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Accepted Manuscript
Mitophagy regulates macrophage phenotype in diabetic nephropathy rats
Yu Zhao, Yinfeng Guo, Yuteng Jiang, Xiaodong Zhu, Yuqiu Liu, Xiaoliang Zhang
PII:
S0006-291X(17)32066-1
DOI:
10.1016/j.bbrc.2017.10.088
Reference:
YBBRC 38705
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 12 October 2017
Accepted Date: 16 October 2017
Please cite this article as: Y. Zhao, Y. Guo, Y. Jiang, X. Zhu, Y. Liu, X. Zhang, Mitophagy regulates
macrophage phenotype in diabetic nephropathy rats, Biochemical and Biophysical Research
Communications (2017), doi: 10.1016/j.bbrc.2017.10.088.
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ACCEPTED MANUSCRIPT
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Mitophagy regulates macrophage phenotype in diabetic nephropathy rats
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Yu Zhao, Yinfeng Guo, Yuteng Jiang, Xiaodong Zhu, Yuqiu Liu Xiaoliang Zhang *.
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Key words: mitophage; macrophage; M1/M2 phenotype; diabetic nephropathy
ABSTRACT
Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic
nephropathy (DN). Macrophages mainly exhibit M1 phenotype, whcih contributes to the
inflammation and fibrosis in DN. Studies indicate that autophage plays an important role in
M1/M2 activation. However, the effect of mitophage on M1/M2 macrophage phenotype
transformation in DN is unknown. This study investigates the role of mitophage on macrophage
polarization in DN. In vivo experiments show that macrophages are exhibited to M1 phenotype
and display a lower level of mitophagy in the kidney of streptozocin (STZ)-induced diabetic rats.
Additionally, inducible nitric oxide synthase (iNOS) expression is positive correlated with the P62
expression, while negative correlated with LC3. Electronic microscope analysis shows
mitochondria swelling, crista decrease and lysosome reduction in DN rats compared with NC rats.
In vitro, RAW264.7 macrophages switch to M1 phenotype under high glucose conditions.
Mitophagy is downregulated in such high glucose induced M1 macrophages. Furthermore,
macrophages tend to switch to the M1 phenotype, expressing higher iNOS and TNF-α when
impaire mitophagy by 3-MA. Rapamycin, an activator of mitophagy, signifcantly blocks
high-glucose induced M1 makers (iNOS and TNF-α) expression, meanwhile enhances M2 makers
(MR and Arg-1) expression. These results demonstrate that mitophage participates in the
regulation of M1/M2 macrophage phenotype in diabetic nephropathy.
Introduction
Diabetic nephropathy (DN) is a chronic inflammatory disease which is characterized by
inflammatory cell infiltration and proinflammatory factor over expression 1. Such inflammation
can drive the classical markers of fibrosis and structural remodeling 2. As a result, resolution of
inflammatory reaction might be critical towards achieving a cure for DN.
Our interest in the regulation of macrophage phenotype in kidney stems from the
overwhelming evidence that diabetic nephropathy (DN) is a chronic inflammatory disease 3.
Macrophages are the key inflammatory cells that play an important role in the progression of DN
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. Presently, mounting results tend to indicate that macrophages phenotype activation finally
determines the evolvement and prognosis of renal injury 4-6. Classically activated macrophages
(M1) have proinflammatory effectors and lead to tissue injury, while, alternatively activated
macrophages (M2) play a role in the inhibition of inflammation and promotion of tissue repair 7, 8.
Our previous studies have shown that kidney tissue injury in DN can be eliminated by promoting
macrophage transformation to M2 phenotype 9, 10. Therefore, further study of the intracellular
mechinsm of macrophage phenotype regulation is highly important.
Autophagy is an intracellular degradation system associated with maintenance of cellular
homeostasis 11, 12. It plays a key role in macrophages function regulation 13. In the current study,
we find that increased macrophages M1 phenotype infiltration is characterized by elevated
expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) in
glomeruli and interstitium of streptozocin (STZ)-induced DN rats. Meanwhile, mitochondria
swelling, crista decrease, lysosome redution and autophagosome reduction are observed in DN
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Institute of Nephrology, Zhong Da Hospital, Southeast University, School of Medicine, Nanjing, Jiangsu,210009,
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rats by electronic microscope. Interstingly, We find a significant correlation between mitophage
and macrophage M1/M2 phenotype in DN rats.
Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria
and maintains cellular homeostasis14-17. Its functions in kidney, as a kind of selective autophagy, is
under intense investigation. Among these investigations, Chuang P.Y. find mitophage is inhibited
in podocyte and, at the same time, the degree of glomerular sclerosis and proteinuria are
significantly increased, in adriamycin-induced glomerulonephritis animal model 18. In vitro, the
expression of LC3 and the colocalization of LC3 and mitochondrial fluorescent probe reduced,
renal tubule apoptosis increased in High glucose stimulated-renal tubular epithelial cells 19. Many
of these studies, such as aboves, suggest that mitophagy abnormalities in innate renal cells
deteriorates the kidney disease. However, until recently, few evidences show the involvement of
mitophagy in pathogenesis of marophage phenotype in DN. In our study, mitophagy level is
decreased in kidney tissue of DN rats. Furthermore, we have observed mitophagy could regulate
macrophage phenotype in diabetic nephropathy rats, which first connects mitophagy and
macrophage phenotype in the DN reaserch field.
Results
1.Animal characteristic
Both blood glucose and body weight did not differ between the two groups at the beginning
of the study. After the injection of STZ, the DN rats developed overt diabetes with higher blood
glucose levels and lower body weights compared with the NC rats beginning at the 4th week after
injection until the end of the experiment (P<0.05, Fig. 1A). Expectedly, the DN rats exhibited
increased proteinuria from the 4th to the 24th week (P<0.05, Fig. 1B). As shown in Fig 1C, DN
rats exhibited severe morphological kidney lesions.The enlargement of the glomerular surface area
and the expansion of the glomerular mesangial matrix increased in DN rats from week 4 to 24.
The baseline and final parameters of DN rats are presented in Table 1. Scr, BUN and the
kidney-to-body weight ratios significantly increased in DN rats at all time points. We also
examined the renal infiltration of CD68-positive macrophages in STZ-induced DN rats. As shown
in Fig 1D, immunohistochemical staining of the CD68 antigen revealed that DN rats exhibited
significant macrophage infiltration in the interstitium at week 8, 12, 18, 24 after STZ induction
compared with non-DN rats. As time went on, the increase in the number of infiltrating
macrophages was even more remarkable.
2. A novel link between autophagy and macrophage M1 phenotype in DN rats
To begin to clarify whether there was diabetic-induced autophagy changes and macrophages
phenotype transformation, we analyzed several specific markers of macrophages phenotype and
autophagy by western blot using protein extracted from kidney tissue. As shown in Figs 1E and 1F
compared with normal control rats, DN rats exhibited significant macrophages infiltration in renal
tissue at week 8 after STZ induction, as illustrated by protein expression of CD68. The peak level
of microtubule-associated protein light chain 3 (LC3), the marker protein of autophagosome, was
achieved at week 8 after STZ injection. Then, LC3 is decreasing gradually along with the progress
of DN (Fig 1G). On the contrary, there is a opposite trend for P62, the marker protein of
autophagosome degradation (Fig 1H). Meanwhile, iNOS, representative of M1 macrophages, was
increased in DN at week 8, and continued to increase until week 24 (Fig 1I). MR, specific markers
for M2 macrophage, had no differences (Fig 1J). The protein expression ratio of iNOS to MR
considered as the proportion of M1/M2 macrophages was also calculated. As showed in Fig 1K,
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the ratio of iNOS/MR was increased gradually. Furthermore, there was a negative correlation
between protein expression of iNOS and LC3 (r=-0.619,P<0.05, Fig 1L),In contrast, protein
expression of iNOS was strong positively correlated with P62 (r=0.837,P<0.05, Fig 1M). Taken
together, the above data demonstrated that there was a strongly link between autophagy and
macrophage phenotype in the renal tissue of DN rats.
3. Mitochondrial morphology changes and mitopagy in DN rats renal tissue.
As shown in Fig.1N and 1O, electronic microscope results showed that mitochondria
swelling, decrease or disappearance of mitochondria crista in kidney of DN rats at week 18. while,
most mitochondria structure was normal in normal control rats. Meanwhile, the complete
autophagic process including the formation and extension of isolating membrane, autophagosome
formation, lysosome chemotaxis to autophagosome , fusion between the mitochondria
autophagosome and lysosome, and degradation in the autolysosome was observed in normal
control rats (Fig.1 P-T). But the complete autophagic process was unobserved and the number of
autophagosomes and lysosome reduced in DN rats (Fig.1U and 1V). In addition, we also found
that the colocalization of VDAC and LC3 decreased in DN rats compared with normal control rats
(Fig.1W).
4. RAW264.7 macrophages switched to M1 phenotype and mitophagy downregulated under
high glucose conditions
To further certify the relationship between macrophage phenotype and mitophagy, we
assessed expression of the marker protein of macrophage phenotype and mitophagy in RAW264.7
cells by western blot and Immunofluorescence Staining. Firstly, RAW264.7 was stimulated with
glucose in a dose and time dependent manner. As showed in Fig.2, the expressions of LC3 and
Beclin-1 significantly decreased from 9h to 36h. On the contrary, the expressions of P62, TNF-α,
iNOS were significantly increased. The expressions of MR,Arg-1, specific markers for M2
macrophages, had no differences. Meanwhile, the expressions of VDAC, the marker for
mitochondria, significantly decreased. We found that the peak level of iNOS and TNF-α
expression in RAW264.7 cells was achieved at 24h intervention. Thus, we used the 30 mM
glucose concentration and 24h time period in later experiments. Then, the colocalization of VDAC
and LC3 was clarified by confocal immunofluorescence in NC group, while co-localization
proteins was evidently reduced in HG group (Fig.3L). These results indicate that mitophagy was
downregulated in high glucose induced M1 macrophages.
5. Regulation of mitophagy changes RAW264.7 macrophage M1/M2 phenotype in high
glucose condition
To determine whether mitophage regulates high glucose-induced macrophage phenotype.
Firstly, in order to select the most appropriate mitophage inhibitor (3-MA) and activator
(rapamycin) intervention concentration, RAW264.7 was stimulated with 3-MA in a dose and
rapamycin in a dose dependent manner. The activity of cells was measured with CCK8. The data
showed that the viabilities of macrophages cultured with 1.0 Mm and 1.5 mM 3-MA had no
differences, but the viabilities of macrophages cultured with 2 mM, 2.5 mM, 5.0 mM obviously
decreased compared with 1.0 mM and 1.5 mM (P<0.05) . Similarly, the viabilities of macrophages
cultured with 50nM and 100 nM RAPA had no differences, but the viabilities of macrophages
cultured with 150 nM, 200 nM, 500 nM obviously decreased compared with 50 nM and 100 nM
(P<0.05) (Fig.3A and 3B) .Thus, we used the 1.5 mM 3-MA concentration and 100 nM rapamycin
in later experiments.
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Then, RAW264.7 cells were cultured with 30mM glucose in 24h with or without 3-MA and
rapamycin intervention. Mitophagy-related proteins expression of LC3, Beclin-1, p62, VDAC, M1
marker (iNOS, TNF-α), and M2 marker (MR, Arg-1) were detected by immunofluorescence and
Western Blot. The result of Western Blot indicated that macrophages tend to switch to the M1
phenotype, expressing higher iNOS and TNF-α when impaired mitophagy by 3-MA. Rapamycin
signifcantly blocked high-glucose induced M1 makers (iNOS and TNF-α) expression, meanwhile
enhanced M2 makers (MR and Arg-1) expression (Figs. 3C-K). The result was consistent with
immunofluorescence (Figs. 3L-N).
At last, the intact mitophagy process was observed in RAW264.7 cells by Electron
Microscopy (Fig. 4).
Discussion
In vivo, mitophage level is decreased while macrophage switching to M1 phenotype. It
suggests the relevance between mitophage and macrophage phenotype in the kidney of DN rats. In
vitro, mitophage activation significantly promote high glucose-induced M1 macrophage to switch
to M2 phenotype. On the contrary, mitophage inhibition significantly promote macrophages to
switch to M1 phenotype. Our results prove that regulating mitophagy can affect macrophage
phenotypic transformation in DN rats.
How the mitophagy level change in DN models is unclear and the findings are inconsistent
and controversial in relevant studies before. Autophage maker, Beclin-1, is increased 8 weeks after
STZ injection in rats 20. However, LC3-Ⅱ/LC3-Ⅰ protein ratio is significantly decreased at the
12th week after STZ injection in rats 21. Consistent with the above two results, mitophage also
have the same change. PINK1 protein, a mitophagy regulation factor, is increased in the renal
cortex of early diabetes (four weeks after diabetes induction) in a rat model of STZ diabetes 22.
This result suggests that mitophagy activity may be elevated in the early stage of diabetes. But
Ming find that the PINK1 protein expression is decreased and fragmented mitochondrias
accumulate in renal tubular epithelial cell of STZ-induced diabetic mice (afetr eight weeks) 19.The
author speculated the reason of above phenomenon is clearance mechanism disturbance of
dysfunctional mitochondria. Our datas show that the mitophage is activated before 8 weeks, then
inhibited after 8weeks. We considerate the process, which starts in early diabetes, that could clear
impaired mitochondria by the activated mitophagy in kidney become overwhelmed gradually. It
leads to impairment of mitophagy, accumulation of fragmented mitochondria and cell death with
the DN progresses. This concept is consistent with Higgins’ opinion 23.
This is the first study that focus on the effect of mitophage on macrophage phenotype under
high glucose condition. Recently, a number of groups have demonstrated a close connection
between autophagy and macrophage24-27. Duan et al. show that increasing of autophagy response
effectively protect macrophages from stress-induced cytotoxicity and improve macrophages
phagocytic capability 28. Guo and his colleagues find that a regulator of autophagy activation
up-regulate autophagy, promote impairment of cutaneous wound healing and enhance the
inflammatory response in db/db mice 29. Inhibition of autophagy regulates the microglial
phenotype which promotes M2 phenotype to switch to the M1 phenotype 30. Consistent with the
results of studies above, we also suggest the possibility that autophage enhances
anti-inflammatory function of macrophage by regulating macrophage phenotype. In our study, we
not only observe the effect of autophage on macrophage phenotype, but also further insight into
mitophage which can affect macrophage phenotype under high glucose condition. Moreover,
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further studies are needed to identify the exact biological role of mitophage on macrophage
phenotype.
There is still much more studies about autophagy awaiting to be explored. Mitophagy is a
dynamic process 31. Recently studies find that increase of autophagosome can not reflect the
autophagy level in nature. It can only reflect the induction of autophagy and the inhibition of
autophagosome. From the point of dynamic process, number of autophagosome is affected by the
formation and removal. Accurate and comprehensive assessment of autophagy not only includes
the detection of autophagosome formation, but also includes autolysosome removal 32. Thus, we
suggest a method that combination of dynamic and static detection shoud be used to obtain
reliable data which can effectively explain the occurrence of autophagy. However, the limitations
of current researches on autophagy include that reagents mainly focussing on autophagy
generation regulator, such as 3-methyl adenine (3-MA), wortmannin, LY294002 and rapamycin,
lack of specificity reagents promoting fusion of autophagosome and lysosome and the degradation
of autophagy lysosomes33. Meanwhile, currently, the researches mainly focus on the reagents of
regulating autophagy, but specific mitophagy regulator needs further study and discovery 31, 34.
In summary, the current study demonstrated the effect of mitophage on M1/M2 phenotype in
DN. Further researchs are needed to gain insights into the molecular mechanisms that connect
mitophagy and macrophage phenotype in DN kidney.
Materials and methods
Animal experiments
All animal care and experimental protocols were in compliance with the Animal Management
Rules of the Ministry of Health of the People’s Republic of China.
Six-week-old healthy male Sprague–Dawley (SD) rats weighing 200–220 g were obtained
from Shanghai Slac Laboratory Animal (Shanghai, China). After one week of acclimation, the rats
were randomly divided into two groups: (1) NC (normal control group, n = 10), (2) DN (DN rats,
n = 40). DN was induced with a single intraperitoneal injection of STZ (sigma) dissolved in 0.1 M
citrate buffer (pH4.5) at 58 mg/kg ,while the control rats received only the 0.1 M citrate buffer
solution. Three days later, the diabetic state was confirmed by measuring tail blood glucose (BG)
level. Rats with a BG level over 16.7 mmol/L were considered as diabetic rats. Blood glucose was
monitored with the blood glucose monitoring system (Bayer) using one drop of tail blood. 24h
urine samples were collected in metabolic cages. Blood samples were taken for measuring
biochemical parameters, and kidneys were collected for histological examination and molecular
assays.
Cellular experiments
Mouse macrophage cell line RAW264.7 was purchased from Shanghai Bogoo Biotechnology
Company (Shanghai, China), were routinely cultured in RPMI 1640 media (containing 11.1 mM
glucose) supplemented with 10% fetal bovine serum (Sciencell, USA) and incubated at 37°C in
5% CO2.
