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• W2621752677 abstract "Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow–derived macrophage cultures and found that YB-1–deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis. Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow–derived macrophage cultures and found that YB-1–deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis. Inflammation of the kidney is a complex phenomenon that is mostly incited to combat injuries or infection. The process encompasses cell recruitment. How cellular infiltrates are regulated and then resolve is of paramount importance for understanding protective mechanisms. Conversely, excessive cellular infiltration and prolonged inflammatory response may aggravate tissue damage, and therefore, detrimentally impact kidney function.1Anders H.J. Ryu M. Renal microenvironments and macrophage phenotypes determine progression or resolution of renal inflammation and fibrosis.Kidney Int. 2011; 80: 915-925Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 2Weidenbusch M. Anders H.J. Tissue microenvironments define and get reinforced by macrophage phenotypes in homeostasis or during inflammation, repair and fibrosis.J Innate Immun. 2012; 4: 463-477Crossref PubMed Scopus (79) Google Scholar Y-box binding protein-1 (YB-1), the prototypic cold shock domain protein, regulates proliferation, matrix protein synthesis, and chemotaxis, and therefore, is a candidate protein involved in the orchestration of inflammatory responses.3Lyabin D.N. Eliseeva I.A. Ovchinnikov L.P. YB-1 protein: functions and regulation.Wiley Interdiscip Rev RNA. 2014; 5: 95-110Crossref PubMed Scopus (207) Google Scholar, 4Hanssen L. Frye B.C. Ostendorf T. et al.Y-box binding protein-1 mediates profibrotic effects of calcineurin inhibitors in the kidney.J Immunol. 2011; 187: 298-308Crossref PubMed Scopus (30) Google Scholar, 5Mertens P.R. Alfonso-Jaume M.A. Steinmann K. Lovett D.H. YB-1 regulation of the human and rat gelatinase A genes via similar enhancer elements.J Am Soc Nephrol. 1999; 10: 2480-2487Crossref PubMed Google Scholar, 6Raffetseder U. Frye B. Rauen T. et al.Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection.J Biol Chem. 2003; 278: 18241-18248Crossref PubMed Scopus (103) Google Scholar, 7Brandt S. Raffetseder U. Djudjaj S. et al.Cold shock Y-box protein-1 participates in signaling circuits with auto-regulatory activities.Eur J Cell Biol. 2012; 91: 464-471Crossref PubMed Scopus (23) Google Scholar, 8Lasham A. Print C.G. Woolley A.G. Braithwaite A.W. YB-1: oncoprotein, prognostic marker and therapeutic target?.Biochem J. 2013; 449: 11-23Crossref PubMed Scopus (164) Google Scholar In resting cells, cytoplasmic YB-1 binds mRNA to regulate translation and splicing. Upon cellular stress, YB-1 translocates to the nucleus, where it binds single-stranded DNA, thus serving as a transcriptional regulator, for example, the chemokine regulated on activation, normal T cell expressed and secreted-chemokine (C-C motif) ligand 5 (CCL5).9van Roeyen C.R. Scurt F.G. Brandt S. et al.Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities.Cell Commun Signal. 2013; 11: 63Crossref PubMed Scopus (36) Google Scholar, 10Kohno K. Izumi H. Uchiumi T. et al.The pleiotropic functions of the Y-box-binding protein, YB-1.Bioessays. 2003; 25: 691-698Crossref PubMed Scopus (432) Google Scholar, 11Swamynathan S.K. Nambiar A. Guntaka R.V. Role of single-stranded DNA regions and Y-box proteins in transcriptional regulation of viral and cellular genes.FASEB J. 1998; 12: 515-522Crossref PubMed Scopus (87) Google Scholar, 12Raffetseder U. Rauen T. Djudjaj S. et al.Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1.Kidney Int. 2009; 75: 185-196Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 13Lindquist J.A. Brandt S. Bernhardt A. et al.The role of cold shock domain proteins in inflammatory diseases.J Mol Med. 2014; 92: 207-216Crossref PubMed Scopus (25) Google Scholar YB-1 mainly acts intracellularly, but it is also secreted. Both YB-1 expression and secretion are induced by proinflammatory factors such as platelet-derived growth factors and lipopolysaccharide (LPS).14Frye B.C. Halfter S. Djudjaj S. et al.Y-box protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen.EMBO Reports. 2009; 10: 783-789Crossref PubMed Scopus (94) Google Scholar Extracellular YB-1 acts as a mitogen and chemoattractant.14Frye B.C. Halfter S. Djudjaj S. et al.Y-box protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen.EMBO Reports. 2009; 10: 783-789Crossref PubMed Scopus (94) Google Scholar, 15Rauen T. Raffetseder U. Frye B.C. et al.YB-1 acts as a ligand for Notch-3 receptors and modulates receptor activation.J Biol Chem. 2009; 284: 26928-26940Crossref PubMed Scopus (78) Google Scholar Mesangial cells play a key role in some inflammatory glomerular diseases, for example, mesangioproliferative (IgA) nephritis, where they assume a macrophage-like phenotype and release proinflammatory factors such as platelet-derived growth factors and CCL5. In this disease, a self-perpetuating inflammatory reaction with enhanced infiltrating immune cells indicates a poor outcome.16Zhu G. Wang Y. Wang J. et al.Significance of CD25 positive cells and macrophages in noncrescentic IgA nephropathy.Ren Fail. 2006; 28: 229-235Crossref PubMed Scopus (7) Google Scholar We identified CCL5 as an YB-1 target gene, thus demonstrating a fundamental role of YB-1 in inflammation.12Raffetseder U. Rauen T. Djudjaj S. et al.Differential regulation of chemokine CCL5 expression in monocytes/macrophages and renal cells by Y-box protein-1.Kidney Int. 2009; 75: 185-196Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Krohn R. Raffetseder U. Bot I. et al.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.Circulation. 2007; 116: 1812-1820Crossref PubMed Scopus (80) Google Scholar Using RNA inhibition to suppress YB-1 expression in rat mesangial cells, we observed reduced CCL5 expressions. Likewise, when we overexpressed YB-1, CCL5 expressions increased, suggesting tight regulation; this was also observed in T cells and monocytes. However, macrophages show a completely different response because CCL5 reporter activity and transcription are inhibited by YB-1. Because macrophages and monocytes are key mediators of inflammation, understanding how YB-1 in these cells contributes to inflammatory disease progression and cell-cell communication is important. Given that conventional YB-1 knockout is embryonically lethal and that heterozygous YB-1–deficient mice provide limited information regarding cell-specific functions, we developed a conditional Ybx1 knockout mouse. YB-1flox/flox mice were crossed with LysMCre mice to generate a noninducible cell-specific knockout in monocytes, macrophages, and neutrophils (YB-1ΔLysM mice), allowing us to investigate the contribution of YB-1 to monocytic cell function. In this study, we characterized the effects of cell-specific YB-1 knockout using a mouse model of renal interstitial fibrosis, namely unilateral ureteral obstruction (UUO). To investigate the mechanism(s) by which YB-1 contributes to disease progression, we utilized bone marrow–derived macrophages (BMDMs). Here, we demonstrate that YB-1 is pivotal to inflammatory cell recruitment after disease induction and reveal a new function of monocytic YB-1 for resolving renal inflammation. Because monocytes-macrophages play a key role in orchestrating kidney disease, we turned our attention to characterizing the role of monocytic YB-1. We generated a conditional deletion of Ybx1 in cells of the myeloid lineage by crossing YB-1flox/flox with LysMCre mice (YB-1ΔLysM mice, Supplementary Figure S1). YB-1ΔLysM mice were born with the expected Mendelian frequency, and no gross abnormalities were noted (Supplementary Table S1A). The Ybx1 deletion was confirmed by polymerase chain reaction (PCR), Western blotting, and flow cytometry (Supplementary Figure S1). Periodic acid-Schiff staining of healthy kidneys from wild-type (WT) and YB-1ΔLysM mice revealed no apparent differences (Figure 1a). Following UUO, tubular atrophy and dilation, as well as interstitial fibrosis, were apparent in WT mice. In comparison, kidneys from YB-1ΔLysM mice showed a significant increase in tubular damage 14 days after UUO, suggesting that lack of