IMMUNOREGULATORY ROLE OF TISSUE

MICROENVIRONMENT IN KIDNEY INJURY

Mark D. Okusa, MD

Chief, Division of Nephrology

University of Virginia Heath System

Charlottesville, VA

Annual Scientific Meeting cum APSN/HKSN CME Course

Hong Kong 29-30 September 2018

Acute Tubular

Injury

Apoptosis

Necrosis

Necroptosis

Ferroptosis

:Pyroptosis

Tubular Obstruction

Backleak

Microvascular

Injury

Vasoconstriction

Leukocyte Adhesion

↑ Permeability

Microvascular

Congestion

Innate

Immunity

Inflammation

DAMPS

Immune Cells

Cytokines

Microenvironment Systemic Response

Hemodynamic

Neural

Immunological

Oxidative Stress

Ischemia-Reperfusion InjuryControlling Inflammation in the Microenvironment

Purinergic signaling – in the Microenvironment

Modified, Lu, AJP-Cell Physiology, 2014

ATP-releasechannels P2X P2Y AR

Renal Interstitial Microenvironment

B. Kaissling, M. Le Hir Histochem Cell Biol. 2008: 130:247-62, 2008

Fibroblasts

CD73

DCs

MCH Class II

CD73

CD39ATP

ADENOSINE

L. Li et al. Kidney Int. 2008 74:1526-37

Mononuclear Phagocytes- contiguous network in kidney

T J Soos et al. Kidney Int. 2006 70:591–596

Green = CX3CR1-GFPRed = MHC Class II

Red = F4/80

Dendritic Cell Microenvironment

Immature DC Mature DC Effector T cellT Reg Cell

Cell to Cell

Contact

Humoral

mediators

(adenosine)

DAMPsPAMPS

Tissue Resident

Autophagy

Filtered Antigens

Tolerance Immunity

CD73 Is a 5’-Nucleotidase Generates Extracellular Adenosine

B. Kaissling, M. Le Hir Histochem Cell Biol. 2008

CD73

MHCCD73

MHC

DAPI

CD73 Expression in PT Cells and Mesangial Cells

S. S. Sung et al. J Am Soc Nephrol. 2017 Mar;28(3):888-902

Ischemia-Reperfusion Injury: Methods

Creatinine is a Marker of Kidney Function

0.0

0.5

1.0

1.5

2.0

Pla

sm

a C

rea

tin

ine, m

g/d

l

Sham IRI

Creatinine --Kidney Injury

22-26 min 24 hr

ReperfusionIschemiaEquilibration

S. S. Sung et al. J Am Soc Nephrol. 2017 Mar;28(3):888-902

Absence of CD73 Sensitizes Mice to IRI

CD73 KO mice was a gift from Linda Thompson

Non-hematopoietic cell CD73 protects kidneys from mild IRI

WT

CD73-/-

WT

CD73-/-

S. S. Sung et al. J Am Soc

Nephrol. 2017 Mar;28(3):888-902

PEPCK Cre Gift of Volker Haase, Vanderbilt UniversityCD73 fl/fl Gift of Dr. Jurgen Schrader, Heinrich-Heine-Universität Düsseldorf

loxP

Genes of interest

Deleted in specific cells

PEPCK

Cre-LoxP SystemConditional KO mice

CD11c Cre CD73fl/fl “DC” CD73 KO

PEPCK Cre CD73fl/fl Proximal Tubule CD73 KO

Floxed CD73 mouse CD11c Cre mouse

PEPCK Cre mouse

CD73

CD73

PEPCK Cre gift of Volker Haase, Vanderbilt

Proximal Tubule Deficient CD73 (PEPCK Cre CD73fl/fl)

PEPCK Cre PEPCK Cre CD73fl/fl

Proximal Tubule CD73 Is Necessary To Protect From IRI

S. S. Sung et al. J Am Soc Nephrol. 2017 Mar;28(3):888-902

CD73 inhibition increases IRI

susceptibility in WT mice

CD73 Protection Due To Its Enzymatic Activity

5’-NT Antagonist:

AMP-CH2-P: 50 mg/Kg/day

5’-NT: 600 U/Kg/day

A2aR Agonist: ATL

5’ Nucleotidase or A2a agonists Rescue CD73 KO Mice

S. S. Sung et al. J Am Soc Nephrol. 2017 Mar;28(3):888-902

CD73

CD39ATP

ADENOSINE

A2aR

ATL

Diabetes

Autoimmunity

Tumor

Foreign Body

Infectious agents

Adapted from G. Wick et al. Annu.

Rev. Immunol. 2013. 31:107–35

Chronic Inflammation and Fibrosis: Microenvironment

B. Kaissling and M. Le Hir,

Histochem Cell Biol

130:247-62, 2008

CD73

CD39ATP

ADENOSINE

CD73-fibroblasts

A2aR

Li Li et al 2012 JCI

Perivascular cell-specific CD73 KO (FoxD1CreCD73fl/fl) mouse model

CD73fl/fl Foxd1Cre CD73fl/fl

CD

73

P

DG

FRβ

CD

31

Sung et. al, JASN 2017

5μm

Proximal tubule

Perivascular cell

Kidney function and collagen deposition in FoxD1CreCD73fl/fl mice

are increased in 14-day unilateral IRI-fibrosis model

***p≤0.001, 2-way ANOVA, t-test

Pla

sm

a c

reatinin

e (

mg/d

l)

Male, 8-12 weeks old, 18-25 g

***

Plasma creatinine and collagen deposition in FoxD1CreCD73fl/fl

mice are increased in 14-day unilateral IRI-fibrosis model

***p≤0.001, 2-way ANOVA, t-test

Pla

sm

a c

reatinin

e (

mg/d

l)

Male, 8-12 weeks old, 18-25 g

***

Lack of CD73 in perivascular cells leads to more myofibroblast (PDGFRβ+ αSMA+) transformation

20μm

N. Goerldt, J. Schrader, M. Okusa et al. unpublished observations 2018

Isolated pericytes are PDGFRβ-positive

20μm

PDGFRβ αSMA

Kidney isolation

Tubular and glomerulardepletion

Positive PDGFRβ selection

N. Goerldt, J. Schrader, M. Okusa et al. unpublished observations 2018

CD73 deficient pericytes/fibroblasts proliferate faster and express

more α-smooth muscle actin

20μm

Proliferation assay (MTT)

**p≤0.01, ****p≤0.0001, 2-way ANOVA

CD73-/-

Wild type

MT

T c

ell

via

bili

ty

(% o

f W

T d

ay 1

)

N. Goerldt, J. Schrader, M. Okusa et al. unpublished observations 2018

Summary/Conclusions

• The kidneys of PT CD73 deficient mice are more susceptible to IRI.• Reconstitution of CD73 KO mice with 5’NT rescues mice to minimize injury.

• Presence of CD73 in perivascular cells protects the kidney from progression to fibrosis after unilateral IRI

• CD73 deficient pericytes/fibroblasts are more prone to transform into myofibroblasts

Acute Kidney Injury

Kidney Fibrosis

CD73 in the kidney microenvironment

ccntrols inflammation and protects kidneys

from AKI and Progressive Fibrosis

Purinergic signaling - overview

ATP-releasechannels P2X P2Y AR

Pannexin1 is an ATP release channel

• Pannexin 1 (PANX1), a transmembrane proteinbelongs to a family of proteins exhibiting a structuralhomology to gap junction-forming invertebrate innexins

• Large, non-selective transmembrane channels thatefficiently release ATP to the extracellular spaceupon activation

• Pannexin 1 is the most ubiquitously expressedof the three pannexins

Penuela, BBA-Biomembranes, 2013

ATP-releasePannexin 1channels

P2X P2Y AR

J. Jankowski et al. J Am Soc Nephrol. 2018 Jul;29(7):1887-1899

W T F /F

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Pla

sm

a c

re

ati

nin

e (

mg

/dL

)

* * * *

s h a m IRI

Panx1 knockout models

Global PT-specific endothelial

+ /+ - / - + /+ - / -

0 .0

0 .5

1 .0

1 .5

2 .0

P

las

ma

cre

ati

nin

e (

mg

/dL

)

s h a m IR I

* * * *

W T F /F

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Pla

sm

a c

re

ati

nin

e (

mg

/dL

)

s h a m IR I

* * * *

protection cell metabolism regulation

Microenvironment in AKI

IL-17

IFN-

Immune Cell Migration

Amplify Tissue Injury

PMN Recruitment

IL-12

Kinsey et al 2012

JASNLi Li et al 2010 JCI

Li Li et al 2007 JI

Lappas et al 2006

JEM

Li Li et al 2012 JCI

ATP/ADP

CD39CD39

AMPCD73

Adenosine Receptors

A1, A2A, A2B, A3

Okusa LabLiping HuangHong YeIsaah Vincent, PhD*Tsuyoshi Inoue, MD, PhDHeather Perry, PhD#

Nicole GörldtJakub JankowskiShinji Tanaka, MD, PhDDebra DruckermanShiqiu Zheng, PhDJunlan Yao, PhDKinga RudnikaJun Li, PhD

Dept. of PharmacologyDiane L. Rosin, PhDChikara Abe, PhDPatrice Guyenet, PhDRuth Stornetta PhD

Dept. of Biomedical EngineeringJohn Hossack, PhDAlexander Klibanov, PhDSushanth GovinahallisathyanarayanaSunil Unnikrishnan, PhD

University of TokyoMasaomi Nangaku, MD, PhDYuichiro Wada, MD, PHDReiko Inagi, PhD

Acknowledgements

Grant Support: MDO (NIH R01 DK105133, NIH SPARC U18EB021787, Coulter Award, Ivy Foundation) and TI and ST (Japanese Society for the Promotion of Science)# NIH K awardee, * T32 trainee

Nephrology/CIIRPeter I. LoboSung-Sang J. SungBert Kinsey#*Amandeep Bajwa#*

Heinrich-Heine-UniversitätDüsseldorfJürgen Schrader

Pannexin 1

ATP

CD 73 AR

AMPADP Adenosine

P2YP2X

Retained ATP

Innate Immunity/Inflammation

Damp

Cell SurvivalCell Death

J. Jankowski et al. J Am Soc Nephrol. 2018 Jul;29(7):1887-1899

ATP