Lipopolysaccharide-induced podocyte injury is mediated by suppression of autophagy

Tan,Xiaofan; Chen,Yuanhan; Liang,Xinling; Yu,Chunping; Lai,Yuxiong; Zhang,Li; Zhao,Xingchen; Zhang,Hong; Lin,Ting; Li,Ruizhao; Shi,Wei

doi:10.3892/mmr.2016.5301

Lipopolysaccharide-induced podocyte injury is mediated by suppression of autophagy

Authors:
- Xiaofan Tan
- Yuanhan Chen
- Xinling Liang
- Chunping Yu
- Yuxiong Lai
- Li Zhang
- Xingchen Zhao
- Hong Zhang
- Ting Lin
- Ruizhao Li
- Wei Shi
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Affiliations: Division of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, P.R. China
Published online on: May 18, 2016 https://doi.org/10.3892/mmr.2016.5301
Pages: 811-818

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Abstract

High-level autophagy has an important role in maintaining the stable state of podocytes. The present study explored the influence of lipopolysaccharide (LPS) on autophagic activity in podocytes and demonstrated its mechanistic involvement in LPS-induced injury. Conditionally immortalized podocytes were cultured in vitro and were treated with chloroquine (CQ), LPS, LPS+rapamycin or LPS+3‑methyladenine (3‑MA). The autophagic vesicles and endoplasmic reticulum were observed using transmission electron microscopy. The tandem mRFP‑GFP‑LC3 adenovirus was used to detect autophagosomes and autolysosomes. The expression levels of light chain 3‑II (LC3 II), beclin‑1, P62, CCAAT‑enhancer‑binding protein homologous protein (CHOP) and podocin were determined by western blot analysis. Autophagic vesicles were detected in podocytes under basic conditions. CQ was found to increase the protein levels of LC3 II in a time‑dependent manner (2, 4 or 6 h), confirming the high activity of autophagy in podocytes. Compared with the control group, LPS induced the expansion of the endoplasmic reticulum and high expression levels of CHOP, while decreasing the protein expression of podocin. Notably, podocytes treated with LPS showed decreases in LC3 II and beclin‑1 levels and autophagosome/autolysosome numbers, which was accompanied by high P62 levels. Furthermore, the autophagy enhancer rapamycin reversed the downregulation of LC3 II and podocin, and the upregulation of CHOP induced by LPS, while the autophagy inhibitor 3‑MA aggravated the effects of LPS. In conclusion, the present study demonstrated that LPS inhibited podocyte autophagy, which contributed to LPS-induced injury of podocytes.

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1	Pavenstadt H, Kriz W and Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev. 83:253–307. 2003. View Article : Google Scholar
2	Greka A and Mundel P: Cell biology and pathology of podocytes. Annu Rev Physiol. 74:299–323. 2012. View Article : Google Scholar
3	Gubler MC: Podocyte differentiation and hereditary proteinuria/nephrotic syndromes. J Am Soc Nephrol. 14(Suppl 1): S22–S26. 2003. View Article : Google Scholar : PubMed/NCBI
4	Patrakka J and Tryggvason K: New insights into the role of podocytes in proteinuria. Nat Rev Nephrol. 5:463–468. 2009. View Article : Google Scholar : PubMed/NCBI
5	Yu CC, Fornoni A, Weins A, Hakroush S, Maiguel D, Sageshima J, Chen L, Ciancio G, Faridi MH, Behr D, et al: Abatacept in B7-1-positive proteinuric kidney disease. N Engl J Med. 369:2416–2423. 2013. View Article : Google Scholar : PubMed/NCBI
6	Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, Giardino L, Rastaldi MP, Calvaresi N, Watanabe H, Schwarz K, et al: Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest. 113:1390–1397. 2004. View Article : Google Scholar : PubMed/NCBI
7	Wei C, Moller CC, Altintas MM, Li J, Schwarz K, Zacchigna S, Xie L, Henger A, Schmid H, Rastaldi MP, et al: Modification of kidney barrier function by the urokinase receptor. Nat Med. 14:55–63. 2008. View Article : Google Scholar
8	Saurus P, Kuusela S, Lehtonen E, Hyvönen ME, Ristola M, Fogarty CL, Tienari J, Lassenius MI, Forsblom C, Lehto M, et al: Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway. Cell Death Dis. 6:e17522015. View Article : Google Scholar : PubMed/NCBI
9	Kumagai T, Baldwin C, Aoudjit L, Nezvitsky L, Robins R, Jiang R and Takano T: Protein tyrosine phosphatase 1B inhibition protects against podocyte injury and proteinuria. Am J Pathol. 184:2211–2224. 2014. View Article : Google Scholar : PubMed/NCBI
10	Zhang B, Shi W, Ma J, Sloan A, Faul C, Wei C, Reiser J, Yang Y, Liu S and Wang W: The calcineurin-NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury. J Mol Med (Berl). 90:1407–1420. 2012. View Article : Google Scholar
11	Zhang B, Xie S, Shi W and Yang Y: Amiloride off-target effect inhibits podocyte urokinase receptor expression and reduces proteinuria. Nephrol Dial Transplant. 27:1746–1755. 2012. View Article : Google Scholar
12	Mizushima N, Levine B, Cuervo AM and Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature. 451:1069–1075. 2008. View Article : Google Scholar : PubMed/NCBI
13	Hartleben B, Gödel M, Meyer-Schwesinger C, Liu S, Ulrich T, Köbler S, Wiech T, Grahammer F, Arnold SJ, Lindenmeyer MT, et al: Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 120:1084–1096. 2010. View Article : Google Scholar : PubMed/NCBI
14	Mundel P, Reiser J, Zúñiga Mejía Borja A, Pavenstädt H, Davidson GR, Kriz W and Zeller R: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res. 236:248–58. 1997. View Article : Google Scholar : PubMed/NCBI
15	Srivastava T, Sharma M, Yew KH, Sharma R, Duncan RS, Saleem MA, McCarthy ET, Kats A, Cudmore PA, Alon US, et al: LPS and PAN-induced podocyte injury in an in vitro model of minimal change disease: changes in TLR profile. J Cell Commun Signal. 7:49–60. 2013. View Article : Google Scholar :
16	Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, Giardino L, Rastaldi MP, Calvaresi N, Watanabe H, Schwarz K, et al: Induction of B7 1 in podocytes is associated with nephrotic syndrome. J Clin Invest. 113:1390–1397. 2004. View Article : Google Scholar : PubMed/NCBI
17	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
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	Klionsky DJ and Emr SD: Autophagy as a regulated pathway of cellular degradation. Science. 290:1717–1721. 2000. View Article : Google Scholar : PubMed/NCBI
20	Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y and Kominami E: Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 441:880–884. 2006. View Article : Google Scholar : PubMed/NCBI
21	Maiuri MC, Zalckvar E, Kimchi A and Kroemer G: Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 8:741–752. 2007. View Article : Google Scholar : PubMed/NCBI
22	Chen YM, Zhou Y, Go G, Marmerstein JT, Kikkawa Y and Miner JH: Laminin beta2 gene missense mutation produces endoplasmic reticulum stress in podocytes. J Am Soc Nephrol. 24:1223–1233. 2013. View Article : Google Scholar : PubMed/NCBI
23	Yu CC, Fornoni A, Weins A, Hakroush S, Maiguel D, Sageshima J, Chen L, Ciancio G, Faridi MH, Behr D, et al: Abatacept in B7 1 positive proteinuric kidney disease. N Engl J Med. 369:2416–2423. 2013. View Article : Google Scholar : PubMed/NCBI
24	Ma J, Zhang B, Liu S, Xie S, Yang Y, Ma J, Deng Y, Wang W, Xu L, Li R, et al: 1,25-dihydroxyvitamin D(3) inhibits podocyte uPAR expression and reduces proteinuria. PLoS One. 8:e649122013. View Article : Google Scholar : PubMed/NCBI
25	Zeng C, Fan Y, Wu J, Shi S, Chen Z, Zhong Y, Zhang C, Zen K and Liu Z: Podocyte autophagic activity plays a protective role in renal injury and delays the progression of podocytopathies. J Pathol. 234:203–213. 2014.PubMed/NCBI
26	Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al: Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 8:445–544. 2012. View Article : Google Scholar : PubMed/NCBI
27	Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H and Johansen T: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol. 171:603–614. 2005. View Article : Google Scholar : PubMed/NCBI
28	Zhou C, Zhong W, Zhou J, Sheng F, Fang Z, Wei Y, Chen Y, Deng X, Xia B and Lin J: Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy. 8:1215–1226. 2012. View Article : Google Scholar : PubMed/NCBI
29	Cybulsky AV, Takano T, Papillon J, Kitzler TM and Bijian K: Endoplasmic reticulum stress in glomerular epithelial cell injury. Am J Physiol Renal Physiol. 301:F496–F508. 2011. View Article : Google Scholar
30	Wu L, Feng Z, Cui S, Hou K, Tang L, Zhou J, Cai G, Xie Y, Hong Q, Fu B and Chen X: Rapamycin upregulates autophagy by inhibiting the mTOR-ULK1 pathway, resulting in reduced podocyte injury. PLoS One. 8:e637992013. View Article : Google Scholar : PubMed/NCBI
31	Seglen PO and Gordon PB: 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA. 79:1889–1892. 1982. View Article : Google Scholar : PubMed/NCBI
32	Mackeh R, Perdiz D, Lorin S, Codogno P and Poüs C: Autophagy and microtubules-new story, old players. J Cell Sci. 126:1071–1080. 2013. View Article : Google Scholar : PubMed/NCBI