Roslyn B. Mannon MD, FASN, FAST Professor of Medicine and Surgery - PowerPoint PPT Presentation

Pre-implantation Kidney Biopsies as a Predictor for Delayed Graft Function Roslyn B. Mannon MD, FASN, FAST Professor of Medicine and Surgery University of Alabama at Birmingham Birmingham AL, USA

Conflicts of Interest • I have no conflicts of interest relevant to this presentation • I will not be discussing the use of off label therapeutics

Outline • Background on deceased donor organs and kidney transplantation • What is delayed graft function and why does it matter? • What are the metrics for kidney organ discard? • What are predictors of recipient allograft function? • How can we impact kidney graft utilization?

The Wait List Declined in 2016 due to More Kidneys Transplanted! American Journal of Transplantation pages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557 http://onlinelibrary.wiley.com/doi/10.1111/ajt.14557/full#ajt14557-fig-0001

Discard Rates are Still Significant Other High Discard Groups: Diabetes • Hypertension • Terminal scr > 1.5 • mg/dL KDPI > 85% • American Journal of Transplantation pages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557 http://onlinelibrary.wiley.com/doi/10.1111/ajt.14557/full#ajt14557-fig-0028

Discard Rates Continue to Rise • Impact of organ allocation sequence since Dec 2014 leading to more sharing, and longer cold times • Metrics for patient and graft survival at one year lead to caution in using deceased donor kidneys – It isn’t enough to say that patient survival improved even with a poorly functioning kidney CT dialysis • Impact of delayed graft function on initial and longer term outcomes

Risk Factors for Delayed Graft Function Fear of delayed allograft function or need for dialysis post transplantation Cost of hospitalization • Complex post management with • dialysis 41% increase in graft loss at • mean of 3.2y and 38% increase in acute rejection (NDT 2009; 24:1039) Less robust kidney function • post transplantation Irish WD. Am Jnl Transplant 2010; 10:2279

The discard rate for biopsied kidneys remained markedly higher than the rate for non-biopsied kidneys ~30% American Journal of Transplantation pages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557 http://onlinelibrary.wiley.com/doi/10.1111/ajt.14557/full#ajt14557-fig-0032

Implantation Biopsy Utility is Disputed • Retrospective single center review: Poor correlation between first and second biopsies; overlapping percent of gs, limited and inconsistent reporting of IF/TA, arteriolar hyalinosis and ATN). Compared to matched controls and contralateral kidney, 1y graft survival was nearly 80% (Kasiske et al. CJASN 2014; 9:562). • Multicenter study of procurement biopsies and ATN: DGF more common with ATN on biopsy but no difference in graft failure rates and ATN only found in 17% of biopsies (Hall; CJASN 2017; 9:573) • Banff Histopathological Consensus Criteria for Pre-Implantation biopsies (Am Jnl Transplant 2017; 17: 140)

What’s Missing? Beyond histology… • Biochemical, immunological and physiological understanding of brain death and impact on post-transplant function • Complex interaction of donor characteristics with clinical management of donor, recipient, surgical implantation, post- operative management, and therapeutics • Call for analysis of discard rate and policies (Kadatz and Gill; CJASN 2018: 13:13)

Goals of UAB Donor Biorepository • To determine the impact of brain death on donor immune activation, graft immune response and recipient allograft function. • To assess pre-donation factors including donor management, donor clinical characteristics, and recipient outcomes (when available) • Assess biological features in discard kidneys

Methods • Under an IRB approval, blood and urine were obtained from deceased donors after brain death (BDD) and cardiac death (DCD), just prior to organ retrieval and the start of cold preservation. • Kidney biopsies were obtained immediately after preservation. • As a control, blood and urine were obtained from healthy volunteers and biopsy tissue from a biospecimen bank at UAB. • Gene expression in kidney biopsies was analyzed by real-time PCR while serum and urine were analyzed by Luminex™ assay and urine values normalized to urine creatinine.

Donor Demographics and Recipient 4 kidneys: Exported 23 kidneys: Discarded 34 BDD Donor age • (68 K) Poor pump numbers • 64 kidneys: Local Organ anatomical damage/defect • Arteriosclerosis • Intimal dissection/surgical cut • 41 kidneys: Transplanted 68 BDD (136 K) Donors Recipients (n=34) (n=41) Mean cold ischemia time (CIT; hours) 21 (1->40) --- Mean age (Years) 45 (17-69) 53 (29-72) African American Race 10 (29%) 27 (66%) Male Gender 23 (68%) 26 (63%) KDPI 58 (2-100) ---

Gene Expression in BD Donor Relative mRNA expression Kidney Biopsies (n=28) 100.00 10.00 1.00 SOD3 IL4 GZMB SMAD7 SMAD2 CCL3 ITGAM HGF LTA CCR2 TNFSF10 TIMP1 C4A SOD2 TLR1 TLR8 IL8 TLR2 TNFRSF13B LTF MPO PTPRC PRF1 GAPDH CD28 0.10 0.01 BDD only ( ≥ 2-fold vs. Normal kidney) ↑ - 41 genes upregulated ↓ - 9 genes downregulated

Functional Analysis of Gene Expression in BDD Kidney Biopsies (n=28) Cytokines/Chemokines Ischemia reperfusion Apoptosis/Necrosis 10.0 100.00 100 ( ≥ 2-fold change vs. Healthy control) 10.00 10 Relative mRNA expression 1.00 1.0 HMOX1 1 HAVCR1 SOD3 SOD2 TLR1 TLR2 TLR4 TLR5 TLR8 LTF MPO 0.10 0.01 0.1 Immune Activators Endothelial injury Matrix/Fibrosis 100 10.0 10.0 10 1.0 HGF SMAD2 SMAD7 TIMP1 COL1A1 IGF1 MMP7 1 1.0 C3 C4A CLU ITGAL ITGAM 0.1

Alteration of Cytokines/Chemokines in Serum and Urine BD Donor Detected by Luminex Urine Serum Serum cytokines 400 60 * Urine cytokine * 300 (pg/mg Ucr) (pg/ml) 40 200 * * 20 100 * 0 0 IL-6 IL-10 IL-15 EGF IL-15 Healthy control, n=11 BDD, n=22(serum), 24(Urine)

Urine MCP-1 Expression in BD Donor Correlates with Recipient Renal Function 100 Urine MCP-1 Cytokine ( pg/mg Ucr) Rho: -0.751, p=0.008 Recipient GFR 6000 80 * at 6-month 60 4000 40 2000 20 0 0 Healthy DBD (n=24) 0 2000 4000 6000 8000 control (n=11) MCP-1 (pg/mg Ucr) Serum MCP-1 1500 Cytokine (pg/ml) 1000 500 0 Healthy DBD (n=22) control (n=11)

MCP-1 Expression Is Enhanced in BDD Kidneys and Urine Levels Predict Delayed Graft Function Control Donors “Low” “Medium” “High” Urine Serum * 2000 2000 2500 NS (pg/ml/mg/mlCr) 2500 MCP-1 (pg/ml) 2000 1500 2000 1500 MCP-1 1500 1500 1000 1000 1000 1000 500 500 500 500 0 0 0 0 control control non-DGF DGF discarded control non-DGF DGF discarded control

Urine Expression of Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Elevated in BD Donors Urine * 400 (ng/ml/mg/mlCr) 300 NGAL * * 200 100 0 control non-DGF DGF discarded Serum * * 600 NGAL (ng/ml) * 400 200 0 control non-DGF DGF discarded

IL-18 is Increased in Urine and Serum of DGF and Discarded Donors Urine * (pg/ml/mg/mlCr) 2500 2000 IL-18 * 1500 1000 500 0 control non-DGF DGF discarded Serum * * 2500 IL-18 (pg/ml) 2000 1500 1000 500 0 control non-DGF DGF discarded

ROC of Best Urine Markers uNGAL uIL-018 Area Under the Curve Area Under the Curve Test Result Variable(s): urine_NGAL Test Result Variable(s): urine_IL18 Asymptotic 95% Asymptotic 95% Confidence Interval Confidence Interval Std. Asymptoti Lower Upper Std. Asymptotic Lower Upper Area Error a c Sig. b Bound Bound Area Error a Sig. b Bound Bound .692 .111 .121 .475 .910 .750 .111 .059 .532 .968 a. Under the nonparametric assumption a. Under the nonparametric assumption b. Null hypothesis: true area = 0.5 b. Null hypothesis: true area = 0.5

High Mobility Group Box 1 (HMGB1) - Introduction Control Hypoxia, ischemia, trauma Damage Associated Molecular Pattern proteins (DAMPs) 200x CsA High Mobility group box 1 (HMGB1) Inflammation, organ injury 200x

HMGB1 Release is Detected in the Urine and Serum of Deceased BD Donors URINE SERUM non-DGF DGF non-DGF DGF * 3 4 * (fold non-DGF) (fold non-DGF) HMGB1 serum HMGB1 urine 3 2 2 1 1 0 0 non-DGF DGF non-DGF DGF

Quantification of HMGB1 in Urine of Brain Dead Donors: correlation with DGF and Discard * 4000 * (pg/ml/mg/mlCr) HMGB1 urine 3000 2000 1000 0 discarded control non-DGF DGF

HMGB1 Translocation is Detected in Kidneys from Brain Dead Donors non-DGF DGF Discarded

HMGB1 Expression in Recipient’s Urine before and after Transplant Recipient 1 Recipient 2 Recipient 3 Recipient 4 Pre-transplant + + + + CTL CTL Post-transplant 0 4 12 0 4 12 0 4 12 0 4 12 (weeks) HMGB1 (urine)

Summary • BD donors demonstrate activation of inflammatory pathways that are frequently systemic. • Among several AKI biomarkers tested, urine MCP-1 and NGAL, as well as serum and urine IL-18, were significantly elevated in donors with DGF or that were later discarded. • Serum and urine TNF α levels were not discriminatory among donor groups. • The extent of HMGB1 flux in the donors could be a biologic marker of kidney injury that predicts donor-related DGF and can be an indicator of graft function in Recipients.