High Glucose Induces Autophagy In Podocytes

Background and Objective:Eukaryote cells carry two major protein degradation pathways-ubiquitin-proteasome system (UPS) and autophagy. Both of these pathways are responsible for the efficient degradation and turnover of proteins within the cells. Failure of either the UPS or autophagy has been associated with disease manifestation, while the upregulation of these processes has been shown to ameliorate certain disease entities. Podocytes are the most important cellular component constituting the glomerular filtration barrier (GFB) with complex and subtle structure. They are highly differentiated cells and play an important role in the pathogenesis of certain renal diseases. Both podocyte injury and loss are key factors in the development of glomerular diseases and progression of renal failure. Podocytes, as an important component of the GFB, are often exposed to various injury factors, which have the potential to induce oxidative stress and/or DNA damage. If a cell is exposed to cytotoxic or phlogogenic macromolecules, it has tendency to form autophagosomes to clear damaged proteins and organelles or results in apoptosis. Diabetic nephropathy is a common podocytopathy. High glucose is known to inflict podocyte injury, but little is reported about the relationship between high concentrations of glucose and autophagy in these cells. The factors involved in the pathogenesis of diabetic nephropathy are multifaceted. Some of these factors are involved in the imbalances between pro-and anti-free-radical processes and the formation of excessive free radicals in podocytes. ROS are known to induce autophagy, so high glucose may promote autophagy in podocytes. Rapamycin and3-methyadenine usually mediate autophagy, but little is known about the change of autophagy that rapamycin and3-methyadenine affect podocytes treated by high glucose. High glucose may induce either autophagy or apoptosis, but the activity of autophagy and apoptosis maybe makes some change in a different period. In the present study, we evaluated the effects of high glucose on the induction of autophagy in podocytes in vivo and in vitro, and we also explored whether the high glucose-induced effect on podocyte is mediated by rapamycin and3-methyadenine. By detecting ROS and related enzymes, we studied the mechanism which high glucose influenced autophagy in podocytes. By observing the change of autophagy and apoptosis in podocytes cultured in high glucose, we studied the relationship of autophagy with apoptosis. Methods:Part I:Sixteen SD rats weighing between170-200g were randomly divided into control group and diabetic group. Diabetes was induced by a single dose of streptozotocin (STZ,65mg/kg, intraperitoneal) in rats. Age-matched control rats received an equal volume of vehicle. Forty-eight hours after injection of STZ, the blood glucose level was measured from the tail vein. Rats with a blood glucose level over16.7mmol/L were considered as diabetic rats. Rats were sacrificed at8weeks and the kidneys were removed. One part of the kidney was fixed in2%glutaraldehyde, followed by epoxy resin embedding for electron microscopic studies.Part II:In vitro, cultured murine immortalized podocytes were treated with normal level of glucose (5mM) or high level of glucose (30mM) for various time periods. After fixation, cells were analysed under electron microscopy (EM). Two groups of conditionally immortalized murine podocytes (CIMPs) transfected with GFP-LC3were detected using fluorescence microscopy. Two groups of CIMPs treated with monodansylcadaverine (MDC) were examined under a confocal microscope, microtubule-associated protein1light chain3(LC3) and Beclin-1were analyzed by Western-blot in two groups of CIMPs.Part III:Equal numbers of CIMPs were incubated in medium containing normal glucose (5mM), high glucose (30mM), or H2O2(100μM, positive control for autophagy) for24h. At the end of the incubation period, cells were harvested and Western blots were prepared and probed for LC3-2and actin. Equal numbers of CIMPs were incubated in medium containing normal glucose (5mM) or high glucose (30mM), NAC (50μM)+normal glucose, or NAC (50μM)+high glucose for24h. At the end of the incubation period, cells were stained with acridine orange and examined under a confocal microscope. Equal numbers of CIMPs were treated with either normal glucose or high glucose in the presence or absence of N-acetylcysteine for24h. Subsequently, cells were harvested and Western blots were prepared and probed for LC3-2and beclin-1. Equal numbers of CIMPs were incubated in serum-free medium containing2’,7’-dichlorofluorescein diacetate (DCFDA,10mM) for40min, followed by incubation in medium containing either normal glucose or high glucose for60min. ROS generation was recorded at the indicated time periods. Equal numbers of CIMPs were incubated in serum-free medium containing either normal levels of glucose or high levels of glucose for24h. At the end of the incubation period, cells were harvested and Western blots were prepared and probed for MnSOD and catalase.Part Ⅳ:Equal numbers of CIMPs were incubated in medium containing normal glucose (5mM) or high glucose (30mM), rapamycin (1ng/ml)+normal glucose, or rapamycin (1ng/ml)+high glucose for24h. At the end of the incubation period, cells were stained with acridine orange and examined under a confocal microscope. LC3-2and Beclin-1were analyzed by Western-blot in four groups of CIMPs. Equal numbers of CIMPs were incubated in medium containing normal glucose (5mM), or high glucose (30mM),3-MA (2mM)+normal glucose, or3-MA (2mM)+high glucose for24h. Subsequently, cells were stained with acridine orange and examined under a confocal microscope. LC3-2and Beclin-1were analyzed by Western-blot in four groups of CIMPs.Part V:CIMPs were incubated in medium containing high glucose. Equal numbers of CIMPs were treated with high glucose for12h,24h,48h, and72h. At the end of the incubation period, cells were harvested and Western blots were prepared and probed for Beclin-1. CIMPs were also incubated in medium containing high glucose and rapamycin or3-MA for12h,24h,48h, and72h. At the end of the incubation period, cells were harvested and apoptosis was evaluated by flow cytometer.Results:Part I:Glomerular podocytes in diabetic rats showed increased number of autophagosomes and also displayed larger size of autophagosomes.Part II:CIMPs treated with high glucose not only showed larger autophagosomes but also presented a higher number of autophagosomes than CIMPs treated with normal glucose. Part Ⅲ:The effect of high glucose was partly inhibited by NAC. High glucose enhanced ROS generation by CIMPs in a time-dependent manner. High glucose-treated CIMPs showed enhanced expression of both MnSOD and catalase.Part Ⅳ:Rapamycin further enhanced high glucose-induced autophagosome formation. The enhanced autophagic effect of high glucose was inhibited by3-Ma.Part V:Autophagy of CIMPs was most obvious after treated with high glucose for24hours and then decreased gradually; Apoptosis of CIMPs was increased after treated with high glucose, and reach at its peak after treatment with high glucose for72hours.Conclusion:High glucose promotes autophagy in podocytes. This effect is mediated by mitochondrial ROS generation. The enhanced effect of high glucose was further exacerbated by rapamycin and inhibited by pretreatment with3-methyladenine. High glucose can induce autophagy and apoptosis. Autophagy occurs early and apoptosis develops in the late course. Rapamycin can enhance autophagy and inhibit apoptosis in podocytes treated with high glucose, but3-MA has the opposite effects.