Results of a Multi-Center Study Identify Mitochondria as Tissue and Blood Damage Associated Molecular Patterns in the Development of Renal Allograft Injury

Abstract

Purpose: Investigating peripheral mitochondrial DNA encoded gene expression and graft tissue anti-mitochondrial protein antibody levels in kidney transplant recipients as novel (donor-specific) allograft damage and/or tolerance associated molecular patterns. Methods: A discovery set of 19 whole blood samples (PaxGene) from Stanford renal transplant recipients included 6 cases of acute rejection (AR), 8 without rejection (no-AR), and 5 tolerant cases (TOL), defined as absence of any immunosuppression and stable function for one year minimum. An independent validation set from Pittsburgh University included 29 whole blood samples from 9 renal AR and 20 no-AR. Expression of mt-DNA encoded genes was analyzed by high throughput QPCR (Fluidigm, Biomark). Negative cross analysis was performed using publicly available gene expression data from 130 patients with autoimmune diseases and 45 healthy controls (HC). In a third set of kidney transplant recipients (11 AR; 11 no-AR) antibody levels against mt-proteins were investigated by ProtoArray (HumanProteinMicorarray v5.0, Invitrogen, Carlsbad, CA). Results: The discovery set revealed upregulation of 11 mt-DNA encoded genes in patients with AR compared to non-AR and TOL (p< 0.05, T-test) independent from time post-transplantation. The average gene expression in TOL patients was lower compared to all other patients. Upregulation of 3 of the 11 genes was validated in the Pittsburgh cohort of AR and no-AR patients, of these, 2 belonged to a gene family known for high mutation rates and currently used in DNA-barcode analyses to distinguish closely related species. Cross-analyses of mt-gene expression in auto-immune diseases showed no differences between diseased patients compared to HC. Subsequent ProtoArray analysis identified 145 post-transplant renal allograft immunogenic mt-proteins (average signal intensity >500). Of these, 4 were encoded by the same gene family upregulated in AR patient whole blood from both discovery and validation set. Conclusions: mt-proteins present novel antigens in transplantation, and together with peripheral mt-DNA encoded genes, may serve as biomarkers for patient monitoring of allograft injury.