Biol Blood Marrow Transplant 19 (2013) 538e546

Mesenchymal Stem Cells Ameliorate Podocyte Injury and

Proteinuria in a Type 1 Diabetic Nephropathy Rat Model ASBMT
American Society for Blood
and Marrow Transplantation

Shuai Wang 1, 2, Yi Li 1, Jinghong Zhao 1, Jingbo Zhang 1, Yunjian Huang 1, *

1
Institute of Nephrology of Chongqing and Department of Nephrology, Xinqiao Hospital, Third Military Medical University,
Chongqing, China
2
Department of Nephrology, Chengdu Military General Hospital, Chengdu, Sichuan, China

Article history:
Received 26 May 2012
Accepted 2 January 2013
Key Words:
Mesenchymal stem cells
Cell therapy
Diabetic nephropathy
Podocyte injury
Proteinuria
Bone morphogenetic protein-7
a b s t r a c t
Mesenchymal stem cells (MSC) attenuate albuminuria and preserve normal renal histology in diabetic mice.
However, the effects of MSC on glomerular podocyte injury remain uncertain. The aim of this study was to
evaluate the effects of MSC on podocyte injury in streptozotocin (STZ)-induced diabetic rats. Thirty days after
diabetes induction by STZ injection (65 mg/kg, intraperitoneally) in Sprague-Dawley rats, the diabetic rats
received medium or 2
106 enhanced green uorescent protein-labeled MSC via the renal artery. In vivo
tracking of MSC was followed by immunouorescence analysis. Diabetes-related physical and biochemical
parameters were measured on day 60 after the MSC infusion. The expression of podocyte markers (nephrin
and podocin), podocyte survival factors (VEGF and BMP-7), and the ultrastructural pathology of podocytes
were also assessed. MSC were only detected in the glomeruli from the left kidney receiving MSC infusion.
Compared with medium-treated diabetic rats, rats treated with MSC showed a suppressed increase in kidney
weight, kidney to body weight index, creatinine clearance rate, and urinary albumin to creatinine ratio;
however, the treatment had no effect on blood glucose or body weight levels. Furthermore, the MSC treat-
ment reduced the loss of podocytes, effacement of foot processes, widening of foot processes, thickening of
glomerular basal membrane (GBM), and loss of glomerular nephrin and podocin. Most important, MSC-
injected kidneys expressed higher levels of BMP-7 but not of VEGF. Our results clearly demonstrated that
intra-arterial administration of MSC prevented the development of albuminuria as well as any damage to or
loss of podocytes, though there was no improvement in blood sugar levels. The protective effects of MSC may
be mediated in part by increasing BMP-7 secretion.
2013 American Society for Blood and Marrow Transplantation.

INTRODUCTION mesenchymal stem cell (MSC) therapy improves micro-

Diabetic nephropathy (DN) is a major complication of
diabetes and represents the leading cause of end-stage renal
disease worldwide [1]. To date, there is no cure for DN. Drugs
that decrease blood glucose, lower blood pressure, or inhibit
the actions of the hormone angiotensin can delay, but not
eliminate, the onset of DN. Thus, the development of novel
therapeutic strategies that could specically target DN is
necessary.
Podocytes are highly specialized cells with important
roles in maintaining the glomerular ltration barrier and
producing growth factors for both the mesangial and endo-
thelial cells [2]. Recent studies have shown that progressive
podocyte injury, called podocytopathy, is one of the key
events in the pathogenesis of DN [3]. Podocyte loss and
injury are associated with the development of albuminuria
and the acceleration of glomerular structural abnormalities
[2,4,5]. Thus, there is a need to clarify the underlying path-
ogenesis of podocyte injury and the associated alterations in
the function of the glomerular ltration barrier to develop
a novel therapeutic strategy for the prevention and amelio-
ration of podocyte injury.
Stem cell therapy is a novel strategy for various diseases
and has the potential to be more effective than single-agent
drug therapies [6]. Several studies have shown that
Financial disclosure: See Acknowledgments on page 545.
* Correspondence and reprint requests: Yunjian Huang, MD, Department
of Nephrology, Xinqiao Hospital, The Third Military Medical University,
Chongqing, 400037, P.R. China.
E-mail address: h55769@yahoo.com.cn (Y. Huang).
1083-8791/$ e see front matter 2013 American Society for Blood and Marrow Transplantation.
http://dx.doi.org/10.1016/j.bbmt.2013.01.001
albuminuria and preserves the normal renal histology of
diabetic mice [7,8]. However, the exact mechanisms under-
lying their therapeutic effects on microalbuminuria have not
been clearly dened. It remains unknown whether the
contribution of MSC to reducing microalbuminuria is asso-
ciated with the improvement of podocyte injury. Based on
these ndings, we hypothesized that the transplantation of
MSC may be a potential therapeutic approach for diabetic
podocyte injury.
In the present study, we aim to evaluate whether MSC
could exert their protective effects against diabetic podocyte
injury. Here, our study provides the rst evidence that MSC
therapy may constitute a new therapeutic intervention to
target podocyte damage.
MATERIALS AND METHODS
Experimental Animal
Adult male Sprague-Dawley (SD) rats (weight, 180 g to 200 g) from the
laboratory animal center of Xinqiao hospital were maintained under specic
pathogen-free conditions at the animal care facilities. After 1 week of
adaptation, diabetes was induced by a single intraperitoneal injection of
streptozotocin (STZ) (65 mg/kg; Sigma, St Louis, MO) in the rats following
overnight fasting. Rats with a blood glucose level over 16.7 mmol/L were
considered diabetes-induced rats and were used in the study. Diabetic rats
received daily injections of long-acting insulin in doses adjusted individually
(ranging from 1 U to 4 U) to maintain blood glucose levels between 16
mmol/L and 28 mmol/L and to avoid ketonuria. All animal procedures were
performed according to the guidelines of the Animal Ethics Committee of
the Third Military Medical University, which are consistent with the NIH
Guide for the Care and Use of Laboratory Animals.
Isolation, Culture, and Characterization of MSC
MSC were obtained from the femurs and tibias of adult male SD rats and
cultured in DMEM/F12 medium (Gibco, Gaithersburg, MD) containing 10%
 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546
fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 mg/mL strepto-
mycin. The phenotypic properties of MSC were performed by ow cytom-
etry analysis using the following markers: CD29, CD44, CD34, and CD45
[9,10]. MSC were expanded in osteogenic or adipogenic differentiation
media (Cyagen Biosciences, Guangzhou, China) in 6-well plates after the
third passage. Osteogenic and adipogenic differentiations were tested using
standard protocol from Cyagen Biosciences Inc.
Cell Labeling for In Vivo Tracking
To assess the intrarenal localization of MSC, we used enhanced green
uorescent protein (EGFP) (Cyagen Biosciences) as a cell tracker in the
tracking experiments. Cells were labeled using transduction with lentiviral-
transduced EGFP, according to the manufacturers protocol. Briey, MSC
after the third passage were seeded at 2
106 cells in 6-well plates 24 hours
before transduction. The next day, 1 mL MSC medium per well containing 8
mg/mL polybrene (Sigma) and viral were added to the cells at 20 multi-
plicities of infection. After viral addition, cells were incubated at 37 C in 5%
CO2 for 6 hours. The medium was replaced by a fresh complete culture
solution, and cells were cultured until further experimentation. Selection
was completed 72 hours after incubation with 10 mg/mL puromycin.
MSC Administration and Experimental Design
Thirty days after the injection of STZ, DN rats were randomly divided
into 2 groups: DN rats treated with medium (DNmedium, n 10) and DN
rats treated with MSC (DNMSC, n 14). Nondiabetic rats were used as the
normal control group (NC, n 6). MSC-treated DN rats were injected with
2
106 MSC (suspended in 1 mL serum-free medium) via the left renal
artery, as described previously [11], and DNmedium rats received an equal
volume of medium. In this procedure, 2 rats that died of anesthesia or during
surgery were excluded; 1 died in each group. Then, the rats were housed
under specic pathogen-free conditions with a standard diet and water for
60 days before they were sacriced (Figure 1). At the end of the experiment,
the surviving rats were sacriced, and the left and right kidneys of MSC-
treated rats (n 9) and medium-treated rats (n 8) were harvested,
weighed, and processed for histological and biochemical evaluation.
EGFP-MSC Detection
At 24 hours and on day 60 after MSC injection, 2 MSC-treated rats were
sacriced, and their kidney tissues were embedded in Tissue-Tek O.C.T.
compound (Sakura Finetek, Torrance, CA) and frozen. The tissues were
sectioned into 8-mm samples using a cryostat microtome at 22 C. After
xation in acetone for 5 minutes, the sections were then covered with
uorescent antifade mounting medium. The 488-nm line of the laser was
used for EGFP uorescence excitation, and renal tissue background auto-
uorescence was detected at 555 nm. We used laser scanning confocal
microscopy (Leica Microsystems, Wetzlar, Germany) for the 2 uorescent
analyses.
Physical and Biochemical Analysis
The body weight, kidney weight, and blood glucose were measured at
the conclusion of the experiment. The serum creatinine levels were
measured in the blood obtained from the retro-orbital sinus at the time of
the sacrice using an automatic biochemistry analyzer (Hitachi, Tokyo,
Japan). The creatinine clearance rate was calculated as urinary creatinine

urine volume/serum creatinine and expressed as milliliters per minute.
Urinary albumin excretion was expressed as the urinary albumin to creati-
nine ratio. Urinary albumin levels were measured in morning spot urine
samples by a protein assay kit (Bio-Rad, Hercules, CA). After diabetes
induction, albumin to creatinine ratios were determined every 30 days.
Renal Morphology
The parafn sections were stained with Periodic Acid Schiff (PAS). The
extent of glomerular damage was expressed as the percentage of glomeruli
Figure 1. Mesenchymal stem cells (MSC) administration and experimental design. To induce diabetes, the rats were injected with 65 mg/kg streptozotocin. Thirty
days later, diabetic nephropathy (DN) was conrmed, and the DN rats received intra-arterial 2
106 MSC (n 14) or an equal volume of control medium (n 10). On
day 90 post-DN induction (60 days after MSC injection), the left and right kidneys of MSC-treated rats and medium-treated rats were harvested, weighed, and
processed for histological and correlative protein evaluation.
539
presenting mesangial expansion [12] or glomerulosclerosis, as described
previously [13]. For ultrastructural evaluation, 1 mm3 pieces of renal cortex
were xed in 2.5% glutaraldehyde in .1 M phosphate buffer (pH, 7.4) for
transmission electron microscopy. Electron micrographs of 5 blocks per
kidney (10 photographs per block) were obtained at a nal magnication of
8900- or 12,500-fold for each rat using a systematic uniform random
sampling protocol. To ensure randomized sampling and avoid repetitious
counting for podocytes in cross-sectional proles, .5-mm sections taken at
least 200 mm (average glomerular diameter) apart were cut and collected.
The average number of podocytes was measured by manually tracing along
GBM on a video screen. Podocytes were dened as those cells residing
within the glomerular tuft but outside the GBM. The podocyte quantity,
expressed as the number of podocytes per 2.5 mm GBM, was calculated by
computerized measurement using the Image Measurement System (Leica
Qwin Lite, Wetzlar, Germany) [14]. At the higher magnication, GBM
thickness was dened as the distance perpendicular to the GBM between
the endothelial and podocyte plasma membranes, measured with the aid of
Image-Pro Plus (Media Cybernetics, Silver Spring, MD). The analysis of
podocyte foot process effacement was expressed as the arithmetic mean of
the podocyte foot processes width (FPW), which was calculated by dividing
the total length of the GBM length by the total number of foot processes
according to published methods [15].

X .X 
FPW p=4
GBM length foot process
A correction factor of p/4 was used to correct for presumed random
variation in the angle of the section relative to the long axis of the
podocyte. All measurements were performed by the same technician
who was unaware of their origin [16]. The podocyte and GBM data were
shown as box-and-whisker plots using the SigmaPlot 12.0 software (Systat,
San Jose, CA).
Immunouorescence
Immunouorescent staining was performed with the specic podocyte
markers nephrin and podocin for localization. Frozen sections (5 mm) were
xed in cold acetone for 10 minutes at 20 C and incubated with wash
buffer containing .1% Triton X-100 at room temperature for 20 minutes to
prevent nonspecic binding. Subsequently, the sections were incubated
overnight at 4 C with polyclonal rabbit anti-nephrin (1:200 dilution; Santa
Cruz Biotechnology, Santa Cruz, CA) and polyclonal rabbit anti-podocin
(1:100 dilution; Santa Cruz Biotechnology) antibodies diluted in bovine
serum albumin at 1%. After being rinsed in phosphate buffered saline (PBS),
the TRITC-labeled goat antirabbit secondary antibody was incubated away
from light at 37 C for 30 minutes. Negative controls were run by replacing
the primary antibody with PBS. Staining was evaluated under laser scanning
confocal microscopy (Leica Microsystems, Wetzlar, Germany).
Western Blot Analysis
The renal cortex was homogenized in lysis buffer (Beyotime Institute of
Biotechnology, Shanghai, China) on ice for 30 minutes. The proteins were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and
electrotransferred to polyvinylidene diuoride nitrocellulose membranes
(Roche Molecular Biochemicals, Mannheim, Germany). After blocking in 5%
nonfat dried milk for 1 hour at room temperature, the membranes were
incubated with polyclonal rabbit antinephrin (1:700 dilution; Santa Cruz)
and polyclonal antipodocin (1:500 dilution; Santa Cruz) antibodies over-
night at 4 C. After washing, the membrane was incubated for 1 hour at room
temperature with goat antirabbit IgG horseradish peroxidase-linked anti-
body (1:2000 dilution; Zhongshan Goldenbridge Biotechnology, Beijing,
China). The membrane-bound antibody detected was incubated with
a chemiluminescent reagent plus (Thermo Fisher Scientic Inc, Rockford, IL)
and captured on Gel Documentation systems (Alpha Innotech, Santa Clara,
540 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546

Figure 2. In vitro differentiation and in vivo tracking of mesenchymal stem cells (MSC). (A) In vitro differentiation of MSC to osteoblasts and adipocytes. MSC in the
primary culture phase shows a broblast-like spindle shape appearance. Magnication
100. The osteogenic differentiation of MSC is indicated by the presence of red
calcium-rich hydroxyapatite stained with Alizarin red. Magnication
100. Adipogenic differentiation is indicated by intracellular mass lipid droplet sediment and by
Oil Red O staining. Magnication
100. Transduction enhanced green uorescent protein (EGFP) into MSC by lentiviral vector resulted in a high transduction
efciency, conrmed by nearly 90% of MSC exhibiting green uorescence 3 days after transduction. Magnication
200. (B) In vivo tracking of EGFP-labeled MSC. The
positive glomeruli, which contained at least 1 to 2 specic GFP uorescence areas, was observed in glomeruli 24 hours and 60 days after MSC injection (arrows).
Autouorescence detection showed signicant autouorescence in tubular areas. Merging both channels showed intrarenal migration of MSC. Magnication
400.
(C) Morphology of adipocytes in MSC-treated rats on day 60 after MSC injection. PAS staining shows the MSC-treated glomeruli contained fat cells (Ca). There was
a focal accumulation of Oil Red O staining in the glomeruli of MSC-treated rats (Cb). Electron microscopy showed the individual large vacuoles as well as several small
droplets within the glomerular podocyte cells (Cc)

CA). Band intensities were quantied by semiquantitative analysis software RESULTS

(Media Cybernetics) and normalized to b-actin.
ELISA Measurement of Cytokines
For the analysis of the growth factor production in MSC cultures, the
supernatant was collected upon conuence in the third passage. The
amounts of VEGF and BMP-7 in cell culture supernatants were quantied by
ELISA (R&D Systems, Minneapolis, MN). The renal cortical homogenate VEGF
and BMP-7 protein levels were also determined using ELISA. All experiments
were performed in triplicate.
Characterization of Rat MSC
Bone marrow-derived MSC from SD rats exhibited typical
broblast-like morphology. MSC identity was proved by
differentiation into osteogenic and adipogenic cells
(Figure 2A). Flow cytometric analysis showed that the MSC
were positive for CD29 (99.97%) and CD44 (99.97%) and
negative for CD34 (1.53%) and CD45 (.43%).

Statistical Analysis MSC Localization

All values are presented as the means  SD. Statistical signicance was
evaluated using 1-way ANOVA with modied t test performed with the
Bonferroni correction. The paired t test was used to compare directly the left
and right kidneys of an animal. A P value <.05 was considered statistically
signicant.
Transduction EGFP into MSC by lentiviral vector resulted
in a high-transduction efciency, conrmed by nearly 90% of
MSC exhibiting green uorescence 3 days after transduction
(Figure 2A). At 24 hours after MSC injection, EGFP-positive
 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546 541

Table 1 extracellular matrix deposition and frequent brin cap

Physical and Metabolic Parameters in Animals formation inside the glomeruli. In contrast, 60 days after MSC
Variable NC (n 6) DNmedium DNMSC injection, there was a signicant decrease in mesangial
(n 8) (n 9) matrix deposition (Figure 4B) and in the glomerulosclerotic
Blood glucose 5.87  .65 26.91  4.71* 24.43  4.03* index (Figure 4C) of the left kidneys of MSC-treated rats,
(mmol/L) compared to the left and right kidneys of medium-treated
y
Kidney weight (g) 1.20  .05 1.56  .06* 1.41  .04*,
rats and the right kidneys of MSC-treated rats.
Body weight (g) 387.0  10.02 203.38  5.90* 210.33  7.45*
Kidney/body 3.09  .09 7.67  .42* 6.68  .36*,
y
Electron microscopy studies were performed to deter-
weight (g/kg) mine the early structural changes in podocyte morphology
y
Creatinine clearance 1.52  .06 1.92  .07* 1.63  .05*, (Figure 5A). Sixty days after MSC injection, compared with
rate (mL/min) the left kidneys of medium-treated rats and the right kidneys
NC indicates normal control; DNmedium, medium-treated rats; DNMSC, of MSC-treated rats, the MSC-injected kidneys exhibited
MSC-treated rats. a signicant improvement in ultrastructural abnormalities,
Data are presented as the means  SD. Kidney weight, kidney/body weight,
such as loss of podocytes (Figure 5B), GBM thickening
and creatinine clearance rate levels were reduced signicantly by mesen-
chymal stem cells (MSC) treatment. Glucose and body weight did not (Figure 5C), and podocyte foot process effacement
change signicantly between the 2 DN rat groups. (Figure 5D).
* P < .05 versus NC group. Oil Red O staining was performed to determine the renal
y
P < .05 versus DNmedium group. accumulation of neutral lipids. As shown in Figure 2C, 60
days after MSC injection, approximately 10% of the MSC-
treated glomeruli contained fat cells (Figure 2Ca). There
cells were only observed in the left kidney receiving EGFP-
was a focal accumulation of Oil Red O staining in the
labeled MSC but not in the contralateral right kidneys (data
glomeruli of MSC-treated rats (Figure 2Cb) and almost no
not shown). EGFP-positive cells were observed in glomeruli
staining in the glomeruli of medium-treated rats or in the
but not in tubulointerstitial areas (data not shown). About
right glomeruli of MSC-treated rats (data not shown). Elec-
20% and 3% glomeruli showed positive uorescence at 24
tron microscopy showed the individual large vacuoles as well
hours and 60 days after MSC infusion, respectively
as several small droplets within the glomerular podocyte
(Figure 2B).
cells (Figure 2Cc).
Effect of MSC on Physical and Biochemical Parameters
Effect of MSC on the Renal Expression of Nephrin and
The blood glucose, body weight, kidney weight, kidney to
Podocin
body weight index, and creatinine clearance rate levels of the
To further reveal the mechanism responsible for the
experimental animals are shown in Table 1. Compared with
prevention of albuminuria in MSC-treated diabetic rats, we
medium-treated DN rats, the MSC treatment suppressed the
studied the expression of nephrin and podocin proteins,
increase in kidney weight, kidney to body weight index, and
which are the 2 major proteins involved in the formation of
creatinine clearance rate levels but had no effect on blood
a slit diaphragm. Comparing the left kidneys of medium-
glucose and body weight levels on day 60 after MSC infusion.
treated rats with the right kidneys of MSC-treated rats,
As shown in Figure 3, the ratio of urinary albumin to
MSC treatment led to a signicant improvement in the loss of
creatinine 30 and 60 days after MSC injection was signi-
glomerular nephrin and podocin staining (Figure 6A).
cantly lower in MSC-treated DN rats than in medium-treated
In line with the results from immunouorescence stain-
rats, suggesting that MSC have an antiproteinuric effect in
ing, western blot analysis showed that MSC injection
DN rats.
signicantly attenuated the diabetic-induced decrease of
glomerular nephrin and podocin expression, compared with
Renal Histopathological and Ultrastructural Changes
the left kidneys of medium-treated rats and the right kidneys
Light microscopy studies (PAS-staining) were performed
of MSC-treated rats (Figure 6B).
to determine the extent of the glomerular damage
(Figure 4A). As shown in Figure 4A, 90 days after STZ injec-
MSC Express High Levels of VEGF and BMP-7
tion, the left and right kidneys of medium-treated rats and
Next, we aimed to evaluate whether MSC could exert
the right kidneys of MSC-treated rats exhibited profound
their protective effects against podocyte damage in part by
increasing VEGF and BMP-7 secretion, which have been
shown to be podocyte survival factors to rescue podocytes
from injury [17,18]. Both factors could be secreted by MSC, as
previously reported [19]. After 24 hours of incubation, MSC
released higher amounts of VEGF (1231  123 pg/mL versus
10  2 pg/mL) and BMP-7 (1868  67 pg/mL versus 49 
12 pg/mL) in vitro in cell culture supernatants than in fresh
medium (Figure 7A).
As shown in Figure 7B, the renal cortical VEGF levels in
the left kidneys from MSC-treated rats (1928  63 pg/mg
protein) did not signicantly increase on day 60 post-MSC
injection compared with the left kidneys of medium-
treated rats (1804  97 pg/mg protein) and the right
kidneys of MSC-treated rats (1838  87 pg/mg protein). In
Figure 3. Urinary albumin excretion. At 30 days and 60 days after mesen-
chymal stem cells injection, the ratio of urinary albumin to creatinine was
contrast, the BMP-7 levels were higher in the left kidneys of
signicantly lower in MSC-treated DN rats than in medium-treated rats. The MSC-treated rats (1431  39 pg/mg protein) than those in the
results are presented as the mean  SD. *P < .05 versus medium-treated rats. left kidneys of medium-treated rats (837  61 pg/mg protein)
542 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546

Figure 4. Mesangial expansion and glomerulosclerosis. (A) Ninety days after streptozotocin injection, the left and right kidneys of medium-treated rats and the right
kidneys of mesenchymal stem cells (MSC)-treated rats exhibited profound extracellular matrix deposition and frequent brin cap formation (arrows) inside the
glomeruli. Sixty days after the MSC injection, there was a signicant decrease in mesangial matrix deposition (B) and glomerulosclerotic index (C) in the left kidneys
of MSC-treated rats, compared to the left kidneys of medium-treated rats and the right kidneys of MSC-treated rats. Data are presented as the mean  SD. *P < .05
versus the kidneys of NC rats; #P < .05 versus the left kidneys of medium-treated rats; :P < .05 versus the right kidneys of MSC-treated rats.

and the right kidneys of MSC-treated rats (889  100 pg/mg
protein), suggesting that the protective effects of MSC on
podocyte injury in DN may be mediated in part by an
increased BMP-7 secretion.
DISCUSSION
MSC are multipotent cells present in bone marrow and in
mesenchymal tissues that can differentiate in vitro into
adipocytic, chondrocytic, and osteocytic lineages. These cells
do not exhibit hemopoietic markers but rather express CD29,
CD44, CD105, and alpha smooth muscle actin [10]. In vitro,
MSC may give rise to insulin-producing cells [20,21]. In vivo,
MSC may differentiate into renal cells [22,23] and reconsti-
tute necrotic segments of damaged kidneys. Thus, bone
marrow-derived MSC have been used successfully in cell
therapy to treat animal models with acute and chronic renal
failure, such as ischemia-reperfusion injury, 5/6 nephrec-
tomy, and unilateral ureteral obstruction, as well as in kidney
transplantation models [24]. The present study provides
clear evidence that although there was no improvement in
blood sugar levels, the injected MSC did prevent the devel-
opment of albuminuria and the loss of podocytes.
The primary advantage of MSC for utilization in cell
therapy is the ease with which they can be harvested from
the bone marrow, isolated by plastic adherence, and
expanded in culture [25]. However, in vitro manipulations
may also alter or inuence their natural phenotypes, leading
to different, as-yet-undened activities and responses. In this
study, we established that adherent, bone marrow-derived,
spindle-shaped cells expressed mesenchymal cell pheno-
types (CD29, CD44) rather than CD34 (hematopoietic cell
marker) and CD45 (leukocyte marker). In addition, the MSC
possessed osteogenic and adipogenic potential; we
conrmed with this approach that the subsequently
administered cells retained the characteristics of MSC.
We found that adequate homing of MSC to the injured
tissue is important for effective therapy. In most studies, MSC
is administered through a standard intravenous route. A
disadvantage of the systemic intravenous delivery of MSC
can be low uptake at the site of injury. Indeed, signicant
engraftment of injured tissue was observed in some studies
[26-28], but not all [29,30]. Schrepfer et al. [31] demon-
strated that the systemic intravenous route of administration
was not appropriate for MSC to reach their site of activation.
Zonta [32] showed that the intra-arterial administration of
MSC were the most effective route to achieve immunomo-
dulating effects in experimental kidney transplantation,
which primarily occurs because large MSC (15 mm to 19 mm)
remain trapped in the capillaries of the small lung lter,
which in turn causes the inadequate homing of MSC to the
 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546 543

Figure 5. Ultrastructural alterations of the podocyte and glomerular basal membrane (A). Sixty days after mesenchymal stem cells (MSC) injection, compared with
the left kidneys of medium-treated rats and the right kidneys of MSC-treated rats, a signicant improvement in ultrastructural abnormalities, such as loss of
podocytes (B), GBM thickening (C), and podocyte foot process effacement (D) was found in MSC-injected kidneys. The degree of podocyte detachment was calculated
as the mean FPW. The podocyte number per 2.5 mm of GBM and GBM thickness are presented as median values using box-and-whisker plots. The results are
presented as the means  SD. M indicates glomerular basal membrane; P, podocyte; FP, foot process; NC, normal control rats. *P < .05 versus the kidneys of NC rats; #P
< .05 versus the left kidneys of medium-treated rats; :P < .05 versus the right kidneys of MSC-treated rats.

injured tissue. However, using the renal artery as the injec-
tion route to administer MSC to treat DN may be associated
with the following 2 major complications: (1) renal infarcts
and loss of function, and (2) ectopic differentiation into
adipocytes within glomeruli [33]. Recently, Ho et al. [34]
demonstrated that multiple intravenous transplantations of
MSC effectively restored the long-term blood glucose
homeostasis during 15 weeks in STZ-induced diabetic mice;
thus, multiple intravenous transplantations of MSC may
serve as a new therapeutic strategy for DM patients. The
following 2 factors may contribute to MSC for the treatment
of DN: (1) blood sugar reduction and (2) renal protective
actions of the MSC within the glomeruli. To examine the
second factor, we could only choose the renal artery as the
injection route instead of systemic administration. In our
study, intra-arterial injection led to 20% of glomeruli con-
taining EGFP cells at 24 hours, as observed in the targeted
kidney but not in the lung, liver, or spleen (data not shown).
However, 60 days after MSC injection, only 3% of glomeruli
containing EGFP cells were found in MSC-treated kidneys.
This low level of engraftment suggests a paracrine, rather
than a cell differentiation, contribution to the benecial
effects of the MSC. However, the positive status of the 3% of
the glomeruli in the kidney on 60 days after injection is still
higher than the 0% in the lung, liver, and spleen and suggests
that there was some targeting of the cell therapy to the
kidney, likely due to the renal artery injection. In addition, on
day 60 after the injection into a renal artery, approximately
10% of the MSC-treated glomeruli contained cells that
exhibited features typical of adipocytes. Electron microscopy
showed that large or multiple small fatty vacuoles were
found within the glomerular podocyte cells. This phenom-
enon had been previously reported by Kunter et al. [33].
Although it seems highly likely that these cells originated
from the transplanted MSC, the exact mechanisms of MSC
transformation are not completely understood. There is
evidence that BMP-7 promotes the differentiation of MSC
into adipocyte cells [35]. In our study, we found that the
expression of BMP-7 was signicantly increased in MSC-
treated kidneys compared to medium-treated kidneys,
which might help explain these results. These phenomena
seem to suggest that the long-term effects of intrarenal,
syngeneic MSC transplantation may result in the maldiffer-
entiation of glomerular MSC into adipocytes, thus calling into
question the potential benet of MSC treatment for DN.
Taken together, our observation is consistent with the
homing capacity of MSC to injured areas via intra-arterial
injection in several animal models [11,36].
Type 1 Diabetes (T1DM), a multifactorial autoimmune
disease involving genetic and environmental factors, is
hallmarked by the T cell- and macrophages-mediated
destruction of pancreatic b-cells, resulting in an irreversible
insulin deciency. STZ or other diabetogenic agents (eg,
alloxan) with b-cell-toxic abilities have also been used for
544 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546

Figure 6. Glomerular expression of nephrin and podocin. (A) The staining scores of nephrin and podocin were signicantly reduced in the left and right kidneys of
medium-treated rats and in the right kidneys of mesenchymal stem cells (MSC)-treated rats. The sources of nephrin and podocin in MSC-injected kidneys were
restored. Magnication
400. (B) Western blot analysis showed that nephrin and podocin expressions were restored in MSC-injected kidneys compared to the left
kidneys of medium-treated rats and the right kidneys of MSC-treated rats. Both proteins expression was quantied by semi-quantitative analysis of the western blots.
The results are presented as the means  SD. *P < .05 versus the kidneys of normal control (NC) rats; #P < .05 versus the left kidneys of medium-treated rats; :P < .05
versus the right kidneys of MSC-treated rats.

