title: Acidic pH inhibits ATP depletion-induced tubular cell apoptosis by blocking caspase-9 activation in apoptosome.
abstract: Tubular cell apoptosis has been implicated in the development of ischemic renal failure. In in vitro models, ATP depletion-induced apoptosis of tubular cells is mediated by the intrinsic pathway involving Bax translocation, cytochrome c release, and caspase activation. While the apoptotic cascade has been delineated, much less is known about its regulation. The current study has examined the regulation of ATP depletion-induced tubular cell apoptosis by acidic pH, a common feature of tissue ischemia. Cultured renal tubular cells were subjected to 3 h of ATP depletion with azide and then recovered in full culture medium. The treatment led to apoptosis in approximately 40% of cells. Apoptosis was significantly reduced, if the pH of ATP depletion buffer was lowered from 7-7.4 to 6-6.5. This was accompanied by the inhibition of caspase activation. However, acidic pH did not prevent Bax translocation and oligomerization in mitochondria. Cytochrome c release from mitochondria was not blocked either, suggesting that acidic pH inhibited apoptosis at the postmitochondrial level. To determine the postmitochondrial events that were blocked by acidic pH, we conducted in vitro reconstitution experiments. Exogenous cytochrome c, when added into isolated cell cytosol, induced caspase activation. Such activation was abrogated, when pH during the reconstitution was lowered to 6 or 6.5. Nevertheless, acidic pH did not prevent the recruitment and association of caspase-9 by Apaf-1, as shown by coimmunoprecipitation. Together, this study demonstrated the inhibition of tubular cell apoptosis following ATP depletion by acidic pH. A critical step blocked by acidic pH seems to be caspase-9 activation in apoptosome.
authors.affiliation: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, 30912, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Brooks
authors.forename: Pimonrat
authors.initials: P
authors.lastname: Ketsawatsomkron
authors.forename: Yang
authors.initials: Y
authors.lastname: Sui
authors.forename: Jinzhao
authors.initials: J
authors.lastname: Wang
authors.forename: Cong-Yi
authors.initials: CY
authors.lastname: Wang
authors.forename: Fu-Shin
authors.initials: FS
authors.lastname: Yu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Dong
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D001711:Biotransformation / Q000502:physiology; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D007684:Kidney Tubules / Q000166:cytology* / Q000648:ultrastructure; D008928:Mitochondria / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D051028:bcl-2-Associated X Protein
chemical_list: C506594:Apaf1 protein, rat; D053477:Apoptotic Protease-Activating Factor 1; C494828:Bax protein, rat; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D045304:Cytochromes c; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases