Effect of ATM on inflammatory response and autophagy in renal tubular epithelial cells in LPS‑induced septic AKI

Zheng,Chenfei; Zhou,Ying; Huang,Yueyue; Chen,Bicheng; Wu,Minmin; Xie,Yue; Chen,Xinxin; Sun,Mei; Liu,Yi; Chen,Chaosheng; Pan,Jingye

doi:10.3892/etm.2019.8115

Effect of ATM on inflammatory response and autophagy in renal tubular epithelial cells in LPS‑induced septic AKI

Authors:
- Chenfei Zheng
- Ying Zhou
- Yueyue Huang
- Bicheng Chen
- Minmin Wu
- Yue Xie
- Xinxin Chen
- Mei Sun
- Yi Liu
- Chaosheng Chen
- Jingye Pan
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Affiliations: Department of Nephrology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Department of Intensive Care Unit, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery, Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
Published online on: October 21, 2019 https://doi.org/10.3892/etm.2019.8115
Pages: 4707-4717
Copyright: © Zheng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to explore the role of ataxia‑telangiectasia mutated (ATM) in lipopolysaccharide (LPS)‑induced in vitro model of septic acute kidney injury (AKI) and the association between ATM, tubular epithelial inflammatory response and autophagy. The renal tubular epithelial cell HK‑2 cell line was cultured and passaged, with HK‑2 cell injury induced by LPS. The effects of LPS on HK‑2 cell morphology, viability, ATM expression and inflammation were observed. Lentiviral vectors encoding ATM shRNA were constructed to knock down ATM expression in HK‑2 cells. The efficiency of ATM knockdown in HK‑2 cells was detected by western blot analysis and reverse transcription‑quantitative PCR (RT‑qPCR). HK‑2 cells transfected with the ATM shRNA lentivirus were used for subsequent experiments. Following ATM knockdown, corresponding controls were set up, and the effects of ATM on inflammation and autophagy were detected in HK‑2 cells using RT‑qPCR, western blotting and ELISA. After LPS stimulation, the HK‑2 cells were rounded into a slender or fusiform shape with poorly defined outlines. LPS treatment reduced cell viability in a partly dose‑dependent manner. LPS increased the expression of tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6, with the levels reaching its highest value at 10 µg/ml. IL‑6 and IL‑1β expression increased with increasing LPS concentration. These findings suggest that LPS reduced HK‑2 cell viability whilst increasing the expression of inflammatory factors. Following transfection with ATM shRNA, expression levels of key autophagy indicators microtubule associated protein 1 light chain 3α I/II ratio and beclin‑1 in the two ATM shRNA groups were also significantly reduced compared with the NC shRNA group. In summary, downregulation of ATM expression in HK‑2 cells reduced LPS‑induced inflammation and autophagy in sepsis‑induced AKI in vitro, suggesting that LPS may induce autophagy in HK‑2 cells through the ATM pathway leading to the upregulation of inflammatory factors.

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1	Bagshaw SM, Uchino S, Bellomo R, Morimatsu H, Morgera S, Schetz M, Tan I, Bouman C, Macedo E, Gibney N, et al: Septic acute kidney injury in critically ill patients: Clinical characteristics and outcomes. Clin J Am Soc Nephrol. 2:431–439. 2007. View Article : Google Scholar : PubMed/NCBI
2	Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, et al: The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 315:801–810. 2016. View Article : Google Scholar : PubMed/NCBI
3	Holthoff JH, Wang Z, Patil NK, Gokden N and Mayeux PR: Rolipram improves renal perfusion and function during sepsis in the mouse. J Pharmacol Exp Ther. 347:357–364. 2013. View Article : Google Scholar : PubMed/NCBI
