The Mechanism Of Intracellular Signal Transduction During Injury Of Renal Tubular Cells Induced By Postasphyxial-serum In Neonates With Asphyxia

Background: Neonatal asphyxia can have its onset in the antepartum, intrapartum or postpartum period. Estimates of the incidence of neonatal asphyxia vary from 5 to 10 per 100 live births. A higher neonatal mortality and multi-organ failure are associated with neonatal asphyxia. Renal injury is a severe and extremely common complication that occurs early in neonates with asphyxia. Reperfusion injury has been suggested as the cause of kidney damage during resuscitation of neonatal asphyxia. Kidney proximal tubule cells develop severe energy deficits and injury during ischemia-reperfusion . Previous studies of ischemia-reperfusion injury demonstrate that inflammatory mechanisms, particularly those involving specific cytokines,are important for cell death. Previous studies have also demonstrated that postasphyxial-serum from neonates with asphyxia may result in apoptosis of renal tubular cells. However, the mechanisms that mediate renal tubular cell apoptosis induced by postasphyxial-serum remain poorly understood.Mitochondrial DNA is particularly sensitive to ischemia-reperfusion injury, and mitochondria play a central role in cell death signaling by releasing pro-apoptotic proteins.However,whether the mitochondria participant postasphyxia-serum induced the injury of renal tubular cells?NF-κB is a pivotal transcription factor for genes that encode proinflammatory cytokines, chemokines, and adhesion molecules. We have previously reported that neonatal postasphyxial-serum is an apoptosis-inducing agent in human renal tubular cells. Pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, can inhibit apoptosis induced by postasphyxial-serum. However,the relation between the activity of NF-κB and mitochondria needed to be forever investigated.In this report we investigate whether postasphyxial-serum treatment induces apoptosis in HK-2 cells through increased Bad and Bax expression, depolarization of mitochondria membrane potential, the release of cytochrome C and AIF from mitochondria to cytosol, and activation of NF-κB.Objective: In this report we investigate the intracellular signal transduction mechanisms that operate during injury of renal tubular cells induced by postasphyxial-serum in neonate.Methods : Cultured human renal proximal tubular cells HK-2 cell were exposed to 10% fetal calf serum (FCS) (normal control), 20% postasphyxial-serum or 20% postasphyxial-serum with pyrrolidine dithiocarbamate (PDTC). The mitochondria membrane potential (△ψm ) was examined by confocal microscopy .The expression of both Bad and Bax in the cytoplasm was detected by immunohistochemistry, and the release of the apoptogenic mitochondrial proteins cytochrome C and AIF was assessed by Western Blot Analysis. Results are expressed as mean±SD. The statistical significance between treatments was analyzed using one-way ANOVA, where p < 0.05 was considered significant.Results: Control cells had"orthodox"configurations, the membrane integrity was preserved and cells were attached to the substrate.After postasphyxial-serum challenge, HK-2 cells showed shrinkage in morphology, decreased growth and only a minority of cells adhered to the substrate. In the presence of PDTC, cells reverted to the"orthodox"state . Loss of mitochondria membrane potential was detected in HK-2 cells treated with 20% postasphyxial-serum as compared to cells in normal serum or PTDC-pretreated cells in 20% postasphyxial-serum. JC-1 fluorescence ratios (590/530nm) for each group is (2.96±0.38),(0.87±0.06) and (2.46±0.52). Control, p<0.01 vs corresponding asphyxia group, asphyxia group, p<0.01 vs corresponding PDTC blocking group .A significant increase of Bad and Bax protein expression was also detected in asphyxia group. Control[(1.97±0.26)and(1.77±0.11)],p<0.01 vs corresponding asphyxia group[(2.73±0.20)and(2.44±0.13)]; asphyxia group, p<0.01 vs corresponding PDTC blocking group [(2.38±0.13)and( 2.17±0.08)].Cytochrome C was not present in the cytosolic fraction of normal cells. Increasing amounts of cytochrome C were detected in the cytosol of postasphyxial-serum treated cells, with a concomitant decrease of cytochrome C in the mitochondrial fraction. In contrast, no significant release of cytochrome C into the cytosol or change in the amount of cytochrome C in the mitochondrial fraction was detected in PDTC-treated cells. The optical density of cytochrome C in control(156.5±3.54,179.5±2.12),p<0.05 vs corresponding asphyxia group(177.5±3.54,155±4.24); asphyxia group, p<0.05 vs corresponding PDTC blocking group (165±4.24,171±5.66). AIF was only detectable in the mitochondrial fraction of normal cells. It was released from mitochondria after postasphyxial-serum challenge, but PDTC inhibited the release of AIF. The optical density of AIF in control(212±4.24),p<0.05 vs corresponding asphyxia group(128±4.24); asphyxia group, p<0.05 vs corresponding PDTC blocking group (201±8.49).Conclusions: Our findings suggest that postasphyxial-serum may induce renal tubular cell apoptosis through the mitochondrial pathway and its intracellular signal transduction mechanism includes the activation of nuclear factor-κB.