benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Acute-Kidney-Injury

Renal tubular cell death in ischemia-reperfusion does not follow the classical apoptosis or necrosis phenotype. We characterized the morphological and biochemical features of injured tubular epithelial cells in ischemic acute kidney injury (AKI).. Ischemic AKI was induced in rats by 60 min of ischemia followed by 24 h of reperfusion. Light and electron microscopic TUNEL (LM-TUNEL and EM-TUNEL), gel electrophoresis of extracted DNA, and caspase-3 involvement were examined during the development of death.. Damaged tubular epithelial cells with condensed and LM-TUNEL-positive (+) nuclei were prominent at 12 and 18 h after reperfusion with DNA 'ladder' pattern on gel electrophoresis. EM-TUNEL+ cells were characterized by nuclei with condensed and clumping chromatin, whereas the cytoplasm showed irreversible necrosis. The protein levels and activity of caspase-3 did not increase in kidneys after reperfusion. In addition, caspase inhibitor (ZVAD-fmk) failed to inhibit DNA fragmentation and prevent tubular epithelial cell death in ischemic AKI.. Caspase-3-independent internucleosomal DNA fragmentation occurs in injured tubular epithelial cells undergoing irreversible necrosis in ischemic AKI. The manner of this cell death may be identical to the cell death termed apoptotic necrosis, aponecrosis, or necrapoptosis. Ischemia-reperfusion injury activates caspase-3-independent endonuclease, which in turn induces irreversible damage of tubular epithelial cells, and may contribute to the initiation and development of AKI.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase 3; DNA Fragmentation; Electrophoresis, Agar Gel; Epithelial Cells; In Situ Nick-End Labeling; Ischemia; Kidney; Kidney Tubules; Male; Microscopy, Electron; Necrosis; Nucleosomes; Rats; Rats, Wistar; Reperfusion Injury

Distant organ effects of acute kidney injury (AKI) are a leading cause of morbidity and mortality. While little is known about the underlying mechanisms, limited data suggest a role for inflammation and apoptosis. Utilizing a lung candidate gene discovery approach in a mouse model of ischemic AKI-induced lung dysfunction, we identified prominent lung activation of 66 apoptosis-related genes at 6 and/or 36 h following ischemia, of which 6 genes represent the tumor necrosis factor receptor (TNFR) superfamily, and another 23 genes are associated with the TNFR pathway. Given that pulmonary apoptosis is an important pathogenic mechanism of acute lung injury (ALI), we hypothesized that AKI leads to pulmonary proapoptotic pathways that facilitate lung injury and inflammation. Functional correlation with 1) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and 2) active caspase-3 (aC3) activity, immunoblotting, and immunohistochemistry (IHC) identified kidney IRI-induced pulmonary apoptosis at 24 h, and colocalization studies with CD34 identified predominantly endothelial apoptosis. Mice were treated with the caspase inhibitor Z-VAD-FMK (0.25 mg ip) or vehicle 1 h before and 8 h after sham or kidney IRI, and bronchoalveolar lavage fluid protein was measured at 36 h as a surrogate for lung leak. Caspase inhibition reduced lung microvascular changes after kidney IRI. The pulmonary apoptosis seen in wild-type control mice during AKI was absent in TNFR(-/-) mice. Using an initial genomic approach to discovery followed by a mechanistic approach to disease targeting, we demonstrate that pulmonary endothelial apoptosis is a direct mediator of the distant organ dysfunction during experimental AKI.

Topics: Acute Kidney Injury; Acute Lung Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Disease Models, Animal; Endothelium; Gene Expression Profiling; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Tumor Necrosis Factor, Type I; Reperfusion Injury

Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Cell Cycle; Cell Death; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Enzyme Inhibitors; Glycerol; Immunohistochemistry; Inflammation; Interleukin-1; Interleukin-18; Male; Rats; Rats, Wistar

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFalpha/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Cytokine TWEAK; Cytokines; Endoplasmic Reticulum; Folic Acid; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Interferon-gamma; Kidney Tubules, Proximal; Mice; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; TWEAK Receptor

Acute renal failure (ARF) is a frequent and serious complication of endotoxemia caused by lipopolysaccharide (LPS) and contributes significantly to mortality. The present studies were undertaken to examine the roles of nitric oxide (NO) and caspase activation on renal peritubular blood flow and apoptosis in a murine model of LPS-induced ARF. Male C57BL/6 mice treated with LPS (Escherichia coli) at a dose of 10 mg/kg developed ARF at 18 h. Renal failure was associated with a significant decrease in peritubular capillary perfusion. Vessels with no flow increased from 7 +/- 3% in the saline group to 30 +/- 4% in the LPS group (P < 0.01). Both the inducible NO synthase inhibitor L-N(6)-1-iminoethyl-lysine (L-NIL) and the nonselective caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD) prevented renal failure and reversed perfusion deficits. Renal failure was also associated with an increase in renal caspase-3 activity and an increase in renal apoptosis. Both L-NIL and Z-VAD prevented these changes. LPS caused an increase in NO production that was blocked by L-NIL but not by Z-VAD. Taken together, these data suggest NO-mediated activation of renal caspases and the resulting disruption in peritubular blood flow are an important mechanism of LPS-induced ARF.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Disease Models, Animal; Enzyme Activation; Interferon-gamma; Kidney Cortex; Lipopolysaccharides; Lysine; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type II; Renal Circulation; Tumor Necrosis Factor-alpha

In previous work, it was demonstrated that apoptosis occurs in the kidney during LPS-induced acute renal failure (ARF). However, the relative importance of apoptosis in LPS-induced ARF remained unproven. Because the caspase enzyme cascade is responsible for carrying out apoptosis, it was hypothesized that treatment with a caspase inhibitor would protect mice from LPS-induced ARF. C57BL/6 mice received an injection of LPS and were treated with either the broad-spectrum caspase inhibitor z-VAD-fmk or vehicle and compared with unmanipulated mice. LPS induced a significant increase in caspase-3 activity in vehicle-treated mice, which was significantly inhibited by z-VAD. Mice that were treated with z-VAD were protected from ARF and demonstrated significantly less apoptosis as measured by both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and DNA laddering. Although apoptosis is classically described as a noninflammatory process, z-VAD treatment significantly attenuated multiple markers of inflammation, such as renal neutrophil infiltration and renal expression of the neutrophil chemotactic factor macrophage inflammatory protein-2. Thus, caspase inhibition may protect against LPS-induced ARF not only by preventing apoptotic cell death but also by inhibiting inflammation. These data raise the possibility that apoptotic kidney cells may actually be a source of this local inflammation, contributing to subsequent nonapoptotic renal injury.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cysteine Proteinase Inhibitors; Endotoxemia; Enzyme Activation; Kidney; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL