Cell Transplantation, Vol. 21, pp. 1727–1741, 2012
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DOI: http://dx/doi.org/10.3727/096368911X623925
E-ISSN 1555-3892
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Adipose Tissue-Derived Stem Cell Treatment Prevents
Renal Disease Progression
Cassiano Donizetti-Oliveira,* Patricia Semedo,* Marina Burgos-Silva,* Marco Antonio Cenedeze,*
Denise Maria Avancini Costa Malheiros,† Marlene A. Reis,‡ Alvaro Pacheco-Silva,*
and Niels Olsen Saraiva Câmara*§
*Experimental and Clinical Immunology Laboratory, Division of Nephrology, Federal University of São Paulo,
Escola Paulista de Medicina, São Paulo, Brazil
†Pathology Department, University of São Paulo, São Paulo, Brazil
‡Pathology Division, UFTM, Uberaba, Minas Gerais, Brazil
§Laboratory of Transplantation Immunobiology, Department of Immunology, University of São Paulo, São Paulo, Brazil
Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that
are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression
of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice
for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 ´ 105 ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval
for the next 6 weeks. Also, animals were treated with 2 ´ 105 ASCs at 6 weeks after reperfusion and sacrificed
4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASCtreated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA
expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4, IL-10, and HO-1 expression
increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys
shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen
deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in
ASC-treated animals was followed by reduced serum levels of TNF-a, KC, RANTES, and IL-1a. Surprisingly,
treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration
of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen
and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early
and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia,
thereby reducing the epithelial–mesenchymal transition.
Key words: Adipose-derived stem cell; Acute kidney injury; Ischemia-reperfusion injury; Fibrosis;
Inflammation
INTRODUCTION
and persistent tubule-interstitial inflammation, leading to
an increase in fibroblast proliferation and the activation
of myofibroblasts and resulting in excessive deposition of
extracellular matrix proteins in kidney (15,31). The epithelial-to-mesenchymal transition (EMT) has been suggested as one the main pathways in fibrosis development
in the progression of AKI to CKD (24,41).
The EMT can be viewed as an adaptive response of
epithelial cells after chronic stress/injury. Hypoxia, oxidative stress, and inflammation can produce and release
several chemokines and cytokines that attract and direct
Despite all efforts to manage acute kidney injury (AKI)
patients, this syndrome is still associated with high mortality and morbidity rates. Moreover, it has a high incidence and may affect approximately 7% of all hospitalized
patients, with the mortality rate reaching near 50% of AKI
patients in intensive care units (ICUs) (34,40). Among
long-term AKI surviving patients (1–10 years), approximately 12.5% are dialysis dependent and 19–31% develop
chronic kidney disease (CKD) (15). This scenario can be
caused by incomplete repair of epithelial cells after AKI
Received October 27, 2010; final acceptance September 20, 2011. Online prepub date: Febuary 2, 2012.
Address correspondence to Niels Olsen Saraiva Câmara, M.D., Nephrology Division, Federal University of São Paulo, 740 Botucatu St.,
04023-900, Vila Clementino, São Paulo, Brazil. Tel: (5511)-59041699; Fax: (5511)-59041894; E-mail: [email protected]
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the influx of inflammatory cells to the tubule-interstitial
space. Infiltrating cells, in turn, activate and produce a
mixture of soluble factors, including proinflammatory and
profibrotic cytokines as well as matrix metallopeptidases
(MMPs) (10,53), creating a hostile microenvironment
for tubular epithelial and endothelial cells. In the early
inflammatory phase of renal fibrosis, cytokines produced
by infiltrating cells, such as interleukin-1 (IL-1), tumor
necrosis factor-a (TNF-a), and interferon-g (IFN-g), profoundly potentiate tubular EMT triggered by transforming growth factor-b (TGF-b) through the upregulation of
the TGF-b receptor (32). TGF-b is the main driving factor
of fibrosis via the promotion of fibroblast proliferation,
extracellular matrix synthesis, and the inhibition of collagenase in multiple organs, all hallmarks of EMT (28).
TNF-a-dependent NF-kB activation also stabilizes the
transcription factor Snail (potent EMT inducer) by blocking its ubiquitination, providing another molecular link
between inflammation and EMT (2,57). The significance
of renal inflammation in initiating and promoting EMT is
also manifested by many observations that renal fibrosis
is almost always preceded by and closely associated with
chronic interstitial inflammation (19,27).
Adipose tissue-derived stem cells (ASCs) are easily isolated adult mesenchymal stem cells (65) that, when influenced by the extracellular milieu in vitro, have the capacity
to differentiate into other cell types such as adipocytes,
myocytes, osteoblasts, and neurons. They are known to
secrete multiple growth factors and therefore have cytoprotective effects in various injury models (3,33). Gimble
et al. have shown that ASCs delivered into an injured tissue
may secrete cytokines and growth factors that stimulate
recovery and clear toxic substances released into the local
environmental lesion, promoting recovery of the surviving
cells (14). Several studies have demonstrated the ability of
ASCs to downregulate immune system responses (11,59).
They can suppress T-cell activation by inhibiting cyclin D2
expression (44), by inducing regulatory T cells (1,16,17) or
by interfering with dendritic cells (1,23). Moreover, ASCs
can act as antiapoptotic factors on target cells, increasing
the recovery of renal function (45).
Recently, stem cell therapies have emerged as new
strategies to halt the progression of chronic kidney diseases. Semedo et al. demonstrated that the administration
of MSCs improved histological and functional parameters
and reduced the fibrotic area in a severe model of renal
ablation. The renal protective actions enabled by MSCs
also attenuated chronic hypoxia, oxidative stress, and the
production of inflammatory cytokines that lead to EMT
and are all presented during CKD (7,50,51). Several other
studies have described the beneficial effects of ASC administration. ASC treatment leads to improved vascular supply (42,45), accelerated healing processes of skin wounds
(39), periodontal tissue regeneration (54), cardiac wall
Donizetti-Oliveira ET AL.
regeneration (36), repair after myocardial infarction (49),
and improvement of cellular function following stroke (25).
In this study, we hypothesize that ASCs can halt renal fibrosis progression in a specific animal model by the modulation
of inflammatory events in different times. Moreover, ASC
therapy also has the potential to improve renal function and
tissue histology when renal scar was already installed.
MATERIALs AND METHODS
Isolation of Adipose Tissue-Derived Stem Cells (ASCs)
Mouse adipose tissues dissected from the inguinal area
were isolated from male C57BL/6J mice and digested
with 0.075% collagenase IA (Sigma Aldrich, St. Louis,
MO, USA) at 37°C for 30 min. The cell suspension was
filtered through 70-µm mesh (BD Biosciences, San Jose,
CA, USA) to remove tissue debris. Collagenase was then
inactivated with fetal calf serum (FCS), and the cell suspension was washed with phosphate-buffered saline (PBS)
solution and centrifuged twice for 5 min at 260 ´ g. The
cell pellet was then suspended in 0.84% NH4Cl to remove
red blood cells. Cells were washed and centrifuged twice
with PBS and cultured on plastic dishes (TPP, Trasadingen,
Switzerland) in low-glucose Dulbecco’s modified Eagle’s
medium (DMEM; Gibco-Invitrogen Corporation, Carlsbad,
CA, USA) supplemented with 10% fetal bovine serum
(FBS; Emcare, Campinas, SP, Brazil), until the cells reached
70–80% confluence. Cells were detached with trypsin and
cultured until the fifth passage in 4 weeks.
ASC Immunophenotyping
Briefly, a cell suspension (1 ´ 105 cells) of ASCs was
incubated for 40 min in saturating concentrations of the
antibodies CD34-fluorescein isothiocyanate (FITC),
CD105-FITC, CD73-phycoerythrin (PE), CD45-peridinin
chlorophyll protein complex (PerCP), and CD90-FITC and
matched isotype controls (all antibodies were purchased
from BD Biosciences Pharmingen, San Diego, CA, USA).
After three washes, the cells were centrifuged at 200 ´ g
for 5 min and ressuspended in ice-cold PBS. Cell fluorescence was evaluated using a flow cytometer (FACSCanto;
Becton Dickinson, Franklin Lakes, NJ, USA).
ASC Differentiation Assay
ASCs were differentiated into adipocytes by treatment
with low-glucose DMEM supplemented with 10% FBS,
dexamethasone (1 µM), isobutylmethylxanthine (0.5 mM),
insulin (10 µg/ml), and indomethacin (100 µM) for 14
days. Osteoblast differentiation was achieved with lowglucose DMEM supplemented with 10% FBS, dexamethasone (0.1 µM), ascorbic acid (0.2 mM), and b-glycerol
phosphate (10 mM) for 28 days. All reagents were purchased from Sigma-Aldrich. These assays were stained
with Oil Red and Von Kossa to visualize lipid vesicles
and calcium deposits, respectively.
Adipose Stem Cell and Renal Disease Progression
Animal Model
Female C57BL/6J mice, 8 weeks of age, were obtained
from CEDEME (Federal University of São Paulo, São
Paulo, Brazil) and subjected to unilateral renal ischemia,
as described by Burne-Taney et al. (4). Mice underwent
abdominal incisions, and their left renal pedicle was
bluntly dissected. A microvascular clamp (Rocca, São
Paulo, SP, Brazil) was placed on the left renal pedicle
for 60 min. Animal temperature was maintained near
37°C. Animals were monitored and maintained for 24 h,
6 weeks, or 10 weeks before sacrifice. The animals groups
was separated as follows: group 1 (n = 5), animals Sham
sacrificed after 24 h; group 2 (n = 6), animals submitted
to unilateral ischemia reperfusion (IR) and sacrificed
24 h later; group 3 (n = 6), animals submitted to unilateral IR treated with ASCs and sacrificed 24 h later; group
4 (n = 5), animals Sham sacrificed after 6 weeks; group 5
(n = 6), animals submitted to unilateral IR and sacrificed
6 weeks later; group 6 (n = 6), animals submitted to unilateral IR treated with ASCs and sacrificed 6 weeks later;
group 7 (n = 5), animals Sham sacrificed after 10 weeks;
group 8 (n = 6), animals submitted to unilateral IR and
sacrificed 10 weeks later; group 9 (n = 6), animals submitted to unilateral IR treated with ASC and sacrificed 10
weeks later. Sham animals underwent the same surgical
procedure without clamping of the renal artery. All experimental procedures were approved by the local research
ethics committee (CEP process no. 0058/2010).
