The secretome analysis of activated human renal fibroblasts revealed beneficial effect of the modulation of the secreted peptidyl-prolyl cis-trans isomerase A in kidney fibrosis

The secretome is an important mediator in the permanent process of reciprocity between cells and their environment. Components of secretome are involved in a large number of physiological mechanisms including differentiation, migration, and extracellular matrix modulation. Alteration in secretome composition may therefore trigger cell transformation, inflammation, and diseases. In the kidney, aberrant protein secretion plays a central role in cell activation and transition and in promoting renal fibrosis onset and progression. Using comparative proteomic analyses, we investigated in the present study the impact of cell transition on renal fibroblast cells secretome. Human renal cell lines were stimulated with profibrotic hormones and cytokines, and alterations in secretome were investigated using proteomic approaches. We identified protein signatures specific for the fibrotic phenotype and investigated the impact of modeling secretome proteins on extra cellular matrix accumulation. The secretion of peptidyl-prolyl cis-trans isomerase A (PPIA) was demonstrated to be associated with fibrosis phenotype. We showed that the in-vitro inhibition of PPIA with ciclosporin A (CsA) resulted in downregulation of PPIA and fibronectin (FN1) expression and significantly reduced their secretion. Knockdown studies of PPIA in a three-dimensional (3D) cell culture model significantly impaired the secretion and accumulation of the extracellular matrix (ECM), suggesting a positive therapeutic effect on renal fibrosis progression

Proteomic analysis of embryonic kidney development: Heterochromatin proteins as epigenetic regulators of nephrogenesis

Elucidation of the mechanisms underlying the nephrogenesis will boost enormously the regenerative medicine. Here we performed 2-D gel-based comparative proteome analyses of rat embryonic kidney from different developmental stages. Out of 288 non-redundant identified proteins, 102 were common in all developmental stages. 86% of the proteins found in E14 and E16 were identical, in contrast only 37% of the identified proteins overlap between E14 and P1. Bioinformatics analysis suggests developmental stage-specific pathway activation and highlighted heterochromatin protein 1 (Cbx1, Cbx3, Cbx5) and Trim28 as potential key players in nephrogenesis. These are involved in the epigenetic regulation of gene silencing and were down-regulated in the course of kidney development. Trim28 is a potential epigenetic regulator of the branching inhibitor Bmp4. Silencing of Trim28 in cultured kidneys resulted in branching arrest. In contrast knockdown of Cbx5 was associated with abnormal ureteric bud growth and slight impairment of branching. ChIP analysis showed that the H3K9me3 distribution on Bmp4 promoters at E14 and E19 inversely correlate with mRNA expression levels. The concentrated expression-pattern of heterochromatin proteins and the negative impact of their silencing on kidney development, suggest an important role in reciprocal and inductive signaling between the ureteric bud and the metanephric mesenchyme

Urine E-cadherin: A Marker for early detection of kidney injury in diabetic patients.

Diabetic nephropathy (DN) is the main reason for end-stage renal disease. Microalbuminuria as the non-invasive available diagnosis marker lacks specificity and gives high false positive rates. To identify and validate biomarkers for DN, we used in the present study urine samples from four patient groups: diabetes without nephropathy, diabetes with microalbuminuria, diabetes with macroalbuminuria and proteinuria without diabetes. For the longitudinal validation, we recruited 563 diabetic patients and collected 1363 urine samples with the clinical data during a follow-up of 6 years. Comparative urinary proteomics identified four proteins Apolipoprotein A-I (APOA1), Beta-2-microglobulin (B2M), E-cadherin (CDH1) and Lithostathine-1-alpha (REG1A), which differentiated with high statistical strength (p < 0.05) between DN patients and the other groups. Label-free mass spectrometric quantification of the candidates confirmed the discriminatory value of E-cadherin and Lithostathine-1-alpha (p < 0.05). Immunological validation highlighted E-cadherin as the only marker able to differentiate significantly between the different DN stages with an area under the curve (AUC) of 0.85 (95%-CI: [0.72, 0.97]). The analysis of the samples from the longitudinal study confirmed the prognostic value of E-cadherin, the critical increase in urinary E-cadherin level was measured 20 ± 12.5 months before the onset of microalbuminuria and correlated significantly (p < 0.05) with the glomerular filtration rate measured by estimated glomerular filtration rate (eGFR)

Impact of the antiproliferative agent ciclopirox olamine treatment on stem cell proteome

AIM: To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. METHODS: Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. RESULTS: Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells. CONCLUSION: The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency

Serum peptide reactivities may distinguish neuromyelitis optica subgroups and multiple sclerosis

Objective: To assess in an observational study whether serum peptide antibody reactivities may distinguish aquaporin-4 (AQP4) antibody (Ab)–positive and -negative neuromyelitis optica spectrum disorders (NMOSD) and relapsing-remitting multiple sclerosis (RRMS). Methods: We screened 8,700 peptides that included human and viral antigens of potential relevance for inflammatory demyelinating diseases and random peptides with pooled sera from different patient groups and healthy controls to set up a customized microarray with 700 peptides. With this microarray, we tested sera from 66 patients with AQP4-Ab-positive (n = 16) and AQP4-Ab-negative (n = 19) NMOSD, RRMS (n = 11), and healthy controls (n = 20). Results: Differential peptide reactivities distinguished NMOSD subgroups from RRMS in 80% of patients. However, the 2 NMOSD subgroups were not well-discriminated, although those patients are clearly separated by their antibody reactivities against AQP4 in cell-based assays. Elevated reactivities to myelin and Epstein-Barr virus peptides were present in RRMS and to AQP4 and AQP1 peptides in AQP4-Ab-positive NMOSD. Conclusions: While AQP4-Ab-positive and -negative NMOSD subgroups are not well-discriminated by peptide antibody reactivities, our findings suggest that peptide antibody reactivities may have the potential to distinguish between both NMOSD subgroups and MS. Future studies should thus concentrate on evaluating peptide antibody reactivities for the differentiation of AQP4-Ab-negative NMOSD and MS

Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-β–mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

Proteomics approaches to fibrotic disorders

This review provides an introduction to mass spectrometry based proteomics and discusses several proteomics approaches that are relevant in understanding the pathophysiology of fibrotic disorders and the approaches that are frequently used in biomarker discovery