Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling in rat renal allografts after prolonged cold ischemia

ABSTRACT: Ischemia/reperfusion injury (IRI) has a major impact on the long-term outcome of renal allografts. However, the mechanisms of IRI related to chronic allograft nephropathy (CAN) are still poorly understood. To address this issue, in a F344 to Lewis transplantation model of the rat, kidneys were either subjected to 20 min. or 24 hours of cold ischemia. A customized cDNA microarray representing 737 immune related genes was used for gene expression analysis after 12 hours and 6 months of engraftment. Thereby showing the 6-month graft function and histology were only moderately deteriorated following short cold ischemic time (20 min.), while prolonged ischemia (24 hrs) accelerated CAN development. Following prolonged cold ischemia already 12 hrs after engraftment about 30 genes were differentially expressed up to >20-fold. This included adhesion molecules, heat shock proteins, transcription factors and chemokines (CXCL10) associated with a strong CD68+ macrophage infiltration. Furthermore we observed a remarkable up-regulation of immunoproteasomes implying a re-organisation of the proteasome complex. This data might explain the higher incidence of alloreactivity following enhanced IRI. After 6 months, the prolonged cold ischemia of 24 hours revealed the significant induction of 4 genes: clusterin, C4, and the CCR7 ligands CCL19/21. This supported the observation of enhanced vasculopathy, humoral alloreactivity and increased tracking of MHC-II+ antigen presenting cells during CAN suggesting them as suitable novel targets for combating IRI. Keywords: transplantation, cold ischemia, inflammation, gene expression profiling Renal allografts from F344 donors were transplanted into unmodified LEW recipients. Before harvesting, grafts were perfused with University of Wisconsin solution at 4Â°C undergoing 20 min. (group A) or 24 hrs (group B) cold ischemia. Engrafted organs were either removed after 12 hrs (group I-A (n=3) and I-B (n=2)) or after 6 months (group II-A (n=3) and II-B (n=2)). Recipient animals of the group II received CyA treatment for 10 days at a dosage of 1.5 mg/kg. Untreated normal kidneys from F344 rats served as control. RNA from transplanted and untreated (control) rat kidneys were labeled by reverse transcription with Cy5 and Cy3 fluorescence, respectively.

ORGANISM(S): Rattus norvegicus

SUBMITTER: A Dernier

PROVIDER: E-GEOD-4441 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Heme oxygenase-1 ameliorates ischemia/reperfusion injury by targeting dendritic cell maturation and migration.

Kotsch Katja K Martins Paulo N A PN Klemz Roman R Janssen Uwe U Gerstmayer Bernhard B Dernier Annelie A Reutzel-Selke Anja A Kuckelkorn Ulrike U Tullius Stefan G SG Volk Hans-Dieter HD

Antioxidants & redox signaling 20071201 12

Ischemia/reperfusion injury (IRI) has a major impact on short- and long-term renal allograft survival by increasing graft immunogenicity. Donor preconditioning by inducing heme oxygenase 1 (HO-1) has been proven to exert cytoprotective and antiinflammatory effects on the graft, thus resulting in reduced graft immunogenicity. The study analyzed the effects and mechanisms of HO-1-mediated cytoprotection in rat kidney transplants exposed to cold preservation. We studied the differential gene-expres ...[more]

PMID: 17854277

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Project description:Fibroblasts are present in every organ. While the role fibroblasts in chronic diseases such as fibrosis or tumor expression has been extensively explored, little is known about their physiological role. The kidney possesses a unique capacity to recover from even severe acute injury. We study molecular mechanisms of this intrinsic repair capacity in the mouse model of ischemia-reperfusion (IR). In this model, the renal artery and vein are clamped for 45 min, leading to acute kidney injury. The kidney spontaneously recovers from such IR injury within 14 days. IR kidney injury is associated with a transient accumulation of fibroblasts in the diseased tissue. We hypothesized that fibroblasts aid the repair of acute IR injury in the kidney. To elucidate how FSP1+ fibroblasts may contribute to the repair of kidney injury, we undertook a global unbiased approach to compare gene expression profiles of fibroblasts isolated from kidneys post-IRI and from control kidneys by FACS sorting. To investigate the role fibroblasts may play in the repair of kidney inhury, we performed ischemia reperfusion injury surgery on transgenic mice in which the FSP-1 promoter drives EGFP expression. Kidney injury peaks at day 3 post-IRI, followed by spontaneous regeration that restores nearly perfect kidney architecture and health by day 10. Fibroblasts are thought to possibly play a role in this process, as they are normally rare in the healthy kidney, acute kidney injury is associated with a transient accumulation of interstitial fibroblasts, but whether they may help repair the acute kidney injury or in fact could contribute to the damage is not known. To compare the gene expression profiles of normal fibroblasts and those from post-ischemic kidneys, we sacrificed control FSP1-GFP mice and the FSP1-GFP mice three days post-IRI. We generated single-cell suspensions from both the post-IRI and control kidneys, and then isolated FSP1-GFP+ cells by FACS sorting that, when cultured on plastic, displayed typical fibroblast morphology. Total RNA was immediately extracted from the sorted cells and amplified to produce enough for a array. We biotinylated five of the samples from post-ischemic kidneys and three of the control (non-ischemic) kidneys and used Affymetrix 3' Arrays to examine differences in gene expression profiles between the two groups that may she some light on what role, if any, fibroblasts play in the spontaneous healing of the kidney after acute kidney injury. We performed ischemia reperfusion surgery in FSP1-GFP mice, and at day 3, we sacrificed the mice, isolated FSP1-GFP positive cells from both IR and normal control kidneys by FACS sorting, extracted total RNA from the isolated FSP1-GFP cells and used Affymetrix Mouse Expression Array 430 2.0 microarrays to perform gene expression profiling of the samples. All told, we performed the FACS Sorting, RNA extration, and hybridization with 5 ischemic kidneys and 3 normal kidneys. Fibroblasts, acute kidney injury, repair, comparative gene expression profiling, microarrays, FACS sorting, role in healing

Dataset Information

Gene expression profiling in rat renal allografts after prolonged cold ischemia

Publications

Heme oxygenase-1 ameliorates ischemia/reperfusion injury by targeting dendritic cell maturation and migration.

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