Additional file 2: of Renal allograft rejection, lymphocyte infiltration, and de novo donor-specific antibodies in a novel model of non-adherence to immunosuppressive therapy

anti-CD20, anti-CD3, and anti-CD68 immunofluorescence costaining of rat spleen follicle. (A) shows anti-CD20-positive B cells in the B cell zone of a splenic follicle (green), anti-CD3-positive T cells in T cell zone of splenic follicle (red), and CD68-positive macrophages (yellow) in the splenic red pulp, also shown in (B) without costaining. (PDF 588 kb

Additional file 3: of Renal allograft rejection, lymphocyte infiltration, and de novo donor-specific antibodies in a novel model of non-adherence to immunosuppressive therapy

anti-CD11b/c and anti-CD68 FACS costain of rat splenocytes. (A) shows unstained cells, (B) shows anti-CD11b/c-PE antibody only, and (C) shows anti-anti-CD11b/c-PE antibody and anti-CD68-APC antibody co-stain of rat splenocytes. (PDF 82 kb

ER-stress induced by Thapsigargin, Tunicamycin or DTT is not attenuated by ALR.

<p>Cultured cells were incubated either for 6 hours with 0.5 μM Thapsigargin, for 16 hours with 10 μg/ml Tunicamycin or 2 mM DTT. (A) Activation of CHOP was analyzed by western blotting of HepG2-sfALR and HepG2 cells treated without or with 100 nM rhALR. (B) Activation of XBP1 in HepG2-sfALR and HepG2 cells treated without or with 100 nM rhALR was analyzed by amplifying XBP1 cDNA by PCR followed by incubation with P<i>st</i>I. Spliced form indicating activation results in a 473 bp amplification product, whereas unspliced form of XBP1 shows digested 290 bp and 183 bp amplification products (one out of three experiments is shown).</p

Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways

<div><p>Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial β-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced β-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.</p></div

Reduced ALR and FOXA2/HNF3β expression in liver tissue of patients with NAFLD.

<p>Expression of (A) ALR mRNA and (B) FOXA2/HNF3β mRNA was analyzed by qRT-PCR in liver tissue samples from patients with hepatic steatosis (n = 27), NASH (n = 29) and normal liver tissue (n = 17). (C) ALR mRNA expression was analyzed by qRT-PCR in liver tissue samples from mice fed a standard diet (SD, lean, n = 5) or high fat diet (HFD, n = 6) for 14 weeks. YWHAZ mRNA expression was determined for normalization (* p < 0.05 differs significantly). (D) HepG2 cells were incubated for 24 hours with 0 mM (vehicle) or 0.4 mM PA and FOXA2/HNF3β translocation from nucleus into cytosol was visualized by immunostaining of FOXA2/HNF3β (one out of three experiments is shown; bar = 10 μm).</p

Uptake rate of [13C] palmitic acid and percentage of 13C-labelled fatty acids to [13C] palmitic acid in Huh7 cells at indicated time points.

<p>Uptake rate of [13C] palmitic acid and percentage of 13C-labelled fatty acids to [13C] palmitic acid in Huh7 cells at indicated time points.</p

Endogenous sfALR reduces PA-induced ER-stress and death receptor expression.

<p>Cultured cells were incubated for 8 hours with indicated concentrations of palmitic acid or vehicle (0 mM PA). (A) Expression of CHOP was analyzed by western blotting lysates of HepG2-sfALR and HepG2 cells treated without or with 100 nM rhALR followed by densitometric analysis. (B) Activation of elF2α by phosphorylation was analyzed by western blotting lysates of HepG2 and HepG2-sfALR cells followed by densitometric analysis. (n = 3; * p < 0.05 differs from corresponding HepG2, # p < 0.05 differs from 0 mM PA). (C) Expression of death receptor 5 (DR5) after 24 hours incubation with PA was analyzed by western blotting. (D) Activation of JNK by phosphorylation was analyzed by western blotting lysates of HepG2 and HepG2-sfALR cells followed by densitometric analysis (n = 3; * p < 0.05 differs from corresponding HepG2, # p < 0.05 differs from 0 mM PA). (E) Activation of XBP1 in HepG2 and HepG2-sfALR cells was analyzed by amplifying XBP1 cDNA by PCR following incubation with P<i>st</i>I. Spliced form of XBP1 indicating activation results in a 473 bp amplification product, whereas unspliced form of XBP1 shows digested 290 bp and 183 bp amplification products (one out of three experiments is shown).</p

Endoenous ALR results in less cellular lipid content, alters lipid metabolism associated genes and enhances ATP levels.

<p>(A) Cultured cells were incubated for 24 hours with indicated concentrations of palmitic acid or vehicle. Cellular lipid content of HepG2 and HepG2-sfALR cells was analyzed by staining lipid droplets using Nile Red following fluorescence microscopy (one out of three experiments is shown; bar = 20 μm). (B) Total TAG levels were analyzed in HepG2-sfALR and HepG2 cells (without or with 100 nM rhALR) incubated with PA for the indicated time. Alteration of total TAG content is shown corresponding to HepG2 cells (n = 3; * p < 0.05 differs from HepG2; # p < 0.05 differs from 0 mM PA). (C) Cultured cells (HepG2-sfALR and HepG2 without or with 100 nM rhALR) were incubated with [13C] PA for the indicated time and analyzed for content of [13C]-labelled fatty acids. Ratio of 14:0/16:0 in percent is shown (n = 3). (D) HepG2-sfALR and HepG2 cells were incubated for 24 hours with palmitic acid or vehicle and analyzed for lipid metabolism related gene expression (CPT1α, ACC, FASN, ELOVL6 mRNA) performing qRT-PCR. YWHAZ mRNA expression was determined for normalization (n = 3). (E) Cell homogenates from cells decribed in (D) were further analyzed by western blotting (one out of three experiments is shown). (F) Cellular ATP content was determined in HepG2 and HepG2-sfALR cells treated for 24 hours with palmitic acid or vehicle (n = 3; * p < 0.05 differs from HepG2; # p < 0.05 differs from corresponding 0 mM PA).</p

ALR is expressed in different isoforms and subcellular localizations (1).

<p>(A) PHH, HepG2 and HepG2 cells stably expressing sfALR (HepG2-sfALR) were separated into mitochondrial, M, and cytosolic, C, fractions followed by western blot analysis of ALR isoform expression. Specificity of subcellular fractions is demonstrated by expression of specific markers for cytosol, HSP70, and mitochondria, CVα. ALR isoforms of 23 kDa and 21 kDa are found in cytosolic as well as mitochondrial fractions, whereas 15 kDa ALR is only detected in cytosolic fractions of PHH with a faint and in HepG2-sfALR cells with an intense signal. (B) Immunocytochemistry of ALR expression in HepG2 and HepG2-sfALR cells. Cells were immuno-stained for ALR (green) and mitochondria (red, Mitotracker) visualizing co-localization by yellow overlap in the merge image. ALR is expressed in the cytosol and in mitochondria with a more intense immune signal in the cytosol of HepG2-sfALR cells.</p

ALR is expressed in different isoforms and subcellular localizations (2).

<p>Immunocytochemistry of ALR expression in HepG2 and HepG2-sfALR cells. Cells were immuno-stained for ALR (green) and no co-localization (yellow overlap in the merge image) was observed with (a) Golgi apparatus (red, Golgi-marker) nor with (B) ER (red, protein disulfide isomerase).</p