Autophagy induced by AAI (10 µM) in NRK-52E cells.

<p>NRK-52E cells were transiently transfected with GFP-LC3 plasmid. After 0 to 24 hrs of AAI (10 µM) incubation, the cells were fixed and analyzed the formation and distribution of GFP-LC3 puncta by immunofluorescence. <b>A</b>, Representative images (×600). Arrows indicated GFP-LC3 puncta (green). Nuclei (blue) were stained by Hoechst33342. <b>B</b>, Percentage of cells with GFP-LC3 puncta. <b>C</b>, Western blot showed an increase in Beclin 1 and LC3-II at early stage of AAI incubation. <b>D</b>, Densitometric analysis of LC3-II in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g001" target="_blank">Fig. 1C</a>. The protein expression level of control (0 hr) group was arbitrarily set as 1 in each blot, and the signals of other conditions in the same blot were normalized with the control to indicate their protein expression level. <b>E</b>, Western blot showed an continue increase in LC3-II with the lysosomal inhibitors E64d (10 µg/ml)+Pepstatin A (10 µg/ml) after AAI incubation. <b>F</b>, Densitometric analysis of LC3-II in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g001" target="_blank">Fig. 1E</a>. <b>B, D</b> and <b>F</b> were expressed as means±SEM of three independent experiments. *and**denote <i>p<0.05</i> and <i>p<0.001</i>, respectively, when compared to control condition.</p

Inhibition of autophagy by Wortmannin and 3-MA increased apoptosis during AAI incubation of NRK52E cells.

<p>NRK52E cells were incubated for 12 hrs in the absence (DMSO control), or presence of AAI (10 µM), Wortmannin (20 nM), 3 MA (5 mM) or AAI plus Wortmannin, AAI plus 3 MA. Wortmannin and 3-MA blockaded the autophagy and increased apoptosis induced by AAI. <b>A</b> and <b>B:</b> Western analysis using antibodies against LC3-II or <i>β</i>-Action. <b>C</b> and <b>D:</b> Densitometric analysis of LC3II in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g003" target="_blank">Fig. 3A and 3B</a>. NRK52E cells were transiently transfected with GFP-LC3 plasmid, then were subjected to the same conditions as described. <b>E</b>, Representative images (×800). Arrows indicated GFP-LC3 puncta (green). Nuclei (blue) were stained by Hoechst33342. <b>F</b>, Graphs represent quantitation analysis of the percentage of cells with GFP-LC3 positive autophagosomes. <b>G</b>, Representative annexinV/PI flow cytometry analysis of NRK52E cells. <b>H</b>, Graphs represented quantitation of analysis of AnnexinV+PI− and AnnexinV+PI+ NRK52E cells by flow cytometry. <b>C</b>, <b>D</b>, <b>F</b> and <b>H:</b> Data was expressed as means±SEM of three independent experiments. *and** denote <i>p<0.05</i> and <i>p<0.001</i>, respectively, when compared to control condition. ^#denote <i>p<0.05</i>,compared to AAI group.</p

AAI induced autophagy via activation ERK1/2 pathway.

<p>NRK52E cells were treated with AAI (10 µM) for 0 to 24 hrs. <b>A</b>, Representative immunoblot images of p-ERK1/2, ERK1/2, JNK1/2, p-JNK1/2, p–p38 and p38 proteins. ERK1/2 pathway was activated at early stage, but not JNK and P38. U0126 inhibited activation of ERK1/2 pathway and accumulation of LC3II induced by AAI. NRK52E cells were incubated for 12 hrs in the absence (DMSO control), or presence of AAI (10 µM), U0126 (5 µM) or AAI plus U0126. <b>B</b>, Representative immunoblot images of p-ERK1/2, ERK1/2, Beclin 1 and LC3II proteins. <b>C</b>, Densitometric analysis of p-ERK1/2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g005" target="_blank">Fig. 5A</a>. <b>D</b>, Densitometric analysis of p-ERK1/2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g005" target="_blank">Fig. 5B</a>. <b>E</b>, Densitometric analysis of LC3II in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030312#pone-0030312-g005" target="_blank">Fig. 5B</a>. <b>F</b>, Representative images (×800). Arrowheads indicate GFP-LC3 puncta (green). Nuclei (blue) were stained by Hoechst33342. <b>G</b>, Graphs represent quantitation analysis of the percentage of cells with GFP-LC3 positive autophagosomes. U0126 increased cell apoptosis induced by AAI. <b>H</b>, Representative annexinV/PI flow cytometry analysis of cells from DMSO control, AAI, U0126 and AAI plus U0126 groups. <b>I</b>, Quantitative analysis of AnnexinV+PI− and AnnexinV+PI+ NRK52E cells by flow cytometry. <b>C</b>,<b>D</b>,<b>E</b>,<b>G</b> and <b>I:</b> Data was expressed as means±SEM of three independent experiments. *and** denote <i>p<0.05</i> and <i>p<0.001</i>, respectively, when compared to control condition. ^#denote <i>p<0.05</i>,compared to AAI group.</p