4	Howell GM, Gomez H, Collage RD, Loughran P, Zhang X, Escobar DA, Billiar TR, Zuckerbraun BS and Rosengart MR: Augmenting autophagy to treat acute kidney injury during endotoxemia in mice. PLoS One. 8:e695202013. View Article : Google Scholar : PubMed/NCBI
5	Bagshaw SM, George C and Bellomo R; ANZICS Database Management Committee, : Early acute kidney injury and sepsis: A multicentre evaluation. Crit Care. 12:R472008. View Article : Google Scholar : PubMed/NCBI
6	Wang F, Zhang G, Lu Z, Geurts AM, Usa K, Jacob HJ, Cowley AW, Wang N and Liang M: Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury. Kidney Int. 88:796–803. 2015. View Article : Google Scholar : PubMed/NCBI
7	Chen LW, Chen W, Hu ZQ, Bian JL, Ying L, Hong GL, Qiu QM, Zhao GJ and Lu ZQ: Protective effects of growth arrest-specific protein 6 (Gas6) on sepsis-induced acute kidney injury. Inflammation. 39:575–582. 2016. View Article : Google Scholar : PubMed/NCBI
8	Yohannes S and Chawla LS: Evolving practices in the management of acute kidney injury in the ICU (Intensive Care Unit). Clin Nephrol. 71:602–607. 2009. View Article : Google Scholar : PubMed/NCBI
9	Gómez H, Kellum JA and Ronco C: Metabolic reprogramming and tolerance during sepsis-induced AKI. Nat Rev Nephrol. 13:143–151. 2017. View Article : Google Scholar : PubMed/NCBI
10	Luo CJ, Luo F, Zhang L, Xu Y, Cai GY, Fu B, Feng Z, Sun XF and Chen XM: Knockout of interleukin-17A protects against sepsis-associated acute kidney injury. Ann Intensive Care. 6:562016. View Article : Google Scholar : PubMed/NCBI
11	Chunzhi G, Zunfeng L, Chengwei Q, Xiangmei B and Jingui Y: Hyperin protects against LPS-induced acute kidney injury by inhibiting TLR4 and NLRP3 signaling pathways. Oncotarget. 7:82602–82608. 2016. View Article : Google Scholar : PubMed/NCBI
12	Xu C, Chang A, Hack BK, Eadon MT, Alper SL and Cunningham PN: TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis. Kidney Int. 85:72–81. 2014. View Article : Google Scholar : PubMed/NCBI
13	Ahn JM, You SM, Lee YM, Oh SW, Ahn SY, Kim S, Chin HJ, Chae DW and Na KY: Hypoxia-inducible factor activation protects the kidney from gentamicin-induced acute injury. PLoS One. 7:e489522012. View Article : Google Scholar : PubMed/NCBI
14	Sutton TA, Hato T, Mai E, Yoshimoto M, Kuehl S, Anderson M, Mang H, Plotkin Z, Chan RJ and Dagher PC: p53 Is renoprotective after ischemic kidney injury by reducing inflammation. J Am Soc Nephrol. 24:113–124. 2013. View Article : Google Scholar : PubMed/NCBI
15	Klionsky DJ and Emr SD: Autophagy as a regulated pathway of cellular degradation. Science. 290:1717–1721. 2000. View Article : Google Scholar : PubMed/NCBI
16	Levine B and Kroemer G: Autophagy in the pathogenesis of disease. Cell. 132:27–42. 2008. View Article : Google Scholar : PubMed/NCBI
17	Leventhal JS, Ni J, Osmond M, Lee K, Gusella GL, Salem F and Ross MJ: Autophagy limits endotoxemic acute kidney injury and alters renal tubular epithelial cell cytokine expression. PLoS One. 11:e01500012016. View Article : Google Scholar : PubMed/NCBI
18	Mei S, Livingston M, Hao J, Li L, Mei C and Dong Z: Autophagy is activated to protect against endotoxic acute kidney injury. Sci Rep. 6:221712016. View Article : Google Scholar : PubMed/NCBI
19	Yu JH, Cho SO, Lim JW, Kim N and Kim H: Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression. Int J Biochem Cell Biol. 60:147–156. 2015. View Article : Google Scholar : PubMed/NCBI
20	Moser BA, Subramanian L, Khair L, Chang YT and Nakamura TM: Fission yeast Tel1 (ATM) and Rad3 (ATR) promote telomere protection and telomerase recruitment. PLoS Genet. 5:e10006222009. View Article : Google Scholar : PubMed/NCBI
