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Jiradej Makjaroen,1 Arthid Thim-Uam,2 Cong Phi Dang,3 Trairak Pisitkun,1 Poorichaya Somparn,1,4 Asada Leelahavanichkul4– 6 1Center of Excellence in Systems Biology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Division of Biochemistry, School of Medical Sciences, University of Phayao, Phayao, Thailand; 3Medical Microbiology, Interdisciplinary and International Program, Graduate School, Chulalongkorn University, Bangkok, Thailand; 4Translational Research in Inflammation and Immunology Research Unit (TRIRU), Department of Microbiology, Chulalongkorn University, Bangkok, Thailand; 5Immunology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 6Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, ThailandCorrespondence: Asada Leelahavanichkul; Poorichaya SomparnImmunology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, ThailandTel +666 2256 4132Email aleelahavanit@gmail.comBackground: Because survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored.Methods: Characteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice.Results: Except for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis.Conclusion: Immunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG’s use in sepsis, IVIG might be beneficial in sepsis with certain conditions.Keywords: sepsis, CLP, proteomics, IVIG

Background Human papillomavirus (HPV) infection causes around 90% of cervical cancer cases, and cervical cancer is a leading cause of female mortality worldwide. HPV-derived oncoprotein E7 participates in cervical carcinogenesis by inducing aberrant host DNA methylation. However, the targeting specificity of E7 methylation of host genes is not fully understood but is important in the down-regulation of crucial proteins of the hallmark cancer pathways. In this study, we aim to link E7-driven aberrations in the host proteome to corresponding gene promoter hypermethylation events in the hope of providing novel therapeutic targets and biomarkers to indicate the progression of cervical cancer. Methods HEK293 cells were transfected with pcDNA3.1-E7 plasmid and empty vector and subjected to mass spectrometry-based proteomic analysis. Down-regulated proteins (where relative abundance was determined significant by paired T-test) relevant to cancer pathways were selected as gene candidates for mRNA transcript abundance measurement by qPCR and expression compared with that in SiHa cells (HPV type 16 positive). Methylation Specific PCR was used to determine promoter hypermethylation in genes downregulated in both SiHa and transfected HEK293 cell lines. The FunRich and STRING databases were used for identification of potential regulatory transcription factors and the proteins interacting with transcription factor gene candidates, respectively. Results Approximately 400 proteins totally were identified in proteomics analysis. The transcripts of six genes involved in the host immune response and cell proliferation (PTMS, C1QBP, BCAP31, CDKN2A, ZMYM6 and HIST1H1D) were down-regulated, corresponding to proteomic results. Methylation assays showed four gene promoters (PTMS, C1QBP, BCAP31 and CDKN2A) were hypermethylated with 61, 55.5, 70 and 78% increased methylation, respectively. Those four genes can be regulated by the GA-binding protein alpha chain, specificity protein 1 and ETS-like protein-1 transcription factors, as identified from FunRich database predictions. Conclusions HPV E7 altered the HEK293 proteome, particularly with respect to proteins involved in cell proliferation and host immunity. Down-regulation of these proteins appears to be partly mediated via host DNA methylation. E7 possibly complexes with the transcription factors of its targeting genes and DNMT1, allowing methylation of specific target gene promoters.

Non-invasive diagnosis of interstitial fibrosis and tubular atrophy (IF/TA), a major cause of chronic allograft dysfunction in post-kidney transplantation (post-KT), is needed.Several candidates of microRNAs (miRs) in plasma exosome or whole plasma were evaluated for IF/TA biomarker.Kidney samples from biopsy and plasma were tested for miRs expression.Expression of miR-21, miR-142-3p and miR-221 in renal histology with high fibrosis score (Banff classification) was higher than the samples with lesser score (n = 17/group). However, expression of these miRs from plasma exosome or from whole plasma of post-KT patients with different severity of IF/TA as determined by percentage of IF/TA including; grade I (5-25%) (n = 15), grade II (26-50%) (n = 15), grade III (≥ 50%) (n = 6) versus stable graft function (no IF/TA) (n = 15) was not different. However, high expression of miR-21 in exosome, but not from whole plasma, was demonstrated in IF/TA grade II and III compared with IF/TA grade I. In contrast, serum creatinine (Scr) and proteinuria, the current standard biomarkers, could not differentiate IF/TA grade I out of grade II/III. There was no correlation between exosome miR-21 versus the current standard renal injury biomarkers, including Scr, blood urea nitrogen and proteinuria, in IF/TA grade II or grade III.High miR-21 in plasma exosome, but not in whole plasma, indicated high grade IF/TA in post-KT patients. This non-invasive monitoring biomarker allows the more frequent evaluation on IF/TA than renal biopsy (a standard but more invasive procedure) resulting in the earlier management. More studies on patients are warrant.

