TY - JOUR
AB - TO007 MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) – A NOVEL ENDOGENEOUS FIBROSIS LIMITING FACTOR Sonja Djudjaj Sonja Djudjaj 1Division of Nephrology RWTH University Aachen Germany 2Institute of Pathology RWTH University Aachen Germany Hongqi Lue Hongqi Lue 3Department of Biochemistry and Molecular Cell Biology RWTH University Aachen Germany Torsten Urzinicok Torsten Urzinicok 4Institute of Experimental Immunology University of Bonn Bonn Germany Daniel Engel Daniel Engel 4Institute of Experimental Immunology University of Bonn Bonn Germany Ina V. Martin Ina V. Martin 1Division of Nephrology RWTH University Aachen Germany Eva M. Buhl Eva M. Buhl 1Division of Nephrology RWTH University Aachen Germany 2Institute of Pathology RWTH University Aachen Germany Jürgen Floege Jürgen Floege 1Division of Nephrology RWTH University Aachen Germany Tammo Ostendorf Tammo Ostendorf 1Division of Nephrology RWTH University Aachen Germany Jürgen Bernhagen Jürgen Bernhagen 3Department of Biochemistry and Molecular Cell Biology RWTH University Aachen Germany Peter Boor Peter Boor 1Division of Nephrology RWTH University Aachen Germany 2Institute of Pathology RWTH University Aachen Germany Abstract Introduction and Aims: Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine with chemokine-like functions, e.g. for macrophages. Experimental studies have almost unequivocally shown that MIF is pro-inflammatory and disease-aggravating in inflammatory diseases including experimental glomerulonephritis. Inhibition of MIF was envisaged as a novel therapeutic approach in these diseases. Surprisingly, we recently showed that MIF was antifibrotic in models of liver fibrosis, in particular via its receptor CD74. This finding prompted us to analyze the role of MIF in renal fibrosis. Methods: We manipulated MIF using genetic deletion (Mif-/- mice), neutralization via MIF antibody (MIF-Ab) or a small molecule MIF inhibitor (ISO-1) and by application of murine recombinant MIF (mrMIF) in three different murine models of renal fibrosis: unilateral ureteral obstruction (UUO), unilateral ischemia/reperfusion induced fibrosis (I/R) and Alport mice. In addition, we investigated the consequences of genetic deletion of the MIF receptor CD74 (CD74-/- mice) in UUO in I/R induced fibrosis. Results: In comparison to wild-type mice (WT), Mif-/- mice had more interstitial fibrosis and macrophage infiltrates in UUO (both day 5 and 10). Confirmatory, compared to isotype matched IgG, MIF-Ab treatment led to aggravated fibrosis and macrophage influx in UUO (day 5). On day 7 of UUO, compared to vehicle treated mice, ISO-1 increased renal fibrosis whereas application of mrMIF reduced fibrosis (even though both treatments were initiated on day 3 after UUO). Treatment with MIF-Ab significantly reduced mortality in Alport mice compared to IgG treated animals. Compared to WT mice, mice lacking the MIF receptor CD74 had increased interstitial fibrosis and macrophage influx in both UUO (day 5) and I/R (day 21), resembling the results of Mif-/- mice. Conclusions: We show that MIF inhibition and CD74 deficiency aggravated whereas MIF application ameliorated renal fibrosis. We thus identified a hitherto unappreciated dual role of MIF in renal disease, which is pro-inflammatory in glomerular disease but anti-fibrotic in the interstitium. TO008 EXTRACELLULAR VESICLES DERIVED FROM ENDOTHELIAL PROGENITOR CELLS INHIBIT PROGRESSION TOWARD CHRONIC KIDNEY DISEASE AND FIBROSIS BY A RNA-DEPENDENT MECHANISM Vincenzo Cantaluppi Vincenzo Cantaluppi 1Nephrology, Dialysis and Renal Transplantation Unit University of Torino Torino Italy Davide Medica Davide Medica 1Nephrology, Dialysis and Renal Transplantation Unit University of Torino Torino Italy Claudio Mannari Claudio Mannari 2Department of Neurosciences University of Pisa Pisa Italy Federico Figliolini Federico Figliolini 1Nephrology, Dialysis and Renal Transplantation Unit University of Torino Torino Italy Massimiliano Migliori Massimiliano Migliori 3Nephrology and Dialysis Unit Versilia Hospital Lido di Camaiore Italy Vincenzo Panichi Vincenzo Panichi 3Nephrology and Dialysis Unit Versilia Hospital Lido di Camaiore Italy Ciro Tetta Ciro Tetta 4Fresenius Medical Care Bad Homburg Germany Giovanni Camussi Giovanni Camussi 1Nephrology, Dialysis and Renal Transplantation Unit University of Torino Torino Italy Abstract Introduction and Aims: Endothelial progenitor cells (EPCs) are bone marrow-derived precursors with pro-angiogenic properties. A decrease of circulating EPCs in patients with chronic kidney disease (CKD) is associated with progression to end stage renal failure. EPCs exert their protective effect by the release of paracrine mediators including extracellular vesicles (EVs), small particles involved in cell-to-cell communication through the transfer of proteins and genetic material. The aim of this study was to evaluate the role of EPC-derived EVs in lowering progression toward CKD and fibrosis in the 5/6 nephrectomy rat model. Methods: EPCs were isolated from peripheral blood and EVs were characterized for protein and RNA content. Wistar rats were subjected to 5/6 nephrectomy and treated at week 4 and 8 with infusion of 30 μg EPC-derived EVs treated with vehicle or 1U/ml RNase. Proteinuria, creatinine clearance and histology were evaluated at 14 weeks. In vitro, we studied the effects of EPC-EVs on human kidney-derived endothelial and tubular epithelial cells cultured with uremic toxins (p-cresyl-sulphate, indoxyl-sulphate, ADMA). Results: EPC-EVs carried different mRNAs and microRNAs involved in angiogenesis, inhibition of apoptosis and fibrogenesis. Administration of EPC-derived EVs in rats subjected to 5/6 nephrectomy reduced proteinuria and preserved renal function with a limitation of histological signs of tubulo-interstitial fibrosis and glomerulosclerosis (decrease of α-SMA and ADMA-positive areas). Moreover, EV-treated nephrectomized rats showed a preserved expression of podocyte (nephrin, synaptopodin) and tubular epithelial (E-cadherin, aquaporin-2) markers and a decrease of capillary rarefaction (RECA-1 staining). These protective effects were not observed when EVs were treated with RNase, suggesting the key role of RNAs shuttled by EVs. In addition, fibroblast-derived EVs did not exert renoprotection. In vitro, EVs induced proliferation, resistance to apoptosis and angiogenesis of kidney-derived endothelial cells incubated with uremic toxins. On tubular cells, EVs decreased de-differentiation, apoptosis and TGF-β secretion induced by uremic toxins. These effects were not observed when EVs were treated with RNase. Conclusions: EPC-EVs decreased proteinuria and lowered progression toward CKD in the 5/6 nephrectomy rat model. The mRNA and microRNA cargo shuttled by EVs to kidney endothelial and tubular epithelial cells protected from capillary rarefaction and progression toward renal fibrosis by inhibiting epithelial-to-mesenchymal transition. TO009 PARIETAL PODOCYTES - TRANSDIFFERENTIATION FROM PARIETAL EPITHELIAL CELLS OR MIGRATION OF PODOCYTES? Kevin Schulte Kevin Schulte 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Katja Berger Katja Berger 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Eva Maria Sicking Eva Maria Sicking 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Peter Boor Peter Boor 2Pathology RWTH University of Aachen Aachen Germany Peggy Jirak Peggy Jirak 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Larissa Thevissen Larissa Thevissen 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Astrid Fuß Astrid Fuß 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Wilhelm Kriz Wilhelm Kriz 3Division of Anatomy Medical Faculty Mannheim / University of Heidelberg Mannheim Germany Jürgen Floege Jürgen Floege 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Bart Smeets Bart Smeets 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Marcus J. Moeller Marcus J. Moeller 1Nephrology and Immunology RWTH University of Aachen Aachen Germany Abstract Introduction and Aims: In adults, it is still unresolved whether parietal epithelial cells (PECs) can potentially act as progenitor cells by differentiating into podocytes. “Parietal podocytes”, i.e. fully differentiated podocytes residing on the inner aspect of Bowman's capsule, are an interesting model in this context. Parietal podocytes have been observed in multiple human