monocytic YB-1 aggravates kidney disease. α-Smooth muscle actin was analyzed as a marker of activated myofibroblasts. As expected, immunohistochemistry showed no differences in contralateral healthy kidneys (Figure 1b). However after inducing UUO, a significant increase in α-smooth muscle actin–positive tubulointerstitial cells indicated an increased number of activated myofibroblasts in YB-1ΔLysM kidneys, which is consistent with the increased tissue damage. Transforming growth factor-β (TGF-β) is a key profibrotic factor that induces the activation of myofibroblasts. Consistent with α-smooth muscle actin staining, tissue lysates show enhanced TGF-β levels in YB-1ΔLysM kidneys compared with those in WT kidneys following disease induction (Figure 1c).Figure 1Monocyte-macrophage– and granulocyte-specific Y-box binding protein-1 (YB-1) ablation enhances unilateral ureteral obstruction (UUO)–dependent tubulointerstitial damage and extracellular matrix deposition. (a) In healthy kidneys from wild-type (WT) and YB-1ΔLysM mice, periodic acid–Schiff (PAS) staining of cortical kidney tissue specimens reveals no apparent dysontogenesis or renal phenotype. Staining of obstructed kidneys on days 6 and 14 of WT and YB-1ΔLysM mice was performed, and representative images were taken. PAS staining shows an increase in tubular damage 14 days after UUO in the YB-1ΔLysM mice compared with that in the WT mice. Quantification of the results obtained with the tubular histology score, which assesses the loss of brush border, tubular dilation, and apoptosis/necrosis of tubular cells. Bar = 50 μm (n = 12). (b) Representative images of the obstructed kidneys on days 6 and 14 after UUO in WT and YB-1ΔLysM mice stained with α-smooth muscle actin (α-SMA). Quantification was performed by assessing a positively stained cortical area (%). Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the relative area (%) of tissue that immunostained positively. Data represent means ± SD, with n = 12 for controls and knockouts. Bar = 50 μm. (c) Ligated or contralateral kidneys were homogenized, and transforming growth factor β (TGF-β) levels were determined after ureteral ligation using flow cytometry–based bead assay (n = 10 mice per group; 6 and 14 days have been pooled). There was significantly more TGF-β in the absence of cell-specific YB-1 after UUO. (d) Representative images of obstructed kidneys on days 6 and 14 in the WT and YB-1ΔLysM mice stained with Sirius red. Quantification was performed by assessing the positively stained cortical area (%). Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. The Sirius red staining for collagen deposits shows an increase in fibrotic material in the YB-1ΔLysM mice compared with that in WT mice. Values indicate the relative area (%) of tissue that stained positively for Sirius red. Bar = 50 μm (n = 12). (e) Increasing the exposure time with a polarization filter allows one to distinguish collagen types I (red) and III (green), both sclerotic proteins. Quantification of collagen types I to III was performed using the ImageProPlus Software. There is a significant increase of collagen type III in the YB-1ΔLysM mice after days 6 and 14 of UUO. Bar = 50 μm. (f) TaqMan analysis of the relative collagen type I alpha and III transcripts in obstructed and healthy contralateral kidneys. There are significantly more collagen type I alpha and III transcripts in the absence of cell-specific YB-1 after both days 6 and 14 of UUO. Data represent means ± SD (n = 12 in each group). *P < 0.05, **P < 0.005. ns, non-significant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Monocyte-macrophage– and granulocyte-specific Y-box binding protein-1 (YB-1) ablation enhances unilateral ureteral obstruction (UUO)–dependent tubulointerstitial damage and extracellular matrix deposition. (a) In healthy kidneys from wild-type (WT) and YB-1ΔLysM mice, periodic acid–Schiff (PAS) staining of cortical kidney tissue specimens reveals no apparent dysontogenesis or renal phenotype. Staining of obstructed kidneys on days 6 and 14 of WT and YB-1ΔLysM mice was performed, and representative images were taken. PAS staining shows an increase in tubular damage 14 days after UUO in the YB-1ΔLysM mice compared with that in the WT mice. Quantification of the results obtained with the tubular histology score, which assesses the loss of brush border, tubular dilation, and apoptosis/necrosis of tubular cells. Bar = 50 μm (n = 12). (b) Representative images of the obstructed kidneys on days 6 and 14 after UUO in WT and YB-1ΔLysM mice stained with α-smooth muscle actin (α-SMA). Quantification was performed by assessing a positively stained cortical area (%). Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the relative area (%) of tissue that immunostained positively. Data represent means ± SD, with n = 12 for controls and knockouts. Bar = 50 μm. (c) Ligated or contralateral kidneys were homogenized, and transforming growth factor β (TGF-β) levels were determined after ureteral ligation using flow cytometry–based bead assay (n = 10 mice per group; 6 and 14 days have been pooled). There was significantly more TGF-β in the absence of cell-specific YB-1 after UUO. (d) Representative images of obstructed kidneys on days 6 and 14 in the WT and YB-1ΔLysM mice stained with Sirius red. Quantification was performed by assessing the positively stained cortical area (%). Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. The Sirius red staining for collagen deposits shows an increase in fibrotic material in the YB-1ΔLysM mice compared with that in WT mice. Values indicate the relative area (%) of tissue that stained positively for Sirius red. Bar = 50 μm (n = 12). (e) Increasing the exposure time with a polarization filter allows one to distinguish collagen types I (red) and III (green), both sclerotic proteins. Quantification of collagen types I to III was performed using the ImageProPlus Software. There is a significant increase of collagen type III in the YB-1ΔLysM mice after days 6 and 14 of UUO. Bar = 50 μm. (f) TaqMan analysis of the relative collagen type I alpha and III transcripts in obstructed and healthy contralateral kidneys. There are significantly more collagen type I alpha and III transcripts in the absence of cell-specific YB-1 after both days 6 and 14 of UUO. Data represent means ± SD (n = 12 in each group). *P < 0.05, **P < 0.005. ns, non-significant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because myofibroblasts produce extracellular matrix, sirius red staining was performed to visualize collagen deposition (Figure 1d and e). A significant increase in interstitial collagen was observed on days 6 and 14 in the YB-1ΔLysM mice. Using a polarization filter, one may distinguish collagen types I (red) and III (green). A significant increase in collagen type I was observed on day 6 after UUO in the YB-1ΔLysM mice. To confirm this, we performed quantitative reverse transcription PCR for collagen types I and III. Our analysis shows significantly more collagen type I alpha transcripts in the absence of YB-1 on both days 6 and 14, whereas mRNA transcripts for collagen type III were significantly reduced in the absence of YB-1 on day 6 after UUO (Figure 1f). As enhanced cell proliferation is a hallmark of tubular cell regeneration and reactivity to damage,18Schocklmann H.O. Lang S. Sterzel R.B. Regulation of mesangial cell proliferation.Kidney Int. 1999; 56: 1199-1207Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar we stained tissue sections for proliferating cell nuclear antigen (PCNA; Figure 2a). In nonobstructed kidneys, only a minor fraction of cells exhibited PCNA positivity, whereas a marked increase in PCNA-positive tubular and interstitial cells was observed after UUO. Compared with that in WT mice, the proliferative response in YB-1ΔLysM mice showed a trend toward higher proliferation rates on day 6 and was significantly lower on day 14.Figure 2Altered cell proliferation rates and enhanced immune cell infiltration is dependent on Y-box binding protein-1 (YB-1). (a) Proliferating cell nuclear antigen (PCNA) immunostaining of obstructed kidneys on days 6 and 14 of unilateral ureteral obstruction (UUO) in the wild-type (WT) and YB-1ΔLysM mice. Numbers of PCNA-positive nuclei were counted in obstructed and healthy contralateral kidneys. Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the number of nuclei positive for PCNA per high-power field. Compared with the WT mice, the proliferative response in YB-1ΔLysM mice is enhanced on day 6 and appears to significantly decline by 14 days of UUO. Bar = 50 μm. Data represent means ± SD (n = 12). (b) Immunostaining of infiltrating monocytes-macrophages was performed using the F4/80 antibody. Quantification was performed by assessing the positively stained cortical area (%). Diagrams show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the relative area (%) of tissue that was positively immunostained. Bar = 50 μm. Data represent means ± SD (n = 12 in each group). (c) Effect of the absence of YB-1 on immune cell infiltration after inducing UUO. C57BL/6 WT and YB-1ΔLysM kidneys were harvested after 2, 6, or 14 days of UUO. Kidney tissues were digested with collagenase, and the resulting single-cell suspension was stained and characterized by flow cytometry; see gating strategy (Supplementary Figure S2). Representative fluorescence-associated cell sorting plots show the number of cluster of differentiation (CD)45+ cells in the healthy contralateral and obstructed kidneys of WT and YB-1ΔLysM mice. After inducing UUO, there is an increase of CD45+ cells in the WT mice, which is even further enhanced using the YB-1ΔLysM mice. Further analysis showed an increase in CD11b+ F4/80+ or CD3+ cells after UUO. Myeloid cells, CD45+ CD11b+, in the kidney are also significantly increased on day 6 after inducing UUO in the YB-1ΔLysM mice. There is a significant increase in F4/80+ macrophages in the absence of YB-1 6 days after UUO. No differences in infiltrating neutrophils (defined by CD45+ CD11b+ GR1+) were observed between the WT and YB-1ΔLysM mice on days 2, 6, or 14 after UUO. All data collection and analyses were performed using the FlowJo software. Controls were used for gating analyses to distinguish between positively and negatively stained cell populations (n = 12 in each group). Cell numbers were calculated from the CD45+ leukocyte population. (d) Automated multidimensional fluorescence microscopy images of sections from the contralateral healthy kidney and from day 14 of UUO kidneys from the WT and YB-1ΔLysM mice. The upper panel depicts the phase contrast (gray), collagen type IV/vimentin (white), and CD45 (cyan) images. The lower panel depicts the CD3 (red), CD11b (green), CD11c (yellow), and F4/80 (magenta) images. Bar = 50 μm (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005. n.s., nonsignificant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Altered cell proliferation rates and enhanced immune cell infiltration is dependent on Y-box binding protein-1 (YB-1). (a) Proliferating cell nuclear antigen (PCNA) immunostaining of obstructed kidneys on days 6 and 14 of unilateral ureteral obstruction (UUO) in the wild-type (WT) and YB-1ΔLysM mice. Numbers of PCNA-positive nuclei were counted in obstructed and healthy contralateral kidneys. Diagrams on the right show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the number of nuclei positive for PCNA per high-power field. Compared with the WT mice, the proliferative response in YB-1ΔLysM mice is enhanced on day 6 and appears to significantly decline by 14 days of UUO. Bar = 50 μm. Data represent means ± SD (n = 12). (b) Immunostaining of infiltrating monocytes-macrophages was performed using the F4/80 antibody. Quantification was performed by assessing the positively stained cortical area (%). Diagrams show data obtained using a computer-based morphometric analysis on days 6 and 14 after inducing UUO in obstructed and healthy contralateral kidneys. Values indicate the relative area (%) of tissue that was positively immunostained. Bar = 50 μm. Data represent means ± SD (n = 12 in each group). (c) Effect of the absence of YB-1 on immune cell infiltration after inducing UUO. C57BL/6 WT and YB-1ΔLysM kidneys were harvested after 2, 6, or 14 days of UUO. Kidney tissues were digested with collagenase, and the resulting single-cell suspension was stained and characterized by flow cytometry; see gating strategy (Supplementary Figure S2). Representative fluorescence-associated cell sorting plots show the number of cluster of differentiation (CD)45+ cells in the healthy contralateral and obstructed kidneys of WT and YB-1ΔLysM mice. After inducing UUO, there is an increase of CD45+ cells in the WT mice, which is even further enhanced using the YB-1ΔLysM mice. Further analysis showed an increase in CD11b+ F4/80+ or CD3+ cells after UUO. Myeloid cells, CD45+ CD11b+, in the kidney are also significantly increased on day 6 after inducing UUO in the YB-1ΔLysM mice. There is a significant increase in F4/80+ macrophages in the absence of YB-1 6 days after UUO. No differences in infiltrating neutrophils (defined by CD45+ CD11b+ GR1+) were observed between the WT and YB-1ΔLysM mice on days 2, 6, or 14 after UUO. All data collection and analyses were performed using the FlowJo software. Controls were used for gating analyses to distinguish between positively and negatively stained cell populations (n = 12 in each group). Cell numbers were calculated from the CD45+ leukocyte population. (d) Automated multidimensional fluorescence microscopy images of sections from the contralateral healthy kidney and from day 14 of UUO kidneys from the WT and YB-1ΔLysM mice. The upper panel depicts the phase contrast (gray), collagen type IV/vimentin (white), and CD45 (cyan) images. The lower panel depicts the CD3 (red), CD11b (green), CD11c (yellow), and F4/80 (magenta) images. Bar = 50 μm (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005. n.s., nonsignificant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tissue damage induces inflammation, which recruits immune cells. To determine the degree of monocyte-macrophage infiltration, F4/80 staining was performed (Figure 2b). In contralateral healthy kidneys, significantly more F4/80+ cells were observed in YB-1ΔLysMmice. A prominent increase in F4/80+ cells was observed on day 6 after UUO, and there was a trend toward significance on day 14 in YB-1ΔLysM mice. To quantify the number of infiltrating cells, organs were harvested and analyzed by flow cytometry (Supplementary Figure S2). After inducing UUO, we observed the expected increase in infiltrating cluster of differentiation (CD)45+ cells in WT mice, which was even further enhanced in YB-1ΔLysM mice (Figure 2c). Among the CD45+ subsets, we observed an increase in infiltrating CD11b+ cells, F4/80+, and CD3+ cells on day 6, which was significantly enhanced by the absence of monocytic YB-1. Unexpectedly, we also observed an increase in the basal numbers of CD45+ CD11b+ and CD45+ F4/80+ cells in YB-1ΔLysM kidneys. While the number of infiltrating granulocytes [gamma response 1 (GR1)+ cells] also increased after inducing the disease, we observed no difference between WT and YB-1ΔLysM mice (Supplementary Table S1B). Automated multidimensional fluorescence microscopy confirmed the differences in infiltrating immune cells observed between WT and YB-1ΔLysM mice. After UUO, YB-1ΔLysM mice demonstrated markedly more F4/80+ and CD11b+ cells than WT mice (Figure 2d). In addition, staining for CD45 and CD3 appeared dispersed in YB-1ΔLysM mice, whereas the cells were more clustered in WT mice. To gain insight into the molecular mechanism(s) underlying the differences in immune cell infiltration, we analyzed RNA extracted from WT and YB-1ΔLysM kidneys before and after inducing the disease for differential gene expression. Multidimensional scaling analysis confirmed a high correlation and reproducibility between the samples (Figure 3a). Bioinformatics analysis focused on the comparison with healthy kidneys. Using an absolute 1.3-fold change cutoff on a log2 scale, we identified 2411 and 3451 genes differentially expressed in WT and YB-1ΔLysM mice, respectively, on day 6; the comparable values on day 14 were 4072 and 5257 genes, respectively (Figure 3b). To facil" @default.
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• W2621752677 title "Inflammatory cell infiltration and resolution of kidney inflammation is orchestrated by the cold-shock protein Y-box binding protein-1" @default.
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