-HCl, pH 7.5, 100mM NaCl, 1% NP-40). Gelatinase
activity was determined by electrophoresis on 8% SDS-PAGE) gels containing 0.1% (w/v)
gelatin. After electrophoresis, the gels were washed in 2.5% Triton X-100 and incubated for 36
hours in zymography developing buffer (Komabiotech, Korea) at 37ÊC. The MMP-2
gelatinolytic activity was detected as clear bands on a blue background after staining the gels with
Coomassie Brilliant Blue R250.
GCTGTATGTCCTGTCGCT CAA CT
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Tissues were fixed in 10% formalin immediately after collection. Thereafter, the tissues were
paraffin-processed and embedded. Immunohistochemistry was performed on formalin-fixed
3 μm thick paraffin embedded sections. Sections were deparaffinized, followed by rehydration
with graded ethanol to water. For immunostaining of FL-MMP-2, the sections were incubated
for 30 minutes with a prediluted monoclonal anti-mouse antibody against the N-terminal
sequence of the FL-MMP-2 protein (ab50441, Abcam, Cambridge, UK), followed by a 30 minute
incubation with biotinylated anti-mouse IgG (Vector). For immunostaining of the
NTT-MMP2 isoform, the sections were incubated overnight at 4ÊC with 5 μg/ml of the isoform-specific
antibody detailed above. After each primary antibody treatment, the sections were incubated for 30
minutes with secondary biotinylated antibody (Vector), followed by an incubation with
Vectastain ABC complex (Vector). Imunohistochemical development was performed using VIP
peroxidase substrate (Vector). All slides were counterstained with methyl green. Semi-quantitative
staining grade was measured. In brief, immunohistochemical staining grade was depicted as
follows: grade 0, negative staining; grade 1, weak patchy staining; grade 2, weak diffuse or dense
patchy staining; grade 3, dense diffuse staining.
Analysis of human diabetic kidney disease
This human study was approved by the institutional review board of the Pusan National
University Hospital (PNUH IRB No: 2014±6) and the need for informed consent from the
participants was waived as patients' records and information were anonymized and de-identified
prior to analysis. All clinical investigation have been conducted according to the principles
expressed in the Declaration of Helsinki. The bio-specimens and data used for this study were
provided by the Biobank of Pusan National University Hospital, a member of the Korean
Biobank Network. In brief, we analyzed the expression of FL-MMP-2 and NTT-MMP-2 in
archival renal biopsy specimens maintained by Pusan National University Hospital (PNUH)
Department of Pathology. The study groups consisted of controls (N = 10) and diabetic kidney
disease group (N = 25). Control samples were obtained mainly from patients with isolated
hematuria, who had normal histopathologic findings. The experimental group (N = 25)
consisted of biopsies from patients with Type 2 diabetes mellitus having evidence for diabetic
nephropathy such as proteinuria, hematuria or declining renal function (Table 2). For
semiquantitative scoring of proximal tubular injury, tubular dilatation, and tubular atrophy, a 4 tier
scoring was done; no change as score 0; 10% of renal tubules as score 1; >10% and 30% of
renal tubules as score 2; >30% of renal tubules as score 3. Immunohistochemical staining for
the MMP-2 isoforms was performed as detailed above. Quantitative expression of FL-MMP-2
and NTT-MMP-2 transcripts was assessed by quantitative RT-PCR following template
extraction from surplus frozen biopsy cores stored at -80 C, using the qPCR method detailed above.
DM (n = 25)
Control (n = 10)
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All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software). The
Mann Whitney U test or Kruskal-Wallis test with Dunn's multiple comparison test were used
to compare among experimental groups as appropriate. Significance was defined as a p-value
of less than 0.05. Results were presented as mean ± standard deviation for all experiments.
High glucose and oxidative stress induce both MMP-2 isoforms in HK2 proximal tubular epithelial cells
In the case of the FL-MMP-2 isoform, gene expression increased over time and peak
expression was approximately 15-fold greater than controls at 48 h (Fig 1, panel A; 15.4±0.7 fold
p<0.001). In the case of the NTT-MMP-2 isoform, gene expression peaked at 24 h in high
glucose medium and was approximately four times greater than controls (Fig 1, panel B; 3.9±0.2
fold p<0.001). With HHE-induced oxidative stress both FL-MMP-2 and NTT-MMP-2
transcripts were significantly elevated in HK2 cells. Specifically, FL-MMP-2 gene expression
slightly increased at 48h (1.2±0.2 fold) compared with controls (Fig 1, panel C). In contrast,
NTT-MMP-2 gene expression was increased by over 5-fold at 24 h and 48 h (Fig 1, panel D;
5.4±1.1, p = 0.001; 5.4±0.7, p = 0.002, respectively).
Confocal immunofluorescent microscopic findings confirmed that both isoforms of
MMP2 are induced by high glucose stimulation of HK2 cells (Fig 2, Panels I, II). However, the
cellular patterns of MMP-2 protein isoform expression differed. Specifically, FL-MMP-2 protein
Fig 1. Induction of MMP-2 isoform expression in cultured HK2 cells by high glucose and HHE
oxidative stress. HK2 cells were cultured in either normal glucose concentrations (5 mM) or high glucose
concentrations (30 mM) and transcript levels of the FL-MMP-2 and NTT-MMP-2 isoforms determined by
qPCR at 2, 24 and 48 hours of incubation. To determine the effect of oxidative stress on MMP-2 isoform
expression, HK2 cells were cultured in the presence or absence of HHE (10 μM) for 2, 24 and 48 hours. All
control cells were harvest at 2hr because the expression of MMP-2 was not significantly different according to
varying time point (data not shown). N = 3 for all study groups. (* p<0.05)
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Fig 2. Immunofluorescent (IF) detection of MMP-2 isoform expression in HK2 cells: effect of culture in normal (5 mM) vs. high
(30 mM) glucose. Panel I. IF staining for FL-MMP-2 isoform of HK2 cells cultured in normal (5 mM) glucose for 24 and 48 hours (A, C).
IF staining for FL-MMP-2 isoform of HK2 cells cultured in high (30 mM) glucose for 24 and 48 hours (B, D). Upper panel x 400; lower
panel x 800. Panel II. IF staining for NTT-MMP-2 isoform of HK2 cells cultured in normal (5 mM) glucose for 24 and 48 hours (A, C). IF
staining for NTT-MMP-2 isoform cultured in high (30 mM) glucose for 24 and 48 hours (B, D). Upper panel x 400; lower panel x 800.
Panels III and IV: Quantitation of IF staining for the FL-MMP-2 isoform (panel III) and NTT-MMP-2 (panel IV) of HK2 cells cultured in
normal (5 mM) and high (30 mM) glucose medium for 24 and 48 hours. (N = 5 for all study groups; *p<0.05).
was diffusely distributed in the cytoplasm, along with concentrated perinuclear staining. In
contrast, NTT-MMP-2 staining was present in a filamentous granular pattern consistent with
our previously reported mitochondrial localization [
]. Quantitative levels of MMP-2 isoform
fluorescence intensity are shown in Fig 2, Panels III and IV, and confirm the impressions
The NF-κB inhibitor PDTC blocks NTT-MMP-2 transcription
First, we tested whether high glucose media upregulates NF-κB expression in HK2 cells.
Western blot analysis showed that NF-κB expression increased greatly as compared with controls
(Fig 3, Panel A). Inclusion of 50μM PDTC in the culture medium had no effect on high
glucose-mediated expression of the FL-MMP-2 isoform, but did significantly suppress expression
of the NTT-MMP-2 isoform to near basal levels (Fig 3, panels B, C). These observations
suggest that high glucose-mediated induction of FL-MMP-2 and NTT-MMP-2 expression are
mediated by distinct signaling pathways.
The FL-MMP-2 and NTT-MMP-2 isoforms are induced in kidneys of diabetic mice
We generated diabetic mice using repetitive low dose streptozotocin injection. Murine kidneys
were examined after 12 and 24 weeks of diabetes. Gelatin zymography demonstrated that total
MMP-2 enzyme activity was increased in renal cortical extracts prepared from 24 week
diabetic mice as compared to controls. Given the relatively limited resolution of gelatin
zymography, with diffusion of areas of lysis, it is not possible to distinguish between the 68 kDa
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Fig 3. Effects of the NF-κB inhibitor, PDTC, on high glucose (30 mM) medium induction of FL-MMP-2 and
NTT-MMP isoform expression. Panel A: Western blot of HK2 cell lysates at 48 hours of culture in control (5 mM) and
high (30 mM) glucose concentrations. Compared to housekeeping GAPDH, there is a significant induction of NF-κB
expression by culture in high glucose medium Panel B: Effect of the NF-κB inhibitor PDTC (50 μM) on FL-MMP-2
transcript levels measured by qPCR after 48 hours culture with control (5 mM) and high (30 mM) glucose. Treatment
with PDTC does not significantly inhibit FL-MMP-2 expression (N = 3; p>0.05). Panel C: Effect of the NF-κB inhibitor
PDTC (50 μM) on NTT-MMP-2 transcript levels measured by qPCR after 48 hours culture with control (5 mM) and high
(30 mM) glucose. Treatment with PCTC significantly inhibits NTT-MMP-2 expression (N = 3; p<0.05).
FL-MMP-2 isoform and the 65 kDa NTT-MMP-2 isoform using this technique (Fig 4, Panel
I). In contrast, there was no change in the level of renal cortical expression of MMP-9. With
quantitative PCR, FL-MMP-2 and NTT-MMP-2 transcripts were upregulated as compared
with non-diabetic control mice. NTT-MMP-2 expression, in particular, was more strikingly
increased as compared with FL-MMP-2 in 24 week diabetic mice (7.2±9.1 fold change,
p = 0.003 vs. 1.8±0.4 fold change, p = 0.002, respectively) (Fig 4, Panel II).
Immunohistochemical studies of murine diabetic kidneys
IHC staining for the FL-MMP-2 isoform revealed low, but detectable levels of protein
expression in the proximal tubular epithelial cells of control mice (Fig 5I-A, B). FL-MMP-2 protein
expression was increased in the tubular epithelial cells of diabetic mice (Fig 5I-C, D).
NTTMMP-2 immunostaining was not detected in the tubular epithelial cells of control mice (Fig
5II-A, B), but was markedly increased in the tubular epithelial cells of the diabetic mice, a
finding consistent with the qPCR assessments (Fig 5II-C, 5D)
Expression of MMP-2 isoforms in human diabetic kidney disease
In human diabetic kidney biopsies analyzed in this study proximal tubular injury, tubular
dilatation, and tubular atrophy scoring were 2.4±0.7, 1.7±1.1, 2.1±0.9, respectively. Both isoforms
of MMP-2 were highly expressed in human diabetic kidney tissue. Specifically, the FL-MMP-2
transcript abundance measured by qPCR was increased about by 12.5±6.4 times compared
with normal control human kidney samples (Fig 6, Panel I). NTT-MMP-2 transcript was
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Fig 4. Induction of renal expression of FL-MMP-2 and NTT-MMP-2 in diabetic mice. Diabetes mellitus
was induced in C57/BL6 male mice by daily injection of streptozotocin (40 mg/kg) for five days. Panel I
A: Gelatin zymography of renal cortical extracts from control and diabetic mice (24 weeks). Zones of lysis
denote gelatin enzymatic activity for MMP-9 (98 kDa) and MMP-2 (68 kDa). MMP-2 enzymatic activity is
increased in the renal cortex of diabetic mice as compared to normoglycemic controls. There is no significant
increase in MMP-9 activity in the renal cortex of diabetic mice as compared to normoglycemic controls. Panel I
B: The band intensity was quantified by measuring pixel intensity. More MMP-2 enzymatic acitivities were
noted in diabetic group as compared to control group. There is no increase in the lytic band corresponding to
MMP-9 in the diabetic samples as compared to controls (*: p<0.05, NS: not significant). Panel II A:
Quantitation of FL-MMP-2 transcript abundance in renal cortices of normoglycemic (control) and diabetic mice
at 12 and 24 weeks. There is a modest, but significant increase in FL-MMP-2 transcript abundance after 24
weeks of diabetes (N = 8;* p<0.05). Panel II B: Quantitation of NTT-MMP-2 transcript abundance in renal
cortices of normoglycemic (control) and diabetic mice at 12 and 24 week. NTT-MMP-2 transcript abundance
is significantly increased at both 12 and 24 weeks of diabetes (N = 8; *p<0.05).
