1. Introduction
Chronic kidney disease (CKD) is a global health issue that leads to lower
quality of life and higher social economic burden [1], especially for patients
who need renal replacement therapy. The advent of dialysis and kidney
transplantation improved the survival rate and quality of life for patients with
End-stage renal disease (ESRD), however, both of the two treatments were costly
and with multiple complications [2, 3]. Thus, effective and affordable treatments
need to be developed to delay or reverse the progression of CKD. Renal fibrosis
is a pathological process and a common final pathway which makes progression to
ESRD [4]. Renal fibrosis is characterized by the imbalanced
deposition/degradation of extracellular matrix (ECM) [5]. Transforming growth
factor-1 (TGF-1) plays critical roles in renal fibrosis via canonical
(Smad-based) and non-canonical signaling pathways [6]. The Smad subfamily has
been classified as receptor Smad (R-Smad), common mediator Smad (Co-Smad) and
inhibitory Smad (I-Smad), among which Smad 7 acts as a key antagonist of TGF-
signaling pathway thereby mediating the R-Smad dephosphorylation or degradation
[7]. The anti-fibrotic effect of Smad 7 has been demonstrated in different kidney
disease models of animals such as diabetic nephropathy, obstructive nephropathy,
and aristolochic acid nephropathy [8]. The E3 ubiquitin ligase Smad
ubiquitination regulatory factor 2 (Smurf 2) can specifically target components
of TGF- signaling family which lead to the ubiquitination and degradation of
smad 7 to deteriorate kidney fibrosis. Previous studies indicated that the
upregulation of Smurf 2 accompanied with downregulation of R-Smads subfamily,
which denoted the activation of TGF- signaling. For example, Smurf 2-transgenic
mice sternal chondrocytes showed an increased Smad 3 degradation [9]. Similarly,
the increased expression of Smurf 2 correlates with decreased Smad 2 expression
in rat glomeruli [10]. However, Smurf 2 also exhibited an effect in facilitating
TGF- response by degrading Smad 7 through a post-transcriptional mechanism. The
role of Smurf 2 under different pathophysiology context remains complicated and
need to be further clarified.
Mesenchymal stem cells (MSCs) are a type of multipotent stem cells derived from
mesoderm and ectoderm which are considered to be a potential therapy in organ
injury, repair and immune response. Liu et al. [11] reported that BMSCs
function as inhibitory role of renal inflammation and fibrosis after injury.
Moreover, Ozbek et al. [12] found that MSCs delivery could significantly
alleviate renal fibrosis via inhibiting epithelial-mesenchymal transition in
unilateral ureteral obstruction (UUO) rats. Although the exact way of BMSCs on
restoring kidney injury remains ambiguous, substantial results demonstrated that
endocrine and paracrine mechanisms are of vital importance in this progression
[13, 14].
Exosomes are 30–100 nm nanoscale lipid bilayer vesicles that can be secreted
from different cells (such as bone marrow mesenchymal stem cells, renal tubular
cells, macrophages, cancer cells) to extracellular space and exert
anti-inflammatory and anti-fibrotic effects [15]. Several studies demonstrated
that BMSC-derived exosome could reduce inflammation and extracellular matrix
deposition in hepatic fibrosis rats via inhibiting TGF-/Smad 2 signaling
pathway [16]. Furthermore, the infiltration of dendritic cells could be
significantly suppressed by MSC-derived exosome in kidney as well as the
expression of inflammatory cytokines in streptozotocin (STZ)-induced diabetic
mice [17]. Up to now, the underlying mechanism of the anti-fibrotic effect of
exosomes still needs further elucidation.
Our current study aims to explore whether BMSC-Exo exerts the anti-fibrosis
effects on a 5/6 nephrectomy model and TGF-1-induced tubular epithelial cells.
In addition, the underlying mechanism of BMSC-Exo mediated anti-fibrosis effects
were investigated and we confirmed that Smurf 2/Smad 7 axis played a vital role
in this progression. Our findings not only lead to a better understanding of the
protective role of BMSC-Exo in fibrosis but also provides a potential therapeutic
strategy for CKD.
2. Materials and methods
2.1 Isolation of BMSC-Exo
Human bone marrow mesenchymal stem cells (BMSCs) were purchased from ScienCell
Research Laboratories (Carlsbad, CA, USA) and cultured in MSC medium supplemented
with 10% exosome-depleted FBS (Thermo Fisher, cat. A25904DG, Waltham, MA, USA).
Primary rat bone marrow mesenchymal stem cells were obtained from rat femur and
cultured in DMEM medium supplemented with 10% exosome-depleted FBS. The
differentiation potential of bone marrow mesenchymal stem cells were identified
using alizarin red S staining and oil red O staining. After 72 h, the conditioned
medium was collected and centrifuged (1000 g for 10 min, 9000
g for 30 min) at 4 C to remove cell debris, followed by
centrifugation at 3000 g for 90 min (4 C) after moving into
the ultrafiltration centrifuge tube. Then Exosome Isolation Reagent (Invitrogen,
Carlsbad, CA, USA) was used in accordance with the manufacturer’s protocol to
isolate exosomes. The exosome-enriched fraction was diluted with PBS and
quantified using a Pierce BCA protein assay kit before further use. BMSCs used in
our study is from 4–6 passages.
2.2 Nanoparticle tracking analysis (NTA) and transmission electron
microscopy
Exosome-enriched suspensions were examined by the ZetaView Particle Metrix (PMX
110, Particle Metrix, Meerbusch, Germany), and particle movement was analyzed
using NTA software ZetaView 8.02.28 (Particle Metrix, Meerbusch, Germany). The
exosome-enriched solution was placed on a copper mesh and stained with a uranyl
acetate solution finally photographed by a Transmission electron microscope (TEM,
Hitachi, Tokyo, Japan).
2.3 Cell culture and treatment of HRPTEpics
Human renal proximal tubular epithelial cells (HRPTEpiC) were purchased from
ScienCell Research Laboratories and cultured in HRPTEpics medium supplemented
with 5% FBS. TGF-1 (10 ng/mL; 7754-BH-025/CF, R&D Systems, USA) was used to
induce fibrosis. The siRNA targeting Smurf 2 (si-Smurf 2) was purchased from
Genechem (Shanghai, China) and transfected into HRPTEpics using the transfection
reagent according to the manufacturer’s protocol. Human BMSC-Exo was used to
stimulate HRPTEpiC.
2.4 Animal models
Briefly, 12-week-old male Sprague-Dawley rats (200 20 g) were purchased
from the Institute of laboratory animal science (Beijing, China), feeding with
unlimited water and food in the temperature-controlled room. Rat BMSC-Exo was
used in in vivo experiments. A total of 15 rats were divided into 3
groups and given different treatments until 16 weeks after the surgery: (1) sham
operation without removal of kidney tissue (group SHAM, n = 5); (2) 5/6
nephrectomy (group SNx, n = 5); (3) 5/6 nephrectomy plus BMSC-Exo (group SNx +
BMSC-Exo, n = 5, tail vein injection, 150 g/week). Each step of 5/6
nephrotomy was based on the established modified process after anesthesia by
pentobarbital [18]. The rat’s body weight was measured at baseline and after 16
weeks of nephrotomy surgery, then the rats were euthanized and samples (serum and
kidney) for the subsequent experiment were collected.
2.5 Rats serum
Rats’ serum samples were collected for analyzing Serum creatinine (Scr) and
blood urea nitrogen (BUN) levels via an automatic biochemical analyzer (AU5421,
Olympus, Tokyo, Japan).
2.6 HE and Masson trichrome staining
Kidney tissues were fixed in 10% formalin, then processed for paraffin
embedding and at 4 m thicknesses. After the procedure of de-paraffinizing,
under the guidance of Hematoxylin and Eosin staining (HE) kit and Masson’s
Trichrome staining kit (Solarbio, Beijing, China), the kidney specimens were
stained. The histopathological and fibrosis observation were picked by optical
microscope (Olympus, Tokyo, Japan).
