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Ischemia followed by reperfusion (I/R) has cardinal implications in the pathogenesis of organ transplantation and rejection. Apoptosis and inflammation are central mechanisms leading to organ damage in the course of renal I/R. General aspects of apoptosis, morphology, induction, and biochemistry are discussed. Activated caspases, the classical effector enzymes of apoptosis, are able to induce not only apoptosis but also inflammation after I/R in experimental models. This redefines the involvement of apoptosis in I/R injury toward a central and functional role in the development of organ damage. Our purpose is to assess aspects of apoptosis and inflammation in terms of involvement in the pathogenesis of I/R-induced organ damage. Moreover, the implications of recent experimental advances for diagnosis and treatment of renal I/R injury in clinical practice will be discussed.

Background: One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R., Methods: The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction., Results: We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion., Conclusions: These data for the first time show that chemokine induction following I/R is dependent on caspase activation.

Background: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury., Methods: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared., Results: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis., Conclusions: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury.

Background: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney., Methods and Results: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion., Conclusions: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Tissue injury as a consequence of ischemia followed by reperfusion is characterized by early as well as late signs of inflammation. The latter, among others, involves IFN-gamma-dependent up-regulation of MHC class I and II Ag expression. Employing a murine model of renal ischemia, we show that renal IL-18 mRNA up-regulation coincides with caspase-1 activation at day 1 following ischemia. IFN-gamma and IL-12 mRNA are subsequently up-regulated at day 6 following ischemia. Combined, but not separate, in vivo neutralization of the IFN-gamma inducing cytokines IL-12 and IL-18 reduces IFN-gamma-dependent MHC class I and II up-regulation to a similar extent as IFN-gamma neutralization, suggesting the involvement of functional IL-12, IL-18, and IFN-gamma protein. These results reveal a novel relationship between tissue injury of nonmicrobial origin and the induction of IL-12 as well as IL-18. The collaboration observed between endogenous IL-12 and IL-18 in the induction of IFN-gamma after renal ischemia/reperfusion, resembles the immune response to bacterial infections.

Background: Ischemia followed by reperfusion is a common clinical event associated with a pro-inflammatory response leading to organ dysfunction. The aim of the present study is to evaluate the interplay between this pro-inflammatory response and apoptosis. We investigated the role of the pro-inflammatory mediator tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory mediator interleukin-10 (IL-10) in inflammation and apoptosis after renal ischemia reperfusion., Methods: Male Swiss mice were subjected to 45 min of ischemia followed by reperfusion and subsequently administered neutralizing Abs against either TNF-alpha (TN3), IL-10 (JES5-2A5) or control., Results: After 1 day of reperfusion, anti-TNF-alpha treatment reduced whereas anti-IL-10 treatment exacerbated postischemic renal injury, inflammation, and, to a lesser extent, apoptosis as measured by changes in blood urea nitrogen content, immunohistologically detectable renal TNF-alpha protein and neutrophils, histological integrity of renal parenchyma, and DNA ladder formation. Furthermore, anti-IL-10 treatment enhanced major histocompatibility complex class I and II expression at day 7 as measured by enzyme immunoassay and immunohistology., Conclusions: These data indicate that the extent of reperfusion-induced apoptosis is modulated by the inflammatory response, during which locally produced TNF-alpha plays a significant role in the development of tissue injury. Subsequently, this pro-inflammatory reaction is followed by endogenous production of the anti-inflammatory cytokine IL-10, which serves as a physiological counterbalance to the effects of TNF-alpha. These novel pathophysiological insights may provide new basis for the development of tools for limiting ischemia and reperfusion injury.