Apoptotic cell death is initiated during normothermic ischemia in human kidneys.

Ischemic damage plays an important role in post-transplant organ failure. Activation of the apoptotic cascade is crucially involved in post-ischemic inflammation resulting in tissue damage and organ dysfunction. Here we investigate the initiation of the apoptotic cascade during normothermic ischemia in human kidneys using a model for normothermic ischemia with kidneys nephrectomized because of renal cell carcinoma. Ex vivo, kidneys were stored at 37 degrees C, and consecutive biopsies were taken from disease-free tissue. Pro- and anti-apoptotic proteins were assessed by Western blotting and immunofluorescence. During normothermic ischemia the pro-apoptotic proteins Bax and activated caspase-9 increased with ischemia time, whereas caspase-8 was not activated. The anti-apoptotic proteins Bcl-2 and cFLIP decreased in time. Data on Bcl-2 and Bax were supported by immunofluorescence for Bcl-2 and activated Bax. However, activation of the central effector caspase-3, essential for execution of the apoptotic process, was not detected. In conclusion, during normothermic ischemia the apoptotic cascade in the human kidney is initiated, but not fulfilled. Our data show that the duration of ischemia significantly correlates with activation of the apoptotic cascade. These findings provide insight in the initiation of apoptotic cell-death during warm ischemia and may be useful in the assessment of ischemic injury.

The mannose-binding lectin-pathway is involved in complement activation in the course of renal ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) is an important cause of acute renal failure (ARF). The complement system appears to be essentially involved in I/R injury. However, via which pathway the complement system is activated and in particular whether the mannose-binding lectin (MBL)-pathway is activated is unclear. This tempted us to study the activation and regulation of the MBL-pathway in the course of experimental renal I/R injury and in clinical post-transplant ARF. Mice subjected to renal I/R displayed evident renal MBL-depositions, depending on the duration of warm ischemia, in the early reperfusion phase. Renal deposition of C3, C6 and C9 was observed in the later reperfusion phase. The deposition of MBL-A and -C completely co-localized with the late complement factor C6, showing that MBL is involved in complement activation in the course of renal I/R injury. Moreover, the degree of early MBL-deposition correlated with complement activation, neutrophil-influx, and organ-failure observed in the later reperfusion phase. In serum of mice subjected to renal I/R MBL-A, levels increased in contrast to MBL-C levels, which dropped evidently. In line, liver mRNA levels for MBL-A increased, whereas MBL-C levels decreased. Renal MBL mRNA levels rapidly dropped in the course of renal I/R. Finally, in human biopsies, MBL-depositions were observed early after transplantation of ischemically injured kidneys. In line with our experimental data, in ischemically injured grafts displaying post-transplant organ-failure extensive MBL depositions were observed in peritubular capillaries and tubular epithelial cells. In conclusion, in experimental renal I/R injury and clinical post-transplant ARF the MBL-pathway is activated, followed by activation of the complement system. These data indicate that the MBL-pathway is involved in ischemia-induced complement activation.

Exogenous alpha-1-acid glycoprotein protects against renal ischemia-reperfusion injury by inhibition of inflammation and apoptosis.

BACKGROUND: Although ischemia-reperfusion (I/R) injury represents a major problem in posttransplant organ failure, effective treatment is not available. The acute phase protein alpha-1-acid glycoprotein (AGP) has been shown to be protective against experimental I/R injury. The effects of AGP are thought to be mediated by fucose groups expressed on the AGP protein inhibiting neutrophil infiltration. However, the precise mechanism of protection remains to be established. We therefore studied the effects of exogenous human AGP (hAGP) in a mouse model of ischemic acute renal failure. METHODS: Mice were subjected to renal I/R and treated with hAGP, fucose-depleted hAGP, or control treated. Also, transgenic mice over-expressing rat AGP or wild-type controls were subjected to renal I/R. RESULTS: Treatment was with hAGP as well as fucose-depleted hAGP protected mice against I/R-induced acute renal failure. Surprisingly, AGP-over-expressing mice were not protected against I/R injury. Both natural and fucose-depleted hAGP inhibited the activation of the complement system, as determined by renal C3 deposition and influx of neutrophils measured by immunohistochemistry and myeloperoxidase-enzyme-linked immunoadsorbent assay. Tubular epithelial cell structure (actin cytoskeleton) and cell-cell interaction (tight-junction architecture) were completely preserved in AGP-treated mice. Also, epithelial caspase activation and apoptotic DNA cleavage were prevented by AGP treatment. CONCLUSIONS: Both natural and fucose-depleted hAGP protect against renal I/R injury by preservation of tubular epithelial structure and inhibition of apoptosis and subsequent inflammation. Therefore, hAGP can be regarded as a potential new therapeutic intervention in the treatment of acute renal failure, as seen after transplantation of ischemically injured kidneys.

Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury.

BACKGROUND: Warm ischemia-reperfusion (I/R) injury plays an important role in posttransplant organ failure. In particular, organs from marginal donors suffer I/R injury. Although iron has been implicated in the pathophysiology of renal I/R injury, the mechanism of iron-mediated injury remains to be established. The authors therefore investigated the role of circulating redox-active iron in an experimental model for renal I/R injury. METHODS: Male Swiss mice were subjected to unilateral renal ischemia for 45 min, followed by contralateral nephrectomy and reperfusion. To investigate the role of circulating iron, mice were treated with apotransferrin, an endogenous iron-binding protein, or iron-saturated apotransferrin (holotransferrin). RESULTS: Renal ischemia induced a significant increase in circulating redox-active iron levels during reperfusion. Apotransferrin, in contrast to holotransferrin, reduced the amount of circulating redox-active iron and abrogated renal superoxide formation. Apotransferrin treatment did not affect I/R-induced renal apoptosis, whereas holotransferrin aggravated apoptotic cell death. Apotransferrin, in contrast to holotransferrin, inhibited the influx of neutrophils. Both apo- and holotransferrin reduced I/R-induced complement deposition, indicating that the effects of transferrin are differentially mediated by its iron and protein moiety. Finally, apotransferrin, in contrast to holotransferrin, dose-dependently inhibited the loss of renal function induced by ischemia. CONCLUSIONS: Redox-active iron is released into the circulation in the course of renal I/R. Reducing the amount of circulating redox-active iron by treatment with apotransferrin protects against renal I/R injury, inhibiting oxidative stress, inflammation, and loss of function. Apotransferrin could be used in the treatment of acute renal failure, as seen after transplantation of ischemically damaged organs.