Björn C. Frye

Recruitment of Podocytes from Glomerular Parietal Epithelial Cells

Loss of a critical number of podocytes from the glomerular tuft leads to glomerulosclerosis. Even in health, some podocytes are lost into the urine. Because podocytes themselves cannot regenerate, we postulated that glomerular parietal epithelial cells (PECs), which proliferate throughout life and adjoin podocytes, may migrate to the glomerular tuft and differentiate into podocytes. Here, we describe transitional cells at the glomerular vascular stalk that exhibit features of both PECs and podocytes. Metabolic labeling in juvenile rats suggested that PECs migrate to become podocytes. To prove this, we generated triple-transgenic mice that allowed specific and irreversible labeling of PECs upon administration of doxycycline. PECs were followed in juvenile mice beginning from either postnatal day 5 or after nephrogenesis had ceased at postnatal day 10. In both cases, the number of genetically labeled cells increased over time. All genetically labeled cells coexpressed podocyte marker proteins. In conclusion, we demonstrate for the first time recruitment of podocytes from PECs in juvenile mice. Unraveling the mechanisms of PEC recruitment onto the glomerular tuft may lead to novel therapeutic approaches to renal injury.

Y‐box protein‐1 is actively secreted through a non‐classical pathway and acts as an extracellular mitogen

Y‐box protein (YB)‐1 of the cold‐shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB‐1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB‐1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB‐1 was secreted through a non‐classical mode resembling that of the macrophage migration inhibitory factor. YB‐1 release requires ATP‐binding cassette transporters, and microvesicles protect YB‐1 from protease degradation. Two lysine residues in the YB‐1 carboxy‐terminal domain are crucial for its release, probably because of post‐translational modifications. The addition of purified recombinant YB‐1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non‐classically secreted YB‐1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

YB-1 Acts as a Ligand for Notch-3 Receptors and Modulates Receptor Activation

Y-box (YB) protein-1 is secreted by mesangial and immune cells after cytokine challenge, but extracellular functions are unknown. Here, we demonstrate that extracellular YB-1 associates with outer cell membrane components and interacts with extracellular Notch-3 receptor domains. The interaction appears to be specific for Notch-3, as YB-1-green fluorescent protein binds to the extracellular domains and full-length forms of Notch-3 but not to Notch-1. YB-1-green fluorescent protein and Notch-3 proteins co-localize at cell membranes, and extracellular YB-1 activates Notch-3 signaling, resulting in nuclear translocation of the Notch-3 intracellular domain and up-regulation of Notch target genes. The YB-1/Notch-3 interaction may be of particular relevance for inflammatory mesangioproliferative disease, as both proteins co-localize in an experimental nephritis model and receptor activation temporally and spatially correlates with YB-1 expression.

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.

Extracellular YB-1 Blockade in Experimental Nephritis Upregulates Notch-3 Receptor Expression and Signaling

Background: Notch receptors are involved in kidney development and pathogenesis of inflammatory glomerular diseases. Given the secretion of Y-box (YB) protein-1 following cytokine stimulation and subsequent extracellular association with membrane receptor Notch-3 in vitro, we elucidated functional effects of YB-1 targeting on the Notch-3 signaling pathway. Methods: Rat mesangial cells were challenged with a monoclonal anti-YB-1 antibody (YB-1-mAb) and analyzed for YB-1 and Notch-3 expression. Notch-3 expression in mice with a targeted disruption of one YB-1 allele (YB-1+/d) was compared with their wild-type littermates. Furthermore, YB-1-mAb was applied during mesangioproliferative anti-Thy1.1 nephritis, and glomerular Notch-3, Notch target genes and YB-1 expression were analyzed by immunohistochemistry, quantitative real-time PCR and immunoblotting. Results: Upon challenge with YB-1-mAb, rat mesangial cells showed an increased expression of YB-1 and Notch-3 protein. Concordantly, we found a significant upregulation of Notch-3 expression in renal cells of YB-1+/d mice. YB-1-mAb treatment in anti-Thy1.1 nephritis resulted in enhanced mesangial Notch-3 expression and differential Notch target gene activation (HES2/Hey-2). Notably, YB-1 mRNA content did not differ between groups; however, glomerular YB-1 protein was significantly increased, suggesting a posttranslational mechanism. Conclusion: Extracellular targeting of YB-1 potently induces glomerular Notch-3 receptor expression, Notch signaling and YB-1 stabilization, most likely via an autoregulatory feedback mechanism.

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-β 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-β 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-β–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-β 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.

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.

Rat Mrp2 gene expression is regulated by an interleukin-1β-stimulated biphasic response with enhanced transcription and subcellular shuttling of YB-1.

INTRODUCTION Expression of hepatobiliary transporters is decreased during endotoxemia. Reduction of Mrp2 is mediated by IL-1β-dependent signals but underlying mechanisms are still unclear. YB-1 is a predominantly cytoplasmic protein that translocates to the nucleus in response to various stimuli. Previously we have shown that YB-1 down-regulates Mrp2 expression in vitro. Therefore we investigated the potential role of YB-1 as regulator of hepatic acute phase genes. METHODS Liver sections from LPS-injected rats (20 h) were stained with YB-1-specific antibodies. Real-time RT-PCR quantification was performed for Mrp2, MMP-2 and YB-1. YB-1 protein was quantified from IL-1β- or TNFα-stimulated rat hepatoma cells (FaO) and the localization of a YFP-YB-1-CFP fusion protein was visualized by confocal microscopy in HepG2 human hepatocellular carcinoma cells. ChIP-assays and EMSA were performed to analyze YB-1 binding to DNA promoter elements. RESULTS In endotoxemic livers Mrp2 mRNA was down-regulated by 80%, while YB-1 mRNA expression increased 2.5-fold. Immunohistochemical staining showed a marked up-regulation and predominant nuclear localization of YB-1 protein in LPS challenged rats. In FAO cells IL-1β incubation increased cytoplasmic YB-1 protein content up to 16h. IL-1β stimulation resulted in a 6-fold up-regulation of endogenous YB-1 in the nuclear compartment, which occurred within 90min. In accord with these findings nuclear fluorescence was detected with a YFP-YB-1-CFP fusion protein introduced in HepG2 cells. In addition to DNA binding studies with endotoxemic rat liver tissue, ChIP assays revealed an IL-1β-dependent increase of YB-1 binding to the Mrp2-promoter in FAO cells. CONCLUSION YB-1 is activated during the hepatic acute phase response. IL-1β promotes a rapid nuclear YB-1 protein shuttling in hepatoma cells within 90 min and a transcriptional induction thereafter. This biphasic response may explain the IL-1β-mediated suppression of Mrp2 expression in endotoxemic rats.

Isolation of Streptococcus urinalis from a human blood culture.

Streptococcus urinalis was isolated from a blood culture of a 60-year-old man with a history of urethral stricture. This species has been recently described as a new member of the pyogenic subgroup of streptococci that cause urinary tract infections.