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 ∼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.

Cytoprotective Effects of Hypoxia against Cisplatin-Induced Tubular Cell Apoptosis

“…Our recent work has shown that acidic pH of 6.5 or lower suppresses tubular cell apoptosis after ATP depletion. A critical step in the apoptotic cascade that is blocked by pH 6 to 6.5 is the assembly of apoptosome, a cytochrome c/Apaf-1/caspase-9 protein complex that is responsible for the initiation of caspase activation (14). In our study, pH decreased from 7.4 to approximately 7.0 during hypoxic incubation.…”

“…In our study, pH decreased from 7.4 to approximately 7.0 during hypoxic incubation. According to our earlier results (14), this pH decrease was not sufficient to block apoptosome formation and apoptosis. Consistently, when the incubation pH was maintained at approximately 7.4 by Hepes buffer, cisplatin-induced apoptosis in hypoxic cells was not increased or restored to the level of normoxic cells (Figure 4).…”

“…As a result, acidic metabolites such as lactate accumulate, leading to cellular acidification. Depending on the cellular context, acidic pH may promote or inhibit cell injury and death (12)(13)(14). In our experiments ( Figure 4A), 16 h of hypoxic incubation lowered the pH of the incubation medium from approximately 7.35 to approximately 7.0, regardless of whether cisplatin was present.…”

“…On the contrary, lower pH protects various types of cells during hypoxic incubation or tissue ischemia (13). In cultured renal tubular cells, our recent work has demonstrated the protective effects of acidic pH against apoptosis after ATP depletion (14). Acidic pH inhibits the formation of apoptosome, the protein complex that is responsible for caspase activation.…”

STAT3 Inhibits Apoptosis of Human Renal Tubular Epithelial Cells Induced by ATP Depletion/Recovery

“…The cells at 80% confluence were used for experiment. The in vitro model of renal ischemic injury was established as reported [18, 19]. Briefly, after the medium was changed to phosphate-buffered saline (PBS) containing 10 µ M antimycin A, 1.5 m M CaCl 2 , and 2 m M MgCl 2 , the cells were incubated for 60 min.…”