Human adipose-derived stem cells modified by HIF-1α accelerate the recovery of cisplatin-induced...

ORIGINAL RESEARCH PAPER

Human adipose-derived stem cells modified by HIF-1aaccelerate the recovery of cisplatin-induced acute renalinjury in vitro

WeiWei Wang • Wei Wang • Yan Jiang •

Zezheng Li • Jin Cheng • Nanmei Liu •

GuoFeng Han • Shi Lu • JinYuan Zhang

Received: 6 April 2013 / Accepted: 8 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Human adipose-derived stem cells (hAS-

Cs) improve renal function in acute kidney injury.

Hypoxia-inducible factor-1a (HIF-1a) was transfected

into hASCs. hASCs modified by lentivirus-mediated

empty-vector and HIF-1a maintained their stem cell

characteristics. The expression of the renal-protective

gene, heme oxygenase-1 and vascular endothelial

growth factor were significantly increased in hASCs

modified by HIF-1a, compared to hASCs modified by

empty-vector. Cellular ultra-structure and TUNEL

staining revealed that hASCs modified by HIF-1apromoted the recovery of apoptotic morphology in

cisplatin-treated human kidney-2 cells (HK-2 cells)

when compared to hASCs modified by empty-vector.

Additionally, hASCs modified by empty-vector inhib-

ited caspase-3 expression and up-regulated Bcl-2

expression in cisplatin-treated HK-2 cells, an effect

even more pronounced with hASCs modified by HIF-

1a. Thus, HIF-1a gene-modified ASCs could be an

effective way to enhance the renal-protective effect.

Keywords Acute kidney injury � Cisplatin �Human adipose-derived stem cells � Human

kidney-2 cells � Hypoxia inducible factor-1a �Renal injury

Introduction

Therapeutic options for acute kidney injury (AKI) are

limited to the use of supportive measures and dialysis.

A recent approach that has sparked great interest and

Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-013-1389-x) contains supple-mentary material, which is available to authorized users.

W. Wang � J. Cheng � N. Liu � G. Han �S. Lu � J. Zhang (&)

Department of Nephrology, Jimin Hospital,

Shanghai 200052, People’s Republic of China

e-mail: jinyuan.zhang@hotmail.com

W. Wang

e-mail: w.vwei@163.com

J. Cheng

e-mail: chenjin455@126.com

N. Liu

e-mail: liunmei@gmail.com

G. Han

e-mail: hanguofeng@yahoo.com.cn

S. Lu

e-mail: shilu_1024@163.com

W. Wang � Y. Jiang � Z. Li

Graduate School, Shanghai University of Traditional

Chinese Medicine, Shanghai 201203, People’s Republic

of China

e-mail: genpichong86@163.com

Y. Jiang

e-mail: 404317488@qq.com

Z. Li

e-mail: 985704026@qq.com

123

Biotechnol Lett

DOI 10.1007/s10529-013-1389-x

gained popularity is the utilization of stem cells to

repair acutely damaged kidneys. Adipose tissue is an

attractive source of multipotent stem cells that can

differentiate into osteogenic, chondrogenic, myo-

genic, neurogenic, hematopoietic or endothelial cells

(Colazzo et al. 2010; Witkowska-Zimny and Walenko

2011). Additionally, ASCs show a high proliferation

rate and low senescence rate even when harvested

from adults, and they do not trigger immune rejection

(Xishan et al. 2013). Thus, adipose tissue is a

promising tissue source for stem cells with powerful

implications for regenerative medicine.

Hypoxia-inducible factor-1a (HIF-1a), one of HIF

family members, is a master regulator that mediates

the adaptive response to hypoxia in cells and tissues.

HIF-1a plays a key protective role against renal

damage, inducing the expression of downstream renal-

protective genes, including heme oxygenase-1 (HO-1)

and vascular endothelial growth factor (VEGF) (Wang

and Zhang 2008; Weidemann et al. 2008).

Under normoxic conditions, HIF-a is rapidly

degraded because it contains an O2-dependent degra-

dation (ODD) domain. Huang et al. (1998) reported that

cells transfected with cDNA encoding HIF-1a in which

the ODD was deleted showed constitutively active HIF-

1a signaling regardless of its O2 tension and that

deletion of the ODD domain [to generate HIF-1a(DODD)] did not affect the function of HIF-1a (Rosen-

berger et al. 2003), a finding which may be beneficial in

exploring the effect of HIF-1a on damaged tissue under

normoxic conditions. Based on these findings, we

investigated the enhanced protective effect of lentivi-

rus-mediated HIF-1a (DODD) over-expression in hAS-

Cs against cisplatin-induced nephrotoxicity in vitro.

Materials and methods

Human adipose-derived stem cells (hASC) culture

and preparation

hASCs, purchased from Cyagen Biosciences Inc.,

were maintained in culture medium including

Dulbecco’s Modified Eagle Medium (DMEM), 10 %

(v/v) fetal bovine serum, 50 U penicillin ml-1 and

50 lg streptomycin ml-1 at 37 �C/5 % CO2. At 80 %

confluence, cells were trypsinized with 0.25 % tryp-

sin/EDTA and passaged into new flasks for further

expansion. The medium was changed every other day.

Lentivirus production

HIF-1a (DODD) was amplified from pcDNA3-HIF-

1a (401D603; a gift from Dr. Franklin) by PCR using

primers containing BamHI and AscI restriction sites.

HIF-1a (DODD) was constructed into lentivirus-

expressing vector containing green fluorescent protein

(GFP) according to the Invitrogen protocol. Viral

particles were harvested and stored at -80 �C. The

empty lentiviral vector was generated using the same

procedure. (HIF-1a over-expression refers to HIF-1a(DODD) over-expression.)

Lentiviral transfection of hASCs

hASCs were plated in 25 cm2 flasks and grown to

80 % confluence (*106 cells). Cells were incubated

overnight with lentivirus at a multiplicity of infection

(moi) of 1 in the presence of 8 lg polybrene ml-1

(Sigma, USA), and the medium was replaced with

5 ml fresh medium the next day. Three days later,

GFP-expressing cells were collected by FACS and re-

plated for further culture. GFP expression of sorted

cells was examined by fluorescence microscopy.

Additionally, immunohistochemistry and western blot

were used to assess the expression of HIF-1a in hASCs

transduced with empty vector and HIF-1a.

Character of hASCs modified by lentivirus-

mediated empty vector and HIF-1a

hASCs modified by lentivirus-mediated empty vector

(EV-hASCs) and HIF-1a (HIF-1a-hASCs) (2 9 106

cells) were trypsinized, washed three times with PBS,

and immunostained for 30 min on ice with monoclo-

nal antibodies against CD29 (FITC-conjugated),

CD44 (FITC-conjugated) and CD105 (phycoery-

thrin-conjugated). Labeled cells were analyzed using

a flow cytometer. Differentiation potential of EV-

hASCs and HIF-1a-hASCs were examined for oste-

ogenic and adipogenic differentiation according to the

manufacture’s protocol (Cyagen).

Cisplatin-induced AKI model in vitro

and treatments

Human kidney-2 (HK-2 cells, ATCC, USA) induced by

cisplatin, was used as an in vitro AKI model. Cisplatin

(Sigma, 1, 2, 3, and 4 lg ml-1) was used to treat HK-2

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123

cells. 3 lg cisplatin ml-1 was the optimal concentration

for inducing apoptosis in HK-2 cells according to

transmission electron microscopy. We designed exper-

iments in which HK-2 cells were cultured in four

different conditions: untreated cells cultured alone

(control); cisplatin-treated cells cultured alone (cisplatin

3 lg ml-1); cisplatin-treated HK-2 cells cultured with

EV-hASCs; and cisplatin-induced HK-2 cells co-cul-

tured with HIF-1a-hASCs. The detailed protocol was as

follows. HK-2 cells were seeded at 2 9 103 cells/cm2 in

six-well plates in keratinocyte serum-free medium

(Invitrogen, USA) supplemented with 2 % (v/v) fetal

bovine serum and incubated for 3 days. Cells were

starved for 12 h to induce synchronization. Control cells

were then cultured in DMEM/F12 supplemented with

5 % (v/v) fetal bovine serum for 6 h, and the three

experimental groups were cultured with DMEM/F12,

5 % (v/v) fetal bovine serum plus cisplatin (3 lg ml-1)

