Background Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum （ER） stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy. Methods Podocyte apoptosis was detected by 4＇,6-diamidio-2-phenylindole （DAPI） stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 （GRP78）, indicators of ER-associated apoptosis C/EBP homologous protein （CHOP）, and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain. Results Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L （10 hours, 13 hours, and 15 hours compared with 0 hour, P 〈0.001） and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours （compared to control, P 〈0.001）. Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control （P 〈0.001）, with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour （P 〈0.001）, with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control. Conclusion Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinu
TAO Jian-lingWEN Yu-bingSHI Bing-yangZHANG HongRUAN Xiong-zhongLI HangLI Xue-meiDONG Wen-jiLI Xue-wang
Background Wallerian degeneration is a self-destructive process of axonal degeneration that occurs after an axonal injury or during neurodegenerative disorders such as Parkinson＇s or Alzheimer＇s disease. Recent studies have found that the activity of the nicotinamide adenine dinucleotide （NAD） synthase enzyme, nicotinamide mononucleotide adenylyltransferase 1 （NMNAT1） can affect the rate of Wallerian degeneration in mice and drosophila. NMNAT1 protects neurons and axons from degeneration. However, the role of NMNAT1 in neurons of central nervous system is still not well understood. Methods We set up the culture of primary mouse neurons in vitro and manipulated the expression level of NMNAT1 by RNA interference and gene overexpression methods. Using electroporation transfection we can up-regulate or down-regulate NMNAT1 in cultured mouse dendrites and axons and study the neuronal morphogenesis by immunocytochemistry. In all functional assays, FK-866 （CAS 658084-64-1）, a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase was used as a pharmacological and positive control. Results Our results showed that knocking down NMNAT1 by RNA interference led to a marked decrease in dendrite outgrowth and branching and a significant decrease in axon growth and branching in developing cortical neurons in vitro. Conclusions These findings reveal a novel role for NMNAT1 in the morphogenesis of developing cortical neurons, which indicate that the loss of function of NMNAT1 may contribute to different neurodegenerative disorders in central nervous system.
