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.

The nucleic acid binding protein YB-1-controlled expression of CXCL-1 modulates kidney damage in liver fibrosis.

Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1+/-) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1+/- mice. YB-1 was identified as a potent transcriptional repressor of the Cxcl1 gene. Precision-cut kidney slices from Yb1+/- mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1+/- mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression.

The YB-1:Notch-3 axis modulates immune cell responses and organ damage in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease and lupus nephritis is a major risk factor for morbidity and mortality. Notch-3 signaling induced by membrane-bound or soluble ligands such as YB-1 constitutes an evolutionarily conserved pathway that determines major decisions in cell fate. Mass spectrometry of extracellular YB-1 in sera from patients with SLE and lupus-prone mice revealed specific post-translational guanidinylation of two lysine residues within the highly conserved cold-shock domain of YB-1 (YB-1-G). These modifications highly correlated with SLE disease activity, especially in patients with lupus nephritis and resulted in enhanced activation of Notch-3 signaling in T lymphocytes. The importance of YB-1:Notch-3 interaction in T cells was further evidenced by increased interleukin (Il)10 expression following YB-1-G stimulation and detection of both, YB-1-G and Notch-3, in kidneys of MRL.lpr mice by mass spectrometry imaging. Notch-3 expression and activation was significantly up-regulated in kidneys of 20-week-old MRL.lpr mice. Notably, lupus-prone mice with constitutional Notch-3 depletion (B6.Faslpr/lprNotch3-/-) exhibited an aggravated lupus phenotype with significantly increased mortality, enlarged lymphoid organs and aggravated nephritis. Additionally, these mice displayed fewer regulatory T cells and reduced amounts of anti-inflammatory IL-10. Thus, our results indicate that the YB-1:Notch-3 axis exerts protective effects in SLE and that Notch-3 deficiency exacerbates the SLE phenotype.

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.

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.

Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension.

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

Complement C5a receptors C5L2 and C5aR in renal fibrosis.

Complement factor C5a has two known receptors, C5aR, which mediates proinflammatory effects, and C5L2, a potential C5a decoy receptor. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key proinflammatory mediators, however, significantly increased in C5L2- compared with C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T cells. Moreover, in C5L2-/- mice, the cytokine and matrix metalloproteinase-inhibitor tissue inhibitor of matrix metalloproteinase-1 was specifically enhanced. Consequently, in C5L2-/- mice the degree of renal fibrosis was similar to wild type (WT), albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR-/- mice had significantly reduced renal fibrosis compared with WT and C5L2-/- mice in UUO. In vitro experiments with primary tubular cells demonstrated that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury and fibrosis markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas the absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.

YB-1 increases glomerular, but decreases interstitial fibrosis in CNI-induced nephropathy.

Calcineurin inhibitors (CNIs) are a cornerstone of the current treatment in solid organ transplantation and autoimmune disease. However, CNIs also bear deleterious effects as they cause glomerular and tubulointerstitial fibrosis in the kidney. We recently identified Y-box protein-1 (YB-1) as a novel downstream effector of CNI-signaling in the cytoplasm of glomerular cells. In the present study, we corroborate the pro-fibrotic role of YB-1 in glomeruli of patients under CNI-treatment. Such effects in glomeruli are significantly mitigated in CNI-treated mice with half-normal YB-1 expression (Yb1+/-). Surprisingly, in the tubulointerstitium we observe an opposite role of the CNI-YB-1 axis. Here, YB-1 is predominantly located to the nuclei and represses transcription of several extracellular matrix genes. Consistently, CNI-treatment in Yb1+/- mice markedly increases pro-fibrotic changes in the tubulointerstitium. In summary, our data provide evidence that fibrotic CNI-induced YB-1 effects in glomerular cells need to be contrasted with beneficial anti-fibrotic effects in the tubulointerstitium.

YB-1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation.

The Y-box-binding protein (YB)-1 plays a non-redundant role in both systemic and local inflammatory response. We analysed YB-1-mediated expression of the immune regulatory cytokine IL-10 in both LPS and sterile inflammation induced by unilateral renal ischaemia-reperfusion (I/R) and found an important role of YB-1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis-regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB-1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB-1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL-10 mRNA expression in mice following LPS challenge and in I/R. Mice with half-maximal expression of YB-1 (Yb1+/- ) had diminished IL-10 expression upon LPS challenge. In I/R, Yb1+/- mice exhibited ameliorated kidney injury/inflammation in the early-phase (days 1 and 5), however showed aggravated long-term damage (day 21) with increased expression of IL-10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context-specific decisions concerning YB-1 function and that a fine-tuning of YB-1, for example, via a post-translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.

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.

Therapeutic nuclear shuttling of YB-1 reduces renal damage and fibrosis.

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. In vitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis in vivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

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.

Calcineurin-mediated YB-1 dephosphorylation regulates CCL5 expression during monocyte differentiation.

Y-box (YB) protein-1 serves as a master regulator in gene transcription and mRNA translation. YB-1 itself is regulated at various levels, e.g. through post-translational modifications. In our previous work, we identified RANTES/CCL5 as a transcriptional target of YB-1. We previously demonstrated that YB-1 protein is transiently up-regulated during monocyte/macrophage differentiation evidenced in monocytic cells (THP-1 cells) that were differentiated using phorbol myristate acetate (PMA). Here we provide evidence that YB-1 phosphorylation, specifically at its serine residue 102 (Ser-102), increases early on in THP-1 cells following PMA treatment as well as in differentiated primary human monocytes. This process is mediated through the Akt signaling pathway. Ser-102-phosphorylated YB-1 displays stronger binding affinity and trans-activating capacity at the CCL5 gene promoter. Notably, Ser-102-phosphorylated YB-1 disappears at later stages of the monocyte/macrophage differentiation process. We demonstrate that serine-threonine phosphatase calcineurin (CN) dephosphorylates YB-1 preventing it from binding to and trans-activating the CCL5 promoter. Co-immunoprecipitation assays prove a direct YB-1/CN interaction. Furthermore, analyses in kidney tissues from mice that were treated with the CN inhibitor cyclosporine A revealed an in vivo effect of CN on the YB-1 phosphorylation status. We conclude that YB-1 phosphorylation at Ser-102 is an important prerequisite for CCL5 promoter activation during macrophage differentiation. Our findings point to a critical role of YB-1 in the resolution of inflammatory processes which may largely be due to CN-mediated dephosphorylation.

YB-1 is an early and central mediator of bacterial and sterile inflammation in vivo.

In vitro studies identified Y-box-binding protein (YB)-1 as a key regulator of inflammatory mediators. In this study, we observed increased levels of secreted YB-1 in sera from sepsis patients. This led us to investigate the in vivo role of YB-1 in murine models of acute peritonitis following LPS injection, in sterile renal inflammation following unilateral ureteral obstruction, and in experimental pyelonephritis. LPS injection enhanced de novo secretion of YB-1 into the urine and the peritoneal fluid of LPS-treated mice. Furthermore, we could demonstrate a significant, transient upregulation and posttranslational modification (phosphorylation at serine 102) of YB-1 in renal and inflammatory cells. Increased renal cytoplasmic YB-1 amounts conferred enhanced expression of proinflammatory chemokines CCL2 and CCL5. Along these lines, heterozygous YB-1 knockout mice (YB-1(+/d)) that display 50% reduced YB-1 levels developed significantly lower responses to both LPS and sterile inflammation induced by unilateral ureteral obstruction. This included diminished immune cell numbers due to impaired migration propensities and reduced chemokine expression. YB-1(+/d) mice were protected from LPS-associated mortality (20% mortality on day 3 versus 80% in wild-type controls); however, immunosuppression in YB-1(+/d) animals resulted in 50% mortality. In conclusion, our findings identify YB-1 as a major, nonredundant mediator in both systemic and local inflammatory responses.

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.

Y-box binding protein-1 mediates profibrotic effects of calcineurin inhibitors in the kidney.

The immunosuppressive calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus are widely used in transplant organ recipients, but in the kidney allograft, they may cause tubulointerstitial as well as mesangial fibrosis, with TGF-ß believed to be a central inductor. In this study, we report that the cold-shock protein Y-box binding protein-1 (YB-1) is a TGF-ß independent downstream effector in CsA- as well as in tacrolimus- but not in rapamycin-mediated activation of rat mesangial cells (rMCs). Intracellular content of YB-1 is several-fold increased in MCs following CNI treatment in vitro and in vivo in mice. This effect ensues in a time-dependent manner, and the operative concentration range encompasses therapeutically relevant doses for CNIs. The effect of CNI on cellular YB-1 content is abrogated by specific blockade of translation, whereas retarding the transcription remains ineffective. The activation of rMCs by CNIs is accomplished by generation of reactive oxygen species. In contrast to TGF-ß-triggered reactive oxygen species generation, hydrogen peroxide especially could be identified as a potent inductor of YB-1 accumulation. In line with this, hindering TGF-ß did not influence CNI-induced YB-1 upregulation, whereas ERK/Akt pathways are involved in CNI-mediated YB-1 expression. CsA-induced YB-1 accumulation results in mRNA stabilization and subsequent generation of collagen. Our results provide strong evidence for a CNI-dependent induction of YB-1 in MCs that contributes to renal fibrosis via regulation of its own and collagen translation.

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.