HUMAN GENE THERAPY 19:807–819 (August 2008)
© Mary Ann Liebert, Inc.
Kallikrein-Modified Mesenchymal Stem Cell Implantation
Provides Enhanced Protection Against Acute Ischemic
Kidney Injury by Inhibiting Apoptosis and Inflammation
Makoto Hagiwara, Bo Shen, Lee Chao, and Julie Chao
Mesenchymal stem cells (MSCs) migrate to sites of tissue injury and serve as an ideal vehicle for cellular gene
transfer. As tissue kallikrein has pleiotropic effects in protection against oxidative organ damage, we investi-
gated the potential of kallikrein-modified MSCs (TK-MSCs) in healing injured kidney after acute
ischemia/reperfusion (I/R). TK-MSCs secreted recombinant human kallikrein with elevated vascular endo-
thelial growth factor levels in culture medium, and were more resistant to oxidative stress-induced apoptosis
than control MSCs. Expression of human kallikrein was identified in rat glomeruli after I/R injury and sys-
temic TK-MSC injection. Engrafted TK-MSCs exhibited advanced protection against renal injury by reducing
blood urea nitrogen, serum creatinine levels, and tubular injury. Six hours after I/R, TK-MSC implantation sig-
nificantly reduced renal cell apoptosis in association with decreased inducible nitric oxide synthase expression
and nitric oxide levels. Forty-eight hours after I/R, TK-MSCs inhibited interstitial neutrophil and mono-
cyte/macrophage infiltration and decreased myeloperoxidase activity, superoxide formation, p38 mitogen-ac-
tivated protein kinase phosphorylation, and expression of tumor necrosis factor-?, monocyte chemoattractant
protein-1, and intercellular adhesion molecule-1. In addition, tissue kallikrein and kinin significantly inhibited
H2O2-induced apoptosis and increased Akt phosphorylation and cell viability in cultured proximal tubular
cells. These results indicate that implantation of kallikrein-modified MSCs in the kidney provides advanced
benefits in protection against ischemia-induced kidney injury by suppression of apoptosis and inflammation.
ulations, an early ischemic insult is believed to cause long-
term renal dysfunction (Chertow et al., 2005; Hertig et al.,
2006). Unfortunately, innovative interventions beyond sup-
portive therapy are currently not available. Mesenchymal
stem cells (MSCs) serve as an ideal vehicle for cellular gene
transfer because they are nonimmunogenic and immuno-
suppressive, and have the ability to migrate to sites of tissue
injury and inflammation to participate in tissue repair
(Yokoo et al., 2003). Studies have shown that implantation of
bone marrow-derived MSCs after renal ischemia/reperfu-
sion (I/R) promoted recovery of renal function and mor-
phological damage, indicating potential promise of healing
damaged kidney after acute I/R injury with MSCs (Lange et
al., 2005; Togel et al., 2005). Moreover, MSCs genetically mod-
ified with the antiapoptotic Akt gene or the antioxidant en-
zyme heme oxygenase (HO)-1 gene were more efficient in
CUTE RENAL FAILURE (ARF) is a common disease with
high morbidity and mortality. In renal transplant pop-
improving cardiac performance and healing damaged my-
ocardium compared with unmodified MSCs by enhancing
stem cell viability via autocrine and paracrine actions (Mangi
et al., 2003; Tang et al., 2005). In addition to the migrating
and homing ability of MSCs (Herrera et al., 2007), the
paracrine effects of MSCs have been attracting a great deal
of attention as the main mechanism of beneficial effects (To-
gel et al., 2005). These experimental results indicate that en-
hanced stem cell therapy by genetic modification provides
advanced benefits in protection against ischemic injury.
Tissue kallikrein is a serine proteinase that processes
kininogen substrates to release vasoactive kinin peptides.
Using a somatic gene transfer approach, we previously dem-
onstrated that tissue kallikrein exhibits renal protection by
antiinflammatory and antifibrotic actions in several animal
models of renal injury (Chao et al., 1998; Murakami et al.,
1998; Bledsoe et al., 2006). Furthermore, we reported that
kallikrein reduced ischemia-induced cardiomyocyte apopto-
sis via Akt-mediated signaling pathways (Yin et al., 2005).
Tissue kallikrein gene delivery exhibits pleiotropic effects in
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425.
protection against oxidative damage in the heart, kidney,
and brain (Xia et al., 2004; Zhang et al., 2004; Li et al., 2007).
Our present study was designed to test the hypothesis that
mesenchymal stem cells genetically modified with the
kallikrein gene exert advanced beneficial effects in protec-
tion against acute renal I/R injury by suppression of apop-
tosis and inflammation.
Materials and Methods
Mesenchymal stem cell isolation
Bone marrow (BM) was obtained from 2-month-old male
Wistar rats (Deng et al., 2003). Briefly, rats were killed, and
femurs and tibias were aseptically removed. BM was flushed
from the shaft of the bone with Dulbecco’s modified Eagle’s
medium (DMEM; Sigma-Aldrich, St. Louis, MO) containing
5% fetal calf serum (FCS; Invitrogen, Paisley, UK) plus peni-
cillin (100 U/ml)–streptomycin (0.1 mg/ml) (Invitrogen),
and then filtered through a 100-?m (pore size) sterile filter
(Falcon; BD Biosciences, San Jose, CA) to produce a single-
cell suspension. MSCs were recovered from BM by their ten-
dency to adhere tightly to plastic culture dishes. Filtered BM
cells were plated in DMEM plus 10% FCS and penicillin (100
U/ml)–streptomycin (0.1 mg/ml) and allowed to adhere.
MSCs were identified by positive immunostaining for vi-
mentin and ?-smooth muscle actin (?-SMA) in passage 2 as
described previously (Davani et al., 2003). Briefly, cells were
washed once and fixed with 3.7% (v/v) formaldehyde in
phosphate-buffered saline (PBS). MSCs were incubated with
the primary antibodies anti-?-SMA (Dako, Glostrup, Den-
mark) and anti-vimentin (Santa Cruz Biotechnology, Santa
Cruz, CA) overnight at 4°C, and then with fluorescein-con-
jugated secondary antibody for 1 hr at room temperature.
Immunostaining was examined with a fluorescence micro-
Generation of genetically modified MSCs expressing
human tissue kallikrein
Ad.CMV-GFP (adenovirus harboring the green fluores-
cent protein [GFP]-encoding gene) and Ad.CMV-TK (ade-
novirus carrying human tissue kallikrein cDNA) were gen-
erated as previously described (Chao et al., 1998). Cultured
MSCs were transduced with Ad.CMV-TK (TK-MSCs) and
Ad.CMV-GFP (GFP-MSCs) at a multiplicity of infection
(MOI) of 50 for 2 hr, followed by a second transduction with
adenovirus at an MOI of 100 for 24 hr. To determine the
transduction efficiency, the ratio of GFP-expressing cells to
total cells was calculated with a fluorescence microscope. Ex-
pression of recombinant human tissue kallikrein in MSCs
was identified by immunostaining and by measuring im-
munoreactive human tissue kallikrein levels secreted into the
culture medium, using a specific enzyme-linked im-
munosorbent assay (ELISA) (Chao et al., 1998). Vascular en-
dothelial growth factor (VEGF) levels in the culture medium
of MSCs 4 days after transduction were measured with an
ELISA kit (R&D Systems, Minneapolis, MN) according to the
Detection of apoptosis caused by H2O2or hypoxia
GFP-MSCs and TK-MSCs were seeded in 6-well plates. At
70% confluency, cells were treated with H2O2(0.5 mM) for
6 hr as previously described (Lim et al., 2006). MSCs were
then incubated with Hoechst 33342 (1 ?g/ml) for 10 min in
the dark. Apoptotic cells were identified by their distinct con-
densed nuclei. Caspase-3 activity in cell lysates was deter-
mined with a fluorometric caspase-3 assay kit (Oncogene,
San Diego, CA) according to the manufacturer’s instructions.
Male Wistar rats (Harlan, Indianapolis, IN) were housed
at a constant room temperature with a 12 hr:12 hr light:dark
cycle and had free access to tap water and rat chow. All pro-
cedures complied with the standards for care and use of an-
imal subjects as stated in the Guide for the Care and Use of Lab-
oratory Animals (Institute of Laboratory Resources, National
Academy of Sciences, Bethesda, MD). Rats were anesthetized
and subjected to renal ischemia/reperfusion (I/R) injury as
previously described (Togel et al., 2005). Briefly, after ab-
dominal laparotomy, kidneys were exposed and renal pedi-
cles were clamped with vascular clamps for 40 min to induce
ischemia. After reflow, the left carotid artery was cannulated
with PE50 tubing. MSCs expressing GFP or TK (1 ? 106) in
200 ?l were injected via the carotid artery within 1 hr. The
same amount of PBS was infused into the control I/R group
by the same route. Thus, rats with I/R were randomly di-
vided into three groups: I/R plus PBS (I/R, n ? 16), I/R plus
GFP-MSCs (I/R ? GFP-MSCs, n ? 16), and I/R plus TK-
MSCs (I/R ? TK-MSCs, n ? 16). Sham-operated control an-
imals (sham, n ? 12) did not undergo occlusion of the renal
arteries. At 6 and 48 hr after I/R, rats were anesthetized and
blood samples were taken from the right atrium. Blood urea
nitrogen (BUN) and serum creatinine levels were measured
with commercial kits (BioAssay Systems, Hayward, CA).
Kidneys were removed and snap frozen in liquid nitrogen
or fixed in formaldehyde for immunohistochemical studies.
Morphological and histological analyses
Kidneys were fixed in 4% formaldehyde, dehydrated, and
paraffin embedded. Sections (4 ?m thick) were stained with
periodic acid–Schiff (PAS) or hematoxylin and eosin (H&E).
Kidney sections were examined in a blinded manner and
scored to evaluate the degree of tubular necrosis (Haq et al.,
1998). The scoring method was as follows: 0, normal kidney;
1, minimal necrosis (?5% involvement); 2, mild necrosis (5
to ?25% involvement); 3, moderate necrosis (25 to ?75% in-
volvement); 4, severe necrosis (?75% involvement). Neu-
trophil infiltration into the interstitium was determined by
counting neutrophils in 10 randomly selected fields at ?400
magnification in the outer stripe of the medulla, the area of
maximal neutrophil migration as described previously (Haq
et al., 1998).
Measurement of nitrate/nitrite, myeloperoxidase activity,
and superoxide formation
Renal tissue was homogenized in lysis buffer (10 mM Tris
[pH 7.4], 100 mM NaCl, 1 mM EDTA, 20 mM Na4P2O7, 2 mM
Na3VO4, and 1% Triton X-100) containing 1:100 protease in-
hibitor cocktail (Sigma-Aldrich). Nitrate/nitrite (NOx) levels
(indicative of NO formation) were measured by a fluoromet-
ric assay (Chao et al., 2007). Myeloperoxidase activity was de-
termined as previously described (Suzuki et al., 1983). Super-
HAGIWARA ET AL. 808
ICAM-1 48 hr after I/R. Inhibition of p38MAPKby implanta-
tion of TK-MSC may lead to suppression of NF-?B activa-
tion and thus expression of proinflammatory cytokines and
cell adhesion molecules, with a resultant decline in renal in-
This work was supported by National Institutes of Health
grants DK-066350, HL-29397, and C06 RR015455 from the
Extramural Research Facilities Program of the National Cen-
ter for Research Resources.
Author Disclosure Statement
The authors state no conflict of interest.
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Address reprint requests to:
Dr. Julie Chao
Department of Biochemistry and Molecular Biology
Medical University of South Carolina
173 Ashley Avenue
Charleston, SC 29425
Received for publication February 7, 2008; accepted after
revision June 8, 2008.
Published online: July 16, 2008.
KALLIKREIN-MODIFIED MSCs IN ARF819