Introduction Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole. Methods Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from −70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (β-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l -lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group. Discussion This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD_DB/Supplementary_Data.xlsx .

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

In patients with active lupus, spontaneous endotoxemia and possibly tolerance to lipopolysaccharide (LPS) is a potentially adverse complication. Similarly, previous reports have demonstrated that FcGRIIb deficient mice (FcGRIIb-/, a lupus mouse model) are susceptible to LPS tolerance-induced decreased cytokine responses that inadequate for the organismal control. Thus, understanding the relationship between FcGRIIb and LPS tolerance could improve the therapeutic strategy for lupus. LPS tolerance can be induced through sequential LPS stimulations in either cells or a model organism. In RAW264.7 (a mouse macrophage cell-line), sequential LPS stimulation induced the secretion of Lipocalin-2 (Lcn-2) despite reduced cytokine secretion and severe energy depletion, as measured by the extracellular flux analysis, typical of LPS tolerance. In contrast, treatment with recombinant Lcn-2 (rLcn-2) attenuated LPS tolerance, as shown by an increase in secreted cytokines and altered macrophage polarization toward M1 (increased iNOS and TNF-&alpha, ) in RAW264.7 cells. These results suggest a role of Lcn-2 in LPS tolerance attenuation. In bone marrow derived macrophages, Lcn-2 level was similar in LPS tolerant FcGRIIb-/- and wild-type (WT) cells despite the increased LPS tolerance of FcGRIIb-/- cells, suggesting relatively low basal levels of Lcn-2 produced in FcGRIIb-/- cells. In addition, attenuation of LPS tolerance effectuated by granulocyte-monocyte colony stimulating factor (GM-CSF) reduced Lcn-2 in both cell types, implying an inverse correlation between Lcn-2 and the severity of LPS tolerance. Consequently, rLcn-2 improved LPS tolerance only in FcGRIIb-/- macrophages and attenuated disease severity of cecal ligation and puncture (CLP) sepsis pre-conditioning with sequential LPS injection (LPS-CLP model) only in FcGRIIb-/- mice, but not in WT mice. To summarize, inadequate Lcn-2 production in FcGRIIb-/- macrophage might, at least in part, be responsible for the inordinate LPS tolerance compared with WT cells. Additionally, supplementation of rLcn-2 attenuates LPS tolerance in FcGRIIb-/- macrophages in vitro, and in FcGRIIb-/- mice with LPS-CLP sepsis in vivo. In conclusion, Lcn-2 secreted by macrophages is possibly an autocrine signal to counter the reduced cytokine secretion in LPS tolerance.

Mosquito-borne flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), are major human pathogens. Among the flaviviral proteins, the nonstructural protein 5 (NS5) is the largest, most conserved, and major enzymatic component of the viral replication complex. Disruption of the common key NS5-host protein-protein interactions critical for viral replication could aid in the development of broad-spectrum antiflaviviral therapeutics. Hundreds of NS5 interactors have been identified, but these are mostly DENV-NS5 interactors. To this end, we sought to investigate the JEV- and ZIKV-NS5 interactomes using EGFP immunoprecipitation with label-free quantitative mass spectrometry analysis. We report here a total of 137 NS5 interactors with a significant enrichment of spliceosomal and spliceosomal-associated proteins. The transcription complex Paf1C and phosphatase 6 were identified as common NS5-associated complexes. PAF1 was shown to play opposite roles in JEV and ZIKV infections. Additionally, we validated several NS5 targets and proposed their possible roles in infection. These include lipid-shuttling proteins OSBPL9 and OSBPL11, component of RNAP3 transcription factor TFIIIC, minichromosome maintenance, and cochaperone PAQosome. Mining this data set, our study expands the current interaction landscape of NS5 and uncovers several NS5 targets that are new to flavivirus biology.

Gastrointestinal (GI) leakage is believed to exacerbate sepsis and new, validated markers of GI barrier performance might benefit clinical decision-making. Serum (1→3)-β-D-glucan (BG) was evaluated as a potential GI leakage marker. Serum BG was tested in several mouse models of GI leakage, including dextran sulfate solution (DSS) administration, endotoxin (LPS) injection, and cecal ligation and puncture sepsis (CLP). Serum BG titer was also evaluated in patients with sepsis and septic shock, for comparison.With 0.75% DSS administration, BG increased only after oral administration of heat-killed C. albicans, but increased spontaneously with 1.5% DSS. In the LPS and CLP models, BG increased as early as 1 h and at 12 h after LPS administration and surgery, respectively. GI leakage was confirmed by orthogonal validation methods including FITC-dextran oral administration in the DSS, LPS, and CLP models and, in the DSS model, with urine sucralose after oral administration and serum endotoxemia. IL-6 increased in parallel with serum BG. Serum BG or IL-6, at 18 h, anticipated sepsis mortality in the CLP model.Analysis of serum BG from patients with febrile neutropenic sepsis (N = 49) and febrile non-neutropenic sepsis (N = 39) demonstrated BG elevation. Patients with bacterial septic shock had serum BG titers similar to levels observed in invasive fungal disease, regardless of febrile neutropenia. Serum BG was lower in less severe cases of bacterial sepsis. Elevated serum IL-6 was associated with GI leakage and elevated serum BG.Serum BG may have potential as a sepsis/septic shock biomarker and further study in this context is warranted.

Proteomics Kidney

Table S1. Carbonylated sites identified by mass spectrometry of carbonylated proteins detected with redox proteomics. (DOCX 15 kb)

Aim miRNA-122 (miR-122) is a new, interesting liver injury biomarker but little is known about its effects when there is an indirect acute liver injury. Methods We investigated this by using indirect liver injury mice models with bilateral ureter obstruction (BUO), bilateral nephrectomy (BiNx) and cecal ligation and puncture (CLP). A direct liver injury model, liver ischemia/reperfusion injury (liver I/R), was performed in parallel. Liver injury (i.e. liver histology, alanine transaminase [ALT]), kidney damage (i.e. serum creatinine) and cytokines (i.e. tumor necrosis factor-α, interleukin [IL]-6, IL-1β, IL-10) were assessed. Results Six hours after BUO/BiNx/CLP, the ALT and serum cytokines were approximately 1.5-fold higher than the baseline whereas miR-122 did not change. After 6 h of BiNx, there were prominent hepatocyte vacuolization but no elevations of miR-122. However, after 24 h of BUO/BiNx/CLP, ALT, hepatocyte vacuolization and miR-122 increased. The cytokines at 6 h might have induced the production of miR-122 at 24 h. The results from the in vitro study with HepG2 cells and each of the cytokines resulted in increased miR-122. On the other hand, when the direct liver injury model was used, there was a fivefold and 22-fold increase in the ALT at 0.5 and 1 h after surgery, respectively, and high serum miR-122 which corroborated the results from the liver histopathology. Conclusion We demonstrated that prior serum cytokine accumulation increased serum miR-122 in indirect liver injury induced by BUO/BiNx and less severe sepsis mouse models. Cytokine accumulation may be responsible for miR-122 expression in these models. The clinical importance of liver injury demonstrated by the discordance between serum miR-122 and ALT was an interesting issue.

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

In the current era of large-scale biology, systems biology has evolved as a powerful approach to identify complex interactions within biological systems. In addition to high throughput identification and quantification techniques, methods based on high-quality mono-specific antibodies remain an essential element of the approach. To assist the large-scale design and production of peptide-directed antibodies for systems biology studies, we developed a fully integrated online application, Ab Designer (http://helixweb.nih.gov/AbDesigner/), to help researchers select optimal peptide immunogens for antibody generation against relatively disordered regions of target proteins. Here we describe Ab Designer in terms of its features, comparing it to other software tools, and use it to design three antibodies against kidney disease-related proteins in human, viz. nephrin, podocin, and apolipoprotein L1.

