The Renal 2',3'-cAMP-Adenosine Pathway

肾脏 2,3-cAMP-腺苷途径

• 批准号: 8282204
• 负责人: EDWIN Kerry JACKSON
• 金额: $ 32.95万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8282204
• 关键词: 2' -adenylic acid; Acute; Adenosine; Anabolism; Apoptosis; Biological Assay; Cells; Chronic Kidney Failure; Cyclic AMP; Cyclic Nucleotides; Disease; Documentation; Electrolytes; Enzymes; Excretory function; Glomerular Filtration Rate; Goals; Histopathology; Inflammation; Injury; Inosine; Ischemia; Isomerism; Kidney; Knockout Mice; Label; Life; Measures; Metabolism; Molecular; Mus; Necrosis; Organ; Pathway interactions; Patients; Pharmaceutical Preparations; Production; Proteinuria; Reaction; Renal Blood Flow; Renal function; Reperfusion Injury; Reporting; Risk; Role; Sampling; Source; Structure; Time; Tissues; Toxin; United States; Urine; Venous; Wild Type Mouse; extracellular; in vivo; indexing; kidney cell; mRNA Transcript Degradation; metabolic poison; mitochondrial permeability transition pore; phosphoric diester hydrolase; receptor; renal ischemia; response

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：2008年，我们发现完整的肾脏产生大量的2',3'-cAMP （3',5'-cAMP的位置异构体），2',3'-cAMP来自mRNA降解，能量消耗大量增加肾脏2',3'-cAMP的生物合成。这是首次在任何器官、组织或细胞中发现内源性2',3'-cAMP。这一激动人心的发现让人灵光一现：2',3'-cAMP可能是细胞外腺苷的一种未被发现但极其重要的来源！我们现在已经证实了这一假设，我们将2',3'- camp的产生，随后通过2'- amp和3'- amp转化为腺苷，称为2',3'- camp -腺苷途径。本应用的重点是2',3'-环核苷酸-3'-磷酸二酯酶（CNPase）在肾脏2',3'- camp -腺苷途径中的作用。CNPase是一种能将2',3'-cAMP转化为2'-AMP的酶，也是已知唯一能促进该反应的酶。为什么我们认为肾CNPase很重要？最近的一份报告表明，细胞内2',3'-cAMP打开线粒体通透性过渡孔（mPTPs），众所周知，mPTPs的打开导致细胞凋亡和坏死。此外，许多研究表明，腺苷通过特异性受体保护肾脏免受缺血/再灌注损伤。因此，CNPase应该具有肾保护作用，因为它可能降低细胞内毒素（2',3'-cAMP）的细胞内水平，并同时增加细胞外保护性代谢物（腺苷）的水平。本提案的主要目标是量化肾CNPase在调节肾脏2',3'-cAMP和腺苷水平中的重要性。我们将采用LC-MS/MS检测2',3'- camp代谢组，使用13c标记的内标来检测：1)来自CNPase -/-， CNPase -/+和CNPase +/+小鼠的完整肾脏和肾细胞对外源性2',3'- camp, 2'- amp,3'- amp，腺苷和肌苷的代谢；2)来自CNPase -/-、CNPase -/+和CNPase +/+小鼠的完整肾脏和肾细胞将内源性（由能量耗竭触发）2‘、3’- camp代谢为2'- amp、3'- amp、腺苷和肌苷。如果CNPase参与了2',3'-cAMP的肾脏代谢，那么这应该反映在外源性和内源性2',3'-cAMP的肾脏代谢组中。最后，我们将量化CNPase -/-、CNPase -/+和CNPase +/+小鼠肾脏缺血/再灌注损伤引起的肾损害。如果CNPase参与细胞中2',3'-cAMP的清除或细胞外腺苷的增加，那么在CNPase缺乏的肾脏中，肾损伤会加重。我们的假设有几个非常重要的含义：1)肾CNPase可能是保护肾脏免受损害的关键酶；2)这种酶的功能障碍（由于疾病或药物）可以解释为什么一些患者在肾损伤后发生急性或慢性肾功能衰竭；3) 2',3'- camp -腺苷途径的药理学或分子操作可能为降低急性和慢性肾功能衰竭的风险提供有效途径，这些疾病是昂贵的，普遍的和改变生活的疾病。