for 6 h. After 6 h, all cells were washed three times with

DMEM. Control and cisplatin-treated cells were then

cultured in DMEM/F12 and 5 % (v/v) fetal bovine

serum for 24 h, and the two co-culture groups were

cultured with EV-hASCs or HIF-1a-hASCs using the

six-well plate transwell co-culture system (Corning,

USA) according to the manufacturer’s instruction.

Briefly, EV-hASCs and HIF-1a-hASCs were first

seeded on polycarbonate inserts (105 cells per insert)

and cultured for 24 h. Inserts were then transferred into

six-well plates containing cisplatin-pretreated HK-2

cells, and cells were cultured together in DMEM/F12

and 5 % (v/v) fetal bovine serum for 24 h. After 24 h,

HK-2 cells were harvested for the following analyses.

Transmission electron microscopy

HK-2 cells were fixed with 2 % (w/v) glutaraldehyde,

dehydrated and embedded in epoxy resin. Ultrathin

sections of 70 nm were prepared and stained with lead

citrate. The ultra-structure of HK-2 cells was observed

using transmission electron microscopy.

Terminal deoxynucleotidyltransferase-mediated

dUTP nick end-labelling (TUNEL) staining

TUNEL staining was performed using the in situ

Apoptosis Detection Kit (Boster Biotech Co, China)

according to the manufacturer’s instruction. Briefly,

HK-2 cells were fixed, endogenous peroxidase was

quenched, and the cells were permeabilised. The cells

were then incubated with the TUNEL reaction

mixture, 3,30-diaminobenzidine tetrahydrochloride

(DAB), and haematein. The apoptotic index was

calculated by the percentage of positive nuclei.

Immunocytochemistry

Immunocytochemical staining was performed using

the SABC system (Boster Biotech Co, China) accord-

ing to the manufacturer’s instruction. Briefly, EV-

hASCs or HIF-1a-hASCs was seeded at 2 9 104 cells/

cm2 on coverslips coated with poly-L-lysine. After

5 days, cells were fixed with acetone for 30 min at

4 �C and washed with PBS three times. Coverslips

were incubated with HIF-1a monoclonal antibody

(Abcam, USA; 1:1,000) overnight at 4 �C followed by

incubation with a suitable secondary antibody. Chro-

mogenic detection was achieved with DAB.

Western blotting

HK-2 cells were rinsed twice with cold PBS and lysed

in whole-cell lysis buffer containing a protease

inhibitor cocktail. Equal amounts of protein were

separated by SDS-PAGE and transferred onto a

nitrocellulose membrane. The membranes were

blocked with 5 % (v/v) nonfat milk in TBS-T

(10 mM Tris-buffered saline with 0.1 % Tween 20)

and incubated with HIF-1a monoclonal antibody

(MAB1536, R&D, USA) overnight at 4 �C. The

membranes were then incubated with the appropriate

secondary antibodies conjugated to horseradish per-

oxidase (HRP; Sigma) (at 1:5,000 dilution) and the

proteins were visualized using enhanced chemilumi-

nescence (ECL) and exposure to X-ray film (Kodak).

Enzyme-linked immunosorbent assay

Culture supernatants from control, model, EV-hASCs

and HIF-1a-hASCs were collected and the level of

HO-1 and VEGF in the medium was determined

according to the manufacturer’s instruction (R&D,

USA).

Analysis of mRNA expression by real time-PCR

Total RNA was isolated from the cells using TRIZOL

and the reverse transcription of the purified RNA was

performed using oligo (dT) priming and superscript II

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reverse transcription, according to the manufacturer’s

instruction (Invitrogen, USA). Real time-PCR was

performed using SYBR green. The primer pairs for the

selected genes are listed in Supplementary Table 1.

Statistical analysis

Samples values are expressed as the mean ± standard

deviation (SD). Data were analyzed by ANOVA using

the SPSS13.0 statistical software package. P-values

less than 0.05 were considered significant.

Results

Efficient transduction of hASCs by lentivirus

mediated-empty vector and HIF-1a

After transient transduction with lentivirus, EV-hAS-

Cs or HIF-1a-hASCs were cultured for 72 h, and then

were observed by phase contrast and fluorescence

microscopy (Fig. 1Aa, b, e, f). After FACS sorting of

GFP-expressing hASCs, we achieved [99 % GFP

positivity as assessed by microscopy at 48 h (Fig. 1Ac,

d, g, h). Expression of HIF-1a was markedly increased

in HIF-1a-hASCs, as demonstrated by western blotting

(Fig. 1B) and immunocytology (Fig. 1C).

Differentiation and surface markers of hASCs

modified by lentivirus-mediated empty vector

and HIF-1a

To evaluate multi-differentiation of EV-hASCs and

HIF-1a-hASCs, both types of hASCs were differentially

induced into osteoblasts and adipocytes, respectively.

After 3 weeks’ incubation, both types of hASCs became

Alizarin Red positive with osteogenic supplementation

(Fig. 2Aa, b) and, were induced with adipogenic

medium, showed Oil Red O positive lipid droplets

(Fig. 2Ac, d). Flow cytometry analysis showed that EV-

hASCs and HIF-1a-hASCs were positive for mesen-

chymal markers CD29, CD44 and CD105 (Fig. 2B).

Evaluation of HO-1 and VEGF expression

To measure the protein expression of HO-1 and VEGF

in the different medium, ELISA was used. HO-1 and

VEGF protein expression in the medium of HK-2 cells

incubated with 3 lg cisplatin ml-1 were significantly

decreased compared to that in the medium of control

cells (P \ 0.05). After co-culture of the cisplatin-

treated HK-2 cells with EV-hASCs or HIF-1a-hASCs,

HO-1 and VEGF protein expression were increased,

and there was a significant difference between both co-

cultured groups and cisplatin-induced cells (P \ 0.05).

Significant differences were also observed between the

cells co-cultured with EV-hASCs and HIF-1a-hASCs

(P \ 0.05) (Fig. 3a and b). Additionally, HO-1 and

VEGF gene expression were also evaluated by real-

time PCR, with the results showing the same trend as

the protein expression (Fig. 3c and d).

Fig. 1 Virus transduction of hASCs. A Transiently transfected

hASCs (a, b and e, f) and stably transfected hASCs (c, d and g, h) in

the EV-hASC and HIF-1a-hASC groups were sorted by FACScan

after 72 h. Western blot (B) and immunocytochemistry (C) anal-

yses were performed for HIF-1a protein expression in the EV-

hASC and HIF-1a-hASC groups (n = 3 each). Scale bar 100 lm

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123

Fig. 2 Characteristics of

EV-hASCs and HIF-1a-

hASCs. A Multi-potential

differentiation of EV-

hASCs and HIF-1a-hASCs,

including osteoblasts

(Alizarin Red, a, b) and

adipocytes (Oil Red O, c, d).

Scale bar 100 lm. B Flow

cytometric analysis showing

EV-hASC and HIF-1a-

hASC positivity for

mesenchymal markers

CD29, CD44, and CD105

(n = 3 each)

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Ultra-structural analysis of HK-2 cells

To determine the optimal cisplatin concentration for

inducing cellular apoptosis in HK-2 cells, transmission

electron microscopy was used to observe the change in

cellular structure. Untreated control HK-2 cells

appeared normal (Fig. 4a). After treatment with 1 lg

cisplatin ml-1 for 6 h, HK-2 cells did not show obvious

alterations in cellular structure (Fig. 4b). HK-2 cells

treated with 2 lg cisplatin ml-1 presented cellular

chromatin condensation (Fig. 4c), and those treated

with 3 lg cisplatin ml-1 showed typical apoptotic

characteristics (Fig. 4d). HK-2 cells treated with 4 lg

cisplatin ml-1 underwent necrosis (Fig. 4e). When HK-

2 cells exposed to 3 lg cisplatin ml-1 were co-cultured

with EV-hASCs, those cells appeared less apoptotic

(Fig. 4f). When HK-2 cells treated with 3 lg cisplatin

ml-1 were co-cultured with with HIF-1a-hASCs, most

cells recovered their normal cellular structure, with only

a few cells exhibiting modest cellular damage, such as

swollen mitochondria or heterochromatic foci (a char-

acteristic of early apoptosis) (Fig. 4g).