21	Abraham RT: Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15:2177–2196. 2001. View Article : Google Scholar : PubMed/NCBI
22	Bencokova Z, Kaufmann MR, Pires IM, Lecane PS, Giaccia AJ and Hammond EM: ATM activation and signaling under hypoxic conditions. Mol Cell Biol. 29:526–537. 2009. View Article : Google Scholar : PubMed/NCBI
23	Wang LT, Chen BL, Wu CT, Huang KH, Chiang CK and Hwa Liu S: Protective role of AMP-activated protein kinase-evoked autophagy on an in vitro model of ischemia/reperfusion-induced renal tubular cell injury. PLoS One. 8:e798142013. View Article : Google Scholar : PubMed/NCBI
24	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
25	Jung G, Roh J, Lee H, Gil M, Yoon DH, Suh C, Jang S, Park CJ, Huh J and Park CS: Autophagic markers BECLIN 1 and LC3 are associated with prognosis of multiple myeloma. Acta Haematol. 134:17–24. 2015. View Article : Google Scholar : PubMed/NCBI
26	Dellepiane S, Marengo M and Cantaluppi V: Detrimental cross-talk between sepsis and acute kidney injury: New pathogenic mechanisms, early biomarkers and targeted therapies. Crit Care. 20:612016. View Article : Google Scholar : PubMed/NCBI
27	Jackson WL Jr: Acute renal failure and sepsis. N Engl J Med. 351:2347–2349. 2004. View Article : Google Scholar : PubMed/NCBI
28	Langenberg C, Wan L, Egi M, May CN and Bellomo R: Renal blood flow in experimental septic acute renal failure. Kidney Int. 69:1996–2002. 2006. View Article : Google Scholar : PubMed/NCBI
29	Fani F, Regolisti G, Delsante M, Cantaluppi V, Castellano G, Gesualdo L, Villa G and Fiaccadori E: Recent advances in the pathogenetic mechanisms of sepsis-associated acute kidney injury. J Nephrol. 31:351–359. 2018. View Article : Google Scholar : PubMed/NCBI
30	Chen L, Yang S, Zumbrun EE, Guan H, Nagarkatti PS and Nagarkatti M: Resveratrol attenuates lipopolysaccharide-induced acute kidney injury by suppressing inflammation driven by macrophages. Mol Nutr Food Res. 59:853–864. 2015. View Article : Google Scholar : PubMed/NCBI
31	Deng SY, Zhang LM, Ai YH, Pan PH, Zhao SP, Su XL, Wu DD, Tan HY, Zhang LN and Tsung A: Role of interferon regulatory factor-1 in lipopolysaccharide-induced mitochondrial damage and oxidative stress responses in macrophages. Int J Mol Med. 40:1261–1269. 2017. View Article : Google Scholar : PubMed/NCBI
32	Li T, Zhao J, Miao S, Xu Y, Xiao X and Liu Y: Dynamic expression and roles of sequestome-1/p62 in LPS-induced acute kidney injury in mice. Mol Med Rep. 17:7618–7626. 2018.PubMed/NCBI
33	Jiao XY, Shen YQ and Li KS: The correlation between cytokine production by cerebral cortical glial cells and brain lateralization in mice. Neuromodulation. 11:23–32. 2008. View Article : Google Scholar : PubMed/NCBI
34	Johnson RL, Murray ST, Camacho DK and Wilson CG: Vagal nerve stimulation attenuates IL-6 and TNFα expression in respiratory regions of the developing rat brainstem. Respir Physiol Neurobiol. 229:1–4. 2016. View Article : Google Scholar : PubMed/NCBI
35	Lee WS, Shin JS, Jang DS and Lee KT: Cnidilide, an alkylphthalide isolated from the roots of Cnidium officinale, suppresses LPS-induced NO, PGE2, IL-1β, IL-6 and TNF-α production by AP-1 and NF-κB inactivation in RAW 264.7 macrophages. Int Immunopharmacol. 40:146–155. 2016. View Article : Google Scholar : PubMed/NCBI
36	Kim KH and Lee MS: Autophagy as a crosstalk mediator of metabolic organs in regulation of energy metabolism. Rev Endoc Metab Disord. 15:11–20. 2014. View Article : Google Scholar
37	Shen HM and Codogno P: Autophagic cell death: Loch Ness monster or endangered species? Autophagy. 7:457–465. 2011. View Article : Google Scholar : PubMed/NCBI
