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

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 are Still Significant

Other High Discard Groups:

• Diabetes
• Hypertension
• Terminal scr > 1.5

mg/dL

• KDPI > 85%

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
• utcomes

Risk Factors for Delayed Graft Function

Irish WD. Am Jnl Transplant 2010; 10:2279

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

The discard rate for biopsied kidneys remained markedly higher than the rate for non-biopsied kidneys

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

~30%

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-

• perative 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

• rgan 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

Donors (n=34) Recipients (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)

• 34 BDD

(68 K) 64 kidneys: Local 4 kidneys: Exported 41 kidneys: Transplanted 23 kidneys: Discarded

• Donor age
• Poor pump numbers
• Organ anatomical damage/defect
• Arteriosclerosis
• Intimal dissection/surgical cut

68 BDD (136 K)

Gene Expression in BD Donor Kidney Biopsies (n=28)

0.01 0.10 1.00 10.00 100.00 MPO SOD3 IL4 GZMB SMAD7 SMAD2 PTPRC CCL3 ITGAM HGF LTA CCR2 PRF1 GAPDH TNFSF10 TIMP1 C4A SOD2 TLR1 TLR8 IL8 CD28 TLR2 TNFRSF13B LTF

BDD only ( ≥ 2-fold vs. Normal kidney)

Relative mRNA expression

↑ - 41 genes upregulated ↓ - 9 genes downregulated

Functional Analysis of Gene Expression in BDD Kidney Biopsies (n=28)

1 10 100

Apoptosis/Necrosis

0.01 0.10 1.00 10.00 100.00

HAVCR1 HMOX1 LTF MPO SOD3 SOD2 TLR1 TLR2 TLR4 TLR5 TLR8

Ischemia reperfusion

0.1 1.0 10.0

Cytokines/Chemokines

1 10 100 C3 C4A CLU

Endothelial injury

1.0 10.0 ITGAL ITGAM

Immune Activators

0.1 1.0 10.0 COL1A1 HGF IGF1 MMP7 SMAD2 SMAD7 TIMP1

Matrix/Fibrosis

Relative mRNA expression ( ≥2-fold change vs. Healthy control)

Alteration of Cytokines/Chemokines in Serum and Urine BD Donor Detected by Luminex

Healthy control, n=11 BDD, n=22(serum), 24(Urine) Serum cytokines (pg/ml) 20 40 60 IL-15 Urine cytokine (pg/mg Ucr) 100 200 300 400 IL-6 IL-10 IL-15 EGF

* * * * Serum Urine *

Urine MCP-1 Expression in BD Donor Correlates with Recipient Renal Function

Cytokine (pg/ml) Serum MCP-1 500 1000 1500 Healthy control (n=11) DBD (n=22) Cytokine ( pg/mg Ucr)

*

Urine MCP-1 2000 4000 6000 Healthy control (n=11) DBD (n=24) 20 40 60 80 100 2000 4000 6000 8000

Rho: -0.751, p=0.008 Recipient GFR

at 6-month

MCP-1 (pg/mg Ucr)

“Low” “Medium” “High”

Donors

Urine

control discarded

500 1000 1500 2000 500 1000 1500 2000 2500

control discarded

500 1000 1500 2000 2500

control non-DGF DGF MCP-1 (pg/ml/mg/mlCr)

*

MCP-1 (pg/ml)

500 1000 1500 2000

control non-DGF DGF

NS

Serum

Control

MCP-1 Expression Is Enhanced in BDD Kidneys and Urine Levels Predict Delayed Graft Function

Urine Expression of Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Elevated in BD Donors

NGAL (ng/ml/mg/mlCr)

Urine

*

100 200 300 400

non-DGF DGF control discarded

*

NGAL (ng/ml)

Serum

200 400 600

non-DGF DGF control discarded

* * * *

IL-18 (pg/ml/mg/mlCr)

IL-18 is Increased in Urine and Serum of DGF and Discarded Donors

IL-18 (pg/ml)

