Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells.
OBJECTIVE
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-alpha (TNF-alpha), 5 and 20 ng/ml of interleukin-1beta (IL-1beta) 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-alpha 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-alpha groups. Fas ligand protein expression did not increase in the low-dose TNF-alpha treated group, but it increased significantly in the high-dose TNF-alpha treated group (p < 0.01), IL-1beta- 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-alpha- and IL-beta-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-alpha and IL-beta 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-alpha, IL-1beta 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.


CONCLUSIONS
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.
Department of Internal Medicine, Korea University Hospital, Anam-dong 5Ka, 136-705 Sungbuk-ku, Seoul, Korea.
Sang Kyung
SK
Jo
Dae Ryong
DR
Cha
Won Yong
WY
Cho
Hyoung Kyu
HK
Kim
Kyung Hyun
KH
Chang
Su Young
SY
Yun
Nam Hee
NH
Won
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D007375:Interleukin-1 / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C505533:FADD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D007375:Interleukin-1; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases