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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
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