Knockdown of augmenter of liver regeneration in HK-2 cells inhibits inflammation response via the mitogen-activated protein kinase signaling pathway.
OBJECTIVE
Augmenter of liver regeneration (ALR) is a growth factor that is ubiquitously expressed in multiple forms among eukaryotes. The present study focused on the role of endogenous ALR on the hypoxia/reoxygenation (H/R)-induced inflammatory response in human kidney 2 (HK-2) cells, and the underlying molecular mechanisms.


METHODS
To determine the relationship between exogenous and endogenous ALR, exogenous ALR was administrated to HK-2 cells, and endogenous ALR protein and mRNA expression was examined by Western blotting and quantitative real-time polymerase chain reaction (qPCR), respectively. In order to knockdown endogenous ALR expression, HK-2 cells were infected with lentiviral shRNA/ALR, after which cell viability was determined by the MTS cell viability assay. Cells were subjected to hypoxia for 6 h and reoxygenation for 12 h. Levels of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR. Cells were harvested, and nuclear and phosphorylated protein extracts were prepared from the HK-2 cell lysates. Nuclear factor κB (NF-κB), and phosphorylated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were analyzed by Western blotting. The translocation of NF-κB was detected by immunofluorescence.


RESULTS
Exogenous ALR inhibited the expression of endogenous ALR. Lentiviral shRNA/ALR markedly downregulated endogenous ALR expression, whereas there were no changes in ALR expression in lentiviral shRNA/control HK-2 cells. The results of the MTS assay showed that silencing ALR expression did not influence cell viability. H/R led to increased production of MCP-1, IL-6, and TNF-α. However, knockdown of ALR attenuated the inflammatory response via inhibition of ERK, p38, and JNK phosphorylation. The translocation of NF-κB into the nucleus was also decreased.


CONCLUSIONS
These results suggest that there is a negative feedback loop involving ALR in HK-2 cells. Knockdown of ALR exerts anti-inflammatory actions via suppression of the mitogen-activated protein kinase signaling pathway.
Department of Nephrology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China, yrysuperman@126.com.
Ruyu
R
Yan
Ling
L
Zhang
Ning
N
Xia
Qi
Q
Liu
Hang
H
Sun
Hui
H
Guo
D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D018925:Chemokines / Q000096:biosynthesis; D003579:Cytochrome Reductases / Q000235:genetics* / Q000502:physiology*; D016207:Cytokines / Q000096:biosynthesis; D055785:Gene Knockdown Techniques; D006801:Humans; D007249:Inflammation / Q000235:genetics* / Q000473:pathology*; D016086:Lentivirus / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D034741:RNA, Small Interfering / Q000235:genetics
D018925:Chemokines; D016207:Cytokines; D034741:RNA, Small Interfering; D003579:Cytochrome Reductases; C500897:GFER protein, human; D050862:Oxidoreductases Acting on Sulfur Group Donors; D020928:Mitogen-Activated Protein Kinases