Fig. 3 The level of HO-1 and VEGF protein and gene expression. The data are expressed as the mean ± SD (n = 5 each) *P \ 0.05,

compared to the model group. #P \ 0.05, compared to the EV-hASCs group

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DNA fragmentation determined by TUNEL assay

To detect DNA fragmentation in situ and calculate the

apoptotic index, TUNEL staining was performed.

Treatment with 3 lg cisplatin ml-1 increased the

apoptotic index in the model and a significant

difference was observed between the model and

control groups (P \ 0.05). However, co-culturing

HK-2 cells with EV-hASCs and HIF-1a-hASCs

lowered the apoptotic index, with a significant

Fig. 4 Ultra-structural change of HK-2 cells. a Control

(untreated cells cultured alone); b incubated with 1 lg cisplatin

ml-1; c incubated with 2 lg cisplatin ml-1; d incubated with

3 lg cisplatin ml-1; e incubated with 4 lg cisplatin ml-1;

f cisplatin (3 lg ml-1)-induced cells co-cultured with EV-

hASCs; g cisplatin (3 lg ml-1)-induced cells co-cultured with

HIF-1a-hASCs. The arrows show cellular alterations (a, normal

cellular shape; b, no obvious changes; c, chromatin condensa-

tion; d, typical apoptotic characteristics; e, necrosis; d and f, the

recovery of cellular structure). Scale bar (a, b, c, g): 10 lm,

Scale bar (d, e, f), 3 lm

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difference between the co-incubated groups and

model group (P \ 0.05) (Fig. 5).

Analysis of caspase-3 and Bcl-2 gene expression

To evaluate the expression of related apoptotic

gene, real time-PCR was used. Cisplatin (3 lg ml-1)

increased the gene expression of caspase-3 and

decreased the gene expression of Bcl-2 in HK-2 cells

compared to control cells (P \ 0.05). Co-culturing

HK-2 cells with EV-hASCs and HIF-1a-hASCs

resulted in decreased levels of caspase-3 expression

and prevented the cisplatin-induced reduction in Bcl-2

gene expression in HK-2 cells. The EV-hASCs and

Fig. 5 Apoptotic analysis.

A TUNEL staining.

a. control (untreated cells

cultured alone); b. incubated

with cisplatin (3 lg ml-1);

c. cisplatin (3 lg ml-1)-

induced cells co-cultured

with h EV-hASCs;

d. cisplatin (3 lg ml-1)-

induced cells co-cultured

with HIF-1a-hASCs. Scale

bar 100 lm. B Apoptotic

index. The data are expressed

as the mean ± SD (n = 5

each) *P \ 0.05, compared

to the model group

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HIF-1a-hASCs groups showed significant differences

compared to the model group (P \ 0.05) (Fig. 6).

Discussion

We have delivered human HIF-1a gene into hASCs

via a lentiviral vector and increased the levels of HIF-

1a expression in vitro. EV-hASCs or HIF-1a-hASCs

maintained their stem cell characteristics, including

the expression of surface antigens and normal differ-

entiation potential. Additionally, EV-hASCs secreted

some renal-protective genes, HO-1 and VEGF, and

HIF-1a-hASCs was obviously promoted the expres-

sion of HO-1 and VEGF.

