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Autophagy protects podocytes from sublytic complement induced injury
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Research article
Autophagy protects podocytes from sublytic complement induced injury Qianying Lv a, Fengjie Yang a, Kun Chen b, Yu Zhang a,n a b
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 10 February 2016 Accepted 12 February 2016
Podocyte injury induced by sublytic complement attack is the main feature of membranous nephropathy (MN). This study aimed at investigating the impact of sublytic complement attack-related autophagy on podocyte injury in vitro. Here, we show that sublytic complement attack enhances MPC5 podocyte autophagy in vitro. Inhibition of autophagy by treatment with 3-methyladenine (3-MA) significantly increased sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but decreased the survival and adhesion of MPC5 podocytes. In contrast, promotion of autophagy by treatment with rapamycin mitigated sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but increased the survival and adhesion of MPC5 podocytes. These data suggest that autophagy may protect podocytes from sublytic complement attack-induced injury in vitro. & 2016 Elsevier Inc. All rights reserved.
Keywords: Autophagy Podocyte Sublytic C5b-9 Complement
1. Introduction Membranous nephropathy (MN) is one of the most common causes of idiopathic nephrotic syndrome. Although about 30% of the patients with MN may undergo spontaneous remission, most of them can progress into end-stage renal failure within 10 years. The pathogenesis of MN is characterized by the deposits of immunocomplex in the lamina rara externa of the glomerular basement membrane (GBM), leading to membrane-like thickness of capillary wall. During the process of MN, podocytes, the glomerular visceral epithelial cells, are damaged because immunocomplex can activate complement cascade by inserting sublytic quantities of C5b-9 into their membranes [1]. Prolonged podocyte injury can accelerate the pathogenesis of kidney diseases, such as glomerulosclerosis [2]. However, the pathological process of podocyte injury and physiological regulation has not been clarified. Autophagy is a highly conserved lysosomal pathway involved in the recycling of cytosol and the removal of superfluous or damaged organelles. Autophagy regulates the survival, differentiation, development and homeostasis of many types of cells. Dysregulated autophagy contributes to the pathogenesis of many diseases, including cancer, neurodegeneration, heart disease and n Correspondence to: Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1095, Jiefang Ave., Wuhan 430030, P. R. China. E-mail address: [email protected] (Y. Zhang).
certain kidney diseases [3]. Autophagy is important to degrade large structures, and critical for the cellular refreshing [4], particularly for quiescent and terminally differentiated cells, such as neurons and glomerular epithelial cells [5]. A previous study has shown high levels of autophagic process in podocytes in animals with MN, passive Heymann nephritis [6]. Inhibition of autophagy deteriorates the pathogenesis of these diseases with severer clinical symptoms, such as proteinuria, extensive foot-process effacement, and loss of podocyte. Accordingly, we hypothesize that autophagy may protect podocytes from the progression of MN. In this study, we employed a cellular model to mimic MN in vitro to determine the role of autophagy in podocyte injury during the process of MN.
2. Materials and methods 2.1. Cell culture Immortalized mouse podocyte cells (MPC5) were kindly provided by Dr. Peter Mundel (Massachusetts General Hospital, Boston, Massachusetts, USA) and were cultured, as previously described [7]. Briefly, podocytes were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, USA). To propagate, the cells were cultured at 33 °C and treated with 10 U/ ml of mouse recombinant γ-interferon (IFNγ, Pepro Tech, England) to stimulate their proliferation. When they reached at 80–90%
http://dx.doi.org/10.1016/j.yexcr.2016.02.009 0014-4827/& 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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confluence, podocytes were harvested, washed and cultured in a plate coated with type I collagen at 37 °C for at least 12 days to allow their differentiation. The differentiated cells were used for the following experiments. 2.2. Induction of sublytic C5b-9 (sC5b-9) attack in podocytes A cell model of MN was established by inducing sC5b-9 attack in podocytes. Podocytes (5 105 cells/well) were incubated at 37 °C for 1 h with 1:100 diluted rabbit anti-MPC5 sera, prepared as described previously [8] and exposed to different dilutions (1:20– 1:400) of human sera from healthy subjects (a complement source) at 37 °C for 1 h. Heat-inactivated human sera served as negative controls. After being centrifuged, the supernatants of incubated podocytes were collected and the concentrations of lactate dehydrogenase (LDH) released by dead cells were determined by measuring the values of optical density (OD) at absorbance of 490 nm using a LDH assay kit, according to the manufacturers' instruction (Biyuantian Biology and Technology, China). The cytolysis rates in individual samples were calculated by the formula, Cytolysis (%)¼ (OD of experimental LDH release – Background OD values)/(Maximum OD values-Background OD values) 100%. A cytolysis rate of r5% was defined as sublytic attack [9]. To induce or inhibit autophagy, podocytes were pre-treated with rapamycin (10 ng/ml) or 3-MA (5 mmol/L, Sigma-Aldrich) for 1 h and treated with rabbit anti-MPC5 sera. Accordingly, there were seven groups of cells, including sC5b-9, sC5b-9/rapamycin, sC5b-9/3-MA, negative control with heat-inactivated human sera, 3-MA alone, vehicle DMSO and untreated control. 2.3. Western blot The levels of LC3-I, LC3-II and p62 in the different groups of cells were determined by Western blotting. Briefly, the cell lysates (30 mg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% fat-free dry milk, the membranes were incubated with rabbit anti-LC3, rabbit anti-p62 (Cell Signaling, Danvers, MA) and rabbit anti-GAPDH (Epitomics, USA). The bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, USA) and visualized using an enhanced chemiluminescent reagent. The relative levels of target to control GAPDH were analyzed by densitometric scanning using WCIF ImageJ software.
groups of podocytes were fixed with pre-warmed 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Subsequently, the cells were stained with rhodamine phalloidin (1:20, Cytoskeleton, USA) for 30 min at room temperature. The cells were evaluated using a confocal microscope (Olympus). 2.6. Cell viability assay The viability of cells was measured by MTT assay. Approximately 2 103 cells were cultured in 96-well plates and treated in triplicate with various reagents (see above) for 48 h. During the last 4 h incubation, the cells were exposed to MTT (5 mg/ml, Sigma-Aldrich) and the resulting formazan in each well was dissolved in 200 μl DMSO, followed by measuring the absorbance at 570 nm using a microplate reader (BioTek, USA). The survival rates of different groups of cells were calculated by (OD values of the experimental samples/OD values of the control) 100%. 2.7. In vitro adhesion assay The impact of autophagy on adhesion of podocytes was determined by adhesion assay, as described previously [11]. Briefly, differentiated podocytes (2 103 cells/well) were cultured in triplicate in complete medium in 96-well plates that had been precoated with collagen I (10 μg/ml) or 1% bovine serum albumin (as control) at 37 °C for 1 h. After being washed with PBS to remove the unbound cells, the adhered cells were fixed in formalin. The numbers of adhesive cells in individual groups were examined by two independent researchers in a blinded manner. The data was expressed as the mean 7SD of each group (12 independent biological replicates) from three separate experiments. 2.8. Apoptosis assay The percentages of apoptosis in the different groups of cells were determined using an FITC-Annexin V/PI Apoptosis Detection Kit, according to the manufacturers' instruction (Nanjing KeyGen Biotech, Nanjing, China). Briefly, the different groups of cells (5 105/tube) were stained in duplicate with FITC-Annexin V/PI for 15 min in the dark and the percentages of apoptotic podocytes were characterized by flow cytometry in a FACScan flow cytometer (BD Biosciences, USA) using Cell Quest TMPro software (BD Biosciences, USA). 2.9. Statistical analysis
2.4. Cell morphology analysis The different groups of podocytes were stained with Wright– Giemsa staining solution (Sigma-Aldrich) and examined under an optical microscope (Olympus, Japan). TEM was also performed, as described previously [10]. The different groups of podocytes were fixed sequentially with 3% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in acetone, and embedded in Epon812. The ultra-thin (80 nm) cellular sections were stained with uranyl acetate/lead citrate and visualized using a Hitachi H-600IV electron microscope (Hitachi Instrument, Japan, 10,000).
Data are present as the means 7SD. All experiments were performed at least 3 times. The difference among the groups was analyzed ANOVA and posthoc Bonferroni test and the difference between two groups was determined by Student's t-test using SPSS version 13.0 statistical software. A p value of o0.05 was considered statistically significant.
3. Results 3.1. Successful induction of sublytic complement attack in podocytes
2.5. Fluorescent staining and confocal microscopy The different groups of podocytes in 6-well plate were stained with 0.05 mmol/L monodansylcadaverine (MDC, Sigma-Aldrich) at 37 °C for 20 min, fixed in 4% paraformaldehyde for 10 min and examined for intracellular autophagic vacuole using a fluorescence microscope (Olympus, Japan). To stain the cytoskeletal proteins of F-actin, the different
The IgG antibodies against membrane surface antigens on podocytes in the subepithelial space form subepithelial immune complexes to trigger activation of the complement cascade and podocyte injury. Podocyte injury is a hallmark of MN, associated with proteinuria and nephrotic syndrome in the clinic. To induce a sC5b-9 attack, podocytes were treated with rabbit anti-MPC5 sera and exposed to different diluted human sera (a source of
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 1. Preparation of sublytic complement attack on podocytes. MPC5 podocytes were treated with rabbit anti-MPC5 sera and different dilutions of human sera (a complement source) and the cytotoxicity of podocytes was determined by LDH assay. Data are expressed as the mean% 7 SD from three separate experiments. The cells were incubated with 1:160 diluted human sera, which resulted in o5% cytotoxicity.