38	Shintani T and Klionsky DJ: Autophagy in health and disease: A double-edged sword. Science. 306:990–995. 2004. View Article : Google Scholar : PubMed/NCBI
39	Levine B and Yuan J: Autophagy in cell death: An innocent convict? J Clin Invest. 115:2679–2688. 2005. View Article : Google Scholar : PubMed/NCBI
40	Casado P, Bilanges B, Rajeeve V, Vanhaesebroeck B and Cutillas PR: Environmental stress affects the activity of metabolic and growth factor signaling networks and induces autophagy markers in MCF7 breast cancer cells. Mol Cell Proteomics. 13:836–848. 2014. View Article : Google Scholar : PubMed/NCBI
41	Shi R, Weng J, Zhao L, Li XM, Gao TM and Kong J: Excessive autophagy contributes to neuron death in cerebral ischemia. CNS Neurosci Ther. 18:250–260. 2012. View Article : Google Scholar : PubMed/NCBI
42	Livingston MJ and Dong Z: Autophagy in acute kidney injury. Semin Nephrol. 34:17–26. 2014. View Article : Google Scholar : PubMed/NCBI
43	Chien WS, Chen YH, Chiang PC, Hsiao HW, Chuang SM, Lue SI and Hsu C: Suppression of autophagy in rat liver at late stage of polymicrobial sepsis. Shock. 35:506–511. 2011. View Article : Google Scholar : PubMed/NCBI
44	Hsieh CH, Pai PY, Hsueh HW, Yuan SS and Hsieh YC: Complete induction of autophagy is essential for cardioprotection in sepsis. Ann Surg. 253:1190–1200. 2011. View Article : Google Scholar : PubMed/NCBI
45	Şen V, Uluca Ü, Ece A, Güneş A, Zeytun H, Arslan S, Kaplan I, Türkçü G and Tekin R: Role of Ankaferd on bacterial translocation and inflammatory response in an experimental rat model of intestinal obstruction. Int J Clin Exp Med. 7:2677–2686. 2014.PubMed/NCBI
46	Sang HS, Lee KE, Kim IJ, Kim O, Kim CS, Choi JS, Choi HI, Bae EH, Ma SK, Lee JU and Kim SW: Alpha-lipoic acid attenuates lipopolysaccharide-induced kidney injury. Clin Exp Nephrol. 19:82–91. 2015. View Article : Google Scholar : PubMed/NCBI
47	Xiang H, Hu B, Li Z and Li J: Dexmedetomidine controls systemic cytokine levels through the cholinergic anti-inflammatory pathway. Inflammation. 37:1763–1770. 2014. View Article : Google Scholar : PubMed/NCBI
48	Kong Y, Yin J, Cheng D, Lu Z, Wang N, Wang F and Liang M: Antithrombin III attenuates AKI following acute severe pancreatitis. Shock. 49:572–579. 2018. View Article : Google Scholar : PubMed/NCBI
49	Lu Z, Cheng D, Yin J, Wu R, Zhang G, Zhao Q, Wang N, Wang F and Liang M: Antithrombin III protects against contrast-induced nephropathy. EBioMedicine. 17:101–107. 2017. View Article : Google Scholar : PubMed/NCBI
50	Chen K, Dai H, Yuan J, Chen J, Lin L, Zhang W, Wang L, Zhang J, Li K and He Y: Optineurin-mediated mitophagy protects renal tubular epithelial cells against accelerated senescence in diabetic nephropathy. Cell Death Dis. 9:1052018. View Article : Google Scholar : PubMed/NCBI
51	Lu B, Capan E and Li C: Autophagy induction and autophagic cell death in effector T cells. Autophagy. 3:158–159. 2007. View Article : Google Scholar : PubMed/NCBI
52	Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Lam HC, Rabinovitch M, et al: Autophagy proteins regulate innate immune response by inhibiting NALP3 inflammasome-mediated mitochondrial DAN release. American Thoracic Society 2011 International Conference, May 13–18, 2011 • Denver Colorado. 2011.A1077
53	Carchman EH, Rao J, Loughran PA, Rosengart MR and Zuckerbraun BS: Heme oxygenase-1-mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice. Hepatology. 53:2053–2062. 2011. View Article : Google Scholar : PubMed/NCBI
54	Kaushal GP: Autophagy protects proximal tubular cells from injury and apoptosis. Kidney Int. 82:1250–1253. 2012. View Article : Google Scholar : PubMed/NCBI