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C3aR complement system
Toxicology Letters 312 (2019) 118–124
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Aristolochic acid I aggravates renal injury by activating the C3a/C3aR complement system
T
Jing Yea,1, Zhizhi Qiana,1, Mei Xueb, Yamin Liuc, Sirui Zhua, Yu Lia, Xiaoli Liua, Danhong Caia, ⁎ Jia Ruia, Liang Zhanga, a
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China College of Preclinical Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China c Department of Pharmacy, Zhongda Hospital, Southeast University, Nanjing, Jiangsu, China b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Complement system Aristolochic acid nephropathy C3a C3aR Epithelial-mesenchymal-transition
Previous studies have reported that the complement system is unconventionally activated in many kinds of glomerulonephritis. Multiple complement components participate in the pathogenic process by triggering immune response or other intracellular signaling pathways. Here, we have investigated the role of C3a and its receptor C3aR in aristolochic acid nephropathy (AAN), which, is featured with progressive interstitial fibrosis. Over release of C3a and increased expression of C3aR parallels to the up-regulation of α-SMA and TGF-β1 in AAN, which appeared to promote epithelial-mesenchymal-transition (EMT). To identify the role of complement activation in AAN, we used an inhibitor of C3aR (C3aRA) to block the coupling of C3a to its receptor. Our results confirmed from decreased EMT, the protective effect of C3aRA in cell apoptosis and inflammatory response induced by aristolochic acid I. These results showed that C3a and its receptor C3aR played pathogenic roles in AAN, and renal tubular epithelial cells were potentially pivotal targets of complement activation that could cause pro-fibrotic effects.
⁎
Corresponding author. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.toxlet.2019.04.027 Received 18 December 2018; Received in revised form 15 April 2019; Accepted 24 April 2019 Available online 29 April 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. C3aRA protected against AAI induced kidney injury. (A) Body weight of mice in different groups. (B) HE and Masson staining in renal tissues. (C–D) Levels of Cr and BUN in serum operated a recovery of renal function in C3aRA group. (E–F) Secretion of IL-6 and TNF-α was analyzed with ELISA detecting kits.. Rseults are shown as mean ± SD, *P < 0.001, *P < 0.01, compared to control group. ##P < 0.01, #P < 0.01 compared to AAI group.
Fig. 2. AAI induced apoptosis and fibrosis is dependent on the activation of C3a/C3aR complement system. (A–B) Detection of C3 and C3a in serum. (C–E) C3aRA reversed the up-regulation of C3aR, Caspase 3, Bax, α-SMA and TGF-β1, along with decreased levels of Bcl-2 and E-cadherin in AAI treated group, which were analyzed with immunoblotting and qPCR. (F) Expression of C3aR, IL-6, TNF-α, α-SMA and E-cadherin in renal tissues detected by immunohistochemistry were reversed in C3aRA treated group. Results are shown as mean ± SD (n = 3) and calculated from at least three independent experiments. *P < 0.001, *P < 0.01, * P < 0.05, compared to control group. ###P < 0.001, ##P < 0.01, #P < 0.05 compared to AAI group.
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Fig. 3. AAI caused activation of C3a/C3aR complement system and over-release of proinflammatory cytokines in HK-2 cells. (A) Cell viability was decreased in 24 h treatment with various concentrations of AAI. (B–D) Enhanced secretion of C3a, IL-6, TNF-α in AAI treated cells and operated a dose-dependent manner. Data are presented as means ± SD, *P < 0.001, *P < 0.01, *P < 0.05, relative to control group. (E–F) Fluorescence of C3aR and α-SMA was detected with immunofluorescence and indicated an increasing trend.
1. Introduction
AAI -induced nephritis. To test this topic, we detected C3a and its receptor C3aR both in vivo and in HK-2 cells after treatment with AAI. These results demonstrate that the complement system is activated in AAN, and that inhibition of C3aR blocks the intracellular signals of C3a, which finally provide a therapeutic mechanism for reducing apoptosis and fibrosis.
Aristolochic acid nephropathy (AAN) is a tubulointerstitial disease caused by over-exposure to aristolochic acids (Baudoux et al., 2018). Plants containing aristolochic acids have been widely applied in traditional medicine for arthritis, eczema, or other diseases in China and surrounding countries (Wang et al., 2018). However, significant sideeffects on renal tissues have limited their use in a clinical environment. It has been reported that aristolochic acid I (AAI) is the highest content in aristolochic acids, which is also demonstrated as the most representative component that can cause severe nephritis in vivo (Pu et al., 2016). The specific mechanism leading to the development of AAN is still unknown, despite multiple studies have been presented. According to clinical observation, immune response is triggered in interstitial inflammation which leads to continuous amplification of the fibrosis signal (Pozdzik et al., 2010). The complement system is typically considered as an important defense mechanism. But recent studies have indicated that the activation of the complement system might play a key role in glomerulonephritis (Noris and Remuzzi, 2017). C3 is a “hub” that regulates cascade activation of complement molecules (Merle et al., 2015). Its bioactive fragment C3a has been reported to act as an anaphylatoxin that can stimulate extravasation of host immune cells or cause matrix deposits via binding to its receptor C3aR (FernandezGodino and Pierce, 2018). As such, we hypothesize that complement activation is related to
2. Materials and methods 2.1. Animals and reagents Pathogen-free,6-to-8-week-old male C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised using standard protocols: temperature 22-25℃, relative humidity 50–60%, and 12 h light-dark cycle. All animals were given food and water in a standard laboratory diet. Depending on their groups, mice were intraperitoneally injected with AAI (5 mg/kg, 0.1 ml/10 g), with C3a receptor antagonist (C3aRA, 0.1 mg/kg, 0.1 ml/ 10 g), or with AAI and C3aRA in combination. The number of each group is ten. In addition, the control group was injected with the solvent of AAI: 10% dimethyl sulfoxide, 20% polyethylene glycol 600, 70% saline. AAI was injected daily and C3aRA injected every other day. After 14 days of treatment, mice were sacrificed and blood was centrifuged for biochemical detection. Kidneys were removed and flushed with NaCl 0.9%. After weighing, kidneys were prepared for further analysis. All animals were treated in accordance with the institutional 120
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Fig. 4. Inhibition of C3aR protected against cell apoptosis and fibrosis in HK-2 cells. (A–B) C3aRA (1μM) decreased apoptosis rate in AAI treated cells. (C–D) Expressions of apoptosis related proteins: Caspase 3, Bax/Bcl-2 and fibrosis related proteins: α-SMA/E-cadherin were down-regulated after treatment of C3aRA in AAI (40μM) treated HK-2 cells. (E) Reduced secretion of C3a, IL-6 and TNF-α in C3aRA treated group. Data are presented as means ± SD. *P < 0.001, *P < 0.01, * P < 0.05, compared to control group. ###P < 0.001, ##P < 0.01, #P < 0.05, relative to AAI group.
dehydration and permeabilization, kidneys were embedded in paraffin and sectioned for hematoxylin-eosin staining or Masson's trichrome staining. Histological analysis was obtained with light microscopy.
animal care guidelines issued by the Experimental Animal Ethical Committee of Nanjing University of Chinese Medicine, China. 2.2. Drugs and reagents
2.4. Immunohistochemistry
Aristolochic acid I was purchased from Henan Institute of Pharmaceutical Sciences (NO.20130201) and its purity≥98%, identified by HPLC. Antagonist of C3aR was from Merck Drugs & Biotechnology, 2730761. Kits for BUN and Cr were obtained from Nanjing Jiancheng Co, Ltd. The following antibodies were obtained from the companies cited: antibody to C3aR (CST, #14472), antibody to TGF-β1 (Biogot Biotechnology Co., Ltd, P03317), antibody to α-SMA (abcam, ab5694); antibody to GAPDH (CST, 8#2118S), and goat antirabbit IgG (CST, 25#7074S). ELISA kits for C3, C3a, IL-6, and TNF-α were brought from Nanjing Jiancheng Co, Ltd. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was obtained from Sigma. Apoptosis detection kits was from Nanjing KeyGEN Biotech Co, Ltd., Trizol from Invitrogen Corporation (15596-026), and transcriptor first strand cDNA synthesis kit from Thermo Fisher.
The immunohistochemistry was performed as described before (Hayashi et al., 2019). Briefly, the kidney sections were then deparaffinized, hydrated, and antigen-retrieved. Endogenous peroxidase was removed by 3% H2O2. Sections were blocked with 10% normal goat serum and incubated with antibodies to IL-6, TNF-α, α-SMA, E-cadherin or C3aR at room temperature for 1 h. Afterward, secondary antibody was applied at room temperature for 1 h. Following incubation with DAB for 10 min, sections were stained with hematoxylin and observed under a microscope.
2.5. Cell lines and cell culture HK-2 cell line was obtained from ATCC, NO: CRL2190™. Cells were cultured in F12-DMEM (WISENTINC, 319075010) medium containing 10% FBS (Hyclone, KPF21570) and 1% penicillin/streptomycin(vol/ vol) at 37℃ in 5% CO2.
2.3. Histopathology The kidneys of mice were fixed in 4% paraformaldehyde. After 121
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Fig. 5. C3aRA reversed AAI induced fibrosis in HK-2 cells. (A–B) C3aRA inhibited fluorescence intensity of α-SMA and C3aR in AAI treated groups. (C) Relative mRNA expression of α-SMA, E-cadherin, TGF-β1 and C3aR in HK-2 cells treated with AAI, C3aRA or co-incubation group. Data are presented as means ± SD. * P < 0.001, *P < 0.01, *P < 0.05, compared to control group. ###P < 0.001, #P < 0.05, relative to AAI group.
2.6. MTT assay
Biosciences AccuriC6).
Cells were seeded in 96-well plates at a density of 5 × 104cells/mL, 200 μL per well. After incubation at 37℃ in 5% CO2 for 12 h, cells were treated with various concentrations of AAI for 24 h. Supernatants were then removed, and 150 μL DMSO was added to each well. Plates were shaken on a table concentrator for 10 min and the absorbance was measured at 490 nm by a microplate reader (TECAN Infinite M200 PRO).
2.9. Biochemical detection
2.7. Immunofluorescence
2.10. Real-time and reverse transcription PCR
Cells were seeded in 24-well plates paved with sterile coverslips at a density of 1.5 × 104cells/mL. When cells grew to 70%–80% confluence, supernatants were removed and different concentrations of AAI were added into each well. After incubation with AAI for 24 h, cells were washed with cold PBS and fixed with 4% paraformaldehyde for 30 min. 0.1% Triton-100 was then applied for permeabilization, and antibody to C3aR or α-SMA was added for incubation at 4 °C overnight. Secondary antibodies were applied at room temperature for 2 h. Finally, DAPI was added for the staining of cell nuclei, and coverslips were observed under a fluorescent microscope (ZEISS inverted fluorescence microscope).
Cell samples and kidneys from mice were extracted with Trizol for total RNA according to manufacturer’s protocols. Concentration and purity of RNA were measured with the ratio of A260/A280. Reverse transcription was performed using abm All-in-one RT MasterMix, with PCR reactions performed as follows: predenaturation at 95℃ for 5 min, denaturation at 94 °C for 15 s, annealing at 60℃ for 60 s, then amplification for 40 cycles. The results were quantified by the ΔΔCt method. The primers used were: Mouse: C3a (forward: 5′-AGCTTCAGGGTCCC AGCTAC-3′; reverse: 5′-CTCTCCAGCCGTAGGACATT-3′); C3aR (forward: 5′-TTTCATCCAGCACCCCAGTG-3′; reverse: 5′-AGATGATGGCAC GAAGGCAA-3′); α-SMA (forward: 5′-GCTACGAACTGCCTGACGG-3′; reverse: 5′-GCTGTTATAGGTGGTTTCGTGGA-3′); E-cadherin (forward: 5′- CAGCCGGTCTTTGAGGGATT-3′; reverse: 5′-TGACGATGGTGTAGG CGATG-3′); TGF-β1 (forward: 5′- CTGCTGACCCCCACTGATAC-3′; reverse: 5′-AGCCCTGTATTCCGTCTCCT); GAPDH (forward: 5′-TATGTC GTGGAGTCTACTGGT-3′; reverse: 5′-GAGTTGTCATATTTCTCGTGG-3′); Human: C3aR (forward: 5′-CAGTGAGGAGCTCACACGTT-3′; reverse: 5′-TAAGAGCCCCTGCTTGTTGG-3′); α-SMA (forward: 5′-GGGACTAAG ACGGGAATCCT-3′; reverse: 5′-AGAGCCATTGTCACACACCA-3′); Ecadherin (forward: 5′-GAAGACAGAAGAGAGACTGGGTTA-3′; reverse: 5′-CAGGTTTTTAGGAAATGGGC-3′); TGF-β1 (forward: 5′-GGCTGTATT
Cell supernatants or blood of mice from different groups were coagulated on ice for 1 h, then centrifuged at 3000 r/min for 10 min. The contents of complement C3, C3a, IL-6, and TNF-α in serum or cell supernatants were measured with ELISA. The levels of serum creatinine (Cr) and urea nitrogen (BUN) were detected with biochemical kits.
2.8. Flow cytometry Annexin V/PI double-staining assay was applied to detect apoptosis rate. Cells were seeded in 6-well plates at a density of 1.5 × 105cells/ mL and incubated with different concentrations of AAI for 24 h. EDTAfree trypsin was then applied for digestion. After being washed twice with PBS, 5 × 105 cells were collected for detection. 5 μL Annexin VFITC and 5 μL propidiumidodide were added for staining. Cells were then incubated for 15 min and analyzed with flow cytometry (BD 122
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supernatants increased in a dose-dependent manner (Fig. 3B–D), as did up-regulation of C3aR and α-SMA on the cell surface (Fig. 3E–F). Additionally, we found an increased apoptosis rate in cells treated with AAI (Fig. 4A–B). As presented in Fig. 4C–D, we also observed increased expression of fibrosis-related proteins and mRNA in 40μM-treated groups. These results suggested that AAI triggeed a series of mechanisms related to cell damage, which also occured in a dose-dependent manner.
TAAGGACACCCGT-3′; reverse: 5′-GACACAGAGATCCGCAGTCC-3′); GAPDH (forward: 5′-CAGTGAGGAGCTCACACGTT-3′; reverse: 5′-TAA GAGCCCCTGCTTGTTGG-3′) 2.11. Immunoblotting Cell or kidney samples were lysed with RIPA lysis buffer. Lysates were then centrifugated, and the concentrations of proteins were analyzed with a BCA detecting kit. After being mixed with loading buffer and denatured in boiling water bath for 5 min, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and reacted overnight with antibodies specific to C3aR, caspase-3, BAX, Bcl-2, E-cadherin, αSMA, TGF-β1, or GAPDH at 4℃. After incubation with secondary antibodies, the immunoblots were visualized with chemiluminescence detection.
3.4. Inhibition of C3aR alleviated AAI-induced apoptosis and fibrosis in HK-2 cells To determine the possible roles of C3a and its receptor C3aR in AAI -induced HK-2 cell injury, we further applied an inhibitor of C3aR (C3aRA, 1μM) in AAI (40 μM)-treated cells (Fig. 5B). Firstly, we observed that cell apoptosis reversed in C3aRA treated groups (Fig. 4A–D). Additionally, down-regulation of α-SMA, TGF-β1 and increased expression of E-cadherin revealed a reduction in fibrosis (Figs. 4C–D, 5 A and C). Protective effect was also found in reduced secretion of IL-6 and TNF-α in cell supernatants (Fig. 4F–G).
2.12. Statistical analyses Results were presented as the mean ± SD. GraphPad Prism 6 software was used for statistical analysis: t-test for two groups, and oneway ANOVA for multiple groups.
4. Discussion
3. Results
The complement system is always considered as a bridge that connects innate and adaptive immunity. As a defensive mechanism against microbial intruders, it functions as an immune sentinel that recognizes and eliminates pathogens, which finally triggers adaptive response (Thorgersen et al., 2019). Recent studies have confirmed that complement participates in the pathogenesis of nephritis, such as lupus nephritis and IgA nephropathy (Cernoch et al., 2018). While the involvement of complement in AAN is still unknown. In the present study, several lines of evidences confirmed the proinflammatory and profibrotic roles of complement fragment C3a and its receptor C3aR in AAI -induced interstitial nephritis. C3a is a product cleaved from C3 and can trigger proinflammatory signal via binding to its G-protein-coupled receptor C3aR (Hansen et al., 2018; Khan and Shamma, 2019). Three biochemical pathways involved in the activation of complement system are converged on C3a, which highlights the functional role of C3a in multiple diseases (Veje et al., 2019). Previous study has demonstrated that C3a and its receptor C3aR contribute to the pathogenesis of pulmonary fibrosis (Gu et al., 2016). C3a-mediated mesenchymal transition of renal tubular epithelial cells has also been confirmed in vitro (Ziyong et al., 2009). In this study, we observed the excessive release of C3 and C3a in serum (Fig. 2A–B), with increased production of IL-6 and TNF-α, which are considered as inflammatory mediators (Fig. 1E–F). Up-regulation of C3aR in renal tissues also suggested the activation of complement system in AAI -induced kidney injury (Fig. 2C–F). α-SMA is a marker of myofibroblasts that barely expressed in normal tissues (Bijkerk et al., 2019). Additionally, E-cadherin-mediated cell adhesion maintains the integrity of cell polarity, which is lacked during fibrosis (Cao et al., 2017; Niño et al., 2018). Over-expression of α-SMA and reduced expression of Ecadherin in renal mesenchyme is consistent with previous studies, which indicated the presence of fibrosis (Fig. 2C–F) (Yi et al., 2018). In accordance with these findings in vivo, we observed similar results in AAI -induced cell injury in HK-2 cells, which also demonstrated that the renal tubular epithelial cell is one of the primary targets of renal tubular injury in AAN (Fig. 3). Blockage of C3aR or C5aR have been considered as a developing therapy method during allograft rejection (Makrides, 1998; Vieyra et al., 2011) for its prevention on tissue fibrosis or chronic tissue injury (Gu et al., 2016). AAN has been defined as a rapidly progressive tubulointerstitial nephritis, as well as interstitial fibrosis, which is featured with overexpressed α-SMA and decreased E-cadherin (Ziyong et al., 2009). Treatment with antagonist to C3aR in the AAI treated groups significantly attenuated fibrosis, marked with the decreased
3.1. Upregulation of C3a and C3aR in AAI-induced kidney injury As shown in Fig. 1A, the body weight of mice in the AAI group was significantly decreased. Renal function was accessed by measuring Cr and BUN in serum, demonstrating an increased trend (Fig. 1C–D). In accordance with biological indicators, histologic analysis indicated significant renal impairment, including dilatation of renal tubules, thickened tubular basement membrane and inflammatory cell infiltration (Fig. 1B). Upregulation of C3aR expression in renal tissues was observed according to both immunoblotting and quantitative real-time PCR (Supplement Fig. 1A). The release of C3a in serum was significantly increased in AAI group and peaked in 14 days (Supplement Fig. 1B). 3.2. Inhibition of C3aR reduces AAI-induced kidney damage To determine the function of C3aR in AAI -induced kidney damage, mice were intraperitoneally injected with C3a receptor antagonist (C3aRA). Compared with the AAI group, the body weight of mice in the C3aRA group significantly increased (Fig. 1A). Further, the serum levels of Cr and BUN, as well as IL-6 and TNF-α were decreased (Fig. 1C–F). According to histologic analysis, inhibition of C3aR also led to less tissue damage (Fig. 1B). Immunohistochemistry for IL-6, TNF-α, and C3aR showed less expression in renal tissues (Fig. 2F). Decreased expression of Bax/Bcl-2 and caspase-3 further demonstrated the protective effect of C3aRA in AAI -induced cell apoptosis (Fig. 2C). Decreased protein or mRNA expression of α-smooth muscle actin (α-SMA) and TGF-β1 was observed in C3aRA group, with elevated E-cadherin, which revealed a downward trend in fibrosis (Fig. 2C–E). These results point to the important role of C3aR in regulating cell apoptosis and renal fibrosis in AAN. 3.3. AAI induces excessive release of C3a and upregulation of C3aR in HK2 cells We further assessed the expression of C3a and C3aR in HK-2 cells. The viability of HK-2 cells was measured with an MTT assay, with the IC50 value of AAI approximately 59.74 μM. After 24 h of treatment, cytotoxicity was observed in only 10, 20, and 40 μM AAI groups (Fig. 3A). As such, we selected these three higher concentrations for the following experiments. Secretion of C3a, IL-6 and TNF-α in cell 123
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expression of α-SMA and TGF-β1 in renal tissues (Fig. 2C–F). We also found that the inhibition of C3aR protected against apoptosis in vivo (Fig. 2C–D) and significantly reduced the apoptosis rate of renal tubular epithelial cells in AAI treated group in vitro (Fig. 4A–B).
lipopolysaccharide-induced early pulmonary fibrosis by targeting ZEB1/2 via p38 MAPK and TGF-β/smad3 signaling pathways. Lab. Invest. 98 (3), 339–359. Cernoch, M., Hruba, P., Kollar, M., Mrazova, P., Stranavova, L., Lodererova, A., Honsova, E., Viklicky, O., 2018. Intrarenal complement system transcripts in chronic antibodymediated rejection and recurrent IgA nephropathy in kidney transplantation. Front. Immunol. 9, 2310. Fernandez-Godino, R., Pierce, E.A., 2018. C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway. Sci. Rep. 8, 9679. Gu, H., Fisher, A.J., Mickler, E.A., Cummings, O.W., Peters-Golden, M., Woodruff, T.M., Wilkes, D.S., Vittal, R., 2016. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis. FASEB J. 30, 2336–2350. Hansen, C.B., Willer, A., Bayarri-Olmos, R., Kemper, C., Garred, P., 2018. Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4 T cells. Immunobiology S0171-2985 (18), 30206–30207. Hayashi, Y., Mikawa, S., Ogawa, C., Masumoto, K., Katou, F., Sato, K., 2019. BMP6 expression in the adult rat central nervous system. J. Chem. Neuroanat. S0891-0618 (19), 30032–30038. Khan, M.A., Shamma, T., 2019. Complement factor and T-cell interactions during alloimmune inflammation in transplantation. J. Leukocyte Biol. 105, 681–694. Makrides, S.C., 1998. Therapeutic inhibition of the complement system. Pharmacol. Rev. 50, 59–87. Merle, N.S., Noe, R., Halbwachs-Mecarelli, L., Fremeaux-Bacchi, V., Roumenina, L.T., 2015. Complement system part II: role in immunity. Front. Immunol. 6, 257. Niño, C.A., Sala, S., Polo, S., 2018. When ubiquitin meets E-cadherin: plasticity of the epithelial cellular barrier. Semin. Cell Dev. Biol. S1084-9521 (18), 30081–30088. Noris, M., Remuzzi, G., 2017. Genetics of immune-mediated glomerular diseases: focus on complement. Semin. Nephrol. 37, 447–463. Pozdzik, A.A., Alix, B., Schmeiser, H.Z., Wassim, M., Christine, D., Salmon, I.L., JeanLouis, V., Nortier, J.L., 2010. Aristolochic acid nephropathy revisited: a place for innate and adaptive immunity? Histopathology 56, 449–463. Pu, X.Y., Shen, J.Y., Deng, Z.P., Zhang, Z.A., 2016. Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat. Exp. Toxicol. Pathol. 68, 315–320. Thorgersen, E.B., Barratt-Due, A., Haugaa, H., Harboe, M., Pischke, S.E., Nilsson, P.H., Mollnes, T.E., 2019. The Role of Complement in Liver Injury, Regeneration and Transplantation. Hepatology, Baltimore, Md.). https://doi.org/10.1002/hep.30508. Veje, M., Studahl, M., Bergström, T., 2019. Intrathecal complement activation by the classical pathway in tick-borne encephalitis. J. Neurovirol. Vieyra, M., Leisman, S., Raedler, H., Kwan, W.H., Yang, M., Strainic, M.G., Medof, M.E., Heeger, P.S., 2011. Complement regulates CD4 T-cell help to CD8 T cells required for murine allograft rejection. Am. J. Pathol. 179, 766–774. Wang, L., Ding, X., Li, C., Zhao, Y., Yu, C., Yi, Y., Zhang, Y., Gao, Y., Pan, C., Liu, S., Han, J., Tian, J., Liu, J., Deng, N., Li, G., Liang, A., 2018. Oral administration of Aristolochia manshuriensis Kom in rats induces tumors in multiple organs. J. Ethnopharmacol. 225, 81–89. Yi, J.H., Han, S.M., Kim, W.Y., Kim, J., Park, M.H., 2018. Effects of aristolochic acid I and/or hypokalemia on tubular damage in C57BL/6 rat with aristolochic acid nephropathy. Korean J. Intern. Med. 33, 763–773. Zi, Y.T., Bao, L., Ellen, H., Sacks, S.H., Sheerin, N.S., 2009. C3a mediates epithelial-tomesenchymal transition in proteinuric nephropathy. J. Am. Soc. Nephrol. 20, 593–603.