Dysfunction of FcGRIIb, the only inhibitory receptor of the FcGR family, is commonly found in the Asian population and is possibly responsible for the extreme endotoxin exhaustion in lupus. Here, the mechanisms of prominent endotoxin (LPS) tolerance in FcGRIIb&minus, /&minus, mice were explored on bone marrow-derived macrophages using phosphoproteomic analysis. As such, LPS tolerance decreased several phosphoproteins in the FcGRIIb&minus, macrophage, including protein kinase C-&beta, type II (PRKCB), which was associated with phagocytosis function. Overexpression of PRKCB attenuated LPS tolerance in RAW264.7 cells, supporting the role of this gene in LPS tolerance. In parallel, LPS tolerance in macrophages and in mice was attenuated by phorbol 12-myristate 13-acetate (PMA) administration. This treatment induced several protein kinase C families, including PRKCB. However, PMA attenuated the severity of mice with cecal ligation and puncture on LPS tolerance preconditioning in FcGRIIb&minus, but not in wild-type cells. The significant reduction of PRKCB in the FcGRIIb&minus, macrophage over wild-type cell possibly induced the more severe LPS-exhaustion and increased the infection susceptibility in FcGRIIb&minus, mice. PMA induced PRKCB, improved LPS-tolerance, and attenuated sepsis severity, predominantly in FcGRIIb&minus, mice. PRKCB enhancement might be a promising strategy to improve macrophage functions in lupus patients with LPS-tolerance from chronic infection.

Background Plasma mycophenolic acid (MPA) concentrations may predict therapeutic response in active lupus nephritis (LN). We determined the efficacy and safety of a concentration-controlled MPA regime in the treatment of severely active LN. Methods In this prospective study, 19 biopsy-proven class III/IV LN patients were treated with mycophenolate mofetil (MMF) for 48 weeks. The MMF dosage was based on maximal plasma MPA concentration at 1-hour post dose (MPA-C1). All patients had plasma MPA-C1 levels monitored weekly until achieving the targeted level of >13 mg/L. A low-dose steroid protocol was started at 0.5 mg/kg/day and rapidly tapered to 5 mg/day. Therapeutic response was evaluated at week 24 and week 48. MPA area-under-the curve (MPA-AUC0–12h) was measured at week 12 to verify the optimum dosage. Results No death or end-stage kidney disease occurred in this study. Seventeen patients (89%) responded to therapy at week 24 with four (21%) patients having complete response. There was no renal relapse at week 48 and four more patients had converted from partial response to complete response. Seventy eight percent of patients achieved the recommended MPA-AUC0–12h level. No association between plasma MPA concentrations and adverse reactions or infections was found. Conclusions MPA-C1 may be a practical monitoring of MPA levels in patients with LN. It is convenient to monitor and may facilitate an optimum estimate of MPA exposure.

Peritoneal dialysis inevitability results in activation of inflammatory processes and its efficiency is highly variable between patients. An improved method to isolate biomarkers and study pathophysiological mechanisms in peritoneal dialysis effluent (PDE) is expected to be of much benefit for the development of this treatment approach and help with patient management. Extracellular vesicles (EVs) are released as part of normal cellular processes. Their proteome is expected to reflect both type and health of their cell of origin. Although there is a significant interest in using EVs for “liquid biopsies”, little is reported of their presence or composition in plentiful dialysis waste fluids, including peritoneal dialysis effluent (PDE). Here we determined the presence of EVs in PDE and subsequently characterized their proteome. EVs were first isolated from PDE using differential centrifugation, then a further enrichment using size exclusion chromatography (SEC) was performed. The presence of EVs was demonstrated using transmission electron microscopy, and their particle counts were investigated using nanoparticle tracking analysis and dynamic light scattering. Using tandem mass spectrometry, marker proteins from three types of EVs i.e. apoptotic bodies, ectosomes, and exosomes were identified. The proteomic results demonstrated that the isolation of EVs by differential centrifugation helped enrich for over 2,000 proteins normally masked by abundant proteins in PDE such as albumin and SEC markedly further improved the isolation of low abundant proteins. Gene ontology analysis of all identified proteins showed the marked enrichment of exosome and membrane-associated proteins. Over 3,700 proteins were identified in total, including many proteins with known roles in peritoneal pathophysiology. This study demonstrated the prominence of EVs in PDE and their potential value as a source of biomarkers for peritoneal dialysis patients.

Background An early sepsis-induced acute kidney injury (sepsis-AKI) biomarker is currently in needed. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is a candidate of sepsis-AKI biomarker but with different cut-point values. Urinary exosomal activating transcriptional factor 3 (uATF3) has been mentioned as an interesting biomarker. Methods We conducted experiments in mice and a prospective, multicenter study in patients as a proof of concept that urine exosome is an interesting biomarker. An early expression of ATF3 in kidney of CD-1 mice at 6 h after cecal ligation and puncture implied the possibility of uATF3 as an early sepsis-AKI biomarker. Increase serum creatinine (Scr) ≥0.3 mg/dL from the baseline was used as an AKI diagnosis and urine was analyzed for uATF3 and uNGAL. Patients with baseline Scr at admission ≥1.5 mg/dL were excluded. Results The analysis showed higher Scr, uNGAL and uATF3 in patients with sepsis-AKI in comparison with patients with sepsis-non-AKI and healthy volunteers. A fair correlation, r2 = 0.47, between uATF3 and uNGAL was showed in sepsis-AKI group with Scr ≥2 mg/dL. To see if uATF3 could be an early sepsis-AKI biomarker, urine sample was collected daily during the first week of the admission. In sepsis-AKI and sepsis-non-AKI groups, uNGAL were 367 ± 43 ng/mL and 183 ± 23 ng/mL, respectively; and uATF3 were 19 ± 4 ng/mL and 1.4 ± 0.8 ng/mL, respectively. With the mean value of uNGAL and uATF3 in sepsis AKI as a cut-off level, AUROC of uNGAL and uATF3 were 64% (95% CI 0.54 to 0.74) and 84% (95% CI 0.77 to 0.91), respectively. Conclusions Urine exosome is an interesting source of urine biomarker and uATF3 is an interesting sepsis-AKI biomarker.

Gastrointestinal (GI) leakage in Clostridium difficile-associated diarrhea (CDAD) is well known but is not routinely assessed in clinical practice. Serum (1→3)-β-D-glucan (BG), a fungal cell wall component used as a biomarker for invasive fungal disease, was tested in a CDAD mouse model with and without probiotics. Higher serum fluorescein isothiocyanate-dextran (FITC-dextran) and spontaneous gram-negative bacteremia, GI leakage indicators, were frequently found in CDAD mice, which died compared with those which survived. BG, serum macrophage inflammatory protein-2 and FITC-dextran but not quantitative blood bacterial count differentiated the clinical severity. Interestingly, a specific dose of Lactobacillus rhamnosus L34 attenuated CDAD and decreased serum BG and FITC-dextran, but not other parameters. BG also showed a higher area under the receiver operating characteristic curve for 7-day mortality than FITC-dextran. Fifty-five percent of CDAD mice with BG ≥ 60 pg/ml (the human negative cut-off value for invasive fungal disease) at 1 day after C. difficile gavage died within 7 days. In conclusion, S: erum BG was elevated in mice with severe CDAD, an established model of GI leakage with a strong association with mortality rate. BG monitoring in patients with CDAD is of interest as both a potential prognostic tool and a therapeutic efficacy indicator.

Mycophenolic acid (MPA) is an effective treatment for active lupus nephritis despite its variable efficacy in different ethnic groups. Here we tested whether pharmacokinetic monitoring may help to optimize dosing of MPA in an Asian population. Patients with biopsy-proven class III or IV lupus nephritis (ISN/RPS category) were treated with mycophenolate mofetil or enteric-coated mycophenolate sodium. One month after initiating treatment we measured plasma MPA levels in eight samples taken over a 12-h period after drug administration. The mean area under the time-dependent curve for MPA of responding patients was significantly higher than those not responding. Successful treatment was seen in patients with areas45 mg h/l. The dosage of the drug was not related to MPA pharmacokinetics. In the mycophenolate mofetil group, however, MPA-area under the curve was positively, and significantly, correlated with trough or 1 h after dose concentrations and associated with a therapeutic response. Thus, our study shows that MPA pharmacokinetics were positively correlated with therapeutic responses of mycophenolate, suggesting that controlling the concentrations may improve its therapeutic efficacy in lupus nephritis. As the absorption and pharmacokinetic peak of enteric-coated tablets is slower, it is important to take different formulations into account when determining optimal MPA concentrations.

Serum neutrophil gelatinase associated lipocalin (sNGAL), a promising acute kidney injury (AKI) biomarker produced by renal and non-renal tissues, might be affected by sepsis. We evaluated sNGAL in zero glomerular filtration rate models [bilateral ureter obstruction (BUO) and bilateral nephrectomy (BiNx)] with subsequent cecal ligation and puncture (CLP)-induced sepsis in 6 to 8-week-old ICR mice. We found that sNGAL increased earlier than serum creatinine (Scr) in BiNx/BUO with and without CLP. The earliest time-point of increased sNGAL in BiNx+CLP was 1 h after surgery. Scr, but not sNGAL, was lower at 18 h after BiNx/BUO+CLP compared with BiNx/BUO alone. Compared with BUO, BiNx had higher, and equal sNGAL at 1 to 18 h and 36 h, respectively. Additionally, similar NGAL expression in internal organs (heart, lung, liver, and spleen) and survival rates indicated the comparable severity of BiNx and BUO. Serum interleukin (IL)-6 was increased and correlated with sNGAL in BiNx/BUO with and without sepsis. In summary, we demonstrated: sNGAL is an early AKI biomarker, which is not affected by sepsis; sNGAL is mainly produced by extrarenal sources as demonstrated by the comparable sNGAL in BiNx and BUO; the saturation of renal NGAL re-absorption in BUO is demonstrated by lower sNGAL in BUO at 1 to 18 h, but not at 36 h when compared with BiNx; and a correlation of sNGAL and IL-6 implied sNGAL is a good sepsis prognostic biomarker. Therefore, sNGAL is a more beneficial sepsis-AKI biomarker than Scr.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

Amphotericin B (Ampho B) is a fungicidal drug that causes cell wall injury. Pharmacological ascorbate induces the extracellular prooxidants, which might enter the Ampho B-induced cell wall porosity and act synergistically. We tested low-dose Ampho B with a short course of pharmacological ascorbate using a mouse model of sepsis preconditioned with an injection of Candida albicans 6 h prior to cecal ligation and puncture (CLP). In this model, candidemia reappeared as early as 6 h after CLP with a predictably high mortality rate. This characteristic mimics sepsis in the phase of immunosuppression in patients. Using the model, at 12- and 18-h post-CLP, we administered isotonic (pH neutralized) pharmacological ascorbate intravenously with low-dose Ampho B or sodium deoxycholate, vehicle-controlled, administered IP. The survival rate of low-dose Ampho B plus ascorbate was 53%, compared with

Nanovesicles present in urine the so-called urinary exosomes have been found to be secreted by every epithelial cell type lining the urinary tract system in human. Urinary exosomes are an appealing source for biomarker discovery as they contain molecular constituents of their cell of origin, including proteins and genetic materials, and they can be isolated in a non-invasive manner. Following the discovery of urinary exosomes in 2004, many studies have been performed using urinary exosomes as a starting material to identify biomarkers in various renal, urogenital, and systemic diseases. Here, we describe the discovery of urinary exosomes and address the issues on the collection, isolation, and normalization of urinary exosomes as well as delineate the systems biology approach to biomarker discovery using urinary exosomes.

Nanovesicles present in urine the so-called urinary exosomes have been found to be secreted by every epithelial cell type lining the urinary tract system in human. Urinary exosomes are an appealing source for biomarker discovery as they contain molecular constituents of their cell of origin, including proteins and genetic materials, and they can be isolated in a non-invasive manner. Following the discovery of urinary exosomes in 2004, many studies have been performed using urinary exosomes as a starting material to identify biomarkers in various renal, urogenital, and systemic diseases. Here, we describe the discovery of urinary exosomes and address the issues on the collection, isolation, and normalization of urinary exosomes as well as delineate the systems biology approach to biomarker discovery using urinary exosomes.

