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. Author manuscript; available in PMC: 2023 Jan 16.
Published in final edited form as: Nat Rev Urol. 2022 Sep 7;19(11):681–687. doi: 10.1038/s41585-022-00642-w

Purine Nucleoside Phosphorylase as a Target to Treat Age-Associated LUT Dysfunction

Lori A Birder 1,2, Edwin K Jackson 2
PMCID: PMC9842101  NIHMSID: NIHMS1860852  PMID: 36071153

Abstract

With age, the lower urinary tract (LUT), including the bladder, urethra and external striated muscle, become dysfunctional. Consequently, many older individuals suffer from Lower Urinary Tract Disorders (LUTDs). By compromising urine storage and voiding, LUTDs degrade quality of life for untold millions. Treatments for LUTDs have been disappointing, thus frustrating both patients and their physicians. Fortunately, emerging evidence suggests that partial inhibition of the enzyme purine nucleoside phosphorylase (PNPase) with 8-aminoguanine (an endogenous PNPase inhibitor that moderately reduces PNPase activity) reverses age-associated defects in the LUT and restores the LUT to that of a younger state. In this Perspective, we provide a review of 1) how 8-aminoguanine was identified as a potential treatment for LUTDs; 2) the effects of 8-aminoguanine on LUT biochemistry, structure and function; and 3) the mechanism of action by which partial inhibition of PNPase by 8-aminoguanine prevents and reverses LUTDs via rebalancing the LUT purine metabolome.

Introduction

Aging is a complex process which is controlled by multiple genetic and environmental factors that results in progressive erosion of cellular, tissue and organ function. The negative impact of age on the urinary system is particularly common, severe and life changing1. In this regard, the prevalence of LUTDs significantly increases in both men and women with age and manifests as overactive bladder (OAB), underactive bladder (UAB), various forms of urinary incontinence (UI) or some combination of these named syndromes1–3. LUTDs are due to loss of bladder compliance/capacity, altered afferent (sensory) or efferent (motor) control, dysfunctional bladder contractility, loss of striated muscle function or, more commonly, some combination of these pathologies4–6. Demographic studies indicate a steep increase in LUTDs beginning in the fifth decade of life in both sexes that worsens with advancing age7,8. Current treatments for LUTDs are disappointing due to lack of efficacy, low tolerability, and inconvenience. Thus, safe, effective, and convenient treatments for age associated LUTDs are a major unmet need.

Although the underlying causes of LUTDs in older adults are controversial, a number of potential biochemical mechanisms have been proposed; one of the most widely accepted of these is increased oxidative stress and associated mitochondrial dysfunction9,10. Although mitochondria generate 95% of all cellular energy in the form of ATP and thus play an essential role in cellular homeostasis, damaged mitochondria generate reactive oxygen species (ROS) that contribute to oxidative stress11. Moreover, malfunctioning mitochondria can promote cellular calcium overload12, apoptosis13 and activation of pro-aging signaling pathways14. Consequently, age-dependent accumulation of damaged mitochondria likely plays an outsized role in the pathological restructuring of the bladder wall, and hence urological symptoms.

This perspective will cover selected aspects of aging that are relevant to LUT function and a recent development of a novel treatment modality, i.e., partial PNPase inhibition with 8-aminoguanine, a naturally occurring and moderately potent PNPase inhibitor. First, we provide the background leading up to the discovery of 8-aminoguanine for LUTDs. Next, we focus on how aging alters fundamental cellular signaling processes that affect the purine metabolome and how a suboptimal purine metabolome leads to pathological changes in the structure and function of LUT cells thus resulting in abnormal urodynamics. Finally, we provide examples of LUT outcome measures in young versus mature versus old-age rats treated with 8-aminoguanine. By ‘rebalancing’ the purine metabolome, 8-aminoguanine elicits multiple effects including simultaneously increasing tissue-protective and decreasing tissue-damaging metabolites (which are a source of ROS) thereby restoring cellular form and function to that of a younger state. We also discuss the implications for use of 8-aminoguanine, which treats cell physiology and not just the disease symptoms, for the urological community.

Endogenous guanosines & guanines

Our hypothesis that partial inhibition of PNPase with 8-aminoguanine has beneficial effects on the aging LUT was motivated by the outcomes of a systematic investigation of the cardiovascular and renal effects of 8-substituted guanosine and guanines as reviewed below.

