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Simple Summary Cold shock Y-box binding protein-1 plays a crucial role in cancerous cell transformation and proliferation. Experimental evidence links autoantibody formation with cancer diseases as well as YB 1 protein levels. Hence, we investigated autoantibody formation targeting YB-1 in cancer patients. Using recombinant proteins and specific peptide arrays, we mapped linear epitopes, which localize in the cold shock and C-terminal domain of the protein, in cancer patients that differ from healthy controls. Furthermore, cancer sera containing autoantibodies that target YB-1 extend the half-life of the YB-1 protein. Since extracellular YB-1 serves as a ligand for receptor Notch3 as well as TNFR1, this may contribute to aberrant signaling that promotes tumor development. In the clinical setting, we envision setting up detection assays for the immune response against YB-1, which may aid in screening for cancer. Abstract Cold shock Y-box binding protein-1 participates in cancer cell transformation and mediates invasive cell growth. It is unknown whether an autoimmune response against cancerous human YB-1 with posttranslational protein modifications or processing develops. We performed a systematic analysis for autoantibody formation directed against conformational and linear epitopes within the protein. Full-length and truncated recombinant proteins from prokaryotic and eukaryotic cells were generated. Characterization revealed a pattern of spontaneous protein cleavage, predominantly with the prokaryotic protein. Autoantibodies against prokaryotic, but not eukaryotic full-length and cleaved human YB-1 protein fragments were detected in both, healthy volunteers and cancer patients. A mapping of immunogenic epitopes performed with truncated E. coli-derived GST-hYB-1 proteins yielded distinct residues in the protein N- and C-terminus. A peptide array with consecutive overlapping 15mers revealed six distinct antigenic regions in cancer patients, however to a lesser extent in healthy controls. Finally, a protein cleavage assay was set up with recombinant pro- and eukaryotic-derived tagged hYB-1 proteins. A distinct cleavage pattern developed, that is retarded by sera from cancer patients. Taken together, a specific autoimmune response against hYB-1 protein develops in cancer patients with autoantibodies targeting linear epitopes.

Y-box binding protein 1 (YB-1) is pivotal for the regulation of cancerogenesis and inflammation. However, its involvement in pregnancy processes such as fetal and placental development remains to be elucidated. We studied Ybx1 (YB-1)+/&minus, heterozygous intercrossings and compared them to YB-1+/+ wild-type (WT) combinations. Additionally, we generated trophoblast-specific YB-1-deficient mice by pairing FVB Cyp19-Cre females to YB-1fl/fl males. YB-1fl/fl-paired FVB WT females served as controls. Serial in vivo ultrasound measurements were performed to assess fetal and placental parameters. After sacrificing the females, implantation and abortion rates were recorded, spiral artery (SA) remodeling was analyzed and fetal and placental weights were determined. Compared to YB-1+/+ counterparts, YB-1+/&minus, females showed reduced implantation areas at gestation day (GD)10, insufficiently remodeled SAs at GD12, increased placental diameter/thickness ratios at GD14 and reduced placental and fetal weights at GD14. Compared to WT, Cyp19-Cre females with YB-1-deficient placentas showed reduced implantation areas at GD8, 10 and 12, decreased placental areas and diameters at GD10 and 12, diminished placental thicknesses at GD12, as well as reduced placental weights at GD12 and 14. In conclusion, our data suggest haploinsufficiency of YB-1 resulting in disturbed fetal and placental development. Moreover, we provide the first evidence for the relevance of trophoblast-specific YB-1 for placentation.

Background. Platelet-derived growth factor (PDGF) is a central mediator of mesangioproliferative glomerulonephritis (GN). In experimental mesangioproliferative GN, PDGF-DD serum levels, unlike PDGF-BB, increased up to 1000-fold. Methods. We assessed disease activity in 72 patients with GN, established a novel PDGF-D ELISA and then determined their PDGF-DD levels. In parallel, we studied renal PDGF-DD mRNA expression by RT-PCR. Results. PDGF-DD serum levels in patients with IgA nephropathy (IgAN) were significantly higher (1.67 ± 0.45 ng/ml) and in patients with lupus nephritis significantly lower (0.66 ± 0.86 ng/ml) compared to healthy controls (1.17 ± 0.46 ng/ml), while patients with focal segmental glomerulosclerosis, membranous GN and ANCA-positive vasculitis did not differ from controls. The subgroup of IgAN patients with elevated PDGF-DD levels (27% of samples) did not differ in their clinical features from those with normal PDGF-DD levels. In IgAN patients with repetitive PDGF-DD determinations, most exhibited only minor fluctuations of serum levels over time. Intrarenal PDGF-DD mRNA expression did not differ between controls and patients, suggesting an extrarenal source of the elevated PDGF-DD in IgAN. Conclusions. Serum PDGF-DD levels were specifically elevated in patients with IgAN, in particular in those with early disease, i.e. preserved renal function. Our data support the rationale for anti-PDGF-DD therapy in mesangioproliferative GN

Hemodialysis (HD) patients are at high risk for all-cause mortality and cardiovascular events. In addition to traditional risk factors, excessive oxidative stress (OS) and chronic inflammation emerge as novel and major contributors to accelerated atherosclerosis and elevated mortality. OS is defined as the imbalance between antioxidant defense mechanisms and oxidant products, the latter overwhelming the former. OS appears in early stages of chronic kidney disease (CKD), advances along with worsening of renal failure, and is further exacerbated by the HD process per se. HD patients manifest excessive OS status due to retention of a plethora of toxins, subsidized under uremia, nutrition lacking antioxidants and turn-over of antioxidants, loss of antioxidants during renal replacement therapy, and leukocyte activation that leads to accumulation of oxidative products. Duration of dialysis therapy, iron infusion, anemia, presence of central venous catheter, and bioincompatible dialyzers are several factors triggering the development of OS. Antioxidant supplementation may take an overall protective role, even at early stages of CKD, to halt the deterioration of kidney function and antagonize systemic inflammation. Unfortunately, clinical studies have not yielded unequivocal positive outcomes when antioxidants have been administered to hemodialysis patients, likely due to their heterogeneous clinical conditions and underlying risk profile.

Peritoneal dialysis (PD) patients manifest excessive oxidative stress (OS) compared to the general population and predialysis chronic kidney disease patients, mainly due to the composition of the PD solution (high-glucose content, low pH, elevated osmolality, increased lactate concentration and glucose degradation products). However, PD could be considered a more biocompatible form of dialysis compared to hemodialysis (HD), since several studies showed that the latter results in an excess accumulation of oxidative products and loss of antioxidants. OS in PD is tightly linked with chronic inflammation, atherogenesis, peritoneal fibrosis, and loss of residual renal function. Although exogenous supplementation of antioxidants, such as vitamins E and C, N-acetylcysteine, and carotenoids, in some cases showed potential beneficial effects in PD patients, relevant recommendations have not been yet adopted in everyday clinical practice.

Glomerular hypertension has been established as a major factor contributing to glomerular scarring. Underlying cellular mechanisms leading to matrix accumulation are largely unknown. The isolated effect of oscillating hyperbaric pressure [OP; Pmax 50mmHg (1mmHg = 0.133kPa), Pmean 24mmHg, with a fixed oscillation of 60/min] on matrix-degrading protease secretion by rat mesangial cells (MCs) was analysed using a pressure chamber model described previously [Mertens, Espenkott, Venjakob, Heintz, Handt and Sieberth (1998) Hypertension 32, 945–952]. MCs were grown under atmospheric pressure (AP) or a controlled OP, and protease synthesis and gene transcription were analysed. A distinct biphasic cellular response to OP with stimulated gelatinase A protein expression and enzyme activity during the initial 24h, and subsequent inhibition, was apparent, as shown by gelatin zymography. Gelatinase B activity remained unchanged. The abundance of gelatinase A transcripts, determined by reverse transcriptase-PCR, indicated a concordant regulation of gene transcription. To elucidate underlying regu latory events, reporter constructs were transfected. In these experiments, a recently identified response element, RE-1, conferred a significant stimulatory effect within the initial 4h of OP. Nuclear protein/RE-1 binding studies revealed additional complexes from 5min up to 3h after OP exposure, with intensities dependent on Pmax. STAT3 was identified as a component of these novel complexes. Down-regulation of cis-activity after 48h of OP exposure was not transferred via the proximal 1686bp of the gelatinase A regulatory sequence. In conclusion, hyperbaric OP elicits time-dependent changes in rat MC gelatinase A gene transcription.

ObjectivesThe aim of this study was to test the hypothesis that, without diagnostic changes in serum creatinine, increased neutrophil gelatinase-associated lipocalin (NGAL) levels identify patients with subclinical acute kidney injury (AKI) and therefore worse prognosis.BackgroundNeutrophil gelatinase-associated lipocalin detects subclinical AKI hours to days before increases in serum creatinine indicate manifest loss of renal function.MethodsWe analyzed pooled data from 2,322 critically ill patients with predominantly cardiorenal syndrome from 10 prospective observational studies of NGAL. We used the terms NGAL(−) or NGAL(+) according to study-specific NGAL cutoff for optimal AKI prediction and the terms sCREA(−) or sCREA(+) according to consensus diagnostic increases in serum creatinine defining AKI. A priori-defined outcomes included need for renal replacement therapy (primary endpoint), hospital mortality, their combination, and duration of stay in intensive care and in-hospital.ResultsOf study patients, 1,296 (55.8%) were NGAL(−)/sCREA(−), 445 (19.2%) were NGAL(+)/sCREA(−), 107 (4.6%) were NGAL(−)/sCREA(+), and 474 (20.4%) were NGAL(+)/sCREA(+). According to the 4 study groups, there was a stepwise increase in subsequent renal replacement therapy initiation—NGAL(−)/sCREA(−): 0.0015% versus NGAL(+)/sCREA(−): 2.5% (odds ratio: 16.4, 95% confidence interval: 3.6 to 76.9, p < 0.001), NGAL(−)/sCREA(+): 7.5%, and NGAL(+)/sCREA(+): 8.0%, respectively, hospital mortality (4.8%, 12.4%, 8.4%, 14.7%, respectively) and their combination (4-group comparisons: all p < 0.001). There was a similar and consistent progressive increase in median number of intensive care and in-hospital days with increasing biomarker positivity: NGAL(−)/sCREA(−): 4.2 and 8.8 days; NGAL(+)/sCREA(−): 7.1 and 17.0 days; NGAL(−)/sCREA(+): 6.5 and 17.8 days; NGAL(+)/sCREA(+): 9.0 and 21.9 days; 4-group comparisons: p = 0.003 and p = 0.040, respectively. Urine and plasma NGAL indicated a similar outcome pattern.ConclusionsIn the absence of diagnostic increases in serum creatinine, NGAL detects patients with likely subclinical AKI who have an increased risk of adverse outcomes. The concept and definition of AKI might need re-assessment.

Kidney disease imparts a vast burden on affected individuals and the overall health care system. Progressive tubulointerstitial fibrosis represents a common final pathway observed in all forms of chronic kidney diseases, characterized by deposition of extracellular matrix, immune cell infiltration, fibroblast proliferation and activation, injury to the tubular epithelium and rarefaction of the peritubular microvasculature. In the recent past, Notch receptors, which are involved in the nephron development, attributed increasingly a significant role in the development of various kidney diseases, and also in the tubulointerstitial fibrosis. In the present work, the involvement of the receptor Notch-3 in the immune cell response and kidney fibrosis was examined. First evidence for an involvement of Notch-3 receptor on the fibrogenesis was shown by in vitro studies in cultured kidney cells. Stimulation with the central profibrotic mediator TGF-beta1 resulted in an increased expression of the receptor Notch-1 and Notch-3 and so on both transcription and protein level. This has been confirmed in vivo in two different animal models of renal damage, the nephrotoxic nephritis (NTN) and the unilateral ureteral obstruction (UUO). The NTN is a primary inflammatory, glomerular and thus proteinuric model whose profile corresponds to a rapidly progressive glomerulonephritis. In addition to the glomerular injury it results in tubulointerstitial immune cell infiltration and fibrosis. The murine UUO model primarily caused damage to the tubular structures and also reflects the hallmarks of tubulointerstitial fibrosis. In both models an activation of the Notch-3 receptor, and increased expression of Notch ligands and target genes was detectable. Striking is, that the increased expression of Notch-3 occurred cell-specific and so after ureteral obstruction detected only in isolated tubular and in some tubulointerstitial. cells. This suggests that the Notch-3-mediated regulatory events are controlled only by subpopulations of cells. We hypothesized that Notch-3 plays a functional relevance in the chemotactic response. Therefore, we studied animals with genetic depletion of Notch-3 receptor after UUO and NTN induction. We could show that Notch-3 knockout (Notch-3 KO) mice were protected from injury and tubular cell loss. The infiltration of inflammatory cells was significantly reduced and delayed, presumably due to a reduced chemokine synthesis and release. The Notch-3 knockout mice also exhibited a very significant reduction in the matrix accumulation that is due to reduced proliferation of interstitial cells. To investigate a direct influence of Notch-3 receptor on the cell proliferation, a cell model was established, which simulates an increased Notch-3 expression/ activation. Overexpression of constitutively active intracellular Notch-3 domain increased proliferation rate of renal tubular cells in vitro. This result was confirmed by incubating the cells with the Notch ligand Jagged-1. In addition to the in vivo and in vitro studies in a third approach, we correlated the expression of members of the Notch receptor family with human renal diseases. Human biopsies of patients with tubular and glomerular kidney diseases have been studied in terms of a gene array where mRNA expression of Notch signal pathways components were measured and hence a heat map was created. It was found that Notch-3 was expressed strongest by all of the Notch family receptors. In summary, our results show that the Notch-3 receptor plays an important role in the orchestration of tubulointerstitial, inflammatory response and in particular the monocytic cell infiltration is affected by Notch-3. This makes the Notch pathway an attractive target for future therapeutic interventions in which an excessive matrix deposition and the consequent destruction of the tissue architecture is to be countered.

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.