Astragaloside IV protest against podocyte injury through upregulating mitophagy via Mfn2/ Pink 1/Parkin axis

doi:10.21203/rs.3.rs-1761014/v1

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Astragaloside IV protest against podocyte injury through upregulating mitophagy via Mfn2/ Pink 1/Parkin axis

https://doi.org/10.21203/rs.3.rs-1761014/v1

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Background

Podocyte injury is the most important pathological hallmark of kidney diseases, Autophagy is the critical factors that involve podocyte injury. here we sought to determine whether astragaloside IV (AS-IV) was able to improve renal function and reverse podocyte injury through regulation of autophagy.

Methods

Using Adriamycin (ADR) mice model and cultured immortalized mouse podocytes, were exposed to AS-IV. Western blotting, immunofluorescence and histochemistry were used to analyze markers of autophagy, mitochondrial dysfunction, podocyte apoptosis and glomerulopathy in the progression of focal segmental glomerular sclerosis.

Results

We observed that AS-IV can inhibit podocyte apoptosis, increased reactive oxygen species (ROS) generation, mitochondrial fragmentation and dysfunction through inducing Mitofusin 2(Mfn2)/ PTEN induced putative kinase 1(PINK1)/Parkin mitophagy pathway both in vivo and in vitro. Over-expression of Mfn2 reduced puromycin aminonucleoside (PAN)-induced podocyte injury, while down regulation of Mfn2 expression limited the renal protective effect of AS-IV through regulating mitophagy.

Conclusion

AS-IV ameliorates renal function and renal pathological changes in ADR mice and inhibits PAN-induced podocyte injury by directly enhancing PINK1/Parkin/Mfn2 associated autophagy.

Astragaloside IV

podocyte

mitophagy

mitofusin 2

Podocytes wrap around the capillaries of the glomerulus, which play an essential role in maintaining the integrity of glomerular filtration barrier [1-2]. Podocyte injury, dedifferentiation and loss are pathological hallmarks of many kidney diseases, such focal segmental glomerular sclerosis (FSGS) and diabetic nephropathy (DN), preceding albuminuria and then exacerbating proteinuria and glomerular sclerosis [3-4]. Being high-energy-requiring cells, podocytes contain abundant mitochondria, and the mitochondrial dynamics and quality control processes are involved in podocyte injury [5]. Autophagy is an evolutionarily conserved lysosomal degradation process of cytoplasmic constituents to maintain homeostasis. Mitophagy, as the selective removal of damaged and dysfunctional mitochondria via autophagy, is an important mechanism of mitochondrial quality control [6-7].

The mitofusin 2 (Mfn2) gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network [8]. Chen et al. reported that Mfn2 is involved in the clearance of damaged mitochondria via selective autophagy [9]. It has also been demonstrated that Mfn2 enhances mitochondrial fusion and promotes mitophagy via enhancing the translocation and phosphorylation of Parkin, which protects cardiomyocyte from angiotensin II-induced injury [5]. In palmitic acid-induced podocyte injury, Jiang et al. found that decrease in Mfn2 expression is associated with inhibition of mitophagy[10].

Astragaloside IV (AS-IV), a bioactive saponin extracted from the Astragalus root, exerts a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor functions [11-13]. Various studies indicated that AS-IV plays an important role in regulating autophagy. AS-IV alleviates podocyte oxidative stress and apoptosis by inhibiting endoplasmic reticulum stress and enhancing autophagy in streptozotocin-induced diabetic mice[14]. AS-IV preserves renal function and morphology by inducing mesangial cell autophagy [15]. A recent study has shown that AS-IV is protective against podocyte injury by enhancing autophagy in a STZ-induced diabetic mouse model.[14] However, the exact mechanism of how AS-IV regulates mitophagy remains unclear. In current study, we aimed to determine whether AS-IV regulates MFN2-mediated mitochondrial fusion and mitophagy, which protects podocytes against injury.

