The Role Of Endoplasmic Reticulum Stress In Proximal Tubular Cell Apoptosis And Its Possible Mechanism

PartⅠAldosterone induced tubular epithelial cells apoptosis and its possible mechanismObjective:Apoptosis contributes to tubular epithelial cell death and atrophy in Aldo-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells.Methods:1. Human kidney-2cells (HK-2cells) were grown in DMEM/F12, containing10%fetal bovine serum (FBS) and1%penicillin/streptomycin. Confluent HK-2cells in6or96well plate were treated with different concentrations of Aldo. Intracellular ROS generation was evaluated by Fluorescence Microscopic and Fluorescent immune microplate reader; Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry, tubular epithelial cell apoptosis was also observed using laser scanning confocal microscope; ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively.2. For knockdown experiments, HK-2cells grown to50%confluence in six-well plates were transiently transfected with CHOP-specific siRNA or control siRNA, a scrambled sequence non-specific for any cellular mRNA. When HK-2cells were treated with Aldo or NAC, cells were transfected with500nM CHOP siRNA or control siRNA for24h before treatment. Apoptosis was detected by Annexin V/propidium iodide staining and flow cytometry.3. HK-2cells were pre-treated with NAC (5mM) for30min and then stimulated with Aldo (100nM) for a further24h. CHOP and GRP78protein were evaluated by Western blot. Results:1. Compared with the control group, Aldo dose-dependently and time-dependently induced ROS generation; Aldo dose-dependently induced HK-2cells apoptosis; Expression of GRP78was markedly upregulated in a time and dose-dependently manner; CHOP protein and mRNA expression were dose-dependently upregulated with treatment of different concentrations of Aldo (P<0.05).2. When HK-2cells were treated with Aldo in the presence of vehicle siRNA, cellular apoptosis was significantly upregulated. However, the expression of CHOP and induction of apoptosis by aldosterone were markedly inhibited by CHOP siRNA and NAC (P<0.05).3. Compared with the control group, GRP78and CHOP proteins were upregulated significantly in the Aldo-induced group. However, these increases could be inhibited by NAC (P<0.05).Conclusion:These observations suggest that aldosterone induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells. Part IIEffects of NAC on aldosterone/salt-induced renal injuryObjective:To observe whether endoplasmic reticulum (ER) stress was activated in aldosterone/salt-induced renal injury and investigate the protective role of NAC on aldosterone/salt-induced renal injury and its potential mechanism.Methods:30male Sprague-Dawley rats at the beginning of the experiments underwent right uninephrectomy under anesthesia with sodium pentobarbital. After2weeks of recovery from surgery, the rats were randomly treated with one of the following for4weeks:group1, tap water plus vehicle (0.5%ethanol, subcutaneously, n=6); group2,1%NaCl in the drinking solution plus vehicle (n=8); group3,1%NaCl in the drinking solution plus aldosterone (0.75μg/h, subcutaneously, n=8); group4,1%NaCl in the drinking solution plus aldosterone plus NAC (100mg/kg/day, by gavage, n=8). Systolic blood pressure (SBP) was measured in conscious rats by tail-cuff plethysmography and24h urine samples were collected in metabolic cages every week. The kidney was perfused with chilled saline solution and fixed in10%buffered paraformaldehyde for histological and TUNEL examination. Malondialdehyde (MDA), superoxide dismutase (SOD) and8-OHdG were determined according to the manufacturer’s instructions. CHOP and GRP78were evaluated by Western blot.Results:Compared with control and1%NaCl-treated group, rats that received aldosterone/1%NaCl exhibited hypertension and severe renal injury. Renal cortical protein levels of CHOP, GRP78, MDA and8-OHdG levels were significantly upregulated, renal cortical sections of aldosterone-induced rats showed increased numbers of TUNEL-positive tubular epithelial cells compared with those in vehicle and vehicle/1%NaCl-treated rats. However, NAC could significantly attenuate these effects of aldosterone and protect renal function (P<0.05). Conclusion:Endoplasmic reticulum (ER) stress was activated in aldosterone/salt-induced renal injury and antioxidant may decrease tubular epithelial cells apoptosis and improve renal function through inhibiting endoplasmic reticulum stress pathway.