Biochemical Pathways

Adenosine and guanosine are purines that exist within cells, in the interstitium and in the circulation. The physiology and pharmacology of adenosine has been extensively investigated, and there is a wealth of information regarding adenosine receptors, adenosine receptor signaling, the role of adenosine in (patho)physiology and applications of adenosinergic compounds in clinical medicine15. Although structurally similar to adenosine, guanosine has received little attention by physiologists or pharmacologists. A compelling reason to investigate the physiology and pharmacology of guanosine is that there are a host of 8-substituted guanosines and guanines that occur in vivo, none of which, until recently, had been characterized pharmacologically. These compounds include 8-hydroxyguanosine, 8-hydroxy-2’-deoxyguanosine, 8-hydroxyguanine, 8-nitroguanosine, 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine16. Because of the dearth of information regarding the physiological/pharmacological actions of these guanosine/guanine derivatives, recently we examined the actions of many of these compounds on the upper urinary system, i.e., the kidneys16.

Renal pharmacology

In the in vivo rat kidney, we observed minimal natriuretic and diuretic activity of intravenously administered guanosine, 8-nitroguanosine and 8-hydroxy-2’-deoxyguanosine and only modest natriuretic and diuretic activity of guanine, 8-nitroguanine, 8-hydroxyguanosine and 8-hydroxyguanine16. Surprisingly, however, intravenous 8-aminoguanosine and 8-aminoguanine augmented the urinary excretion of sodium by 26.6-fold and 17.2-fold, respectively, and the urinary excretion of glucose by 12.1-fold and 12.2-fold, respectively16. Even more remarkable, both compounds suppressed potassium excretion by 69.1% and 71.0%, respectively16. These results in the upper urinary system motivated us to examine the effects of 8-aminoguanine in the lower urinary tract (see “8-Aminoguanine in LUTDs” below)17.

Encouraged by the striking effects of 8-aminoguanosine/8-aminoguanine on renal excretory function, we also examined the effects of oral administration of 8-aminoguanosine and/or 8-aminoguanine on several disease models. 8-Aminoguanosine and/or 8-aminoguanine exerted antihypertensive effects in several models of hypertension18, prevented strokes and prolonged lifespan in Dahl salt sensitive rats on a high salt diet18, decreased HbA1c levels in rats with the metabolic syndrome18 and improved lung histopathology and right ventricular function in rats with pulmonary hypertension18. We also found that addition of 8-aminoguanosine to red blood cells obtained from sickle cell patients attenuated hypoxia-induced sickling18. These findings indicated that 8-aminoguanosine/8-aminoguanine may have utility for treating a number of serious diseases.

Mechanism of action

When injected into the circulation, PNPase rapidly metabolizes 8-aminoguanosine to 8-aminoguanine19; therefore, we hypothesized that 8-aminoguanosine is a “prodrug” of 8-aminoguanine. We investigated this concept and confirmed that systemically administered 8-aminoguanosine is quickly converted to 8-aminoguanine and that the diuretic and natriuretic effects of 8-aminoguanosine are indeed mediated, for all intents and purposes, exclusively by conversion to 8-aminoguanine20. In contrast, we observed that both 8-aminoguanosine and 8-aminoguanine exert intrinsic antikaluretic actions that are independent of PNPase inhibition20. In vitro studies show that 8-aminoguanosine and 8-aminoguanine are low and moderate affinity inhibitors, respectively, of PNPase (Ki of 8-aminoguanosine for PNPase is approximately 7 µmol/L, whereas that for 8-aminoguanine is 0.8 µmol/L21). Thus, we entertained the hypothesis that most of the renal actions of 8-aminoguanine (as well as 8-aminoguanosine via its conversion to 8-aminoguanine) are due to moderate inhibition of PNPase. Our subsequent studies demonstrated that the natriuretic, diuretic and glucosuric actions of 8-aminoguanine – the active drug – are accompanied by increases in PNPase substrates and decreases in PNPase products in the urine – suggesting inhibition of renal PNPase by 8-aminoguanine in vivo22. We also observed that a similar level of PNPase inhibition with an alternative synthetic PNPase inhibitor mimicked 8-aminoguanine’s effects on the excretion of urine, sodium and glucose, but not potassium22. Both 8-aminoguanosine and 8-aminoguanine appeared to inhibit, although not potently, Rac1 (a small G-protein); and NSC2376 – a selective inhibitor of Rac1 – mimicked the antikaluretic effects of 8-aminoguanine22. Together, our results suggest that in the circulation PNPase – which is richly expressed in many organ systems and in red blood cells (see below) – converts systemically administered 8-aminoguanosine to 8-aminoguanine, and 8-aminoguanine subsequently is delivered to the kidney where it blocks PNPase in the urinary system and thereby causes diuresis, natriuresis and glucosuria by altering the renal purine metabolome. Unlike 8-aminoguanosine, administration of 8-aminoguanine directly inhibits PNPase and induces diuresis, natriuresis and glucosuria without the need for systemic metabolic activation. Rac1 is known to stimulate mineralocorticoid receptors in principle cells in the collecting duct23,24, leading to excretion of potassium. Thus, the antikaluretic mechanism of action of 8-aminoguanosine and 8-aminoguanine may involve reduced Rac1 signaling; however, because the effects of 8-aminoguanosine and 8-aminoguanine on Rac1 are quite modest22, this hypothesis remains less secure and requires further investigation to assess its validity.

8-Aminoguanine in LUTDs

The impressive efficacy of 8-aminoguanine in the upper urinary system motivated our hypothesis that 8-aminoguanine might be efficacious in treating or reversing diseases of the lower urinary system, more specifically age-associated LUTDs. This hypothesis was further encouraged by the fact that a major component of 8-aminoguanine’s pharmacological effects in the kidney are due to inhibition of PNPase. To explain our rationale for testing 8-aminoguanine in LUTDs, we next describe the structure, localization and enzymology of PNPase as well as the potential uroprotective effects of PNPase substrates and urodamaging effects of PNPase products.

PNPase

For a comprehensive review of the structure, enzymology and biochemistry of PNPase see Bzowska et al.25. Here, we briefly describe key aspects of mammalian PNPases that relate to the pharmacology of 8-aminoguanine in the urinary system.

Structure of PNPase.

The mRNA for human PNPase consists of 6 exons which code for a PNPase monomer of 289 amino acids with a molecular weight of 32 kDa25. In mammals the main active form of PNPase is a homotrimer with a molecular weight of 80 to 100 kDa25, although other stoichiometries in mammals have been reported25. X-ray crystallographic studies have provided detailed information regarding the 3-dimentional structure of the homotrimer; moreover, the role of specific amino acids in active-site binding and catalysis have been elucidated in detail25.

Localization of PNPase.

PNPase is primarily a cytosolic enzyme25; however, PNPase is also found extracellularly, for example in the plasma26 and cerebral spinal fluid27. Although PNPase is ubiquitously expressed, the expression of PNPase is particularly high in the urinary bladder, kidneys, gastrointestinal tract, spleen, bone marrow, brain, endocrine tissues, reproductive organs, erythrocytes, lymphocytes, and granulocytes25.

Enzymology of PNPase.

PNPase is a phosphorylase that in the presence of inorganic orthophosphate reversibly breaks the N-glycosidic bond of ribo- and deoxyribonucleosides containing a purine nucleobase25. Mammalian PNPase cleaves only ribo- and deoxyribonucleosides containing the nucleobases hypoxanthine and guanine, and therefore metabolizes inosine to hypoxanthine and guanosine to guanine25. However, some bacterial forms of PNPase also metabolize adenosine to adenine25. PNPase is critically important for the purine salvage pathway25, i.e., PNPase allows available purine bases to be converted to purine nucleosides.

Uro-damaging effects.

Current evidence highlights an important role for increased ROS and oxidative stress-induced damage in a broad range of LUTDs10. While there are several sources of ROS, it is well established that the enzyme xanthine oxidase generates ROS as a by-product of metabolism of hypoxanthine to xanthine and xanthine to uric acid28. Importantly, PNPase directly produces hypoxanthine from inosine25. Also, PNPase converts guanosine to guanine25, and guanine deaminase (guanase) metabolizes guanine to xanthine29. Consequently, PNPase would likely promote ROS production by supplying xanthine oxidase with ample supplies of substrates, both hypoxanthine and xanthine. PNPase can provide xanthine oxidase with ROS-generating substrates within a given cell; alternatively, hypoxanthine and xanthine generated remotely by PNPase can be delivered to and transported into distance cells. This remote route of delivery would be relevant to LUTDs since hypoxanthine and xanthine generated by PNPase in the kidneys, as well as other organ systems, would reach the LUT via the urine. Thus, PNPase products generated either within the target LUT cell or remotely could damage LUT cells via ROS production. In support of this concept, our recent work confirms that urinary hypoxanthine levels are elevated in aged rats as well as in urines from patients with LUTDs as compared to controls without underlying urologic disorders. We have also found that administration of hypoxanthine to rats for several weeks induces severe structural and functional damage to the LUT.

Uroprotective effects.