producing chemically induced T1DM models through their
administration in a large dose or repeated low doses for
several days. However, although the STZ model results in
hyperglycemia and insulinopenia, it does not bear strong
autoimmune features [37-39], suggesting that the STZ-
induced diabetic model is not the equivalent of T1DM.
Although an ideal experimental model of T1DM does not
exist, we believe that the use of this STZ model may allow us
to obtain valuable information about understanding the
molecular bases that contribute to the induction of T1DM in
humans.
Recent studies have shown that the systemic adminis-
tration of MSC into STZ-induced type 1 diabetic C57BL/6
mice [7] and type 2 diabetic NOD/scid mice [8] reduced
microalbuminuria and preserved normal renal histology. In
contrast, untreated diabetic mice presented glomerular

Figure 7. The expression of VEGF and BMP-7 in cultured mesenchymal stem cells (MSC) and kidneys of diabetic nephropathy rats. (A) After 24 hours of incubation,
MSC released high amounts of VEGF and BMP-7 in vitro in cell culture supernatants (black bars) as opposed to in fresh medium (white bars). xP < .05 versus fresh
medium. (B) Sixty days after MSC injection, there were no signicant differences in the renal cortical VEGF levels between the 2 groups. In contrast, BMP-7 levels
were signicantly higher in the MSC-treated kidneys than in the left kidneys of medium-treated rats or the right kidneys of the MSC-treated rats. The results were
presented as the means  SD. *P < .05 versus the kidneys of normal control rats; #P < .05 versus the left kidneys of medium-treated rats; :P < .05 versus the right
kidneys of MSC-treated rats.
 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546
hyalinosis and mesangial expansion. The observed renopro-
tection was associated with a decrease in glycemic levels that
could be explained by beta-pancreatic islet regeneration
resulting from MSC administration. However, in these
studies, neither group demonstrated a signicant histologic
amelioration of podocyte damage in response to MSC injec-
tion; furthermore, their data could not completely distin-
guish these benecial effects from a reduction in the serum
glucose levels or direct protection of the podocytes. In this
study, we successfully established an experimental DN,
similar to human type 1 DN and characterized by hypergly-
cemia and albuminuria, that was also associated with the
loss of podocytes, effacement of foot process, widening of
foot process, and thickening of the GBM [1,40-42]. These
changes contribute to progressive glomerulosclerosis, which
is a feature of experimental DN. More important, our results
revealed that MSC effectively ameliorated the phenotypic
changes of podocytes in this animal model. Another inter-
esting nding of the present study was that the renopro-
tective effect of MSC in podocyte injury was only found in the
left kidneys, not in the right kidneys, of MSC-treated DN rats.
In addition, the renoprotection of MSC were not attributable
to glycemic control because MSC via intra-arterial injection
had no impact on the elevated serum glucose levels in the
diabetic rats. As shown in Table 1, MSC administration by
intra-arterial injection reversed neither hyperglycemia nor
body weight loss, the latter of which was associated with
poor blood glucose control. Thus, our study clearly suggested
that MSC themselves may have had a direct benecial effect
on the ultrastructural alterations in podocytes.
Podocytes play a major role in maintaining the integrity
and permeability of the glomerular ltration barrier [43,44].
In DN, the effacement of podocyte foot processes and the loss
of slit diaphragm proteins result in a leakage of albumin and
proteinuria [45,46]. The results of the present study showed
that the levels of urinary protein were signicantly increased
in diabetic rats and could be signicantly lowered by MSC. To
identify the molecular basis of the observed phenomena, we
performed immunostaining for nephrin and podocin, which
are constituents of the slit diaphragm and act as a barrier for
glomerular capillary walls [47]. Our study revealed that MSC
administration could restore the expression of nephrin and
podocin in diabetic rats. Hence, our data suggested that the
antiproteinuric effects of MSC in diabetes-induced protein-
uria may be associated with the restoration of the expres-
sional patterns of podocyte-associated proteins and the
maintenance of podocyte foot process in the glomerular
ltration barrier.
What are the mechanisms underlying the benet of MSC
in our model? Several recent studies suggest that MSC may
mediate their benecial effects, in part, through paracrine
and/or endocrine activity, not through a differentiation
mechanism as had been described in some acute renal
damage models [48]. To elucidate the potential mechanism
of MSC, we focused on evaluating the expression of VEGF and
BMP-7 in vitro and in vivo, which are podocyte survival
factors secreted by MSC. In this respect, it was noteworthy
that our MSC released high amounts of VEGF and BMP-7
in vitro. Our ndings were in line with the data of others
showing that MSC release VEGF and BMP-7, exerting bene-
cial effects in chronic kidney disease and acute kidney
injury models [19,49,40]. We found that the expression of
BMP-7 was signicantly increased in MSC-treated kidneys
compared to medium-treated kidneys. There were small
increases in VEGF in MSC-treated kidneys. However, these
545
did not reach statistical signicance. These data suggested
that MSC exert their protective effects against podocyte
damage possibly by increasing BMP-7 secretion. Both in vitro
and in vivo studies have indicated that BMP-7 is important
for the maintenance of podocyte viability and differentiation
[18,51,52]. In adult rodent glomeruli in vivo, BMP-7 and its
receptors are expressed in podocytes [18]. In cultured mouse
podocytes, high glucose decreases BMP-7 expression, and
the treatment of podocytes with rhBMP-7 restores podocin
and synaptopodin expression [51]. In STZ-induced diabetic
mice, the maintenance of renal and glomerular podocyte
BMP-7 levels by means of a transgene reduces the onset and
progression of nephropathy, especially podocyte dropout
[52]. Moreover, the ability of MSC to contribute to podocyte
regeneration has been recently shown in a mouse model of
Alport syndrome [53]. In addition to BMP-7 and VEGF, MSC
can secrete a number of factors, such as HGF, bFGF, and IGF-I,
all known to improve renal function in acute kidney injury,
mediated by their antiapoptotic, mitogenic, and other cyto-
kine actions [49,50]. Based on these observations, we deduce
that the mechanisms that mediate the protective effects of
MSC may be primarily paracrine actions.
In summary, our current study provided further evidence
that MSC could not only exert antialbuminuric effects but
also, more important, prevent early phenotypic changes in
podocytes and, subsequently, glomerulosclerosis. We believe
that the successful treatment of DN with MSC demonstrated
herein holds substantial promise for the development of
novel, MSC-based interventions that can prevent the devel-
opment of DN. However, because our study is an animal
study, these ndings must be conrmed after further study,
such as clinical trial, on human subjects.
ACKNOWLEDGMENTS
We thank the laboratory animal center of Xinqiao hospital
for the excellent rat care. We also thank Dr. Xiaohua Guo
(University of Utah) for his invaluable advice.
Financial disclosure: This study was supported by
the National Natural Science Foundation of China (No.
30570871) and the Natural Science Foundation of Chongqing
(No. 2007BB1017).
Conict of Interest Statement: There are no conicts of
interest to report.
REFERENCES
1. Molitch ME, DeFronzo RA, Franz MJ, et al. Nephropathy in diabetes.
Diabetes Care. 2004;27(Suppl 1):S79-S83.
2. Steffes MW, Schmidt D, McCrery R, Basgen JM. Glomerular cell number
in normal subjects and in type 1 diabetic patients. Kidney Int. 2001;59:
2104-2113.
3. Ziyadeh FN, Wolf G. Pathogenesis of the podocytopathy and protein-
uria in diabetic glomerulopathy. Curr Diabetes Rev. 2008;4:39-45.
4. Dalla Vestra M, Masiero A, Roiter AM, et al. Is podocyte injury relevant
in diabetic nephropathy? Studies in patients with type 2 diabetes.
Diabetes. 2003;52:1031-1035.
5. White KE, Bilous RW. Structural alterations to the podocyte are related
to proteinuria in type 2 diabetic patients. Nephrol Dial Transplant. 2004;
19:1437-1440.
6. Togel F, Westenfelder C. Adult bone marrow-derived stem cells for
organ regeneration and repair. Dev Dyn. 2007;236:3321-3331.
7. Ezquer FE, Ezquer ME, Parrau DB, et al. Systemic administration of
multipotent mesenchymal stromal cells reverts hyperglycemia and
prevents nephropathy in type 1 diabetic mice. Biol Blood Marrow
Transplant. 2008;14:631-640.
8. Lee RH, Seo MJ, Reger RL, et al. Multipotent stromal cells from human
marrow home to and promote repair of pancreatic islets and renal
glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci U S A. 2006;103:
17438-17443.
546 S. Wang et al. / Biol Blood Marrow Transplant 19 (2013) 538e546
9. Jiang TS, Cai L, Ji WY, et al. Reconstruction of the corneal epithelium
with induced marrow mesenchymal stem cells in rats. Mol Vis. 2010;
16:1304-1316.
10. Ma P, Hindocha S, Ma R, et al. Adult mesenchymal stem cells and cell
surface characterization - a systematic review of the literature. Open
Orthop J. 2011;5:253-260.
11. Kunter U, Rong S, Djuric Z, et al. Transplanted mesenchymal stem cells
accelerate glomerular healing in experimental glomerulonephritis.
J Am Soc Nephrol. 2006;17:2202-2212.
12. Lee EA, Seo JY, Jiang Z, et al. Reactive oxygen species mediate high
glucose-induced plasminogen activator inhibitor-1 up-regulation in
mesangial cells and in diabetic kidney. Kidney Int. 2005;67:1762-1771.
13. Thallas-Bonke V, Thorpe SR, Coughlan MT, et al. Inhibition of NADPH
oxidase prevents advanced glycation end product-mediated damage in
diabetic nephropathy through a protein kinase C-alpha-dependent
pathway. Diabetes. 2008;57:460-469.
14. Macedo CS, Lerco MM, Capelletti SM, et al. Reduction of podocytes
number in late diabetic alloxan nephropathy: prevention by glycemic
control. Acta Cir Bras. 2007;22:337-341.
15. van den Berg JG, van den Bergh Weerman MA, Assmann KJ, et al.
Podocyte foot process effacement is not correlated with the level of
proteinuria in human glomerulopathies. Kidney Int. 2004;66:
1901-1906.
16. Gundersen HJ, Seefeldt T, Osterby R. Glomerular epithelial foot
processes in normal man and rats. Distribution of true width and its
intra- and inter-individual variation. Cell Tissue Res. 1980;205:147-155.
17. Ma J, Matsusaka T, Yang HC, et al. Induction of podocyte-derived VEGF
ameliorates podocyte injury and subsequent abnormal glomerular
development caused by puromycin aminonucleoside. Pediatr Res.
2011;70:83-89.
18. Mitu GM, Wang S, Hirschberg R. BMP7 is a podocyte survival factor and
rescues podocytes from diabetic injury. Am J Physiol Renal Physiol.
2007;293:F1641-F1648.
19. Ninichuk V, Gross O, Segerer S, et al. Multipotent mesenchymal stem
cells reduce interstitial brosis but do not delay progression of chronic
kidney disease in collagen4A3-decient mice. Kidney Int. 2006;70:
121-129.
20. Neshati Z, Matin MM, Bahrami AR, Moghimi A. Differentiation of
mesenchymal stem cells to insulin-producing cells and their impact on
type 1 diabetic rats. J Physiol Biochem. 2010;66:181-187.
21. Sun Y, Chen L, Hou XG, et al. Differentiation of bone marrow-derived
mesenchymal stem cells from diabetic patients into insulin-
producing cells in vitro. Chin Med J (Engl). 2007;120:771-776.
22. Huls M, Russel FG, Masereeuw R. Insights into the role of bone
marrow-derived stem cells in renal repair. Kidney Blood Press Res.
2008;31:104-110.
23. Morigi M, Imberti B, Zoja C, et al. Mesenchymal stem cells are reno-
tropic, helping to repair the kidney and improve function in acute renal
failure. J Am Soc Nephrol. 2004;15:1794-1804.
24. Asanuma H, Meldrum DR, Meldrum KK. Therapeutic applications of
mesenchymal stem cells to repair kidney injury. J Urol. 2010;184:
26-33.
25. Meirelles Lda S, Fontes AM, Covas DT, Caplan AI. Mechanisms involved
in the therapeutic properties of mesenchymal stem cells. Cytokine
Growth Factor Rev. 2009;20:419-427.
26. Ezquer F, Ezquer M, Simon V, et al. Endovenous administration of
bone-marrow-derived multipotent mesenchymal stromal cells
prevents renal failure in diabetic mice. Biol Blood Marrow Transplant.
2009;15:1354-1365.
27. Morigi M, Introna M, Imberti B, et al. Human bone marrow mesen-
chymal stem cells accelerate recovery of acute renal injury and prolong
survival in mice. Stem Cells. 2008;26:2075-2082.
28. Semedo P, Correa-Costa M, Antonio Cenedeze M, et al. Mesenchymal
stem cells attenuate renal brosis through immune modulation and
remodeling properties in a rat remnant kidney model. Stem Cells. 2009;
27:3063-3073.
29. Walczak P, Zhang J, Gilad AA, et al. Dual-modality monitoring of tar-
geted intraarterial delivery of mesenchymal stem cells after transient
ischemia. Stroke. 2008;39:1569-1574.
30. Mouiseddine M, Francois S, Souidi M, Chapel A. Intravenous human
mesenchymal stem cells transplantation in NOD/SCID mice preserve
liver integrity of irradiation damage. Methods Mol Biol. 2012;826:
179-188.
31. Schrepfer S, Deuse T, Reichenspurner H, et al. Stem cell trans-
plantation: the lung barrier. Transplant Proc. 2007;39:573-576.
32. Zonta S, De Martino M, Bedino G, et al. Which is the most suitable and
effective route of administration for mesenchymal stem cell-based
immunomodulation therapy in experimental kidney transplantation:
endovenous or arterial? Transplant Proc. 2010;42:1336-1340.
33. Kunter U, Rong S, Boor P, et al. Mesenchymal stem cells prevent
progressive experimental renal failure but maldifferentiate into
glomerular adipocytes. J Am Soc Nephrol. 2007;18:1754-1764.
34. Ho JH, Tseng TC, Ma WH, et al. Multiple intravenous transplantations
of mesenchymal stem cells effectively restore long-term blood
glucose homeostasis by hepatic engraftment and beta cell differen-
tiation in streptozosin-induced diabetic mice. Cell Transplant. 2012;
21:997-1009.
35. Neumann K, Endres M, Ringe J, et al. BMP7 promotes adipogenic but
not osteo-/chondrogenic differentiation of adult human bone marrow-
derived stem cells in high-density micro-mass culture. J Cell Biochem.
2007;102:626-637.
36. Yoo JH, Park C, Jung DI, et al. In vivo cell tracking of canine allogenic
mesenchymal stem cells administration via renal arterial catheteriza-
tion and physiopathological effects on the kidney in two healthy dogs.
J Vet Med Sci. 2011;73:269-274.
37. Rees DA, Alcolado JC. Animal models of diabetes mellitus. Diabet Med.
2005;22:359-370.
38. Lenzen S. The mechanisms of alloxan- and streptozotocin-induced
diabetes. Diabetologia. 2008;51:216-226.
39. Szkudelski T. The mechanism of alloxan and streptozotocin action in B
cells of the rat pancreas. Physiol Res. 2001;50:537-546.
40. Menini S, Iacobini C, Oddi G, et al. Increased glomerular cell (podocyte)
apoptosis in rats with streptozotocin-induced diabetes mellitus: role in
the development of diabetic glomerular disease. Diabetologia. 2007;50:
2591-2599.
41. Barutta F, Corbelli A, Mastrocola R, et al. Cannabinoid receptor 1
blockade ameliorates albuminuria in experimental diabetic nephrop-
athy. Diabetes. 2010;59:1046-1054.
42. Sohn E, Kim J, Kim CS, et al. Extract of the aerial parts of Aster kor-
aiensis reduced development of diabetic nephropathy via anti-
apoptosis of podocytes in streptozotocin-induced diabetic rats. Bio-
chem Biophys Res Commun. 2010;391:733-738.
43. Saleem MA. Biology of the human podocyte. Nephron Exp Nephrol.
2003;95:e87-e92.
44. Kawachi H, Miyauchi N, Suzuki K, et al. Role of podocyte slit diaphragm
as a ltration barrier. Nephrology (Carlton). 2006;11:274-281.
45. Ihalmo P, Wessman M, Kaunisto MA, et al. Association analysis of
podocyte slit diaphragm genes as candidates for diabetic nephropathy.
Diabetologia. 2008;51:86-90.
46. White KE. Research into the glomerular podocyteeis it relevant to
diabetic nephropathy? Diabet Med. 2006;23:715-719.
47. Kerjaschki D. Caught at-footed: podocyte damage and the molecular
bases of focal glomerulosclerosis. J Clin Invest. 2001;108:1583-1587.
48. Masereeuw R. Contribution of bone marrow-derived cells in renal
repair after acute kidney injury. Minerva Urol Nefrol. 2009;61:373-384.
49. Togel F, Hu Z, Weiss K, et al. Administered mesenchymal stem cells
protect against ischemic acute renal failure through differentiation-
independent mechanisms. Am J Physiol Renal Physiol. 2005;289:
F31-F42.
50. Togel F, Weiss K, Yang Y, et al. Vasculotropic, paracrine actions of
infused mesenchymal stem cells are important to the recovery from
acute kidney injury. Am J Physiol Renal Physiol. 2007;292:F1626-F1635.
51. De Petris L, Hruska KA, Chiechio S, Liapis H. Bone morphogenetic
protein-7 delays podocyte injury due to high glucose. Nephrol Dial
Transplant. 2007;22:3442-3450.
52. Wang S, de Caestecker M, Kopp J, et al. Renal bone morphogenetic
protein-7 protects against diabetic nephropathy. J Am Soc Nephrol.
2006;17:2504-2512.
53. Prodromidi EI, Poulsom R, Jeffery R, et al. Bone marrow-derived
cells contribute to podocyte regeneration and amelioration of renal
disease in a mouse model of Alport syndrome. Stem Cells. 2006;24:
2448-2455.