Emodin prevents renal ischemia-reperfusion injury via suppression of CAMKII/DRP1-mediated mitochondrial fission

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Abstract

Acute kidney injury (AKI) is a serious threat to human health. Clinically, ischemia-reperfusion (I/R) injury is considered one of the most common contributors to AKI. Emodin has been reported to alleviate I/R injury in the heart, brain, and small intestine in rats and mice through its anti-inflammatory effects. The present study investigated whether emodin improved AKI induced by I/R and elucidated the molecular mechanisms. We used a mouse model of renal I/R injury and human renal tubular epithelial cell model of hypoxia/reoxygenation (H/R) injury. Ischemia/reperfusion resulted in renal dysfunction. Pretreatment with emodin ameliorated renal injury in mice following I/R injury. Emodin reduced mitochondrial-mediated apoptosis, suppressed the overproduction of mitochondrial reactive oxygen species and accelerated the recovery of adenosine triphosphate both in vivo and in vitro. Emodin prevented mitochondrial fission and restored the balance of mitochondrial dynamics. The phosphorylation of dynamin-related protein 1 (DRP1) at Ser616, a master regulator of mitochondrial fission, was upregulated in both models of I/R and H/R injury, and this upregulation was blocked by emodin. Using computational cognate protein kinase prediction and specific kinase inhibitors, we found that emodin inhibited the phosphorylation of calcium/calmodulin-dependent protein kinase II (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1554), thereby inhibiting its kinase activity and reducing the phosphorylation of DRP1 at Ser616. The results demonstrated that emodin pretreatment could protect renal function by improving mitochondrial dysfunction induced by I/R.

Introduction

Acute kidney injury (AKI) is an essential contributor to the development of chronic kidney disease (CKD) (Chawla et al., 2014), characterized by the accumulation of end products of nitrogen metabolism (urea and creatinine) and the rapid loss of renal function. Ischemia and reperfusion (I/R) injury can occur after surgical procedures. It is one of the most common causes of AKI and associated with substantial mortality and morbidity(Cao et al., 2017). However, there are currently no effective therapeutic strategies for the treatment of AKI after I/R injury.

Accumulating evidence indicates that mitochondria play a vital role in the progression of AKI. The proximal renal tubule is dilated, and the brush border is lost in clinical biopsies of AKI. Renal proximal tubule cells also exhibit mitochondrial dysfunction(Basile et al., 2012;Linkermann et al., 2014). Mitochondrial dynamics are disrupted, thereby contributing to mitochondrial fragmentation(Andrade−Oliveira et al., 2015). Fragmented mitochondria are potential sources of reactive oxygen species (ROS), cytochrome C, mitochondrial DNA, and other potentially injurious molecules(Emma et al., 2016) that can induce tubular cell death(Yang et al., 2016). Therefore, mitochondria might be a therapeutic target for AKI(Emma et al., 2016). Studies revealed that stimulated mitochondrial biogenesis reduced necrosis and improved renal function in mice that were subjected to I/R-induced AKI(Jesinkey et al., 2014). The inhibition of mitochondrial fission conferred significant protection against I/R-induced AKI(Brooks et al., 2009). Therefore, the early protection of mitochondrial function may be a therapeutic alternative to suppress oxidative stress and inflammation and prevent tubular necrosis(Szeto et al., 2011).

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative from the rhizome of Rheum palmatum L. It is the main active monomer of Da Huang (Radix et Rhizoma Rhei) (Liu et al., 2015). Emerging evidence indicates that emodin has a wide range of pharmacological properties, such as antibacterial(Li et al., 2016), anti-inflammatory(Xia et al., 2019), antioxidant(Xia et al., 2019), immunosuppressive(Qiu et al., 2017), and anti-renal fibrosis(Ma et al., 2018) effects. Accumulating evidence shows that emodin inhibits abnormal intrinsic renal changes and plays a role in protecting the kidneys. A randomized clinical trial reported that emodin decreased the gluconeogenesis of renal tubular cells and decreased adenosine triphosphate (ATP) content in epithelial mitochondria(Khan et al., 2014). Another study found that emodin reduced the activity of Na+/K+-ATPase and Ca2+-ATPase in epithelial cells(Zhou and Chen, 1988). Emodin was also reported to prevent H/R-induced apoptosis in human renal tubular cells along with the restoration of the Bcl-2/Bax ratio(Chen et al., 2017). However, the effect of emodin on renal I/R injury has not been well documented.

Therefore, the present study investigated whether emodin protected against renal I/R injury and explored the underlying mechanisms. Our results showed that emodin exerted renoprotective effects by restoring the dynamic balance of mitochondrial fusion and fission, maintaining mitochondrial homeostasis, and reducing tubular cell death by inhibiting the activity of calcium/calmodulin-dependent protein kinase II (CAMKII) and downregulating the phosphorylation of dynamin-related protein 1 (DRP1) at Ser616.

Section snippets

Reagents

HK2 (human kidney proximal tubular cells) cells were purchased from Cell Culture Centre, Institute of Basic Medical Science Chinese Academy of Medical Sciences (Beijing, China). Emodin was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). DRP1 antibody, phospho-DRP1 (Ser616) antibody, phospho-DRP1 (Ser637) antibody, MFN2 antibody, OPA1 antibody, PINK1 antibody, PARKIN antibody, CASPASE-3 antibody, Cleaved CASPASE-3 antibody, BAX antibody, BCL2 antibody were purchased from Cell

Emodin ameliorated functional and histological I/R-induced renal injury

To investigate the effects of emodin on I/R-induced AKI, it was injected intraperitoneally at doses of 1, 3, 10 mg/kg for 7 days before renal I/R surgery. In mice with I/R injury, serum creatinine concentrations, and urea nitrogen (BUN) levels were elevated. Pretreatment with 1, 3, and 10 mg/kg emodin dose-dependently decreased serum creatinine and BUN levels (Fig. 1A and B). KIM-1, a biomarker of renal injury, was upregulated in the I/R group, and significantly downregulated in the

Discussion

The present study found that emodin ameliorated I/R-induced renal injury by regulating mitochondrial homeostasis in renal tubular epithelial cells. Emodin prevented I/R-induced renal injury and protected renal tubular epithelial cells from I/R-induced apoptosis. Emodin also restored the dynamic balance of mitochondrial fusion and fission and maintained mitochondrial homeostasis in renal tubular epithelial cells. Furthermore, emodin downregulated the phosphorylation of DRP1 at Ser616 in a

CRediT authorship contribution statement

Yanqing Wang: Investigation, Data curation, Writing – original draft. Qian Liu: Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Jiaying Cai: Investigation, Data curation, Visualization. Pin Wu: Investigation, Data curation. Di Wang: Investigation, Data curation. Yundi Shi: Investigation, Data curation. Tianru Huyan: Investigation, Data curation, Writing – original draft. Jing Su: Investigation, Resources, Funding acquisition. Xuejun Li:

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (No. 81974506, 81673486, 81373405 and 30901803 to L. T., No. 81802245 to J. S., No. 81874318, 82073878 and 81673453 to X.-J. L.), the Beijing Natural Science Foundation (No. Z200019 and 7172119), and the Interdisciplinary Medicine Seed Fund of Peking University (BMU2020MX032).

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