Genomic Profiling of Kidney Ischemia-Reperfusion Reveals Expression of Specific Alloimmunity-Associated Genes: Linking “Immune” and “Nonimmune” Injury Events

Abstract

Increased organ ischemia time leads to delayed graft function (DGF), increased acute rejection (AR), enhanced chronic allograft nephropathy (CAN) and reduced long term allograft survival. The mechanisms by which IRI predisposes to AR and CAN are unknown. We hypothesized that gene expression profiling of IRI-affected kidney would identify how IRI predisposes to AR and CAN. Furthermore, we examined how current immunosuppressive drug molecular targets are altered by IRI.C57BL/6J mice were exposed to 30 (n=3) or 60 (n=3) min of bilateral kidney ischemia or sham surgery (n=5). At 36 hr kidney tissue was collected and analyzed using Affymetrix 430MOEA (22626 genes) array and GC-RMA-SAM pipeline. Genes with the false discovery rate (q<1%) and ±50% fold change (FC) were considered affected by IRI. Genes coding for histocompatibility and antigen presenting factors, calcineurin and mTOR pathway-associated proteins were selected using Gene Ontology (GO) analysis. GO analysis identified 10 and 17 alloimmunity-related genes affected by IRI induced by 30 and 60 min of ischemia, respectively including Traf6 (FC=2.99) and H2-D1 (FC=2.58). We also detected significant IRI genomic responses in calcineurin and mTOR pathways represented by Fkbp5 (FC=4.18) and Fkbp1a (FC=2.0); Eif4ebp1 (FC=16.8) and Akt1 (FC=3.64), respectively.These data demonstrated that IRI upregulates expression of several alloimmunity-associated genes, which can in turn enhance alloimune responses. Our discovery of IRI-induced upregulation of genes associated with calcineurin and mTOR pathways are consistent with clinical observations that FK506 and Rapamycin can alter course of DGF. Further validation and dissection of these pathways can lead to novel approaches by which improved management of early "non-immune" transplant events can decrease susceptibility to more classic "immune" changes and CAN.

Kidney Injury Accelerates Cystogenesis via Pathways Modulated by Heme Oxygenase and Complement

“…13 This comparison of the published literature and our own re-analyses of these transcriptional data using the same bioinformatic approach 14 revealed significant overlap between cystogenesis and AKI, dominated by NFkB-regulated genes such as C3, Lcn2, Saa3, Lyz, Serpine1, Cd14 and Ccl2. To ascertain whether HO activity regulates the expression of these NFkB-regulated genes in Cys1 cpk/cpk kidneys, we determined the expression of major NFkB targets in Cys1 cpk/cpk kidneys with induced versus inhibited HO activity.…”

“…To ascertain whether HO activity regulates the expression of these NFkB-regulated genes in Cys1 cpk/cpk kidneys, we determined the expression of major NFkB targets in Cys1 cpk/cpk kidneys with induced versus inhibited HO activity. In addition to Lcn2 (an AKI marker NGAL-encoding gene), we analyzed expression of AKI markers Ccl2 (an MCP-1-encoding gene), 22 Cd14, 13,23 and C3 (a complement component 3-encoding gene; reviewed by Sheerin et al 24 ). Ccl2 and Cd14 are also emerging as markers and/or predictors of renal cystogenesis.…”

“…In addition, the levels of C3 overexpression in this cystic model and AKI (36 hours after 30-minute ischemia) are similar, with a four-fold 14 versus five-fold increase. 13 Because local C3 production is essential for the pathogenesis of adverse AKI and other renal injury outcomes, 24,27,28 renal C3 expression and the ensuing amount of available C3 substrate may represent a limiting factor in renal activation of the complement system and its subsequent adverse injury effects. In cystic kidneys, C3 that is locally produced and activated 14 may have similar adverse effects.…”

“…[10][11][12] Although AKI is an important trigger for cystogenesis, it remains unknown whether milder chronic renal injury, such as that caused by PKD gene defects, also accelerates cystogenesis. The similarity of the renal injury responses during AKI and PKD gene defect-induced cyst formation, as reflected by genome-wide transcriptional analyses, 13,14 suggests a central role of PKD gene mutation-induced injury in renal cystogenesis. For example, of 22 genes found to be most highly overexpressed in mice with rapidly versus slowly progressing cystic kidney disease, 14 18 were also found to be overexpressed in kidneys of mice subjected to ischemia-reperfusion injury 13 (i.e., Hp, C3, Lcn2, Saa3, Ctss, Sprr2f, Lrg1, E430024C06Rik, Ccl6, Lyz, Niban, 1200016E24Rik, Socs3, Serpine1, C1qb, Timp1, Cd14, and F13a).…”

“…The similarity of the renal injury responses during AKI and PKD gene defect-induced cyst formation, as reflected by genome-wide transcriptional analyses, 13,14 suggests a central role of PKD gene mutation-induced injury in renal cystogenesis. For example, of 22 genes found to be most highly overexpressed in mice with rapidly versus slowly progressing cystic kidney disease, 14 18 were also found to be overexpressed in kidneys of mice subjected to ischemia-reperfusion injury 13 (i.e., Hp, C3, Lcn2, Saa3, Ctss, Sprr2f, Lrg1, E430024C06Rik, Ccl6, Lyz, Niban, 1200016E24Rik, Socs3, Serpine1, C1qb, Timp1, Cd14, and F13a). Likewise, transcriptome analysis indicates substantial overlap between biologic processes associated with cystogenesis and with AKI.…”