Improved mechanistic understanding of renal cell death in acute kidney injury (AKI) has generated new therapeutic targets. Clearly, the classic lesion of acute tubular necrosis is not adequate to describe the consequences of renal ischemia, nephrotoxin exposure, or sepsis on glomerular filtration rate. Experimental evidence supports a pathogenic role for apoptosis in AKI. Interestingly, proximal tubule epithelial cells are highly susceptible to apoptosis, and injury at this site contributes to organ failure. During apoptosis, well-orchestrated events converge at the mitochondrion, the organelle that integrates life and death signals generated by the BCL2 (B-cell lymphoma 2) protein family. Death requires the ‘perfect storm’ for outer mitochondrial membrane injury to release its cellular ‘executioners’. The complexity of this process affords new targets for effective interventions, both before and after renal insults. Inhibiting apoptosis appears to be critical, because circulating factors released by the injured kidney induce apoptosis and inflammation in distant organs including the heart, lung, liver, and brain, potentially contributing to the high morbidity and mortality associated with AKI. Manipulation of known stress kinases upstream of mitochondrial injury, induction of endogenous, anti-apoptotic proteins, and improved understanding of the timing and consequences of renal cell apoptosis will inevitably improve the outcome of human AKI.

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up-or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor ; (PPAR;) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPAR; in HCC, PPAR; expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPAR; activation and inhibition were compared. PPAR; expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPAR; inhibitors and PPAR; small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPAR; inhibitors had no effect on initial B B1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPAR; inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPAR; overexpression is present in HCC. Inhibition of PPAR; function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPAR; inhibitors represent a potential novel treatment approach to HCC.

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3␤ (GSK3␤) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3␤ promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3␤, Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3␤ mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference-mediated knockdown of GSK3␤ promoted cell survival. Furthermore, RNA interference-mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3␤. In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine 163 site targeted by stress-activated GSK3␤. In rats, TDZD-8 inhibited ischemia-induced activation of GSK3␤, Bax, and caspase 3; ameliorated tubular and epithelial cell damage; and significantly protected renal function. Taken together, GSK3␤-mediated Bax activation induces apoptosis and tubular damage that contribute to acute ischemic kidney injury.

) and (2ЈZ,3ЈE)-6-bromoindirubin-3Ј-oxime (BIO), two glycogen synthase kinase-3␤ (GSK3␤) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway. These studies demonstrate that Li ϩ and BIO activate Wnt signaling as indicated by the following changes: phosphorylation (inhibition) of GSK3␤; decreased phosphorylation of ␤-catenin (a GSK3␤ substrate); nuclear translocation of ␤-catenin; specific transcriptional activation of Tcf/catenin-responsive pTopflash constructs; and an increase in the expression of cyclin D1 (indicative of a promitogenic cell response). In addition, Li ϩ or BIO significantly increases the phosphorylation (activation) of Akt, an anti-apoptotic protein, and inhibits apoptosis (decreases both annexin-V staining and caspase-3 activation), during serum deprivation. Inhibition of phosphatidylinositol 3-kinase (responsible for Akt activation) either by wortmanin or LY-294002 prevented Li ϩ