Mol Cell Biochem (2014) 394:261–273 DOI 10.1007/s11010-014-2102-7

Crosstalk between protective autophagy and NF-jB signal in high glucose-induced podocytes Miaomiao Wei • Zhigui Li • Zhuo Yang

Received: 2 January 2014 / Accepted: 15 May 2014 / Published online: 24 June 2014 Ó Springer Science+Business Media New York 2014

Abstract Despite a great deal of recent studies focused on the pivotal role of autophagy in maintaining podocyte energy homeostasis, the mechanisms of autophagy in regulating transcriptional factors under high glucose (HG) condition are not fully understood. Here, we evaluated the effect of HG on nuclear factor-kappa B (NF-jB) signaling and autophagic process. The results showed that HG promoted autophagy in podocytes. Bafilomycin A1 (Baf A1) further enhanced this effect, but 3-methyadenine (3-MA) inhibited it. The proautophagic effects of HG manifested in the form of enhanced podocyte expression of light chain 3 (LC3)-II. In these cells, blockade of NF-jB signal by ammonium pyrrolidinethiocarbamate constrained in effectively reducing LC3-II up-regulation and increasing podocyte apoptosis. Furthermore, the autophagy inhibitors, such as Baf A1 and 3-MA, significantly enhanced HGinduced NF-jB activation and increased apoptosis. Thus, we conclude that the accumulation of autophagosomes results from enhancement of the autophagic flux, but not the blockage of autophagosome–lysosome fusion by HG. We also prove that HG-induced apoptosis, autophagy, and NF-jB signal are in a close crosstalk through a yet undetermined mechanism in podocytes. Keywords Nuclear factor-kappa B  Autophagy  Apoptosis  Podocytes  High glucose

Miaomiao Wei and Zhigui Li have contributed equally to this work. M. Wei  Z. Li  Z. Yang (&) College of Medicine, Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China e-mail: [email protected]

Introduction Diabetic nephropathy (DN) is a major microvascular complication in patients with type 1 and type 2 diabetes mellitus (T1DM and T2DM), which is characterized by progressive proteinuria due to compromised glomerular filtration barrier [1]. The morphologically peculiar podocyte and its slit diaphragm structure are of primary importance to the integrity of glomerular filtration barrier [2]. Podocyte loss precedes and predicts the occurrence of proteinuria [3], and may be an early pathological manifestation of DN [4, 5]. Therefore, understanding mechanisms of podocyte injury is of great significance for the prevention and treatment of kidney disease. Autophagy involves sequestration of proteins and cell organelles in autophagosomes (double-membrane structures), which directs them to lysosomes [6]. The formation of autophagosomes is dependent on the induction of several genes including light chain 3 (LC3), phosphatidylinositide 3-kinase, Beclin 1, and Atgs [7]. Recent reports have suggested that autophagy is upregulated and plays a protective role in kidney disease [8]. A more recent study demonstrated that the autophagy level was much higher in podocytes than that in other renal cells, and podocytes depended on autophagy to maintain homeostasis [9]. However, the functional significance of autophagy in podocyte dysfunction under diabetic condition has remained unknown. Autophagy and apoptosis constitute the two processes through which injured/aged cells or organelles are eliminated [10–14]. In several settings, autophagy is a mode of stress adaptation that suppresses apoptosis, whereas in other scenarios, autophagy provides an alternative pathway to cell death and is described as an autophagic cell death, or programmed cell death (PCD) type II. It appears that the

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same stimuli can induce either autophagy or apoptosis depending on the threshold [10]. Autophagy, a highly regulated lysosomal pathway involved in the recycling of cytosol and the removal of superfluous or damaged organelles, is essential for the survival, differentiation, development, and homeostasis of cells [15]. On the other hand, apoptosis removes damaged or unwanted cells. Since autophagy serves as a defense mechanism by clearing damaged cytoplasmic organelles [12], it plays an important role in the cellular refreshing, which is particularly important in quiescent and terminally differentiated cells [16]. At present, DN is considered as an inflammatory disease and the factors involved in the pathogenesis of DN are multifaceted [17]. An increasing number of studies have shown that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) is closely related to the pathophysiological mechanisms of diabetes. NF-jB signaling pathway is involved in the regulation of multiple cellular functions, such as apoptosis, autophagy, cellular proliferation, differentiation, metabolism, and adaptive and innate immunity responses. There is a substantial literature indicating that both NF-jB signaling and autophagy are reciprocally involved in the control of cellular survival, although the detailed characterization has remained elusive. Moreover, it seems that autophagy and apoptosis are controlled in cooperation with certain common regulatory proteins, for example, Bcl-2, Bcl-xl, and BNIP3 [18]. It is also known that autophagy is an important regulator of inflammatory responses, in particular via the inflammasomes. Currently, it is not known whether the regulation mechanisms of autophagy, apoptosis, and inflammasomes are linked to each other, and how this crosstalk can determine cell fate under DN condition. In the present study, we evaluated the effects of high glucose (HG) on the induction of autophagy and apoptosis in mouse podocytes. We also studied the crosstalk of NFjB signaling and autophagy in triggering cell apoptosis by inhibiting the NF-jB [using pyrrolidinethiocarbamate (PDTC)] and the autophagy process [using 3-methyladenine (3-MA) and Baf A1]. In addition, we determined the potential role of the crosstalk in HG-induced podocyte injury.

Materials and methods Materials RPMI 1640 culture medium was purchased from Life Technologies (Gibco BRL, Grand Island, NY, USA). Fetal bovine serum (FBS), D-glucose, Hoechst 33342, ammonium PDTC, 3-MA and 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT), and acridine orange were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1 (Baf A1) was purchased from Rubico, Germany. b-Actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LC3 antibody was purchased from MBL International (Woburn, MA, USA). Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis detection kit was from Bipec Biopharma Corporation (Massachusetts, MA, USA). The NF-jB activation-nuclear transportation kit was purchased from Beyotime Biotechnology (Nantong, China). All reagents were of the highest purity commercially available. Cell culture and drug treatment The conditionally immortalized mouse podocyte (CIMPs) line was kindly provided by Dr. Peter Mundel (Mount Sinai School of Medicine, New York, NY, USA) and was described previously [19]. Cells were cultured at the permissive temperature (33 °C) in RPMI-1640 medium (Gibco, USA) supplemented with 10 % FBS (Sigma, USA),10 U/ml recombinant murine interferon-c (Sigma, USA),100 U/ml penicillin G, and 100 mg/ml streptomycin in the presence of 5 % CO2. To induce differentiation, podocytes were grown under nonpermissive conditions at 37 °C for 10–14 days in the absence of interferon-c. After serum starvation for 24 h, cells were exposed to the high concentration of D-glucose (30 mM) in low-serum medium (2 % FBS) for the indicated time periods. The NF-jB inhibitor PDTC (4 lM) and specific inhibitors of autophagy, such as 3-MA (10 mM) and Baf A1 (150 and 300 nM) were administered 30 min before applying HG. All experiments were performed on differentiated podocytes. Cell viability assay CIMPs activity was assessed by MTT assay. This assay is a nonradioactive cell proliferation assay that identifies living cells, and is based on the cellular conversion of a tetrazolium salt into a formazan product, a chromophore, which can be quantified by spectrophotometry. Briefly, cells were plated at a density of 1 9 104 cells per well in 200 ll of RPMI 1640 medium on 96-well plates, and cultured for 24 h. Then, cells were exposed to 30 mM HG and incubated for 24 and 48 h. 3-MA (10 mM) and Baf A1 (150, 300 nM) were administered for 30 min and remained in contact with the cells subsequently for a 24–48 h exposure to the HG. As osmotic control, podocytes were cultured in medium containing 5.6 mmol/l D-glucose and 30 mmol/l D-mannitol (Man). After treatments, cells were treated with 500 lg/ml of MTT and cultured for 3 h in a CO2 incubator. Cells with a functional mitochondrial succinate dehydrogenase can

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convert MTT to formazan. The formazan crystals formed were solubilized in DMSO (Sigma) and measured with a microplate reader (Multiskan Mk3; Thermo Labsystems, Helsinki, Finland) at k = 580 nm. Each experiment was repeated at least three times.

observation. NF-jB p65 protein (green, magnification 4009) and cell nucleus (blue, magnification 4009) were photographed using a fluorescence microscope (FV1000SIX81; Olympus, Tokyo, Japan). All experiments were performed a minimum of three times.

Annexin V-FITC and propidium iodide (PI) staining

Acridine orange staining

CIMPs were pretreated with or without PDTC (4 lM) or specific inhibitors of autophagy for 30 min, and the inhibitors were remained in contact with the cells subsequently for a 24–48 h exposure to the HG at concentrations of 30 mM. After treatment, 1 9 106 cells were harvested and washed three times with PBS. For cell apoptosis analysis, cells were fixed in 400 ll of binding buffer and stained with 5 ll PI and 5 ll FITC-labeled Annexin V for 10 min at room temperature in the dark. Cell fluorescence was analyzed by the Cell Quest software (BD Biosciences).

Acridine orange is a fluorescent dye which stains acidic compartments (such as lysosomes and autolysosomes) by orange/red, while stains cytoplasm andnucleus by bright green. After 24 h of drug treatment, podocytes were washed with PBS and then stained with 1 lM acridine orange for 15 min at 37 °C. Excess acridine orange was washed away with PBS, and cells were immediately analyzed under a Leica TCS SP5 laser-scanning confocal microscope (magnification 8009). All experiments were performed a minimum of three times.

Hoechst 33342 staining

Immunofluorescence analysis

Apoptosis was measured by staining with Hoechst 33342. Differentiated podocytes were plated at a density of 5 9 104 cells/cm2 for Hoechst staining. After treatment, cells were fixed in 4 % paraformaldehyde and mounted in 10 lM Hoechst 33342 for 20 min. Then, cells were analyzed by the laser-scanning confocal microscope (FV1000S-IX81; Olympus, Tokyo, Japan). Apoptosis was defined as the presence of nuclear condensation on Hoechst staining, and the percentage of the cells with nuclear condensation was calculated on at least 300 consecutive cells (magnification 4009). All experiments were performed a minimum of three times.

CIMPs growing on glass coverslips were fixed in 4 % paraformaldehyde and permeabilized in 0.5 % Triton X-100 in PBS. Fixed cells were blocked in 10 % normal goat serum (NGS) in PBS before incubation with primary antibody diluted at 1:500 at 4 °C overnight. After extensive washing, slides were incubated 4 h at room temperature with 1:1,000 red fluorescent Alexa Fluor 594 dye-labeled anti-mouse IgG (Invitrogen, San Diego, CA). Coverslips were mounted in DAPI for 15 min. Finally, the fluorescent signals were examined using a Leica TCS SP5 laserscanning confocal microscope (magnification 8009). All experiments were performed a minimum of three times.

Determination of NF-jB activation-nuclear transportation

Western blotting assay

Translocation of NF-jB p65 from the cytoplasm to the nucleus was determined using the cellular NF-jB translocation assay (Beyotime Biotech) according to manufacturer’s instructions [20]. Briefly, podocytes were cultured in 12-well plates until reaching confluence and then pretreated with PDTC (4 lM), 3-MA (10 mM) and Baf A1 (150 and 300 nM) for 30 min. Then, the inhibitors were remained in contact with the cells subsequently for a 24–48 h exposured to the HG at concentrations of 30 mM. After that cells were fixed for 10 min at room temperature. After three washes with PBS, the fixed cells were blocked for 1 h to reduce non-specific binding. Next, cells were probed with the primary NF-jB p65 antibody for 1 h, followed by incubating with Alexa 488-conjugated (working dilution 1:1000) anti-rabbit IgG for 1 h and with 40 ,6-diamidino-2-phenylindole (DAPI) for 5 min before

The protein expression was measured by western blot analysis. Confluent CIMPs were washed three times with phosphate-buffered saline (PBS) and scraped in 200 ll RIPA lysis buffer. Following centrifugation at 12,000g at 4 °C for 5 min, proteins were extracted and ready for western blot analysis. The protein concentration of the supernatant was measured by the BCA protein assay (Beyotime Biotech). For western blot analysis, proteins were separated by 8 % reducing sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) by electroblotting. Non-specific background was blocked by incubating the membranes with 5 % non-fat dried milk for 1 h. Membranes were incubated overnight at 4 °C using anti-LC3 antibody (1:2,000, Woburn, MA, USA) and anti-beta-actin antibody (KangChen, Bio-Tech, China), and then incubated with the

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Mol Cell Biochem (2014) 394:261–273 b Fig. 1 High glucose-induced injury in CIMPs. CIMPs were treated

with or without 30 mM high glucose for a range of time periods. a The cell apoptotic morphological detection was assessed by staining with Hoechst 33342 after culturing in the presence of high glucose for 12, 24 and 48 h. Magnification 9400. b The percentage of apoptotic cells treated with high glucose by Hoechst 33342 staining. c The cell death was assessed by flow cytometry following staining with propidium iodide and annexin V after culturing in the presence of high glucose for 24, 48 and 72 h. Dot plots indicate intensity of annexin V-FITC fluorescence on the X-axis and PI fluorescence on the Y-axis. Percentages of cells presented in areas of respective quadrant profiles. d Apoptotic ratios (including the early and late apoptotic ratios) were analyzed by flow cytometry. e The cell viability was decreased gradually in a time-dependent manner. Man D-mannitol. Representative data are expressed as mean ± SD (N = 4); *P \ 0.05 versus control group, **P \ 0.01 versus control group

appropriate secondary antibody (goat anti-mouse, Pierce, New York, USA) at 1/5,000 dilution for 1 h at room temperature. In all experiments, protein loading was standardized by preliminary experiments in which actin expression was quantitated. Blots were then probed for protein of interest band intensity assessed by densitometry. Statistical analysis Results are shown as the mean ± SD, and N represents the number of experiments. Statistical analysis was performed using SPSS11.5 software (Chicago, IL, US). Pooled results from replicate quantitative experiments were compared by analysis of variance (ANOVA). Two-tailed P values of less than 0.05 were taken as indicating statistical significance.

Results High glucose inhibits cell viability and induces apoptosis in CIMPs To determine whether HG induces injury in CIMPs, cells were incubated in medium containing 30 mM HG for 24, 48, and 72 h, respectively. The cell viability was examined using the MTT assay, and results showed that HG reduced the cell viability compared to that of control cells (P \ 0.01, N = 4, Fig. 1e). The Hoechst dye was able to diffuse through intact membranes of podocytes and stain DNA. As shown in Fig. 1a, the nuclei exhibited dispersed and weak fluorescence in normal cells. In contrast, podocytes treated with different time periods of HG showed significantly condensed chromatin, some of which were assembled at the nuclear membrane, and showed the typical form of a half-moon, as white arrows pointed in Fig. 1a and b indicated that HG-induced podocyte apoptosis in a time-dependent manner (P \ 0.01, N = 4). The apoptosis of podocytes was tested by annexin V/PI staining

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assay, as shown in Fig. 1c and d. The apoptotic rates of podocytes were significantly increased from 5.86 ± 2.97 % in control group to 18.78 ± 4.82 % (P \ 0.01, N = 4, Fig. 1d) after 72 h exposure to HG. Effects of 3-methyladenine and bafilomycin A1 on high glucose-induced autophagy in podocytes To determine whether HG promotes autophagy, CIMPs were incubated in medium containing 30 mM HG for 24 and 48 h, respectively, followed by preparation of western blotting assay. Western blotting analysis showed that the expression of autophagy-related protein LC3-II was evident in HG-treated podocytes. Cumulative data of four sets of experiments were shown in Fig. 2c and d (P \ 0.01, N = 4). As a further verification of the activation effect of HG on autophagy, western blotting assay with an LC3 antibody was used to monitor the conversion of LC3-I to LC3-II after 3-MA or Baf A1 pretreatment. To determine the effect of 3-MA on autophagy, CIMPs were incubated in medium containing 30 mM HG with 10 mM 3-MA pretreatment for 24 and 48 h, respectively. 3-MA inhibited the formation of autophagosomes by inhibiting the activity of the conversion of MAP1LC3A-I to MAP1LC3A-II. As shown in Fig. 2c and d, the enhanced effect of HG was inhibited by 3-MA. Meanwhile, we observed higher levels of the protein in the presence of Baf A1 and HG, which further activated the conversion of LC3-I to LC3-II than in the presence of HG alone. With Baf A1, the degradation of autophagosomes was reduced, thus resulting in an accumulation of LC3-II. The conversion of LC-3 induced by HG was the cause of autophagy, whereas the accumulation of LC-3 was the effect of blocking autophagy by Baf A1. When both drugs were present at the same time, autophagy was induced by HG and blocked by Baf A1 at the end of the metabolic pathway. In this last condition, LC-3 accumulation was found in cells. By adding Baf A1 into the above HG study, the number of LC3-II was not consistent with the HG alone group, but showed a clear increase in LC3-I to LC3-II conversion. To further address autophagy induction, cells were treated for 24 h with the same drug treatment, followed by the preparation of immunofluorescence assay of LC3. As shown in Fig. 2a, in untreated cells, LC3 staining was diffuse and rather weak. When cells were stimulated with HG, bright LC3 spots indicative for autophagosome formation became visible. Correlating to our western blot data, bright LC3 dots were weakened by 3-MA and did further stronger by Baf A1 pretreatment. Next, induction of autophagy was confirmed by acridine orange staining. As seen in Fig. 2b, green fluorescence with minimal orange/red fluorescence was primarily displayed in control group, indicating a lack of acidic vesicular organelles. But HG-treated cells showed an obvious

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Fig. 2 Effects of 3-MA, Baf A1 and PDTC on autophagy during high glucose treatment. CIMPs were cultured for 24 and 48 h with 30 mM high glucose in the absence or presence of 150 nM Baf A1,10 mM 3-MA or 4 lM PDTC. a Cells were stained for LC3 (red) and DAPI (blue). Representative pictures are shown for immunofluorescence staining of LC3 of 24 h. Magnification 9800.

b Immunofluorescence microscopy of acridine orange-stained podocytes treated for 24 h with indicated drug treatment. Magnification 9800. c Immunoblot analysis of LC3 of 24 and 48 h drugs treatment. d Densitometry of LC3-II signals in immunoblots. Data in d are expressed as mean ± SD (N = 4); **P \ 0.01 versus control group, ## P \ 0.01 versus high glucose-only group. (Color figure online)

increase in orange/red fluorescent compared with that of control group. Consistent with previous experiments, orange/red fluorescence intensity was weakened by 3-MA and significantly enhanced by Baf A1. Together with the results from protein analysis, this indicated that HG accelerated the production of autophagosomes rather than inhibited the degradation of autophagosomes.

facilitated HG-induced NF-jB activation. After pretreatment of cells with 3-MA, NF-jB activity was remarkably enhanced. Blocking autophagy by Baf A1 also significantly increased p65 nuclear protein levels in a dose-dependent manner within 24 h (P \ 0.01, N = 4, Fig. 4a, b). Taken together, these results demonstrated that the decline in autophagy can stimulate NF-jB signaling and generate chronic inflammation.

Inhibition of autophagy facilitates high glucose-induced NF-jB activation To investigate the effects of HG on the NF-jB activationnuclear translocation level, CIMPs were measured using the specific assay. As shown in Fig. 3a and b, in untreated cells, p65 was mainly found in the cytoplasm. 30 mM HG treatment for 24 h induced nuclear translocation of NF-jB, and the intracellular activated NF-jB level increased obviously at 48 h. And pretreatment of cells with 4 lM PDTC significantly reduced HG-induced nuclear import of NF-jB p65 in podocytes (P \ 0.01, N = 4, Fig. 3a, b). We next determined whether the inhibition of autophagy

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Inhibition of autophagy enhances high glucose-induced CIMP apoptosis To evaluate the effect of autophagy on podocytes, cells were pretreated with 3-MA or Baf A1, respectively, followed by 30 mM HG for 24 and 48 h, respectively, and then examined the cell viability using the MTT assay. As shown in Fig. 5e (P \ 0.01, N = 4), MTT assay indicated that 3-MA or Baf A1 alone had no significant effects on the cell viability. When combined with HG, 3-MA and Baf A1 exhibited greater inhibition of the cell growth in a timedependent manner. Based on the results of the MTT assay,

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Fig. 3 High glucose-induced NF-jB p65 protein nuclear translocation. CIMPs were treated with or without 30 mM high glucose. After 24 and 48 h of high glucose treatment, the cells were incubated with p65 antibody and Alexa 488-conjugated anti-rabbit IgG, and nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI). The images were obtained by confocal laser microscopy and overlay; the cyan fluorescence indicates the location of p65 protein in nuclei. a The overlaid images indicate the location of p65 protein in nuclei. Magnification 9400. b p65 fluorescence intensity value in nuclei was calculated. P ammonium PDTC. Data in b are expressed as mean ± SD (N = 4); **P \ 0.01 versus control group, ##P \ 0.01 versus high glucose-only group

we observed the effects of autophagy inhibition on HGinduced apoptosis in podocytes by flow cytometry of cells stained with PI/Annexin V-FITC. It was observed that early apoptosis increased markedly in podocytes that were treated with HG in combination with 3-MA compared with the group treated with HG alone (P \ 0.01, N = 4, Fig. 5a, c). Meanwhile, apoptotic and necrotic cells increased significantly in podocytes treated with Baf A1 compared to that in HG or 3-MA-treated podocytes (P \ 0.01, N = 4, Fig. 5a, c). We next treated CIMPS with similar conditions for 12 and 24 h, and examined apoptotic chromatin

condensation by Hoechst 33342 staining. Compared with HG group, apoptotic chromatin condensation was induced and clearly observed in podocytes in HG ? 3-MA group (P \ 0.01, N = 4, Fig. 5b, d). Inhibition of NF-jB suppressed high glucose-induced autophagy and increased apoptosis Addition of PDTC, an inhibitor of NF-jB, sensitized CIMPs to the induction of the cell death by HG conditions. The combination of PDTC and HG killed most, if not all,

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Fig. 4 Nuclear translocation of the NF-jB p65 subunit induced by inhibition of autophagy. Cells were stimulated with 30 mM high glucose for 24 h in the absence or presence of 150, 300 nM Baf A1 or 10 mM 3-MA. The nuclear translocation of the NF-jB p65 subunit was assessed by indirect immunofluorescence confocal microscopy using anti-p65 subunit antibodies and Alexa 488-conjugated anti-rabbit IgG, and nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI). a Immunofluorescence colocalization illustrated p65 immunoreactivity was presented in podocytes. Fields illustrated on the right represented the nuclear stained DAPI. The middle panel showed p65 localization and the left panel showed the merge of nuclear and p65. Magnification 9400. b p65 fluorescence intensity value in nuclei was calculated. Data in b are expressed as mean ± SD (N = 4); **P \ 0.01 versus high glucose-only group

podocytes, exhibiting a clear synergism. When pretreated with PDTC, the cell viability was significantly reduced (P \ 0.01, N = 4, Fig. 6e) and HG-induced podocyte apoptosis was effectively increased (P \ 0.01, N = 4, Fig. 6c, d). The apoptotic nuclear was observed and the nuclear condensation or fragmentation characteristic of apoptosis was shown in podocytes (Fig. 6a, b). We next investigated the impact of HG together with PDTC on autophagy in podocytes. As shown in Fig. 2c and d (P \ 0.01, N = 4), there was a clear decrease of LC3-I to LC3-II conversion when combination of HG and PDTC. In

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parallel sets of experiments, cells were treated under similar conditions and then prepared for immunofluorescence assay and acridine orange staining. As shown in Fig. 2a and b, both of LC3 dots and orange/red fluorescent intensity became weakened. These remind that under HG circumstances, NF-jB is a crucial inducer of autophagy and inhibition of NF-jB activation blocked the autophagic response and augmented cell death. Furthermore, when cells acquired the apoptosis increase, they were devoid of autophagy expression, which was perhaps a result of the suppression of autophagy protective effect.

