1. Contribute to Oxidant Stress and
TRPM2 Channels Activate Rac1 and
Contribute to Oxidant Stress and
Ischemic AKI
Brian Reeves Division of Nephrology
Penn State College of Medicine

2. TRPM2 channels Permeable to sodium and calcium Widely expressed
– 1500 aa (170 kDa)
Long intracellular N- terminus
C-terminus ADPR
hydrolase activity
Mediate H2O2-
induced cell death
Sumoza-Toledo and Penner, J Physiol 2011

3. Early events in renal ischemia/reperfusion
Why TRMP2?
Early events in renal ischemia/reperfusion
Cellular calcium and sodium overload
TRPM2 permeable to sodium, calcium and potassium
Activation of DNA repair mechanisms with
resulting ADPR production
TRPM2 gated by ADPR
Production of reactive oxygen species
TRPM2 gated by H2O2
Production of TNF-a
TRPM2 activated by TNF (probably indirect)
Injury exacerbated by high temperature
TRPM2 activated by high temperature
Injury reduced by acidosis
TRPM2 gating inhibited by acidosis

4. Preparation of TRPM2 KO Mice
Transmembrane Domain
WT TRPM2
Ca2+ Pore
Neo
Conditional
Adenovirus EIIa
promoter Cre Recombinase
(targets early embryo)
TRPM2 KO X
loxP
loxP =
Result
Whole Animal KO

5. Expression of TRPM2 in mouse kidney
WT-TRPM2/DAPI
WT-TRPM2/LTL
TRPM2 KO

6. TRPM2 KO mice are resistant to renal I/R injury
140
1.2
WT I/R
WT I/R
KO I/R
WT sham KO sham
KO I/R
WT sham KO sham
120
1.0
creatinine (mg/dl)
BUN (mg/dl)
100
0.8 * * 80 *
0.6 * 60 *
0.4 40 * 20
0.2
* P<0.02 vs WT
0.0 24
hours 48 24
hours 48

7. TRPM2 KO mice are resistant to renal I/R injury
WT Sham TRPM2KO Sham
WT IR 24h
TRPM2KO IR 24h

8. Apoptotic pathways are suppressed in TRPM2 KO kidneys

9. Localization of TRPM2 in kidney
sham I/R 6 h no 1o Ab
TRPM2 mRNA expression after I/R (fold change) 1
0.8
0.6
0.4
0.2
MicroArray Realtime
PCR

10. Parenchymal TRPM2 Mediates Ischemia/reperfusion Injury
2.5
180
160
140
120
100 80 60 40 20
BUN
creatinine
2.0
Creatinine (mg/dl)
BUN (mg/dl)
1.5
* P<0.001 vs WT>WT
1.0 * * * *
0.5
0.0
WT>WT
KO>WT WT>KO
KO>KO

11. Histologic changes in TRPM2 chimeric mice
WT>WT
KO>WT
WT>KO
KO>KO

12. Pharmacologic inhibition of TRPM2:
Effect of clotrimazole on I/R injury
160
1.6
vehicle I/R clotrimazole I/R
vehicle
clotrimazole
140
1.4
vehicle sham
clotrimazole sham
120
1.2
creatinine (mg/dl)
BUN (mg/dl)
100
1.0 80
0.8 60
0.6 * * * * * * 40
0.4 20
0.2
0.0 24
time (hrs) 48 72 24
time (hrs) 48 72
* P<0.02 vs vehicle

13. Pharmacologic inhibition of TRPM2: Effect of 2-APB on I/R injury
2 . 0
c r e a t in in e ( m g /d l)
1 . 5 *
1 . 0 **
0 . 5 **
0 . 0
v e h i c l e
1 h r a f t e r
6 h r a f t e r
1 h r b e f o r e
1 2 h r a f t e r
2 - A P B
a d m i n i s t r a t i o n
2-APB (2-aminoethoxydiphenyl borate)