Urine Serum

500 1000 1500 2000 2500 500 1000 1500 2000 2500

non-DGF DGF control discarded non-DGF DGF control discarded

* * * *

Area Under the Curve

Test Result Variable(s): urine_IL18 Area Std. Errora Asymptotic Sig.b Asymptotic 95% Confidence Interval Lower Bound Upper Bound .750 .111 .059 .532 .968

• a. Under the nonparametric assumption
• b. Null hypothesis: true area = 0.5

uIL-018 uNGAL

Area Under the Curve

Test Result Variable(s): urine_NGAL Area Std. Errora Asymptoti c Sig.b Asymptotic 95% Confidence Interval Lower Bound Upper Bound .692 .111 .121 .475 .910

• a. Under the nonparametric assumption
• b. Null hypothesis: true area = 0.5

ROC of Best Urine Markers

High Mobility Group Box 1 (HMGB1)

• Introduction

Damage Associated Molecular Pattern proteins (DAMPs) High Mobility group box 1 (HMGB1) Hypoxia, ischemia, trauma Inflammation, organ injury

200x 200x

Control CsA

HMGB1 Release is Detected in the Urine and Serum of Deceased BD Donors

1 2 3 4

non-DGF DGF

URINE HMGB1 urine (fold non-DGF)

non-DGF DGF

1 2 3

SERUM HMGB1 serum (fold non-DGF)

non-DGF DGF

non-DGF DGF * *

1000 2000 3000 4000

HMGB1 urine (pg/ml/mg/mlCr) non-DGF DGF control discarded

* * Quantification of HMGB1 in Urine of Brain Dead Donors: correlation with DGF and Discard

DGF Discarded non-DGF

HMGB1 Translocation is Detected in Kidneys from Brain Dead Donors

HMGB1 Expression in Recipient’s Urine before and after Transplant

Recipient 2 HMGB1 (urine)

Pre-transplant Post-transplant (weeks)

Recipient 1 Recipient 4 Recipient 3

+ + + + 4 12 4 12 4 12 4 12

CTL CTL

• 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 TNFα 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.

Summary

Conclusion

Specific biomarkers to predict kidney injury in recipients following transplantation would provide important information for clinical management and further enhance organ utilization.

Acknowledgments

• UAB Laboratory

– Anna Zmijewska – Jianguo Chen – Jarek Zmijewska – Michael Seifert – Miriam Bernard – John Murphy

• Extramural Colleagues

– Arthur Matas and the DeKAF Study Group – CTOT-10/12/15/19/21 Study Groups

• UAB Surgery

– Jayme Locke – Carlton Young – Michael Hanaway – Joseph Tector – Jared White – Devin Eckhoff – Stephen Gray

MCP-1 Expression Is Upregulated in the Kidney

• f BD Donors

Normal

“Low” “Medium” “High”

BD Donors

Cytosolic Interstitial Luminal

Summary

• In brain dead donor kidneys, prior to reperfusion and

preservation, there is an enhanced expression of genes associated with apoptosis, inflammation, ischemia, and endothelial injury, and upregulation of molecules associated with fibrogenesis.

• Serum levels of IL-6, IL-10, IL-15, and EGF and Urine IL-15

were significantly increased in BD donors compared to normal healthy individuals.

• Urine levels of MCP-1 were significantly elevated compared

to normal healthy individuals, but serum levels were not.

• There was a strong negative association between donor urine

MCP1 and recipient eGFR at 6months.

• Immunohistochemical staining demonstrated that MCP-1 was

enhanced, predominantly expressed in renal tubular epithelial cells, greater in the cortex than medulla.

Conclusions

• BD donors demonstrate activation of inflammatory

pathways that are frequently systemic. In the case

• f MCP-1, localized production in the kidney is

enhanced following BD. Further investigation into this pathway may shed light on innate immune activation in the allograft.

• Donor urinary MCP-1 may be a useful noninvasive

marker for screening donors and predicting graft

• utcomes in kidney transplant patients.