Cell Treatment
It used sex-mismatched cells for transplantation. After
4 h of surgery, female mice received male ASCs intra­
peritoneally and were sacrificed 24 h or 6 weeks after surgery. Another group received the same ASC treatment 6
weeks after surgery and were sacrificed at 10th week post­treatment. Cells (2 ´ 105) were administered to each animal.
Assessment of Renal Function
Blood was collected from inferior vena cava for serum
creatinine and urea measurements. Serum creatinine was
measured by Jaffé’s modified method. Serum urea was
measured using a Labtest Kit (Labtest, Lagoa Santa,
MG, Brazil).
Morphology
Kidneys obtained 6 and 10 weeks postischemia were
analyzed by Masson and Picrosirius Red staining. For histological examinations, kidneys were fixed with 10% buffered formaldehyde for 24 h, washed with 70% ethanol for
24 h, and then embedded in paraffin. Sections were cut to a
thickness of 4 µm. To evaluate the extent of renal interstitial
expansion, the fraction of the renal cortex that stained positively for extracellular matrix components (collagen) was
quantitatively evaluated in Masson-stained sections by a
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point-counting technique in consecutive microscopic fields
at a final magnification of 100´ under a 176-point grid.
Picrosirius Red staining was measured at a magnification of
20´ using the NIS Elements program of Nikon microscopy
with at least 20 consecutive fields. Acute tubular necrosis (ATN) was measured in kidney sections stained with
hematoxylin–eosin by a blinded reviewer. The extension of
tubular necrosis (ATN) morphology and patterns of regeneration in the kidney was examined by blinded morphometric assessment using a computer-assisted image system
with an Olympus BX40F-3 microscope (Olympus Optical
Company, Tokyo, Japan) and the software program KS300
program (Zeiss, Göttingen, Germany). The results were
expressed as a ratio of the injured area related to total area
(1,458,467 mm2). Twenty-five random fields were counted
at an original magnification of 835´, almost covering the
whole kidney, corresponding to an area of 36,461,675 mm2,
for each animal, per lamina. For morphometric analyses,
ATN and regeneration were analyzed at 24 h after reperfusion. Tubular necrosis pattern included loss of cytoplasmatic and nuclear membranes integrity, loss of brush border,
vacuolization of tubular epithelial cells, and presence of
intratubular debris. Tubular regeneration or repair was characterized by attenuated epithelium enclosing dilated lumens
filled with proteinaceous fluid or granular material.
Real-Time PCR
Kidney samples were quickly frozen in liquid nitrogen. Total RNA was isolated using the TRIzol Reagent
(Invitrogen, Carlsbad, CA, USA), and RNA concentrations were determined by Nanodrop. First-strand cDNAs
were synthesized using the MML-V reverse transcriptase
(Promega, Madison, WI, USA). Reverse transcriptase
polymerase chain reaction (RT-PCR) was performed using
TaqMan (Applied Biosystem, USA) for the following
molecules: collagen-1 (Col-1) (00801666_g1), connective
tissue growth factor (CTGF) (Mm01192931_g1), IL-1b
(Mm01336189_m1), IL-4 (Mm00445259_m1), IL-6
(00561420_m1), IL-10 (Mm00439616_m1), inducible
nitric oxide synthase (iNOS) (Mm01309902_m1), hemeoxygenase-1 (HO-1) (Mm00516004_m1), hypoxanthine
phosphoribosyltransferase (HPRT) (Mm00446968_m1),
sex-determining region Y (SRY) (Mm00441712_s1),
TNF-a (Mm00443258_m1), vascular endothelial
growth factor (VEGF) (Mm01281449-m1), and vimentin (00449201_m1). Cycling conditions were as follows:
10 min at 95°C followed by 45 cycles of 20 sec at 95°C, 20
at 58°C, and 20 at 72°C. RT-PCR was performed using the
SYBR Green real-time PCR assay (Applied Biosystem,
USA) for the following molecules: HPRT (sense) 5¢-CTC
ATG GAC TGA TTA TGG ACA GGA C-3¢, (antisense)
5¢-GCA GGT CAG CAA AGA ACT TAT AGC C-3¢; bone
morphogenetic protein-7 (BMP-7) (sense) 5¢-AGA ATT
CTT CCA CCC TCG ATA CC-3¢, (antisense) 5¢-TCC TTA
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TAG ATC CTG AAT TCG GCT-3¢; and TGF-b (sense)
5¢-AAC TAT TGC TTC AGC TCC ACA GAG A-3¢, (antisense) 5¢-GTT GGC ATG GTA GCC CTT G-3¢. Sequence
Detection Software 1.9 (SDS) was used for analysis, and
mRNA expression was normalized to HPRT abundance.
The values are expressed relative to a reference (the calibrator) from the sham-operated samples. The Ct (threshold
cycle) for the target gene and the Ct for the internal control were determined for each sample. Samples were run in
triplicate. The relative expression of mRNA was calculated
by 2-DDCt. All the experimental samples are expressed as an
n-fold difference relative to the calibrator.
