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Cold Storage Disrupts the Proteome Landscape in Rat Kidney Transplants

ABSTRACT: Approximately 70% of kidney grafts are obtained from deceased donors, and these grafts must be preserved in hypothermic conditions to prolong their viability until transplantation. However, prolonged cold storage (CS) of kidneys results in poor long-term outcomes after transplantation. We reported previously that CS of rat kidneys for 18 h prior to transplant impaired proteasome function, disrupted protein homeostasis, and reduced graft function. The goal of the present study was to identify the renal proteins that are dysregulated by this CS-induced injury. Isolated donor Lewis rat kidneys were subject to 18-h CS and transplanted into recipient Lewis rats (CS+Tx). Autotransplantation (ATx: transplant with 0-h CS) or Sham (right nephrectomy) surgeries served as controls. The proteome of kidney homogenates was analyzed with tandem mass-tag mass spectrometry to identify CS-induced abnormalities in kidney grafts. CS injury disrupted the renal phosphoproteome in kidney grafts and dysregulated numerous signaling pathways. Integrated analysis of global proteomes and phosphoproteomes identified 15 proteins that were significantly regulated in a CS-specific manner. In particular, proteins and pathways such as complement and coagulation cascades were upregulated, while antioxidant pathways, such as glutathione, were suppressed in CS+Tx groups compared to ATx and Sham controls. This study, for the first time, provides deeper insight into the disruption of the renal graft proteome caused by CS injury and provides a novel set of pathways and molecules that can be investigated to delineate their specific role in renal transplant outcomes, ultimately improving outcomes for patients with end-stage kidney disease.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Rattus Norvegicus (ncbitaxon:10116)

SUBMITTER: Nirmala Parajuli

PROVIDER: MSV000094566 | MassIVE | Thu Apr 18 10:52:00 BST 2024

SECONDARY ACCESSION(S): PXD051567

REPOSITORIES: MassIVE

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Project description:The clinical results obtained with organ allografts from marginal or extended donors are less satisfactory over both the short-term and long-term than the outcome of grafting from living donors. Early postoperative graft function depends on several pretransplant factors, with the major donor influences being brain death (BD) and ischemia/reperfusion(I/R) injury. The effects of BD and I/R injury not only reduce the number of functioning nephrons, but also trigger a host immune response to the grafted kidney. It has been hypothesized that allografts from marginal sources may not be biologically inert at the time of surgery, but may already be programmed to initiate or amplify a host response. To exclude the effects of allogenicity, we established a rat renal isograft model using dead donors and compared the results with those obtained after grafting from living donors. In this study, we performed an analysis of the changes of gene expression in rat kidney isografts using samples obtained at 0(pre-transplant) and 1 hour time points since transplantation procedure. The results enhanced our insight into the pathways and cascades that are activated or down-regulated by BD and/or I/R injury. Better knowledge of the key components of BD or I/R injury will provide clues to the factors triggering progression that leads to a decline of organ viability, as well as ideas on how to overcome such graft failures. Such data may also allow us to identify novel biomarkers as predictors of adverse outcomes. Experiment Overall Design: Inbred male Lewis rats were used for these experiments as recipients and donors. The left kidney was transplanted orthotopically by end-to-end anastomosis. The contralateral right kidney was removed at the time of transplantation. The time of operative ischemia for transplantation was 30 min, which did not vary between animals. Donor animals were divided into two groups. The experimental group received kidneys from BD rats, while kidneys from living donors were used in the control group. Brain death was produced by gradually increasing the intracranial pressure that led to brain stem herniation. The rats were mechanically ventilated for a period of 6 hours. We compared the gene expression profiles of renal isografts from BD donors and grafts from living donors using a high-density oligonucleotide microarray that contained approximately 20,500 genes. Experiment Overall Design: For DNA microarray experiments, 200 ng aliquots of total RNA were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Product) according to the manufacturerâ??s instructions. RNA purified from each kidney graft was used for microarray analysis (Cy3-labeled), with pooled RNA derived from normal kidneys as a template control (Cy5-labeled). After checking the labeling efficiency, 1ï?g aliquots of Cy5-labeled normal control RNA and Cy3-labeled RNA from individual grafts (control 0 hour, 1 hour, BD 0 hour, BD 1 hour, n=3/group) were mixed, and then hybridized to Agilent Rat Oligo Microarrays (Product No. G4130A). After washing, the microarray slides were analyzed with an Agilent Microarray scanner and software (scanner model G2565BA). Data analysis was performed using Agilent Feature Extraction software (Ver. A.7.1.1), and Excel 2003 (Microsoft). The data were imported into GeneSpring 7.0 (Silicon Genetics, Redwood City, CA), with per spot, per chip, and intensity dependent (lowess) normalization being applied for each array. The ratio of the normalized channels (Cy3/Cy5) was used to assess the level of expression.

Project description:Microarray analysis of human kidneys with acute kidney injury (AKI) has been limited because such kidneys are seldom biopsied. However, all kidney transplants experience AKI, and early kidney transplants without rejection are an excellent model for human AKI: they are screened to exclude chronic kidney disease, frequently biopsied, and have extensive follow-up. We used histopathology and microarrays to compare indication biopsies from 28 transplants with AKI to 11 pristine protocol biopsies of stable transplants. Kidneys with AKI showed increased expression of 394 injury-repair response associated transcripts, including many known epithelial injury molecules (e.g. ITGB6, LCN2), tissue remodeling molecules (e.g. VCAN), and inflammation molecules (S100A8, ITGB3). Many other genes also predict the phenotype, depending on statistical filtering rules, including AKI biomarkers as HAVCR1 and IL18. Most mouse orthologs of the top injury-repair transcripts were increased in published mouse AKI models. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score AKI kidneys correlated with reduced function, future recovery, brain death, and need for dialysis, but not future graft loss. In contrast, histologic features of "acute tubular injury" did not correlate with function or with the molecular changes. Thus the injury-repair associated transcripts represent a massive coordinate injury-repair response of kidney parenchyma to AKI, similar to mouse AKI models, and provide an objective measure for assessing the severity of AKI in kidney biopsies and validation for the use of many AKI biomarkers. AKI biopsies sample names and CEL files are from GSE21374. All consenting renal transplant patients undergoing biopsies for cause as standard of care between 09/2004 and 10/2007 at the university of Alberta or between 11/2006 and 02/2007 at the University of Illinois were included in the analysis. In addition to the cores required for standard histopathology, we collected one core for gene expression studies. the relationship between gene expression in the biopsy and subsequent graft loss was analyzed. This dataset is part of the TransQST collection.

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Cold Storage Disrupts the Proteome Landscape in Rat Kidney Transplants

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