Publication

Abstract : We recently reported that necrotic renal proximal tubular cells (RPTC) can induce the death of renal interstitial fibroblasts. Since autophagy plays either cytoprotective or cytodestructive roles depending on the experimental condition, the present study was carried out to investigate whether necrotic RPTC would induce autophagy of renal interstitial fibroblasts and, if so, whether autophagy would contribute to cell death or exert a protective effect. Exposure of necrotic RPTC supernatant (RPTC-Sup) induced autophagy in renal interstitial fibroblast cells (NRK-49F) in a time- and dose-dependent manner, and its induction was earlier than caspase-3 activation. Inhibition of autophagy with 3-methyladenine (3-MA) or knockdown of Beclin-1, a molecule involved in the initiation of autophagosome formation, with small interference RNA (siRNA) significantly enhanced necrotic RPTC-Sup-induced cell death. Necrotic RPTC-Sup induced phosphorylation of extracellular signal-regulated kinases (ERK1/2), p38, c-Jun NH2-terminal kinases (JNKs), and AKT. Treatment with an ERK1/2 pathway inhibitor, but not with specific inhibitors for p38, JNKs, or AKT pathways, blocked NRK-49F autophagy and cell death upon exposure to necrotic RPTC-Sup. Furthermore, knockdown of MEK1 with siRNA also reduced autophagy along with cell death in NRK-49F exposed to necrotic RPTC-Sup. In contrast, overexpression of MEK1/2 increased RPTC-Sup-induced fibroblast cell death without enhancing autophagy. Collectively, this study demonstrates that necrotic RPTC induce both autophagy and cell death and that autophagy plays a cytoprotective or prosurvival role in renal fibroblasts. Furthermore, necrotic RPTC-induced autophagy and cell death in renal fibroblasts is mediated by the activation of the MEK1-ERK1/2 signaling pathway. autophagy is a normal cellular process that is responsible for removal and recycling of bulk cytoplasmic constituents, misfolded proteins, and damaged intracellular organelles to maintain cellular homeostasis. Generally, cells exhibit a low basal rate of autophagy, but autophagy is upregulated in response to nutrient or growth factor deprivation in order to replenish amino acids and glucose for cellular function (3, 7, 23). Autophagy is also a self-adaptive process that protects cells during periods of profound cellular stress like prolonged starvation, hypoxia, endoplasmic reticulum stress, and pathogen infection. Typically, autophagy fulfills a prosurvival function, but this rescue function turns deleterious under certain circumstances (7, 9). Autophagy is a tightly controlled multistep process. It is initiated by the formation of phagophore (also called the isolation membrane), which engulfs the targets in cytoplasm and then transforms into autophagosome. During autophagy, the formation of autophagosomes requires redistribution of LC3 from the cytosol to autophagic vesicles by conversion of cytosolic LC3 I to the phospholipid conjugate form, LC3 II. In addition, autophagy involves Beclin-1, a core component of initiation, and a ubiquitin-like system composed of autophagy-related gene (ATG) proteins, the Atg12-Atg5 conjugation system (7, 18). Expression of Atg12-Atg5 and conversion of LC3 I to LC3 II are widely used to monitor autophagy. It is generally accepted that specific stimuli may trigger unique signaling pathways that are then integrated into the core regulatory mechanisms to induce autophagy. Among the numerous signaling pathways involved in this process, the mitogen-activated protein kinase (MAPK) family of protein kinases has been shown to play an important role at different checkpoints of the autophagy process. This family is composed of three pathways: extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and p38 (5, 8, 13, 16). ERK1/2 and p38 pathways are reported to be involved in maturation of autophagosome (8, 13), and JNK leads to autophagy through upregulation of Beclin-1 and LC3 expression (16, 22). Autophagy has been implicated in various diseases, including urinary unilateral obstruction (UUO)-induced renal fibrosis (14), glomerular diseases (25), polycystic kidney disease (1), and acute kidney injury (AKI) (11, 12, 15, 27). During the early stage of AKI, damaged or injured cells release various cytokines and cellular contents into the intercellular environment, where they may induce death of interstitial cells like peritubular fibroblasts. This could be related to the reduced number of peritubular fibroblasts in the injured kidney, as the result of a variety of insults (i.e., ischemia, ureteric obstruction, and focal needle stick injury), mostly in the area adjacent to the damaged tubular epithelium (17). Using a necrotic renal proximal tubular cell (RPTC) and renal interstitial fibroblast coculture system, we have provided evidence that necrotic RPTC can directly induce death of renal interstitial fibroblasts through an ERK-dependent mechanism (20, 21). However, it remains unknown whether necrotic RPTC would cause autophagy of renal fibroblasts and, if so, whether it regulates death of this cell type. In this study, we addressed these issues.