complement) to mimic the characteristics of MN. The complement-mediated podocyte injury was determined by measuring the levels of LDH. We found that control cells with heat-inactivated human sera had 2.5 70.5% of cytolysis while treatment with antibodies at 1:160 dilution and different dilutions of human sera caused varying levels of podocyte cytolysis in a complement dosedependent manner (Fig. 1). Treatment with 1:160 dilutions of human sera caused r5% of podocyte cytolysis and this concentration of sera was used for the following experiments. 3.2. Sublytic C5b-9 attack promotes autophagy in podocytes To evaluate the effect of sC5b-9 attack on autophagy, podocytes were treated with 1:160 diluted normal rabbit sera alone as the control, antibodies and 1:160 diluted human sera as the sC5b-9 attack, antibodies and heat-inactivated human sera (1:160) as the complement inactivation control for 48 h. Subsequently, the different groups of cells were stained with MDC and the autophagic vacuoles in the cells were detected by a confocal microscope. There was obviously more blue punctate staining in the sC5b-9 treated cells than in the control cells (Fig. 2A), which reduced in the heat-inactivated human sera-treated cells. Similarly, TEM analysis revealed that there were more autophagosomes (double membrane encapsulated portions of the cytoplasm and/or organelles) in the sC5b-9 treated cells than in the control cells, but autophagosomes were not detectable in the heat-inactivated human sera-treated cells and the normal rabbit sera treated cells (Fig. 2B). Furthermore, the relative ratios of LC3-II to LC3-I was significantly greater in the sC5b-9 treated cells than in the control cells, the heat-inactivated human sera-treated cells and the normal rabbit sera treated cells. Simultaneously, the levels of p62 significantly decreased in the sC5b-9 treated cells (Fig. 2C). These three lines of evidence clearly indicated that sC5b-9 attack induced autophagy in podocytes. 3.3. Inhibition of autophagy enhances sC5b-9 induced injury To determine the consequence of autophagy, podocytes were pre-treated with, or without 3-MA to inhibit autophagy and induced sC5b-9 attack, following examining their morphology, adhesion and apoptosis. Treatment with 3-MA inhibited the conversion of LC3-I to LC3-II, significantly reduced the ratios of LC3-II
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to LC3-I in podocytes, but increased the levels of p62, demonstrating that 3-MA inhibited podocyte autophagy (Fig. 3A). Histologically, while well-differentiated control podocytes exhibited in an arborized-shape with long processes and spindle-like projections, sC5b-9 attack caused the differentiated MPC5 cells to look smaller in size, round in shape, with fewer branches, but without the normal stress fibrotic structure (Fig. 3B and C). These indicated that sC5b-9 attack induced morphological and stress fiber changes in podocytes. Furthermore, pre-treatment with 3-MA obviously enhanced the damage induced by the sC5b-9 attack in podocytes. In contrast, treatment with inactive complement did not induce podocyte damage. In addition, treatment with 3-MA also deteriorated the reduced capacity of podocytes to adhere on collagen or BSA-coated plates (Fig. 3D) and increased the percentages of apoptotic cells (Fig. 3E). As a result, pre-treatment with 3-MA significantly reduced the survival rates of podocytes in vitro (Fig. 3F). Together, our data indicated that inhibition of autophagy enhanced the sC5b-9 attack-induced injury of podocytes in vitro. 3.4. Rapamycin attenuates podocyte injury caused by sC5b-9 attack Rapamycin can target the mTORC1 and promote autophagy [12]. To look additional line of evidence, podocytes were pretreated with 10 ng/ml of rapamycin and subjected to sC5b-9 attack, followed by examining their autophagy, morphology, apoptosis and survival in vitro. Treatment with rapamycin significantly increased the ratios of LC3-II to LC3-I and decreased the levels of p62, demonstrating that rapamycin enhanced autophagy in podocytes (Fig. 4A). Pre-treatment with rapamycin mitigated the sublytic C5b-9 attack-induced morphological changes in podocytes (Fig. 4B and C). Finally, pre-treatment with rapamycin enhanced the survival and adhesion rates, but reduced the percentages of apoptotic podocytes in vitro (Fig. 4D–F). These data further indicated that autophagy protected from sC5b-9 attack-induced podocyte injury in vitro.
4. Discussion Complement-mediated sublethal podocyte injury plays a major role in the pathogenesis of MN. Podocytes are highly differentiated cells, and they have a limited ability of proliferation. Thus, maintenance of podocyte healthy and function is crucial for the function of glomerular filtration and the kidney. A previous study has shown that mature podocytes have high levels of autophagic activity [13]. In this study, we examined the regulatory effect of autophagy on sublytic complement attack-induced podocyte injury in vitro. We found that sublytic complement attack enhanced autophagy in MPC5 podocytes in vitro. Evidentially, increased ratios of LC3-II to LC3-I proteins, decreased levels of p62, and more autophagosomes, the hallmarks of autophagy [14], were detected in sublytic complement-treated MPC5 cells, but not inactive complement-treated cells. Interestingly, we also observed that sublytic complement attack induced injury-related morphological changes and cytoskeleton redistribution and apoptosis of MPC5 podocytes. Furthermore, sublytic complement attack significantly reduced the adhesion and survival rates of MPC5 podocytes. These data indicated that sublytic complement attack induced podocyte injury and enhanced podocyte autophagy. A previous study has shown that complement C5b-9 membrane attack complex can induced ER stress in both cultured podocytes and an animal model of passive Heymann nephritis [15]. A recent study supports the link of ER stress and autophagy [16]. Therefore, we speculate that sC5b-9 attack may induce autophagy by inducing ER stress response. Given that enhanced autophagy activity is usually associated with supporting the survival of many types of cells, the
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 2. Sublytic C5b-9 attack stimulates autophagy activity in podocytes. MPC5 podocytes were treated with anti-MPC5 sera and 1:160 diluted human sera for 48 h and the contents of autophagic vacuoles (blue spots) were stained with MDC, followed by examination using confocal microscopy. Furthermore, the cells were subjected to TEM examination and the relative levels of LC3-II/I and p62 expression were characterized by Western blot assays. Data are representative images or expressed as the mean7 SD from three separate experiments. (A) MDC staining of autophagic vacuoles. A total of 100 cells from each group were evaluated by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with punctate blue staining (white arrows mark positive cells). n¼ 3. (B) TEM analysis of autophagosomes (black arrows mark autophagosomes). Bar graph represents the quantitative data of autophagic vacuoles (n¼ 3). (C) Western blot analysis of the relative levels of LC3 and p62 expression. *Po 0.05 vs the control group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; NRS, negative control with normal rabbit sera.
