Edwin K. Jackson - US grants

For any given frequency of sympathetic nerve stimulation, a greater vasoconstrictor response is observed in spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto control rats (WKY). This enhanced noradrenergic neurotransmission in SHR is due in part to some unknown defect in the control of norepinephrine exocytosis, such that for a given impulse frequency a greater quantity of norepinephrine is released from noradrenergic varicosities in SHR than in WKY. Sympathetic nerve stimulation increases the production rate of prostaglandins (PGs) by postjunctional elements and PGE-2, PGD-2 and, less potently, PGI-2 inhibit neurotransmitter release. Therefore, a PG negative feedback loop controlling norepinephrine release may be operative. In a recent study Martineau et al. (25) determined, using highly specific methodology, that in vivo synthesis of PGE-2 and PGI-2 is markedly depressed in SHR. Defective synthesis of PGs in SHR could interrupt the PG negative feedback loop controlling norepinephrine release and account for the enhanced neurotransmission characteristic of SHR. In pilot experiments the activity of the PG negative feedback loop in SHR and WKY was assessed by examining the consequences of interrupting the PG feedback loop by blocking PG biosynthesis. In WKY, inhibition of PG biosynthesis increased by 100% the vascular response to sympathetic nerve stimulation, without influencing the response to exogenous norepinephrine. In contrast in SHR, blockade of PG biosynthesis did not influence the vascular response to either sympathetic nerve stimulation or norepinehrine. The implication from this experiment is that in WKY the PG negative feedback loop controlling norepinephrine release is functional; whereas, in SHR this system is defective. The goal of this proposal is to further assess the hypothesis that the PG negative feedback loop controlling norepinephrine release is defective in SHR. To achieve this goal the in vivo activity of the PG negative feedback loop controlling norepinephrine release will be determined and compared in SHR and WKY using four different approaches: 1) The effects of cyclooxygenase inhibition on vascular responses to sympathetic nerve stimulation will be determined in the in situ blood perfused mesentery and kidney of SHR and WKY. 2) The effects of cyclooxygenase inhibition on the neuronal spillover of norepinephrine will be measured in the mesentery and kidney of SHR and WKY. 3) The effects of PGE-2, PGD-2 and PGI-2 on noradrenergic neurotransmission will be assessed in the mesentery and kidney of SHR and WKY. 4) The effects of sympathetic nerve stimulation on PGE-2 and 6-keto-PGF-1Alpha biosynthesis will be determined in the mesentery and kidney of SHR and WKY.

The purpose of this proposal is to test the hypothesis that angiotensin II (AII) stimulates adenosine release and that the adenosine release by AII serves to inhibit renin release and to attenuate at least some of the biological effects of AII. Stated differently, I wish to examine the physiological role of AII- induced adenosine release as a modulator of the renin-angiotensin system. The significance of the hypothesis is that adenosine may play an important role in restraining the activity of the renin- angiotensin system, so that blockade of this adenosine-mediated restraint, e.g., caffeine consumption, could have adverse cardiovascular effects. The rationale for this hypothesis is provided by several observations recently made in my laboratory: (1) Adenosine is a potent inhibitor of renal ischemia-induced renin release; (2) Adenosine attenuates, whereas AII potentiates, noradrenergic neurotransmission in vivo; (3) Plasma levels of adenosine are elevated six-fold in renovascular hypertension, yet are normal in genetic hypertension; (4) Caffeine, a widely consumed adenosine receptor antagonist, markedly exacerbates renovascular hypertension, but not genetic hypertension; (5) Caffeine causes a sustained elevation of plasma renin activity in renovascular, but not genetic, hypertension; and (6) Caffeine augments the slow- pressor effect of low-dose infusions of AII in normal, but not sympathectomized, animals. These data suggest that activation of the renin-angiotensin system enhances adenosine production, and adenosine acts both to limit renin release and to limit the effects of AII on the sympathetic nervous system. The physiological role of AII-induced adenosine release will be assessed in vivo in several ways. First, I will determine if AII can release adenosine from the pulmonary, mesenteric and renal vascular beds in vivo following either acute infusions of exogenous AII or during chronic activation of the endogenous renin- angiotensin system. Second, I will determine if adenosine serves to limit the ability of AII to enhance noradrenergic neurotransmission. Initially, I will determine if exogenous adenosine can modify AII-induced potentiation of sympathetically-mediated vascular contractions and norepinephrine release. Subsequently, I will compare the ability of exogenous and endogenous AII to facilitate noradrenergic neurotransmission in the absence and presence of specific and novel adenosine receptor antagonists. Finally, I will determine if adenosine mediates the "short-loop" negative feedback control of renin release by examining this feedback loop in the absence and presence of adenosine receptor blockade.

Past results from this ongoing research endeavor strongly support the theory that endogenous adenosine plays a significant physiological role to restrain the release of renin in response to a variety of stimuli. Two important questions regarding this theory, referred to as the adenosine brake hypothesis, linger: 1) Does the adenosine brake exist? and, 2) How does it operate? The first objective is to conduct a critical test of the adenosine brake hypothesis that should corroborate the in vivo existence of this regulatory system. Since adenosine inhibits renin release via A1 adenosine receptors, if endogenous adenosine functions to limit the renin release response to stimuli, then selective A1 receptor, but not A2 receptor, blockade should augment the renin release response to agents such as isoproterenol and PGI2. This prediction will be tested by using a series of novel A1 and A2 receptor antagonists that recently were evaluated and validated in this laboratory. The second objective is to investigate the mechanism of the adenosine brake on renin release. In this regard, the transmembrane negative feedback loop hypothesis will be tested as a potential explanation for how the adenosine brake on renin release operates. This hypothesis proposes that: 1) hormonal activation of adenylate cyclase (AC) stimulates renin release via cAMP-induced activation of protein kinase A; 2) stimulation of AC causes egress of cAMP out of juxtaglomerular cells and/or neighboring cells; 3) extracellular cAMP is degraded locally to AMP and hence to adenosine; 4) adenosine, via an A1 adenosine receptor, inhibits AC and consequently brakes the renin release response to the original hormonal stimulus. This hypothesis would be addressed in part by the same experiments used to test the existence of the adenosine brake, and will be further tested by determining: 1) whether A1 receptor blockade does not alter the renin release response to 8-bromo-cAMP; 2) whether PGI2 and isoproterenol increase renal cortical interstitial levels of cAMP, AMP, adenosine, inosine and hypoxanthine and whether these increases are appropriately altered by pharmacological agents; 3) whether cAMP and AMP inhibit renin release and whether this inhibition is appropriately modified by pharmacological agents; 4) whether infusions of cAMP and AMP appropriately alter renal cortical interstitial levels of purines and whether these changes are appropriately altered by pharmacological agents; and 5) whether inhibition of cAMP-dependent protein kinase attenuates the effects of isoproterenol and PGI2 on renin release and prevents A1 receptor antagonists from potentiating renin release responses to isoproterenol and PGI2.

For any given frequency of sympathetic nerve stimulation, a greater vasoconstrictor response is observed in spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto control rats (WKY). This enhanced noradrenergic neurotransmission in SHR is due in part to some unknown defect in the control of norepinephrine exocytosis, such that for a given impulse frequency a greater quantity of norepinephrine is released from noradrenergic varicosities in SHR than in WKY. Sympathetic nerve stimulation increases the production rate of prostaglandins (PGs) by postjunctional elements and PGE-2, PGD-2 and, less potently, PGI-2 inhibit neurotransmitter release. Therefore, a PG negative feedback loop controlling norepinephrine release may be operative. In a recent study Martineau et al. (25) determined, using highly specific methodology, that in vivo synthesis of PGE-2 and PGI-2 is markedly depressed in SHR. Defective synthesis of PGs in SHR could interrupt the PG negative feedback loop controlling norepinephrine release and account for the enhanced neurotransmission characteristic of SHR. In pilot experiments the activity of the PG negative feedback loop in SHR and WKY was assessed by examining the consequences of interrupting the PG feedback loop by blocking PG biosynthesis. In WKY, inhibition of PG biosynthesis increased by 100% the vascular response to sympathetic nerve stimulation, without influencing the response to exogenous norepinephrine. In contrast in SHR, blockade of PG biosynthesis did not influence the vascular response to either sympathetic nerve stimulation or norepinehrine. The implication from this experiment is that in WKY the PG negative feedback loop controlling norepinephrine release is functional; whereas, in SHR this system is defective. The goal of this proposal is to further assess the hypothesis that the PG negative feedback loop controlling norepinephrine release is defective in SHR. To achieve this goal the in vivo activity of the PG negative feedback loop controlling norepinephrine release will be determined and compared in SHR and WKY using four different approaches: 1) The effects of cyclooxygenase inhibition on vascular responses to sympathetic nerve stimulation will be determined in the in situ blood perfused mesentery and kidney of SHR and WKY. 2) The effects of cyclooxygenase inhibition on the neuronal spillover of norepinephrine will be measured in the mesentery and kidney of SHR and WKY. 3) The effects of PGE-2, PGD-2 and PGI-2 on noradrenergic neurotransmission will be assessed in the mesentery and kidney of SHR and WKY. 4) The effects of sympathetic nerve stimulation on PGE-2 and 6-keto-PGF-1Alpha biosynthesis will be determined in the mesentery and kidney of SHR and WKY.

Adenosine is an endogenous nucleoside that exerts profound effects on the heart, blood vessel and kidneys via specific cell surface receptors. Hence, a detailed understanding of mechanisms regulating adenosine levels may provide insights into physiological processes and may suggest new therapeutic modalities for cardiovascular and renal diseases. During the past grant period, we identified a novel biochemical pathway that importantly contributes to the modulated production of adenosine. This mechanism, called the cAMP-adenosine pathway, entails the conversion of extracellular cAMP to AMP via ecto-phosphodiesterase (ecto-PDE), followed by the metabolism of AMP to adenosine via ecto-5' nucleotidase (ecto-5'-NT). Our studies indicate that the cAMP-adenosine pathway is operative in aortic vascular smooth muscle cells, pre-glomerular microvascular smooth muscle cells, mesangial cells, cardiac atrial and ventricular fibroblasts, isolated microvessels, perfused vascular beds and intact animals, Having established the existence of the cAMP- adenosine pathway, our next two objectives are to determine how the cAMP- adenosine pathway is regulated and elucidate the physiological/ pathophysiological roles of the cAMP-adenosine pathway. With regard to regulatory mechanisms, recent studies by Kitakaze et al. establish that in cardiomyocytes the alpha1-adrenoceptor agonist methoxamine markedly stimulates ecto-5-NT activity via the Gq/PI-PLC/PKC cascade, and pilot studies in our laboratory suggest that in the rat renal vascular bed methoxamine stimulates ecto-PDE activity by 500%. These findings support the hypothesis that the Gq/PI-PLC/PKC cascade enhance the cAMP-adenosine will be addressed by determining the effects of agents that activate the Gq/PI-PLC/PKC cascade on ecto-enzyme activity and isoproterenol-induced adenosine production and be determining whether these effects are blocked by selective PI-PLC and PKC inhibitors. With regard to the physiological roles of the cAMP-adenosine pathway, we have obtained evidence that the cAMP-adenosine pathway importantly contributes to renal sympathetic nerve activation-induced renal vasoconstriction, a novel concept that will also be addressed in the current proposal.

[unreadable] DESCRIPTION (provided by applicant): Research in our laboratory corroborates that hypertension in the spontaneously hypertensive rat (SHR) is mediated in part by an enhanced renovascular response to angiotensin II (Ang II). Recently, we discovered that the enhanced renovascular response to Ang II in SHR is caused by potentiation of Ang II-induced renal vasoconstriction by the Gi signal transduction pathway. The finding of an enhanced coincidence signaling between the Ang II signal transduction pathway and the Gi signal transduction pathway in the renal microcirculation of SHR has important implications regarding the pathophysiology of genetic hypertension. Renal sympathetic nerves release two major neurotransmitters, norepinephrine (NE) and neuropeptide Y (NPY). NE and NYP activate postjunctional alpha 2-adrenoceptors and Y1 receptors, respectively, and both receptors are coupled nearly exclusively to the Gi signal transduction pathway. Our research suggests that an important contributing cause to genetic hypertension is release of NE and NPY from renal sympathetic nerves with subsequent activation of postjunctional alpha 2-adrenoceptors and Y1 receptors leading to significant stimulation of the Gi signal transduction pathway and, therefore, potentiation of renovascular responses to Ang II. To test this hypothesis we will determine in normotensive and hypertensive rats: 1) whether a Y1 receptor agonist potentiates renovascular responses to Ang II (studies with an alpha 2- adrenoceptor agonist were completed in preliminary studies); 2) whether near threshold activation of alpha 2-adrenoceptors combined with near threshold activation of Y1 receptors enhances renovascular responses to Ang II; 3) whether renal sympathetic activation potentiates Ang II-induced renal vasoconstriction by a mechanism involving co-activation of alpha 2-adrenoceptors and Y1 receptors; 4) whether blockade of the Gi signal transduction pathway prevents enhancement of Ang II-induced renal vasoconstriction by activation of alpha 2-adrenoceptors, by activation of Y1 receptors, by co-activation of alpha 2-adrenoceptors and Y1 receptors and by renal nerve stimulation; and 5) the expression of Y1 receptors in the renal microcirculation. We also will address the mechanism of coincidence signaling between the Ang II signal transduction pathway and the Gi signal transduction pathway in the renal microcirculation by testing the hypothesis that coincidence signaling between Ang II and Gi is mediated by RhoA/Rho kinase/PLD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Because the renal cortex expresses high affinity type 1 adenosine receptors that modulate preglomerular microvascular tone, renin release and sodium reabsorption, it is important to understand how renal cortical interstitial levels of adenosine are regulated. Because our comprehension of the regulation of renal cortical interstitial adenosine is rudimentary, the overall purpose of this proposal is to further our knowledge in this regard. The venous drainage of the pancreas empties directly into the portal circulation, an anatomical arrangement that maximizes concentrations, and therefore effects, of pancreatic hormones on hepatocytes. Glucagon is a pancreatic hormone secreted into the portal circulation. Importantly, glucagon is a powerful stimulant of hepatic adenylyl cyclase, and activation of hepatic adenylyl cyclase causes release of large quantities of cyclic AMP into the systemic circulation. We hypothesize that systemic cyclic AMP (secreted from the liver in response to glucagon) is delivered to the renal cortex via the dense peritubular capillary network and is metabolized in the renal cortex to adenosine via the sequential actions of ectophosphodiesterase (converts cyclic AMP to AMP) and ecto-5'-nucleotidase (converts AMP to adenosine). In this view, the liver secretes an endocrine pro-hormone (cyclic AMP) that is metabolized locally in the target tissue (renal cortical interstitial space) to a biologically active hormone (adenosine) via a specific set of enzymes (ecto-phosphodiesterase and ecto-5'-nucleotidase). The specific goal of this proposal is to test this innovative hypothesis using our newly developed and unique LC/MS ion trapping assay for purines. The proposed mechanism will be addressed both in vitro and in vivo. In vitro we will determine whether cyclic AMP added to the basolateral aspect of proximal convoluted tubules is rapidly metabolized to adenosine by the proposed enzymes. In vivo we will determine whether the appropriate maneuvers appropriately influence the levels of cyclic AMP and adenosine in the renal cortical interstitial compartment by a mechanism involving the proposed enzymes. This work may identify a novel pathway by which the pancreas and liver regulate renal cortical interstitial levels of adenosine and may reveal important mechanistic insights into diseases such as the hepatorenal syndrome and the metabolic syndrome X. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our results during the last funding period indicate that activation of renovascular Y1 receptors (Y1Rs) dramatically enhances renovascular responses to physiological levels of angiotensin II (Ang II) in kidneys of spontaneously hypertensive rats (SHR). However, Y1Rs have little effect on Ang ll-induced responses in kidneys of normotensive Wistar-Kyoto rats (WKY). In stark contrast, activation of Y2 receptors (Y2Rs) has, at most, only a minor effect on renovascular responses to Ang II in SHR kidneys, with no effect whatsoever in WKY kidneys. These data indicate that endogenous agonists of Y1 Rs, but not Y2Rs, would potentiate Ang ll-induced renal vasoconstriction in genetically-susceptible kidneys, provided these endogenous agonists could reach the renal microcirculation. Are there endogenous agonists of Y1 Rs that could activate Y1Rs in the kidney microcirculation? The answer is yes. A fatty meal releases peptide YY1-36 (PYY1-36) into the systemic circulation from endocrine L-cells in the small bowel, colon and rectum producing physiologically active levels of PYY1-36 in plasma that are 500% to 1000% above basal circulating levels, and this circulating PYY1-36 would be delivered promptly to the renal microcirculation via the blood stream (humoral input to kidney microcirculation). Renal sympathetic nerves release neuropeptide Y1-36 (NPY1- 36) in response to CNS-mediated activation of renal sympathetic nerves (neural input to kidney microcirculation), resulting in high local levels of NPY1-36 in sympathetically-innervated renal microvessels during renal sympathetic activation. Because both PYY1-36 and NPY1-36 are potent Y1R agonists, physiological processes that increase PYY1-36 release from the gut, NPY1-36 release from renal sympathetic nerves or both simultaneously would activate Y1Rs in the renal microcirculation, which in genetically-susceptible kidneys would enhance Ang ll-induced renal vasoconstriction. It is conceivable, however, that stimulation of Y1Rs by physiological processes that increase the exposure of the renal microcirculation to PYY1-36 and NPY1-36 is diminished by the activity of vascular dipeptidyl peptidase IV (DPP IV) residing in the walls of blood vessels that comprise the renal microcirculation. DPP IV converts PYY1-36 to PYY3-36 and NPY1-36 to NPY3-36 by cleaving two amino acids from the N-terminus of either PYY1-36 or NPY1-36. Whereas PYY1-36 and NPY1-36 are potent Y1R agonists, PYY3-36 and NPY3-36 are inactive at Y1Rs but are potent and selective Y2R agonists. These facts suggest the hypothesis that DPP IV in the renal vasculature is a critical determinant of the extent to which PYY1-36 and NPY1-36 enhance renovascular responses to Ang II in genetically-susceptible kidneys. The testing of this hypothesis is the focus of this application. The proposed hypothesis is absolutely novel. The concept that DPP IV exists in the renal vasculature and regulates renal blood flow has never been proposed or tested.

DESCRIPTION (provided by applicant): The term "cAMP" is used universally to refer to 3',5'-cyclic adenosine monophosphate (3',5'- cAMP), the famous "second messenger" discovered by Dr. Earl Southerland. Importantly, the vast majority of investigators measure 3',5'-cAMP using various commercially available assay kits, and our group is the ONLY group in the world that routinely measures 3',5'-cAMP using high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS). Dr. Jackson's lab focuses mostly on the role of purines in renal physiology/pharmacology, and routinely employs LC-MS/MS, rather than assay kits, to measure 3',5'-cAMP. In experiments unrelated to the brain, Dr. Jackson's lab serendipitously discovered that the kidney produces more 2',3'-cAMP (a positional isomer of 3',5'- cAMP) than 3',5'-cAMP. Dr. Kochanek's lab focuses primarily on mechanisms of traumatic brain injury (TBI), and has discovered that purines play a major role in protecting the brain from TBI. Because of Dr. Jackson's expertise in measuring purines and Dr. Kochanek's interest in the role of purines in TBI, a natural and productive collaboration evolved between these investigators. After the serendipitous discovery of 2',3'-cAMP in kidneys, Drs. Jackson and Kochanek decided to investigate whether 2',3'-cAMP exists in the brain. In preliminary studies, LC-MS/MS analysis of 44 samples of cerebral spinal fluid (CSF) from TBI patients showed that 2',3'-cAMP is a major constituent of "cAMP" in CSF from patients with TBI. In addition to 2',3'-cAMP, the levels of 2'-AMP, adenosine and inosine (metabolite of adenosine) were also measured in these same samples. Importantly, there was an large and significant correlation between 2',3'-cAMP and 2'-AMP, 2'-AMP and adenosine and 2'-AMP and inosine in human CSF. These surprising discoveries suggested a critical need to explore the role of 2',3'-cAMP in TBI pathophysiology, particularly with respect to the role of 2',3'-cAMP as a precursor for adenosine. Accordingly, the purpose of this exploratory project is to begin to characterize the significance of 2',3'-cAMP in TBI by testing the innovative concept that 2',3'-cAMP is involved in a "CNPase Neuroprotection Mechanism" in which brain injury leads to release of 2',3'-cAMP from mRNA, and 2',3'-cAMP is then metabolized to 2'-AMP by CNPase followed by conversion of 2'-AMP to adenosine. PUBLIC HEALTH RELEVANCE: TBI is a major source of mortality, morbidity and life-long impairment in both the civilian and military populations, with limited treatment options and generally dismal outcomes. The CNPase Neuroprotective Mechanism could be extremely important in producing extracellular adenosine during TBI, thus providing this protective, "retaliatory" metabolite to mitigate cellular damage. Manipulation of this mechanism by drugs could provide novel approaches to prevent and treat TBI.

During the previous funding period we discovered that endogenous adenosine by activating A1 receptors is a major determinant of the renovascular response to renal sympathetic nerve stimulation (RSNS). This conclusion is based on our observations that selective A1-receptor antagonism in rats and A1-receptor deletion in mice suppresses renovascular responses to RSNS. Moreover, we discovered that there are three reasons A1 receptors importantly contribute to RSNS-induced renal vasoconstriction: 1) RSNS triggers adenosine formation; 2) Preglomerular microvessels express high levels of vasoconstrictor A1 receptors; and 3) In the renal vasculature, the Gi signaling pathway (which A1 receptors engage) converges with the Gq signaling pathway (which ?1-adrenoceptors engage) to trigger ?coincident signaling? at phospholipase C leading to augmentation by adenosine of the vasoconstrictor response to released norepinephrine (NE). Because ATP is released from noradrenergic varicosities, as well as from vascular smooth muscle and endothelial cells, the main precursor of adenosine in the sympathetic neuroeffector junction is likely ATP. CD39 catalyzes the metabolism of ATP to ADP and ADP to AMP, and CD73 metabolizes AMP to adenosine; thus these twin ecto-enzymes acting in tandem are considered the most important mechanism for producing extracellular adenosine from ATP. Surprisingly, however, our experiments show that neither pharmacological inhibition of CD39 nor genetic deletion of CD73 attenuates renovascular responses to RSNS. Instead, our preliminary findings suggest that inhibition of kidney non-specific alkaline phosphatase (KAP) markedly attenuates both renovascular responses to RSNS and adenosine release by RSNS. We hypothesize that renovascular KAP, rather than CD39/CD73, is necessary for the formation of the pool of adenosine that participates in RSNS-induced renal vasoconstriction. If our hypothesis is correct then KAP inhibition, knockdown (KAP-/+) or knockout (KAP-/-) should attenuate RSNS- and NE-induced renovascular responses and purine release. Moreover, our hypothesis predicts that A1-receptor stimulation should rescue (reverse) the suppression of renovascular responses to NE induced by KAP inhibition or knockdown/knockout. These predictions will be tested in Aims #1 and #2, respectively. We have strong preliminary evidence that coincident signaling between A1 receptors and NE not only enhances renal vasoconstriction but also induces the renovascular release of soluble KAP ? which could further enhance adenosine formation and potentiate renovascular responses to NE. Aim #3 of this proposal will determine whether and how A1-receptor stimulation augments RSNS- or NE-induced release of soluble KAP. Using the world?s first A1-receptor knockout Dahl salt sensitive rat, our final aim (Aim #4) will ?put our hypothesis to work? by determining whether A1-receptor knockout and long-term KAP inhibition attenuate in vivo RSNS-induced renal vasoconstriction and chronically lower salt-induced hypertension and target organ damage.

DESCRIPTION (provided by applicant): Dipeptidyl peptidase IV (DPPIV) inhibitors are a new class of drugs for treatment of type 2 diabetes. Because drugs in this class [e.g., sitagliptin (Januvia)] afford sustained reductions in HbA1c with a low risk of hypoglycemia and little effect on body weight, it is likely that DPPIV inhibitors will be extensively employed to manage the world-wide pandemic of type 2 diabetes and the metabolic syndrome. Indeed, sitagliptin is already the 2nd leading branded oral antidiabetic agent in the USA. In the near future, tens of millions of patients will be taking DPPIV inhibitors, many for the rest of their lives; thus, we should strive to fully understand the risks, both short-term and long-term, associated with DPPIV inhibition. Based on their mechanism of action, we anticipate that DPPIV inhibitors will express adverse effects. DPPIV metabolizes incretin hormones [e.g., glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP)], and consequently DPPIV inhibitors raise circulating levels of incretins and thereby exert antidiabetic actions by increasing insulin release, inhibiting glucagon secretion and retarding gastric emptying. However, DPPIV metabolizes at least 35 endogenous substrates, and the pharmacological consequences of inhibiting the metabolism of these substrates are mostly unknown. Of particular concern to us is the fact that DPPIV converts neuropeptide Y1-36 (NPY1-36) and peptide YY1-36 (PYY1-36), which is Y1 receptor (Y1R) agonists, to NPY3-36 and PYY3-36, respectively, which are selective Y2 receptor (Y2R) agonists. Indeed, DPPIV could just as logically be named NPY Converting Enzyme because the kcat/Km of DPPIV for NPY1-36 is approximately 36-fold and 73-fold greater for NPY1-36 compared with GLP-1 and GIP, respectively. Clearly DPPIV inhibitors may alter the balance between Y1R and Y2R stimulation, and this may have adverse renal consequences. For example, our previously published work shows that DPPIV inhibition augments angiotensin II-induced renal vasoconstriction in genetically-susceptible kidneys via a Y1R mediated action. Moreover, our recently obtained pilot data suggest that NPY1-36 and PYY1-36 stimulate (via Y1R activation) proliferation of, and extracellular matrix production by, preglomerular vascular smooth muscle cells (PGVSMCs) and glomerular mesangial cells (GMCs) obtained from genetically-susceptible kidneys and that inhibition of DPPIV augments these effects. Our pilot data also suggest that the scaffold protein RACK1 is responsible for the greater effects of Y1R activation and DPPIV inhibition in PGVSMCs and GMCs from genetically-susceptible kidneys. These preliminary findings motivate us to test the following hypothesis: Inhibition of DPPIV in PGVSMCs and GMCs prevents the local metabolism of NPY1-36 and PYY1-36, thus increasing Y1R activation in PGVSMCs and GMCs. In PGVSMCs and GMCs from kidneys that are genetically-susceptible, this mechanism leads to RACK1 mediated enhancement of cellular proliferation and extracellular matrix production, thus increasing the risk of glomerulosclerosis and renal dysfunction. PUBLIC HEALTH RELEVANCE: Inhibitors of DPPIV represent a novel class of antidiabetic drugs for treatment of Type 2 diabetes, and drugs in this class, for example sitagliptin (Januvia(R); recently FDA approved), afford significant and sustained reductions in HbA1c with a low risk of hypoglycemia and little effect on body weight. These characteristics of DPPIV inhibitors, along with the emerging uncertainty regarding the safety of thiazolidinediones, make it highly likely that DPPIV inhibitors will be extensively employed to manage the world-wide pandemic of type 2 diabetes; indeed, the DPPIV inhibitor sitagliptin is the 2nd leading branded oral antidiabetic agent in the USA. Because in the near future >100 million patients yearly will be taking DPPIV inhibitors and because patients who are prescribed DPPIV inhibitors will continue to consume them for the remainder of their lives, there is some urgency to more fully understand the long-term risks associated with DPPIV inhibition. The long-term risks of DPPIV inhibitors in the setting of hypertension and the metabolic syndrome are of particular concern because frequently these conditions are co-morbidities in type 2 diabetics. The present proposal examines the critical issue as to whether inhibition of renal DPPIV has adverse effects on the kidneys of animals with hypertension, with and without the metabolic syndrome that could accelerate the development of diabetic renal disease.

? DESCRIPTION (provided by applicant): In 1999, Zou and colleagues made the fundamental discovery that in the kidney, adenosine A2 receptors vasodilate the medullary vasculature leading to natriuresis/diuresis. During the previous funding period for HL109002, we discovered that extracellular guanosine remarkably increases extracellular levels of adenosine by inhibiting the disposition of extracellular adenosine (we call this the guanosine-adenosine mechanism). We also discovered that 8-aminoguanosine [inhibitor of purine nucleoside phosphorylase (PNPase - the enzyme that metabolizes guanosine)] increases extracellular guanosine and thereby activates the guanosine- adenosine mechanism, i.e., increases adenosine levels. Combining Zou's conclusions with our findings prompted us to conduct preliminary experiments examining the effects of 8-aminoguanosine on renal function; and the results of these preliminary experiments suggest that 8-aminoguanosine does indeed induce natriuresis/diuresis and administered chronically is powerfully antihypertensive. Because 8-aminoguanine is 10-fold more potent as a PNPase inhibitor compared to 8-aminoguanosine, we also postulated that the renal effects of 8-aminoguanosine are mediated by conversion to 8-aminoguanine, a postulate that has withstood preliminary testing. Finally, because there are reports of 8-aminoguanosine in tissues (produced from peroxynitrite reacting with guanosine moieties followed by reduction of 8-nitroguanosine to 8- aminoguanosine), we developed a mass spectrometry-based assay for 8-aminoguanosine and 8- aminoguanine and demonstrated their existence both in kidneys and urine. Thus our preliminary findings support the exciting possibility that we have discovered a previously unrecognized system that regulates renal excretory function and blood pressure. In a nutshell, our hypothesis is that 8-aminoguanine is an endogenous purine that exerts natriuretic, diuretic, and antihypertensive activity and that 8- aminoguanine acts by blocking renal PNPase, which engages the guanosine-adenosine mechanism in the kidney. We propose to test this hypothesis in rats by: 1) Characterizing the diuretic/natriuretic, renal hemodynamic effects and effects on urinary purines of administering 8-aminoguanosine and 8-aminoguanine either intravenously (IV) or directly into the renal artery (IRA); 2) Determining using LC-MS/MS the effects of IV and IRA 8-aminoguanosine and 8-aminoguanine on renal interstitial levels and kidney tissue levels of 8- aminoguanosine, 8-aminoguanine, guanosine, adenosine, and inosine; 3) Determining whether 9- deazaguanine (specific PNPase inhibitor) can mimic the effects of 8-aminoguanosine and 8-aminoguanine on renal excretory function; 4) Determining, using knockout rats, whether the renal excretory effects of 8- aminoguanosine/8-aminoguanine require A2A or A2B, but not A1, receptors; 5) Testing whether peroxynitrite production determines the levels of endogenous 8-aminoguanosine/8-aminoguanine; and 6) Determining, using radiotelemetry, the long-term antihypertensive activity of oral 8-aminoguanosine/8-aminoguanine.

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.

PROJECT SUMMARY (See instructions): The Single Nephron and Metabolomics Core will provide an important national resource for investigators who wish to define the expression, localization and functional characteristics of transport and other relevant proteins in single nephron tubules or defined renal epithelial cells. Moreover, metabolomics services will be available to measure changes in levels of small molecules involved in regulating nephron function. It is expected that the data generated from this Core will be complemented by analyses performed in other Center Cores, such as the Cellular Physiology and Kidney Imaging Cores. The Single Nephron and Metabolomics Core aims to offer an integrated approach including functional (including in vitro microperfusion of isolated segments, measurements of transepithelial ion/solute fluxes, fluorescence functional imaging of single tubular cells), biochemical (microassays of enzyme/transporter activity), molecular (single tubule quantitative PCR and immunoblotting), and analytical (renal metabolomics) strategies applied to microdissected tubules to address relevant questions proposed by users. Furthermore the Core will provide expertise in design and implementation of single nephron/cell studies and instruction in the technical aspects of all services offered by the Core. The specific objectives of the Core are to: (1) provide microdissected tubules for quantification of mRNA abundance (real time PCR) and protein expression (immunoblotting), immunolocalization, and enzyme/transporter microassays; (2) perform functional fluorescence assays of channel/transporter function in isolated tubules microperfused in vitro; (3) perform measurements of transepithelial ion/solute fluxes across isolated tubules microperfused in vitro; (4) quantify mRNA and protein abundance in urinary exosomes; (5) perform metabolomics analyses of microdissected tubules and perfusate; and (6) provide training in all of the above.

DESCRIPTION (provided by applicant): The term cAMP usually refers to the second messenger 3',5'-cyclic adenosine monophosphate. We serendipitously discovered that organ systems can produce (from mRNA degradation) and export to the extracellular compartment a positional isomer of 3',5'-cAMP, namely 2',3'-cAMP. We showed that organ systems convert extracellular 2',3'-cAMP to 2'-AMP + 3'-AMP and can metabolize 2'-AMP and 3'-AMP to adenosine. We refer to this pathway as the 2',3'-cAMP-adenosine pathway. We also showed that extracellular 2',3'-cAMP increases greatly post-traumatic brain injury (TBI) in brain in rodents and humans; and that when the pathway is impaired, TBI outcomes worsen in rodents. Intracellular 2',3'-cAMP opens mitochondrial permeability transition pores while extracellular adenosine is neuroprotective. Thus the 2',3'- cAMP-adenosine pathway may be important in TBI because it eliminates an intracellular neurotoxin (export of 2',3'-cAMP) and generates an extracellular neuroprotectant (conversion of 2',3'-cAMP to adenosine). We also identified the enigmatic myelin protein 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) to be the major enzyme that metabolizes extracellular 2',3'-cAMP to 2'-AMP (a key step toward conversion into adenosine). KO mice lacking CNPase produce less extracellular adenosine post-TBI, are more susceptible to injury and develop axonal degeneration with age despite no gross myelin abnormalities. Hypothesis: the 2',3'-cAMP- adenosine pathway is an endogenous cytoprotective mechanism after TBI. We will elucidate which CNS cell types produce 2',3'-cAMP, what kinds of injury trigger 2',3'-cAMP production, how 2',3'-cAMP is transported out of cells, how downstream AMPs are converted to adenosine, and if manipulating the 2',3'-cAMP-adenosine pathway alters secondary damage. Specific Aim 1: To determine which CNS cell types produce 2',3'-cAMP after injury. Because in vivo TBI increases extracellular 2',3'-cAMP, it is important to determine which CNS cells produce 2',3'-cAMP and whether the effect is injury-type dependent. Aim 1 will determine if metabolic stress, hypoxia or mechanical injury enhances 2',3'-cAMP production by astrocytes, microglia, neurons or oligodendrocytes. Specific Aim 2: To determine whether Multidrug Resistance Protein 4 (MRP4) mediates egress of 2',3'-cAMP. Because 2',3'-cAMP is an intracellular toxin, it is critical to elucidate how 2',3'-cAMP is extrude from CNS cells. Aim 2 will test the hypothesis that MRP4 exports 2',3'-cAMP. Specific Aim 3: To determine if Tissue Alkaline Phosphatase (TAP) participates in the extracellular metabolism of 2'-AMP and 3'- AMP (downstream metabolites of 2',3'-cAMP) to adenosine. Because extracellular adenosine is neuroprotective it is essential to understand how extracellular 2'-AMP and 3'-AMP are converted to extracellular adenosine. Specific Aim 4: To test the hypothesis that the 2',3'-cAMP-adenosine pathway is an endogenous protective mechanism post-TBI. Aim 4 will further test the hypothesis that the 2',3'-cAMP- adenosine pathway is cytoprotective by determining the effect of inhibiting or augmenting it on TBI outcomes.

? DESCRIPTION (provided by applicant): Acute kidney injury (AKI) is a common and devastating clinical problem. Despite the morbidity, mortality, and financial drain associated, there are no established therapies outside of dialytic treatment. Hypotension or sepsis with associated ischemia is the most common cause of human AKI, and the ensuing injury to the kidney tubular epithelium is of central importance to the pathophysiology of AKI. Recent work has identified adaptive responses in the tubular epithelium to cope with ischemia-reperfusion injury (IRI). Unfortunately, these adaptive responses are often suboptimal. There is a critical need to identify how adaptive responses are regulated in AKI so that therapeutic interventions to prevent and treat AKI can be developed. Hypoxia inducible factor-1 (HIF-1) is a transcription factor regarded as the most significant mediator of cellular adaptive responses to hypoxic insult. There is emerging evidence that the transmembrane glycoprotein mucin 1 (human MUC1 or rodent Muc1) expressed on the apical surface of kidney epithelia plays a novel and important role in enhancing HIF-1 activity. MUC1 is cleaved at the cell surface and its cytoplasmic tail is targeted to the nucleus where it binds and stabilizes HIF-1? and thereby transactivates the HIF-1 protective pathway. We have strong preliminary data suggesting that genetic deletion of Muc1 from mouse tubular epithelial cells exacerbates damage from IRI, limits adaptive HIF-1 responses, alters tubular metabolism, and inhibits recovery. The central hypothesis of this application is that epithelial Muc1 is an important modulator of tubular epithelial adaptive and regenerative responses during AKI. The specific aims are: (1) To determine if Muc1 protects the kidney during ischemia-reperfusion injury (IRI) by enhancing the HIF-1? protective pathway. We will test the hypothesis that Muc1 is protective in IRI through transactivation of the HIF-1 protective pathway. Experiments are designed to determine if Muc1 is protective in a mouse model of IRI, and if this protection results through transduction of the HIF-1 protective pathway by Muc1 stabilization of HIF-1?. (2) To determine if increased levels of Muc1 in the kidney enhances protection during IRI. As female mice are more resistant to IRI than males, and females express more Muc1 in the proximal tubule than male mice, we will test the hypothesis that increased levels of Muc1 in the kidney make the kidney more resistant to IRI. Sensitivity of kidneys to bilateral IRI will be assessed 24 h after varying times of ischemia using male and female mice either (i) overexpressing Muc1/MUC1 (Tg-MUC1), (ii) with reduced Muc1 levels (heterozygotes Muc1-/+), or (iii) injected with a PPAR? agonist to induce Muc1 expression. The overall goal of our proposed experiment is to understand the mechanism of Muc1 protection of the kidney during IRI and determine whether increased levels of Muc1 are protective. This information can be used to design therapies to induce MUC1 and limit the severity of AKI.

Inhibitors of dipeptidyl peptidase 4 (DPP4) represent a novel class of antidiabetic drugs for treatment of type 2 diabetes. Because DPP4 inhibitors afford sustained reductions in HbA1c with a low risk of hypoglycemia and little effect on body weight, DPP4 inhibitors are widely employed to manage the world-wide pandemic of type 2 diabetes. For example, the DPP4 inhibitor sitagliptin is among the top 10 prescribed drugs in the USA (>100 million prescriptions per year) and is the 2nd leading branded oral antidiabetic agent in the USA. Alarmingly, evidence is accumulating that chronic DPP4 inhibition increases heart failure risk in type 2 diabetic patients. However, due to a complete lack of a theoretical model that could explain the biochemical basis of this clinical finding and due to the absence of an adequate animal model to recapitulate the clinical results, the mechanism of the increased heart failure risk in patients taking DPP4 inhibition remains unknown. Therefore, at present we do not know how or why DPP4 inhibitors cause adverse cardiac effects, which patients are at risk or how to prevent the risks of DPP4 inhibitors while sustaining the benefits. The overarching goal of this application is to remedy this situation. In this application, we propose a comprehensive model for how DPP4 inhibitors cause cardiac (and renal) fibrosis. Previously, we discovered that full-length neuropeptide Y and peptide YY [NPY(1-36) and PYY(1-36), respectively] exert pro-growth effects (i.e., cell proliferation and extracellular matrix production) on cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), glomerular mesangial cells (GMCs). We also found that DPP4 inhibition augments the pro-growth effects of NPY(1-36) and PYY(1-36) because DPP4 normally inactivates NPY(1-36) and PYY(1-36) by removing two N-terminal amino acids. Recently, we performed preliminary studies to determine whether DPP4 substrates other than NPY(1- 36) and PYY(1-36) could also affect proliferation of, and extracellular matrix production by, CFs, PGVSMCs and GMCs and whether DPP4 inhibition augments the effects of these peptides. Our preliminary data suggest that CXCL12(1-68) may indeed activate CFs, PGVSMCs and GMCs. Moreover, these effects of CXCL12(1- 68) appear to be augmented by DPP4 inhibition and synergize with NPY(1-36) and PYY(1-36). This makes sense because CXCL12(1-68) is an excellent substrate for DPP4 and is metabolized by DPP4 to CXCL12(3- 68). CXCL12(1-68) is a potent agonist at CXCR4 receptors, while CXCL12(3-68) is not. Herein we propose (and describe experiments to test) the model that chronic DPP4 inhibition results in cardiac, and perhaps renal, fibrosis by blocking the metabolism of NPY(1-36), PYY(1-36) and CXCL12(1-68). Moreover, we propose, and have evidence supporting, the concept that insulin degrading enzyme (IDE) inhibitors (under development for type 2 diabetes) synergize with DPP4 inhibitors to worsen organ fibrosis. We will test our hypothesis in a comprehensive set of vertically-integrated (in vitro to in vivo) experiments.

The Animal and Translational Core (Core B) provides an important regional, national, and international resource for investigators who wish to study mechanisms of, and possible treatments for, kidney diseases at an integrative level of biological hierarchy in preparation for direct translational studies in humans. To achieve these goals, the Animal and Translational Core provides a comprehensive panel of techniques for evaluating kidney parameters and associated critically important cardiovascular and autonomic variables. Because hypertension and the metabolic syndrome are both causes and consequences of kidney disease, the Core also offers a wide range of unique female and male animal model systems ideally suited for the task at hand. Ageing is perhaps the greatest risk factor for acute kidney injury (AKI) and chronic kidney disease (CKD), and mitochondrial dysfunction is considered a fundamental process underlying age-related kidney disease. Accordingly, the Animal and Translational Core has developed a suite of reliable analytical approaches to measure key energy molecules and biomarkers of mitochondrial health/dysfunction. These techniques are not widely available; thus the Animal and Translational Core will provide within a single facility the analytical services needed to deepen our understanding of kidney pathophysiology and the pharmacology of renoprotective drugs. Basic knowledge of mechanisms of kidney disease and possible treatments thereof is key to improving kidney outcomes; yet per se fundamental discoveries do not decrease the burden of kidney disease. Basic knowledge must be linked to human translational studies to provide meaningful change for societies. The first barrier to translational studies is the development and deployment of quality drug assay systems to define the pharmacokinetics of novel therapeutic entities in animals and humans, which is prerequisite to designing and interpreting both preclinical studies and phase 1, 2 and 3 clinical trials. Dr. Jackson, a co-director of the Animal and Translational Core, is a trained analytical chemist and a clinical pharmacologist with a track record in drug development. Using ultra-performance-tandem mass spectrometry, Dr. Jackson develops high-quality drug assays for users of the Animal and Translational Core and provides intellectual resources to help guide translational studies to fruition. To provide a comprehensive array of services for kidney researchers, the Animal and Translational Core has developed strong relationships with several other facilities at the University of Pittsburgh including the Transgenic and Gene Targeting Core, the Innovative Technologies Development Core, the Center for Metabolism and Mitochondrial Medicine, and the newly expanded Aging Institute. Core B is thus positioned to expedite the discovery and development of new therapies for kidney diseases.

We have discovered that 8-aminoguanine (8A-Guanine), a naturally-occurring 8-aminopurine, has a unique pharmacological profile, i.e., it exerts diuretic, natriuretic, glucosuric, antikaluretic and antihypertensive activity. In addition, 8A-Guanine protects against target-organ damage and increases the lifespan of Dahl SS rats on a high salt diet, an effect due to prevention of salt-induced strokes. Because 8-aminoguanosine (8A- Guanosine) is converted to 8A-Guanine in the systemic circulation, this 8-aminopurine has similar effects to 8A-Guanine. The mechanism of action of 8A-Guanine (and 8A-Guanosine via its metabolism to 8A-Guanine) is mostly via inhibition of purine nucleoside phosphorylase (PNPase). Importantly, in preliminary experiments we observed that in Dahl SS rats a high salt diet (4%) reduced endogenous renal interstitial levels of 8A- Guanosine and 8A-Guanine by 85% and 100%, respectively. These preliminary studies suggest that a high salt intake induces 8-aminopurine deficiency, at least in Dahl SS rats; but this finding must be confirmed in Dahl SS rats and tested in other models of hypertension. It occurred to us that 8-aminoinosine (8A-Ino) and 8-aminohypoxanthine (8A-HX) have chemical structures very similar to 8A-Guanosine and 8A-Guanine, respectively, and are analogues of naturally-occurring inosine and hypoxanthine, respectively; therefore we reasoned that these compounds too may be endogenous 8-aminopurines with beneficial biological activities. Because no one has ever examined the biological effects of either 8A-Ino or 8A-HX, we conducted preliminary renal studies with these compounds. These preliminary studies suggest that both 8A-Ino and 8A-HX may have effects on renal function similar to those of 8A-Guanosine and 8A-Guanine, but may be even more efficacious in this regard. However, these findings must be confirmed. Also, it is unknown: 1) whether the effects of 8A-Ino are mediated via its metabolism to 8A-HX; 2) whether 8A-Ino and 8A-HX have antihypertensive and organ- protective effects; 3) whether 8A-Ino and 8A-HX, like 8A-Guanosine and 8A-Guanine, are naturally-occurring; and 4) whether their biosynthesis is also suppressed by a high salt diet. Together, our published and preliminary findings motivate our ?8-AMINOPURINE HYPOTHESIS?, which postulates that: 1) 8A-Guanosine, 8A-Guanine, 8A-Ino and 8A-HX comprise a naturally-occurring 8-aminopurine system that is natriuretic, antihypertensive and organ-protective; 2) 8-aminopurine deficiency contributes to salt- sensitive hypertension, target-organ damage and mortality; and 3) 8-aminopurine deficiency can be corrected by oral treatment with 8-aminopurines. Here we propose to further test this hypothesis by: 1) elucidating the renal effects of 8A-Ino and 8A-HX; 2) determining whether a high salt diet induces a deficiency in all 4 8-aminopurines; and 3) determining whether 8A-Ino and 8A-HX, like 8A-Guanosine and 8A-Guanine, have antihypertensive activity and prevent target organ damage. Finally, we will explore whether the mechanism of action of 8A-Ino and 8A-HX involves not only inhibition of PNPase, but also of xanthine oxidase.