Bio-Plex
A Bio-Plex mouse 23-Plex cytokine assay kit (Bio-Rad
Laboratories, Inc., Hercules, CA, USA) was used to test
samples for the presence of 23 cytokines. The assay was
read on the Bio-Plex suspension array system, and the data
were analyzed using Bio-Plex Manager software version
4.0. Standard curves ranged from 32,000 to 1.95 pg/ml.
Immunohistochemistry
Collagen-I (diluted 1:200; COL-1, AbCAM, Cambridge,
MA, USA), fibroblast specific protein-1 (FSP-1) (diluted
1:400, S100A4, DAKO), proliferating cell nuclear antigen (PCNA) (diluted 1:300; clone PC10, DAKO), and
Hypoxyprobe (diluted 1:500; 121 Middlesex Turnpike,
Burlington, MA USA) staining were performed in paraffinembedded sections. Previously, the slides were deparaffinized,
rehydrated, and submitted to Tris–EDTA (pH 9) antigen
retrieval solution treatment at 95°C. For Hypoxyprobe, the
antigen retrieval solution used was citrate buffer (10 mM) at
55°C. The endogenous peroxidase activity was blocked with
3% hydrogen peroxide, and sections were blocked with protein block solution (DAKO, Carpinteria, CA, USA). Slides
were then incubated with primary antibody or negative control reagent, followed by incubation with the labeled polymer
Envision (DAKO), using two sequential 30-min incubations
at room temperature. Staining was completed by a 1- to 3-min
incubation with 3,3¢-diaminobenzidine (DAB)+ substrate–
Donizetti-Oliveira ET AL.
chromogen, which results in a brown-colored precipitate
at the antigen site. Hematoxylin counterstaining was also
completed. To analyze tissue hypoxia, we administered
HypoxyprobeTM-1 (pimonidazole HCl) solution at a dosage
of 60 mg/kg body weight (IP) approximately 25 min before
sacrifice. Following injection, HypoxyprobeTM-1 (HPI, Inc.,
Burlington, MA, USA) is distributed to all tissues but forms
adducts with thiol-containing proteins only in those cells
that have oxygen partial pressure lower than 10 mmHg
(pO2 ≤ 10 mmHg) at 37°C.
Statistical Analysis
Data were expressed as the mean ± SD. Differences
between the groups were evaluated for statistical significance using ANOVA followed by the Bonferroni test and
Student’s t test. Differences were considered significant
at p < 0.05.
RESULTS
Isolation of ASCs and Characterization
ASCs were characterized by immunephenotype assays
using several cell surface markers described in the litera­
ture (65). CD34, CD73, CD105, and CD45 were assessed. Expression of CD73 was 24.9% ± 40.9%, CD105
71.2% ± 19.7%, CD34 6.0% ± 3.8%, CD45 6.9% ± 1.9%,
and CD90 81.7% ± 1.5%. These results were expected
based on the already described ASC immunephenotype
profile (18,35,65). First passage cells were submitted to
parallel adipocyte and osteocyte differentiation assays.
Under supplemented specific medium, cells differentiated
to adipocytes were identified by the visualization of lipid
droplets inside cells by Oil Red staining. Osteocyte differentiation was also performed, and after 28 days under
osteocyte medium, calcium deposits were observed via
Von Kossa staining (data not shown).
ASC Treatment Leads to In Situ and Systemic
Immune Modulation
In this model, severe ischemia and reperfusion is achieved
in only one kidney. However, due to long ischemic time
FACING PAGE
Figure 1. Effects of intraperitoneal adipose-derived stem cell (ASC) treatment in mice 24 h after unilateral ischemia. Female
mice C57BL/6J were subjected to ischemia and reperfusion injury (IRI) and sacrificed after 24 h. After 4 h of ischemia induction, one group received 2 ´ 105 ASCs isolated from male mice and administered intraperitoneally. Blood and renal tissue were
collected and used to assess renal function, mRNA expression, and histology. (A) Serum urea level. (B) Necrosis areas in kidney.
(C) Regeneration areas in kidney. (D) Representative photomicrograph illustrating acute tubular necrosis area in kidney tissue
of an untreated animal and (E) an ASC-treated animal. (F–H) Proliferating cell nuclear antigen (PCNA)-positive nuclei were
more frequent in treated animals, which was likely due to increased regeneration events. (F) Representative photomicrograph
illustrating PCNA expression in kidney tissue from an ASC-treated animal and (G) an untreated animal. (H) Quantification
of PCNA staining. (I–M) qRT-PCR from kidney tissue values normalized to hypoxanthine phosphoribosyltransferase (HPRT);
(I) interleukin-6 (IL-6) mRNA expression. (J) Tumor necrosis factor-a (TNF-a) mRNA expression. (K) IL-1b mRNA expression.
(L) IL-4 mRNA expression. (M) IL-10 mRNA expression. In (N), we analyzed the sex-determining region Y (SRY) expression
by qualitative polymerase chain reaction (PCR) in ASC-treated kidneys and in male mice C57Bl/6 kidney like control. qRT-PCR
data are expressed as mean of 2–DDCt ± SD; *p < 0.05 (Sham n = 5, untreated and ASC-treated animals group n = 6). ND, not defined.
Scale bar: 25 mm.
Adipose Stem Cell and Renal Disease Progression
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(1 h), kidney functional parameter was altered. Although no
difference was found in creatinine levels (data not shown),
urea serum levels were increased in animals subjected to
this severe ischemia when compared to sham animals, but
ASC treatment presented less renal dysfunction (Fig. 1A).
These findings correlated with acute tubular necrosis (ATN)
analyses as the kidney submitted to ischemia had a higher
area of necrosis after 24 h (Fig. 1B), whereas ATN was
decreased in ASC-treated animals. Moreover, regeneration
patterns were higher in ASC-treated animals (Fig. 1C, E)
as confirmed by PCNA staining (Fig. 1F–H).
Inflammation has an important role in kidney ischemiareperfusion outcomes (8,21,22). In addition, it is known
that ASCs leads to immune modulation. Therefore, we
analyzed mRNA expression of several cytokines after
ASC treatment. IL-6 mRNA expression was reduced
in the kidney tissue of ASC-treated animals (Fig. 1I).
TNF-a and IL-1b mRNA expression were also quantified,
but no statistical differences were observed (Fig. 1J, K),
although ASC-treated animals had reduced expression.
Surprisingly, anti-inflammatory mRNA cytokines IL-4
and IL-10 were augmented in the kidney tissue of ASCtreated animals (Fig. 1L, M).
Systemically, we again found the same immunomodulatory pattern demonstrated in kidney tissue. Serum proinflammatory cytokines, such as IL-1a, IL-1b, IL-12 (p70),
and keratinocyte-derived chemokine (KC), were decreased
in ASC-treated group (Table 1). IL-6 and IL-13 levels were
also assessed, but no differences were observed (Table 1).
Levels of cytokine regulated upon activation normal T cell
expressed and presumably secreted (RANTES or CCL5)
were also reduced in ASC-treated animals (Table 1). In
this multiplex assay, Granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating
factor (GM-CSF), IL-12 (p40), monocyte chemotactic
protein-1 (MCP-1), macrophage inflammatory protein-1b
(MIP-1b), and TNF-a levels did not differ between ASCtreated animals and untreated animals. Moreover, IL-2,
IL-3, IL-4, IL-10, IL-17, MIP-1a, and IFN-g were not
detected with this assay.
To track ASCs within kidneys after treatment, SRY
mRNA expression was amplified by real-time PCR (Fig. 1N).
We did not detect SRY mRNA intensities, indicating the
absence of these cells within the tissue at this time point.
Donizetti-Oliveira ET AL.
Table 1. Serum Determined Cytokine Levels 24 h After
Reperfusion
Cytokine
Sham
IL-1a
2.85 ± 0.03
IL-1b
27.02 ± 1.02
IL-12(p70) 2.18 ± 0.05
KC
14.35 ± 0.13
IL-6
1.69 ± 0.06
IL-13
53.93 ± 2.03
RANTES
4.41 ± 0.12
Unilateral IR
Unilateral
IR + ASC
23.35 ± 5.14
17.32 ± 1.63*
146.52 ± 41.31 124.06 ± 20.00
8.12 ± 1.84
5.74 ± 0.23*
59.93 ± 25.00
37.38 ± 3.95
34.91 ± 9.05
22.29 ± 1.22*
312.54 ± 119.07 464.65 ± 113.32
5.34 ± 1.48
2.87 ± 0.19*
A multiplex bead-based immunoassay kit was used to test the serum.
Data are expressed as mean ± SD; * p < 0.05 (Sham n = 5, untreated and
ASC-treated animals group n = 6). ASC, adipose-derived stem cell; IL,
interleukin; KC, keratinocyte-derived chemokine; RANTES, regulated
upon activation normal T cell expressed and presumably secreted.
Halting Fibrosis Progression by Downregulating
Inflammation After Severe Ischemia
In this model of severe ischemia and reperfusion
injury, it was observed that the kidney submitted to ische­
mia was shrunken after 6 weeks when compared to the
contralateral kidney in untreated animals. Surprisingly,
the ischemic kidneys of ASC-treated animals were not
reduced in size (Fig. 2A). Despite it, creatinine and urea
levels did not change with ASC treatment, probably due
to the presence of the contralateral kidney, which can
gradually compensate for the injured kidney’s functional loss (Fig. 2B, C). These shrunken kidneys showed
higher areas of fibrosis, as demonstrated by Masson
Trichrome and Picrosirius Red stainings (Fig. 2D–F).
No relevant fibrosis was seen in the sham-­operated
group.
We also quantified mRNA expression of some profibrotic molecules to strengthen our quantification of collagen deposition data. Type 1 collagen (Col-1), vimentin,
and connective tissue growth factor (CTGF) expression were assessed in renal tissue after 6 weeks. Again,
reduced mRNA expression of those markers was observed
in the kidney tissue of ASC-treated animals (Fig. 3A–C).
Furthermore, to confirm these results, protein analyses
were completed using immunohistochemistry assays for
fibroblast-specific protein 1 (FSP-1) and Col-1. As seen
in Figure 3D, there is more pronounced staining of FSP-1
and Col-1 in the group of untreated animals, indicating
FACING PAGE
Figure 2. Effects of intraperitoneal ASCs in mice 6 weeks after unilateral ischemia. (A) (a) Kidney necroscopy following unilateral IR
after 6 weeks. The right kidney subjected to severe unilateral IRI appeared shrunken when compared to the contralateral kidney that
was not subjected to IRI. (b) Kidneys from animal treated with ASCs. No differences in kidney size were observed in these animals.
(B) Serum creatinine levels. (C) Serum urea levels. (D) Representative image of kidneys from the sham animals (Sham), untreated
(IR 6 weeks) or ASC-treated (IR 6 weeks + ASC) stained with Masson’s trichrome (a, b, c) and Picrosirius Red (d, e, f), respectively.
Polarized light analyses of Picrosirius Red staining showing only type I collagen fibers (g, h, i). (E) Quantification of Masson’s
trichrome staining and (F) Picrosirius Red staining. Picrosirius Red and Masson’s trichrome was quantified by image analysis using
NIS Elements (Nikon); however, Picrosirius Red was visualized using polarized light (g, h, i). For Sham, n = 5 mice/group; untreated
and ASC-treated animals group, n = 6 mice/group; *p < 0.05. Results are shown as mean values ± SD. Scale bar: 20 mm.
Adipose Stem Cell and Renal Disease Progression
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Donizetti-Oliveira ET AL.
Figure 3. Profibrotic-related molecule expression in the renal tissue after 6 weeks of injury. Gene expression in unilateral severe
ischemia-reperfusion (IR) injury kidneys of ASC-treated and untreated animals, generated by referencing each gene to HPRT as
an internal control. (A) Collagen (Col-1) mRNA expression. (B) Vimentin mRNA expression. (C) Connective tissue growth factor
(CTGF) mRNA expression. (D) Representative photomicrograph illustrating fibroblast-specific protein-1 (FSP-1) and Col-1 immuno­
histochemistry expression in the renal tissue after 6 weeks. Sham, n = 5 mice/group; untreated and ASC-treated animals group, n = 6
mice/group; *p < 0.05. Data expressed as the mean of 2–DDCt ± SD. Scale bar: 20 mm.
FACING PAGE
Figure 4. Immunemodulatory-related molecule expression in animals at 6 weeks after reperfusion. Gene expression in unilateral severe
IRI kidneys of ASC-treated and untreated animals, generated by referencing each gene to HPRT as an internal control. (A) TNF-a mRNA
expression. (B) IL-6 mRNA expression. (C) Heme-oxygenase-1 (HO-1) mRNA expression. (D) Bone morphogenetic protein-7 (BMP-7)/
transforming growth factor-b (TGF-b) mRNA expression in kidney tissue 6 weeks after treatment. Serum was assayed for cytokine levels
using a multiplex bead-based immunoassay kit. Serum cytokine levels for (E) IL-1a, (F) TNF-a, (G) keratinocyte-derived chemokine (KC),
(H) RANTES, and (I) IL-6 were determined in sham-operated animals, animals only subjected to IR, and animals subjected to unilateral
IR injury treated with ASC. (J) Representative photomicrograph illustrating Hypoxyprobe immunohistochemistry expression in the renal
tissue after 6 weeks. (K) Vascular endothelial growth factor (VEGF) mRNA expression. (L) Inducible nitric oxide synthase (iNOS) mRNA
expression in kidney tissue 6 weeks after treatment. Gene expression data expressed as the mean of 2–DDCt ± SD. Protein data expressed as
the mean ± SD; Sham, n = 5 mice/group; untreated and ASC-treated animals group, n = 6 mice/group; *p < 0.05. Scale bar: 20 mm.
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an enlarged population of fibroblasts with greater extracellular matrix protein deposition.
Systemic Immunomodulation After ASC Treatment
As early as 24 h after severe ischemia and reperfusion
injury, we observed an important modulation of the inflammatory response in ASC-treated animals. As this reduced
inflammation at the early time point could be implicated
in the progression of fibrosis, we further investigated the
inflammatory patterns inside the kidney and at 24 h and
6 weeks after therapy. Moreover, via real-time PCR some
protective and angiogenic molecules related to tissue repair
was quantified. At 6 weeks posttreatment, the expression
of renal proinflammatory cytokines, such as TNF-a and
IL-6, was still reduced in animals subjected to severe ische­
mia (Fig. 4A, B) and treated with ASCs. In addition, hemeoxygenase-1 (HO-1) levels and BMP-7/TGF-b ratios were
increased in those animals as compared to untreated animals
(Fig. 4C, D). ASC therapy also led to systemic immunomodulation, as serum cytokine levels were reduced in ASCtreated animals. IL-1a, TNF-a, KC, and RANTES levels
were also significantly reduced in those animals when compared to untreated animals (Fig. 4E–H). Levels of IL-6 did
not differ between these two groups, although a tendency
toward a reduction in this cytokine was observed in ASCtreated animals (Fig. 4I).
Protective Effects of ASC Against Chronic Hypoxia
Chronic hypoxia due to the rarefaction of capillaries is
one possible theory to explain the continuous progression
of renal fibrosis, as low oxygen tension can be a major
factor for the promotion of EMT. Therefore, we analyzed
whether tissue hypoxia was reduced in renal tissue by
using a probe that detects lower areas of pO2. Kidneys of
ASC-treated animals demonstrated lower areas of hypoxia
as compared to untreated animals (Fig. 4J). Moreover, the
expression of iNOS was augmented in kidney tissue in
ASC-treated animals, and there were a tendency toward
an augmentation in VEGF expression (Fig. 4K, L). The
higher expression of these molecules in ASC-treated animals can be due to an improvement in kidney vascularization due to ASC angiogenic properties.
Reversion of Renal Fibrosis by ASC Treatment
The role of ASC in reverting established renal fibrosis was tested. ASCs were administrated at 6 weeks after
ischemia, and animals were followed up to the 10th week
postischemia treatment. Surprisingly, the kidneys of ASCtreated animals showed reduced areas of fibrosis, as analyzed by Picrosirius Red staining (Fig. 5A, B). Moreover,
a functional amelioration of kidney was also observed and
correlated with these reduced areas of fibrosis (Fig. 5C, D).
Confirming these data, mRNA expression of vimentin
was reduced in ASC-treated animals when compared to
Donizetti-Oliveira ET AL.
the untreated group (Fig. 6A). Type I collagen mRNA
expression was also decreased in ASC-treated animals, but
no significant difference was observed (Fig. 6B). In addition, the immunohistochemistry assay for type I collagen
and FSP-1 correlated with gene analysis studies (Fig. 6C).
Furthermore, the mRNA expression levels of IL-10 and
BMP-7 were increased after ASC treatment (Fig. 6D, E).
Again, despite the positive effect achieved, ASCs could not
be visualized in kidney tissue, as analyzed by SRY mRNA
expression (Fig. 6F).
DISCUSSION
In 2002, ASCs were first described by Zuk et al. (64),
after which, the number of ASC studies increased exponentially. The regenerative properties of the ASCs are of
great interest in the treatment of many diseases (63). This
is particularly true for fibroproliferative diseases where
tissue destruction caused by extracellular matrix deposition leads to organ function loss. Of further importance
is the consideration that the interaction of the epithelial
tubular cell and extracellular matrix is crucial to determine kidney function.
CKD is emerging as a worldwide health problem.
The possible therapies to end-stage renal failure include
dialysis and transplantation; however, these treatments
do not always meet patient needs (30). Several comorbitities may cause CKD, but one aspect is continually
overlooked: persistent inflammation after AKI events
(47,58). Inflammation triggers fibrosis progression in the
kidney and leads to the transformation of an epithelial
cell to a myofibroblast, the so-called EMT (24). Whether
EMT occurs in vivo is not known, the role of inflammation on fibrosis scar formation is not questioned. Despite
functional amelioration after AKI, residual inflammation
may contribute to epithelial injury leading to fibrosis.
In this model of severe unilateral ischemia, the remaining inflammation causes kidneys to shrink due to fibrosis scar formation. It is noteworthy to emphasize that no
ideal mouse model for CKD exist that can reproduce all
features see in humans. However, this model allows us
to study hypoxia, inflammation, and ultimately its consequence, renal fibrosis.
Here, we propose that ASCs may inhibit EMT by
decreasing inflammation. At 24 h posttreatment, animals
had better outcomes that were functionally correlated
with downregulation of inflammation. The consequences
of this immunomodulation are reflected at 6 weeks posttreatment when lower fibrosis areas are seen.
Our group has shown that a single episode of severe unilateral renal ischemia leads to inflammation that results in
the development of fibrosis (12). The main mechanism of
action for adult stem cells is immunoregulation (23,55). This
occurs predominantly through a paracrine effect mediated
by ASCs called touch and go, where these cells are thought
Adipose Stem Cell and Renal Disease Progression
Figure 5. Delayed ASC administration positively restores renal function and reverses fibrosis.
(A) Representative image of kidneys of untreated animals (IR 10 weeks) (a, c) and animals treated
with ASCs (IR 10 weeks + ASC) (b, d) with Picrosirius Red staining. Top (a, b) shows strong staining,
and bottom (c, d) shows the same image using polarized light (magnification: ´40). (B) Picrosirius
Red staining quantified by image analysis using NIS Elements (Nikon). (C) Serum creatinine levels.
(D) Serum urea levels. Sham, n = 5 mice/group; untreated and ASC-treated animals group, n = 6 mice/
group; *p < 0.05. Results are shown as mean values ± SD. Scale bar: 20 mm.
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Donizetti-Oliveira ET AL.
Figure 6. Tissue cytokine expression in animals 10 weeks after reperfusion. Gene expression in IR kidneys generated by referencing each gene to HPRT as an internal control.
(A) Vimentin mRNA expression and (B) Col-1 mRNA expression of profibrotic factors.
(C) Representative photomicrograph illustrating FSP-1 and Col-1 immunohistochemistry expression
in the renal tissue after 10 weeks. (D) IL-10 mRNA expression and (E) BMP-7 mRNA expression
of protective factors. (F) SRY expression by qualitative polymerase chain reaction (PCR) in ASCtreated kidneys and in male mice C57Bl/6 kidney-like control. Data are expressed as the mean of
2–DDCt ± SD. Sham, n = 5 mice/group; untreated and ASC-treated animals group, n = 6 mice/group.
*p < 0.05. Scale bar: 20 mm.
Adipose Stem Cell and Renal Disease Progression
to migrate to the injury area, to secrete immune factors and
later, to leave the tissue, and/or to die. This might explain
why ASCs were not seen in the kidney tissues of ASC-treated
animals. At early time, we found that ASC therapy modulates inflammation, with decreased expression of renal and
systemic levels of Th1 cytokines (IL-1b, IL-6, and TNF-a)
and increased protective renal Th2 cytokine (IL-4 and IL-10)
expression. This modulation reflected in an improvement in
histological and functional organ parameters.