Cell culture

Immortalized differentiated murine podocytes (MPC5) were obtained from Dr. Peter Mundel (Mount Sinai School of Medicine, New York, USA) and maintained in RPMI-1640 (Gibco BRL, Grand Island, New Jersey , USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and 100 U/ml, interferon (IFN)-γ (PeproTech, Inc., Rocky Hill, New [41] Jersey , USA) at 33°C with 5% CO2 for proliferation. To induce differentiation, podocytes were cultivated without IFN-γ at 37 °C for 10 days. For subsequent experiments, differentiated podocytes were treated with 50 μM puromycin aminonucleoside (PAN) (MedChemExpress New Jersey ,USA) with or without AS-IV （MedChemExpress New Jersey ,USA）for 24 h.

Cell viability assay.

Cell viability was measured using a CCK-8 assay (Beyotime ShangHai，China).The podocytes were seeded in 96-well plate cultured for 24h, then incubated with different concentrations of AS-IV with or without PAN at 37˚C, 5% CO2 for 24 h. Then 10 µl of CCK-8 was added to each well, and the cells were incubated at 37˚C for 1 -1.5h The absorbance was detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Small Interfering RNA Transfection

non-targeting siRNA Pool (Control-si) or Mfn2-specific siRNA pool were ordered from GenePharma (Shanghai, China). Mfn2-siRNA and control-siRNA were transfected into MPC5 using Opti-MEMI Reduced Serum Medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Lipofectamine 3000 transfection kit ( Invitrogen New York, USA). The expression of Mfn2 was examined by western-blot.

Lentiviral infection

Lentiviral carrying Mfn2 or empty vector (GenePharma ,Shanghai, China) were infected MPC5, then incubated for 12 h. Cells were then switched to normal culture medium. The expression of Mfn2 was examined by western blotting.

Western blot assay

Cells and kidney tissues were lysed with cell lysis buffer ( Beyotime ，Shanghai, China) on ice for 30 minutes and then centrifuged at 12000 × g for 10 minutes at 4 °C. The protein concentration was determined by BCA Protein Assay Reagent Kit (Beyotime, Shanghai, China). Protein was loaded in each lane of SDS-polyacrylamide electrophoresis gel. Then transferred to polyvinylidene difluoride membrane, which was then blocked with 5% bovine serum albumin for 1 h at room temperature and further incubated with a specific primary antibody and horseradish peroxidase-conjugated second antibodies. Results were Quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA)

Anti-pink1, anti-parkin, anti-Mfn2, anti-LC3,anti-podocin and anti-gapdh were purchased from Proteintech ( No.23274-1-AP, No.14060-1-AP, No.12186-1-AP, No.18725-1-AP,No.10494-1-AP,No.20384-1-AP),Anti-Synaptopodin,anti-nephrin were ordered from Abcam (No.ab224491,No.58968.).Anti-P62 was order from Abclonal( No.A7758).

Measurement of Oxidative Stress

Hydrogen peroxide, indicative of ROS generation, were measured with DCFH-DA (Beyotime, Shanghai, China) After the treatment, podocytes were incubated with 20 μM DCFH-DA at 37°C in the dark for 30 min. ROS generation was detected by flow cytometry and observed by the fluorescence microscope (ZEISS, Germany).

Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential (MMP) was determined using the lipophilic cationic dye JC-1 (Beyotime, Shanghai, China). Podocytes were loaded with JC-1 in the dark for 20 min and fluorescence was measured by flow cytometry and observed by the fluorescence microscope (ZEISS, Germany). The ratio of red to green (590/520) fluorescence reflects the MMP.

Apoptosis assay.

Apoptosis was assessed by flow cytometry after cell staining with Annexin V-FITC Apoptosis Detection Kit I (Beyotime, Shanghai, China). Briefly, Cells were washed with PBS and following which 1x106cells were suspended in 195 µl of binding buffer. the solution then 5 µl of Annexin V and 10 µl of propidium iodide (PI) were added; the mixture was gently vortexed and incubated for 20 min at room temperature in the dark. Subsequent flow cytometric analysis was performed within 1 h.

Establishment of the experimental model

BALB/c mice male 10-12w [Grade SPF II, Certificate Number SCXK (Jing) 2016–0006] weighing 18–20 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai China). All mice were housed with drinking water and standard chow ad libitum, and were randomly divided into control group (n = 8), Adriamycin( ADR) group (n = 8 ), and ADR with AS-IV treatment group (n =8). FSGS was induced by giving a single dose of ADR (10.4mg/kg, MedChemExpress, USA) intravenous injection via tail vein, and normal saline was administered in control group. One week after adriamycin injection, mice in AS-IV treatment group started to received AS-IV (25 mg/kg, MedChemExpress, USA) by gavage daily for 8 weeks, and the other two groups of mice also received same volume of saline by gavage. By the end of the nine-week protocol, mice were euthanatized, and the renal tissues were fixed for further research. All animal procedures were performed according to the National Institutes of Health guidelines (Guide for the Care and Use of the Laboratory Animals). The animal protocols were reviewed and approved by the ethics committee of the Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University.

Serum analysis

Mice were anaesthetized and blood samples were collected from the orbital sinus. Blood was centrifuged and serum was isolated. Serum creatinine (Scr) and blood urea nitrogen (BUN) were analyzed using FUJI DRI-CHEM CRE and BUN slides in a FUJI DRI-CHEM 7000i biochemistry analyser (Fujifilm, Tokyo, Japan).

Urine analysis:

Quantitative collection of overall urine was performed using metabolic cages for 12h. Albumin and creatinine concentrations were measured using ELISA (Abcam)

Immunofluorescence analysis

Kidney tissues were fixed with 4% paraformaldehyde overnight and dehydrated with 30% sucrose for 24 hours. Finally, fixed tissues were embedded in OCT compound and cut into 5-μm sections at -20℃ for further staining. Cells were grown on glass covers slips, then fixed with 4% paraformaldehyde for further process. The tissue sections and cells were blocked with 5% bovine serum albumin for 1 h at room temperature and further incubated with a specific primary antibody and FITC and Cy3–labeled secondary antibody. Nuclei was stained with DAPI for 5 min. Images were then acquired by laser scanning confocal microscopy (Leica Microsystems)

For mitochondria staining in cultured podocytes, cells were grown on glass coverslips and mitochondrial morphology was visualized by labeling the cells. with 50 nmol/L MitoTracker Red CMXRos (Thermo Fisher Scientific, USA) in the dark for 30 min at 37°C.

Electron microscopy:

Kidneys and cells were perfused and fixed with glutaraldehyde and then cut into 1mm³ tissue blocks for epoxy embedding. Transmission electron microscopy was performed by standard procedures.

Statistical analysis

GraphPad Prism and SPSS 19.0 software were used for the statistical analysis. Data were presented as mean values ± standard deviation (Mean ± SD) for independent experiments. For two groups, t-test was performed. Comparisons among more than two groups were assessed by one-way analysis of variance (ANOVA) followed. P<0.05 was considered statistically significant.

1.Effects of AS-IV on PAN-induced podocyte injury and apoptosis

To examine the effect of AS-IV on PAN induced podocyte injury, we initially determined podocyte proliferation in response to increased doses of AS-IV. MPC5 cells were exposed to PAN (50 µM) and different doses of AS-IV (50 nM, 100 nM, 1 µM, 5 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, and 60 µM) for 24 hours, and cell proliferation were measured by CCK-8 assay. As shown in Fig. 1A, PAN inhibited MPC5 cell proliferation, and this was rescued by administration of 5 µM As-IV for 24h. Therefore, 5 µM was chosen as the target concentration of AS-IV used for the following experiments.

