Stage lighting on PAR-1: a step further in the understanding of acquired focal and segmental glomerulosclerosis.

The pathogenic mechanisms of acquired focal and segmental glomerular sclerosis are only partially known and represent a medical challenge in nephrology. The article by May et al. sheds additional light on previous data indicating the key role of the protease-activated receptor 1. The new evidence is based on in vivo studies in relevant animal models and on patient biopsies and represents a significant step forward in the understanding of this pathologic condition.

Proteomics of Plasma and Plasma-Treated Podocytes: Application to Focal and Segmental Glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid-binding proteins, generally associated with lipoproteins, was found to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.

Circulating plasmablasts and high level of BAFF are hallmarks of minimal change nephrotic syndrome in adults.

BACKGROUND: The recent success achieved with the use of B cell-depleting agents in some patients with minimal change nephrotic syndrome (MCNS) suggests an unexpected role for B lymphocytes in the pathogenesis of this immune-mediated glomerular disease. Nevertheless, no extensive B-cell phenotyping analysis has ever been performed in untreated adult patients soon after MCNS diagnosis. METHODS: We investigated the distribution of the different B-cell subpopulations in 22 untreated adult patients with biopsy-proven MCNS [MCNS relapse (MCNS-Rel)]. We compared these data with those for 24 healthy controls, 13 MCNS patients in remission (with no specific treatment) and 19 patients with idiopathic membranous nephropathy (IMN). RESULTS: Patients with MCNS-Rel or IMN had higher proteinuria and lower serum albumin and gammaglobulin levels (P < 0.0001 for all comparisons) than MCNS patients in remission. Plasmablasts were the only B-cell subsets present at significantly higher levels in MCNS-Rel patients than in the patients of the other three groups (P < 0.05 for all comparisons). The lower albumin levels and higher proteinuria levels were positively correlated with the percentage of circulating plasmablasts (Spearman test's ρ = -0.54, P = 0.01 and ρ = 0.65, P = 0.002, respectively). Similarly, the increase of immunoglobulin M (IgM) and the decrease of IgG levels were significantly associated with the percentage of plasmablasts in MCNS-Rel patients (Spearman's ρ = 0.36, P = 0.01 and Spearman's ρ = -0.60, P = 0.01, respectively). Increased production of interleukin (IL)-21, IL-6 and B-cell activating factor (BAFF) in the serum of MCNS-Rel patients was found significantly correlated with the percentage of plasmablasts (ρ = 0.72, P = 0.0002, ρ = 0.49, P = 0.04 and ρ = 0.62, P = 0.009, respectively). CONCLUSIONS: An increase in the proportion of circulating plasmablasts seems to be a hallmark of untreated MCNS in adult patients. Further studies are required to more precisely determine the phenotype and functions of these cells.

A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome.

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

Gangliosides in Podocyte Biology and Disease.

Gangliosides constitute a subgroup of glycosphingolipids characterized by the presence of sialic acid residues in their structure. As constituents of cellular membranes, in particular of raft microdomains, they exert multiple functions, some of them capital in cell homeostasis. Their presence in cells is tightly regulated by a balanced expression and function of the enzymes responsible for their biosynthesis, ganglioside synthases, and their degradation, glycosidases. The dysregulation of their abundance results in rare and common diseases. In this review, we make a point on the relevance of gangliosides and some of their metabolic precursors, such as ceramides, in the function of podocytes, the main cellular component of the glomerular filtration barrier, as well as their implications in podocytopathies. The results presented in this review suggest the pertinence of clinical lipidomic studies targeting these metabolites.

Lipidomics Conquers a Niche, Consolidates Growth.

Sixteen years after the first published article in which the term "lipidomics" was stated [...].

Kidney Lipidomics by Mass Spectrometry Imaging: A Focus on the Glomerulus.

Lipid disorders have been associated with glomerulopathies, a distinct type of renal pathologies, such as nephrotic syndrome. Global analyses targeting kidney lipids in this pathophysiologic context have been extensively performed, but most often regardless of the architectural and functional complexity of the kidney. The new developments in mass spectrometry imaging technologies have opened a promising field in localized lipidomic studies focused on this organ. In this article, we revisit the main works having employed the Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) technology, and the few reports on the use of TOF-Secondary Ion Mass Spectrometry (TOF-SIMS). We also present a first analysis of mouse kidney cortex sections by cluster TOF-SIMS. The latter represents a good option for high resolution lipid imaging when frozen unfixed histological samples are available. The advantages and drawbacks of this developing field are discussed.