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Fig. 5 Inhibition of autophagy showed the increase in apoptosis during high glucose treatment. CIMPs were incubated with 30 mM high glucose in the absence or presence of 150 nm, 300 nM Baf A1 or 10 mM 3-MA. a The apoptosis was confirmed by annexin V/PI staining assay at 24 and 48 h. b The cells were stained with Hoechst 33342 to examine the cell and nuclear morphology to analyze apoptosis at 12 and 24 h. Representative images of nuclear staining.

Magnification 9400. c The apoptotic ratios (including the early and late apoptotic ratios) were analyzed by flow cytometry. d The percentage of cells showing typical apoptotic morphology by Hoechst 33342 staining. e The cell viability was examined by MTT assay at 24 and 48 h. Data in c, d and e are mean ± SD of four separate experiments; *P \ 0.05 versus control group, **P \ 0.01 versus control group and ##P \ 0.01 versus high glucose-only group

Discussion

chronic microvascular complication DN has become the most important factor leading to end-stage renal disease (ESRD) in the world. Due to its complexity and its postmitotic nature, podocytes represent the most fragile component of the glomerular filtration barrier. Failures of reparative mechanisms in podocytes promote persistent proteinuria and the development of DN [21]. Our study has shown the induction of podocyte apoptosis (Fig. 1a–d) and autophagy (Fig. 2a–d) by HG, which is consistent with several recent studies [22, 23]. The connection between autophagy and apoptosis or other forms of cell death is a burgeoning area of research. There is a substantial body of literature suggesting that under certain circumstance autophagy can promote cell death. Recent evidence indicates that autophagy itself may be a mechanism of cell death [24–26], but the exact mechanism between autophagy and apoptosis or other forms of cell death in podocytes

The present study demonstrates that HG enhances autophagy in CIMPs. Baf A1 further enhances HG-induced autophagy, whereas 3-MA inhibits HG-induced autophagy. HG also induces NF-jB signal in podocytes in a timedependent manner. Interestingly, the NF-jB inhibitor PDTC inhibits HG-induced autophagy and increases cell apoptosis. Moreover, the inhibition of autophagy enhances HG-induced apoptosis and activates the NF-jB signal, which suggests that the autophagy and NF-jB signal are essential to determine podocytes fate. These findings also indicate that HG-induced podocyte autophagy, apoptosis, and NF-jB signal are in close crosstalk, and the crosstalk plays a crucial role in HG-induced podocyte injury. Given the increasing prevalence of diabetes in both developed and developing countries, it is likely that its

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Mol Cell Biochem (2014) 394:261–273 b Fig. 6 Blockade of NF-jB signaling reduced the cell viability and

increased the apoptosis. CIMPs were cultured with 30 mM high glucose in the absence or presence of 4 lM PDTC for a range of time periods. a The apoptotic morphological detection was assessed by Hoechst 33342 after culturing in the presence of high glucose for 12 and 24 h. Magnification 9400. b The percentage of typical apoptotic morphology cells by Hoechst 33342 staining. c The ppoptosis was confirmed by annexin V/PI staining assay at 24 and 48 h. d Apoptotic ratios (including the early and late apoptotic ratios) were analyzed by flow cytometry. e The cell viability was examined by MTT assay at 24 and 48 h. P ammonium PDTC. Data in b, d and e are mean ± SD of four separate experiments; *P \ 0.05 versus control group, **P \ 0.01 versus control group and ##P \ 0.01 versus high glucose-only group

is still unclear. In our study, the apoptotic rate of Baf A1treated podocytes was significantly higher than that of podocytes treated with 3-MA (Fig. 5a–d). These data indicate that the accumulation of autophagosomes induces apoptotic cell death. However, apoptotic rate was lower in podocytes treated with HG alone than that of Baf A1treated cells (Fig. 5a–d). This finding suggests that HGinduced autophagosome accumulation, by itself, may not be sufficient to promote apoptotic cell death. Similarly, previous studies found that the apoptotic cell death was promoted through the accumulation of a sufficient amount of autophagosomes [27, 28]. These findings suggested a relationship between autophagy and apoptosis, and a certain level of autophagosome accumulation may be needed to promote podocyte cell death and apoptosis. Several recent studies have shown that the activation of the NF-jB signal plays an important role in mediating podocyte function [29–35]. NF-jB, consisting of p65 and p50 subunits, is activated in response to numerous stimuli and then translocated into the nucleus by the degradation of the inhibitor protein IjB. Ammonium PDTC is a specific inhibitor of NF-jB with anti-oxidation, anti-bacterial and fungal, and anti-tumor properties [36]. Ha et al. [37] and Zhang and co-workers [38] have reported the activation of NF-jB in HG-induced mesangial cells. Herein, we report a similar activation of NF-jB in HG-induced podocytes. And we firstly focus on the delicate role of NF-jB between autophagy and apoptosis in podocytes. We showed that the inhibition of autophagy with 3-MA and Baf A1 in podocytes caused an activation of NF-jB (Fig. 4a, b) and an increase of cell apoptosis (Fig. 5a–d). These results indicate a potential protective role of NF-jB in HG-induced podocytes. As we know that autophagy is to be a self-repair process of the cellular [18]. So the inhibition of the autophagy blocks the self-repair process under stress condition, which causes a NF-jB mediated shift to self-killing result ‘‘cell apoptosis’’. In fact, Gonza´lez-Polo et al. [27] and Boya et al. [28] have reported the linkage of autophagy and apoptosis. Their results showed that the inhibition of autophagy, either at