It was believed that patients who survived an initial AKI
would have a complete return of renal function. Today, however, data from animal models and from clinical studies have
put on check the long-term consequences of AKI (4,12,43).
In our model, we observed a long-term effect of treatment
with ASCs, where the initial immunomodulation was still
persistent with enhanced expression of protective factors
such as HO-1 and BMP-7. BMP-7 is a competitor of TGF-b
receptor signaling. By calculating the ratio of BMP-7 and
TGF-b, we found a protective effect of ASC administration,
where the expression of BMP-7 was higher than TGF-b on
treated animals. TGF-b is seen as the most important factor
to induce fibrosis through activation of resident fibroblasts
and by inducing EMT. These findings correlate to the quantification of fibrosis area found in animals after treatment,
to a decreased expression of profibrotic factors (vimentin
and type 1 collagen), to a decrease in fibroblasts infiltration
(FSP-1-positive cells), and to a lower tissue collagen deposition. We can assume that early modulation of inflammation and TGF-b signaling pathway resulted in less fibrosis.
HO-1 is a protective gene involved in heme degradation, resulting in end products with anti-inflammatory,
antioxidant, and antiapoptotic actions (5). Its upregulation correlates the cytoprotection found after treatment
with ASCs. This decrease in inflammation seen by both
the reduction of inflammatory factors and increase of protective factors is closely linked to less formation of fibrosis, since inflammation is one of the factors that induce
EMT. Upregulation of HO-1 was already demonstrated
to inhibit EMT (26,52) in animal models where fibrosis
development was modulated (12,56).
Hypoxia may also induce EMT (62). Among CKD progression theories, chronic hypoxia can trigger fibrosis in
part due to vessels rarefaction. Here, we observed that ASC
therapy may prevent the capillary loss. By using a probe
that detects lower areas of pO2, a Hypoxyprobe, we found
that hypoxic areas were reduced in ASC-treated animals as
compared to controls. This might be due to an increased
expression of both HO-1 and VEGF molecules. With
the breakdown of heme by HO-1, biliverdin and carbon
monoxide are released, which might be responsible for an
increase in microcirculation and vasodilatation (5). In some
conditions, iNOS expression is associated with inflammation (production of nitrogen reactive species). However,
the production of NO by iNOS can also be associated with
1739
vasodilatation that ultimately decreases hypoxic areas,
while it has angiogenic actions by positively modulating
VEGF expression (6,29,37). VEGF is a key factor for vessel growth and normal glomerular and vascular development (48). The higher expression of these molecules in
ASC-treated animals illustrates the angiogenic characteristics of stem cells with consequent improvement in vascularization and in reduction of chronic hypoxia.
Although no ideal experimental model of CKD exists,
we believe that the use of this severe ischemia model allows
us to study the consequences of sustained inflammation and
permits us to treat in a specific point and later access the
improvement. For this purpose, we administrated the ASC 6
weeks after reperfusion, since at this point, our previous data
together with corroborating literature show us the presence
of interstitial fibrosis (4,12). Following the administration of
the stem cells, we analyzed the results after 4 weeks. The
results were startling. We found a higher mRNA expression
of protective factors (IL-10 and BMP-7) and a lower expression of profibrotic factors, which were followed by less
fibrosis quantified by histological and protein analysis.
Several studies already showed the possibility of
fibrotic regression (13,38,46). Inhibitors of angiotensin
play an important role in the progression and potential
regression of glomerulosclerosis (9,20,61). VEGF-A treatment also resulted in less injury and enhanced endothelial
cell proliferation and capillary repair in models of acute
glomerulonephritis or thrombotic microangiopathy (48)
and ameliorated development of glomerulosclerosis and
tubuleinterstitial fibrosis. Treatment with BMP-7 can
reverse TGF-induced renal fibrosis in mice and repair
severely damaged renal tubular epithelial cells, in association with reversal of chronic renal injury (60). So here,
we present the possibility of a fibrotic regression with
ASC treatment. We believe that the decrease of fibrosis is the result of a series of factors resulting from stem
cells’ biological properties. EMT inducers are activated
in an inflammatory microenvironment, in which both
TGF-b and hypoxia can induce the expression of specific
transcription factors. The modulation of inflammation
together with the increased expression of protective antifibrotic, angiogenic factors would lead to a blockade of
EMT, where cells returned to epithelial and endothelial
characteristics, through a continuous bidirectional event.
In summary, we propose that systemic ASC treatment
halts and even reverses fibrosis instigated by a severe ischemia via the downregulation of inflammation and hypoxia.
ACKNOWLEDGMENTS: This work was supported by the
Brazilian Foundation–FAPESP (Fundação de Apoio à Pesquisa
do Estado de São Paulo, grants 09/13251-6, 07/07139-3), CNPq
(473844/2009-5 and International Associated Laboratory CNPq/
Inserm), DECIT/Ministério da Saúde, and Complex Fluids INCT.
The authors would like to thank Meire Hiyane, Claudia Silva
Cunha, and Bernardo Paulo Abe for their technical support. The
authors declare no conflicts of interest.
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Donizetti-Oliveira ET AL.
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