In 2008, we discovered that intact kidneys produce large quantities of 2',3'-cAMP (a positional isomer of 3',5'-cAMP), that 2',3'-cAMP derives from mRNA degradation and that energy depletion massively increases renal 2',3'-cAMP biosynthesis. This was the FIRST documentation of endogenous 2',3'-cAMP in any organ, tissue or cell. This exciting discovery triggered an eureka moment: 2',3'-cAMP may be an unrecognized, but critically important, source of extracellular adenosine! We have now confirmed this hypothesis, and we refer to the production of 2',3'-cAMP, followed by its conversion to adenosine, via 2'-AMP and 3'-AMP, as the 2',3'- cAMP-Adenosine Pathway. The focus of this application is the role of 2',3'-cyclic nucleotide-3'- phosphodiesterase (CNPase) in the renal 2',3'-cAMP-adenosine pathway. CNPase is an enzyme that can convert 2',3'-cAMP to 2'-AMP and is the ONLY enzyme known to promote this reaction. Why do we think that renal CNPase is important? A recent report demonstrates that intracellular 2',3'-cAMP opens mitochondrial permeability transition pores (mPTPs), and it is well known that opening of mPTPs leads to apoptosis and necrosis. Moreover, many studies demonstrate that adenosine, via specific receptors, protects the kidney from ischemia/reperfusion injury. Therefore, CNPase should be renoprotective because it likely reduces the intracellular levels of an intracellular toxn (2',3'-cAMP) and concomitantly increases the extracellular levels of a protective metabolite (adenosine). The main goal of the present proposal is to quantify the importance of renal CNPase in regulating renal levels of 2',3'-cAMP and adenosine. We will employ a LC-MS/MS assay for the 2',3'-cAMP metabolome using a 13C-labeled internal standard to examine: 1) the metabolism of exogenous 2',3'-cAMP to 2'-AMP, 3'-AMP, adenosine and inosine by intact kidneys and renal cells from CNPase -/-, CNPase -/+ and CNPase +/+ mice;and 2) the metabolism of endogenous (triggered by energy depletion) 2',3'-cAMP to 2'-AMP, 3'-AMP, adenosine and inosine by intact kidneys and renal cells from CNPase -/-, CNPase -/+ and CNPase +/+ mice. If CNPase is involved in the renal metabolism of 2',3'-cAMP, then this should be reflected in the renal metabolome of exogenous and endogenous 2',3'-cAMP. Finally, we will quantify renal damage induced by ischemia/reperfusion injury in kidneys from CNPase -/-, CNPase -/+ and CNPase +/+ mice. If CNPase is involved in ridding cells of 2',3'-cAMP or increasing extracellular adenosine, then renal injury should be exacerbated in kidneys deficient in CNPase. Our hypothesis has several critically important implications: 1) Renal CNPase may be a key enzyme in protecting the kidneys from insults;2) Malfunction of this enzyme (because of disease or drugs) could explain why some patients develop acute or chronic renal failure in response to renal injury;and 3) Pharmacological or molecular manipulation of the 2',3'- cAMP-adenosine pathway may offer an effective approach to reducing the risk of acute and chronic renal failure, diseases which are costly, prevalent and life altering.

Public Health Relevance

We hypothesize that 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) protects the kidneys from injury. Our hypothesis, if correct, has several critically important implications: 1) renal CNPase should be viewed as a key enzyme in protecting the kidneys from insults;2) malfunction of this enzyme (because of disease or drugs) could explain why some patients develop acute or chronic renal failure in response to renal injury;and 3) pharmacological or molecular manipulation of the 2',3'-cAMP-adenosine pathway may offer an effective approach to reducing the risk of acute and chronic renal failure, diseases which are costly, prevalent and life altering.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK068575-09
Application #
8479345
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
Rys-Sikora, Krystyna E
Project Start
2012-06-15
Project End
2017-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2013
Total Cost
$317,980
Indirect Cost
$108,092
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Jackson, Edwin K; Gillespie, Delbert G; Mi, Zaichuan et al. (2014) Role of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the renal 2',3'-cAMP-adenosine pathway. Am J Physiol Renal Physiol 307:F14-24
Jackson, Edwin K; Mi, Zaichuan (2014) The guanosine-adenosine interaction exists in vivo. J Pharmacol Exp Ther 350:719-26
Jackson, Edwin K; Cheng, Dongmei; Verrier, Jonathan D et al. (2014) Interactive roles of CD73 and tissue nonspecific alkaline phosphatase in the renal vascular metabolism of 5'-AMP. Am J Physiol Renal Physiol 307:F680-5
Schuler, P J; Saze, Z; Hong, C-S et al. (2014) Human CD4+ CD39+ regulatory T cells produce adenosine upon co-expression of surface CD73 or contact with CD73+ exosomes or CD73+ cells. Clin Exp Immunol 177:531-43
Jackson, Edwin K; Gillespie, Delbert G (2013) Extracellular 2',3'-cAMP-adenosine pathway in proximal tubular, thick ascending limb, and collecting duct epithelial cells. Am J Physiol Renal Physiol 304:F49-55
Saze, Zenichiro; Schuler, Patrick J; Hong, Chang-Sook et al. (2013) Adenosine production by human B cells and B cell-mediated suppression of activated T cells. Blood 122:9-18
Erickson, Catherine E; Gul, Rukhsana; Blessing, Christopher P et al. (2013) The ?-blocker Nebivolol Is a GRK/?-arrestin biased agonist. PLoS One 8:e71980
Jackson, Edwin K; Cheng, Dongmei; Mi, Zaichuan et al. (2013) Role of CD73 in Renal Sympathetic Neurotransmission in the Mouse Kidney. Physiol Rep 1:
Jackson, Edwin K; Gillespie, Delbert G (2013) Regulation of Cell Proliferation by the Guanosine-Adenosine Mechanism: Role of Adenosine Receptors. Physiol Rep 1:e00024
Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G et al. (2013) Role of CNPase in the oligodendrocytic extracellular 2',3'-cAMP-adenosine pathway. Glia 61:1595-606

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