enhanced autophagy by sublytic complement attack may reflect a feedback negative regulation of autophagy on sublytic complement attack-induced podocyte injury. Interestingly, we found that treatment with 3-MA to inhibit autophagy significantly increased sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but decreased the survival and adhesion of MPC5 podocytes. Our data were consistent with previous observations that mice lacking autophagy-related protein 5 (Atg5) in podocytes were more susceptible to glomerular disease [17] and that autophagy promotes the recovery from puromycin amino nucleoside (PAN)-induced injury
[18]. In contrast, promotion of autophagy by treatment with rapamycin mitigated sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but increased the survival and adhesion of MPC5 podocytes. The protective effects of rapamycin on podocyte injury were in agreement with previous findings that treatment with a low dose of rapamycin inhibits podocyte injury and the development of diabetic nephropathy [19] and that rapamycin reduces puromycin amino nucleotideinduced podocyte injury by increasing autophagy activity [20]. It is possible that low dose of rapamycin inhibits partial mTOR1 activity and reduces its inhibitory effect on autophagy in podocytes [21,22].
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 3. Inhibition of autophagy enhances sC5b-9-induced podocyte injury. MPC5 podocytes were pre-treated with, or without, 3-MA and subjected to sublytic complement attack for 48 h. Subsequently, the relative levels of LC3-II/I and p62 expression were determined by Western blot and the cells were stained with Wright–Giemsa or rhodamine phalloidin, followed by examining under a light or fluorescent microscope, respectively. In addition, the viability, adhesion and apoptosis of MPC5 cells were determined by MTT, adhesion and apoptosis assays, respectively. Data are representative images or expressed as the mean 7SD of each group from three separate experiments. (A) The relative levels of LC3-II/I and p62 (n¼ 3). (B) Wright–Giemsa staining of cells ( 400). (C) The F-actin distribution in cells. One hundred cells were evaluated from each group by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with disrupted cytoskeleton (n¼ 3). (D) The adhesion rate of podocytes on collagen-I and BSA. (E) The percentages of apoptotic cells, including early apoptotic cells (Annexin V-positive/PI-negative) and late apoptotic cells (Annxin V-positive/PI-positive). (F) The survival rates of cells. *Po 0.05 vs the control group, ΔP o0.05 vs the sC5b-9 group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; 3-MA þ sC5b-9, 3-MA plus sC5b-9; 3-MA, 3-MA alone.
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Fig. 4. Promotion of autophagy by Rapamycin attenuates sC5b-9-induced podocyte injury. MPC5 podocytes were pre-treated with, or without, rapamycin and subjected to sublytic complement attack for 48 h. Subsequently, the relative levels of LC3-II/I and p62 expression were determined by Western blot and the cells were stained with Wright–Giemsa or rhodamine phalloidin, followed by examining under a light or fluorescent microscope, respectively. In addition, the viability, adhesion and apoptosis of MPC5 cells were determined by MTT, adhesion and apoptosis assays, respectively. Data are representative images or expressed as the mean7 SD of each group from three separate experiments. (A) The relative levels of LC3-II/I and p62 (n¼ 3). (B) Wright–Giemsa staining of cells ( 400). (C) The F-actin distribution in cells. One hundred cells were evaluated from each group by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with disrupted cytoskeleton (n¼ 3). (D) The adhesion rate of podocytes on collagen-I and BSA. (E) The percent of apoptotic cells, including early and late apoptotic cells. (F) The survival rates of cells. *P o 0.05 vs the control group, ΔP o0.05 vs the sC5b-9 group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; DMSO, DMSO alone; rapamycin þ sC5b-9, rapamycin plus sC5b-9.
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Collectively, our findings and those of others support the notion that autophagy protects podocytes from sublytic complement attack-induced injury. We are interested in further investigating molecular mechanisms by which autophagy regulates sublytic complement attack-induced podocyte injury. Our data indicated that sublytic complement attack induced podocyte injury and enhanced autophagy. While inhibition of autophagy significantly enhanced sublytic complement attack-induced podocyte injury, promotion of autophagy mitigated sublytic complement attack-induced podocyte injury in vitro. These data clearly indicated that autophagy protected podocytes from sublytic complement attack-induced injury in vitro. Hence, our data may provide new insights into the regulatory effect of autophagy on podocyte injury. Therapeutic promotion of autophagy may delay the progression of complement-dependent podocytopathies.
Conflict of interest The authors declare no conflicts of interest.
Fund This work was supported by the Grant from the National Natural Science Foundation of China (No. 81100514).
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Research article
Autophagy protects podocytes from sublytic complement induced injury Qianying Lv a, Fengjie Yang a, Kun Chen b, Yu Zhang a,n a b
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
art ic l e i nf o
a b s t r a c t
Article history: Received 7 August 2015 Received in revised form 10 February 2016 Accepted 12 February 2016
Podocyte injury induced by sublytic complement attack is the main feature of membranous nephropathy (MN). This study aimed at investigating the impact of sublytic complement attack-related autophagy on podocyte injury in vitro. Here, we show that sublytic complement attack enhances MPC5 podocyte autophagy in vitro. Inhibition of autophagy by treatment with 3-methyladenine (3-MA) significantly increased sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but decreased the survival and adhesion of MPC5 podocytes. In contrast, promotion of autophagy by treatment with rapamycin mitigated sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but increased the survival and adhesion of MPC5 podocytes. These data suggest that autophagy may protect podocytes from sublytic complement attack-induced injury in vitro. & 2016 Elsevier Inc. All rights reserved.
Keywords: Autophagy Podocyte Sublytic C5b-9 Complement
1. Introduction Membranous nephropathy (MN) is one of the most common causes of idiopathic nephrotic syndrome. Although about 30% of the patients with MN may undergo spontaneous remission, most of them can progress into end-stage renal failure within 10 years. The pathogenesis of MN is characterized by the deposits of immunocomplex in the lamina rara externa of the glomerular basement membrane (GBM), leading to membrane-like thickness of capillary wall. During the process of MN, podocytes, the glomerular visceral epithelial cells, are damaged because immunocomplex can activate complement cascade by inserting sublytic quantities of C5b-9 into their membranes [1]. Prolonged podocyte injury can accelerate the pathogenesis of kidney diseases, such as glomerulosclerosis [2]. However, the pathological process of podocyte injury and physiological regulation has not been clarified. Autophagy is a highly conserved lysosomal pathway involved in the recycling of cytosol and the removal of superfluous or damaged organelles. Autophagy regulates the survival, differentiation, development and homeostasis of many types of cells. Dysregulated autophagy contributes to the pathogenesis of many diseases, including cancer, neurodegeneration, heart disease and n Correspondence to: Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1095, Jiefang Ave., Wuhan 430030, P. R. China. E-mail address: [email protected] (Y. Zhang).
certain kidney diseases [3]. Autophagy is important to degrade large structures, and critical for the cellular refreshing [4], particularly for quiescent and terminally differentiated cells, such as neurons and glomerular epithelial cells [5]. A previous study has shown high levels of autophagic process in podocytes in animals with MN, passive Heymann nephritis [6]. Inhibition of autophagy deteriorates the pathogenesis of these diseases with severer clinical symptoms, such as proteinuria, extensive foot-process effacement, and loss of podocyte. Accordingly, we hypothesize that autophagy may protect podocytes from the progression of MN. In this study, we employed a cellular model to mimic MN in vitro to determine the role of autophagy in podocyte injury during the process of MN.
2. Materials and methods 2.1. Cell culture Immortalized mouse podocyte cells (MPC5) were kindly provided by Dr. Peter Mundel (Massachusetts General Hospital, Boston, Massachusetts, USA) and were cultured, as previously described [7]. Briefly, podocytes were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, USA). To propagate, the cells were cultured at 33 °C and treated with 10 U/ ml of mouse recombinant γ-interferon (IFNγ, Pepro Tech, England) to stimulate their proliferation. When they reached at 80–90%
http://dx.doi.org/10.1016/j.yexcr.2016.02.009 0014-4827/& 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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confluence, podocytes were harvested, washed and cultured in a plate coated with type I collagen at 37 °C for at least 12 days to allow their differentiation. The differentiated cells were used for the following experiments. 2.2. Induction of sublytic C5b-9 (sC5b-9) attack in podocytes A cell model of MN was established by inducing sC5b-9 attack in podocytes. Podocytes (5 105 cells/well) were incubated at 37 °C for 1 h with 1:100 diluted rabbit anti-MPC5 sera, prepared as described previously [8] and exposed to different dilutions (1:20– 1:400) of human sera from healthy subjects (a complement source) at 37 °C for 1 h. Heat-inactivated human sera served as negative controls. After being centrifuged, the supernatants of incubated podocytes were collected and the concentrations of lactate dehydrogenase (LDH) released by dead cells were determined by measuring the values of optical density (OD) at absorbance of 490 nm using a LDH assay kit, according to the manufacturers' instruction (Biyuantian Biology and Technology, China). The cytolysis rates in individual samples were calculated by the formula, Cytolysis (%)¼ (OD of experimental LDH release – Background OD values)/(Maximum OD values-Background OD values) 100%. A cytolysis rate of r5% was defined as sublytic attack [9]. To induce or inhibit autophagy, podocytes were pre-treated with rapamycin (10 ng/ml) or 3-MA (5 mmol/L, Sigma-Aldrich) for 1 h and treated with rabbit anti-MPC5 sera. Accordingly, there were seven groups of cells, including sC5b-9, sC5b-9/rapamycin, sC5b-9/3-MA, negative control with heat-inactivated human sera, 3-MA alone, vehicle DMSO and untreated control. 2.3. Western blot The levels of LC3-I, LC3-II and p62 in the different groups of cells were determined by Western blotting. Briefly, the cell lysates (30 mg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% fat-free dry milk, the membranes were incubated with rabbit anti-LC3, rabbit anti-p62 (Cell Signaling, Danvers, MA) and rabbit anti-GAPDH (Epitomics, USA). The bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, USA) and visualized using an enhanced chemiluminescent reagent. The relative levels of target to control GAPDH were analyzed by densitometric scanning using WCIF ImageJ software.