14. Summary – part 1
TRPM2 KO mice are viable and have normal kidney function/histology
TRPM2 KO mice are resistant to ischemia/reperfusion renal injury
Effects of TRPM2 are referable to its expression in parenchymal
cells
TRPM2 KO mice exhibit better preserved renal histology, less renal apoptosis and neutrophil infiltration after I/R
Apoptotic pathways are suppressed in TRPM2 KO kidneys after
I/R
Pharmacologic blockade of TRPM2 protects against renal ischemic reperfusion injury in vivo

15. No change in kidney electrolyte content

16. TRPM2 increases oxidant stress
NADPH Oxidase

17. Rac1 and oxidative stress
Rac1 is a subunit of NAPDH oxidase.
When GTP bound, activated Rac1 and phosphorylated p47phox & p67phox translocate to membrane to form the NAPDH complex to initiate production of reactive oxygen species
Another TRP channel, TRPC5, forms a complex with Rac1 and Rac1 enhances membrane level of TRPC5.
NAPDH oxidase
membrane
p47 phox
p40phox
p67 phox
Rac1
GDP
NADPH + 2O2
GTP ℗
Rac1
GDP
GTP
NADP+ + 2O2°- + H+
Bosco et al, Chem Biol. 2012; Bezzerides V, et al. Nat Cell Biol. 2004; Christophe M and Nicolas S. Curr Pharm Des, 2006.

18. TRPM2-dependent Rac1 activation

19. - - TRPM2 associates with active Racl 1Hci02 + - + + + - - - - - ++ -EV
Myc-Rac1- VT V5-TRPMl H
Rae
++ -
- - EV
Myc-Ra-c1..:\\fT
V5-TRPM2
H:i
+ + +
EV +
Myc-Rac - vr MY c-Rac1-CA
My1c-Rac1l-DN
V5-T1RPMl
1Hci + -
- - + +
++ ++
- - + - +
TRPM2
PAb Ract TRPM1 -
Aliltf-V5 tg;G
TRPMl
IP:Arntii-MYic
TIRPMl
TRPMl
illl pllJt
Rac1
illlplllt
Rac1

20. TRPM2 associates with Rac1 after I/R
sham
I/R 6 hr

21. Rac1 inhibition reduces I/R injury

22. Rac1 dependent oxidant stress in I/R
vehicle
NSC23766
Blot Ab
Anti-4 Hydroxynonenal antibody
cleaved caspase-3

23. Summary- part 2
Less oxidative stress in TRPM2 KO mice kidney after I/R
Rac1 is activated in renal I/R injury
Rac1 activation is blunted in TRPM2 KO mice
Rac1 interacts with TRPM2 in vivo and in vitro. Activation of Rac1 enhances TRPM2/Rac1 interactions
Rac1 increases membrane localization of TRPM2 (not
shown)
Pharmacologic inhibition of Rac1 protects against renal ischemia reperfusion injury and reduces oxidative stress

24. TRPM2-mediated Rac1 and NADPH activation in oxidative stress
NAPDH ?
TRPM2
membrane
p47
p67
p40phox phox phox Rac1
℗ ℗
GTP
Oxidative stress
GTP
Rac1
GDP
Apoptosis inflammatory responses
Apoptosis inflammatory responses

25. Conclusions
TRPM2 is an important ‘death channel’ which mediates renal ischemia reperfusion injury in vivo and hypoxic epithelial cell death in vitro.
Pharmacologic inhibition of TRPM2 may be effective for the prevention of ischemic AKI
Rac1 is activated in I/R in a TRPM2-dependent fashion
Pharmacologic inhibition of Rac1 may be
effective for the prevention of ischemic AKI

26. Future directions Location of relevant TRPM2 expression: endothelial
cell, tubule cells
Location of cellular TRPM2: plasma membrane,
endosome, lysosome, or mitochondria?
Pharmacologic inhibitors of TRPM2: screening assay
Mechanisms for TRPM2 activation
How does TRPM2 activate Rac1? •
How does TRPM2 suppress Akt activation?

27. Acknowledgements Barbara Miller, Division of Pediatric
Hematology/Oncology Guofeng Gao
Raghu Tadagavadi Weiwei Wang Nicole Briley Michael Love
Genzyme (Renal Innovations Program grant)