Single and combination toxic metal exposures induce apoptosis in cultured murine podocytes exclusively via the extrinsic caspase 8 pathway.
Arsenite, cadmium, and mercury are among the most abundant toxic metals (TM) in the environment. Although the most common renal manifestation of TM toxicity is proximal tubular dysfunction, significant glomerular injury can also occur. We hypothesized that glomerular injury following TM exposure results from TM-induced apoptosis of podocytes. To test this hypothesis we examined the extent of apoptosis and the apoptotic pathways induced in cultured murine podocytes incubated for three days with arsenite, cadmium, or mercury, and with equimolar combinations of all three metals. Apoptosis was detected by DNA laddering, and the number of apoptotic nuclei determined by Tunel assay. Treatment for three days with each TM resulted in DNA laddering and induced a dose-dependent increase in apoptotic nuclei. In contrast, treatment with equimolar combinations of TM induced significantly fewer apoptotic nuclei than individual TM treatments. Apoptosis induced by each TM was associated with a significant (approximately 400%) increase in caspase 8 activity, but no change in caspase 9 activity, and Western analyses revealed a marked up-regulation of Fas (approximately 500%) and FADD (approximately 300%) with no change in expression of Bax, Bcl-2, or Bcl-xL. Similar to the apoptotic response, combinations of TM induced less caspase 8 activity and Fas/FADD expression than individual TM treatments. Collectively, these results demonstrate that (1) TM induced apoptosis in cultured murine podocytes via the extrinsic Fas-FADD caspase 8 pathway, rather than the mitochondrial apoptotic pathway, and (2) combination TM exposure induced less apoptosis than individual TM, indicating an antagonistic rather than an additive or synergistic toxicity.
Eichler
Tad
T
Pediatric Nephrology Division, C. S. Mott Children's Hospital, University of Michigan, Ann Arbor, Michigan 48109, USA.
Ma
Qing
Q
Kelly
Caitlin
C
Mishra
Jaya
J
Parikh
Samir
S
Ransom
Richard F
RF
Devarajan
Prasad
P
Smoyer
William E
WE
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis; D000818:Animals; D017209:Apoptosis; D018053:Arsenites / Q000633:toxicity*; D002104:Cadmium / Q000633:toxicity*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D053938:DNA Fragmentation; D004347:Drug Interactions; D053200:Fas-Associated Death Domain Protein; D020287:In Situ Nick-End Labeling; D008628:Mercury / Q000633:toxicity*; D051379:Mice; D050199:Podocytes / Q000187:drug effects* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D019014:fas Receptor
D048868:Adaptor Proteins, Signal Transducing; D018053:Arsenites; C505534:Fadd protein, mouse; C491899:Fas protein, mouse; D053200:Fas-Associated Death Domain Protein; D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor; D002104:Cadmium; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D008628:Mercury; C015001:arsenite