Pre-implantation Kidney Biopsies as a Predictor for Delayed Graft Function

Roslyn B. Mannon MD, FASN, FAST

Professor of Medicine and SurgeryUniversity 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 Transplantationpages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557http://onlinelibrary.wiley.com/doi/10.1111/ajt.14557/full#ajt14557-fig-0001

American Journal of Transplantationpages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557http://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

outcomes

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 Transplantationpages 18-113, 2 JAN 2018 DOI: 10.1111/ajt.14557http://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-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

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

SMAD

7

SMAD

2

PTPR

C

CCL3

ITGA

M

HGF

LTA

CCR2

PRF1

GAPD

H

TNFSF10

TIMP1 C4A

SOD2

TLR1

TLR8 IL8

CD28

TLR2

TNFRSF13B

LTF

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

Relative

mRN

A e

xpre

ssion

↑ - 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

HAV

CR1

HMOX1 LTF

MPO

SOD3

SOD2

TLR1

TLR2

TLR4

TLR5

TLR8

Ischemiareperfusion

0.1

1.0

10.0

Cytokines/Chemokines

1

10

100

C3 C4A CLU

Endothelialinjury

1.0

10.0

ITGAL ITGAM

ImmuneActivators

0.1

1.0

10.0

COL1A1 HGF

IGF1

MMP7

SMAD

2SM

AD7

TIMP1

Matrix/Fibrosis

Relative

mRN

A e

xpre

ssion

( ≥2

-fold

chan

ge v

s. H

ealthy

con

trol)

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

Luminex

Healthy control, n=11

BDD, n=22(serum), 24(Urine)

Seru

m c

ytok

ines

(p

g/ml)

0

20

40

60

IL-15

Urine

cyt

okine

(p

g/mg

Ucr

)

0

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

Cyto

kine

(pg

/ml) Serum MCP-1

0

500

1000

1500

Healthy control (n=11)

DBD (n=22)

Cyto

kine

(pg

/mg

Ucr

)*

Urine MCP-1

0

2000

4000

6000

Healthy control (n=11)

DBD (n=24)0

20406080

100

0 2000 4000 6000 8000

Rho: -0.751, p=0.008

Recipien

tGF

R at

6-m

onth

MCP-1 (pg/mg Ucr)

“Low” “Medium” “High”

Donors

Urine

control discarded0

500

1000

1500

2000

0500

1000150020002500

control discarded0

5001000150020002500

control non-DGF DGF

MCP

-1

(pg/

ml/m

g/m

lCr) *

MCP

-1 (p

g/m

l)0

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

NG

AL

(ng/

ml/m

g/m

lCr)

Urine

*

0

100

200

300

400

non-DGF DGFcontrol discarded

*

NG

AL

(ng/

ml)

Serum

0

200

400

600

non-DGF DGFcontrol discarded

** *

*

IL-1

8 (p

g/m

l/mg/

mlC

r)

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

IL-1

8 (p

g/m

l)

Urine

Serum

0500

1000150020002500

0

500

1000

1500

2000

2500

non-DGF DGFcontrol discarded

non-DGF DGFcontrol discarded

*

*

*

*

Area Under the CurveTest Result Variable(s): urine_IL18

AreaStd.

ErroraAsymptotic

Sig.b

Asymptotic 95% Confidence IntervalLower Bound

Upper Bound

.750 .111 .059 .532 .968a. Under the nonparametric assumptionb. Null hypothesis: true area = 0.5

uIL-018 uNGAL

Area Under the CurveTest Result Variable(s): urine_NGAL

AreaStd.

ErroraAsymptoti

c Sig.b

Asymptotic 95% Confidence IntervalLower Bound

Upper Bound

.692 .111 .121 .475 .910a. Under the nonparametric assumptionb. 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

0

1

2

3

4

non-DGF DGF

URINE

HM

GB

1 ur

ine

(fold

non

-DG

F)

non-DGF DGF0

1

2

3

SERUM

HM

GB

1 se

rum

(fold

non

-DG

F)

non-DGF DGF

non-DGF DGF

**

0

1000

2000

3000

4000HM

GB1

urin

e(p

g/m

l/mg/

mlC

r)

non-DGF DGFcontrol discarded

**

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

DGF Discardednon-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 4Recipient 3

+ + + +

40 12 40 12 40 12 40 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 of 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 of 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 outcomes in kidney transplant patients.