Ystem. Moreover, it can be seen from the scatter plots of CKDN2A vs eGFR at 1 year that renal function deteriorates significantly at CDKN2A expression levels above 1.8. ECD kidneys occupy both category III and category IV Table 4. Multivariate model outcome for eGFR at 6 months.Standardised Independent Variable Coefficients (Beta) CDKN2A ECD Kidney Recipient Glomerulonephritis 20.397 20.211 20.p-value 0.034 0.233 0.The model approaches statistical significance using the strict Bonferroni correction (p = 0.021). doi:10.1371/journal.pone.0068133.tPre-Transplant CDKN2A Predicts Renal FunctionTable 5. Multivariate model outcome for eGFR at 1 year.Table 6. Suggested Donor Kidney Classification system incorporating CDKN2A as the biomarker of ageing and ECD kidney criteria.Standardised Independent Variable Coefficients (Beta) CDKN2A ECD Kidney Recipient Glomerulonephritis 20.428 20.236 20.p-value 0.019 0.166 0.061 Category I Category II Category III Category IV SCD Kidney and CDKN2A expression levels ,1.8 SCD Kidney and CDKN2A expression levels .1.8 ECD Kidney and CDKN2A expression levels ,1.8 ECD Kidney and CDKN2A expression levels .1.The model explains 27.1 of the eGFR p = 0.008. doi:10.1371/journal.pone.0068133.tin this pre-transplant scoring tool meaning that ECD status carries a poorer prognosis than CDKN2A itself. The allocation of CDKN2A to a higher tier in this scoring system would require further studies to strengthen the correlations observed above. Since DCA 18204824 forms part of 1315463 ECD criteria, it was not used as a single determinant of transplant function in multivariate analysis or the categorical scoring system. A further benefit from our data, is that strategies to mitigate the rate of biological ageing applied to living donors would be expected to have impact on post-transplant outcomes. Reduction of psychological and psychosocial stress and improved lifestyle via changes to diet and exercising might readily be considered. [14,27,28] Biomarkers, specifically CDKN2A, may well expand the field of octogenarian donation for example, by discriminating organs with “less miles on the clock”. Larger multicentre studies are needed to strengthen the hypothesis and the proposed scoring system suggested in this report. It is envisaged that the biomarker CDKN2A will be integrated into a similar, robust and validated pre-transplant scoring system for all kidneys and other transplanted organs in the near future.(SCD ?Standard Criteria Donors, ECD ?Extended Criteria Donors). Predicted kidney function and incidence of graft failure increases with higher category placement. doi:10.1371/journal.pone.0068133.tHuman Renal Biopsies and RNA/DNA ExtractionRenal biopsies were obtained on the surgical backbench via wedge resection or needle biopsy according to the surgeon’s preference. All biopsies were obtained from “donation after brain death” (DBD) and “donation after cardiac death” (DCD) donors. All samples were stored in `RNA later’ solution (Ambion, MedChemExpress Ergocalciferol Austin, TX, USA) at ?0uC until processing. RNA was extracted using Trizol reagent (Invitrogen, Paisley, UK) following manufacturer’s guidelines. The MaxwellH 16 DNA purification robot kits by get Lecirelin Promega were used for for DNA isolation.Delayed Graft Function (DGF)DGF was defined as failure of serum creatinine to fall by half within seven days of the transplant, or need for dialysis within seven days of the transplant, except dialysis performed for fluid overload or elevated serum potassium levels [29].MDRD 4.Ystem. Moreover, it can be seen from the scatter plots of CKDN2A vs eGFR at 1 year that renal function deteriorates significantly at CDKN2A expression levels above 1.8. ECD kidneys occupy both category III and category IV Table 4. Multivariate model outcome for eGFR at 6 months.Standardised Independent Variable Coefficients (Beta) CDKN2A ECD Kidney Recipient Glomerulonephritis 20.397 20.211 20.p-value 0.034 0.233 0.The model approaches statistical significance using the strict Bonferroni correction (p = 0.021). doi:10.1371/journal.pone.0068133.tPre-Transplant CDKN2A Predicts Renal FunctionTable 5. Multivariate model outcome for eGFR at 1 year.Table 6. Suggested Donor Kidney Classification system incorporating CDKN2A as the biomarker of ageing and ECD kidney criteria.Standardised Independent Variable Coefficients (Beta) CDKN2A ECD Kidney Recipient Glomerulonephritis 20.428 20.236 20.p-value 0.019 0.166 0.061 Category I Category II Category III Category IV SCD Kidney and CDKN2A expression levels ,1.8 SCD Kidney and CDKN2A expression levels .1.8 ECD Kidney and CDKN2A expression levels ,1.8 ECD Kidney and CDKN2A expression levels .1.The model explains 27.1 of the eGFR p = 0.008. doi:10.1371/journal.pone.0068133.tin this pre-transplant scoring tool meaning that ECD status carries a poorer prognosis than CDKN2A itself. The allocation of CDKN2A to a higher tier in this scoring system would require further studies to strengthen the correlations observed above. Since DCA 18204824 forms part of 1315463 ECD criteria, it was not used as a single determinant of transplant function in multivariate analysis or the categorical scoring system. A further benefit from our data, is that strategies to mitigate the rate of biological ageing applied to living donors would be expected to have impact on post-transplant outcomes. Reduction of psychological and psychosocial stress and improved lifestyle via changes to diet and exercising might readily be considered. [14,27,28] Biomarkers, specifically CDKN2A, may well expand the field of octogenarian donation for example, by discriminating organs with “less miles on the clock”. Larger multicentre studies are needed to strengthen the hypothesis and the proposed scoring system suggested in this report. It is envisaged that the biomarker CDKN2A will be integrated into a similar, robust and validated pre-transplant scoring system for all kidneys and other transplanted organs in the near future.(SCD ?Standard Criteria Donors, ECD ?Extended Criteria Donors). Predicted kidney function and incidence of graft failure increases with higher category placement. doi:10.1371/journal.pone.0068133.tHuman Renal Biopsies and RNA/DNA ExtractionRenal biopsies were obtained on the surgical backbench via wedge resection or needle biopsy according to the surgeon’s preference. All biopsies were obtained from “donation after brain death” (DBD) and “donation after cardiac death” (DCD) donors. All samples were stored in `RNA later’ solution (Ambion, Austin, TX, USA) at ?0uC until processing. RNA was extracted using Trizol reagent (Invitrogen, Paisley, UK) following manufacturer’s guidelines. The MaxwellH 16 DNA purification robot kits by Promega were used for for DNA isolation.Delayed Graft Function (DGF)DGF was defined as failure of serum creatinine to fall by half within seven days of the transplant, or need for dialysis within seven days of the transplant, except dialysis performed for fluid overload or elevated serum potassium levels [29].MDRD 4.