At the same time that PNPase products (i.e., hypoxanthine and guanine via xanthine) are urodamaging, PNPase substrates (i.e., inosine and guanosine) are likely uroprotective. As reviewed by Haskó et al.30, inosine is strongly anti-inflammatory and inhibits the release of pro-inflammatory cytokines from a variety of immune cells including macrophages, lymphocytes and neutrophils; moreover, inosine is protective in animal models of inflammation such as sepsis, ischemia/reperfusion and autoimmunity30. In addition to exhibiting anti-inflammatory activity, inosine also reduces oxidative stress in a number of experimental animal models31–36. Like inosine, guanosine also is an anti-inflammatory purine that protects against oxidative stress34,37–54. Given the important roles of inflammation and ROS in age-related LUTDs55,56, these pharmacological actions of inosine and guanosine support the concept that inosine and guanosine would protect tissues and organs, including the LUT from damage. Indeed, administration of inosine protects the urinary bladder against damage induced by either obstruction57 or spinal cord injury58. Also, inosine stimulates neural axonal outgrowth and regeneration of connections after stroke59, an effect that would improve bladder motor and sensory functions, which are diminished in aging. Although guanosine has not been specifically tested for protection of the urinary bladder, studies show that guanosine protects against stroke60–63 and acute kidney injury64.

Rationale for 8-aminoguanine use.

Even though PNPase plays a key role in the purine salvage pathway and a complete lack of PNPase activity leads to immunodeficiency65, there is a strong rationale to propose that partial inhibition of PNPase would benefit patients with LUTDs. As described above, PNPase consumes potentially uroprotective substrates – inosine and guanosine – while generating urodamaging products – hypoxanthine and guanine (via conversion of guanine to xanthine). This concept is illustrated in Figure 1. PNPase deficiency is an autosomal recessive disorder, and parents of affected homozygous offspring are healthy66. This suggests that partial inhibition of PNPase is safe. Indeed, studies in patients with partial PNPase deficiency indicate that only 8 to 11% of normal PNPase activity is required for healthy neurological development and immunity67. Thus, an optimal PNPase inhibitor for treatment of LUTDs may be a moderately potent one, such as 8-aminoguanine, rather than a highly potent one, such as forodesine. A moderately potent inhibitor would reduce PNPase activity yet leave sufficient PNPase activity for health; in contrast a highly potent inhibitor may dangerously lower PNPase activity.

Figure 1. Graphical Overview:

Figure 1.

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The PNPase inhibitor 8-aminoguanine ‘rebalances’ the purine metabolome- thereby restoring aging-associated abnormalities in LUT form and function.

8-Aminoguanine: Effects on LUT health span

Animals chosen for investigation of age-related phenomena should have certain characteristics, and in terms of rodents, several strains and lines of rats are commonly used in gerontologic studies (including that of the cardiovascular, neurological, and urological systems). In this regard, the aged Fischer 344 rat is widely used in preclinical gerontological investigations. Using the aged Fischer 344 rat and harnessing the ability of 8-aminoguanine to inhibit PNPase has yielded an exciting experimental outcome; 8-aminoguanine actually restores/reverses a number of age-associated alterations in rodent LUT structure and function.

Most senescent cells express the tumor suppressor P16INK4a (referred to as p16);68 thus p16 is a key biomarker for cellular senescence and biologic aging.69 There are indications that clearing such types of senescent cells (which often are associated with secretion of inflammatory mediators) may actually enhance health-span.70 Our studies show that in aged Fischer 344 rats 8-aminoguanine treatment for 6weeks reduces to levels similar to that in younger rats, bladder expression of the senescence biomarker p16 and other factors involved in promoting oxidative stress and mitochondrial dysfunction. Consequently, age-dependent accumulation of damaged mitochondria likely plays an outsized role in the pathological restructuring of the bladder wall, and hence urological dysfunction (see Table 1 for summary of Evidence of Uro-protection by 8-Aminoguanine) Thus, it is possible that 8-aminoguanine, by normalizing a dysfunctional purine signaling cascade, may enhance the LUT ‘health-span’. This is the first study showing that a pharmacologic treatment can improve (or even restore) the underlying bladder health of bladder cells, a result that could lead to an improvement of symptoms.17