kidney diseases, in particular in atubular glomeruli. Methods: First, a mouse model to generate atubular glomeruli by electrocoagulation was established and characterized in different genetic backgrounds. Results: Parietal podocytes were observed exclusively in atubular glomeruli. In addition, glomerular cysts were formed particularly in young mice of the Sv129 genetic background. Next, PECs or podocytes were traced in the above-described model for detubularization by irreversible genetic tagging in triple transgenic mice (PEC- or Pod-rtTA/LC1/R26R). Our results showed conclusively that PECs undergo apoptosis after detubularisation and that visceral podocytes migrate onto Bowman's capsule. No direct transdifferentiation from PECs towards podocytes was observed. This finding was confirmed in the unilateral ureter obstruction (UUO) model. Immunohistochemical stainings of human kidney biopsies always showed a sharpedged border between PECs and podocytes on Bowman's capsule of atubular glomeruli. No gradual differentiation of PECs into podocytes could be observed, supporting that no transdifferentiation of PECs into podocytes occurred also in humans. Conclusions: In summary, there are two surprising findings of this study: 1. Detubularisation leads to acute ablation of PECs and 2. visceral podocytes migrate onto Bowman's capsule in atubular glomeruli. Transdifferentiation from PECs to parietal podocytes did not occur in this model. TO010 NECROTIC GLOMERULAR CELLS RELEASE HISTONES THAT TRIGGER GLOMERULAR INFLAMMATION AND CRESCENT FORMATION IN GLOMERULONEPHRITIS V. R. Santhosh Kumar V. R. Santhosh Kumar 1Klinische Biochemie Medizinische Klinik und Poliklinik IV Munich Bayern Germany Onkar P. Kulkarni Onkar P. Kulkarni 1Klinische Biochemie Medizinische Klinik und Poliklinik IV Munich Bayern Germany Narayana Murthy Darisipudi Narayana Murthy Darisipudi 1Klinische Biochemie Medizinische Klinik und Poliklinik IV Munich Bayern Germany Shrikant Ramesh Mulay Shrikant Ramesh Mulay 1Klinische Biochemie Medizinische Klinik und Poliklinik IV Munich Bayern Germany Hans-Joachim Anders Hans-Joachim Anders 1Klinische Biochemie Medizinische Klinik und Poliklinik IV Munich Bayern Germany Abstract Introduction and Aims: Extracellular histones, released from dying cells have the potential to kill endothelial cells and to activate Toll like receptor (TLR)-2 and -4, which was shown to drives tubulointerstitial inflammation in septic or postischemic acute kidney injury. However, their contribution to glomerulonephritis is yet unknown. We speculated that extracellular histones elicit similar pathogenic effects also in glomerulonephritis. Methods: C57BL/6 male mice were procured from Jackson Laboratories (Bar Habour, MA).All experimental procedures were approved by the local government authorities. Glomerulonephritis was induced in mice with single intravenous injection of 100 µl of a sheep anti-GBM antiserum. Groups of mice were treated i.p. either with 20 mg/kg of control IgG or anti-histone antibody (BWA3)that has the potential to neutralize the effects of extracellular histones in vitro and in vivo. After 7 days animals were sacrificed and kidneys were collected for further data analysis by immunostaining, flow cytometry, ELISA and RT-PCR. Proteinuria was assessed as albumin to creatinine ratio in spot urine samples. Results: Intravenous injection of antiserum in control mice induced proteinuria, increased urine albumin/creatinine ratio, plasma creatinine and BUN levels. This was associated with reduced number of podocytes, crescentic glomerulonephritis, and with infiltration of neutrophils and macrophages into the kidney. Anti-histone antibody treatment significantly reduced proteinuria and increased the numbers of WT-1/nephrin positive podocytes. This was associated with lower plasma creatinine and BUN levels as well as with less neutrophil and macrophage infiltrates into the kidney. Histone blockade also significantly reduced renal mRNA expression of TNF-alpha, which was associated with less glomerulosclerosis, crescents, and tubular atrophy. Conclusions: We conclude that the release of histones from dying cells contribute to renal immunopathology and dysfunction