Artesunate relieves acute kidney injury through inhibiting macrophagic Mincle‐mediated necroptosis and inflammation to tubular epithelial cell

Abstract Artesunate is a widely used derivative of artemisinin for malaria. Recent researches have shown that artesunate has a significant anti‐inflammatory effect on many diseases. However, its effect on acute kidney injury with a significant inflammatory response is not clear. In this study, we established a cisplatin‐induced AKI mouse model and a co‐culture system of BMDM and tubular epithelial cells (mTEC) to verify the renoprotective and anti‐inflammatory effects of artesunate on AKI, and explored the underlying mechanism. We found that artesunate strongly down‐regulated the serum creatinine and BUN levels in AKI mice, reduced the necroptosis of tubular cells and down‐regulated the expression of the tubular injury molecule Tim‐1. On the other hand, artesunate strongly inhibited the mRNA expression of inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α), protein levels of inflammatory signals (iNOS and NF‐κB) and necroptosis signals (RIPK1, RIPK3 and MLKL) in kidney of AKI mouse. Notably, the co‐culture system proved that Mincle in macrophage can aggravate the inflammation and necroptosis of mTEC induced by LPS, and artesunate suppressed the expression of Mincle in macrophage of kidney in AKI mouse. Overexpression of Mincle in BMDM restored the damage and necroptosis inhibited by artesunate in mTEC, indicating Mincle in macrophage is the target of artesunate to protect tubule cells in AKI. Our findings demonstrated that artesunate can significantly improve renal function in AKI, which may be related to the inhibition of Mincle‐mediated macrophage inflammation, thereby reducing the damage and necroptosis to tubular cells that provide new option for the treatment of AKI.


| INTRODUC TI ON
Acute kidney injury (AKI) is a complex syndrome with high morbidity and mortality. 1 Recent study reported that the morbidity of AKI in hospitalized patients is as high as 10%-15%, and in ICU patients even exceeds 50%. 2 Normally, the major causes and pathophysiological mechanisms for AKI include renal hypoperfusion, cardiorenal syndrome, nephrotoxin exposure, sepsis, major surgery, intra-abdominal hypertension, rapidly progressive glomerulonephritis and acute interstitial nephritis. 3 As AKI seriously threatens the health of patients and can significantly increase the risk of CKD, more and more studies have begun to pay attention to the pathogenesis and treatment of AKI. Notably, AKI has been known to be linked to intrarenal and systemic oxidative stress 4 and inflammation. 5,6 Accumulating evidence has proved that inflammation plays an important role in the development of AKI, which aggravates the injury of renal tubular epithelial cell and damages the renal function in a rapid time. [7][8][9] Therefore, exploring the cellular and molecular mechanisms of inflammation in kidney of AKI has an important role for the treatment of AKI. In this process, almost all immune cells are believed to be involved in the inflammation of AKI kidney, especially of dendritic cells, monocytes/macrophages, neutrophils, T lymphocytes and B lymphocytes, 10 in which macrophage has been proved to play an important role in the process of renal inflammation. 11,12 The deletion of macrophage in kidney protects renal function from IRI injury. 13 Therefore, macrophages are an important target cell for research and treatment of inflammationinduced injury in kidney of AKI.
Macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor, which can recognize damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPS), and mainly expressed on membrane of monocyte, macrophage and dendritic cell. 14 At first, Mincle was considered to be an active receptor for a variety of pathogenic bacteria, 15 even it is currently believed to enhance the response to Streptococcus pneumoniae in mice and aggravate inflammation. 16 However, more and more studies have shown that Mincle is also involved in various other pathological and physiological activities, including promoting fibrosis, 17 tumour formation 18 and infection. 19 A recent study demonstrated that Mincle is essential for maintaining the M1 phenotype of macrophage in kidney of AKI, and down-regulation of Mincle in macrophage relieved the renal injury, suggesting that Mincle is a key promotor for macrophagic inflammation in AKI. 20 Our previous research also proved that inhibition of Mincle-related signal pathway protects kidney from AKI injury. 21 Therefore, drug intervention targeting Mincle may be the key to the treatment of AKI.
Artemisinins are a class of sesquiterpene trioxane lactone drugs widely used in anti-malaria. 22 Artesunate, artemether and arteether are the mainly used derivatives of artemisinin for malaria in worldwide. 23 Among them, artesunate (ART) is the most studied artemisinin because of its better water solubility and higher oral bioavailability. 24 In addition to anti-malarial effect, in recent years, more and more studies have reported that artesunate has significant anti-inflammatory, antioxidant and anti-autophagy effects. A recent study demonstrated that the inhibitory effect of artesunate on ulcerative colitis is associated with its suppressing excessive ER stressmediated intestinal barrier damage and inflammatory response. 25 On the other hand, artesunate could be used to treat airway remodelling by regulating PPARγ/TGF-β1/Smad signalling in the context of chronic obstructive pulmonary disease. 26 Artesunate can also against hepatic ischaemia/reperfusion-induced inflammasomopathy by interrupting cross-talk of inflammatory and oxidative stress trajectories signifies. 27 In view of the significant anti-inflammatory effect of artesunate, we hope to explore whether artesunate can be used to against renal inflammation induced by acute kidney injury, as well as its improvement of kidney function and potential mechanisms.

| Isolation of BMDM and mTEC
Bone marrow-derived macrophages (BMDM) were isolated from the tibia and femur of C57BL/6 mice and differentiated in lowglucose DMEM medium containing 30% supernatant of L929 cell for 7 days following the protocol in our previous study. 21 To obtain the primary tubular epithelial cells (mTEC) from mouse, the kidney was digested with 3 mg/ml Collagen 4 at 37℃ for 15min, then filtered the suspension on a 70μm cell strainer and centrifuged at 120 g for 5 min, followed by culturing the tubular epithelial cells in F12/DMEM medium with 5% foetal bovine serum containing 50 ng/ml epidermal growth factor and 5 μl/ml ITS-G for 5 days.
All cells were incubated in a 37℃ incubator containing a constant 5% CO 2 . | 8777 LEI Et aL.

| Cell culture
The primary BMDM cells were cultured in Dulbecco modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 30% L929 supernatant, 10% foetal bovine serum (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin at 37℃ with 5% CO 2 . To establish the macrophage inflammatory model, BMDM were incubated with 200 ng/ml lipopolysaccharide (LPS) for protein extraction for 24 h and RNA extraction for 6 h. The primary mTEC cells were cultured in F12/DMEM (Sigma-Aldrich) supplemented with 5% FBS, 100 U/ ml penicillin and 100 mg/ml streptomycin at 37℃ with 5% CO 2 . The concentration of artesunate used in the cellular experiments was 2 and 10 μg/ml.

| MTT
A total of 5000 BMDM of mTEC cells were seeded in a well of 96-well plate. The next day, replace the medium with drug-containing culture medium for 24 h culture, followed by discarding the supernatant and adding 0.5% MTT-containing medium for 4 h. After using DMSO to dissolve the purple formazan, we measure the absorbance by a microplate reader at 570 nm with a reference absorbance at 630 nm.

| Detection of renal function
Serum creatinine and BUN of each group were detected by renal function detection kits ordered from Jiancheng Bioengineering Institute following the product manual. The acute tubular necrosis score was obtained by evaluating casts, brush border loss, tubular dilation, necrosis and calcification in kidney of each group from H&E staining. 29

| ELISA
Enzyme-linked immunosorbent assay kits were used to measure the

| Pathological staining
After fixing the kidney samples with 4% paraformaldehyde and embedding in paraffin, samples were cut into 4μm serial sections.
Subsequently, the samples were dewaxed in xylene and rehydrated in ethanol gradients and then stained with Haematoxylin and Eosin (Beyotime) for H&E staining, as well as 1% periodic acid and Periodic Acid-Schiff reaction for PAS staining. Images were captured by a light microscope (Eclipse 80i, Nikon).

| Real-time PCR
Total RNA was obtained from cells and kidney by using TRIzol reagent. Total RNA of each sample was reverse-transcribed to cDNA by using Reverse Transcription Kit. The mRNA expression levels of each gene were determined by Mastercycler EP Realplex2 real-time PCR system. PCR amplification was carried out for 40 cycles. The sequences of each primer are listed in Table 1.

| Western blot
Proteins from kidney and cells were isolated by using RIPA lysis buffer with PMSF. Briefly, samples treated with RIPA for 30 min on ice and followed by discarding the supernatant after 13,000 g centrifugation. at 4℃ overnight. Subsequently, membranes were incubated with the corresponding secondary antibodies (peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG) at RT for 1 h. Images of bands were captured by using a chemiluminescence imaging system (ChemiScope 6200, Clinx), and the grey intensity of the bands was calculated by ImageJ software.

| Co-culture system
BMDM and mTEC cells were co-cultured by using Millicell ® Hanging Cell Culture Inserts system (Millipore). mTEC cells were seeded in the well of 6-well or 12-well plates, and the next day, collected BMDM cells were added into the upper compartment (insert) to make physically separation of BMDM and mTEC.
Subsequently, co-culture of these two types of cells with LPS and/ or ART for 6 h to isolate RNA and 24 h to extract protein from mTEC, respectively.

| Statistics
Data were presented as the mean ± standard deviation. Data analysis was performed with one-way analysis of variance test by using SPSS software 21.0 (SPSS); p value <0.05 was considered to be statistically significant.

| Artesunate ameliorated renal dysfunction in cisplatin-induced AKI
In this study, we established a cisplatin-induced mouse model of AKI to observe the therapeutic effect of ART on renal dysfunction against cisinduced AKI in vivo. After 72 h of cisplatin injection, the results showed a significant increase in Scr and BUN levels. Dramatically, the enhanced Scr and BUN levels were strikingly decreased by ART treatment ( Figure 1A,B). The results of acute tubular necrosis score indicated that ART can effectively attenuated Cis-induced renal injury ( Figure 1C).
Kidney injury molecule-1 (Kim-1, also known as Tim-1) is an emblematic kidney injury biomarker, whose mRNA and protein levels were remarkably reduced by ART in kidney of AKI mouse, suggesting that ART significantly improved renal tubular injury ( Figure 1D-F). To further observe the effect of ART on renal dysfunction induced by cisplatin, we performed H&E and PAS staining, and the results demonstrated that ART significantly decreased renal damage ( Figure 1G). These findings verified that ART ameliorated renal dysfunction in Cis-induced AKI.

| Artesunate suppressed renal inflammation and renal necroptosis in Cisinduced AKI
Numerous previous studies have shown that inflammation plays an important role in acute kidney injury. To examine whether ART was able to improve the Cis-induced inflammatory response, we employed real-time PCR to detect the expression of inflammatory cytokines in kidney. As we expected, the mRNA levels of IL-1β, IL-6, TNFα and Cyclin D1 in kidney tissues dramatically increased with Cis stimulation, while the mRNA levels of these cytokines were remarkably reduced after ART treatment (Figure 2A-D). Meanwhile, we detected the protein level of iNOS, p-p65 and p-p50 in kidney of AKI. As a result, we found that ART can down-regulate the protein levels of iNOS, and activation of p-p65 and p-p50 in kidney of AKI ( Figure 2E,F). In addition, we performed Western blotting to detect the activation of p-MLKL, p-RIPK1 and p-RIPK3 ( Figure 2G Figure 3E-H). In summary, we found that ART inhibited Mincle, which maintained M1 macrophage activation in kidney of AKI.

| Artesunate improved the morphology of LPSstimulated BMDM
The chemical structure of ART is shown in Figure 4A.

| Artesunate improved inflammation and necroptosis of mTEC by inhibiting Mincle in BMDM
To further investigate the mechanism of ART on LPS-stimulated BMDM and

| DISCUSS ION
In the present study, we explored the potentially protective effects of artesunate on cisplatin-induced AKI mouse as well as be the essential mechanism of improving AKI by artesunate. In summary, artesunate relieves cis-induced AKI through inhibiting Minclemediated macrophagic inflammatory response.
In addition to its significant anti-malarial effect, artesunate also has other antiviral effects, such as anti-herpes virus 30 and hepatitis B virus. 31 In addition, artesunate also has an antiparasitic effect, including anti-trypanosoma, 32 toxoplasma gondii 33 and schistosoma. 34 In recent years, the anti-inflammatory effects of artesunate have attracted more and more attention, including anti-autoimmune diseases (rheumatoid arthritis, 35 inflammatory bowel disease 36 and systemic lupus erythematosus 37 ), allergic inflammation, 38 septic inflammation 39 and Alzheimer's disease. 40 However, there is no report on the role of artesunate in inflammatory disease of AKI. Therefore, we hope to verify whether artesunate has a protective effect on kidney of AKI and its potential mechanism. In view of the immune effect of artesunate, we hope to start with the inhibition of artesunate on the inflammation of infiltrated macrophage in kidney of AKI, and explore its possible renoprotective mechanism.
It is well known that inflammation is the dominating pathogenesis of cisplatin-induced AKI. 41  In summary, this study found that artesunate can significantly reduce renal damage and necroptosis and improve renal dysfunction and inflammation in kidney of AKI mouse model, and its mechanism is mainly related to inhibit macrophagic Minclemediated necroptosis and inflammation to tubular epithelial cell.
In short, artesunate inhibits the activation of M1 macrophages and the RIPK1/RIPK3/MLKL signalling pathway by down-regulating the expression of Mincle, thus reducing the inflammatory response and necroptosis, and further improving the renal injury of AKI. These findings provide a new theoretical direction for AKI treatment.

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available.