Then we determine the expression of several critical slit diaphragm proteins, such as nephrin and podocin. PAN significantly inhibited nephrin and podocin expression, which was reversed by AS-IV(Fig. 1B, and C). We further assessed the role of AS-IV on PAN-induced cell apoptosis. Flow cytometry analysis showed that AS-IV treatment significantly reduced apoptosis induced by PAN. (Fig. 1D). These data indicated that AS-IV protected podocytes from PAN-induced damage and apoptosis.

2. As-IV Protects Podocyte From Pan-induced Mitochondrial Damage By Inducing Cell Mitophagy

Elevated ROS production and loss of mitochondrial membrane potential (MMP)are hallmarks of mitochondrial damage. As shown in Fig. 2A-B, ROS production was increased significantly by PAN treatment, as measured by DCFH-DA fluorescence and flow cytometry and this was inhibited by AS-IV. As indicated by JC-1 red fluorescence, MMP in podocytes was also maintained by AS-IV when challenged by PAN.

Both MitoTracker Red staining and transmission electron microscopy (TEM) were used to examine podocytes mitochondrial morphological changes. As shown in Fig. 2C-D, mitochondria exhibited a filamentous shape in control cells. 24-hour PAN treatment resulted in mitochondrial network breakdown into short rods. Less mitochondrial network broke down in MPC5 that received AS-IV pretreatment. As detected under TEM, mitochondria showed irregular swelling and decreased cristae in cells treated with PAN, and pretreating cells with AS-IV reduced these changes.

Mitochondria are highly dynamic organelles, and their morphology is dependent on a balance between mitochondrial fusion and fission events. We therefore evaluated the expression of Mfn2. Western-blot showed that PAN reduced Mfn2 expression, which was reversed by AS-IV. All these indicated that PAN induce mitochondrial damage accompanied with the decrease of Mfn2 expression, AS-IV can elevate Mfn2 expression inhibit PAN induce mitochondrial damage in podocyte (Fig. 1B) .

In order to identify the effect of PAN exposure activates mitophagy and if the protect effect of AS-IV associated with mitophagy. We detected the protein expression of LC3 and P62 in MPC5, and we initially found that PAN inhibited the expression of LC3II/I, elevated P62 expression which mean PAN inhibited mitophagy activation. AS-IV through activated mitophagy to protect podocytes against PAN induced injury. (Fig. 1B). TEM also shown that in AS-IV pretreated group, we can find out more classical autophagosome (Fig. 2E)

3. Mfn2 overexpression activated mitophagy and prevented PAN-induced podocytes injury.

To further elucidate whether podocyte damage is directly regulated by Mfn2, we overexpressed Mfn2 in MPC5 cells using a lentiviral vector (Fig. 3A), and ROS production, MMP and apoptosis were determined. Mfn2 overexpression significantly decreased PAN-induced ROS production and apoptosis rate. JC-1 staining showed that the overexpression of Mfn2 mitigated PAN-induced reduction in MMP. (Fig. 4A-D)

Slit diaphragm proteins expression (nephrin) and mitophagy associated protein (LC3 and P62) was measured by Western blot and IF. As shown in Fig. 3B and 3C, Mfn2 overexpression mitigated mitophagy inhibition and injury of podocyte induced by PAN treatment. LC3, and nephrin were increased, P62 was decreased compared with control-LV + PAN group.

Previous study has indicated that Mfn2 regulates Pink1/Parkin translocation and expression, inducing mitophagy. The expression of Pink1 and Parkin significantly decreased after PAN treated and overexpression Mfn2 increased these proteins expression (Fig. 3B) These results indicated that Mfn2 through regulating PINK1/Parkin pathway enhanced podocyte mitophagy and improved mitochondrial quality, prevented PAN induced podocyte damage.