Our preclinical studies also reveal that within 6 weeks of oral 8-aminoguanine treatment, aged rats (near the end of their lifespan) have a marked improvement in a number of outcome measures that are typically linked with the negative effects of aging. For example, aging can influence a number of features of the peripheral nervous system. Both clinical and animal studies have reported a loss of myelinated and unmyelinated nerve fibers in older subjects; and sensation often deteriorates with age.71 Further, aging can also impede the regeneration of neural fibers after injury. In our studies, we found that aged rats are far less responsive to tactile (e.g., mechanical) stimuli as compared to younger animals which matches that reported in the elderly population.71 We also found that 8-aminoguanine treatment of aged animals dramatically increases tactile sensitivity closer to that of a younger state.17

Aging can also lead to a significant decline in microvascular function- which can play an important role in aging-associated end organ damage. One of the primary mechanisms involved in microvascular dysfunction is augmented ROS and oxidative stress. Oxidative stress may be produced in part by bladder ischemia and repeated ischemia/reperfusion during a micturition cycle. Transrectal color Doppler ultrasonography in elderly LUTS patients reveal a significant decrease in LUT blood flow.72 Our preclinical data show aging is associated with decreased areas of bladder perfusion and an increase in tortuous vessels.17 Consistent with our findings, clinical studies show that in elderly populations, a significant increase in vessel tortuosity (along with reduction in blood flow) is associated with advanced age. 73 With 8-aminoguanine treatment, we observed a reduction in the number of tortuous blood vessels (those exhibiting abnormal twists and turns) throughout the bladder and the tissue no longer appeared ischemic, resembling that of young bladder.17

Age-related changes in the extracellular matrix (ECM) may also have an impact on the function of various cell types within the bladder wall and play a key role in urinary continence. An age-induced decline in bladder and outflow tract contractile function could result from remodeling of connective tissues (such as collagen) within component LUT tissues. Characteristics of the ECM, including the quantity and organization of collagen fibers, help to determine bladder wall passive mechanical properties. These passive mechanical properties influence normal pressure-volume relationships during bladder filling and the distribution of tension in the bladder wall that is generated by the smooth muscle and required for efficient voiding. Our prior work shows that maintenance of normal bladder compliance requires coordinated recruitment (straightening) of collagen fibers during bladder filling.74 In the older adult, premature fiber recruitment at a lower stretch limits the ability of the bladder to expand during the filling phase. As illustrated in Figure 2, our studies show that aging is associated with decreased collagen fiber tortuosity (a measure of elasticity) and an associated increased bladder stiffness.17 8-Aminoguanine treatment reverses these collagen abnormalities in aged Fischer 344 rats to that of a younger state.17

Figure 2. Aging leads to increased collagen fiber ‘stiffness’- restored to a younger state with 8-aminoguanine.

Figure 2.

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Illustrated are concurrent multi-photon imaging and mechanical testing of bladder collagen fibers (in red) in the detrusor layer of young (panel A), aged (panel B) and aged rats treated with 8-aminoguanine (panel C). Compared to the younger rat, there is a substantial decrease in ‘tortuosity’ or waviness in the detrusor layer of aged bladders which results in a premature collagen fiber recruitment and in turn, early shift to a stiff phase (panel D). This pathology is reversed with 8-aminoguanine treatment resulting in a recovery of collagen fiber tortuosity and a right shift of the stress-strain curve- closer to that of a younger, healthy state.17

Taken together, these findings are dramatic evidence that aging results in a dysregulation of the LUT purine ‘metabolome’ that impacts bladder filling and storage, and that inhibition of PNPase may be protective and prevent a number of adverse bladder health outcomes common in the older adult.

Conclusions and Perspectives

In this perspective, we provide an overview of the pharmacology of PNPase inhibitors and examples by which partial inhibition of PNPase can ‘restore’ bladder cellular health closer to that of a younger, healthy state. PNPase inhibitors have long been of interest as a primary target for chemotherapeutic intervention as well as having antitumor and antiviral activities. However, during the past few years, recent developments have shifted the attention toward the use of PNPase inhibitors for the treatment of various disorders including that of age-related benign urological conditions. This is of particular interest given the preponderance of benign conditions as the population ages. In this regard, LUTS problems are often taken for granted and undertreated (especially in the older population). The current line of pre-clinical investigation strongly suggests that inhibition of PNPase may reverse or restore bladder cellular dysfunction to a younger and healthier state, unlike many current treatments that focus on symptom management and suppression. In addition, PNPase inhibitors may also have benefit as an adjunctive therapy given along with existing pharmacologic treatments, which could improve the response in patients with inadequate response to an existing therapy and lead to a decrease incidence of prominent adverse effects especially in the older adult. While certainly further evidence of safety and efficacy is required, PNPase inhibitors could be an effective treatment for age associated LUTDs.

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