Association Between High NK-Cell Count and Remission of Primary Membranous Nephropathy: A Retrospective Chart Review and Pilot Study.

PURPOSE: Primary membranous nephropathy (PMN) is the most frequent cause of nephrotic syndrome in adults. Rituximab monotherapy has emerged as a front-line treatment for patients with PMN, but potential markers for predicting the response to rituximab are unknown. METHODS: In this single-arm retrospective pilot study, 48 patients with PMN without previous immunosuppressive therapy were enrolled. All patients were treated with rituximab and were followed up for at least 6 months. The primary end point was the achievement of complete or partial remission at 6 months. The subsets of lymphocytes were collected at baseline, 1 month, 3 months and 6 months to identify prognostic factors for achieving remission of PMN with rituximab therapy. FINDINGS: A total of 58.3% of patients (28/48) achieved remission. Lower serum creatinine, greater serum albumin, and greater phospholipase A2 receptor antigen detected in kidney biopsy at baseline were found in the remission group. After multiple adjustments, a high percentage of natural killer (NK) cells at baseline, especially ≥15.7%, was strongly associated with remission (relative risk = 1.62; 95% CI, 1.00-2.62; P = 0.049), and patients with a response to rituximab had a greater mean percentage of NK cells during the follow-up period compared with nonresponders. Analysis using a receiver operating characteristic curve indicated prognostic value of the NK-cell percentage at baseline, with an area under the curve of 0.716 (95% CI, 0.556-0.876; P = 0.021). IMPLICATIONS: The findings from this retrospective pilot study suggest that a high percentage, especially ≥15.7%, of NK cells at baseline might predict a response to rituximab treatment. These findings provide a basis for designing larger-scale studies to test the predictive value of NK cells in patients with PMN undergoing rituximab treatment.

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects.

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4 ×109 cells/L; non-AKI: 10.1±2. 9 ×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.

Reevaluation of Lung Injury in TNF-Induced Shock: The Role of the Acid Sphingomyelinase.

Tumor necrosis factor (TNF) is a well-known mediator of sepsis. In many cases, sepsis results in multiple organ injury including the lung with acute respiratory distress syndrome (ARDS). More than 20-year-old studies have suggested that TNF may be directly responsible for organ injury during sepsis. However, these old studies are inconclusive, because they relied on human rather than conspecific TNF, which was contaminated with endotoxin in most studies. In this study, we characterized the direct effects of intravenous murine endotoxin-free TNF on cardiovascular functions and organ injury in mice with a particular focus on the lungs. Because of the relevance of the acid sphingomyelinase in sepsis, ARDS, and caspase-independent cell death, we also included acid sphingomyelinase-deficient (ASM-/-) mice. ASM-/- and wild-type (WT) mice received 50 µg endotoxin-free murine TNF intravenously alone or in combination with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD) and were ventilated at low tidal volume while lung mechanics were followed. Blood pressure was stabilized by intra-arterial fluid support, and body temperature was kept at 37°C to delay lethal shock and to allow investigation of blood gases, lung histopathology, proinflammatory mediators, and microvascular permeability 6 hours after TNF application. Besides the lungs, also the kidneys and liver were examined. TNF elicited the release of inflammatory mediators and a high mortality rate, but failed to injure the lungs, kidneys, or liver of healthy mice significantly within 6 hours. Mortality in WT mice was most likely due to sepsis-like shock, as indicated by metabolic acidosis, high procalcitonin levels, and cardiovascular failure. ASM-/- mice were protected from TNF-induced hypotension and reflex tachycardia and also from mortality. In WT mice, intravenous exogenous TNF does not cause organ injury but induces a systemic inflammatory response with cardiovascular failure, in which the ASM plays a role.

Cold shock protein YB-1 is involved in hypoxia-dependent gene transcription.

Hypoxia-dependent gene regulation is largely orchestrated by hypoxia-inducible factors (HIFs), which associate with defined nucleotide sequences of hypoxia-responsive elements (HREs). Comparison of the regulatory HRE within the 3' enhancer of the human erythropoietin (EPO) gene with known binding motifs for cold shock protein Y-box (YB) protein-1 yielded strong similarities within the Y-box element and 3' adjacent sequences. DNA binding assays confirmed YB-1 binding to both, single- and double-stranded HRE templates. Under hypoxia, we observed nuclear shuttling of YB-1 and co-immunoprecipitation assays demonstrated that YB-1 and HIF-1α physically interact with each other. Cellular YB-1 depletion using siRNA significantly induced hypoxia-dependent EPO production at both, promoter and mRNA level. Vice versa, overexpressed YB-1 significantly reduced EPO-HRE-dependent gene transcription, whereas this effect was minor under normoxia. HIF-1α overexpression induced hypoxia-dependent gene transcription through the same element and accordingly, co-expression with YB-1 reduced HIF-1α-mediated EPO induction under hypoxic conditions. Taken together, we identified YB-1 as a novel binding factor for HREs that participates in fine-tuning of the hypoxia transcriptome.

IL-6 Trans-Signaling Drives Murine Crescentic GN.

The role of IL-6 signaling in renal diseases remains controversial, with data describing both anti-inflammatory and proinflammatory effects. IL-6 can act via classic signaling, engaging its two membrane receptors gp130 and IL-6 receptor (IL-6R). Alternatively, IL-6 trans-signaling requires soluble IL-6R (sIL-6R) to act on IL-6R-negative cells that express gp130. Here, we characterize the role of both pathways in crescentic nephritis. Patients with crescentic nephritis had significantly elevated levels of IL-6 in both serum and urine. Similarly, nephrotoxic serum-induced nephritis (NTN) in BALB/c mice was associated with elevated serum IL-6 levels. Levels of serum sIL-6R and renal downstream signals of IL-6 (phosphorylated signal transducer and activator of transcription 3, suppressor of cytokine signaling 3) increased over time in this model. Simultaneous inhibition of both IL-6 signaling pathways using anti-IL-6 antibody did not have a significant impact on NTN severity. In contrast, specific inhibition of trans-signaling using recombinant sgp130Fc resulted in milder disease. Vice versa, specific activation of trans-signaling using a recombinant IL-6-sIL-6R fusion molecule (Hyper-IL-6) significantly aggravated NTN and led to increased systolic BP in NTN mice. This correlated with increased renal mRNA synthesis of the Th17 cell cytokine IL-17A and decreased synthesis of resistin-like alpha (RELMalpha)-encoding mRNA, a surrogate marker of lesion-mitigating M2 macrophage subtypes. Collectively, our data suggest a central role for IL-6 trans-signaling in crescentic nephritis and offer options for more effective and specific therapeutic interventions in the IL-6 system.

Gp130-dependent signaling in the podocyte.

Renal inflammation, in particular glomerular, is often characterized by increased IL-6 levels. The in vivo relevance of IL-6 signaling in glomerular podocytes, which play central roles in most glomerular diseases, is unknown. Here, we show that in normal mice, podocytes express gp130, the common signal-transducing receptor subunit of the IL-6 family of cytokines. Following systemic IL-6 or LPS injection in mice, podocyte IL-6 signaling was evidenced by downstream STAT3 phosphorylation. Next, we generated mice deficient for gp130 in podocytes. Expectedly, these mice exhibited abrogated IL-6 downstream signaling in podocytes. At the age of 40 wk, they did not show spontaneous renal pathology or abnormal renal function. The mice were then challenged using two LPS injury models as well as nephrotoxic serum to induce crescentic nephritis. Under all conditions, circulating IL-6 levels increased markedly and the mice developed the pathological hallmarks of the corresponding injury models such as proteinuria and development of glomerular crescents, respectively. However, despite the capacity of normal podocytes to transduce IL-6 family signals downstream, there were no significant differences between mice bearing the podocyte-specific gp130 deletion and their control littermates in any of these models. In conclusion, under the different conditions tested, gp130 signaling was not a critical component of the (patho-)biology of the podocyte in vivo.

High prevalence of Y-box protein-1/p18 fragment in plasma of patients with malignancies of different origin.

BACKGROUND: Expression of the cold shock protein Y-box protein 1 (YB-1) is associated with deleterious outcome in various malignant diseases. Our group recently showed that the detection of an 18 kDa YB-1 fragment (YB-1/p18) in human plasma identifies patients with malignant diseases. We now tested the prevalence, clinical, and diagnostic value of YB-1/p18 detection in common tumors. METHODS: A newly established monoclonal YB-1 antibody was used to detect YB-1/p18 by immunoblotting in plasma samples from 151 unselected tumor patients, alongside established tumor markers and various diagnostic measures, during evaluation for a cancerous disease and in follow-up studies after therapeutic interventions. RESULTS: Circulating YB-1/p18 was detected in 78% of patients having a tumor disease. YB-1/p18 positivity was highly prevalent in all examined malignancies, including lung cancer (32/37; 87%), breast cancer (7/10; 70%), cancer of unknown primary (CUP; 5/5, 100%) or hematological malignancies (42/62; 68%). Positivity for YB-1/p18 was independent of other routine laboratory parameters, tumor stage, or histology. In comparison to 13 established tumor markers (cancer antigens 15-3, 19-9, 72-4, and 125; carcinoembryonic antigen; cytokeratin fragments 21-1; neuron-specific enolase; alpha-fetoprotein; beta-2-microglobulin; squamous cell carcinoma antigen; thymidine kinase; tissue polypeptide antigen; pro-gastrin-releasing peptide), YB-1/p18 detection within serum samples was the most sensitive general parameter identifying malignant disorders. YB-1/p18 concentrations altered during therapeutic interventions, but did not predict prognosis. CONCLUSIONS: Plasma YB-1/p18 detection has a high specific prevalence in malignancies, thereby providing a novel tool for cancer screening independent of the tumor origin.

Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities.

BACKGROUND: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s). RESULTS: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter. CONCLUSION: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

Notch-3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury.

Kidney diseases impart a vast burden on affected individuals and the overall health care system. Progressive loss of renal parenchymal cells and functional decline following injury are often observed. Notch-1 and -2 receptors are crucially involved in nephron development and contribute to inflammatory kidney diseases. We specifically determined the participation of receptor Notch-3 following tubulointerstitial injury and in inflammatory responses. Here we show by heat map analyses that Notch-3 transcripts are up-regulated in human kidney diseases. A similar response was corroborated with kidney cells following TGF-ß exposure in vitro. The murine unilateral ureteral obstruction (UUO) model mirrors hallmarks of tubulointerstitial injury and damage. A subset of tubular and interstitial cells demonstrated up-regulated Notch-3 receptor expression in diseased animals. We hypothesized a relevance of Notch-3 receptors for the chemotactic response. To address this question, animals with genetic ablation of receptor Notch-3 were analysed following UUO. As a result, we found that Notch-3-deficient animals are protected from tubular injury and cell loss with significantly reduced interstitial collagen deposition. Monocytic cell infiltration was significantly reduced and retarded, likely due to abrogated chemokine synthesis. A cell model was set up that mimics enhanced receptor Notch-3 expression and activation. Here a pro-mitogenic response was seen with activated signalling in tubular cells and fibroblasts. In conclusion, Notch-3 receptor fulfils non-redundant roles in the inflamed kidney that may not be replaced by other Notch receptor family members. Thus, specific blockade of this receptor may be suitable as therapeutic option to delay progression of kidney disease.

Cold shock Y-box protein-1 participates in signaling circuits with auto-regulatory activities.

The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-ß, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways.

Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1.

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at -28/-10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.

Y-box binding protein-1 controls CC chemokine ligand-5 (CCL5) expression in smooth muscle cells and contributes to neointima formation in atherosclerosis-prone mice.

BACKGROUND: The CC chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) is upregulated in mononuclear cells or deposited by activated platelets during inflammation and has been implicated in atherosclerosis and neointimal hyperplasia. We investigated the influence of the transcriptional regulator Y-box binding protein (YB)-1 on CCL5 expression and wire-induced neointimal hyperplasia. METHODS AND RESULTS: Analysis of the CCL5 promoter revealed potential binding sites for YB-1, and interaction of YB-1 with a sequence at position -204/-173 was confirmed by DNA binding assays. Both YB-1 expression and CC chemokine ligand-5 (CCL5) mRNA expression were increased in neointimal versus medial smooth muscle cells, as analyzed by real-time polymerase chain reaction. Overexpression of YB-1 in smooth muscle cells (but not macrophages) enhanced CCL5 transcriptional activity in reporter assays, mRNA and protein expression, and CCL5-mediated monocyte arrest. Carotid arteries of hyperlipidemic apolipoprotein E-deficient mice were subjected to intraluminal transfection with a lentivirus encoding YB-1 short hairpin RNA or empty vector directly after wire injury. Double immunofluorescence revealed YB-1 expression in neointimal smooth muscle cells but not macrophages and colocalization with neointimal CCL5, which was downregulated by YB-1 short hairpin RNA. Neointima formation was decreased significantly after YB-1 knockdown compared with controls and was associated with a diminished content of lesional macrophages. A reduction of lesion formation by YB-1 knockdown was not observed in apolipoprotein E-deficient mice deficient in the CCL5 receptor CCR5 or after treatment with the CCL5 antagonist Met-RANTES, which indicates that YB-1 effects were dependent on CCL5. CONCLUSIONS: The transcriptional regulator YB-1 mediates CCL5 expression in smooth muscle cells and thereby contributes to neointimal hyperplasia, thus representing a novel target with which to limit vascular remodeling.

Mesangial cell expression of proto-oncogene Ets-1 during progression of mesangioproliferative glomerulonephritis.

BACKGROUND: Ets-1 is a transactivator of matrix-associated genes and key factor in neoangiogenesis. The expression of Ets-1 mRNA and protein was analyzed in healthy rat kidney and in a model for mesangioproliferative disease without and with inhibition of platelet-derived growth factor-B (PDGF-B) activity. METHODS: Immunohistochemistry was performed using a specific noncrossreacting anti-Ets-1 antibody and included a counterstain with alpha-smooth muscle actin (alpha-SMA). Nuclear proteins from isolated glomeruli were analyzed by Western blotting. Changes in Ets-1 mRNA levels were detected by in situ hybridization and Northern blotting. PDGF-B antagonism was performed in nephritic rats by specific aptamers. RESULTS: The distribution of Ets-1-positive cells in healthy rats was heterogenous with exclusively nuclear staining of glomerular, tubular, and vascular cells. Profound changes were detected in the anti-Thy 1.1 glomerulonephritis. Nuclear Ets-1 staining was intense in mesangial cells, whereas podocyte and endothelial cell staining was unchanged. The strongest signal was seen on days 2 to 7. By Western blotting of glomerular proteins a single 52 kD band was detected in healthy rats, which was increased 4.5-fold after disease induction. At the same time a 54 kD band appeared that most likely represents phosphorylated Ets-1. Ets-1 transcripts were detected in mesangial cells that include exon IV but lack exon VII. A concordant 6.4-fold up-regulation of mRNA was detected in glomeruli. Specific PDGF-B antagonism by aptamer treatment from days 3 to 7 after disease induction led to reduced Ets-1 expression on day 7, correlating with decreased mesangial cell numbers. CONCLUSION: A temporal increase of mesangial cell Ets-1 expression correlates with mesangial cell activation in mesangioproliferative glomerulonephritis. PDGF-B may partially contribute to the increased expression.

Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection.

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.