4. Mfn2 Was Required For As-iv Induced Activation Of Mitophagy

To determine the role of Mfn2 in the AS-IV-induced mitophagy activation in podocytes, we used small interference RNA (siRNA) to knock-down the Mfn2 protein level in podocytes (Fig. 5A). We found that down regulated Mfn2 expression remarkably inhibited the protective effect of AS-IV, as indicated by a decrease of nephrin and podocin expression. We further detected the mitophagy associated protein, found out that the expression of LC3 II/I, Pink1 and Parkin decreased, P62 increase, which means the inhibition of Pink1/Parkin associated mitophagy pathway. All the experimental results show that Mfn2 play an important role in AS-IV induced activation of mitophagy. (Fig. 5B).We also detected ROS and JC-1inhibited the expression of Mfn2, AS-IV treated cannot inhibited ROS generation and the decrease of membrane potential.(Fig. 5C-E)

5. AS-IV alleviates glomerulosclerosis reduces nephrotic proteinuria and improves kidney dysfunction.

To further verify the role of AS-IV on protecting podocyte injury in vivo, we established the ADR-induced FSGS model with or without AS-IV intervention.

Urine albumin to creatinine ratio (UACR) was calculated to assess urine albumin excretion, BUN and serum creatinine were measured to monitor general renal function. As expected, compared with the ADR-mice group, AS-IV treatment group showed diminished UACR, BUN and serum creatinine. (Fig. 6A)

Ultrastructural changes of podocyte foot processes were revealed using TEM, the extensive fusion of foot processes was evident in ADR mice. Treatment of the ADR mice with AS-IV maintained the normal shape of the foot process. (Fig. 6B) Additionally, AS-IV also restored the protein levels of both synaptopodin and podocin. ( Fig. 6C,D)

Kidney tissue Pathological staining showed that ADR caused remarkable segmental glomerulosclerosis with hyaline deposits in glomeruli, inflammatory cell infiltration in the interstitium, and increased dilatation of proximal tubules compared with the control. However, AS-IV significantly reduced these effects. (Fig. 6E)

6. AS-IV through Mfn2/PINK1/Parkin pathway active podocyte mitophagy and prevents mitochondrial dysfunction in ADR-induced mice nephropathy.

To verify the role of AS-IV on regulating mitophagy in vivo, podocytes mitochondrial ultrastructure in ADR mice detected by TME, in ADR mice group, mitochondria shown as punctate and round the ridge disappeared, whereas in AS-IV-treated group most mitochondria remain in the normal form. We also discovered more classical autophagosomes in AS-IV group. (Fig. 6B,F) as reflected by western blotting and immunofluorescence analysis, Mfn2 was mainly expressed on podocyte(Fig. 6G), Mfn2, Pink1, Parkin, LC3II/I expression significantly decreased and accompanied by increased of P62 expression in ADR group ,which indicated the inhibition of mitophagy ,while AS-IV reverse these changes. These results suggest that AS-IV through activate Mfn2/Pink1/Parkin pathway, inducing active mitophagy, plays a protective role in kidney (Fig. 6D)

Podocytes, the highly specialized epithelial cells that line the urinary surface of the glomerular capillary tuft, are the primary targets in both MCD and FSGS [1, 16–17]. Podocyte injury results in proteinuria. It is widely accepted that a loss of glomerular podocytes is a key feature of the progression of renal diseases. As a kind of highly differentiated cell, autophagy plays a critical role in supporting podocytes. Many studies had clarified the renal protection effect of autophagy in podocytes under the pathological conditions suggesting the therapeutic potential of autophagy induction. [18] Astragalosides have a wide range of pharmacological properties, such as anti-inflammatory, antiviral, and immunomodulatory activities (19–20). AS-IV, one of the saponins from Astragalus membranaceus, Several studies have shown the effect on autophagy in various cells, [21, 22] including the podocyte. In this study, we investigated the renal protective effect of AS-IV on mitochondrial dynamics-related and autophagy-related proteins, as well as podocyte apoptosis and morphological changes in PAN-induced podocyte and ADR mice model. The main findings of our study were that AS IV ameliorated podocyte injury and mitochondrial dysfunction through regulation Mfn2/PINK1/Parkin pathways. These findings might provide a promising experimental basis for further investigate of AS-IV.