miRNA-122 (miR-122) is a new, interesting liver injury biomarker but little is known about its effects when there is an indirect acute liver injury.We investigated this by using indirect liver injury mice models with bilateral ureter obstruction (BUO), bilateral nephrectomy (BiNx) and cecal ligation and puncture (CLP). A direct liver injury model, liver ischemia/reperfusion injury (liver I/R), was performed in parallel. Liver injury (i.e. liver histology, alanine transaminase [ALT]), kidney damage (i.e. serum creatinine) and cytokines (i.e. tumor necrosis factor-α, interleukin [IL]-6, IL-1β, IL-10) were assessed.Six hours after BUO/BiNx/CLP, the ALT and serum cytokines were approximately 1.5-fold higher than the baseline whereas miR-122 did not change. After 6 h of BiNx, there were prominent hepatocyte vacuolization but no elevations of miR-122. However, after 24 h of BUO/BiNx/CLP, ALT, hepatocyte vacuolization and miR-122 increased. The cytokines at 6 h might have induced the production of miR-122 at 24 h. The results from the in vitro study with HepG2 cells and each of the cytokines resulted in increased miR-122. On the other hand, when the direct liver injury model was used, there was a fivefold and 22-fold increase in the ALT at 0.5 and 1 h after surgery, respectively, and high serum miR-122 which corroborated the results from the liver histopathology.We demonstrated that prior serum cytokine accumulation increased serum miR-122 in indirect liver injury induced by BUO/BiNx and less severe sepsis mouse models. Cytokine accumulation may be responsible for miR-122 expression in these models. The clinical importance of liver injury demonstrated by the discordance between serum miR-122 and ALT was an interesting issue.

Background Hyperlipidemia occurs in up to 50% of kidney transplant (KT) recipients who take everolimus (EVL). As a result of this, statins are the most commonly prescribed lipid-lowering drugs among these patients. However, we are concerned whether there are any drug interactions between EVL and statins, because both of these drugs use the same pharmacokinetics pathway. Therefore, we assessed the effects of concomitant use of EVL and atorvastatin. Methods In this randomized, open-label, crossover study, 20 KT patients were assigned (1:1) to receive EVL with or without 20 mg atorvastatin for 1 month. One-month washout period was used before crossover. Plasma EVL concentrations were measured by homogeneous particle-enhanced turbidimetric immunoassay. Twelve-hour area under the time-concentration curve (AUC0–12) of EVL was calculated with the use of whole-blood EVL concentrations from 10 different time points (0, 0.5, 1, 1.5, 2, 2.5, 4, 6, 8, and 12 hours). Results The mean (SD) AUC0–12 for EVL and EVL plus atorvastatin was 155.9 (41.6) ng·h/mL and 151.3 (51.4) ng·h/mL, respectively (P > .05; paired t test). No difference of EVL Cmax or Tmax was found after atorvastatin coadministration. Even though the EVL AUC0–12 levels were not affected by atorvastatin coadministration in one-half of the subjects, for the rest of the patients, there were unpredictable changes in the EVL AUC0–12 levels. This may be due to the high intrapatient variability of EVL drug concentration (coefficient of variation ranges from 9.8% to 34.1%). Conclusions Coadministration of atorvastatin with EVL in KT recipients did not affect the pharmacokinetics of EVL.

AIM: To study the differential protein profile in serum of hepatitis B patients. METHODS: Serum samples were obtained from patients with chronic hepatitis B who were receiving peginterferon alfa-2b. The serum samples were subjected to albumin depletion and analyzed by two-dimensional gel electrophoresis (2-DE). Differentially expressed protein spots were identified by electrospray ionization-quadrupole time-of-flight mass spectrometry. Alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen were further analyzed by an enzyme-linked immunosorbent assay and immunonephelometry. RESULTS: Nineteen patients with HBeAg-positive chronic hepatitis B (CHB) were studied. These patients were followed for at least 1 year after treatment and were classified according to their treatment response: responders (n = 9) and non-responders (n = 10). 2-DE and MS/MS analysis were performed to compare the serum proteins before initiating peginterferon alfa-2b. From the quantitative analysis of the 2-D gel, 7 proteins were detected between the two groups at different levels before treatment. Among these potential candidates, serum levels of alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen-like precursor were further analyzed. In the validation phase, 23 subjects, 9 sustained responders and 14 non-responders, were recruited. Interestingly, the levels of alpha-2-HS-glycoprotein and complement component C3c were elevated in the serum of the non-responders compared to the responders. CONCLUSION: Serum alpha-2-HS-glycoprotein and complement component C3c may be potential serum biomarkers in predicting the treatment response of peginterferon alfa-2b in patients with CHB prior to treatment.