during crescentic glomerulonephritis. This may either relate to their direct toxic effects on endothelial and epithelial cells or to their potential to activate innate immunity via TLR2 and TLR4. TO011 INVOLVEMENT OF HEPARANASE IN THE PATHOGENESIS OF GLOMERULAR DISEASES Suheir Assady Suheir Assady 1Rambam Health Care Campus Haifa Israel 2Rappapot Faculty of Medicine, Technion-IIT Haifa Israel Joel Alter Joel Alter 1Rambam Health Care Campus Haifa Israel 2Rappapot Faculty of Medicine, Technion-IIT Haifa Israel Michael Litvak Michael Litvak 1Rambam Health Care Campus Haifa Israel Neta Ilan Neta Ilan 2Rappapot Faculty of Medicine, Technion-IIT Haifa Israel Israel Vlodavsky Israel Vlodavsky 2Rappapot Faculty of Medicine, Technion-IIT Haifa Israel Zaid Abassi Zaid Abassi 1Rambam Health Care Campus Haifa Israel 2Rappapot Faculty of Medicine, Technion-IIT Haifa Israel Abstract Introduction and Aims: Heparanase (HPA), an endoglycosidase that cleaves heparan sulfate (HS), is involved in various processes, including: Extracellular matrix (ECM) turnover, inflammation, angiogenesis, and metastasis. Although an association between HPA and glomerular injury was suggested, a role for HPA in the pathogenesis of severe proteinuria has not yet been fully elucidated. Therefore, the present study examines the contribution of HPA to the pathogenesis of Adriamycin-induced nephrotic syndrome (ADR-NS) in a mouse model. Methods: Wild-type (wt) BALB/c mice (n=7) and HPA over-expressing transgenic mice (hpa-TG; n=7), were tail-vein injected with either ADR (10mg/kg) or vehicle (controls). At days 0, 7 and 14, mice were housed in metabolic cages for daily urine collection to determine albumin to creatinine ratio (UACR). Injected mice and their controls were sacrificed at day 15, and kidneys were harvested for various analyses: 1) Heparanase enzymatic activity; 2) Western blot (WB) and RT-PCR; 3) histomorphology using hematoxylin and eosin, PAS and Masson's trichrome staining; 4) immunofluorescence (IF); and 5) electron microscopy (EM). Results: While ADR-injected wt mice developed severe albuminuria, hpa-TG mice showed only a mild elevation in UACR, compared with their uninjected counterparts (0.19±0.029 vs. 42.24±1.39 mg/mg, p<0.0001, wt sham vs. wt ADR, respectively; and 0.19±0.02 vs. 0.40±0.041 mg/mg, p=0.004, hpa-TG sham vs. hpa-TG ADR, respectively, at 14 days after ADR injection). In parallel, when incubated with sulfate-labeled ECM, homogenate of cortex from wt ADR mice showed elevated HPA enzymatic activity, and resulted in release of high amounts of HS degradation fragments. Semiquantitave RT-PCR analysis revealed a significant reduction in expression of podocyte-specific markers, such as: Podocalyxin, NEPH1, FAT1, nephrin, actinin-4, and podocin, in ADR-treated wt mice, but not in ADR-treated hpa-TG mice. These results were solidified by WB and IF analysis of nephrin and podocin protein expression, both known as key slit diaphragm components. The reduction in podocyte markers expression was secondary to podocyte loss as revealed by EM, where injected wt mice displayed a massive podocyte loss, and foot process effacement. In contrast, hpa-TG mice maintained integrity of podocyte architecture. Moreover, light microscopy of stained cross sections of kidneys from ADR-injected wt mice, but not hpa-Tg mice, showed mild to severe glomerular and tubular damage, with strong hyaline staining inside the glomeruli and tubular lumen. Conclusions: The present study provides new insights into the involvement of HPA in the pathogenesis of proteinuria.Specifically, our results suggest that HPA may have a nephroprotective role in ADR-NS, most likely via a mechanism independent of HS degradation. Moreover, hpa-TG mice comprise an invaluable in vivo platform to investigate the interplay between HPA and glomerular injury. © The Author 2013. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. 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TI - Glomerular injury
JO - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gft168
DA - 2013-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/glomerular-injury-4uWCsUp0lI
SP - i46
EP - i47
VL - 28
IS - suppl_1
DP - DeepDyve
ER -