Mitochondrial fission and fusion are regulated coordinately to maintain mitochondrial homeostasis and normal function of cells [23]. Mfn2 located on the Mitochondrial outer membrane, play an important role in regulate mitochondrial homeostasis. Decreased expression of MFN2 causes mitochondrial dysfunction, changes calcium homeostasis, increases translocation of Bax to mitochondria and delay apoptosis [24]. The Mfn2-PINK1-Parkin signaling pathway plays a critical role in regulating mitophagy.[25] Chen’s group reported that in genetically normal cardiomyocytes, mitochondrial uncoupling with FCCP resulted in Parkin redistribution into intracellularly mitochondria-rich regions, whereas in Mfn2-depleted cells, Parkin diffused in the cytoplasm, indicating that endogenous Mfn2 promotes the need for Parkin localization to depolarize mitochondria[9]. Xiong once reported that Mitofusin 2 promotes Parkin translocation and phosphorylation, inducing mitophagy to clear damaged mitochondria, participates in mitophagy and mitochondrial fusion against Ang II-induced cardiomyocyte injury.[5]. Bucha also reported E2F1–Mfn2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders in HeLa cells.[26]

In our study, we found out PAN induced ROS production, cell apoptosis, podocyte injury which were accompanied by a decrease in Mfn2 and mitophagy. AS-IV effective reverse PAN induced podocyte injury through elevating Mfn2 expression and inducing mitophagy. In order to further to further identified if AS-IV through directly regulating Mfn2 expression participate in regulating mitophagy then play a podocyte protective role.

Through upregulate or downregulate mfn2 expression in MPC5, we discovered that, Overexpress Mfn2 also can alleviate PAN induced podocyte injury through activating mitophagy, while disturbed Mfn2 expression can inhibit Mfn2/PINK1/Parkin associated mitophagy and reduced the protective effect of AS-IV. Our results indicated that AS-IV can directly regulated mfn2 expression take part in regulating mitophagy.

We further built ADR mice model and treated with AS-IV to identify our point. In vivo, AS-IV alleviates glomerulosclerosis and podocyte injury, reduces nephrotic proteinuria and improves kidney dysfunction. Further mechanism studies we discovered that, AS-IV through inducing Mfn2/PINK1/Parkin pathway to activate podocyte mitophagy and preventing mitochondrial dysfunction, to play a protective role in podocytes.

In summary, our study suggests that AS-IV ameliorates renal function and renal pathological changes in ADR mice and inhibits PAN-induced podocyte injury by directly enhancing PINK1/Parkin/Mfn2 associated autophagy. These findings provide a potential therapeutic target of AS-IV on glomerular diseases.

Ethics approval and consent to participate

All animal testing was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Review Board of Zhejiang University School of Medicine

Consent for publication

Not applicable

Availability of data and materials

All datasets generated for this study are included in the article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no potential conflicts of competing interests.

Funding

This is supported by Natural Science Foundation of Zhejiang Province (LQ20H290003) ; National Natural Science Foundation of China (NO. 82104586) .

Authors' contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; XJ and LTS conducted the experiments, YYD conduct the animal experiments, YYL wrote the manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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Astragaloside IV protest against podocyte injury through upregulating mitophagy via Mfn2/ Pink 1/Parkin axis

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Abstract

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Introduction

Method

Results

1.Effects of AS-IV on PAN-induced podocyte injury and apoptosis

2. As-IV Protects Podocyte From Pan-induced Mitochondrial Damage By Inducing Cell Mitophagy

4. Mfn2 Was Required For As-iv Induced Activation Of Mitophagy

Discussion

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