AIM: TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). METHODS: PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-alpha, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. RESULTS: We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-alpha and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. CONCLUSION: Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells.
AIM: TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). METHODS: PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-alpha, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. RESULTS: We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-alpha and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. CONCLUSION: Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells.
Authors: M Balasubramanyam; S Aravind; K Gokulakrishnan; P Prabu; C Sathishkumar; H Ranjani; V Mohan Journal: Mol Cell Biochem Date: 2011-01-20 Impact factor: 3.396
Authors: Sang Heon Suh; Ko Eun Lee; In Jin Kim; Ok Kim; Chang Seong Kim; Joon Seok Choi; Hoon-In Choi; Eun Hui Bae; Seong Kwon Ma; Jong Un Lee; Soo Wan Kim Journal: Clin Exp Nephrol Date: 2014-03-19 Impact factor: 2.801