Follistatin-like 1 Prevents Renal Ischemia-Reperfusion Injury by Inhibition of Apoptosis via Upregulating AMPK/PPAR-δ Pathway

doi:10.21203/rs.3.rs-2478700/v1

Download PDF

Research Article

Follistatin-like 1 Prevents Renal Ischemia-Reperfusion Injury by Inhibition of Apoptosis via Upregulating AMPK/PPAR-δ Pathway

https://doi.org/10.21203/rs.3.rs-2478700/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Follistatin-like 1 (FSTL-1), a secreted glycoprotein, is upregulated in the serum of patients with acute kidney injury. However, it is unknown whether it protects against renal ischemia-reperfusion (I/R) injury. Our present study found that treatment with FSTL-1 (100 mg/kg) intravenous injection alleviated renal injury, as evidenced by reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels, along with reduced histopathological kidney damage. Moreover, FSTL-1 treatment reduced the number of apoptotic cells and the accumulation of reactive oxygen species (ROS) during I/R injury. The protective effect of FSTL-1 was via AMPK/PPAR-δ pathway, because, after blockade of AMPK/PPAR-δ pathway by individual inhibitor (GSK0660, a PPAR-δ antagonist, or compound C, an AMPK inhibitor), the protective effects of FSTL-1 on oxidative stress and apoptosis were blocked. Taken together, our results reveal that FSTL-1 attenuates renal I/R injury by inhibiting apoptosis in renal tubular epithelial cells, which is meditated by activating AMPK/PPAR-δ pathway.

Follistatin-like 1

kidney

ischemia-reperfusion injury

AMPK

PPAR-δ

Acute kidney injury (AKI) is a serious clinical complication, which causes rapid deterioration of renal function and even renal failure, resulting in a poor prognosis for patients[1]. Renal ischemia-reperfusion (I/R) injury is the leading cause of AKI, which often occurs in a variety of clinical treatments and pathophysiological processes, including kidney transplantation, trauma, and shock[2]. A large number of basic and clinical studies have been carried out to prevent and treat renal I/R injury, and related therapeutic strategies have been established, such as reducing the generation of reactive oxygen species (ROS) and improving mitochondrial energy metabolism[3, 4], but the effects remain to be further confirmed. Therefore, it is urgent to develop new treatment approaches.

Follistatin-like 1 (FSTL-1), also known as transforming growth factor beta1-stimulated clone 36 (TSC-36), is a secreted glycoprotein that belongs to the follistatin family. It exists abundantly in human tissues, including the heart, kidney, skeletal muscle and vascular endothelium[5]. It has been reported that circulating levels of FSTL-1 are higher in patients with cardiac diseases and AKI, and the levels of FSTL-1 in circulation are associated with a poor prognosis[6, 7]. However, FSTL-1 treatment improves cardiac function in dogs with heart failure and myocardial I/R injury[8, 9]. Meanwhile, FSTL-1 reduces renal damage in mice following subtotal nephrectomy[10]. Thus, it is of high importance to evaluate the benefits of FSTL-1 administration in renal I/R injury. In this study, we investigated whether FSTL-1 treatment prevented renal I/R damage, and found that FSTL-1 exerted its protective effects by reducing apoptosis, which were regulated by the 5’ adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-δ (PPAR-δ) pathway.

2.1 Animal and Renal I/R Injury Model

C57BL/6J mice (20–25 g) at the age of 10–12 weeks were obtained from the Experimental Animal Center of Daping Hospital (Chongqing, China). Mice were kept at a constant temperature of 21°C with a 12-h light cycle and had access to food and water ad libitum.

The renal I/R injury model in mice was performed as previously described previously[11]. Briefly, C57BL/6J mice were anesthetized with sodium pentobarbital (50 mg/kg) and placed on a heating pad to maintain body temperature. After an abdominal laparotomy, the right kidney was freed and removed. The left kidney was used for ischemia and reperfusion, and the left renal artery was clamped by an artery clip for 45 min, followed by removal of the vascular clamp. Sham-operated mice underwent nephrectomy and isolation of the left kidney only. FSTL-1 was dissolved in normal saline (100 mg/kg, R&D system, Minneapolis, USA) and injected by tail vein 5 min before ischemia in the FSTL-1 group, while saline was used instead of FSTL-1 in the control group, which was consistent with previous study[9]. Twenty-four hours after renal reperfusion, kidneys and blood samples were collected for further analysis.

2.2 Renal Function

The collected blood samples were centrifuged at 3,000 rpm for 30 minutes to collect serum. The renal function, including serum creatinine (Scr) and blood urea nitrogen (BUN), was measured by a creatinine assay kit and a BUN assay kit by enzymatic colorimetric methods (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Concentrations of Scr and BUN were measured by determining the absorbance with a spectrometer plate reader at 546 nm and 640 nm, respectively, according to the manufacturer’s protocol (ThermoFisher Scientific, Waltham, MA, USA).

2.3 Histological Staining

Kidney tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The samples were cut into 4 µm thick sections and subsequently stained using a Hematoxylin and Eosin (H&E) kit (Beyotime Biotechnology, China). All of the sections were obtained from six mice in each group. The images were obtained using the viewpoint M8 Digital Scanning Microscopy System software (PreciPoint, Freising, Germany). Subsequently, each sample section was examined by a pathologist from the Department of Pathology of Daping Hospital in a blind manner. Ten microscope fields (×400) were randomly chosen from each section for histological analysis. To determine the extent of renal damage, morphological changes in the tubules were evaluated. These changes included tubular epithelial cell swelling, vacuolization, cast formation and desquamation. The pathological score ranged from 0 to 5 points, depending on the area of involvement of the injury. The scale is as follows: 0, normal; 1, histological changes involving < 10% of injury area; 2, similar changes involving > 10% but < 25% of injury area; 3, similar changes involving > 25% but < 50% of injury area; 4, similar changes involving > 50% but < 75% of injury area; 5, similar changes involving > 75% of injury area[11, 12].

The Periodic Acid-Schiff (PAS) staining was performed using a PAS staining kit (Solarbio Biotechnology, China) according to the manufacturer’s instruction. Briefly, sections were incubated with oxidizing agent for 6.5 min, then, stained with Schiff’s reagent for 15 min, followed by counterstaining with hematoxylin solution for 2 min. Finally, sections in each group were captured by the viewpoint M8 Digital Scanning Microscopy System software (PreciPoint, Freising, Germany) and ten microscope fields (×400) were randomly chosen from each section for histological analysis.

2.4 Cell Culture and H/R Treatment

Human kidney 2 (HK-2) cells (RRID: CVCL_7048, ATCC, Manassas, VA, USA) were cultured at 37℃ in DMEM/F-12 (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) for 48 h. Experiments were performed with HK-2 cells at 80–90% confluence. For hypoxia/reoxygenation (H/R) treatment, cells were exposed to 24 h of hypoxia (5% CO₂, 1% O₂, and 94% N₂) followed by 3 h of reoxygenation[13, 14]. For drug treatment, the cells were incubated before hypoxia using FSTL-1, Compound C (5 µmol/L, MCE), or GSK0660 (500 nmol/L, MCE) for 24 h, which is consistent with previous studies[15, 16].

2.5 Cell Viability Assay

Cell viability was assayed using the CCK-8 kit (Beyotime Biotechnology, China). Briefly, cells were cultured in a 96-well plate and treated with FSTL-1 (1, 2, 5, 10, 15, 25 ng/ml) or without FSTL-1 for 24 h. After normoxia or H/R treatment, CCK-8 solution was added and cellular dehydrogenase activity was measured with a spectrometer plate reader at 450 nm (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, the cell viability was calculated using the followingformula:

$$\text{C}\text{ell viability }\left(\text{%}\right)\text{ = }\frac{\text{ODe - ODb}}{\text{ODc - ODb}}\text{ × 100%}$$

Where ODe is the absorbance value of the experimental well (DMEM with cells, CCK-8 and FSTL-1), ODc is the absorbance value of the control well (DMEM with cells, CCK-8, and without FSTL-1) and ODb is the absorbance value of the blank well (DMEM with CCK-8, and without cells and FSTL-1). The data were then normalized by control group.

2.6 TUNEL Assay

TUNEL assays were performed with an In Situ Cell Death Detection kit (Beyotime Biotechnology, China). In brief, sections were incubated in 10 µg/ml protease K (Beyotime Biotechnology, China) for 30 min at 37℃. The slides were then rinsed with phosphate buffered saline. 50 µl of TUNEL reaction mixture were added to the slices and incubated for 60 minutes at 37℃ in the absence of light. Subsequently, slides were incubated with 4’, 6-diamino-2-phenyl-indole to visualize all nuclei (DAPI). All of the sections were obtained from six mice in each group and the images were captured by the Olympus DP80 digital camera (Olympus, Tokyo, Japan). Ten microscope fields (×400) were randomly chosen from each section for histological analysis and the percentage of TUNEL-positive cells was calculated by the imageJ software (NIH, Bethesda, MD, USA) as previously described[17].