groups of podocytes were fixed with pre-warmed 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Subsequently, the cells were stained with rhodamine phalloidin (1:20, Cytoskeleton, USA) for 30 min at room temperature. The cells were evaluated using a confocal microscope (Olympus). 2.6. Cell viability assay The viability of cells was measured by MTT assay. Approximately 2 103 cells were cultured in 96-well plates and treated in triplicate with various reagents (see above) for 48 h. During the last 4 h incubation, the cells were exposed to MTT (5 mg/ml, Sigma-Aldrich) and the resulting formazan in each well was dissolved in 200 μl DMSO, followed by measuring the absorbance at 570 nm using a microplate reader (BioTek, USA). The survival rates of different groups of cells were calculated by (OD values of the experimental samples/OD values of the control) 100%. 2.7. In vitro adhesion assay The impact of autophagy on adhesion of podocytes was determined by adhesion assay, as described previously [11]. Briefly, differentiated podocytes (2 103 cells/well) were cultured in triplicate in complete medium in 96-well plates that had been precoated with collagen I (10 μg/ml) or 1% bovine serum albumin (as control) at 37 °C for 1 h. After being washed with PBS to remove the unbound cells, the adhered cells were fixed in formalin. The numbers of adhesive cells in individual groups were examined by two independent researchers in a blinded manner. The data was expressed as the mean 7SD of each group (12 independent biological replicates) from three separate experiments. 2.8. Apoptosis assay The percentages of apoptosis in the different groups of cells were determined using an FITC-Annexin V/PI Apoptosis Detection Kit, according to the manufacturers' instruction (Nanjing KeyGen Biotech, Nanjing, China). Briefly, the different groups of cells (5 105/tube) were stained in duplicate with FITC-Annexin V/PI for 15 min in the dark and the percentages of apoptotic podocytes were characterized by flow cytometry in a FACScan flow cytometer (BD Biosciences, USA) using Cell Quest TMPro software (BD Biosciences, USA). 2.9. Statistical analysis
2.4. Cell morphology analysis The different groups of podocytes were stained with Wright– Giemsa staining solution (Sigma-Aldrich) and examined under an optical microscope (Olympus, Japan). TEM was also performed, as described previously [10]. The different groups of podocytes were fixed sequentially with 3% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in acetone, and embedded in Epon812. The ultra-thin (80 nm) cellular sections were stained with uranyl acetate/lead citrate and visualized using a Hitachi H-600IV electron microscope (Hitachi Instrument, Japan, 10,000).
Data are present as the means 7SD. All experiments were performed at least 3 times. The difference among the groups was analyzed ANOVA and posthoc Bonferroni test and the difference between two groups was determined by Student's t-test using SPSS version 13.0 statistical software. A p value of o0.05 was considered statistically significant.
3. Results 3.1. Successful induction of sublytic complement attack in podocytes
2.5. Fluorescent staining and confocal microscopy The different groups of podocytes in 6-well plate were stained with 0.05 mmol/L monodansylcadaverine (MDC, Sigma-Aldrich) at 37 °C for 20 min, fixed in 4% paraformaldehyde for 10 min and examined for intracellular autophagic vacuole using a fluorescence microscope (Olympus, Japan). To stain the cytoskeletal proteins of F-actin, the different
The IgG antibodies against membrane surface antigens on podocytes in the subepithelial space form subepithelial immune complexes to trigger activation of the complement cascade and podocyte injury. Podocyte injury is a hallmark of MN, associated with proteinuria and nephrotic syndrome in the clinic. To induce a sC5b-9 attack, podocytes were treated with rabbit anti-MPC5 sera and exposed to different diluted human sera (a source of
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 1. Preparation of sublytic complement attack on podocytes. MPC5 podocytes were treated with rabbit anti-MPC5 sera and different dilutions of human sera (a complement source) and the cytotoxicity of podocytes was determined by LDH assay. Data are expressed as the mean% 7 SD from three separate experiments. The cells were incubated with 1:160 diluted human sera, which resulted in o5% cytotoxicity.
complement) to mimic the characteristics of MN. The complement-mediated podocyte injury was determined by measuring the levels of LDH. We found that control cells with heat-inactivated human sera had 2.5 70.5% of cytolysis while treatment with antibodies at 1:160 dilution and different dilutions of human sera caused varying levels of podocyte cytolysis in a complement dosedependent manner (Fig. 1). Treatment with 1:160 dilutions of human sera caused r5% of podocyte cytolysis and this concentration of sera was used for the following experiments. 3.2. Sublytic C5b-9 attack promotes autophagy in podocytes To evaluate the effect of sC5b-9 attack on autophagy, podocytes were treated with 1:160 diluted normal rabbit sera alone as the control, antibodies and 1:160 diluted human sera as the sC5b-9 attack, antibodies and heat-inactivated human sera (1:160) as the complement inactivation control for 48 h. Subsequently, the different groups of cells were stained with MDC and the autophagic vacuoles in the cells were detected by a confocal microscope. There was obviously more blue punctate staining in the sC5b-9 treated cells than in the control cells (Fig. 2A), which reduced in the heat-inactivated human sera-treated cells. Similarly, TEM analysis revealed that there were more autophagosomes (double membrane encapsulated portions of the cytoplasm and/or organelles) in the sC5b-9 treated cells than in the control cells, but autophagosomes were not detectable in the heat-inactivated human sera-treated cells and the normal rabbit sera treated cells (Fig. 2B). Furthermore, the relative ratios of LC3-II to LC3-I was significantly greater in the sC5b-9 treated cells than in the control cells, the heat-inactivated human sera-treated cells and the normal rabbit sera treated cells. Simultaneously, the levels of p62 significantly decreased in the sC5b-9 treated cells (Fig. 2C). These three lines of evidence clearly indicated that sC5b-9 attack induced autophagy in podocytes. 3.3. Inhibition of autophagy enhances sC5b-9 induced injury To determine the consequence of autophagy, podocytes were pre-treated with, or without 3-MA to inhibit autophagy and induced sC5b-9 attack, following examining their morphology, adhesion and apoptosis. Treatment with 3-MA inhibited the conversion of LC3-I to LC3-II, significantly reduced the ratios of LC3-II
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to LC3-I in podocytes, but increased the levels of p62, demonstrating that 3-MA inhibited podocyte autophagy (Fig. 3A). Histologically, while well-differentiated control podocytes exhibited in an arborized-shape with long processes and spindle-like projections, sC5b-9 attack caused the differentiated MPC5 cells to look smaller in size, round in shape, with fewer branches, but without the normal stress fibrotic structure (Fig. 3B and C). These indicated that sC5b-9 attack induced morphological and stress fiber changes in podocytes. Furthermore, pre-treatment with 3-MA obviously enhanced the damage induced by the sC5b-9 attack in podocytes. In contrast, treatment with inactive complement did not induce podocyte damage. In addition, treatment with 3-MA also deteriorated the reduced capacity of podocytes to adhere on collagen or BSA-coated plates (Fig. 3D) and increased the percentages of apoptotic cells (Fig. 3E). As a result, pre-treatment with 3-MA significantly reduced the survival rates of podocytes in vitro (Fig. 3F). Together, our data indicated that inhibition of autophagy enhanced the sC5b-9 attack-induced injury of podocytes in vitro. 3.4. Rapamycin attenuates podocyte injury caused by sC5b-9 attack Rapamycin can target the mTORC1 and promote autophagy [12]. To look additional line of evidence, podocytes were pretreated with 10 ng/ml of rapamycin and subjected to sC5b-9 attack, followed by examining their autophagy, morphology, apoptosis and survival in vitro. Treatment with rapamycin significantly increased the ratios of LC3-II to LC3-I and decreased the levels of p62, demonstrating that rapamycin enhanced autophagy in podocytes (Fig. 4A). Pre-treatment with rapamycin mitigated the sublytic C5b-9 attack-induced morphological changes in podocytes (Fig. 4B and C). Finally, pre-treatment with rapamycin enhanced the survival and adhesion rates, but reduced the percentages of apoptotic podocytes in vitro (Fig. 4D–F). These data further indicated that autophagy protected from sC5b-9 attack-induced podocyte injury in vitro.
4. Discussion Complement-mediated sublethal podocyte injury plays a major role in the pathogenesis of MN. Podocytes are highly differentiated cells, and they have a limited ability of proliferation. Thus, maintenance of podocyte healthy and function is crucial for the function of glomerular filtration and the kidney. A previous study has shown that mature podocytes have high levels of autophagic activity [13]. In this study, we examined the regulatory effect of autophagy on sublytic complement attack-induced podocyte injury in vitro. We found that sublytic complement attack enhanced autophagy in MPC5 podocytes in vitro. Evidentially, increased ratios of LC3-II to LC3-I proteins, decreased levels of p62, and more autophagosomes, the hallmarks of autophagy [14], were detected in sublytic complement-treated MPC5 cells, but not inactive complement-treated cells. Interestingly, we also observed that sublytic complement attack induced injury-related morphological changes and cytoskeleton redistribution and apoptosis of MPC5 podocytes. Furthermore, sublytic complement attack significantly reduced the adhesion and survival rates of MPC5 podocytes. These data indicated that sublytic complement attack induced podocyte injury and enhanced podocyte autophagy. A previous study has shown that complement C5b-9 membrane attack complex can induced ER stress in both cultured podocytes and an animal model of passive Heymann nephritis [15]. A recent study supports the link of ER stress and autophagy [16]. Therefore, we speculate that sC5b-9 attack may induce autophagy by inducing ER stress response. Given that enhanced autophagy activity is usually associated with supporting the survival of many types of cells, the
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 2. Sublytic C5b-9 attack stimulates autophagy activity in podocytes. MPC5 podocytes were treated with anti-MPC5 sera and 1:160 diluted human sera for 48 h and the contents of autophagic vacuoles (blue spots) were stained with MDC, followed by examination using confocal microscopy. Furthermore, the cells were subjected to TEM examination and the relative levels of LC3-II/I and p62 expression were characterized by Western blot assays. Data are representative images or expressed as the mean7 SD from three separate experiments. (A) MDC staining of autophagic vacuoles. A total of 100 cells from each group were evaluated by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with punctate blue staining (white arrows mark positive cells). n¼ 3. (B) TEM analysis of autophagosomes (black arrows mark autophagosomes). Bar graph represents the quantitative data of autophagic vacuoles (n¼ 3). (C) Western blot analysis of the relative levels of LC3 and p62 expression. *Po 0.05 vs the control group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; NRS, negative control with normal rabbit sera.
enhanced autophagy by sublytic complement attack may reflect a feedback negative regulation of autophagy on sublytic complement attack-induced podocyte injury. Interestingly, we found that treatment with 3-MA to inhibit autophagy significantly increased sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but decreased the survival and adhesion of MPC5 podocytes. Our data were consistent with previous observations that mice lacking autophagy-related protein 5 (Atg5) in podocytes were more susceptible to glomerular disease [17] and that autophagy promotes the recovery from puromycin amino nucleoside (PAN)-induced injury
[18]. In contrast, promotion of autophagy by treatment with rapamycin mitigated sublytic complement attack-induced changes in the injury-related morphology, stress fiber, and podocyte apoptosis, but increased the survival and adhesion of MPC5 podocytes. The protective effects of rapamycin on podocyte injury were in agreement with previous findings that treatment with a low dose of rapamycin inhibits podocyte injury and the development of diabetic nephropathy [19] and that rapamycin reduces puromycin amino nucleotideinduced podocyte injury by increasing autophagy activity [20]. It is possible that low dose of rapamycin inhibits partial mTOR1 activity and reduces its inhibitory effect on autophagy in podocytes [21,22].
Please cite this article as: Q. Lv, et al., Autophagy protects podocytes from sublytic complement induced injury, Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.02.009i
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Fig. 3. Inhibition of autophagy enhances sC5b-9-induced podocyte injury. MPC5 podocytes were pre-treated with, or without, 3-MA and subjected to sublytic complement attack for 48 h. Subsequently, the relative levels of LC3-II/I and p62 expression were determined by Western blot and the cells were stained with Wright–Giemsa or rhodamine phalloidin, followed by examining under a light or fluorescent microscope, respectively. In addition, the viability, adhesion and apoptosis of MPC5 cells were determined by MTT, adhesion and apoptosis assays, respectively. Data are representative images or expressed as the mean 7SD of each group from three separate experiments. (A) The relative levels of LC3-II/I and p62 (n¼ 3). (B) Wright–Giemsa staining of cells ( 400). (C) The F-actin distribution in cells. One hundred cells were evaluated from each group by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with disrupted cytoskeleton (n¼ 3). (D) The adhesion rate of podocytes on collagen-I and BSA. (E) The percentages of apoptotic cells, including early apoptotic cells (Annexin V-positive/PI-negative) and late apoptotic cells (Annxin V-positive/PI-positive). (F) The survival rates of cells. *Po 0.05 vs the control group, ΔP o0.05 vs the sC5b-9 group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; 3-MA þ sC5b-9, 3-MA plus sC5b-9; 3-MA, 3-MA alone.
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Fig. 4. Promotion of autophagy by Rapamycin attenuates sC5b-9-induced podocyte injury. MPC5 podocytes were pre-treated with, or without, rapamycin and subjected to sublytic complement attack for 48 h. Subsequently, the relative levels of LC3-II/I and p62 expression were determined by Western blot and the cells were stained with Wright–Giemsa or rhodamine phalloidin, followed by examining under a light or fluorescent microscope, respectively. In addition, the viability, adhesion and apoptosis of MPC5 cells were determined by MTT, adhesion and apoptosis assays, respectively. Data are representative images or expressed as the mean7 SD of each group from three separate experiments. (A) The relative levels of LC3-II/I and p62 (n¼ 3). (B) Wright–Giemsa staining of cells ( 400). (C) The F-actin distribution in cells. One hundred cells were evaluated from each group by two independent researchers in a blinded manner. The quantitative data represents the percentages of podocytes with disrupted cytoskeleton (n¼ 3). (D) The adhesion rate of podocytes on collagen-I and BSA. (E) The percent of apoptotic cells, including early and late apoptotic cells. (F) The survival rates of cells. *P o 0.05 vs the control group, ΔP o0.05 vs the sC5b-9 group. sC5b-9, sublytic C5b-9 group; complement-I, negative control with heat-inactivated human sera; control, untreated control cells; DMSO, DMSO alone; rapamycin þ sC5b-9, rapamycin plus sC5b-9.
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Collectively, our findings and those of others support the notion that autophagy protects podocytes from sublytic complement attack-induced injury. We are interested in further investigating molecular mechanisms by which autophagy regulates sublytic complement attack-induced podocyte injury. Our data indicated that sublytic complement attack induced podocyte injury and enhanced autophagy. While inhibition of autophagy significantly enhanced sublytic complement attack-induced podocyte injury, promotion of autophagy mitigated sublytic complement attack-induced podocyte injury in vitro. These data clearly indicated that autophagy protected podocytes from sublytic complement attack-induced injury in vitro. Hence, our data may provide new insights into the regulatory effect of autophagy on podocyte injury. Therapeutic promotion of autophagy may delay the progression of complement-dependent podocytopathies.
Conflict of interest The authors declare no conflicts of interest.
Fund This work was supported by the Grant from the National Natural Science Foundation of China (No. 81100514).
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