The single insult of hypoxic preconditioning induces an antiapoptotic response in human proximal tubular cells, in vitro, across cold storage.

OBJECTIVE: To examine whether hypoxia (one of the many components of ischaemic preconditioning) can induce a protective response in culture renal tubular cells, and thus determine if non-lethal periods of hypoxia could confer protection against apoptotic injury to human proximal tubular cells during cold storage and subsequent cytotoxic insult, and establish the cellular mechanisms by which this protection is induced. MATERIALS AND METHODS: Human proximal tubular cells (HK-2) were pre-incubated for 24 h in normoxic or hypoxic conditions and then incubated at 4 degrees C for 6 h to mimic cold storage, before being returned to normal conditions and exposed to varying concentrations of cyclosporine A (CSA). Cell viability and apoptosis were measured using propidium iodide staining and flow cytometry. The expression of heat-shock protein (HSP)-70 was determined by Western blotting. RESULTS: Hypoxia had no effect on cell viability or apoptosis. Pre-exposure of cells to hypoxia significantly protected against CSA-induced damage even after a period of cold storage. Western blotting analysis showed that hypoxia up-regulated the anti-apoptotic protein HSP-70. HK-2 cells over-expressing HSP-70 mimicked hypoxia preconditioning, in that they were protected during cold storage and CSA-induced apoptosis. CONCLUSION: Exposure of renal tubular cells to a sequential model of cold storage, reperfusion and incubation with CSA resulted in apoptotic cell death. Preconditioning these cells with hypoxia induced a protective response and up-regulation of the anti-apoptotic protein HSP-70. There was a similar response in non-preconditioned cells over-expressing HSP-70. Further understanding of the cellular changes occurring during this period of preconditioning will allow the development of more targeted, clinically relevant methods of preconditioning in renal transplantation.

Biosensor developments: application to prostate-specific antigen detection.

Prostate-specific antigen (PSA) is the best serum marker currently available for the detection of prostate cancer and is the forensic marker of choice for determining the presence of azoospermic semen in some sexual assault cases. Most current assays for PSA detection are processed on large analyzers at dedicated testing sites, which require that samples be sent away for testing. This leads to delays in patient management and increased administration costs. The recent emphasis placed on the need for point-of-care patient management has led to the development of novel biosensor detection strategies that are suitable for the miniaturization of assays for various targets including PSA. This review highlights the current and novel analytical technologies used for PSA detection, which will benefit clinicians, patients and forensic workers in the future.

Heat shock-induced protection of renal proximal tubular epithelial cells from cold storage and rewarming injury.

Cold storage and reperfusion injury to transplanted kidneys contributes to increased incidence of delayed graft function and may have a negative impact on graft survival. This study examined the mechanisms by which previous heat shock protects against cell death in an in vitro model of kidney storage. Cold storage is mimicked by incubating human renal proximal tubular epithelial (HK-2) cells in University of Wisconsin solution at 4 degrees C with and without subsequent rewarming. Heat shock was induced by incubation of cells at 42 degrees C for 1 h. Altered protein expression was measured by Western blot, and cell viability and apoptosis were measured by propidium iodide DNA staining using flow cytometry. The specific role of heat-shock protein 70 (HSP-70) was determined both by siRNA knockdown and by stable overexpression approaches. Cold storage and rewarming-induced cell death was associated with decreased expression of HSP-70, HSP-90, HSP-27, and Bcl-2. Previous heat shock significantly reduced HK-2 cell death after cold storage and rewarming and was associated with the maintenance of HSP-70, HSP-27, and Bcl-2 protein levels. Blocking heat stress-induced HSP-70 with siRNA did not significantly block the protective effect of heat stress against cold storage and rewarming cell death; however, overexpression of HSP-70 protected HK-2 cells from this stress. It is concluded that previous heat shock protects HK-2 cells from cold storage and rewarming injury. siRNA inhibition of HSP-70 induction did not block the protective effect of heat shock, indicating that HSP-70 is not essential to the heat stress-induced protective effect reported in this study.

Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction.

Ureteric obstruction is frequently encountered in primary care urology and can lead to damage to the ipsilateral kidney. Relief of all types of obstruction generally leads to the normalization of any deterioration in renal function noted at diagnosis. However, some evidence from animal models suggests that obstruction can cause progressive deleterious effects on renal function and blood pressure control, especially in the presence of preexisting pathologies such as essential hypertension. The last 10 years have seen a proliferation of studies in rodents wherein complete unilateral ureteric obstruction has been used as a model of renal fibrosis. However, the relevance of the findings to human obstructive uropathy has, in many cases, not been the primary aim. In this review, we outline the major events linking damage to the renal parenchyma and cell death to the evolution of fibrosis following obstruction. Special focus is given to the role of apoptosis as a major cause of cell death during and post-complete ureteric obstruction. Several interventions that reduce tubular apoptosis are discussed in terms of their ability to prevent subsequent progression to end-organ damage and fibrosis.

TGF-beta1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation.

Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.

Polyunsaturated and monounsaturated fatty acids increase neutral lipid accumulation, caspase activation and apoptosis in a neutrophil-like, differentiated HL-60 cell line.

We report here that monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs) provoke the accumulation of neutral lipids and apoptosis in retinoic acid-treated HL-60 cells in a concentration- and time-dependent manner. The PUFAs (arachidonic acid, docosahexanoic acid and eicosapentaenoic acid) provoked higher levels of HL-60 apoptosis compared with the monounsaturated oleic acid or the saturated palmitic acid. Cell size and granularity were also altered by fatty acid treatment. The PUFA-induced apoptosis was correlated with increased activity of caspase 3 and caspase 9. Lipid peroxidation was also increased in the presence of PUFAs, but was not responsible for activating cell apoptosis. Lipid derived metabolites may be responsible for activation of caspases and induction of cell apoptosis.

Glucose, but not glutamine, protects against spontaneous and anti-Fas antibody-induced apoptosis in human neutrophils.

Neutrophils are phagocytic cells of the innate immune system that use a combination of reactive oxygen species and anti-microbial toxins to kill and destroy ingested micro-organisms. Once they have performed their function, neutrophils die by apoptosis, which is important for the effective resolution of the inflammatory response. Both glucose and glutamine are important fuels for neutrophils, yet little has been done to investigate the comparative effects of glucose and glutamine on neutrophil apoptosis. We hypothesized that glucose and/or glutamine significantly alter rates of spontaneous and anti-Fas antibody-induced apoptosis of human neutrophils cultured ex vivo. Neutrophil apoptosis was reduced by increasing the extracellular concentration of glucose, but was unaffected by glutamine concentration. The protective effect of glucose appeared to correlate with the rate of glucose utilization. The addition of a competitive inhibitor of glycolysis, 2-deoxy-D-glucose (10 mM), attenuated the protective effect of 5.5 mM glucose, indicating that glucose metabolism is essential for its protective effect against apoptosis. There was a significant (P<0.05) reduction in the intracellular ATP concentration of neutrophils incubated in the absence of extracellular glucose compared with cells incubated in the presence of 5.5 mM glucose. The protective effect of glucose against apoptosis may be mediated by maintenance of the intracellular ATP concentration.