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early or late stage of the process, may lead to the apoptosis as a result of the failure to adapt to starvation. Researchers may consider that aggravating the apoptosis by inhibiting NF-jB is a validation of the protective effect of NF-jB on HG-induced podocytes. Concordantly, we found that the inhibition of the NF-jB aggravated cell apoptosis (Fig. 6c, d) under HG condition, which exactly supported our hypothesis. Furthermore, the inhibition of the NF-jB alleviated the autophagy level (Fig. 2c, d), which indicated a crosstalk of NF-jB and autophagy. This crosstalk may play a crucial role in triggering cell apoptosis under HG-induced podocytes. This appears to be a mechanism that with the inhibition of the NF-jB, the autophagy was downregulated, indicating a blockage of the self-repair process under HG condition, and it subsequently leading to a shift cell apoptosis. Collectively, these results indicate a delicate role of NFjB between autophagy and apoptosis in podocytes. With a crosstalk of NF-jB and autophagy, the NF-jB presents a protected role in anti-apoptosis in HG-induced podocytes. It needs to point out that there is significant crosstalk between autophagy and apoptosis in controlling podocyte dysfunction. We find NF-jB to be a coordinator role between them in HG-induced podocyte. Further study for what and how other potential factors are functional among NF-jB, autophagy, and apoptosis is needed. In that regard, several studies have indicated a potential role that p62 (sequestosome-1) may play in these signaling. TRAF6 is a lysine 63 (K63) E3 ubiquitin ligase involved in NF-jB activation [39]. Importantly, the interaction of p62 with TRAF6 promotes its oligomerization and subsequent activation, which leads to K63 polyubiquitination of TRAF6 resulting in the activation of NF-jB [40, 41]. Mice lacking Atg7 are impaired in autophagy, leading to the appearance in the brain and liver of polyubiquitinated aggregates that colocalize with p62 [42]. In mice lacking both Atg7 and p62, these aggregates are completely absent, suggesting that p62 plays a structural role in their formation [42]. These findings place p62 at critical decision points that control cell death and survival.

Conclusion In summary, our studies suggest that autophagy may play an essential protective role in maintaining the homeostasis of podocytes, and HG milieu promotes autophagy in podocytes. This effect of HG is in a close crosstalk with NF-jB signal. The defective autophagy may elicit overwhelming progression of podocyte injury and in turn leads to DN. Overall, these results may provide a promising therapeutic modality for treatment of DN in the future.

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Acknowledgments This work was supported by grant from the National Natural Science Foundation of China (31271074), the National Basic Research Program of China (2011CB944003) and State Key Laboratory of Medicinal Chemical Biology. Conflict interest interest.

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The authors declare that they have no conflict of 21.

References 1. Haraldsson B, Nystro¨m J (2012) The glomerular endothelium: new insights on function and structure. Curr Opin Nephrol Hypertens 21:258–263 2. Pavenstadt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307 3. Zhang Y, Chen B, Hou X-H, Guan G-J, Liu G, Liu H-Y, Li X-G (2007) Effects of mycophenolate mofetil, valsartan and their combined therapy on preventing podocyte loss in early stage of diabetic nephropathy in rats. Chin Med J 120:988–995 4. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW (1997) Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Investig 99:342–348 5. Meyer T, Bennett P, Nelson R (1999) Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 42:1341–1344 6. Ohsumi Y, Mizushima N (2004) Two ubiquitin-like conjugation systems essential for autophagy. Semin Cell Dev Biol 15:231–236 7. Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9:1102–1109 8. Hartleben B, Go¨del M, Meyer-Schwesinger C, Liu S, Ulrich T, Ko¨bler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT (2010) Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Investig 120:1084–1096 9. Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y (2003) MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis. FASEB J 17:1165–1167 10. Baehrecke EH (2005) Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol 6:505–510 11. Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217–245 12. Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Investig 115:2679–2688 13. Meijer AJ, Codogno P (2004) Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol 36:2445–2462 14. Mizushima N (2007) Autophagy: process and function. Genes Dev 21:2861–2873 15. Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721 16. Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330:1344–1348 17. Blazquez-Medela AM, Lopez-Novoa JM, Martinez-Salgado C (2010) Mechanisms involved in the genesis of diabetic nephropathy. Curr Diabetes Rev 6:68–87 18. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752 19. Mundel P, Reiser J, Borja AZMa, Pavensta¨dt H, Davidson GR, Kriz W, Zeller R (1997) Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of

123

22.

