ABSTRACT
Objective:To investigate the effects of Klotho on autophagy of human renal tubule cells under high glucose through AMP-activated protein kinase (AMPK) and extracellular signal regulated kinase (ERK) pathways.Methods:Human renal tubular epithelial cells cultured in vitro were divided into control group and high glucose group (HG group, added with 30 mmol/L glucose); According to the transfection of pcDNA3.1-vector or pcDNA3.1-Klotho, they were divided into two groups: Vector group and Klotho group; According to whether AMPK inhibitor compound C or ERK inhibitor curcumin was added after pcDNA3.1-Klotho transfection and high glucose stimulation, they were divided into four groups: HG+ Vector group, HG+ Klotho group, HG+ Klotho+ compound C group and HG+ Klotho+ curcumin group. The expression of Klotho was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot; The relative expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK/AMPK and p-ERK/ERK were detected by Western blot; Changes of autophagosome in human renal tubular epithelial cells observed by transmission electron microscope. Results:The protein and mRNA expression of Klotho in human renal tubular epithelial cells of HG group was significantly lower than that in the control group (all P<0.05); The LC3-Ⅱ/LC3-Ⅰ in Klotho group was significantly higher than that in Vector group ( P<0.05); The number of autophagosomes in Klotho group was also significantly higher than that in Vector group ( P<0.05); p-AMPK/AMPK in Klotho group was significantly higher than that in Vector group ( P<0.05), while p-ERK/ERK in Klotho group was significantly lower than that in Vector group ( P<0.05). The protein relative expression of p-AMPK/AMPK in HG+ Klotho+ compound C group (0.44±0.04) was significantly lower than that in HG+ Klotho group (0.79±0.08) ( P<0.01); The protein relative expression of p-ERK/ERK in HG+ Klotho+ curcumin group (1.05±0.12) was significantly higher than that in HG+ Klotho group (0.56±0.05) ( P<0.01). The relative expression of LC3-Ⅱ/LC3-Ⅰ protein in HG+ Klotho+ compound C group and HG+ Klotho+ curcumin group (0.79±0.12; 0.68±0.09) were significantly lower than that in HG+ Klotho group (1.65±0.20) (all P<0.01). Conclusions:Klotho can enhance autophagy of human renal tubular epithelial cells under high glucose condition by activating AMPK and inhibiting ERK pathway.
ABSTRACT
Fluorocarbon emulsion, a substitute of blood, 20 ml/kg were injected once into dogs intravenously. As a result, part of the substitute deposited in the lympho-retic-ular system and was phagocytized by fixed and free histiocytes (rnonophagocytic system), and the histiocytes then converted into foaming cells.One month after injection, the fluorocarbon's deposits in the foaming cells remained at the peak level in the liver, spleen and other organs of the lympho-reti-cuJar system. After six months,no deposits could be found in all viscera and no signs of any pathological changes, except the spleen under a light microscope.After twelve months, no foaming cells were found in the spleen, liver, and kidney under light cmicroscope,yet but foaming cells could be found in the spleen and liver with the help of an electron microscope and we believed that this sign had no pathological significance in clinic practice.The target cells only showed the action of phagocitizing and depositing.According to our observation, the fluorocarbon emulsion is a non cytoplasmic toxin and an inert biologic substance, so there are no secondary histo-pathologic changes caused by fluorocarbon deposition. It seems that 20 ml/kg of fluorocarbon emulsion (equal to 1200 ml for adult human)injected intravenously is rather safe.