Microvascular inflammation and endothelial cell activation in kidney

Microvascular inflammation and endothelial cell activation in kidney antibody mediated rejection

Michael Mengel Department of Laboratory Medicine and Pathology University of Alberta, Edmonton, Canada

Three Pathways to Antibody-Mediated Injury Antibody Alone

Complement Mediated

Cell Mediated (FcR)

Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

Role of C4d in antibody-mediated rejection Classical complement pathway activation: Antibody + Antigen

C1 C4

C4a + C4b 

Mannose binding lectin/MASP1

C4d binds covalently to local site

Helmut Feucht Clin Exp Immunol 86:464, 1991

Detection of C4d is crucial for diagnosing antibody mediated rejection

Microcirculation inflammation in AMR Kidney

CD3

Heart

CD68

Phenotype of glomerulits CD15 – early AMR

CD68 – late AMR

Diagnosis of AMR

Mengel M et al. Transpl Int. 2012 Jun;25(6):611-22

Follow up of C4d positive biopsies and the development of TX-Glomerulopathy • Significant more often associated with Transplant Glomerulopathy (53% vs. 14%) • Significant more often associated with Transplant Capillaropathy (71% vs. 13%) • Significantly associated with progression of Transplant Glomerulopathy in follow-up biopsy (82% vs. 27%, median after 23 months)

Regele et al.

Pathogenesis of Capillaropathy

Transplant-Capillaropathy

antibody-mediated injury and microcirculation inflammation glomerulitis

glomerulopathy

Sequential development of CHR in non-human primates No CHR

106

Stage I

Stage II

182

225

Stage III

352

Stage IV

371

Days post-transplant

Smith et al (Boston) AJT 8:1662, 2008

C4d versus microcirculation inflammation in biopsies prior to AMR treatment (1996-2001)

Verghese et al. Clin. Transplant 2013 in press

DeKAF Study Biopsies for late graft dysfunction C4d-DSA-

C4d-DSA+ C4d+DSAC4d+DSA+

Months post-bx

N=173 Gaston et al Transplant 90:68,2010

Graft Survival Banff lesions

unsupervised Principal Component Analysis

Limited specificity of microcirculation inflammation Fahim et al. Am J Transplant. 2007 Feb;7(2):385-93.

% cases with capillaritis

Gibson et al. Am J Transplant. 2008 Apr;8(4):819-25. 80 70 60 50 40 30 20 10 0

68.4 45.7

14.1

no rejection (n=453)

borderline (n=105)

TCMR (n=76)

The association of TG (D) (n=44) with antibody (A), PTCBMML (B), and C4d (C). TG phenotype*

A

B

C

D

n (%)

“ABCD”

+

+

+

+

10 (27)

“ABD”

+

+

-

+

12 (32)

“ACD”

+

-

+

+

2 (5)

“AD”

+

-

-

+

1 (3)

“BCD”

-

+

+

+

1 (3)

“BD”

-

+

-

+

9 (24)

“CD”

-

-

+

+

1 (3)

“D”

-

-

-

+

1 (3)

73% of Tg cases show some signs of humoral rejection Sis et al. AJT 2007; 7: 1743-1752

Loupy et al: Subclinical progressive microcirculation injury in presensitized patients, despite C4d negativity

Potential causes for C4d negativity

Complement activation Complement dependent cell injury Endothelial activation Recruitment and activation of leukocytes

C4d+ ABMR

Is C4d deposited in low amounts (Thus not detectable by current methods) Treatment effects?

? C4d negative ABMR

Do HLA antibodies in a complementindependent way cause EC activation and subsequent inflammation and Fc receptor mediated graft injury?

?

Three Pathways to Antibody-Mediated Injury Antibody Alone

Complement Mediated

Cell Mediated (FcR)

Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

endothelial genes are increased in AMR Gene Symbol VWF CAV1 RHOJ MCAM CDH5 SELE PALMD PECAM1 KLF4 CYYR1 CD34 TEK SOX18 ZNF521 RASIP1 HOXD4 RAI14 PODXL DLC1 Also not in our strict FGD5 definition of ENDAT list, FOXF2 but increased in ABMR: EMCN CDH13 KDR Duffy blood group CETP SOX7 MAOB THBD MALL

N Normalized 1.05 1.17 0.99 1.12 1.00 1.06 0.93 0.93 1.11 1.04 1.05 1.04 1.02 0.92 0.98 1.02 0.95 1.11 0.95 0.88 1.02 1.01 0.94 1.04 0.97

Signal 115.36 206.07 27.60 192.50 75.34 23.25 78.15 294.26 236.31 162.34 138.33 190.45 13.48 37.82 80.80 372.75 273.72 1489.49 273.83 212.06 12.59 700.89 307.06 26.61 1450.68

ABMR Normalized 6.39 5.35 2.94 2.93 2.58 2.23 2.12 2.00 1.52 1.52 1.50 1.47 1.46 1.38 1.37 1.33 1.21 1.06 0.98 0.97 0.86 0.85 0.77 0.72 0.69

Signal 738.30 857.90 85.27 476.93 201.54 55.26 182.35 638.35 318.70 238.68 198.87 275.89 19.92 55.56 114.53 495.27 347.63 1404.16 287.39 233.58 10.61 600.32 244.78 18.27 1031.30

Red arrows indicate genes that are known to be involved in endothelial cell activation

TCMR Normalized 3.39 2.99 1.65 2.02 1.61 1.20 1.36 1.60 1.00 1.15 1.12 1.04 1.08 0.80 0.97 1.02 0.87 0.74 0.74 0.71 0.99 0.64 0.60 1.06 0.52

Signal 419.08 494.11 53.58 326.45 123.20 28.66 122.61 514.29 216.59 182.55 148.89 198.66 14.41 33.75 80.99 378.73 256.99 1067.37 221.40 170.90 12.25 459.15 197.55 29.67 795.14

Welch t test FDR 0.05

Sis et al. AJT 2009;9:2312-23

Endothelial Cell-Associated Transcripts correlate with pathologic features of AMR (in 173 biopsies) Endothelial Transcripts Correlation coefficient

p

C4d deposition

.376

p