2.7 DHE Staining

For detection of the superoxide anions, HK-2 cells were incubated with the oxidative fluorescent dye dihydroethidium (DHE, 2.5 µmol/L) for 30 min at 37°C. After washing with phosphate buffered saline (PBS), the images were captured by the Olympus DP80 digital camera (Olympus, Tokyo, Japan) and ten microscope fields (×400) were randomly chosen from each section for histological analysis. The fluorescence intensity was used to reflect the ROS levels and was calculated by the imageJ software (NIH, Bethesda, MD, USA). The data were then normalized by control group.

2.8 LDH Detection

Lactate dehydrogenase (LDH) is a stable cytosolic enzyme that is released upon membrane damage in injured cells[18], HK-2 cells were cultured and then subjected to H/R treatment with FSTL-1 (10 ng/ml) in 6-well plates. The LDH detection kit was used to measure the amount of LDH in the culture supernatant released from the cells according to the manufacturer's instructions by chemical colorimetry (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was measured at 450 nm using a spectrometer plate reader (ThermoFisher Scientific, Waltham, MA, USA).

2.9 SOD Activity Assay

The superoxide dismutase (SOD) assay was performed on renal tissue and cells according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the concentrations of proteins were measured using a BCA protein assay kit (Beyotime Biotechnology, China). Then, the inhibition of water-soluble tetrazolium-1 (WST-1) reduction caused by the xanthine-xanthine oxidase system as the superoxide generator was used to measure SOD activity in tissue and cell homogenates. One unit of SOD was defined as the amount of enzyme required to inhibit the WST-1 reduction rate by 50%. The absorbance was measured at 450 nm using a spectrometer plate reader (ThermoFisher Scientific, Waltham, MA, USA). The SOD activity was expressed as units/mg protein (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.10 MDA level Assay

The malondialdehyde (MDA) assay was performed on renal tissue and cells according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the concentrations of proteins were measured using a BCA protein assay kit (Beyotime Biotechnology, China). Then, the MDA concentration was determined using the thiobarbituric acid (TBA) method. To determine MDA concentrations, the amount of lipid peroxides (LPO), a byproduct of MDA, was measured. The absorbance was measured at 532 nm using a spectrometer plate reader (ThermoFisher Scientific, Waltham, MA, USA).

2.11 Western Blot Analysis

Western blotting was performed according to standard protocols described previously[19]. Briefly, proteins were extracted from kidney tissues or HK-2 cells using ice-cold RIPA lysis buffer. The protein lysates were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes. The blots were blocked with 5% non-fat dry milk in TBS (Tris-buffered Saline) with 0.5% Tween-20 for 90 min at room temperature and the membranes were incubated with primary antibodies against phosphorylated AMPKα at Thr¹⁷² (1:1,000; Cell Signaling Technology, Danvers, MA, USA), PPAR-δ (1:1,000, ProteinTech, Wuhan, China), t-AMPKα (1:1,000, ProteinTech, Wuhan, China), BCL-2 (1:1,000, ProteinTech, Wuhan, China), BAX (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and Active caspase 3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:10,000, ProteinTech, Wuhan, China) antibodies at 4°C overnight. After the washing blots, the membranes were incubated for 1 h with fluorescent-labeled goat anti-rabbit IgG (1:15,000) or fluorescent-labeled goat anti-mouse IgG (1:15,000, LI-COR Biotechnology, Lincoln, Nebraska, NE, USA), and the bands were visualized by the Odyssey Western Blot Detection System (LI-COR Biotechnology, Lincoln, Nebraska, NE, USA). The images were analyzed using the Odyssey Application Software to obtain integrated intensities.

2.12 Statistical Analysis

The data are expressed as mean ± SEM. The data were analyzed using GraphPad PRISM software version 8.0 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way ANOVA followed by Bonferroni post hoc tests for multi-group (> 2) comparison and Student’s t-test was used for two-group comparison. Value of P < 0.05 was considered significant.

3.1 FSTL-1 Improves Renal Function and Alleviates Pathological Damage after Renal I/R Injury

To determine the protective effect of FSTL-1 on renal I/R injury, FSTL-1 (100 mg/kg) was intravenously administrated through the tail vein 5 min before ischemia, and renal I/R injury mouse model was constructed (Fig. 1A). We discovered that FSTL-1 reduced kidney damage, as evidenced by representative images of mouse kidney specimens (Fig. 1B). As compared with the I/R injury group, FSTL-1 significantly reduced Scr and BUN levels after I/R injury (Fig. 1C, D). FSTL-1 also reduced the expression of KIM-1 during I/R injury, which is a marker for tubule injury[20] (Fig. 1E, F). Furthermore, the H&E and PAS staining demonstrated that FSTL-1 reduced renal pathological damage caused by I/R injury (Fig. 1G, H). These results demonstrate that FSTL-1 treatment significantly attenuates renal pathological damage, preserves tubular structures and improves renal function.

3.2 FSTL-1 Alleviates Oxidative Stress and Apoptosis Induced by I/R Injury in vivo and in vitro

Oxidative stress is prominent as a result of reoxygenation following ischemia in the kidney, which leads to excessive apoptosis and results in a functional imbalance of the entire organ[21]. We then investigated the role of FSTL-1 in I/R-induced oxidative stress and apoptosis. ROS levels in renal tissues were detected with MDA and ROS levels. The increased ROS levels in I/R-induced mouse renal tissues were reduced by FSTL-1 treatment (Fig. 2A, B). Furthermore, the TUNEL assay demonstrated that FSTL-1 significantly reduced I/R-induced cell apoptosis (Fig. 2C, D).

The protective effect of FSTL-1 was confirmed in an in-vitro experiment. FSTL-1 increased HK-2 cell viability under H/R injury in a concentration-dependent manner (Fig. 3A). The significant effect occurred in 10 ng/ml FSTL-1. Therefore, we used 10 ng/ml FSTL-1 as the working concentration in the following experiments. Subsequently, our results showed that FSTL-1 decreased the release of LDH from HK-2 cells following H/R injury (Fig. 3B). Consistent with in vivo experiments, FSTL-1 could lower ROS accumulation, reduce HK-2 cell apoptosis in HK-2 during H/R injury (Fig. 3C-H). These data suggest that FSTL-1 protects the kidney from oxidative damage and apoptosis by reducing ROS accumulation.

3.3 AMPK/PPAR-δ Pathway is Involved in the Protective Effect of FSTL-1 on Apoptosis Against H/R Injury.

It has been reported that activation of AMPK by FSTL-1 protects myocardial I/R injury from apoptosis[22]. Meanwhile, AMPK as a transcription factor increases the expression of PPAR-δ[23], and PPAR-δ has long been thought to play a protective effect during myocardial and renal I/R injury[24–26]. As a result, we further investigated whether the AMPK/PPAR-δ pathway contributed to the protective effect of FSTL-1 against renal I/R injury. Our data showed that FSTL-1 increased the downregulated AMPKα phosphorylation (p-AMPK) at Thr¹⁷² and PPAR-δ expressions during I/R injury (Fig. 4A, B). However, in the presence of compound C (5 µmol/L, an AMPK inhibitor) or GSK0660 (500 nmol/L, a PPAR-δ inhibitor), the protective effect of FSTL-1 on HK-2 cells during H/R injury was blocked, indicating the importance of AMPK and PPAR-δ on the FSTL-1 signaling pathway (Fig. 4C). Our further study found that, in the presence of compound C, FSTL-1-mediated increased AMPK phosphorylation and PPAR-δ expressions were inhibited. However, GSK0660 administration had no effect on FSTL-1 mediated upregulation of AMPK phosphorylation (Fig. 4D, E), indicating that PPAR-δ is downstream to AMPK. Moreover, apoptosis-related indicators were detected and the results showed FSTL-1 significantly decreased BAX and active caspase-3 expressions and increased BCL-2 expression, which were all reversed by the co-incubation with compound C or GSK0660 (Fig. 4F, G). Therefore, FSTL-1 depends on the AMPK/PPAR-δ pathway to inhibiting apoptosis to protect renal I/R injury.

The pathogenesis of renal I/R injury is still unclear, which leads to a lack of effective treatment approach[27]. Therefore, finding a novel strategy to block renal I/R injury would have tremendous benefits in clinical practice. Previous studies have found several natural products and cytokines from organs such as fat, muscle and stomach to improve renal I/R injury through anti-inflammatory and antioxidant effects[28, 29], but there has been little success in clinical trials. FSTL-1, as a member of the follistatin family, is involved in many cardiovascular and renal diseases[9, 30]. FSTL-1 is mainly located in the renal tubular epithelium and the interstitial compartment, and its expression level is relatively low in the normal kidney, however, in chronic kidney diseases an increased expression can be detected[31, 32]. In the present study, we find that FSTL-1 attenuates I/R-induced renal pathological damage and dysfunction, indicating that FSTL-1 might be a potential drug that could treat renal I/R injury.

Excessive oxidative stress and apoptosis are key features of the pathological renal remodeling that occurs in response to I/R injury. The mitochondria are overwhelmed by the rapid restoration of oxygen, resulting in massive ROS production, lipid peroxidation, and the formation of toxic intermediate aldehydes. These toxic products lead to the inactivation of subcellular components and the opening of mitochondrial permeability transition pores (MPTP), which accelerates cell death, aggravates oxidative damage and finally results in tissue dysfunction[33, 34]. Studies have demonstrated the anti-oxidative effect of FSTL-1 in myocardial I/R injury and doxorubicin-induced cardiomyopathy[35, 36]. Meanwhile, FSTL-1 administration decreases neuronal and cardiomyocyte apoptosis and improves their functions during myocardial I/R injury and middle cerebral artery occlusion[22, 37]. Our study also found that I/R induced a significant increase in tubular cell apoptosis in the kidney in vivo, which was reduced by FSTL-1 treatment.

It has been reported that FSTL-1 regulates a variety of signaling pathways in human diseases, such as ameliorating apoptosis and cardiomyocyte hypertrophy via the AMPK pathway[6]. AMPK is important in the regulation of cellular energy[38]. Extensive studies have shown that AMPK exerts its therapeutic effects by protecting against H₂O₂-induced cardiomyocyte injury and hepatic I/R injury. PPAR-δ, as the downstream of AMPK, has been confirmed to play a protect effect rat renal I/R injury[26]. In our investigation, we found that in the presence of GSK0660 (a PPAR-δ antagonist), or compound C (an AMPK inhibitor), the protective effects of FSTL-1 on oxidative stress and apoptosis were blocked, indicating the importance of AMPK/PPAR-δ pathway on the FSTL-1 protective effects.

Our study demonstrated that FSTL-1 attenuated I/R injury by inhibiting apoptosis via activating AMPK/PPAR-δ pathway. Thus, administration of FSTL-1 might be a useful adjunctive therapy for renal I/R injury.

6 Availability of Data and Materials

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

7 Author Contributions

A ll authors contributed to the study conception and design. Wenbin Luo, Ziyue Zhang and Ken Chen designed the study. Wenbin Luo, Chunyu Zeng and Ken Chen wrote the manuscript. Chunyu Zeng, Hao Luo and Ken Chen acquired funding for the study. Wenbin Luo, Ziyue Zhang, Shengnan Chen, Hao Luo, Xiaoping Li, Shuang Qu, Shuo Zheng, Duofen He and Ken Chen acquired and analyzed the data.

8 Funding

This work was supported by grants from the National Natural Science Foundation of China (82170447).

9 Ethics Approval and Consent to Participate

This study was approved by the Committee on Animal Care of the Third Military Medical University and was carried out in accordance with the NIH guidelines for the care and use of laboratory animals.

10 Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Kellum, John A., Paola Romagnani, Gloria Ashuntantang, Claudio Ronco, Alexander Zarbock, and Hans-Joachim Anders. 2021. Acute kidney injury. Nature Reviews Disease Primers 7: 52. https://doi.org/10.1038/s41572-021-00284-z.
Chatauret, N., L. Badet, B. Barrou, and T. Hauet. 2014. Ischemia-reperfusion: From cell biology to acute kidney injury. Progrès en Urologie 24: S4–S12. https://doi.org/10.1016/S1166-7087(14)70057-0.
Zhao, Meng, Yizhuo Wang, Ling Li, Shuyun Liu, Chengshi Wang, Yujia Yuan, Guang Yang, et al. 2021. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics 11: 1845–1863. https://doi.org/10.7150/thno.50905.
Ma, Haijian, Xiaozhen Guo, Shichao Cui, Yongmei Wu, Yangming Zhang, Xiaoyan Shen, Cen Xie, and Jingya Li. 2022. Dephosphorylation of AMP-activated protein kinase exacerbates ischemia/reperfusion-induced acute kidney injury via mitochondrial dysfunction. Kidney International 101: 315–330. https://doi.org/10.1016/j.kint.2021.10.028.
Li, Xinxin, Lian Li, Yue Chang, Wen Ning, and Xinqi Liu. 2019. Structural and functional study of FK domain of Fstl1. Protein Science 28: 1819–1829. https://doi.org/10.1002/pro.3696.
Mattiotti, Andrea, Stuti Prakash, Phil Barnett, and Maurice J. B. van den Hoff. 2018. Follistatin-like 1 in development and human diseases. Cellular and Molecular Life Sciences 75: 2339–2354. https://doi.org/10.1007/s00018-018-2805-0.
Lang, Hong, Hang Zhang, Mengqing Ma, Xin Wan, Yuyang Chen, Qing sun, and Changchun Cao. 2022. Serum Fstl1, a novel biomarker screened based on protein array technology, predict acute kidney injury and major renal adverse events after cardiac surgery: a prospective cohort study. Clinica Chimica Acta: S0009898122013663. https://doi.org/10.1016/j.cca.2022.11.008.
Seki, Mitsuru, Jeffery C. Powers, Sonomi Maruyama, Maria A. Zuriaga, Chia-Ling Wu, Clara Kurishima, Lydia Kim, et al. 2018. Acute and Chronic Increases of Circulating FSTL1 Normalize Energy Substrate Metabolism in Pacing-Induced Heart Failure. Circulation: Heart Failure 11: e004486. https://doi.org/10.1161/CIRCHEARTFAILURE.117.004486.
Ogura, Yasuhiro, Noriyuki Ouchi, Koji Ohashi, Rei Shibata, Yoshiyuki Kataoka, Takahiro Kambara, Tetsutaro Kito, et al. 2012. Therapeutic Impact of Follistatin-Like 1 on Myocardial Ischemic Injury in Preclinical Models. Circulation 126: 1728–1738. https://doi.org/10.1161/CIRCULATIONAHA.112.115089.
Hayakawa, Satoko, Koji Ohashi, Rei Shibata, Yoshiyuki Kataoka, Megumi Miyabe, Takashi Enomoto, Yusuke Joki, et al. 2015. Cardiac Myocyte-Derived Follistatin-Like 1 Prevents Renal Injury in a Subtotal Nephrectomy Model. Journal of the American Society of Nephrology 26: 636–646. https://doi.org/10.1681/ASN.2014020210.
Liu, Chao, Ken Chen, Huaixiang Wang, Ye Zhang, Xudong Duan, Yuanzheng Xue, Hongye He, et al. 2020. Gastrin Attenuates Renal Ischemia/Reperfusion Injury by a PI3K/Akt/Bad-Mediated Anti-apoptosis Signaling. Frontiers in Pharmacology 11: 540479. https://doi.org/10.3389/fphar.2020.540479.
Wang, Zhen, Yukai Liu, Yu Han, Weiwei Guan, Xun Kou, Jinjuan Fu, Di Yang, et al. 2013. Protective effects of aliskiren on ischemia–reperfusion-induced renal injury in rats. European Journal of Pharmacology 718: 160–166. https://doi.org/10.1016/j.ejphar.2013.08.038.
Yu, Wenli, Mingwei Sheng, Rubin Xu, Jianjian Yu, Kang Cui, Jingkai Tong, Liying Shi, Hengchang Ren, and Hongyin Du. 2013. Berberine protects human renal proximal tubular cells from hypoxia/reoxygenation injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways. Journal of Translational Medicine 11: 24. https://doi.org/10.1186/1479-5876-11-24.
Kwon, Dae Sik, Chae Hwa Kwon, Jae Ho Kim, Jae Suk Woo, Jin Sup Jung, and Yong Keun Kim. 2006. Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells. European Journal of Cell Biology 85: 1189–1199. https://doi.org/10.1016/j.ejcb.2006.06.001.