increased 3.1±1.7 fold (p<0.001). Immunohistochemical staining grade for both FL-MMP-2
and NTT-MMP-2 was higher in diabetic kidney disease as compared to controls (FL-MMP-2:
1.8±0.9 vs. 2.5±0.5, p = 0.03; NTT-MMP-2: 0.9±0.7 vs. 2.4±0.6, p<0.001) (Fig 6, Panel II). IHC
staining for both the FL-MMP-2 and NTT-MMP-2 isoforms was most prominent within
dilated tubules associated with tubulointerstitial mononuclear cell infiltration (Fig 6, panel
We performed IHC for the FL-MMP-2 and NTT-MMP-2 isoforms on serial sections of
human diabetic renal tissue to determine whether these isoforms were expressed within the
same tubular segments. Representative results are shown in Fig 7, and confirm co-expression
of the two MMP-2 isoforms in the same tubular segments.
The principal findings of the current study are the demonstration that two discrete MMP-2
isoforms are induced by a simulated diabetes mellitus milieu in vitro and in a widely used
murine model of diabetic renal disease. Further, we confirmed expression of both the
FL-MMP-2 and NTT-MMP-2 isoforms in archival renal biopsy specimens of human diabetic
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Fig 5. Immunohistochemical staining of renal cortices for FL-MMP-2 (panel I) and NTT-MMP-2 (panel II) of
normoglycemic and diabetic mice (24 weeks). Panel I: There is detectable FL-MMP-2 in the renal cortex of
control mice (A, B; x 200 and x 400 respectively) and this is increased in renal cortex of mice following 24 weeks of
diabetes (C, D; x 200 and x 400 respectively). Panel II: NTT-MMP-2 protein expression is not detectable in the renal
cortex of control mice (A, B; x 200 and x 400 respectively) but is greatly increased in the renal cortex of mice with 24
weeks of diabetes (C, D; x 200 and x 400 respectively). Note strong NTT-MMP-2 staining in necrotic tubular
epithelial cells in tubular lumen of diabetic kidneys (D, arrow).
Fig 6. FL-MMP-2 and NTT-MMP-2 expression in renal biopsies of human controls and diabetic nephropathy. Panel I: qPCR measurement
of FL-MMP-2 and NTT-MMP-2 transcript abundance in renal biopsies from controls and patients with diabetic nephropathy (DM). The transcript of
both MMP-2 isoforms is significantly increased in the diabetic kidneys (N = 25; * p<0.05). Panel II: Semiquantitative scoring of
immunohistochemistry staining of the FL-MMP-2 and NTT-MMP-2 isoforms as detailed in Materials and Methods. There is a statistically
significant increase in both MMP-2 isoform protein expression in kidneys with diabetic nephropathy (N = 25; * p<0.05). Panel III: As compared to
control kidney biopsies (A: FL-MMP-2; B: NTT-MMP-2) expression of both MMP-2 isoforms is increased in kidneys with diabetic nephropathy,
particularly in dilated and atrophic tubules associated with interstitial inflammation (x 200).
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Fig 7. The FL-MMP-2 and NTT-MMP-2 isoforms are expressed within the same tubular segments. Serial sections of kidneys with diabetic
nephropathy were stained with PAS (A) and by immunohistochemistry for FL-MMP-2 (B) and NTT-MMP-2 (C). Staining for both isoforms is prominent
within dilated tubules and occurs in the same tubular segments (arrows, x200).
nephropathy. Both isoforms are expressed within the same tubular segments and these
frequently demonstrated evidence for tubular injury, including dilatation and inflammation.
The specific role(s) of matrix metalloproteinases in diabetic nephropathy are undefined
We note that there is a large and very inconsistent literature concerning the expression of
multiple individual matrix metalloproteinases in both experimental and clinical diabetic
]. This inconsistency also applies to the specific matrix metalloproteinase, MMP-2.
Potential explanations derive from cell culture experiments employing a wide variety of
conditions and cell types, as well as from the use of widely varying animal models of diabetes
mellitus. Further, much of the attention, at least in terms of human diabetic nephropathy, has been
placed in changes in matrix metalloprotease expression in glomeruli rather than on tubular
epithelial cell injury and inflammation [
Characterization of the NTT-MMP-2 isoform
We initially characterized the NTT-MMP-2 isoform in isolated mitochondria from a murine
model of systolic heart failure and accelerated atherogenesis [
]. In addition, we detected the
NTT-MMP-2 isoform in mitochondria from aged mice. We demonstrated that the
NTTMMP-2 isoform is generated by oxidative stress-mediated activation of an alternate promoter
located within the first intron of the MMP-2 gene and that translation is initiated from M77 in
the second exon. The NTT-MMP-2 isoform is present within the mitochondrial
intramembranous space and induces mitochondrial-nuclear stress signaling with induction of a defined
transcriptome of pro-inflammatory innate immunity and cell death-associated genes [
Relationship between MMP-2 expression and oxidative stress
We recently reported that both MMP-2 isoforms are expressed in archival biopsies from
patients with renal transplant delayed graft function, and that expression of the NTT-MMP-2
isoform was most closely linked with tubular epithelial cell necrosis [
]. Oxidative stress is a
common feature of renal transplant delayed graft function, which represents a defined form of
acute ischemia/reperfusion injury. Prior transcriptional regulatory studies of the FL-MMP-2
promoter have shown that transcription is potently enhanced in the setting of oxidative stress
by an AP-1 binding enhancer [16±18]. Similarly, the NTT-MMP-2 alternate promoter is
activated primarily by oxidative stress via an NF-κB enhancer element (DHL, unpublished
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observations), which presumably explains the selective suppression of NTT-MMP-2
transcription by the anti-oxidant, NF-κB inhibitor, PDTC.
Within the setting of experimental and human diabetes mellitus, acute and chronic
hyperglycemia are associated with enhanced oxidative stress in multiple organs, including the
]. Tubular epithelial cell injury, with resultant tubular atrophy, peritubular
inflammation and fibrosis is a common feature of diabetic nephropathy and is associated with
an enhanced sensitivity to superimposed ischemia/reperfusion injury . We noted that
tubular epithelial cell expression of the MMP-2 isoforms occurred in the same renal tubular
segments which frequently showed evidence for tubule cellular injury and inflammation.
Based on our prior studies with renal tubule-specific transgenic expression of the FL-MMP-2
isoform, we suggest that the final pathologic phenotype of diabetic nephropathy is induced by
the combined actions of the FL-MMP-2 isoform on the tubular basement membrane and on
the induction of tubular cell necrosis and inflammation by the NTT-MMP-2 isoform.
Potential therapeutic aspects of the current study
Our work suggests that selective targeting of the two MMP-2 isoforms could represent a
potential novel therapeutic approach for the prevention or treatment of diabetic nephropathy, in
addition to the recognized approaches of stringent control of blood glucose and lipids. While
clinical trials primarily using anti-oxidant vitamins have not shown benefit, it is possible that
anti-oxidant strategies employing more potent agents, such as PDTC, could possibly have
benefit through the inhibition of NTT-MMP-2 synthesis.
The current study is based on an analysis of FL-MMP-2 and NTT-MMP-2 expression in an in
vitro model of diabetes mellitus using HK-2 tubular epithelial cells and with the streptozotocin
murine model of Type I diabetes mellitus. Further, our human renal biopsy archival studies
were not powered to determine a relationship between MMP-2 isoform expression and clinical
outcomes. Future studies are planned to address these important issues and to validate the
MMP-2 isoforms as high yield therapeutic targets.
This study was supported by Biomedical Research Institute Grant (2015±3), Pusan National
University Hospital. The bio-specimens and data used for this study were provided by the
Biobank of Pusan National University Hospital, a member of the Korean Biobank Network.
DHL was supported by Department of Veterans Affairs Merit Review Award I-1BX000593
and National Institutes of Diabetes, Digestive and Kidney Disease grant RO1DK39776. DHL's
contributions are the result of work supported with the resources and the use of facilities at the
San Francisco VA Medical Center.
Conceptualization: DHL SSK SHS MYL NS SSB.
Data curation: DHL SSK SHS MYL NS SSB.
Formal analysis: SHS NS HR IYK EYS DWL SBL ISK.
Funding acquisition: SHS DHL.
Investigation: SSK SHS MYL NS.
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Methodology: DHL SSK SHS MYL NS SSB.
Project administration: SHS DHL MYL.
Resources: SHS NS HR IYK EYS DWL SBL ISK.
Software: SHS MYL.
Supervision: SHS DHL.
Validation: SHS DHL MYL.
Visualization: SHS DHL MYL.
Writing ± original draft: NS SSK SHS DHL.
Writing ± review & editing: NS SSK SHS DHL.
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