2.7 Immunohistochemistry
The sectioned and embedded specimens of kidney tissue were gradually going
through dewax, dehydrate, antigen repair and block peroxidase activity. Then the
specimens were blocked with 2% bovine serum albumin. Subsequently, primary
antibodies against fibronectin (1:100, ab2413, Abcam, Cambridge, UK), Collagen-I
(1:200, ab34710, Abcam, Cambridge, UK) and -SMA (1:200, ab5694, Abcam,
Cambridge, UK) were added to incubate overnight. Secondary antibody specific for
primary antibody was added on the second day for 1 h. At last, the specific
proteins were colored by 3,3-diaminobenzidine tetrahydrochloride (DAB) kit
(P0203, Beyotime, Shanghai, China) and the nucleus was colored by hematoxylin.
The quantification of immunohistochemistry was performed by Image J software
(National Institutes of Health, NIH, Bethesda, MD, USA).
2.8 Western blot
Total protein was extracted from cells or tissues using the Radio
Immunoprecipitation Assay (RIPA) lysis buffer and kept at 4 C for 30
min with vortex shock every 10 min. The primary antibodies against Collagen-I
(1:1000, ab34710, Abcam, Cambridge, UK), -SMA (1:1000, ab5694, Abcam,
Cambridge, UK), E-cadherin (1:1000, ab40772, Abcam, Cambridge, UK), Smad 7
(1:500, ab216428, Abcam, Cambridge, UK), Smurf 2 (1:1000, 12024S, Cell Signaling
Technology, Boston, USA), GAPDH (1:4000, ab181602, Abcam, Cambridge, UK) were
incubated overnight for the first day and specific horseradish peroxidase
(HRP)-conjugated secondary antibody were incubated 1 hour for the second day.
GAPDH was used to normalize the densitometry values of the targeted protein. The
primary antibodies were listed in Supplementary Table 1.
2.9 Statistical analysis
Data are shown as mean standard deviation (SD). Statistical analysis was
performed using Prism 8.0 software (Graph Pad, San Diego, CA, USA). The
significant difference between the two groups was determined by Student’s
t-test and one-way factorial ANOVA. p-value less than 0.05 was
considered as statistically significant.
3. Results
3.1 BMSC-Exo were internalized by HRPTEpiC
In our study, both alizarin red S staining and oil red O staining revealed
positive results, indicating the highly differentiated ability of BMSCs
(Supplementary Fig. 1). Three methods were applied to identify exosomes
secreted by BMSCs. The transmission electron microscope (TEM) indicated that the
extracellular vesicles of the samples were “bowl-shaped” (Fig. 1A). NTA showed
that the particle size of BMSC-Exo was approximately 30–150 nm (Fig. 1B).
Western blot was used for detecting the exosome marker (CD9, CD63, and CD81) and
the nuclear marker (Histone H3), the results showed that the expression of CD9,
CD63, and CD81 were enriched in the isolated fractions while Histone H3 were
absent (Fig. 1C). Subsequently, we aimed to demonstrate the internalization of
exosomes. PKH67 labeled BMSC-Exo were added to HRPTEpiC and incubated for
different times (30 min, 24 h), and the labeled exosomes (green fluorescence)
were found around the nucleus after being incubated for 24 h (Fig. 1D).
Fig. 1.
Identification and internalization of BMSC-Exo. (A) TEM revealed
the ultrastructure of BMSC-Exo (left panel scale bar = 0.5 m, right panel
scale bar = 0.1 m). (B) BMSC-Exo was analyzed by NTA. (C) Exosome markers
(CD9, CD63, and CD81) and the nuclear marker (Histone H3) were detected by
Western Blot. (D) The internalization of labeled-BMSC-Exo (green fluorescence) by
HRPTEpiC.
3.2 BMSC-Exo improved renal function and histomorphological damage
in the 5/6 SNx rat
To evaluate the effect of BMSC-Exo on renal function and histomorphological
damage, a stable 5/6 nephrectomy rat model was established. The kidney of the
group SHAM had a normal broad bean-like morphology with a smooth surface. The
size of the kidney was significantly bigger under the administration of BMSC-Exo
compared with SNx group after 16 weeks of the surgery, indicating BMSC-Exo
treatment improved the atrophy of the kidney (Fig. 2A). Moreover, HE staining
showed that the regular structure of the kidney was destroyed and displayed
glomerular sclerosis, tubular vacuoles, interstitial fibrosis and inflammatory
cell infiltration (Fig. 2B) in the SNx group. With the continuous treatment of
BMSC-Exo, the aforementioned histological changes were significantly improved,
leading to the recovery of renal tissue after injury (Fig. 2B). To further
validate the protective role of exosomes, Scr (Serum Creatine), BUN (Blood urea
nitrogen) and body weight were measured. Versus the SHAM group, Scr and BUN
levels were raised in the SNx group which significantly decreased after
administration of BMSC-Exo. For the SHAM group, SNx group and the SNx + Exo
group, the level of Scr was 53.01 3.256, 110.9 5.465, 72.53
3.361 respectively, paralleled with the same trend of BUN (16.65
4.571, 37.55 1.005 and 19.60 0.6809). Moreover, the body weight
of SNx + Exo group was higher than that of the SNx group (Fig. 2C). The decreased
Scr and BUN levels and the increased body weight caused by BMSC-Exo treatment
suggesting the explicit therapeutic effect on histomorphological damage and renal
function.
Fig. 2.
BMSC-Exo administration improved renal function and
histomorphological damage in the SNx rat. (A) Images of kidney in different
treatment groups. (B) The structure of kidneys was observed under HE staining
(scale bar = 100 m. Black Arrow 1: glomerular sclerosis; Black Arrow 2:
tubular vacuoles; Black Arrow 3: interstitial fibrosis; Black Arrow 4:
inflammatory cell infiltration. (C) Comparison of Scr and BUN concentration in
rats. (D) Changes of bodyweight in SHAM group, SNx group and SNx + Exo group.
***p 0.001, ****p 0.0001.
3.3 BMSC-Exo alleviates renal fibrosis in the 5/6 SNx rats
To evaluate the effect of BMSC-Exo on renal fibrosis, the Masson-trichrome
staining, immunohistochemical staining and western blot were performed. As shown
in Fig. 3A, the blue area indicated collagen generation (fibrotic area) was
significantly increased after 5/6 nephrectomy. However, the application of
BMSC-Exo through tail vein injection significantly reduced the blue-stained
collagen deposition area. Consistent with the pathological changes,
immunohistochemical staining showed that the positive staining of fibronectin,
Collagen-I and -SMA were significantly decreased in the SNx + Exo group
compared with the SNx group (Fig. 3B). Moreover, the protein expression level of
fibrotic markers Collagen-I and -SMA were decreased while E-cadherin
expression was increased after the application of BMSC-Exo compared with SNx
group (Fig. 3C). With the evidence above, the therapeutic effect of BMSC-Exo on
renal fibrosis is reliable.
Fig. 3.
BMSC-Exo alleviated renal fibrosis in the SNx rats. (A) MASSON
staining of rat renal tissues (scale bar = 100 m). (B) The expression of
Collagen-I, Fibronection and -SMA was detected by immunohistochemical
staining (scale bar = 100 m). (C) Protein expression level of Collagen-I,
-SMA and E- cadherin in rats was detected by western blot. *p 0.05, **p 0.01, ***p 0.001.
3.4 BMSC-Exo alleviates
TGF-1-induced fibrosis in HRPTEpiCs
TGF-1 plays a crucial role in the progression of fibrosis. To further
demonstrate the anti-fibrotic effect of BMSC-Exo, HRPTEpiCs were incubated with
10 ng/mL TGF-1 (TGF- group) or with TGF-1 plus 100 ug/mL BMSC-Exo (TGF- +
Exo group) for 48 hours. The ovoid HRPTEpiC was elongated and turned into a
spindle shape after TGF-1 incubation observed under the microscope. The results
of western blot showed that TGF-1 stimulation evidently increased the
expression of -SMA, Collagen-I and decreased the expression of
E-cadherin; which were reversed by the administration of BMSC-Exo (Fig. 4A). The
results emphasized that BMSC-Exo treatment alleviated TGF-1-induced
fibrosis in vitro.
Fig. 4.
BMSC-Exo alleviated renal fibrosis through Smurf 2/Smad 7 axis.
(A) Protein expression level of Collagen-I, -SMA and E- cadherin in
HRPTEpiC was detected by western blot. (B) The expression of Smad 7 and Smurf 2
was detected in rats under treatment of BMSC-Exo. (C) The expression of Smad 7
and Smurf 2 was detected in HRPTEpiC after administration of BMSC-Exo.
*p 0.05, **p 0.01, ***p 0.001,
****p 0.0001.
3.5 BMSC-Exo improves renal fibrosis via inhibiting TGF-/Smurf 2/Smad 7 pathway
As we have mentioned in the Introduction section, Smad 7 and Smurf 2 play an
important role in TGF- signaling pathway. In addition, the role of Smurf 2
under different pathophysiology context remains complicated and need to be
further clarified. In this study, the two molecules Smad 7 and Smurf 2 were
measured by western blot both in vivo and in vitro. Firstly, we
demonstrated that the expression of Smad 7 was obviously decreased while Smurf 2
was increased after the establishment of 5/6 nephrectomy compared with the SHAM
group. However, the application of BMSC-Exo showed a reverse effect as Smad 7
level was up-regulated and Smurf 2 level was down-regulated compared with the SNx
group (Fig. 4B). The same trend was also found in the in vitro study. As
shown in Fig. 4C, the expression of Smad7 was significantly decreased while Smurf
2 was increased after the incubation of TGF-1, which could be reversed by
BMSC-Exo. These results confirmed that BMSC-Exo could partly improve renal
fibrosis through regulating Smurf 2/Smad 7 axis.
We further verify the characteristic of BMSC-Exo on Smurf 2/Smad 7 axis.
Firstly, the siRNA that specifically targeted Smurf 2 (si-Smurf 2) was used to
knock down the expression of Smurf 2. As shown in Fig. 5A, the protein expression
of Smurf 2 was significantly decreased by si-Smurf 2, while the expression of
Smad 7 was increased. Furthermore, the administration of BMSC-Exo enhanced these
effects.
Fig. 5.
BMSC-Exo enhanced the inhibitory effect of si-Smurf 2 on renal
fibrosis. (A) The protein expression level of Smad 7 and Smurf 2 after
transfected with si-Smurf 2. (B) The protein expression level of fibrotic markers
Collagen-I, -SMA and E-cadherin was measured after transfected with
si-Smurf 2. **p 0.01 compared with si-NC group, ***p
0.001 compared with si-NC group, ****p 0.0001 compared with si-NC
group; #p 0.05 compared with TGF- + si-NC group, ##p 0.01 compared with TGF- + si-NC group, ###p 0.001 compared
with TGF- + si-NC group, ####p 0.0001 compared with TGF- +
si-NC group; &p 0.01 compared TGF- + si-Smurf 2.
Next, we detected the expression levels of fibrotic markers Collagen-I,
-SMA and E-cadherin after silencing Smurf 2. The results demonstrated
that after transfection of si-Smurf 2, the expression of Collagen-I,
-SMA significantly decreased while E-cadherin expression increased
(Fig. 5B), indicating the protective effect of si-Smurf 2 on renal fibrosis.
Furthermore, the decreased expression of Collagen-I, -SMA and the
increased expression of E-cadherin in group TGF- + si-Smurf 2 were
significantly enhanced after administration of BMSC-Exo. Taken together, these
aforementioned results confirmed that the BMSC-Exo could inhibit TGF-1-induced
renal fibrosis, partially, by regulating the Smurf 2/Smad 7 axis (Fig. 6).
Fig. 6.
Graphical abstract.
4. Discussion
Nowadays, the treatment for ESRD is costly and has substantial morbidity. Thus,
the new strategy in treating renal fibrosis which is the main pathological
process of CKD should be taken into attention. Under this context, the appearance
and application of BMSCs-Exo showed a potential role in treating renal fibrosis.
Studies have shown that MSC-exosomes from different sources have completely
different functions [19]. However, the research on the effect of BMSC-Exo on
renal fibrosis is still limited.
Exosomes are 30–150 nm microvesicles with the lipid bilayer, which contains
abundant quantities of mRNA, LncRNA and microRNA to exert multiple effects
[20, 21]. Exosomes secreted by MSCs have been proved to have therapeutic effects
in several complex diseases including in kidney. In the ischemia-induced acute
kidney injury model, administration of MSC-derived exosomes evidently alleviates
renal injury, improves renal function by inhibiting oxidative stress through
regulating NADPH oxidase [22]. Also, human umbilical cord mesenchymal stem
cell-derived exosomes can enhance autophagy via modulation ATG16L in the
cisplatin-induced acute kidney injury (AKI) [19]. Furthermore, exosomes derived
from MSCs also exert an anti-inflammation effect in diabetic nephropathy [17]. In
the UUO model, miR-let7C from MSC-derived exosomes could attenuate the process of
EMT in TGF-beta induce renal tubular epithelial cells [20]. Collectively, the
above studies partly demonstrated the role of exosomes in improve renal injury
and fibrosis.
However, because of the complexity of the containing molecules and the
polytropic effect in different pathological circumstances, further studies should
be done to clarify the therapeutic effect and the underlying mechanisms of
BMSC-derived exosomes. In this study, we used primary BMSCs that are more stable
and functionally closer to the original tissue to obtain exosomes, but the
disadvantage is that they come from a very limited number of donors. A 5/6
nephrectomy rat model and TGF-1 treated HRPTEpiCs were established to further
investigate the role of BMSC-derived exosomes. In our in vivo study, we
found that the administration of BMSC-Exo significantly improved atrophic and
histomorphological damage of the kidney and alleviated the impaired renal
function. Consistent with the previous report, the exosomes also exert an
anti-fibrotic role in the 5/6 nephrectomy rat, for reducing the fibrotic area as
well as the fibrotic markers. In the nephrectomy rats, TGF-1 plays a crucial
role in triggering renal fibrogenesis by breaking the balance of ECM
synthesis/degradation [23]. TGF-1 also promotes renal tubular epithelial cells
emerging a fibrotic phenotype thus accelerate the process of renal fibrosis [24].
BMSC-Exo restored the decreased E-cadherin expression and inhibited
-SMA and Collagen-I expression compared with TGF-1 single treatment.
Taken together, BMSC-Exo treatment can inhibit the fibrotic process in the 5/6
nephrectomy rat model and TGF-1 treated HRPTEpiCs.
TGF-1 acts as a pro-fibrotic molecular mainly through stimulating the
downstream Smad signaling pathway to trigger and facilitate subsequent
fibrogenesis procession [25]. Previous studies have clearly demonstrated the
underlying mechanisms in regulating the TGF-1/Smad signaling pathway [3, 26].
Among the Smad subfamily, Smad 7 was considered as a negative regulator of
TGF-1/Smad signaling transduction via blocking the recruitment and
phosphorylation of the Smad 2-Smad 3 complex [27]. The crucial role of Smad 7 in
renal fibrosis has been proved in different kidney disease models, such as the
UUO model, 5/6 nephrectomy model, and ischemia-perfusion model, both of them
showed the downregulation of Smad 7 in the injured kidney, on the contrary,
restored Smad 7 significantly inhibit renal fibrosis in the above models [28, 29].
In our present study, the decreased expression of Smad 7 in the 5/6 nephrectomy
rats and TGF-1 treated HRPTEpics were observed. However, the administration of
BMSC-Exo partly restored the Smad 7 expression and accompanied with decreasing
expression of fibrotic markers -SMA and Collagen-I and increasing
expression of E-cadherin. As previous research demonstrated, Smad 7 can also
exert an anti-inflammation role via upregulating the expression of
IB thus inhibiting the activity of the NF-B
signaling pathway [30]. Therefore, the role of BMSC-Exo in inflammation should
also be further elucidated in renal fibrosis.
The ubiquitin-proteasome system strictly controlled the downstream action of the
Smad subfamily and the signal transduction of TGF- pathway [31]. The subsequent
activation of protein degradation by E1-E3 ubiquitin ligases depends on the
activation of the ubiquitin-proteasome system. Smad ubiquitination regulatory
factor-2 (Smurf 2) is an E3 ubiquitin ligase that has been proved to physically
interact with Smad 7 to induce its ubiquitination and degradation [32, 33].
Furthermore, Smurf 2 could also recruit intranuclear Smad 7 and form Smurf 2-Smad
7 complex then binding to TGF- type I receptor and leading the subsequent
protein degradation, finally inhibit the TGF-/Smad signaling pathway [34].
Thus, under different pathological contexts, the role of Smurf 2 in regulating
TGF-/Smad pathway is still controversial. In our study, the results showed that
the expression of Smurf 2 was significantly inhibited and Smad 7 was increased by
BMSC-Exo both in 5/6 nephrectomy rats and TGF-1 induced HRPTEpics. Furthermore,
knockdown of Smurf 2 induced upregulation of Smad 7 level and downregulated the
expression level of -SMA and Collagen-I, and these effects could be
enhanced by BMSC-Exo. With the evidence above, we indicated that BMSC-Exo has an
inhibitive effect on renal fibrosis, to some extent, by regulating the Smurf
2/Smad 7 axis. Although the benefits of BMSC-Exo in treating fibrotic diseases
have been proved in several studies as well as ours, some problems are remained
to be further solved, such as the long-term safety of the exosomes and the
optimal timing for the treatment of CKD.
5. Conclusions
In summary, BMSC-Exo could improve renal fibrosis in 5/6 nephrectomy rats and
inhibit TGF-1-induced fibrotic changes of HRPTEpics, to some extent, via
antagonism of the Smurf 2/Smad 7 axis. This study not only reveals the
significant role of the Smurf 2/Smad 7 axis in regulating renal fibrosis but also
expands our understanding of the regulatory effects of BMSC-Exo on diseases with
complex mechanisms such as CKD-VC.
Abbreviations
BMSC-Exo, Bone marrow mesenchymal stem cells-derived exosome; SNx, 5/6 subtotal
nephrotomy; HRPTEpiCs, human renal proximal tubular epithelial cells; CKD,
Chronic kidney disease; TGF-1, Transforming growth factor-1; R-Smad, receptor
Smad; Co-Smad, common mediator Smad; I-Smad, inhibitory Smad; Smurf 2, Smad
ubiquitination regulatory factor 2; MSCs, Mesenchymal stem cells; TEM,
Transmission electron microscope; si-Smurf 2, siRNA targeting Smurf 2; Scr, Serum
creatinine; BUN, Blood urea nitrogen; HE, Hematoxylin and Eosin staining; GAPDH,
Glyceraldehyde 3-phosphate dehydrogenase; AKI, acute kidney injury; ESRD,
End-stage renal disease; UUO, unilateral ureteral obstruction.
Author contributions
WL, XC and YL provided the concept and designed
the study; YL and YG performed experiments; XC and YL interpreted the results; YL prepared figures; XC drafted the manuscript; XC, YL, WG and WL edited and revised the manuscript. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
All procedures involving animal samples were approved and supervised by the
animal ethics committee of Beijing Friendship Hospital, Capital Medical
University (21-1005). The treatment of animals in all experiments conforms to the
ethical standards of experimental animals.
Acknowledgment
Not applicable.
Funding
This work was supported by the financial support from the Beijing Municipal
Administration of Hospitals Clinical Medicine Development of Special Funding
Support (No. ZYLX201824).
Conflict of interest
The authors declare no conflict of interest.