23. 24. 25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36. 37.

38.

conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236:248–258 Wang G-F, Wu S-Y, Xu W, Jin H, Zhu Z-G, Li Z-H, Tian Y-X, Zhang J-J, Rao J-J, Wu S-G (2010) Geniposide inhibits high glucose-induced cell adhesion through the NF-jB signaling pathway in human umbilical vein endothelial cells. Acta Pharmacol Sin 31:953–962 D’agati V (2007) Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts). Kidney Int 73:399–406 Susztak K, Raff AC, Schiffer M, Bo¨ttinger EP (2006) Glucoseinduced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 55:225–233 Ma T, Zhu J, Chen X, Zha D, Singhal PC, Ding G (2013) High glucose induces autophagy in podocytes. Exp Cell Res 319:779–789 Debnath J, Baehrecke EH, Kroemer G (2005) Does autophagy contribute to cell death? Autophagy 1:66–74 Kroemer G, Levine B (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9:1004–1010 Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH, Lenardo M (2006) Autophagic programmed cell death by selective catalase degradation. Proc Natl Acad Sci USA 103:4952–4957 Gonza´lez-Polo R-A, Boya P, Pauleau A-L, Jalil A, Larochette N, Souque`re S, Eskelinen E-L, Pierron G, Saftig P, Kroemer G (2005) The apoptosis/autophagy paradox: autophagic vacuolization before apoptotic death. J Cell Sci 118:3091–3102 Boya P, Gonza´lez-Polo R-A, Casares N, Perfettini J-L, Dessen P, Larochette N, Me´tivier D, Meley D, Souquere S, Yoshimori T (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040 Sanchez-Nin˜o MD, Sanz AB, Ihalmo P, Lassila M, Holthofer H, Mezzano S, Aros C, Groop P-H, Saleem MA, Mathieson PW (2009) The MIF receptor CD74 in diabetic podocyte injury. J Am Soc Nephrol 20:353–362 Ding M, Cui S, Li C, Jothy S, Haase V, Steer BM, Marsden PA, Pippin J, Shankland S, Rastaldi MP (2006) Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice. Nat Med 12:1081–1087 Lee EY, Chung CH, Khoury CC, Yeo TK, Pyagay PE, Wang A, Chen S (2009) The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-b, increases podocyte motility and albumin permeability. Am J Physiol Renal Physiol 297:F85–F94 Katsuyama K, Fujinaka H, Yamamoto K, Nameta M, Yaoita E, Yoshida Y, Tomizawa S, Uchiyama M, Yamamoto T (2009) Expression of the chemokine fractalkine (FKN/CX3CL1) by podocytes in normal and proteinuric rat kidney glomerulus. Nephron Exp Nephrol 113:e45–e56 Sayyed S, Ha¨gele H, Kulkarni O, Endlich K, Segerer S, Eulberg D, Klussmann S, Anders H-J (2009) Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes. Diabetologia 52:2445–2454 Barisoni L, Nelson PJ (2007) Collapsing glomerulopathy: an inflammatory podocytopathy? Curr Opin Nephrol Hypertens 16:192–195 Tipping PG (2008) Are podocytes passive or provocative in proteinuric glomerular pathology? J Am Soc Nephrol 19:651–653 Hart SP, Dransfield I, Rossi AG (2008) Phagocytosis of apoptotic cells. Methods 44:280–285 Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB (2002) Role of high glucose-induced nuclear factor-jB activation in monocyte chemoattractant protein-1 expression by mesangial cells. J Am Soc Nephrol 13:894–902 Deb DK, Chen Y, Zhang Z, Zhang Y, Szeto FL, Wong KE, Kong J, Li YC (2009) 1,25-Dihydroxyvitamin D3 suppresses high

Mol Cell Biochem (2014) 394:261–273 glucose-induced angiotensinogen expression in kidney cells by blocking the NF-jB pathway. Am J Physiol Ren Physiol 296:F1212–F1218 39. Moscat J, Diaz-Meco MT, Albert A, Campuzano S (2006) Cell signaling and function organized by PB1 domain interactions. Mol Cell 23:631–640 40. Sanz L, Diaz-Meco MT, Nakano H, Moscat J (2000) The atypical PKC-interacting protein p62 channels NF-jB activation by the IL-1-TRAF6 pathway. EMBO J 19:1576–1586

273 41. Moscat J, Diaz-Meco MT, Wooten MW (2007) Signal integration and diversification through the p62 scaffold protein. Trends Biochem Sci 32:95–100 42. Komatsu M, Waguri S, Koike M, Sou Y-S, Ueno T, Hara T, Mizushima N, Iwata J-I, Ezaki J, Murata S (2007) Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131:1149–1163

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