The main forms of cisplatin-induced renal cell

injury are apoptosis and necrosis and the pathway of

cell death is concentration dependent, with low

concentrations of cisplatin primarily inducing apop-

tosis and high concentrations predominantly inducing

necrosis. In this study, 3 lg cisplatin ml-1 was

regarded as optimal for inducing apoptosis in HK-2

cells. Unlike necrosis, apoptosis is mediated by the

active participation of the dying cells. Therefore,

apoptosis appears much easier to reversal by some

methods compared with unreversible necrosis.

Stem cells, especially mesenchymal stem cells

(MSCs), have protective effects against AKI arising

from chemical (glycerol and cisplatin) and ischemia–

reperfusion injury by secreting beneficial factors and

activating signal proteins (Kim et al. 2012; Zarjou and

Agarwal 2012). Conversely, beneficial effects were

not reported for CsA-induced renal injury (Chung

et al. 2013). Our study demonstrates that EV-hASCs

may improve the cellular morphology of co-cultured

HK-2 cells incubated with cisplatin suggesting that

EV-hASCs exert their renal protective function

through an autocrine mechanism, such as HO-1 and

VEGF expression. Zarjou et al. (2011) study indicated

that the conditioned medium of HO-1?/?MSCs

rescued the functional and morphological changes

associated with cisplatin-induced AKI, whereas an

HO-1-/--conditioned medium was ineffective. In

addition, Togel et al. (2009) study showed that VEGF

is an important mediator of the early and late phase of

renoprotective action after AKI within the context of

stem cell treatment. We also explored the mechanism

for the EV-hASC-mediated protection of cisplatin-

treated HK-2 cells. Caspase-3 is activated by mito-

chondrial injury leading to an increase in cytochrome c

release into the cytoplasm, a process that was precisely

regulated by members of the Bcl-2 family (Zheng et al.

2013). In our study, EV-hASCs suppressed the

cisplatin-induced activation of caspase-3 suggesting

that EV-hASCs may act upstream of caspase-3 to

block apoptosis. Based on our observations, a decrease

in caspase-3 expression correlated well with an

increase in anti-apoptotic Bcl-2 expression.

Genetically-modified stem cells over-expressing

particular genes can have an enhanced protective

effect on damaged cells and tissues, perhaps through a

paracrine mechanism (Lu et al. 2008; Tadagavadi and

Fig. 6 Analysis of caspase-3 and BcL-2 gene expression. The

data are expressed as the mean ± SD (n = 5 each) *P \ 0.05,

compared to the model group. #P \ 0.05, compared to the EV-

hASCs group

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Reeves 2010). In our study, HIF-1a-hASCs improved

the cellular morphology of cisplatin-treated HK-2

cells to a greater degree than the parental hASCs.

Elsewhere, activation of HIF by DMOG halted the

progression of proteinuria, attenuated structural dam-

age and decreased oxidative stress, inflammation, and

fibrosis in a remnant kidney model (Zarjou et al. 2011)

and TRC160334, a novel HIF hydroxylase inhibitor,

stabilized HIF-a and activated HIF, a situation that led

to a significant reduction in renal injury and serum

creatinine and the improvement of urine output in AKI

(Jamadarkhana et al. 2012). Additionally, activation of

HIF by cobalt or DMOG attenuated renal dysfunction,

proteinuria, and structural damage through a reduction

of oxidative stress, inflammation, and apoptosis in

renal tubular epithelial cells in gentamicin-induced

AKI (Ahn et al. 2012). The mechanisms through

which hASCs protected HK-2 cells from cisplatin-

induced cell apoptosis included promoting the secre-

tion of HO-1 and VEGF, decreasing activation of

caspase-3 and increasing expression of Bcl-2, which

showed an effect even more pronounced with hASCs

modified by HIF-1a.

In conclusion, our study indicated that hASCs and

HIF-1a-modified hASCs show a protective effect on

cisplatin-induced AKI in vitro and HIF-1a-modified

hASC was more efficacious in treating AKI in vitro.

Acknowledgments This research was supported by National

Natural Science Foundation of China (No. 81100493), Key

Project of Basic Research of Science and Technology of Shanghai

(12DJ1400203), Shanghai Rising-Star Program (09QA1407500)

and Project of Shanghai Excellent Young Doctor (XYQ2011012).

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