Objective Mycophenolic acid (MPA) has become the first-line drug therapy for proliferative lupus nephritis, a common and serious complication of systemic lupus erythematosus. Although a sufficient MPA exposure is required, a high interindividual variability in the pharmacokinetics of MPA has been observed. The knowledge of MPA pharmacokinetics in lupus nephritis patients is limited, especially in Asian patients. This study aimed to develop a population pharmacokinetic model for MPA and determine the population pharmacokinetic parameters and their interindividual variability in Thai patients with lupus nephritis. Methods A total of 112 MPA plasma concentrations from 14 adult lupus nephritis patients (International Society of Nephrology/Renal Pathology Society Class III/IV) receiving mycophenolate mofetil were included in this study. The data was analyzed using NONMEM. The model evaluation was performed by the bootstrap approach and visual predictive check. Results A two-compartment model with a lag time best described the data. The estimated mean apparent clearance (CL/F) was 14.5 l/h with an interindividual variability of 45.2%. The estimated mean CL/F was found to be lower than the values previously reported. The estimated mean apparent volume of the central compartment was 12.2 l with an interindividual variability of 166%. None of the covariates were found to significantly influence MPA pharmacokinetics. Conclusion In this study, a population pharmacokinetic model of MPA in severe lupus nephritis patients was successfully developed. The mean pharmacokinetic parameters were estimated and a high interindividual variability of MPA in this population was observed. This provides evidence to show that individualizing dosage regimens in this population is crucial. The model developed in this study could be used to obtain initial information for MPA dose adjustments in Thai and Asian patients with lupus nephritis. Further studies are required to validate the results and clarify the influence of covariates on MMF pharmacokinetics.

Although renal histopathology is the gold standard for the diagnosis and prognosis of lupus nephritis (LN), the invasiveness of renal biopsy warrants the discovery of novel non-invasive diagnostic and prognostic biomarkers. In the present study, urine samples from 10 LN patients (5 active and 5 inactive) were analyzed by two-dimensional gel electrophoresis (2-DE) to screen for potential biomarkers of active LN. Quantitative analysis and statistics revealed 16 protein spots whose levels significantly differed between groups. These proteins were successfully identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Among these potential candidates, differential levels of urinary Zn-α2-glycoprotein (ZA2G) and prostaglandin H(2)D-isomerase (PGDS) were further validated by enzyme-linked immunosorbent assay (ELISA) in another independent group of 78 subjects, including 30 active LN, 26 inactive LN, 14 non-LN glomerular diseases, and 8 healthy normal individuals. Whereas ZA2G levels were elevated in urine of patients with active LN and non-LN glomerular diseases, PGDS was elevated only in the urine of the active LN group. Urinary PGDS, not ZA2G, may serve as a biomarker for active LN and upon validation in larger studies, may become the non-invasive test to evaluate the disease activity in future management of LN.

The antioxidant activities of curcumin, its natural demethoxy derivatives (demethoxycurcumin, Dmc and bisdemethoxycurcumin, Bdmc) and metabolite hydrogenated derivatives (tetrahydrocurcumin, THC; hexahydrocurcumin, HHC; octahydrocurcumin; OHC) were comparatively studied using 2,2-diphenyl-1-picrylhydrazyl (DDPH) radical, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) induced linoleic oxidation and AAPH induced red blood cell hemolysis assays. Hydrogenated derivatives of curcumin exhibited stronger DPPH scavenging activity compared to curcumin and a reference antioxidant, trolox. The scavenging activity significantly decreased in the order THC>HHC=OHC>trolox>curcumin>Dmc>>>Bdmc. Stronger antioxidant activities toward lipid peroxidation and red blood cell hemolysis were also demonstrated in the hydrogenated derivatives. By the model of AAPH induced linoleic oxidation, the stoichiometric number of peroxyl radical that can be trapped per molecule (n) of hydrogenated derivatives were 3.4, 3.8 and 3.1 for THC, HHC and OHC, respectively. The number (n) of curcumin and Dmc were 2.7 and 2.0, respectively, which are comparable to trolox, while it was 1.4 for Bdmc. The inhibition of AAPH induced red blood cell hemolysis significantly decreased in the order OHC>THC=HHC>trolox>curcumin=Dmc. Results in all models demonstrated the lower antioxidant activity of the demethoxy derivatives, suggesting the ortho-methoxyphenolic groups of curcumin are involved in antioxidant activities. On the other hand, hydrogenation at conjugated double bonds of the central seven carbon chain and beta diketone of curcumin to THC, HHC and OHC remarkably enhance antioxidant activity.