Albumin-induced Apoptosis In Tubular Epithelial Cells Through Mitogen-activated Protein Kinase Pathways

The pathologic changes of many forms of chronic proteinuric renal diseases are characterized as glomerular sclerosis and tubulointerstitial fibrosis. Howerer, the recent studys showed a closer link between tubulointerstitial lesions and prognosis of renal damages. It has been proven that the degree of proteinuria correlates closely with the rate of progression of chronic renal failure. A great numbers of clinical observations as well as animal models of protein-overload indicated that urinary proteins directly resulted in tubulointerstitial damages, however,the mechanisms remain largely unknown. In addition, there are no effective therapeutic strategies on tubulointerstitial injury resulting from proteinuria at present. Therefore, this study is to evaluate whether albumin induces apoptosis in tubular cells and the relevant signal transduction, whether taurine protects the tubular cells from apoptosis induced by albumin, and whether tubular cells apoptosis is present in patients with heavy proteinuria.To explore the mechanisms by which urinary proteins damage tubular cells, we incubated rat tubular cells(NRK-52E) with various concentrations (10, 20, 30mg/ml) of delipidated, endotoxin-free bovine serum albumin(BSA) for 6, 12, 18 and 24 hours respectively. Trypan blue exclusive test showed the ratio of necrotic cells less than 10%. Then, the apoptosis was evaluated by fluorescence microscope, transmission electron microscope, scanning electron microscope, confocal laser scanning microscope (CLSM) and flow cytometry. The results showed the albumin-induced tubular cell apoptosis in a dose - and time- dependent manner.Apoptosis is programm cell death regulated by gene modulation. Many signal pathways including mitogen-activated protein kinases (MAPKs) are involved in the apoptotic effect. MAPKs mediate intra- and extracellular signal transduction. p38 kinase and c-Jun N-terminal kinases (JNKs) are activated by proinflammatory cytokines, heat shock, hyperosmolarity and other cellular stresses, and implicated in apoptoticprocesses. The extracellular signal-regulated kinases(ERKs) are activated by mitogens and growth factors and are mainly involved in cell proliferation and differentiontion. We hypothesed that MAPKs may mediate albumin-induced tubular cell apoptosis. NRK-52E cells were treated with BSA 30mg/ml for 15, 30, 60 and 120 minutes respectively. Activity of p38, JNK and ERK were assessed by Western blot analyses. To assess the roles of p38, JNK and ERK activity in albumin-induced apoptosis SB202190 (20uM, p38 inhibitor), SP600125 (10uM, JNK inhibitor) or PD098059 (20uM, ERK inhibitor) were added to the NRK-52E cells in the presence of albumin for 24 hours. Then, apoptosis was assayed by the methods as mentioned above. Results showed that albumin stimulated p38 and JNK and inhibited ERK. SB202190 and SP600125 ameliorated tubular cells apoptosis but PD098059 treatment enhanced the apoptosis.Tubular cell apoptosis in proteinuric status may be one of the major causes leading to tubulointerstitial damages, so it is important to seek the therapeutic approach to prevent apoptosis . Taurine, a sulfur-containing amino acid, plays an important role in several essential biological processes, such as development of the central nervous system and the retina, calcium modulation, membrane stabilization, reproduction, and immunity. Taurine confers a cytoprotective effect and reduces the apoptosis during ischemia. To investigate the anti-apoptotic effect of taurine on albumin-induced apoptosis, NRK-52E cells were treated with various concentrations (10, 20, 30 and 40mM) of taurine and BSA (20mg/ml or 30mg/ml). By detection of apoptosis and assay for p38, JNK, ERK, we demonstrated a decreased cell apoptosis and activity of p38 and JNK, but an increased ERK activity after taurine treatment.To further investigate proteinuria-related cell apoptosis, the renal biopsy specimen from patients with heavy proteinuria were evaluated with light microscope. We demonstrated apoptosis of proximal tubular epithelial cells in rena...