Korean J Physiol Pharmacol.  2018 Mar;22(2):135-143. 10.4196/kjpp.2018.22.2.135.

Tumor necrosis factor α-converting enzyme inhibitor attenuates lipopolysaccharide-induced reactive oxygen species and mitogen-activated protein kinase expression in human renal proximal tubule epithelial cells

Affiliations
  • 1Department of Internal Medicine, Chonnam National University Medical School, Gwangju 61707, Korea. skimw@chonnam.ac.kr
  • 2Global Desalination Research Center (GDRC), School of Environmental Science and Engineering (SESE), Gwangju Institute of Science and Technology (GIST), Gwnagju 61005, Korea.

Abstract

Tumor necrosis factor-α (TNFα) and the angiotensin system are involved in inflammatory diseases and may contribute to acute kidney injury. We investigated the mechanisms by which TNFα-converting enzyme (TACE) contributes to lipopolysaccharide (LPS)-induced renal inflammation and the effect of TACE inhibitor treatment on LPS-induced cellular injury in human renal proximal tubule epithelial (HK-2) cells. Mice were treated with LPS (10 mg/kg, i.p.) and HK-2 cells were cultured with or without LPS (10 µg/ml) in the presence or absence of a type 1 TACE inhibitor (1 µM) or type 2 TACE inhibitor (10 µM). LPS treatment induced increased serum creatinine, TNFα, and urinary neutrophil gelatinase-associated lipocalin. Angiotensin II type 1 receptor, mitogen activated protein kinase (MAPK), and TACE increased, while angiotensin-converting enzyme-2 (ACE2) expression decreased in LPS-induced acute kidney injury and LPS-treated HK-2 cells. LPS induced reactive oxygen species and the down-regulation of ACE2, and these responses were prevented by TACE inhibitors in HK-2 cells. TACE inhibitors increased cell viability in LPS-treated HK-2 cells and attenuated oxidative stress and inflammatory cytokines. Our findings indicate that LPS activates renin angiotensin system components via the activation of TACE. Furthermore, inhibitors of TACE are potential therapeutic agents for kidney injury.

Keyword

Acute kidney injury; Angiotensin-converting enzyme-2; Lipopolysaccharide; Mitogen activated protein kinase; Oxidative stress; TNFα-converting enzyme

MeSH Terms

Acute Kidney Injury
Angiotensins
Animals
Cell Survival
Creatinine
Cytokines
Down-Regulation
Epithelial Cells*
Humans*
Inflammation
Kidney
Lipocalins
Mice
Necrosis
Neutrophils
Oxidative Stress
Protein Kinases*
Reactive Oxygen Species*
Receptor, Angiotensin, Type 1
Renin-Angiotensin System
Tumor Necrosis Factor-alpha*
Angiotensins
Creatinine
Cytokines
Lipocalins
Protein Kinases
Reactive Oxygen Species
Receptor, Angiotensin, Type 1
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 Effect of LPS on serum creatinine, TNFα, and NGAL. Serum creatinine, tumor necrosis factor-α (TNFα), and urinary neutrophil gelatinase-associated lipocalin (NGAL) were higher in lipopolysaccharide (LPS)-treated mouse than in control mice (n=7). Results are presented as mean±SEM of three individual experiments. *p<0.05 vs. control.

  • Fig. 2 Effects of LPS on the renin-angiotensin system. Protein expression levels of Ang II/III, ACE, ACE2, AT1R, and TACE were higher, while that of ACE2 was lower in LPS-treated mice than in controls (A, B). Results are presented as mean±SEM. *p<0.05 vs. control.

  • Fig. 3 Effects of LPS on the oxidative stress, inflammation and apoptosis. The protein expression of HO-1 were higher in LPS-treated mice compared to control (A). The phosphorylation level of p38 and JNK was increased in LPS-treated mice compared to control (B). Protein expression of Bax was higher, while Bcl2 was lower in LPS-treated mice than in control. Ratio of Bax to Bcl2 was increased in LPS-treated mice (C). Results are presented as mean±SEM. *p<0.05 vs. control.

  • Fig. 4 ROS generation detected using the ROS-sensitive fluorescent dye DCF. LPS caused an increase in DCF fluorescence after incubation for 8 h, which was attenuated by a TACE type 1 inhibitor (TAPI1) and TACE type 2 inhibitor (TAPI2) (A, B). Protein expression of HO-1 increased in LPS-treated HK-2 cells, which was attenuated by TAPI1 and TAPI2 (C). A cell proliferation assay using WST-1 showed a marked decreased in cell proliferation in LPS-treated HK-2 cells, which was attenuated by TAPI1 and TAPI2 (D). *p<0.05 vs. control. #p<0.05 vs. LPS.

  • Fig. 5 Real-time PCR. Effects of TAPI1 and TAPI2 on the IL-6, TNFα, iNOS and MCP-1 in LPS-treated HK-2 cells. mRNA expression levels of IL-6, TNFα, iNOS and MCP-1 were higher in LPS-treated HK-2 cells than control, and were attenuated by TAPI1 and TAPI2. Results are presented as mean±SEM. *p<0.05 vs. control. #p<0.05 vs. LPS.

  • Fig. 6 Effects of TAPI1 and TAPI2 on the renin-angiotensin system in LPS-treated HK-2 cells. Protein expression levels of Ang II/III, AT1R, and TACE were higher in LPS-treated HK-2 cells than in controls, and were attenuated by TAPI1 and TAPI2. The protein expression of ACE2 was decreased in LPS-treated HK-2 cells compared to control, which was counter-regulated by TAPI1 and TAPI2 (A, B). Immunofluorescence for TACE shows increased expression in LPS-treated HK-2 cells (C). TACE activity was increased in LPS-treated HK-2 cells, which was attenuated by TAPI1 and TAPI2 treatment (D). Results are presented as mean±SEM. *p<0.05 vs. control. #p<0.05 vs. LPS.

  • Fig. 7 Effects of LPS on the MAPK pathway in LPS-treated HK-2 cells. Expression of pP38 tended to increase in LPS-treated mice and phosphorylation of P38, c-Jun N-terminal kinase (pJNK), and extracellular signal-regulated kinase (pERK 1/2) increased in LPS-treated HK-2 cells, and was attenuated by TAPI1 and TAPI2 treatment. Increased phosphorylation of ERK 1/2 was not affected by TAPI1 treatment. Results are presented as mean±SEM of three individual experiments. *p<0.05 vs. control. #p<0.05 vs. LPS.

  • Fig. 8 Effects of LPS on apoptosis. Protein expression of Bax was increased, while protein expression of Bcl2 was decreased in LPS treated HK-2 cells compared to controls (A). The ratio of Bax to Bcl2 was increased in LPS-treated HK-2 cells, and was attenuated by TAPI1 and TAPI2 (B). DAPI staining shows increased apoptotic cells in LPS-treated HK-2 cells, which was attenuated by TAPI1 and TAPI2 treatment (C). FACS showed apoptotic cells in LPS-treated HK-2 cells, which was attenuated by TAPI1 and TAPI2 treatment (D). *p<0.05 vs. control. #p<0.05 vs. LPS.


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