Chemico-Biological Interactions 277 (2017) 110e118

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Angiotensin II induces calcium-mediated autophagy in podocytes

through enhancing reactive oxygen species levels
Na Gao a, c, 1, Hui Wang b, d, 1, Hongqiang Yin a, Zhuo Yang a, *
a
School of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials Ministry of Education, Nankai University,
Tianjin 300071, PR China
b
College of Life Sciences, Nankai University, Tianjin 300071, PR China
c
Tianjin Medical University Cancer Institute and Hospital, Tianjin 300200, PR China
d
School of Mathematical Sciences, Nankai University, Tianjin 300071, PR China

a r t i c l e i n f o a b s t r a c t

Article history: As well known, abnormalities of Angiotensin II (Ang II) is closely related with glomerular damage. This
Received 1 May 2017 study was to investigate whether Ang II could affect autophagy in podocytes via oxidative stress, and
Received in revised form whether autophagy had a positive role in protecting podocytes impaired by Ang II. 3-(4,5-
25 July 2017
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podo-
Accepted 11 September 2017
Available online 12 September 2017
cyte death. The measurements of malondialdehyde (MDA) and H2O2 levels, and flow cytometry assay
revealed that Ang II considerably increased reactive oxygen species (ROS) generation in podocytes.
Meaningfully, treatment with ROS scavenger N-(mercaptopropionyl)-glycine (N-MPG) could inhibit
Keywords:
Ang II
podocyte death and attenuate accumulation of ROS induced by Ang II. The patch-clamp experiments
Podocytes indicated that Ang II increased the current of transient receptor potential canonical 6 (TRPC6). Moreover,
TRPC6 measurement of Fluo-3 image showed that Ang II increased intracellular Ca2þ level, as N-MPG and La3þ
Autophagy impeded Ang II induced Ca2þ influx. Acridine orange staining indicated that Ang II induced accumulation
ROS of acidic vacuoles. Beclin-1 and LC3 are essential for autophagosome formation. Furthermore, as one of
the selective substrates for autophagy, P62 plays a key role in the formation of cytoplasmic proteinaceous
inclusion. Western blot assay presented that Ang II obviously elevated LC3-II/LC3-I ratio and expression
of beclin-1, and reduced expression of P62. Meanwhile, N-MPG expectedly down-regulated autophagy in
Ang II-treated podocytes. Rapamycin can enhance the level of autophagy by inhibiting mTOR, and 3-
methyladenine (3-MA) can inhibit autophagosome formation through blocking class III phosphatidyli-
nositol 3-kinase. MTT assay exhibited that rapamycin significantly enhanced the cell viability, while 3-
MA considerably reduced it in Ang II-treated podocytes. Consequently, this study demonstrated that
Ang II could increase TRPC6 induced Ca2þ influx and enhance autophagy through increasing ROS levels in
podocytes, and autophagy could protect Ang II-treated podocytes. Improving TRPC6 channels and
autophagy may become a new targeted therapy to relieve glomerular damage induced by Ang II.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction glomerular endothelial cells compose glomerular filtration bar-

rier. The foot processes of neighboring podocytes form the slit
Podocytes, the glomerular basement membrane, and diaphragm. Through the slit diaphragm, podocytes constitute a
molecular and electric barrier to maintain the function and
structural integrity of glomerular filtration barrier and prevent
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
the protein leakage [1,2]. Under pathological conditions, podo-
mide; 3-MA, 3-methyladenine; Ang II, Angiotensin II; MDA, malondialdehyde; N- cytes become the target cells of injury in several conditions of
MPG, N-(mercaptopropionyl)-glycine; ROS, reactive oxygen species; TRPC6, tran- glomerular injury [3]. Because podocytes are terminally differ-
sient receptor potential canonical 6. entiated cells with limited ability of proliferation, podocyte loss
* Corresponding author. School of Medicine, Nankai University, Tianjin 300071,
becomes an important event in glomerulosclerosis and end-stage
PR China. Tel.: þ86 22 23504364; fax: þ86 22 23502554.
E-mail address: zhuoyang@nankai.edu.cn (Z. Yang). kidney disease [4,5]. Transient receptor potential canonical 6
1
Na Gao and Hui Wang have contributed equally to this work. (TRPC6) channels as Ca2þ and Naþ-permeable channels are highly

