Key driver genes as potential therapeutic targets in renal allograft rejection
Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang
Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang
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Research Article Transplantation

Key driver genes as potential therapeutic targets in renal allograft rejection

Abstract

Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. Molecules with regulatory control among immune pathways of AR that are inadequately suppressed, despite standard-of-care immunosuppression, could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative, network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators — the key driver genes (KDGs) — within dysregulated AR pathways. A 982–meta-gene signature with differential expression in AR versus non-AR was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across 7 data sets. Fourteen KDGs were derived from this signature. Interrogation of 2 publicly available databases identified compounds with predicted efficacy against individual KDGs or a key driver–based gene set, respectively, which could be repurposed for AR prevention. Minocycline, a tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline attenuated the inflammatory profile of AR compared with controls and when coadministered with immunosuppression prolonged graft survival. This study demonstrates that a network-based strategy, using expression and genotype data to predict KDGs, assists target prioritization for therapeutics in renal allograft rejection.

Authors

Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang

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Figure 7

Minocycline treatment reduced the inflammatory profile of AR in a murine heterotopic heart transplant model.

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Minocycline treatment reduced the inflammatory profile of AR in a murine...
(A) C57BL/6 mice were transplanted with hearts from BALB/c mice, representing a full MHC mismatch. Recipient mice received intraperitoneal injections of minocycline (n = 9) or saline (n = 9) twice daily starting 36 hours before the allogeneic cardiac transplant, through to postoperative day 4 (POD4) and euthanized; all allografts were still viable (with palpable beat) at this time. Four syngeneic cardiac transplants (C57BL/6 donors to C57BL/6 recipients, no treatment) were used as controls. (B and C) The mRNA transcript expression (Rel. mRNA Exp., presented on a log scale) was measured by real-time PCR. Four of the 14 KDGs, including the top-ranked key driver, Caspase-1, were significantly reduced in the allografts of the minocycline-treated animals compared with saline-treated animals. Significant attenuation of allograft mRNA expression of several proinflammatory chemokines and cytokines with minocycline treatment was observed most prominently with IFN-γ and IL-2; TNF-α expression was not significantly different. (D) Protein concentrations of IFN-γ and TNF-α (Prot. conc. pg/mL) in sera were measured with array beads, and the results were in line with the allograft expression of the corresponding transcripts. (E and F) Representative H&E and immunohistochemistry staining for CD3 (T cells) and galectin-3 (macrophages) (both stained brown) in cardiac allografts demonstrated substantially less inflammatory cell infiltrate in the minocycline-treated animals compared with those given saline (n = 9 in each group), which was quantified by the Positive Pixel Count Algorithm (Pos. Pix. Ct/mm2, Version 9, Leica Biosystems). Furthermore, infiltration of both cell types in the allogeneic allografts of minocycline-treated mice was not significantly different from that in the syngeneic grafts (n = 3). Relative expression of mRNA transcripts was calculated using the ΔΔCT method, normalized to the expression in the syngeneic transplant group, with GAPDH as the housekeeping gene. Scale bars: 400 μm (H&E and CD3), 200 μm (galectin-3). For mRNA and serum protein data, Student’s 2-tailed t test was used to analyze differences between the 2 groups for normally distributed data, and the Mann-Whitney U test was used for non-normally distributed data. Cell infiltrate quantification was compared using 1-way ANOVA and Tukey’s multiple comparisons test. All data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). IP, intraperitoneal; POD, postoperative day; minoc, minocycline-treated allografts; saline, saline-treated allografts; syn, syngeneic grafts.