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
Objective To analyze the clinical features of hemodialysis patients complicated by infective endo carditis. Methods The clinical features of six such patients admitted to Peking Union Medical College Hospital during the year 1990 to 2009 were analyzed. All of them were diagnosed based on Chinese Children Diagnostic Criteria for Infective Endocarditis. Results The average age of the six patients was 52.3±19.3 years old. Four were males. Vascular accesses at the onset of infective endocarditis were as follows： permanent catheters in three, temporary catheters in two, and arteriovenous fistula in one. Three were fbund with mitral valve involvement, two with aor tic valve involvement, and one with both. Five vegetations were found by transthoracic echocardiography, and one by transesophageal echocardiography. Four had positive blood culture results. The catheters were all removed. Four of the patients were improved by antibiotics treatment, in which two were still on hemodialy sis in the following 14-24 months and the other two were lost to follow-up. One patient received surgery, but died of heart failure alter further hemodialysis for three months. One was well on maintenance hemodi alvsis for three months after surgery. Conclusions Infective endocarditis should be suspected when hemodialysis patients suffer from long-term fever, for which prompt blood culture and transthoracic echocardiography confirmation could be performed. Transesophageal echocardiography could be considered even when transthoracic echocardiography produces negative findings. With catheters removed, lull course of appropriate sensitive antibiotics and surgery if indicated could improve the outcome of chronic hemodialysis patients complicated by infective cndocarditis.
Objective To investigate the role of acyl-coenzyme A:cholesterol acyltransferase inhibitor(ACATI) in apoptosis induced by lipids and whether lipids-induced apoptosis is accompanied by increase of free cholesterol in endoplasmic reticulum(ER),in order to further understand the mechanism of lipids-induced apoptosis in advanced atherosclerosis.Methods Human vascular smooth muscle cells(VSMCs) and phorbol 12-myristate 13-acetate(PMA) differentiated THP-1 macrophages were used.Tritiated thymidine incorporation was applied to detect cell proliferation.Cytotoxicity was assessed by lactate dehydrogenase(LDH) release.4',6-diamidino-2-phenylindole(DAPI) staining,caspase-3,-7 assay,and Annexin-V/propidium iodide(PI) staining were used to detect apoptosis.High performance liquid chromatography was used in intracellular free cholesterol and cholesterol ester assay.ER free cholesterol was quantified.Results Different lipids had different effects on proliferation and cytotoxicity of VSMCs.25-hydroxycholesterol(25OHC) had biphasic effects on the proliferation of VSMCs.At low concentration,it stimulated cell proliferation,but turned to proliferation inhibition as concentration reached 15 μg/mL.25OHC and acetylated low density lipoprotein(AcLDL) could respectively induce apoptosis in human VSMCs and PMA differentiated THP-1 macrophages,which was aggravated by ACATI,accompanied by increase of intracellular free cholesterol content.There was also an increase of cholesterol content in ER with AcLDL-induced apoptosis in THP-1 macrophages.Conclusions Lipids could induce apoptosis,accompanied by increase of intracellular free cholesterol content,which could be augmented by ACATI,suggesting that insults resulting in ER free cholesterol rise might be the initiator of apoptosis.
Jian-ling TaoXiong-zhong RuanHang LiXue-mei LiXue-wang Li
Background Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease Macrophage death in advanced atherosclerosJs promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum （ER） stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate （PMA） for five days as an in vitro model, to investigate if acetylated low-density lipoprotein （AcLDL） loading could also induce apoptosis and its underlying mechanisms. Methods Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol （FC） and cholesterol ester （CE） assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed. Results Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 ug/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER. Conclusion ER stress is involved in AcLDL induced apoptosJs in THP-1 macrophages with free cholesterol accumulation in the ER.
TAO Jian-lingRUAN Xiong-zhongLI HangLI Xue-meiJohn E MoorheadZac VargheseLI Xue-wang