http://dx.doi.org/10.1016/j.cbi.2017.09.010
0009-2797/© 2017 Elsevier B.V. All rights reserved.
 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118
expressed in podocytes and play an important role in the patho-
genesis of renal diseases [6e10].
Angiotensin II (Ang II), as an important active substance of
renin-angiotensin system, combined with angiotensin receptor
plays some biological roles, such as contraction of blood vessels and
increasing blood pressure [11,12]. Within the glomerulus, Ang II
decreases the ultrafiltration coefficient, modulates glomerular
capillary permselectivity [13], and affects both inflammatory and
fibrotic processes [14]. Ang II can increase intracellular calcium
activity, cause a reactive oxygen species (ROS)-mediated F-actin
cytoskeleton rearrangement, and lead to podocyte injury [15,16].
The oxidative stress is an imbalance between ROS and the cell
antioxidant capacity [17]. The increase of oxidative stress usually
accompanied with the increase of malondialdehyde (MDA) and
H2O2 levels. MDA, a by-product of lipid peroxidation, is a sensitive
index of oxidative damage. The concentration of MDA reflects the
proportional to lipid peroxidation and oxidant stress [18]. H2O2 is
naturally produced in organisms as a by-product of oxidative
metabolism. The increase of oxidative stress usually accompanied
with the increase of H2O2 level.
Autophagy plays an important role in maintaining a well-
controlled balance between anabolism and catabolism, which is
necessary to normal cell growth and development [19]. Under
normal conditions, a basal level of autophagy can maintain pro-
tein quality control and remove damaged proteins, organelles and
lipids, and avoid harming normal cellular function. Autophagy
also plays an essential role during starvation, cellular differenti-
ation, cell death and aging [20]. It permits cells to eliminate un-
wanted or unnecessary proteins and organelles, and to reuse the
components [21]. Furthermore, the autophagy-related proteins,
such as LC3 and Beclin-1, are necessary for autophagosome for-
mation in various type of cells [22e24]. As autophagy inhibitors,
3-methyladenine (3-MA) can inhibit autophagosome formation
through blocking class III phosphatidylinositol 3-kinase [25]. It
has been reported that autophagy has a protective function
against renal damage induced by hypoxia, ischemia, aging and
anticancer drugs [26e32]. While the mechanisms underlying the
development of glomerular injury are greatly complex [33], the
activation of the intrarenal renin-angiotensin system may be a
possible mechanism [34]. The purpose of this study was to assess
whether Ang II can affect TRPC6 channels and autophagy in
podocytes via oxidative stress. Meanwhile, we evaluated whether
autophagy had a positive role in protecting podocytes impaired by
Ang II. It may provide an interesting potential clinical application
for chronic glomerular disease.
2. Materials and methods
2.1. Materials
RPMI 1640 cell culture medium was purchased from GIBCO
Invitrogen. The mouse recombinant interferon-gamma (IFN-g),
Rapamycin, 3-MA, fetal bovine serum (FBS), 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),
N-(mercaptopropionyl)-glycine (N-MPG) and Fluo-3/AM fluores-
cent dye were purchased from Sigma Chemical Co., St Louis, MO.
The primary antibodies used in the analysis, anti-P62 and anti-
Beclin-1ab were purchased from Cell Signaling Technology, USA.
Mouse monoclonal anti-LC3 IgG primary antibody was purchased
from MBL and rabbit monoclonal anti-b-actin IgG primary antibody
was purchased from Santa Cruz Biotechnology, Inc. CA, USA. The
reagent kits using for measurement MDA and H2O2 were purchased
from Nanking Jiancheng Institute of Biological Engineering Inc.
(Nanking, China).
111
2.2. Podocyte culture
Conditionally immortalized mouse podocyte cell lines were
cultured at 33  C in RPMI 1640 medium containing 10% FBS,
100 mg/ml penicillin plus 100 mg/ml streptomycin (Sigma) and 20
U/ml of IFN-g in a humidified atmosphere of 5% CO2. To induce
differentiation, podocytes were cultured at 37  C with a IFN-g-free
medium for two weeks. The conditionally immortalized mouse
podocyte cell line was established by Prof. Peter Mundel [35].
2.3. Cell viability of podocytes with Ang II and Ang II þ N-MPG
The cell viability was assessed with MTT assay, which was based
on the reduction of the dye MTT to formazan crystals, an insoluble
intracellular blue product, by cellular dehydrogenases. Briefly, cells
of 1  106 cells/mL were seeded into each well in 96-well culture
plates and incubated with different concentrations of Ang II (0,
108, 107 and 106 mol/L) for 36 h. Subsequently, 20 ml MTT (5 mg/
ml) was added to each culture well. Following cultured at 37  C for
4 h, the medium was removed carefully and 150 ml DMSO was
added in each well to dissolve the formazan product. Finally, for-
mazan absorbance was assessed by a microplate reader (Multiskan
Mk3; Thermo Labsystems Helsinki, Finland) with a wave length of
492 nm. Meanwhile, the concentrations of Ang II used in assays of
oxidative stress and autophagy were based on the results of the
MTT test. The cell viability was expressed as a percentage of the
viability in control culture. Cell viability (%) ¼ (the viability of Ang
II-treated group/the control group) x 100.
2.4. Measurement of MDA and H2O2 levels
The MDA and H2O2 levels in podocytes were evaluated by MDA
and H2O2 assay kit. The podocytes were incubated in 6-well plates
for 24 h for stabilization. Then normal cell culture medium, Ang II
(107 mol/L) and Ang II (107 mol/L) þ N-MPG (1 mM) were added
for 36 h, the levels of MDA and H2O2 were measured according to
the manufacturer instructions. The levels of MDA and H2O2 in
control group were defined as 100%, while the levels of MDA and
H2O2 in the rest groups were expressed as a percentage of the MDA
and H2O2 levels in control group.
2.5. Measurement of ROS generation
Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to
detect the intracellular ROS level. The cells were plated in 6-well
plates. After reaching 60%e70% confluent, they were treated with
normal cell culture medium, Ang II (107 mol/L) and Ang II
(107 mol/L) þ N-MPG (1 mM) for 36 h. After incubation, the cells
were washed with PBS (pH 7.4). Following stained with DCFH-DA
(Genmed Scientifics Inc., Wilmington, DE, USA) for 20 min at
37  C, the cells were detected and analyzed by flow cytometry. The
level of ROS generation was calculated as follows: the level of ROS
(%) ¼ the percentage of DCF-positive cells in M1 region.
2.6. Whole-cell patch-clamp recording
The conventional whole-cell patch-clamp recording was carried
out at room temperature (22e24  C). Experiments were performed
in podocytes attached to glass coverslips and transferred to a
recording chamber on a stage of Olympus inverted microscope
immediately before recording. For whole-cell recording TRPC6
channel currents, the bath solution contained (mM): 130 NaCl, 4
KCl, 1 MgCl2, 2 CaCl2 and 10 HEPES, pH 7.3. The pipette solution
contained (mM): 130 CsOH, 130 L-aspartic acid, 0.3 CaCl2, 2 MgCl2,
10 HEPES, 10 EGTA, 3 ATP-Mg, 0.2 GTP-Na, pH 7.3. The currents
112 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118
were induced by a 150-ms voltage ramp protocol from 100 mV to
100 mV with a holding potential 60 mV. The patch pipettes had a
resistance ranged from 3 to 6 MU. The cell capacitance ranged from
100 to 250 pF in individual podocytes.
2.7. Ca2þ level detection
Podocytes were seeded in 6-well plates and treated with normal
cell culture medium, Ang II (107 mol/L), Ang II (107 mol/L) þ N-
MPG (1 mM) and Ang II (107 mol/L) þ La3þ(100 mM) respectively
for 36 h. The stock solutions of Fluo-3/AM were diluted with HBSS
that contained (mM): 138 NaCl, 5.3 KCl, 0.3 NaH2PO4, 0.4 KH2PO4,
4.2 NaHCO3, 5.6 D-glucose and 10 HEPES. The podocytes were
loaded with 5 mM Fluo-3/AM at 37  C for 30 min. Then, the inves-
tigated cells were washed by HBSS, and visualized under a confocal
laser scanning microscope at room temperature (22e24  C). The
calcium-dependent fluorescence was excited at 488 nm laser line
and the fluorescence signal was acquired at 522 nm.
2.8. Acridine orange staining
Cellular acidic vesicular organelles were examined by acridine
orange staining. Acridine orange, as a fluorescent molecule, can
interact with DNA emitting green fluorescence and accumulate in
acidic organelles in which it becomes protonated forming aggre-
gates that emit bright red fluorescence [36]. Podocytes were seeded
in 6-well plates and treated with normal cell culture medium, Ang
II (107 mol/L) and Ang II (107 mol/L) þ N-MPG (1 mM) for 36 h.
After treatment, the cells were stained with acridine orange at 37  C
for 15 min. After washing with PBS, the cells were immediately
visualized by a Leica TCS SP5 laser-scanning confocal microscope
for detecting acidic vesicular organelles.
2.9. Western blot analysis
Podocytes were treated with normal cell culture medium, Ang II
(107 mol/L) and Ang II (107 mol/L) þ N-MPG (1 mM) for 36 h. The
method of cell lysates and Western blotting was modified on the
basis of previous studies [37]. Podocytes were washed with PBS (pH
7.4), then harvested and lysed in lysis buffer (50 mM Tris-HCl [pH
7.4], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulphate
[SDS], 0.5% deoxycholic acid sodium salt [DOC]) at 0  C. The lysates
were centrifuged at 12000 rpm at 4  C for 10 min. The supernatant
was mixed with loading buffer (ratio is 4:1) and boiled at 95e100  C
for 20 min. Total proteins were subjected to electrophoresis in a 10
%e13% SDS-PAGE gel. After separation on polyacrylamide gel, the
proteins were transferred to polyvinylidene fluoride (PVDF)
membranes. The PVDF membranes were blocked with 5% zero fat
milk powder in TBS with 0.05% Tween 20 at room temperature for
1 h. Then the PVDF membranes were incubated with primary an-
tibodies. After washing with TBST, the PVDF membranes were
incubated with horseradish peroxidase-labeled secondary anti-
bodies. The blots were developed with a chemiluminescence
detection kit (Pierce) and exposed to X-ray film (Eastman Kodak,
Rochester, NY). Equal protein loading and the protein transfer were
confirmed by immunoblotting for determination of actin protein
using b-actin antibody (1:1000 Santa cruz) on the same Western
blot assay.
2.10. Cell viability of podocytes with autophagy regulators
The effects of rapamycin and 3-MA, which were known as
autophagy regulators, were detected by the MTT assay. Following
procedures of standard method described above, the cells were
treated with normal cell culture medium, rapamycin (10 ng/ml), 3-
MA (10 mM), Ang II (107 mol/L), Ang II (107 mol/L) þ rapamycin
(10 ng/ml), Ang II (107 mol/L) þ 3-MA (10 mM) for 36 h.
2.11. Statistical analysis
The current data were acquired using an EPC10 amplifier (HEKA,
Germany), which connected to a computer and stored on a com-
puter hard disk using pulse 8.52 software (HEKA) analyzed off-line
using the pCLAMP 9.0 and Origin 8.0. All data were presented as
mean ± S.D. The statistical significance was assessed by one-way
analysis of variance (ANOVA) and Tukey's multiple comparison
post-test using the SPSS (16.0) software. MTT assay, Western blot
assay, measurement of MDA and H2O2 were repeated thrice; flow
cytometry was repeated 4 times; whole-cell patch-clamp recording
was repeated 6 times. In Ca2þ level detection and acridine orange
staining, we randomly selected 20 cells for analysis of fluorescent
intensity. Significant differences were taken when P < 0.05.
3. Results
3.1. Cell viability assay
The viability of differentiated podocytes was determined by
MTT assay. Podocytes were treated with medium containing
different concentrations (0, 108, 107, and 106 mol/L) of Ang II for
36 h. As shown in Fig. 1a, the cell viability was decreased when the
concentration increased.
In order to detect the effect of Ang II induced oxidative stress on
cell viability, podocytes were treated with Ang II (107 mol/L) and
Ang II (107 mol/L) þ N-MPG (1 mM) for 36 h. It was found that the
viability of podocytes was statistically reduced to 61.56 ± 9.04% in
107 mol/L Ang II group (P < 0.01, Fig. 1b). Moreover, the cell
viability of Ang II þ N-MPG group was significantly enhanced to
87.46 ± 6.60% (P < 0.05).
3.2. Measurement of MDA and H2O2 levels
The effects of N-MPG on the intracellular MDA and H2O2 levels
in Ang II-treated podocytes were measured by using the MDA and
H2O2 test kits (Fig. 1c and d). It was found that the cellular level of
MDA was remarkably increased in Ang II group (129.69 ± 7.29%,
P < 0.05, Fig. 1c), and it was significantly decreased in Ang II þ N-
MPG group (108.59 ± 3.65%, P < 0.05). Additionally, it could be seen
that the cellular level of H2O2 was remarkably increased in Ang II
group (250.88 ± 20.69%) compared to that in control group
(P < 0.01, Fig. 1d). Moreover, it was significantly decreased in Ang
II þ N-MPG group (184.89 ± 8.90%, P < 0.05).
3.3. Measurement of ROS generation
DCFH-DA was used to detect the intracellular ROS level. Fig. 1h
showed that the ratio of DCF-positive cells was 4.97 ± 0.88% in
control group. Furthermore, the DCF-positive cell ration in Ang II
group was significantly higher (21.45 ± 2.23%, P < 0.001) than that
in control group. In addition, it was much lower in Ang II þ N-MPG
group (7.65 ± 0.70%, P < 0.001) compared to that in Ang II group.
3.4. Whole-cell recording of TRPC6 currents
In order to detect the effect of Ang II induced oxidative stress on
TRPC6 currents, Whole-cell patch-clamp technology was used.
Whole-cell TRPC6 currents were evoked by 200 ms voltage ramp
protocol from a holding potential of 60 mV. In this series of ex-
periments, the patch was depolarized from 100 to 100 mV. Pre-
vious studies have shown that these protocols can isolate
 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118 113

Fig. 1. N-MPG attenuated Ang II-induced cell death and oxidative stress in podocytes. a The viability of podocytes was determined by MTT assay. Podocytes were treated with
different concentrations of Ang II (0, 108, 107 and 106 mol/L) (n ¼ 3/group). b N-MPG increased the cell viability of podocytes with Ang II. The cell viability of 107 mol/L Ang II
group and Ang II (107 mol/L) þ N-MPG (1 mM) group was determined by MTT assay (n ¼ 3/group). c The effects of N-MPG on the intracellular MDA levels in 107 mol/L Ang II
treated podocytes were measured by using the MDA test kits (n ¼ 3/group). d The effects of N-MPG on the intracellular H2O2 levels in 107 mol/L Ang II treated podocytes were
measured by using H2O2 test kits (n ¼ 3/group); Measurement of ROS generation in podocytes (e-h, n ¼ 4/group). The cells were treated with different culture medium for 36 h. e
control, f Ang II (107 mol/L), g Ang II (107 mol/L) þ N-MPG (1 mM), h Statistical analysis of flow cytometry. Each data represents the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001
vs. control group; #P < 0.05, ###P < 0.001 vs. Ang II group.

macroscopic TRPC6 currents [38]. Ang II increased the amplitude of 3.5. Ca2þ influx detection
TRPC6 currents, and increased the slope of IeV curve (Fig. 2b). Ang
II increased mean currents at all membrane potentials, especially To evaluate the effect of Ang II induced oxidative stress on the
from 20 to 80 mV (P < 0.01). Ca2þ influx, we used Ca2þ-sensitive dye Fluo-3/AM in podocytes.
114 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118

Fig. 2. N-MPG decreased Ang II-induced currents of TRPC6 channel in podocytes. a Examples of TRPC6 currents evoked from a holding potential of 60 mV in differentiated
podocytes. b IeV curves of control group and Ang II (107 mol/L) group (n ¼ 6/group). c Representative microscopic images of intracellular Ca2þ using Fluo 3/AM staining. Scale bar,
50 mm. d Fluorescent intensity of intracellular Ca2þ in podocytes under different conditions (n ¼ 20/group). Each data represents the mean ± S.D. **P < 0.01, ***P < 0.001 vs. control
group; ###P < 0.001 vs. Ang II group.

The fluorescence of Fluo-3/AM in podocytes in these different
groups were shown in Fig. 2c. Changes of cytosolic calcium were
visualized by confocal laser scanning microscopy. The results
showed that Ang II caused elevation of the intracellular Ca2þ con-
centration in podocytes. Clearly, N-MPG and La3þ (TRPC6 blocker)
could significantly decrease Ang II induced Ca2þ influx (Fig. 2d).
3.6. Acridine orange staining
Acridine orange was used to stain cellular acidic vesicular or-
ganelles, which reflected the level of autophagy. As shown in
Fig. 3a, green fluorescence and a spot of orange fluorescence were
displayed in control group, and in the Ang II-treated podocytes the
orange fluorescence displayed most. However, orange fluorescence
of Ang II þ N-MPG-treated podocytes was markedly less than Ang II
-treated podocytes. As shown in Fig. 3b, our data provide evidence
that the intensity of red fluorescence increased when the cells were
treated with Ang II than that in control group (P < 0.001), mean-
while, the red fluorescent intensity decreased in Ang II þ N-MPG
group contrast to Ang II group (P < 0.001). In addition, there was no
statistical difference of green fluorescent intensity among these
three groups.
were P62 (Fig. 3d), beclin-1 (Fig. 3e) and the ratio of LC3 II/LC3 I
(Fig. 3f). It was shown that the ratio of LC3 II/LC3 I and the level of
beclin-1 were significantly increased in Ang II group compared
with those of control group (P < 0.05), and decreased in Ang II þ N-
MPG group compared with those of Ang II group (P < 0.05).
Meanwhile, it could be seen that the level of P62 was significantly
decreased in Ang II group compared with that of control group
(P < 0.01) and increased in Ang II þ N-MPG group compared with
that of Ang II group (P < 0.01).
3.8. Cell viability of podocytes with autophagy regulators
In order to confirm the protective effect of Ang II induced
autophagy, rapamycin and 3-MA were used in this study. As shown
in Fig. 4, the viability of podocytes was statistically reduced to
63.42 ± 1.79% in Ang II group (P < 0.001). Meanwhile, the cell
viability was decreased in rapamycin group (86.40 ± 3.73%) and 3-
MA group (87.71 ± 2.90%) than that in control group (P < 0.05).
Moreover, the cell viability was further significantly reduced to
47.90 ± 5.40% in Ang II þ 3-MA group (P < 0.01), but risen to
74.82 ± 2.04% in Ang II þ rapamycin group (P < 0.01) compare to
Ang II group.

3.7. Western blot analysis 4. Discussion

The autophagy levels were assessed by measurements of LC3 II Ang II can contribute to the progression of renal injury through
to LC3 I ratio and beclin-1 and P62 levels. The results were obtained its hemodynamic effects [11], and its direct effects on kidney cells
from the experiments in autophagy molecular markers, which [39]. Ang II is known to induce oxidative stress in a variety of renal
 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118 115

Fig. 3. N-MPG decreased Ang II-induced autophagy. a Cellular acidic vesicular organelles were examined by acridine orange staining. Podocytes were treated with Ang II
(107 mol/L) and Ang II (107 mol/L) þ N-MPG (1 mM) for 24 h. When stained with acridine orange, DNA emits green fluorescence and acidic vesicular organelles emit red
fluorescence. Scale bar, 50 mm. b Statistical analysis of relative fluorescent intensity (n ¼ 20/group). c immunoblots from single representative experiments of P62, Beclin-1 and LC3.
d P62 band density was measured (n ¼ 3/group). e Beclin-1 band density was measured (n ¼ 3/group). f LC3-II/LC3-I band density ratio was measured (n ¼ 3/group). Each data
represents the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Ang II group. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

that Ang II inhibited the viability of podocytes in a concentration-

Fig. 4. Rapamycin enhanced and 3-MA inhibited the viability in Ang II-treated
podocytes. The viability of podocytes was determined by MTT assay. Podocytes were
treated with rapamycin (10 ng/ml), 3-MA (10 mM), Ang II (107 mol/L), Ang II
(107 mol/L) þ rapamycin (10 ng/ml), Ang II (10-7 mol/L) þ 3-MA (10 mM). Each data
represents the mean ± S.D. n ¼ 3/group. *P < 0.05, ***P < 0.001 vs. control group;
##
P < 0.01 vs. Ang II group.
cells [40]. On the other hand, autophagy, as one of the major cellular
mechanisms, has been related to oxidative stress [41]. In our pre-
sent study, we found that Ang II could increase TRPC6 currents by
increasing the level of ROS in podocytes. Moreover, TRPC6 medi-
ating Ca2þ influx induced protective autophagy in podocytes. It is
indicated that regulation of autophagy might be a positive way to
attenuate the Ang II-induced oxidative damage in podocytes.
In this study, the viabilities of podocytes incubated with
different concentrations of Ang II were investigated. It was found
dependent manner. Our previous study has shown that Ang II can
increase the generation of ROS in podocytes [42]. Meanwhile, the
viability of podocytes treated with ROS scavenger N-MPG was
obviously increased. MTT results showed that when pretreated
with N-MPG, the cell viability increased from 61.56% to 87.46% in
107 mol/L Ang II-treated group. These results showed that N-MPG
could reduce the cell damage by Ang II (p < 0.05). These results
demonstrated that Ang II might induce the death of podocytes by
enhancement of intracellular ROS generation, while N-MPG could
improve the cellular viability and reduce the damage caused by Ang
II. The MDA and H2O2 levels of podocytes cultured with 107 mol/L
Ang II were obviously increased. In contrast to the Ang II group,
MDA and H2O2 levels of the group treated with N-MPG were
decreased from 129.69% to 108.59% (P < 0.05) and from 250.88% to
184.89%, respectively. Furthermore, flow cytometry assay revealed
that Ang II considerably increased ROS generation of podocytes,
and N-MPG alleviated it. The present study indicated that Ang II
was able to inhibit podocyte growth and induce apoptosis with the
increased ROS levels, while N-MPG decreased ROS levels in Ang II
treated podocytes.
Some study has shown that Ang II increases free cytosolic cal-
cium in podocytes by calcium releasing from intracellular stores
and an infiux from the extracellular space [43]. In this study, the
currents of TRPC6 channel were measured by whole-cell patch-
clamp to detect the effects of Ang II in podocytes. TRPC6 is an
essential component of the podocyte foot processes and slit dia-
phragm and co-localizes with a number of podocyte structural
proteins, including nephrin and podocin [44e47]. It was demon-
strated that mutation of TRPC6 protein might cause dysfunction of
podocytes and lead to focal segmental glomerulosclerosis
[10,48,49]. Results indicated that Ang II increased the current
amplitude and the slope of IeV curve of TRPC6 channel. Some
116 N. Gao et al. / Chemico-Biological Interactions 277 (2017) 110e118
research found that Ang II let Ca2þ influx increase in podocytes and
led to depolarization [38,50]. Our previous study has shown that
Ang II can increase the generation of ROS in podocytes [42]. In order
to detect the effect of ROS on TRPC6 channel, we measured Ca2þ
influx. It was found that ROS scavenger N-MPG inhibited Ang II
induced the increase of intracellular Ca2þconcentration, which
suggested that ROS could induce the opening of TRPC6 channels.
Results of acridine orange staining showed that Ang II induced
accumulation of vesicular organelles, which was attenuated by N-
MPG. The morphological alterations that are suggestive of cell
death like blebbing of the plasma membrane could be seen in the
cells undergoing autophagic death [51,52]. The data provided evi-
dence that the number of acidic vesicular organelles increased
when the cells were treated with Ang II, which meant activation of
autophagy process, and N-MPG could inhibit the autophagy.
Furthermore, Western blot assay was employed to measure the
expression of autophagy-related proteins. During autophagy, sol-
uble form LC3 (LC3 I) is converted to the lipidated form of LC3 (LC3
II), and the LC3 II/LC3 I ratio is increased. It is important to measure
the level of autophagy by detecting LC3 conversion (LC3 I to LC3 II)
[53]. In addition, beclin-1 and P62 are important proteins in auto-
phagy process. Researches have shown that beclin-1 regulates each
major step in the autophagic pathways, such as autophagosome
