Leukocyte accumulation in graft blood vessels during self-limiting acute rejection of rat kidneys Immunobiology
ABSTRACT: Acute rejection episodes trigger chronic renal allograft vasculopathy. Numerous leukocytes, predominantly monocytes, accumulate in graft blood vessels during reversible acute rejection preceding chronic rejection of rat kidneys. We speculate that they contribute to transplant vasculopathy and set out to characterize them. Allogeneic renal transplantation was performed in the Fischer 344 to Lewis rat strain combination, Lewis isografts served as controls. Leukocytes were harvested by intensive perfusion of graft blood vessels and subjected to flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Kidneys of LEW and F344 rats were transplanted in LEW rats. Five biological replicates were performed for both isogenic and allogenic transplantation. Transcriptomes of allogenics were compared to isogenics on 5 dual-color hybridizations.
Project description:Objective: Apolipoprotein E (Apo E) is a multifunctional protein, originally described in the context of lipoprotein metabolism and cardiovascular disease. More recently, anti-inflammatory functions of ApoE have been documented. ApoE was studied in the context of several inflammatory disorders, but its role in the pathogenesis of acute organ rejection is unknown. In this study, we test the hypothesis that ApoE attenuates acute renal allograft rejection. Materials and methods: The Dark Agouti (DA) to Lewis (Lew) and the Brown Norway (BN) to Lew rat strain combinations were used to investigate fatal acute rejection. In addition, Fischer 344 (F344) kidneys were transplanted to Lew rats to study reversible acute rejection. Isograft recipients and untreated Lew rats were used as controls. ApoE mRNA expression was quantified in intravascular leukocytes accumulating in the blood vessels of renal grafts and in graft tissue. Apo E protein levels were assessed in blood plasma. To test the protective potential of ApoE, recipients of BN kidneys were treated with ApoE-mimetic peptide. Results: Intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA, whereas during fatal rejection, ApoE expression remained unchanged in the BN to Lew rat strain combination or was significantly reduced when DA rats were used as donors of the kidney. On the protein level, no changes in ApoE were seen in plasma. However, we do not know if local leukocytic ApoE expression results in increased ApoE concentrations inside graft blood vessels. Peptide treatment of allograft recipients reversed fatal rejection and significantly improved animal survival. Conclusions: ApoE plays a protective role in acute organ rejection. Further studies are needed to understand the exact mechanism how ApoE reverses acute rejection. dual-color balanced dye-swap design with 4 biological replicates, hybridized on 4 arrays
Project description:WKY and LEW strains have been widely studied for their differential susceptibility to experimental glomerulonephritis. In particular these strains show strong variations in the macrophage activation. This dataset measures expression of macrophages in backcross population of WKY DC and LEW rats and includes a few control origninating from the WKY DC strain.
Project description:The frequency of delayed function of kidney transplants varies greatly and is associated with the quality of graft, donor age, and the duration of cold ischemia time. Body weight differences between donor and recipient can affect primary graft function. The underlying mechanism is poorly understood. Here, we have transplanted kidney grafts from commensurate body weight (L-WD) or reduced body weight (H-WD) donor rats into syngeneic or allogeneic recipients. 24 hours post-transplantation, serum creatinine level in H-WD recipients was significantly higher compared to that of L-WD recipients indicating impaired primary graft function. We detected a 10 fold higher transcription of IL-6 and dramatically increased tubular destruction in grafts from H-WD recipients. This was accompanied by decreased expression of genes associated with kidney function and an up-regulation of other genes such as cytochrome P450 isoforms, FosL and Trib3 as revealed by DNA microarray analysis. A single application of IL-6 into L-WD recipients is sufficient to impair primary graft function and to cause tubular damage. Whereas, immediate neutralization of IL-6 receptor signaling rescued primary graft function resulting in low serum creatinine levels, well-preserved kidney graft architecture and a normalized gene expression profile. These findings have strong clinical implication as anti-IL6R treatment of patients receiving grafts from lower-weight donors could be used to improve primary graft function. The dataset comprises eight samples divided into four sample groups. Each group represents rat kidneys collected after allogeneic transplantation under a certain condition and includes two biological replicates. The first group is characterized by a high body weight difference between donor and recipient, rats in the second group exhibit a low weight difference. Group three and four are similar to group one, but underwent an additional treatment with anti-IL6R mAb or prednisolone immediately after transplantation.
Project description:Acute rejection episodes trigger chronic renal allograft vasculopathy. Numerous leukocytes, predominantly monocytes, accumulate in graft blood vessels during reversible acute rejection preceding chronic rejection of rat kidneys. We speculate that they contribute to transplant vasculopathy and set out to characterize them. Allogeneic renal transplantation was performed in the Fischer 344 to Lewis rat strain combination, Lewis isografts served as controls. Leukocytes were harvested by intensive perfusion of graft blood vessels and subjected to flow cytometry, quantitative RT-PCR and genome-wide transcriptional profiling. Overall design: Kidneys of LEW and F344 rats were transplanted in LEW rats. Five biological replicates were performed for both isogenic and allogenic transplantation. Transcriptomes of allogenics were compared to isogenics on 5 dual-color hybridizations.
Project description:To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs IS. But IS such as calcineurin inhibitors CNI can interfere with tolerance induction. We investigated the effect of an additional CNI treatment on anti-CD4 mAb-induced tolerance induction upon rat kidney transplantation. Additional CNI treatment induced deteriorated graft function and chronic rejection characterised by alloantibody production, intragraft plasma cells and C3d deposition. Microarray analysis revealed enhanced intragraft expression of the B cell chemokine CXCL13 upon additional CNI treatment. In contrast PNOC, a B cell-related gene highly expressed in operational tolerant kidney transplant recipients, was decreased in grafts of anti-CD4 mAb+CsA-treated recipients suggesting an altered balance of B regulatory genes. Transient B cell depletion or transfer of Tregs three weeks after transplantation could inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection. These data represent unexpected findings and should be taken into consideration when designing new clinical trials. To safe the benefit of CNI in controlling memory T cell responses we need strategies for a therapeutic optimization. We show here that early B cell depletion or transfer of Tregs may be one approach to arrest B cell accumulation, activation and graft destruction. comparative analysis of different immunosuppressive regimens on renal allograft gene expression versus untreated allografts
Project description:The goal is to identify new molecules implicated in tolerance, to determine the implication of these molecules in immune responses to transplantation by gene expression comparison of 27,088 individual rat genes between tolerated kidney allotransplant and syngeneic kidney transplant. In this study 27,088 individual rat genes expression from total mRNA of 3 tolerated allogeneic kidney transplants by anti-classII, were compared to 3 syngeneic kidney transplants at day 100 post transplantation.
Project description:We have extended our investigation to differential immunogenicity between tolerogenic PVG rats and immunogenic LEW rats by analyzing gene expression in adipose-derived mesenchymal stem cells (ASCs) with LPS stimulation. Furthermore, to establish a direct link between gene expression and immunogenic functional annotation, ASCs from LEW and PVG rats were obtained, and the effects of inherent difference and LPS treatment on global gene expression were evaluated using microarray analyses. We analyzed microarray gene expression profiles of ASCs from 3 LEW and 3 PVG rats of 8-week age and separated ASCs from each individual into four conditions, 24h and 48h control, 24h and 48h with 1 μg/ml LPS stimulation. The total RNA samples of triplicate ASCs isolated from LEW and PVG with and without LPS treatment were pooled in each condition and the global gene expression profiles were analyzed using microarray.
Project description:The goal is to identify molecules involved in the accumulation of blood MDSCs in tolerant kidney allografted recipients when compared to syngeneic grafted recipients. In this study 27,088 individual rat genes expression from total mRNA of blood MDSCs from 3 tolerated allogeneic kidney transplanted recipient by anti-CD28, were compared to MDSCs from 3 syngeneic kidney transplanted recipient at day 100 post transplantation.
Project description:The effect of cafeteria (CAF) diet in PBMC gene expression was analyzed in two inbred rat strains the Wistar Kyoto(WKY) and Lewis (LEW) rat PBMCs were isolated following a CAF or standard diet and subjected to whole genome expression analysis Rats from each strain were either fed with standard chow diet (n=5) or CAF (n=5). After 7 weeks of the indicated diet, rats were fasted for 9 hours, culled and PBMCs isolated from whole blood collected from the abdominal aorta
Project description:Femoral neck bone mineral density and structure candidate gene analysis in Fischer 344 (F344) and Lewis (LEW) rats; Hip fracture is the most devastating osteoporotic fracture type with significant morbidity and mortality. Previously, we identified that the region of 4q21-q41 on chromosome (Chr) 4 uniquely harbors multiple femoral neck quantitative trait loci (QTLs) in inbred Fischer 344 (F344) and Lewis (LEW) rats. In this study we identified the candidate genes for femoral neck density and structure by correlating gene expression in the proximal femur with the femoral neck phenotypes linked to the QTLs on Chr 4. Microarray analysis was performed using RNA extracted from proximal femora of 4-week-old rats from F344, LEW and two other strains. A total of 104 genes in the 4q21-q41 region were differentially expressed (p<0.05) among all strains of rats with a false discovery rate (FDR) less than 10%. These 104 genes were then ranked based on the proportion of variation in femoral neck phenotypes in F2 animals homozygous for a particular strain’s allele at the Chr 4 QTL explained by the expression level of the gene in that strain. A total of 37 genes, including 21 candidate genes and 16 predicted genes, were strongly correlated (r2>0.50) with different femoral neck phenotypes and prioritized for further analysis. Ingenuity pathway analysis revealed several direct or indirect relationships among the candidate genes related to bone metabolism including pathways related to beta-estradiol, interleukin 6, insulin growth factor 2, androgen receptor and tumor necrosis factor. Experiment Overall Design: Comparison of differentially expressed genes at the chromosome 4 QTL identified in F344 and LEW F2 rats.