Serum and urine chemistry analyses
Blood urea nitrogen (BUN), creatinine (Scr) were analyzed by an automatic biochemistry
analyzer (Hitachi, Japan).Urinary proteinuria was measured using an ELISA Kit (Jiancheng,
Nanjing, China) according to the manufacturer’s method .
Renal histology analyses
Kidney sections were stained with periodic acid-Schiff (PAS) and masson trichrome staining
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then examined by light microscopy (magnification, ×400) in a blinded manner. We analyzed 20
randomly selected non-overlapping PAS or masson-stained glomeruli from each rat.
Electron Microscopy
Tissues blocks (1 mm3) and cells fixed in 2% glutaraldehyde in 0.1 M potassium phosphate
sodium buffer at 4◦C. After postfixation with 2% osmium tetroxide, the samples were dehydrated
in a series of graded ethanol solutions. Ethanol was then substituted for propylene oxide, and the
samples were embedded in epoxy resin. Ultrathin sections were double stained with uranyl acetate
and lead citrate. Sections were examined using a JEM1200EX electron microscope (HITACHI,
JAPAN) at 80 keV.
Immunohistochemistry
Immunohistochemistry was performed on paraffin sections using a microwave-based antigen
retrieval technique. Sections were incubated with primary mouse anti-CD68 (Santa Cruz,
SC-59103) followed by appropriate secondary antibody incubation. The immunostaining was
visualized using diaminoben zidine tetrahydrochloride, and the slides were counterstained with
hematoxylin.
Immunofluorescence Staining
The sections of renal tissues or RAW264.7 cells were fxed and blocked. Then slides were
incubated with primary antibodies: anti-LC3 antibody (Sigma, L7543), anti-VDAC antibody
(Abcam, ab14734), anti-iNOS antibody (Abcam, ab15323), anti-MR antibody (Abcam, ab64693),
respectively, overnight at 4 °C. Then, renal tissues or cells were washed and incubated with
appropriate secondary antibody in darkness for 4h or 30min at room temperature. After staining
nuclei with DAPI, renal tissues or cells were visualized using a IX70 fluorescence microscope
(OLYMPUS, Japan).
Western blot
Proteins that from renal tissues or RAW264.7 cells were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose
membrane.After blocking, membranes were incubated with the primary antibodies against LC3
(abcam, ab63817), Beclin-1(abcam, ab217179), P62 (Cell Signaling, 5114), iNOS (Abcam,
ab15323), TNF-α (Santa, SC-1350), MR (Abcam, ab64693), Arg-1 (Santa Cruz, SC-20150),
VDAC (santa curze, sc-390996) and β-actin at 4°C overnight. After three washes with PBST/5
min, the nitrocellulose membranes were incubated with horseradish peroxidase-conjugated
secondary antibody for 1-2 h. Finally, the membranes were visualized with an enhanced
chemiluminescence advanced system (GE Healthcare, UK) and captured on X-ray film.
Immunoreactive bands were quantified with densitometry using Image J software (NIH, USA)
Statistical analysis
All experiments were repeated at least three times. The data were expressed as the mean and
standard deviation (SD) and were analyzed with SPSS 19.0. The differences of LC3, Beclin-1,
P62, VDAC, iNOS, TNF-α, MR, Arg-1, VDAC among different groups were analyzed by
one-way ANOVA. A difference was considered significant if the value was less than 0.05.
Funding
This work was supported by grants from the National Natural Science Foundation of China
(No. 81570612 and No. 81370826), the Clinical Medical Research Center Program of Jiangsu
Province (BL2014080). Address all correspondence and requests for reprints to Xiaoliang Zhang,
Institute of Nephrology, Zhong Da Hospital, Southeast University, School of Medicine, Nanjing,
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Jiangsu 210009, China, E-mail Address: tonyxlz@163.com, Tel: 86–25–83262441, Fax:
86–25–83285132.
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Figure Legends
Table 1. Parameters of the experimental groups of rats
BG: blood glucose; KW/BW: kidney weight/body weight; Scr: serum creatinine; BUN: blood urea nitrogen. Date
are presented as mean±SD (n = 8). *P<0.05 vs NC at the same time point, respectively.
Fig.1. The general parameters and the change of mitophagy and macrophage phenotype in
experimental animals.