Liu, Fang, Shaohong Fang, Xinxin Liu, Ji Li, Xuedong Wang, Jinjin Cui, Tao Chen, et al. 2020. Omentin-1 protects against high glucose-induced endothelial dysfunction via the AMPK/PPARδ signaling pathway. Biochemical Pharmacology 174: 113830. https://doi.org/10.1016/j.bcp.2020.113830.
Luo, Hao, Cong Lan, Chao Fan, Xue Gong, Caiyu Chen, Cheng Yu, Jialiang Wang, et al. Down-regulation of AMPK/PPARd signalling promotes endoplasmic reticulum stress-induced endothelial dysfunction in adult rat offspring exposed to maternal diabetes: 13.
Luo, Wenbin, Yu Tao, Shengnan Chen, Hao Luo, Xiaoping Li, Shuang Qu, Ken Chen, and Chunyu Zeng. 2022. Rosmarinic Acid Ameliorates Pulmonary Ischemia/Reperfusion Injury by Activating the PI3K/Akt Signaling Pathway. Frontiers in Pharmacology 13: 860944. https://doi.org/10.3389/fphar.2022.860944.
Bronikowska, Joanna, Ewelina Szliszka, Dagmara Jaworska, Zenon P. Czuba, and Wojciech Krol. 2012. The Coumarin Psoralidin Enhances Anticancer Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL). Molecules 17: 6449–6464. https://doi.org/10.3390/molecules17066449.
Luo, Hao, Caiyu Chen, Li Guo, Zaicheng Xu, Xiaoyu Peng, Xinquan Wang, Jialiang Wang, et al. 2018. Exposure to Maternal Diabetes Mellitus Causes Renal Dopamine D1 Receptor Dysfunction and Hypertension in Adult Rat Offspring. Hypertension (Dallas, Tex.: 1979) 72: 962–970. https://doi.org/10.1161/HYPERTENSIONAHA.118.10908.
Han, Won K., Veronique Bailly, Rekha Abichandani, Ravi Thadhani, and Joseph V. Bonventre. 2002. Kidney Injury Molecule-1 (KIM-1): A novel biomarker for human renal proximal tubule injury. Kidney International 62: 237–244. https://doi.org/10.1046/j.1523-1755.2002.00433.x.
Rovcanin, Branislav, Branislava Medic, Gordana Kocic, Tatjana Cebovic, Marko Ristic, and Milica Prostran. 2016. Molecular Dissection of Renal Ischemia-Reperfusion: Oxidative Stress and Cellular Events. Current Medicinal Chemistry 23: 1965–1980. https://doi.org/10.2174/0929867323666160112122858.
Ogura, Yasuhiro, Noriyuki Ouchi, Koji Ohashi, Rei Shibata, Yoshiyuki Kataoka, Takahiro Kambara, Tetsutaro Kito, et al. 2012. Therapeutic Impact of Follistatin-Like 1 on Myocardial Ischemic Injury in Preclinical Models. Circulation 126: 1728–1738. https://doi.org/10.1161/CIRCULATIONAHA.112.115089.
Narkar, Vihang A., Michael Downes, Ruth T. Yu, Emi Embler, Yong-Xu Wang, Ester Banayo, Maria M. Mihaylova, et al. 2008. AMPK and PPARδ Agonists Are Exercise Mimetics. Cell 134: 405–415. https://doi.org/10.1016/j.cell.2008.06.051.
Kapoor, Amar, Massimo Collino, Sara Castiglia, Roberto Fantozzi, and Christoph Thiemermann. 2010. ACTIVATION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-β/δ ATTENUATES MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN THE RAT. Shock 34: 117–124. https://doi.org/10.1097/SHK.0b013e3181cd86d6.
Papatheodorou, Ioanna, Eleftheria Galatou, Georgios-Dimitrios Panagiotidis, Táňa Ravingerová, and Antigone Lazou. 2021. Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation, Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production. International Journal of Molecular Sciences 22: 6399. https://doi.org/10.3390/ijms22126399.
Collino, Massimo, Elisa Benetti, Gianluca Miglio, Sara Castiglia, Arianna Carolina Rosa, Manuela Aragno, Christoph Thiemermann, and Roberto Fantozzi. 2011. Peroxisome proliferator-activated receptor β/δ agonism protects the kidney against ischemia/reperfusion injury in diabetic rats. Free Radical Biology and Medicine 50: 345–353. https://doi.org/10.1016/j.freeradbiomed.2010.10.710.
Zhao, Hailin, Azeem Alam, Aurelie Pac Soo, Andrew J.T. George, and Daqing Ma. 2018. Ischemia-Reperfusion Injury Reduces Long Term Renal Graft Survival: Mechanism and Beyond. EBioMedicine 28: 31–42. https://doi.org/10.1016/j.ebiom.2018.01.025.
Machado, Douglas Ikedo, Eloiza de Oliveira Silva, Sara Ventura, and Maria de Fatima Fernandes Vattimo. 2022. The Effect of Curcumin on Renal Ischemia/Reperfusion Injury in Diabetic Rats. Nutrients 14: 2798. https://doi.org/10.3390/nu14142798.
Oh, Jieun, and Hamid Rabb. 2013. Adiponectin: an enlarging role in acute kidney injury. Kidney International 83: 546–548. https://doi.org/10.1038/ki.2012.479.
Shimano, M., N. Ouchi, K. Nakamura, B. van Wijk, K. Ohashi, Y. Asaumi, A. Higuchi, et al. 2011. Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy following pressure overload. Proceedings of the National Academy of Sciences 108: E899–E906. https://doi.org/10.1073/pnas.1108559108.
Maksimowski, Nicholas A., Xuewen Song, Eun Hui Bae, Heather Reich, Rohan John, York Pei, James W. Scholey, and Nephrotic Syndrome Study Network (NEPTUNE). 2021. Follistatin-Like-1 (FSTL1) Is a Fibroblast-Derived Growth Factor That Contributes to Progression of Chronic Kidney Disease. International Journal of Molecular Sciences 22: 9513. https://doi.org/10.3390/ijms22179513.
Feng, Daniel, Cindy Ngov, Nathalie Henley, Nadia Boufaied, and Casimiro Gerarduzzi. 2019. Characterization of Matricellular Protein Expression Signatures in Mechanistically Diverse Mouse Models of Kidney Injury. Scientific Reports 9: 16736. https://doi.org/10.1038/s41598-019-52961-5.
Bugger, Heiko, and Katharina Pfeil. 2020. Mitochondrial ROS in myocardial ischemia reperfusion and remodeling. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1866: 165768. https://doi.org/10.1016/j.bbadis.2020.165768.
Xiao, Mengqing, Huiqin Zhong, Lin Xia, Yongzhen Tao, and Huiyong Yin. 2017. Pathophysiology of mitochondrial lipid oxidation: Role of 4-hydroxynonenal (4-HNE) and other bioactive lipids in mitochondria. Free Radical Biology and Medicine 111: 316–327. https://doi.org/10.1016/j.freeradbiomed.2017.04.363.
Szabó, Márton Richárd, Márton Pipicz, Tamás Csont, and Csaba Csonka. 2020. Modulatory Effect of Myokines on Reactive Oxygen Species in Ischemia/Reperfusion. International Journal of Molecular Sciences 21: 9382. https://doi.org/10.3390/ijms21249382.
Zhao, Yintao, Jingjing Sun, Wei Zhang, Meng Peng, Jun Chen, Lu Zheng, Xiangqin Zhang, Haibo Yang, and Yuan Liu. 2020. Follistatin-Like 1 Protects against Doxorubicin-Induced Cardiomyopathy through Upregulation of Nrf2. Oxidative Medicine and Cellular Longevity 2020: 1–11. https://doi.org/10.1155/2020/3598715.
Liang, Xiping, Qin Hu, Bo Li, Devin McBride, Hetao Bian, Pierre Spagnoli, Di Chen, Jiping Tang, and John H. Zhang. 2014. Follistatin-Like 1 Attenuates Apoptosis via Disco-Interacting Protein 2 Homolog A/Akt Pathway After Middle Cerebral Artery Occlusion in Rats. Stroke 45: 3048–3054. https://doi.org/10.1161/STROKEAHA.114.006092.
Qi, Dake, and Lawrence H. Young. 2015. AMPK: energy sensor and survival mechanism in the ischemic heart. Trends in Endocrinology & Metabolism 26: 422–429. https://doi.org/10.1016/j.tem.2015.05.010.

No competing interests reported.

Fullimageofgels.pptx

Download PDF

Version 1

posted

You are reading this latest preprint version

Follistatin-like 1 Prevents Renal Ischemia-Reperfusion Injury by Inhibition of Apoptosis via Upregulating AMPK/PPAR-δ Pathway

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Animal and Renal I/R Injury Model

2.2 Renal Function

2.3 Histological Staining

2.4 Cell Culture and H/R Treatment

2.5 Cell Viability Assay

2.6 TUNEL Assay

2.7 DHE Staining

2.8 LDH Detection

2.9 SOD Activity Assay

2.10 MDA level Assay

3 Results

3.1 FSTL-1 Improves Renal Function and Alleviates Pathological Damage after Renal I/R Injury

3.2 FSTL-1 Alleviates Oxidative Stress and Apoptosis Induced by I/R Injury in vivo and in vitro

3.3 AMPK/PPAR-δ Pathway is Involved in the Protective Effect of FSTL-1 on Apoptosis Against H/R Injury.

4 Discussion

5 Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1