NEPHRUTIX: A randomized, double-blind, placebo vs Rituximab-controlled trial assessing T-cell subset changes in Minimal Change Nephrotic Syndrome.

Minimal-change nephrotic syndrome (MCNS) is an immune-mediated glomerular disease. We have analyzed the modifications on T-cell subsets in twenty-three patients who were highly steroid/calcineurin inhibitor and/or mycophenolate mofetil-dependent for frequently relapsing nephrotic syndrome (FRNS) and who were enrolled in a multicenter, double-blind, randomized, placebo vs Rituximab-controlled trial. Patients with FRNS entered the trial at remission and were randomly assigned to receive either Rituximab or placebo. In both groups, patient blood samples were analyzed at inclusion and then monthly until six months post-perfusion. Disclosure of patient's allocation code occurred in relapse or at the end of the trial. All patients under placebo displaying relapse were subsequently treated with Rituximab. Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group, except one (n = 9/10). On the other hand, relapses occurred within a few weeks (means ≈ 7.3 weeks) in all patients receiving placebo (n = 13). At inclusion, before rituximab therapy, the frequency of different T-cell subsets were highly similar in both groups, except for CD8+ and invariant TCRVα24 T-cell subsets, which were significantly increased in patients of the Placebo group ((p = 0,0414 and p = 0.0428, respectively). Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group (n = 10), except one. Relapses were associated with a significant decrease in CD4+CD25highFoxP3high Tregulatory cells (p = 0.0005) and IL2 expression (p = 0.0032), while CMIP abundance was significantly increased (p = 0.03). Remissions after Rituximab therapy were associated in both groups with significant decrease in the frequency of CD4+CD45RO+CXCR5+, invariant natural killer T-cells (INKT) and CD4-CD8- (double-negative, DN) T-cells expressing the invariant Vα24 chain (DN-TCR Vα24) T-cells, suggesting that MCNS involves a disorder of innate and adaptive immune response, which can be stabilized by Rituximab treatment.

Repression of CMIP transcription by WT1 is relevant to podocyte health.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

A new workflow for proteomic analysis of urinary exosomes and assessment in cystinuria patients.

Cystinuria is a purely renal, rare genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases, patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow-up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible, and noninvasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study conducted on eight cystinuria patients versus 10 controls highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins, and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking the potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinary exosomes in different renal diseases and to search for diagnostic/prognostic markers. Data are available via ProteomeXchange with identifier PXD001430.

Inhibition of the VEGF signalling pathway and glomerular disorders.

Anti-cancer therapeutic approaches targeting the vascular endothelial growth factor (VEGF) ligand (anti-VEGF) or inhibiting its receptors (RTKI) have recently been developed. In spite of the promising results achieved, a serious drawback and dose-limiting side effect is the development, among others, of renal complications. This encompasses two glomerular pathological entities, namely minimal change/focal segmental glomerulosclerosis and thrombotic micro-angiopathy, involving two distinct cell types, podocytes and endothelial cells, respectively. The mechanisms that link anti-cancer therapy by RTKI to podocyte dysfunction and nephrotic level proteinuria are still poorly understood. Nevertheless, recent findings strongly suggest a central role of RelA, the master subunit of NF-κB and c-mip, an active player in podocyte disorders. RelA, which is up-regulated following anti-VEGF therapy, is inactivated by RTKI, leading to c-mip over-expression in the podocyte. This results in severe alterations in the architecture of podocyte actin cytoskeleton and subsequent severe proteinuria. Hence, clarifying the mechanisms linking c-mip and RelA as key pathogenic factors represents a critical goal in the understanding of different glomerulopathies. In the context of VEGF-targeted anti-cancer therapy, the study of these mechanisms along with the molecular cross-talk between podocyte and endothelial cell constitutes the basis for the emerging field of onconephrology.

Lipidomics by Supercritical Fluid Chromatography.

This review enlightens the role of supercritical fluid chromatography (SFC) in the field of lipid analysis. SFC has been popular in the late 1980s and 1990s before almost disappearing due to the commercial success of liquid chromatography (LC). It is only 20 years later that a regain of interest appeared when new commercial instruments were introduced. As SFC is fully compatible with the injection of extracts in pure organic solvent, this technique is perfectly suitable for lipid analysis and can be coupled with either highly universal (UV or evaporative light scattering) or highly specific (mass spectrometry) detection methods. A short history of the use of supercritical fluids as mobile phase for the separation oflipids will be introduced first. Then, the advantages and drawbacks of SFC are discussed for each class of lipids (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, polyketides) defined by the LIPID MAPS consortium.

Expression patterns of RelA and c-mip are associated with different glomerular diseases following anti-VEGF therapy.

Renal toxicity constitutes a dose-limiting side effect of anticancer therapies targeting vascular endothelial growth factor (VEGF). In order to study this further, we followed up 29 patients receiving this treatment, who experienced proteinuria, hypertension, and/or renal insufficiency. Eight developed minimal change nephropathy/focal segmental glomerulopathy (MCN/FSG)-like lesions and 13 developed thrombotic microangiopathy (TMA). Patients receiving receptor tyrosine kinase inhibitors (RTKIs) mainly developed MCN/FSG-like lesions, whereas TMA complicated anti-VEGF therapy. There were no mutations in factor H, factor I, or membrane cofactor protein of the complement alternative pathway, while plasma ADAMTS13 activity persisted and anti-ADAMTS13 antibodies were undetectable in patients with TMA. Glomerular VEGF expression was undetectable in TMA and decreased in MCN/FSG. Glomeruli from patients with TMA displayed a high abundance of RelA in endothelial cells and in the podocyte nuclei, but c-mip was not detected. Conversely, MCN/FSG-like lesions exhibited a high abundance of c-mip, whereas RelA was scarcely detected. RelA binds in vivo to the c-mip promoter and prevents its transcriptional activation, whereas RelA knockdown releases c-mip activation. The RTKI sorafenib inhibited RelA activity, which then promoted c-mip expression. Thus, our results suggest that c-mip and RelA define two distinct types of renal damage associated with VEGF-targeted therapies.

Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect.

We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.

Upregulation of c-mip is closely related to podocyte dysfunction in membranous nephropathy.

Membranous nephropathy is a glomerular disease typified by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathology involves the generation of autoantibodies against antigen targets on the surface of podocytes. The mechanisms of nephrotic proteinuria, which reflect a profound podocyte dysfunction, remain unclear. We previously found a new gene, c-mip (c-maf-inducing protein), that was associated with the pathophysiology of idiopathic nephrotic syndrome. Here we found that c-mip was not detected in the glomeruli of rats with passive-type Heymann nephritis given a single dose of anti-megalin polyclonal antibody, yet immune complexes were readily present, but without triggering of proteinuria. Rats reinjected with anti-megalin develop heavy proteinuria a few days later, concomitant with c-mip overproduction in podocytes. This overexpression was associated with the downregulation of synaptopodin in patients with membranous nephropathy, rats with passive Heymann nephritis, and c-mip transgenic mice, while the abundance of death-associated protein kinase and integrin-linked kinase was increased. Cyclosporine treatment significantly reduced proteinuria in rats with passive Heymann nephritis, concomitant with downregulation of c-mip in podocytes. Thus, c-mip has an active role in the podocyte disorders of membranous nephropathy.

GSH monoethyl ester rescues mitochondrial defects in cystic fibrosis models.

Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response. mtGSH depletion was confirmed in colonic epithelium of CFTR-null mice and in CFTR-mutated human epithelial cells. GSH uptake experiments performed on isolated mitochondria suggest that mtGSH depletion is not due to a defective GSH transport capacity by CF mitochondria, despite the decreased expression of two mtGSH carriers, oxoglutarate carrier and dicarboxylate carrier. CM-H(2)DCFDA [5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] fluorescence and aconitase activity showed an increase in reactive oxygen species levels in CFTR-defective cells and a pro-oxidative environment within CF mitochondria. The activities of respiratory chain complexes were further examined. Results showed a selective loss of Complex I (CI) function in CF models associated with an altered mitochondrial membrane potential (Δψ(m)). CI analysis showed normal expression but an overoxidation of its NADH-ubiquinone oxidoreductase Fe-S protein 1 subunit. GSH monoethyl ester (GSH-EE) significantly enhanced mtGSH levels in the IB3-1/C38 model and reversed CI inhibition, suggesting that mtGSH depletion is responsible for the loss of CI activity. Furthermore, GSH-EE attenuated Δψ(m) depolarization and restored normal IL-8 secretion by CFTR-defective cells. These studies provide evidence for a critical role of a mtGSH defect in mitochondrial dysfunction and abnormal IL-8 secretion in CF cells and reveal the therapeutic potential of mitochondria-targeted antioxidants in CF.

9-O Acetylated Gangliosides in Health and Disease.

Glycosphingolipids comprise a lipid class characterized by the presence of sugar moieties attached to a ceramide backbone. The role of glycosphingolipids in pathophysiology has gained relevance in recent years in parallel with the development of analytical technologies. Within this vast family of molecules, gangliosides modified by acetylation represent a minority. Described for the first time in the 1980s, their relation to pathologies has resulted in increased interest in their function in normal and diseased cells. This review presents the state of the art on 9-O acetylated gangliosides and their link to cellular disorders.

Pharmacology of Tyrosine Kinase Inhibitors: Implications for Patients with Kidney Diseases.

Tyrosine kinase inhibitors (TKI) have introduced a significant advancement in cancer management. These compounds are administered orally, and their absorption holds a pivotal role in determining their variable efficacy. They exhibit extensive distribution within the body, binding strongly to both plasma and tissue proteins. Often reliant on efflux and influx transporters, TKI undergo primary metabolism by intestinal and hepatic cytochrome P450 enzymes, with nonkidney clearance being predominant. Owing to their limited therapeutic window, many TKI display considerable intraindividual and interindividual variability. This review offers a comprehensive analysis of the clinical pharmacokinetics of TKI, detailing their interactions with drug transporters and metabolic enzymes, while discussing potential clinical implications. The prevalence of kidney conditions, such as AKI and CKD, among patients with cancer is explored in their effect on TKI pharmacokinetics. Finally, the potential nephrotoxicity associated with TKI is also examined.

A role for 12R-lipoxygenase in MUC5AC expression by respiratory epithelial cells.

Eicosanoids are metabolites of arachidonic acid produced by cyclooxygenases (COXs) or lipoxygenases (LOXs). They mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases. The gel-forming mucin MUC5AC is over-expressed in the airways of patients with these diseases. MUC5AC expression is mediated by an extracellular signal-regulated kinase (ERK)/Sp1 dependent mechanism. Our aim was to study the role of eicosanoids and their signalling pathways in MUC5AC expression. Inhibitors of 12-LOX, but not those of COX, 5-LOX or 15-LOX, reduce MUC5AC expression induced by phorbol myristate acetate (PMA) in the bronchial epithelial cell line NCI-H292. These inhibitors also abrogate the production of whole mucus by cell monolayers. Two forms of 12-LOX (R and S) exist in mammals. Using siRNAs we show that 12R-LOX but not 12S-LOX is involved in MUC5AC expression induced by PMA, lipopolysaccharide or transforming growth factor-α. 12R-LOX also participates in MUC2 and MUC5B expression, although to a lesser extent than for MUC5AC. Contrarily, 12R-LOX silencing does not modify interleukin-8 production. 12-LOX inhibitors reduce ERK activation and Sp1 translocation induced by PMA. Moreover, the 12R-LOX product 12(R)-hydroxyeicosatetraenoic acid, induces MUC5AC expression, ERK activation and Sp1 translocation. 12R-LOX is involved in MUC5AC expression. This occurs via ERK- and Sp1-signalling pathways.

Plasma lipidomics reveals potential prognostic signatures within a cohort of cystic fibrosis patients.

Cystic fibrosis (CF) is associated with abnormal lipid metabolism. We have recently shown variations in plasma levels of several phosphatidylcholine (PC) and lysophopshatidylcholine (LPC) species related to disease severity in CF patients. Here our goal was to search for blood plasma lipid signatures characteristic of CF patients bearing the same mutation (F508del) and different phenotypes, and to study their correlation with forced expiratory volume in 1 s (FEV1) and Pseudomonas aeruginosa chronic infection, evaluated at the time of testing (t = 0) and three years later (t = 3). Samples from 44 F508del homozygotes were subjected to a lipidomic approach based on LC-ESI-MS. Twelve free fatty acids were positively correlated with FEV1 at t = 0 (n = 29). Four of them (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) were also positively correlated with FEV1 three years later, along with PC(32:2) and PC(36:4) (n = 31). Oleoylethanolamide (OEA) was negatively correlated with FEV1 progression (n = 17). Chronically infected patients at t = 0 showed lower PC(32:2), PC(38:5), and C18:3n-3 and higher cholesterol, cholesterol esters, and triacylglycerols (TAG). Chronically infected patients at t = 3 showed significantly lower levels of LPC(18:0). These results suggest a potential prognostic value for some lipid signatures in, to our knowledge, the first longitudinal study aimed at identifying lipid biomarkers for CF.