Inflammatory Cytokines and Lipopolysaccharide Induce Fas-Mediated Apoptosis in Renal Tubular Cells

Abstract

Background/Aims: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known. In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions. Methods: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-α (TNF-α), 5 and 20 ng/ml of interleukin-1β (IL-1β) and 30 ng/ml LPS for 24 h. Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis. Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. Results: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-α 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-α groups. Fas ligand protein expression did not increase in the low-dose TNF-α treated group, but it increased significantly in the high-dose TNF-α treated group (p< 0.01), IL-1β- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02). The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-α- and IL-β-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-α and IL-β treated group, it did not show statistically significant differences. In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09). Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-α, IL-1β and LPS treated cells. The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups. Conclusion: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia.