Introduction and Aims: Chronic kidney disease (CKD) is a major risk factor for atherosclerotic cardiovascular diseases. Recently it has been reported that apoptosis of aortic smooth muscle cells (AoSMCs) may relate to the atherosclerosis with plaque formation or calcification. Therefore, in the present study, we examined the effect of indoxyl sulfate (IS), which is thought to be one of uremic toxin, for apoptosis in cultured rat AoSMCs. Methods: The induction of apoptosis was quantitated by assay of the caspase CPP32, which plays a direct role in the execution of cell death. And the activity of SAPK/JNK and P38 MAP kinase, which is known as apoptosis-inducing signal transduction, was assessed by standard immunoblot using phospho-specific antibodies. Results: Twenty five μg/ml of IS, which is compatible with the concentration of IS in the serum of end-stage renal failure patients, induced 3.9±2.7-fold increase in the caspase CPP32 activity responding to serum withdrawal for 12 hours. The blockade of organic anion transporter (OAT) by 0.5mM probenecid (Pb) abolished the effect of IS on the apoptosis in AoSMCs (relative increase in the caspase CPP32: Pb-, IS-; 1±0.1, Pb-, IS+; 2.4±0.2, Pb+, IS-; 0.9±0.1, Pb+, IS+; 1.2±0.1). Indoxyl sulfate activated SAPK/JNK in AoSMCs that was significantly elevated by 30 minutes and sustained for over 2 hours, although it did not affect the activation of P38 MAP kinase. Conclusions: These results indicate that IS accelerates apoptosis induced by serum withdrawal in rat AoSMCs, which is mediated by cellular transport of IS via the OAT and may also be related to the activation of SAPK/JNK pathway. The induction of apoptosis by the accumulation of IS in blood due to CKD may play an important role in atherosclerotic lesion formation.

We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.

Mitochondrial outer-membrane permeabilization by pro-apoptotic Bcl-2 family members plays a crucial role in apoptosis induction. However, whether this directly causes the release of the different mitochondrial apoptogenic factors simultaneously is currently unknown. Here we report that in cells or with isolated mitochondria, pro-apoptotic Bcl-2 proteins cause the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not endonuclease G (EndoG) and apoptosis-inducing factor (AIF). In cells treated with Bax/Bakdependent pro-apoptotic drugs, neither the caspase inhibitor zVAD-fmk nor loss of Apaf-1 affected the ef¯ux of cytochrome c, Smac/Diablo and HtrA2/Omi, but both prevented the release of EndoG and AIF. Our ®ndings identify the mitochondrial response to pro-apoptotic stimuli as a selective process leading to a hierarchical ordering of the effectors involved in cell death induction. Moreover, as in Caenorhabditis elegans, EndoG and AIF act downstream of caspase activation. Thus EndoG and AIF seem to de®ne à caspase-dependent' mitochondria-initiated apoptotic DNA degradation pathway that is conserved between mammals and nematodes.

Mitochondrial fusion may regulate mitochondrial morphogenesis and underlie complementation between mitochondrial genomes in mammalian cells. The nuclear encoded mitochondrial proteins Mfn1 and Mfn2 are human homologues of the only known protein mediators of mitochondrial fusion, the Drosophila Fzo GTPase and Saccharomyces cerevisiae yFzo1p. Although the Mfn1 and Mfn2 genes were broadly expressed, the two genes showed different levels of mRNA expression in different tissues. Two Mfn1 transcripts were detected at similar levels in a variety of human tissues and were dramatically elevated in heart, while Mfn2 mRNA was abundantly expressed in heart and muscle tissue but present only at low levels in many other tissues. Human Mfn1 protein localized to mitochondria and participated in a high molecular weight, detergent extractable protein complex. Forced expression of Mfn1 in cultured cells caused formation of characteristic networks of mitochondria. Introduction of a point mutation in the conserved G1 region of the predicted GTPase domain(Mfn1K88T) dramatically decreased formation of mitochondrial networks upon Mfn1 overexpression, suggesting that network formation required completion of the Mfn1 GTPase cycle. Conversely, a protein variant carrying a point mutation in the G2 motif of the Mfn1 GTPase domain acted as a dominant negative: overexpression of Mfn1T109A resulted in fragmentation of mitochondria. We propose that Mfn1T109A interferes with fusion activity of endogenous Mfn1 protein, possibly by binding necessary cofactors,so to allow unopposed mitochondrial fission. Thus, Mfn1 appears to be a key player in mediating mitochondrial fusion and morphology in mammalian cells.