formation and autophagosome maturation, and overexpression of
beclin-1 can up-regulate class III phosphoinositide 3-kinase, and
finally lead to autophagy activity. P62 can reflect autolysosomal
lytic activity and autophagy flux, the expression of this protein is
decreased when autophagy is promoted [54]. In this study, Western
blot results indicated that the ratio of LC3-II/LC3-I and expression
level of beclin-1 were enhanced by Ang II, which could confirm that
there was an increasing number of autophagosomes. Interestingly,
Ang II enhanced autophagy in podocytes, and N-MPG down-
regulated autophagy in Ang II-treated podocytes.
Autophagy is a homeostatics cytoprotective mechanism, as
different types of cells have unequal capacity against stress envi-
ronment [55]. However, a massive and uncontrolled autophagy can
result in cell death, which is known as autophagic cell death [56]. To
further elucidate the protective function of autophagy, which could
be altered by regulating autophagy level, MTT assay was used to
measure the effects of autophagy regulators on Ang II-induced cell
death. We employed the rapamycin as inducer and 3-MA as in-
hibitor of autophagy. Rapamycin can enhance the level of auto-
phagy by inhibiting mTOR, which is a negative regulator of
autophagy induction [57]. As autophagy inhibitor, 3-MA can inhibit
autophagosome formation through blocking class III phosphatidy-
linositol 3-kinase [25]. According to the cell viability analysis, it was
found that rapamycin rescued cell death from Ang II, however, 3-
MA aggravated Ang II-induced cell death. The data clearly
showed that up-regulated autophagy could reduce Ang II-induced
cell death. It appears that the induction of autophagy may be a
default mechanism to prevent Ang II-induced podocyte injury.
Oxidative stress is an imbalance between ROS production and
scavenging, resulting in tissue and cell oxidative damage. During
oxidative stress, the ROS level is higher than the capacity of the
antioxidant machinery, thus proteins, lipids and DNA are oxidized.
Autophagy, as a highly regulated process, is related to long-lived
proteins and distinct organelles [58,59]. Autophagy involves
sequestration of proteins and organelles in autophagosomes, which
directs sequestered proteins and organelles to lysosomes [60]. It
was well known that, there is a close relationship between oxida-
tive stress and autophagy. Several studies have shown that oxida-
tive stress plays an important role in the induction of autophagy
[58,61,62]. However, the mechanism of ROS induced autophagy is
not well understood. The majority of research indicated that
elevated cytosolic Ca2þ concentrations were shown to promote the
autophagic process [63e65]. The activation of calmodulin (CaM)
activates CaMKKb, followed by AMPK-dependent inhibition of
mTOR [66]. Moreover, as a fast and potent intracellular Ca2þ buffer,
BAPTA-AM could prevent the induction of autophagy, indicating
the importance of cytosolic Ca2þ [67]. In this study, we found that
Ang II induced ROS could regulate TRPC6 channels to mediate Ca2þ
influx. Therefore, we suggested that Ang II induced ROS, then
induced autophagy in podocytes via activating TRPC6 currents.
However, there is a significant crosstalk between autophagy and
ROS. In that regard, several studies reported that there were several
signaling pathways, such as Akt/mTOR pathway and p38-NF-kB
pathway, which played a potential role in the autophagy/ROS
signaling [68,69]. Obviously, a further study is needed to investigate
any clear signaling network between those. Moreover, in this study,
we found that Ang II could induce both cell death and protective
autophagy in podocytes via ROS generation. It will be interesting to
investigate the underlying mechanisms of Ang II-induced cell
death, like necrosis, apoptosis.
5. Conclusion
The present study showed that Ang II could induce oxidative
stress in podocytes. The increase of ROS level activated autophagy
through enhancing TRPC6 mediated Ca2þ influx. Autophagy could
protect against Ang II-induced podocytes death. By regulating
TRPC6 channels and autophagy may become a new way to relieve
Ang II-induced renal injury. It may become a new targeted therapy
to slow the development of glomerular injury.
Competing interests
The authors have declared that no competing interest exists.
Acknowledgments
This work was supported by grant from the National Natural
Science Foundation of China (81571804, 81771979) and Tianjin
Research Program of Application Foundation and Advanced Tech-
nology (14JCZDJC35000).
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