A, Body weight. B, Proteinuria. C, PAS and masson stains of rats' renal tissues as indicated (×400). D
Immunohistochemical staining of CD68+ macrophages in interstitium (×400). E–M, Representative Western
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mitochondria. Scale bar indicates 0.5 µm. P-T, Intact mitochondrial autophagy is observed in normal rat kidney
tissue. P: The isolation film formation and extension. Q: Mitochondria autophagosome. R: Lysosome chemotaxis
to mitochondria autophagosome. S: Fusion between the mitochondria autophagosome and lysosome. T:
autophagolysosome. Scale bar indicates 0.5µm . U-V, Mitochondrial autophagy is observed in DN rat kidney tissue.
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Red arrow indicates Mitochondria autophagosome. White arrow indicates autophagolysosome. Black arrow
indicates lysosome. Scale bar indicates 0.5 µm. W, Mitophagy is observed by immunolabeling and confocal
microscopy imaging detection in rat kidney tissue (×400).
Fig.2. The effect of high glucose on macrophage phenotype and mitophagy in RAW264.7 cells.
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A-I, RAW264.7 cells were stimulated with glucose in a time dependent manner. Representative Western blotting
analysis (A) and quantification of LC3 (B), Beclin-1 (C), P62 (D), TNF-α (E), iNOS (F), MR (G) , Arg-1 (H),
VDAC (I). J-R, RAW264.7 cells were stimulated with glucose in a dose dependent manner. Representative
Western blotting analysis (J) and quantification of LC3 (K), Beclin-1 (L), P62 (M), TNF-α (N), iNOS (O), MR (P),
Arg-1 (Q), VDAC (R). β-Actin was used as an internal control. Data are presented as mean± SD (n = 3). *P<0.05
vs NC, respectively.
Fig.3. The effect of autophagy regulator on macrophage phenotype and mitophagy in RAW264.7
cells.
A and B, Viabilities of macrophages exposed to indicated culture conditions were determined by CCK8 assay. Data
are presented as mean± SD (n = 3). *P<0.05 vs 0mM or 0nM group, respectively.#P < 0.05 vs. 1.0mM or 50nM
group. C-K, Western blotting analysis (C) and quantification of quantification of LC3 (D), Beclin-1 (E), P62 (F),
TNF-α (G), iNOS (H), MR (I), Arg-1 (G), VDAC (K). L-N, Mitochondrial autophagy is observed by
immunolabeling and confocal microscopy imaging detection in RAW264.7 cells under different intervention
conditions. Data are presented as mean± SD (n = 3). *P<0.05 vs NC group, #P<0.05 vs HG group.
Fig.4. Intact mitochondrial autophagy is observed in RAW264.7 cells.
A: The isolation film formation and extension. B: Mitochondria autophagosome. C: Lysosome chemotaxis to
mitochondria autophagosome. D: Fusion between the mitochondria autophagosome and lysosome. E:
autophagolysosome. A and B, Scale bar indicates 0.1 µm; C-D, Scale bar indicates 0.2 µm.
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(r=0.837,P<0.05) (M) expression. N-O, Mitochondrial morphology in renal tissue of rats. White arrows indicate
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ratio (K). L and M, correlations between M1 macrophage phenotypes and LC3 (r=-0.619,P<0.05) (L) or P62
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blotting analysis (E) and quantification of CD68+ (F) and LC3 (G), P62 (H), iNOS (I), MR (G) and iNOS to MR
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NC
DN(8w)
*
DN(12w)
30.02±2.47
DN(18w)
*
DN(24w)
32.43±3.19 *
BG (mmol/L)
6.24±0.81
31.37±3.33
KW/BW (mg/g)
3.04±0.37
5.26±0.43 *
6.56±0.82 *
6.63±0.51 *
6.72±0.96 *
Scr (µmol/L)
32.01±8.34
52.13±6.06*
86.52±9.95 *
92.13±6.42 *
100.13±5.77 *
BUN (mmol/L)
5.93±0.88
14.10±2.50 *
16.61±2.37 *
17.05±2.08 *
17.73±3.43 *
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31.16±3.65
*
Table 1
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Highlights
1. A novel link between autophagy and macrophage M1 phenotype in DN
rats.
downregulated under high glucose conditions.
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2. RAW264.7 macrophages switched to M1 phenotype and mitophagy
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phenotype in high glucose condition.
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3. Regulation of mitophagy changes RAW264.7 macrophage M1/M2
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