3’,5’-cAMP

CELL

3’,5’-c

AMP

5’-AMP

ADO

ATP

AC

Ecto-3’,5’-PDE

Ecto-5’-NT

ParacrineAutocrine

Transp

orter

Extracellular 3’,5’-cAMP-AdenosinePathway

Jackson, E.K.: Annual Review of Pharmacology and Toxicology 31: 1-35, 1991.

UPLC

ION SOURCE

1st Quadrupole

PARTICLE DETECTOR

Separates Analytes in Time Domain According To Their Physicochemical Properties

Electrospray Ion Source to Gently Ionize Analytes In Sample to Generate Parent Ions

Serves as a Mass Filter to Pass Only the Parent Ion of Interest to Next Part of Mass Spec

2nd QuadrupoleServes as a Collision Cell to Fragment Parent Ion

To Daughter Ions

3rd QuadrupoleServes as a Mass Filter to Pass Only the Daughter

Ion of Interest to Particle Detector

Advantage: Very specific, accurate and sensitive assay

Ultra-Performance Liquid ChromatographCoupled to a Triple Stage Quadrupole Mass Spectrometer

Isolated Perfused Rat Kidney

Tyrode’sSolution

PumpDrugsBubble

Trap

Physiograph

Ureter

Artery

Vein

H2O37°C

WaterCirculator

KIDNEY

Pressure Transducer

Applied LC-MS/MS assay to measure 3’,5’-cAMP in renal venous perfusatefrom isolated, perfused rat kidneys

0 1 2 3 4 5 6 7 8

0

10

20

30

40

50

60

70

80

90

100

3’,5’-cAMP

What the #!%&

is this?!

Rela

tive

Abun

danc

e(F

ull S

cale

: 6.5

x 1

03 )

Retention Time (minutes)

330 m/z (3’,5’-cAMP + H+) → 136 m/z (Adenine + H+)

The unknown and 3’,5’-cAMP had same m/z

Fragmentation of the unknown and 3’,5’-cAMP yielded daughter ions with

same m/z

N

HNN

NH+

NH2

OP

O

O

HO

O

HO

N

NN

NH+

NH2

H

H

OHO

O

HO

N

NN

N

NH2

O

P

O

3’,5’-cAMP

O

N

NN

N

NH2

HO

O O

P

O

HO

HYPOTHESISUnknown = 2’,3’-cAMP

0 1 2 3 4 5 6 7 8 9 100

20

40

60

80

100

Rela

tive

Abun

danc

e(F

ull S

cale

: 1.3

x 1

04 )

Retention Time (minutes)

330 m/z → 136 m/z3’,5’-cAMP

2’,3’-cAMP

0 1 2 3 4 5 6 7 8 9 100

20

40

60

80

100

Unknown

330 m/z → 136 m/z

Rela

tive

Abun

danc

e(F

ull S

cale

: 1.0

x 1

04 )

Renal Venous Perfusate From Isolated, Perfused Rat Kidney: During Isoproterenol

Injected authentic 2’,3’-cAMP into LC-MS/MSand monitored the 330 m/z to 136 m/z transition

Ren, J., Mi, Z., Stewart, N., and Jackson, E.K.: Journal of Pharmacology and Experimental Therapeutics 328: 855-865, 2009.

CELL2’,3

’-cAMP

2’-AMP + 3’-AMP

ADO

mRNA

Ecto-2’,3’-PDEs

Ecto-2’/3’-NTs

ParacrineAutocrine

2’,3’-cAMP

Transp

orter

RNases

Extracellular 2’,3’-cAMP-AdenosinePathway

Jackson, E.K., Ren, J., and Mi, Z. Journal of Biological Chemistry 284, 33097-33106, 2009

2’,3’-cAMP, 3’-AMP and 2’-AMP Are Biological Active

O

N

NN

N

NH2

HO

O O

P

O

HO

2’,3’-cAMP 2’,3’-cGMP

HN

NN

N

OH

N

HN

NH2N

HN

O

2’,3’-cIMP

NH

O

HN

O

2’,3’-cUMP

NH

O

N

H2N

2’,3’-cCMP

All possible 2’,3’-cNMPs, except 2’,3’-cTMP, exist

GuanineUracil

HypoxanthineCytosin

e

Bacteria --- Plants --- Animals --- Humans

Confirms mRNA as source

Conserved, therefore important !

CSF Levels of 2’,3’-cAMP TBI Patients

Renal Venous Perfusate Levels of 2’,3’-cAMP in

Energy-Deprived Kidneys

Even Tobacco Leaves Release 2’,3’-cAMP in Response to Injury!!

Big Questions is: Does the Release of 2’,3’-cAMP Promote or Ameliorate Organ Injury?

CELL2’,3

’-cAMP

2’-AMP + 3’-AMP

ADO

mRNA

Ecto-2’,3’-PDEs

Ecto-2’/3’-NTs

TissueProtection

2’,3’-cAMP

Transp

orter

RNases

mPTPs

Apotosis

Azarashvili et al.AJP-Cell 2009, 296:1428

Hypothesis: 2’,3’-cAMP Release Ameliorates Organ Injury

How to test this hypothesis?

Need somewayto block the

pathway!

2’,3’-Cyclic Nucleotide-3’-Phosphodiesterase (CNPase)

Myllykoski et al.JMB 2013, 425: 4307

Crystallographic Analysis of the Reaction Cycle of 2 ,3 -Cyclic ′ ′Nucleotide 3 -Phosphodiesterase, a Unique Member′

of the 2H Phosphoesterase Family

Adenosine

mRNA

2’,3’-cAMP

2’,3’-cAMP

2’-AMP

Opening of mPTP

(Pro-Apoptotic;

Pro-Necrotic)

Adenosine 2’-AMP

(Cell Membrane)

CNPase

CNPase

Does Attenuating the 2’,3’-cAMP-Adenosine PathwayEnhance or Promote Organ Injury?

CNPase = 2’,3’-Cyclic Nucleotide-3’-Phosphodiesterase

XX

CNPase -/- Mouse Expresses Neurodegeneration With Age

WHAT ABOUT THE 2’,3’-cAMP-ADENOSINEPATHWAY BRAIN INJURY?

CNPase 1CNPase 2

GAPDH

CNPase +/+ at 24 hours post injury CNPase -/- at 24 hours post injury

CNPase +/+ at 7 days post injury CNPase -/- at 7 days post injury

CNPase 1 (46 kDa)

PGVSMCsGMCs

Whole Kidney

MWMarkers

20 kDa

30 kDa

40 kDa

50 kDa60 kDa

80 kDa100 kDa120 kDa

CNPase 2 (48 kDa)

β-actin (42 kDa)

22.76 ± 0.10 24.25 ± 0.11 Ct for CNPaseGMCs PGVSMCs

14.58 ± 0.04 15.11 ± 0.11 Ct for β-actin

IS CNPase Expressed In the Kidney?

Examined CNPase Expression in Rat Whole Kidney, GMCs and PGVSMCs

MWMarkersPGVECsCDCsPTCsTALCs

MWMarkers

CNPase 1 (46 kDa)CNPase 2 (48 kDa)

β-actin (42 kDa)

20 kDa

30 kDa

40 kDa50 kDa60 kDa

80 kDa100 kDa120 kDa

23.64 ± 0.24 23.30 ± 0.06

TALCs PTCs

23.34 ± 0.01 24.32 ± 0.01

CDCs PGVECs

Ct for CNPase

Ct for β-actin 14.24 ± 0.20 14.88 ± 0.02 15.15 ± 0.04 16.28 ± 0.01

IS CNPase Expressed In the Kidney?

Examined CNPase Expression in RatTALCs, PTCs, CDCs and PGVECs

IS CNPase Expressed In the Kidney?

Darlot et al., Topology of Schwann cells and sympathetic innervation along preglomerular vessels: a confocal microscopic study in protein S100B/EGFP transgenic mice.

Am J Physiol Renal 295: F1142-1148, 2008.

Is CNPase Involved in the Metabolism of 2’,3’-cAMP in the Intact Kidney?

Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered 2’,3’-cAMP and measured renal venous levels of

2’,3’-cAMP and 2’-AMP

Is the Metabolism of Endogenous 2’,3’-cAMP Altered in Kidneys from CNPase -/- Mice?

Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered metabolic poisons, dropped kidneys directly into liquid nitrogen,

extracted purines and measured total kidney levels of purines.

Is the Metabolism of 2’,3’-cAMP Altered in Kidneys from CNPase -/- Mice?

Isolated and perfused kidneys from CNPase +/+ vs -/- mice, administered metabolic poisons, dropped kidneys directly into liquid nitrogen,

extracted purines and measured total kidney levels of purines.

0 1 2 3 4 5 60

20

40

60

80

100

0 1 2 3 .4 5 6

0

20

40

60

80

100

2’,3’-cAMP

3’,5’-cAMP

CNPase -/-2’,3’-cAMP

3’,5’-cAMP

CNPase +/+

Rela

tive

Abun

danc

e

Retention Time (Minutes)

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

15 min

Induced AKI in CNPase +/+ versus CNPase -/- mice and examined renal function and structure 48 hours later.

Grenz et al., 2011 www.jove.com

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

Are CNPase -/- Mice More Susceptible to Acute Kidney Injury?

B

C

A

ICAM

MCP1

Bottom Line:Knocking Out CNPase Attenuates

Acute Kidney Injury!!

BUT WHY??

CELL2’,3

’-cAMP

2’-AMP

ADO

mRNA

Ecto-2’,3’-PDEs

Ecto-2’/3’-NTs

TissueProtection

Extracellular 2’,3’-cAMP-Adenosine Pathway

2’,3’-cAMP

Transp

orter

RNases

mPTPs

Apotosis

Bad Stuff

CNPaseX2’-AMP + 3’-AMP

Mitohorm

esis

Does 2’-AMP Cause Bad Stuff?

Yes, 2’-AMP is a Potent RenalVasconstrictor!

2’-AMP is Renal Vasoconstrictor

2’-AMP Also Reduces GFR

Inhibition of Alkaline Phosphatase Induces AKI

CELL2’,3

’-cAMP

2’-AMP

ADO

mRNA

Tissue Protection

2’,3’-cAMP

Transp

orter

RNases

mPTPs

Renal Vasoconstriction

CNPase

Mitohorm

esis

Alkaline Phosphatase

CNPase

Inhibitor

Inhibitor o

f 2’-A

MP-

Induced Renal

Vasoco

nstricti

on

AlkalinePhosphatase

Opportunities for Treating/Preventing AKI

Induce

Mitohorm

esis

Alkaline Phosphatase for Sepsis-Induced AKI

Heemskerk et al.Critical Care Medicine 2009 Feb;37(2):417-423

Alkaline phosphatase treatment improves renal function in severe sepsis or septic shock patients.

Pickkers et al.Critical Care 2012 Jan 23;16(1):R14.

Alkaline phosphatase for treatment of sepsis-induced acute kidney injury: a prospective randomized double-blind placebo-controlled trial.

Future Directions?

What is the mechanism of 2’-AMP-induced renal vasoconstriction? Can we block 2’-AMP-induced renal vasoconstriction?

Would blockade of 2’-AMP-induced vasoconstriction prevent/treat AKI?

Can we develop a selective CNPase inhibitor?Would pharmacological inhibition of CNPase prevent/treat AKI?

Would administration of alkaline phosphatase prevent/treat AKI?

What happens to renal mitochondria when CNPase is blocked?Would inducing mitohormesis prevent AKI?