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target

Effects of LPS on HMGB1 release in HMrSV5 cells.

<p>(A) Cells were treated with LPS at various concentrations for 48 hr. Cell culture media were collected and analyzed by immunoblotting with HMGB1 antibody. (B) Densitometry of HMGB1 proteins in immunoblots. (C) Cell viability was evaluated by MTT assay after treatment with LPS at the indicated concentrations for 48 hr. (D) Immunoblot analysis of HMGB1 in cell culture supernatants following 2 µg/ml LPS stimulation for the indicated time. (E) Quantitative determination of the relative abundance of HMGB1 proteins among different groups. (F) Cell viability was assayed by MTT at different time following LPS incubation. Data in B, C, E and F are expressed as mean ± SE (<i>n = </i>6). *<i>P</i><0.05 <i>versus</i> control group.</p

HMGB1 nuclear-cytoplasmic translocation in LPS-induced HMrSV5 cells.

<p>Cells were treated with 2 µg/ml LPS for the indicated time period. (A) Representative confocal microscopic images showed the cellular localization of HMGB1 (red) and nuclear staining (blue) by indirect immunofluorescence staining in cells. Original magnification×400. (B) HMGB1 content in cytoplasm and nuclear fractions after LPS stimulation were assessed by Western blot analysis. (C) Quantitative determination of the relative abundance of HMGB1 in the cytoplasm and the nucleus among different groups. Data are expressed as mean±SE of three experiments. *<i>P<</i>0.01 <i>versus</i> negative control in the cytoplasm; #<i>P<</i>0.01 <i>versus</i> negative control in the nucleus.</p

Patients Characteristics.

<p>Note: ESRD, end-stage renal disease; PD, peritoneal dialysis; BMI, body mass index; Hb, haemoglobin; hsCRP, high-sensitivity C-reactive protein; Kt/V, solute clearance as a dialysis adequacy index; rGFR, residual glomerular filtration rate calculated by mean of creatinine and urea clearance.</p><p>Characteristics are presented as the mean ± SE or as median (interquartile range) for continuous variables and percentages for categorical variables.</p>a<p><i>p<0.05, vs</i> no peritonitis group.</p>b<p><i>p<0.05,vs</i> Gram-positive peritonitis group.</p

HMGB1 was secreted via lysosome-mediated secretory pathway in response to LPS stimulation.

<p>(A). HMGB1 was present in vesicles cofractionating with lysosomes after LPS administration. Lysosome fractions were untreated (lanes 1 and 2, Try) or solubilized (lanes 3 and 4, TyrTx) with Triton X-100 (TX) before trypsin (Try) digestion and subjected to Western blot analysis with anti-Cathepsin D and anti-HMGB1 antibodies. (B) Densitometry of HMGB1 content in different groups. Data are expressed as mean ± SE, <i>n = </i>3 per treatment, *<i>P</i><0.05 <i>versus</i> Try treated only group. (C) Cells were treated with LPS (2 ug/ml) for 24 hr. Representative immunofluorescence analysis of cellular localization of HMGB1 (green) and LAMP2a (red), a maker of lysosome in cells. Original magnification×400. (D) HMGB1 protein contents in lysosome fractions following LPS treatment were determined by Western blotting. (E) Quantitative determination of the relative abundance of HMGB1 among different groups. Data are expressed as mean ± SE, <i>n = </i>3 per treatment, *<i>P</i><0.05 <i>versus</i> control group, # <i>P</i><0.05 <i>versus</i> LPS treated for 24 hr.</p