5. Conclusion In summary, our results in this study have revealed the activation of complement system in aristolochic acid induced nephritis. These findings supported the detrimental effects of C3a and its receptor C3aR in the development of fibrosis and inflammation in AAN. Furthermore, our observations offer new insights into the immunologic mechanisms involved in AAI -induced kidney injury, which can influence local inflammation and cell destiny. We hold the opinion that complementbased therapeutic intervention deserves further development in the treatment of AAN. 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. Transparency document The Transparency document associated with this article can be found in the online version. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.04.027. References Baudoux, T., Husson, C., De, P.E., Jadot, I., Antoine, M.N., Nortier, J.L., Hougardy, J.M., 2018. CD4 and CD8 T cells exert regulatory properties during experimental acute aristolochic acid nephropathy. Sci. Rep. 8, 5334. Bijkerk, R., Au, Y.M., Stam, W., Duijs, J.M.G.J., Koudijs, A., Lievers, E., Rabelink, T.J., van Zonneveld, A.J., 2019. Long Non-coding RNAs Rian and Miat mediate myofibroblast formation in kidney fibrosis. Front. Pharmacol. 10, 215. Cao, Y., Liu, Y., Ping, F., Yi, L., Zeng, Z., Li, Y., 2017. miR-200b/c attenuates
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Aristolochic acid I aggravates renal injury by activating the C3a/C3aR complement system
T
Jing Yea,1, Zhizhi Qiana,1, Mei Xueb, Yamin Liuc, Sirui Zhua, Yu Lia, Xiaoli Liua, Danhong Caia, ⁎ Jia Ruia, Liang Zhanga, a
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China College of Preclinical Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China c Department of Pharmacy, Zhongda Hospital, Southeast University, Nanjing, Jiangsu, China b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Complement system Aristolochic acid nephropathy C3a C3aR Epithelial-mesenchymal-transition
Previous studies have reported that the complement system is unconventionally activated in many kinds of glomerulonephritis. Multiple complement components participate in the pathogenic process by triggering immune response or other intracellular signaling pathways. Here, we have investigated the role of C3a and its receptor C3aR in aristolochic acid nephropathy (AAN), which, is featured with progressive interstitial fibrosis. Over release of C3a and increased expression of C3aR parallels to the up-regulation of α-SMA and TGF-β1 in AAN, which appeared to promote epithelial-mesenchymal-transition (EMT). To identify the role of complement activation in AAN, we used an inhibitor of C3aR (C3aRA) to block the coupling of C3a to its receptor. Our results confirmed from decreased EMT, the protective effect of C3aRA in cell apoptosis and inflammatory response induced by aristolochic acid I. These results showed that C3a and its receptor C3aR played pathogenic roles in AAN, and renal tubular epithelial cells were potentially pivotal targets of complement activation that could cause pro-fibrotic effects.
⁎
Corresponding author. E-mail address: [email protected] (L. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.toxlet.2019.04.027 Received 18 December 2018; Received in revised form 15 April 2019; Accepted 24 April 2019 Available online 29 April 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. C3aRA protected against AAI induced kidney injury. (A) Body weight of mice in different groups. (B) HE and Masson staining in renal tissues. (C–D) Levels of Cr and BUN in serum operated a recovery of renal function in C3aRA group. (E–F) Secretion of IL-6 and TNF-α was analyzed with ELISA detecting kits.. Rseults are shown as mean ± SD, *P < 0.001, *P < 0.01, compared to control group. ##P < 0.01, #P < 0.01 compared to AAI group.
Fig. 2. AAI induced apoptosis and fibrosis is dependent on the activation of C3a/C3aR complement system. (A–B) Detection of C3 and C3a in serum. (C–E) C3aRA reversed the up-regulation of C3aR, Caspase 3, Bax, α-SMA and TGF-β1, along with decreased levels of Bcl-2 and E-cadherin in AAI treated group, which were analyzed with immunoblotting and qPCR. (F) Expression of C3aR, IL-6, TNF-α, α-SMA and E-cadherin in renal tissues detected by immunohistochemistry were reversed in C3aRA treated group. Results are shown as mean ± SD (n = 3) and calculated from at least three independent experiments. *P < 0.001, *P < 0.01, * P < 0.05, compared to control group. ###P < 0.001, ##P < 0.01, #P < 0.05 compared to AAI group.
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Fig. 3. AAI caused activation of C3a/C3aR complement system and over-release of proinflammatory cytokines in HK-2 cells. (A) Cell viability was decreased in 24 h treatment with various concentrations of AAI. (B–D) Enhanced secretion of C3a, IL-6, TNF-α in AAI treated cells and operated a dose-dependent manner. Data are presented as means ± SD, *P < 0.001, *P < 0.01, *P < 0.05, relative to control group. (E–F) Fluorescence of C3aR and α-SMA was detected with immunofluorescence and indicated an increasing trend.
1. Introduction
AAI -induced nephritis. To test this topic, we detected C3a and its receptor C3aR both in vivo and in HK-2 cells after treatment with AAI. These results demonstrate that the complement system is activated in AAN, and that inhibition of C3aR blocks the intracellular signals of C3a, which finally provide a therapeutic mechanism for reducing apoptosis and fibrosis.
Aristolochic acid nephropathy (AAN) is a tubulointerstitial disease caused by over-exposure to aristolochic acids (Baudoux et al., 2018). Plants containing aristolochic acids have been widely applied in traditional medicine for arthritis, eczema, or other diseases in China and surrounding countries (Wang et al., 2018). However, significant sideeffects on renal tissues have limited their use in a clinical environment. It has been reported that aristolochic acid I (AAI) is the highest content in aristolochic acids, which is also demonstrated as the most representative component that can cause severe nephritis in vivo (Pu et al., 2016). The specific mechanism leading to the development of AAN is still unknown, despite multiple studies have been presented. According to clinical observation, immune response is triggered in interstitial inflammation which leads to continuous amplification of the fibrosis signal (Pozdzik et al., 2010). The complement system is typically considered as an important defense mechanism. But recent studies have indicated that the activation of the complement system might play a key role in glomerulonephritis (Noris and Remuzzi, 2017). C3 is a “hub” that regulates cascade activation of complement molecules (Merle et al., 2015). Its bioactive fragment C3a has been reported to act as an anaphylatoxin that can stimulate extravasation of host immune cells or cause matrix deposits via binding to its receptor C3aR (FernandezGodino and Pierce, 2018). As such, we hypothesize that complement activation is related to
2. Materials and methods 2.1. Animals and reagents Pathogen-free,6-to-8-week-old male C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised using standard protocols: temperature 22-25℃, relative humidity 50–60%, and 12 h light-dark cycle. All animals were given food and water in a standard laboratory diet. Depending on their groups, mice were intraperitoneally injected with AAI (5 mg/kg, 0.1 ml/10 g), with C3a receptor antagonist (C3aRA, 0.1 mg/kg, 0.1 ml/ 10 g), or with AAI and C3aRA in combination. The number of each group is ten. In addition, the control group was injected with the solvent of AAI: 10% dimethyl sulfoxide, 20% polyethylene glycol 600, 70% saline. AAI was injected daily and C3aRA injected every other day. After 14 days of treatment, mice were sacrificed and blood was centrifuged for biochemical detection. Kidneys were removed and flushed with NaCl 0.9%. After weighing, kidneys were prepared for further analysis. All animals were treated in accordance with the institutional 120
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Fig. 4. Inhibition of C3aR protected against cell apoptosis and fibrosis in HK-2 cells. (A–B) C3aRA (1μM) decreased apoptosis rate in AAI treated cells. (C–D) Expressions of apoptosis related proteins: Caspase 3, Bax/Bcl-2 and fibrosis related proteins: α-SMA/E-cadherin were down-regulated after treatment of C3aRA in AAI (40μM) treated HK-2 cells. (E) Reduced secretion of C3a, IL-6 and TNF-α in C3aRA treated group. Data are presented as means ± SD. *P < 0.001, *P < 0.01, * P < 0.05, compared to control group. ###P < 0.001, ##P < 0.01, #P < 0.05, relative to AAI group.
dehydration and permeabilization, kidneys were embedded in paraffin and sectioned for hematoxylin-eosin staining or Masson's trichrome staining. Histological analysis was obtained with light microscopy.
animal care guidelines issued by the Experimental Animal Ethical Committee of Nanjing University of Chinese Medicine, China. 2.2. Drugs and reagents
2.4. Immunohistochemistry
Aristolochic acid I was purchased from Henan Institute of Pharmaceutical Sciences (NO.20130201) and its purity≥98%, identified by HPLC. Antagonist of C3aR was from Merck Drugs & Biotechnology, 2730761. Kits for BUN and Cr were obtained from Nanjing Jiancheng Co, Ltd. The following antibodies were obtained from the companies cited: antibody to C3aR (CST, #14472), antibody to TGF-β1 (Biogot Biotechnology Co., Ltd, P03317), antibody to α-SMA (abcam, ab5694); antibody to GAPDH (CST, 8#2118S), and goat antirabbit IgG (CST, 25#7074S). ELISA kits for C3, C3a, IL-6, and TNF-α were brought from Nanjing Jiancheng Co, Ltd. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was obtained from Sigma. Apoptosis detection kits was from Nanjing KeyGEN Biotech Co, Ltd., Trizol from Invitrogen Corporation (15596-026), and transcriptor first strand cDNA synthesis kit from Thermo Fisher.
The immunohistochemistry was performed as described before (Hayashi et al., 2019). Briefly, the kidney sections were then deparaffinized, hydrated, and antigen-retrieved. Endogenous peroxidase was removed by 3% H2O2. Sections were blocked with 10% normal goat serum and incubated with antibodies to IL-6, TNF-α, α-SMA, E-cadherin or C3aR at room temperature for 1 h. Afterward, secondary antibody was applied at room temperature for 1 h. Following incubation with DAB for 10 min, sections were stained with hematoxylin and observed under a microscope.
2.5. Cell lines and cell culture HK-2 cell line was obtained from ATCC, NO: CRL2190™. Cells were cultured in F12-DMEM (WISENTINC, 319075010) medium containing 10% FBS (Hyclone, KPF21570) and 1% penicillin/streptomycin(vol/ vol) at 37℃ in 5% CO2.
2.3. Histopathology The kidneys of mice were fixed in 4% paraformaldehyde. After 121
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Fig. 5. C3aRA reversed AAI induced fibrosis in HK-2 cells. (A–B) C3aRA inhibited fluorescence intensity of α-SMA and C3aR in AAI treated groups. (C) Relative mRNA expression of α-SMA, E-cadherin, TGF-β1 and C3aR in HK-2 cells treated with AAI, C3aRA or co-incubation group. Data are presented as means ± SD. * P < 0.001, *P < 0.01, *P < 0.05, compared to control group. ###P < 0.001, #P < 0.05, relative to AAI group.
2.6. MTT assay
Biosciences AccuriC6).
Cells were seeded in 96-well plates at a density of 5 × 104cells/mL, 200 μL per well. After incubation at 37℃ in 5% CO2 for 12 h, cells were treated with various concentrations of AAI for 24 h. Supernatants were then removed, and 150 μL DMSO was added to each well. Plates were shaken on a table concentrator for 10 min and the absorbance was measured at 490 nm by a microplate reader (TECAN Infinite M200 PRO).
2.9. Biochemical detection
2.7. Immunofluorescence
2.10. Real-time and reverse transcription PCR
Cells were seeded in 24-well plates paved with sterile coverslips at a density of 1.5 × 104cells/mL. When cells grew to 70%–80% confluence, supernatants were removed and different concentrations of AAI were added into each well. After incubation with AAI for 24 h, cells were washed with cold PBS and fixed with 4% paraformaldehyde for 30 min. 0.1% Triton-100 was then applied for permeabilization, and antibody to C3aR or α-SMA was added for incubation at 4 °C overnight. Secondary antibodies were applied at room temperature for 2 h. Finally, DAPI was added for the staining of cell nuclei, and coverslips were observed under a fluorescent microscope (ZEISS inverted fluorescence microscope).
Cell samples and kidneys from mice were extracted with Trizol for total RNA according to manufacturer’s protocols. Concentration and purity of RNA were measured with the ratio of A260/A280. Reverse transcription was performed using abm All-in-one RT MasterMix, with PCR reactions performed as follows: predenaturation at 95℃ for 5 min, denaturation at 94 °C for 15 s, annealing at 60℃ for 60 s, then amplification for 40 cycles. The results were quantified by the ΔΔCt method. The primers used were: Mouse: C3a (forward: 5′-AGCTTCAGGGTCCC AGCTAC-3′; reverse: 5′-CTCTCCAGCCGTAGGACATT-3′); C3aR (forward: 5′-TTTCATCCAGCACCCCAGTG-3′; reverse: 5′-AGATGATGGCAC GAAGGCAA-3′); α-SMA (forward: 5′-GCTACGAACTGCCTGACGG-3′; reverse: 5′-GCTGTTATAGGTGGTTTCGTGGA-3′); E-cadherin (forward: 5′- CAGCCGGTCTTTGAGGGATT-3′; reverse: 5′-TGACGATGGTGTAGG CGATG-3′); TGF-β1 (forward: 5′- CTGCTGACCCCCACTGATAC-3′; reverse: 5′-AGCCCTGTATTCCGTCTCCT); GAPDH (forward: 5′-TATGTC GTGGAGTCTACTGGT-3′; reverse: 5′-GAGTTGTCATATTTCTCGTGG-3′); Human: C3aR (forward: 5′-CAGTGAGGAGCTCACACGTT-3′; reverse: 5′-TAAGAGCCCCTGCTTGTTGG-3′); α-SMA (forward: 5′-GGGACTAAG ACGGGAATCCT-3′; reverse: 5′-AGAGCCATTGTCACACACCA-3′); Ecadherin (forward: 5′-GAAGACAGAAGAGAGACTGGGTTA-3′; reverse: 5′-CAGGTTTTTAGGAAATGGGC-3′); TGF-β1 (forward: 5′-GGCTGTATT
Cell supernatants or blood of mice from different groups were coagulated on ice for 1 h, then centrifuged at 3000 r/min for 10 min. The contents of complement C3, C3a, IL-6, and TNF-α in serum or cell supernatants were measured with ELISA. The levels of serum creatinine (Cr) and urea nitrogen (BUN) were detected with biochemical kits.
2.8. Flow cytometry Annexin V/PI double-staining assay was applied to detect apoptosis rate. Cells were seeded in 6-well plates at a density of 1.5 × 105cells/ mL and incubated with different concentrations of AAI for 24 h. EDTAfree trypsin was then applied for digestion. After being washed twice with PBS, 5 × 105 cells were collected for detection. 5 μL Annexin VFITC and 5 μL propidiumidodide were added for staining. Cells were then incubated for 15 min and analyzed with flow cytometry (BD 122
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supernatants increased in a dose-dependent manner (Fig. 3B–D), as did up-regulation of C3aR and α-SMA on the cell surface (Fig. 3E–F). Additionally, we found an increased apoptosis rate in cells treated with AAI (Fig. 4A–B). As presented in Fig. 4C–D, we also observed increased expression of fibrosis-related proteins and mRNA in 40μM-treated groups. These results suggested that AAI triggeed a series of mechanisms related to cell damage, which also occured in a dose-dependent manner.
TAAGGACACCCGT-3′; reverse: 5′-GACACAGAGATCCGCAGTCC-3′); GAPDH (forward: 5′-CAGTGAGGAGCTCACACGTT-3′; reverse: 5′-TAA GAGCCCCTGCTTGTTGG-3′) 2.11. Immunoblotting Cell or kidney samples were lysed with RIPA lysis buffer. Lysates were then centrifugated, and the concentrations of proteins were analyzed with a BCA detecting kit. After being mixed with loading buffer and denatured in boiling water bath for 5 min, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and reacted overnight with antibodies specific to C3aR, caspase-3, BAX, Bcl-2, E-cadherin, αSMA, TGF-β1, or GAPDH at 4℃. After incubation with secondary antibodies, the immunoblots were visualized with chemiluminescence detection.
3.4. Inhibition of C3aR alleviated AAI-induced apoptosis and fibrosis in HK-2 cells To determine the possible roles of C3a and its receptor C3aR in AAI -induced HK-2 cell injury, we further applied an inhibitor of C3aR (C3aRA, 1μM) in AAI (40 μM)-treated cells (Fig. 5B). Firstly, we observed that cell apoptosis reversed in C3aRA treated groups (Fig. 4A–D). Additionally, down-regulation of α-SMA, TGF-β1 and increased expression of E-cadherin revealed a reduction in fibrosis (Figs. 4C–D, 5 A and C). Protective effect was also found in reduced secretion of IL-6 and TNF-α in cell supernatants (Fig. 4F–G).
2.12. Statistical analyses Results were presented as the mean ± SD. GraphPad Prism 6 software was used for statistical analysis: t-test for two groups, and oneway ANOVA for multiple groups.
4. Discussion
3. Results
The complement system is always considered as a bridge that connects innate and adaptive immunity. As a defensive mechanism against microbial intruders, it functions as an immune sentinel that recognizes and eliminates pathogens, which finally triggers adaptive response (Thorgersen et al., 2019). Recent studies have confirmed that complement participates in the pathogenesis of nephritis, such as lupus nephritis and IgA nephropathy (Cernoch et al., 2018). While the involvement of complement in AAN is still unknown. In the present study, several lines of evidences confirmed the proinflammatory and profibrotic roles of complement fragment C3a and its receptor C3aR in AAI -induced interstitial nephritis. C3a is a product cleaved from C3 and can trigger proinflammatory signal via binding to its G-protein-coupled receptor C3aR (Hansen et al., 2018; Khan and Shamma, 2019). Three biochemical pathways involved in the activation of complement system are converged on C3a, which highlights the functional role of C3a in multiple diseases (Veje et al., 2019). Previous study has demonstrated that C3a and its receptor C3aR contribute to the pathogenesis of pulmonary fibrosis (Gu et al., 2016). C3a-mediated mesenchymal transition of renal tubular epithelial cells has also been confirmed in vitro (Ziyong et al., 2009). In this study, we observed the excessive release of C3 and C3a in serum (Fig. 2A–B), with increased production of IL-6 and TNF-α, which are considered as inflammatory mediators (Fig. 1E–F). Up-regulation of C3aR in renal tissues also suggested the activation of complement system in AAI -induced kidney injury (Fig. 2C–F). α-SMA is a marker of myofibroblasts that barely expressed in normal tissues (Bijkerk et al., 2019). Additionally, E-cadherin-mediated cell adhesion maintains the integrity of cell polarity, which is lacked during fibrosis (Cao et al., 2017; Niño et al., 2018). Over-expression of α-SMA and reduced expression of Ecadherin in renal mesenchyme is consistent with previous studies, which indicated the presence of fibrosis (Fig. 2C–F) (Yi et al., 2018). In accordance with these findings in vivo, we observed similar results in AAI -induced cell injury in HK-2 cells, which also demonstrated that the renal tubular epithelial cell is one of the primary targets of renal tubular injury in AAN (Fig. 3). Blockage of C3aR or C5aR have been considered as a developing therapy method during allograft rejection (Makrides, 1998; Vieyra et al., 2011) for its prevention on tissue fibrosis or chronic tissue injury (Gu et al., 2016). AAN has been defined as a rapidly progressive tubulointerstitial nephritis, as well as interstitial fibrosis, which is featured with overexpressed α-SMA and decreased E-cadherin (Ziyong et al., 2009). Treatment with antagonist to C3aR in the AAI treated groups significantly attenuated fibrosis, marked with the decreased
3.1. Upregulation of C3a and C3aR in AAI-induced kidney injury As shown in Fig. 1A, the body weight of mice in the AAI group was significantly decreased. Renal function was accessed by measuring Cr and BUN in serum, demonstrating an increased trend (Fig. 1C–D). In accordance with biological indicators, histologic analysis indicated significant renal impairment, including dilatation of renal tubules, thickened tubular basement membrane and inflammatory cell infiltration (Fig. 1B). Upregulation of C3aR expression in renal tissues was observed according to both immunoblotting and quantitative real-time PCR (Supplement Fig. 1A). The release of C3a in serum was significantly increased in AAI group and peaked in 14 days (Supplement Fig. 1B). 3.2. Inhibition of C3aR reduces AAI-induced kidney damage To determine the function of C3aR in AAI -induced kidney damage, mice were intraperitoneally injected with C3a receptor antagonist (C3aRA). Compared with the AAI group, the body weight of mice in the C3aRA group significantly increased (Fig. 1A). Further, the serum levels of Cr and BUN, as well as IL-6 and TNF-α were decreased (Fig. 1C–F). According to histologic analysis, inhibition of C3aR also led to less tissue damage (Fig. 1B). Immunohistochemistry for IL-6, TNF-α, and C3aR showed less expression in renal tissues (Fig. 2F). Decreased expression of Bax/Bcl-2 and caspase-3 further demonstrated the protective effect of C3aRA in AAI -induced cell apoptosis (Fig. 2C). Decreased protein or mRNA expression of α-smooth muscle actin (α-SMA) and TGF-β1 was observed in C3aRA group, with elevated E-cadherin, which revealed a downward trend in fibrosis (Fig. 2C–E). These results point to the important role of C3aR in regulating cell apoptosis and renal fibrosis in AAN. 3.3. AAI induces excessive release of C3a and upregulation of C3aR in HK2 cells We further assessed the expression of C3a and C3aR in HK-2 cells. The viability of HK-2 cells was measured with an MTT assay, with the IC50 value of AAI approximately 59.74 μM. After 24 h of treatment, cytotoxicity was observed in only 10, 20, and 40 μM AAI groups (Fig. 3A). As such, we selected these three higher concentrations for the following experiments. Secretion of C3a, IL-6 and TNF-α in cell 123
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expression of α-SMA and TGF-β1 in renal tissues (Fig. 2C–F). We also found that the inhibition of C3aR protected against apoptosis in vivo (Fig. 2C–D) and significantly reduced the apoptosis rate of renal tubular epithelial cells in AAI treated group in vitro (Fig. 4A–B).
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5. Conclusion In summary, our results in this study have revealed the activation of complement system in aristolochic acid induced nephritis. These findings supported the detrimental effects of C3a and its receptor C3aR in the development of fibrosis and inflammation in AAN. Furthermore, our observations offer new insights into the immunologic mechanisms involved in AAI -induced kidney injury, which can influence local inflammation and cell destiny. We hold the opinion that complementbased therapeutic intervention deserves further development in the treatment of AAN. 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. Transparency document The Transparency document associated with this article can be found in the online version. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.04.027. References Baudoux, T., Husson, C., De, P.E., Jadot, I., Antoine, M.N., Nortier, J.L., Hougardy, J.M., 2018. CD4 and CD8 T cells exert regulatory properties during experimental acute aristolochic acid nephropathy. Sci. Rep. 8, 5334. Bijkerk, R., Au, Y.M., Stam, W., Duijs, J.M.G.J., Koudijs, A., Lievers, E., Rabelink, T.J., van Zonneveld, A.J., 2019. Long Non-coding RNAs Rian and Miat mediate myofibroblast formation in kidney fibrosis. Front. Pharmacol. 10, 215. Cao, Y., Liu, Y., Ping, F., Yi, L., Zeng, Z., Li, Y., 2017. miR-200b/c attenuates
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