The Dark Side of Extracellular ATP in Kidney Diseases

Abstract

Intracellular ATP is the most vital source of cellular energy for biologic systems, whereas extracellular ATP is a multifaceted mediator of several cell functions via its interaction, in an autocrine or paracrine manner, with P2 purinergic receptors expressed on the cell surface. These ionotropic and metabotropic P2 purinergic receptors modulate a variety of physiologic events upon the maintenance of a highly sensitive “set point,” the derangement of which may lead to the development of key pathogenic mechanisms during acute and chronic diseases. Growing evidence suggests that extracellular ATP signaling via P2 purinergic receptors may be involved in different renal pathologic conditions. For these reasons, investigators and pharmaceutical companies are actively exploring novel strategies to antagonize or block these receptors with the goal of reducing extracellular ATP production or accelerating extracellular ATP clearance. Targeting extracellular ATP signaling, particularly through the P2X₇ receptor, has considerable translational potential, given that novel P2X₇-receptor inhibitors are already available for clinical use (e.g., CE224,535, AZD9056, and GSK1482160). This review summarizes the current evidence regarding the involvement of extracellular ATP and its P2 purinergic receptor–mediated signaling in physiologic and pathologic processes in the kidney; potential therapeutic options targeting extracellular ATP purinergic receptors are analyzed as well.

ATP is the most important source of energy for intracellular reactions,¹ including synthesis and degradation of biologic molecules, muscle contraction, and membrane transport.² Intracellular ATP (iATP) levels, which are regulated by mitochondrial oxidative phosphorylation,³ reflect cell activity and viability, and a decline in iATP levels is associated with cell death.⁴ ATP is then exported from the mitochondrial matrix to the cytoplasm, across the inner mitochondrial membrane by the ADP/ATP carrier protein.³ The release of cytoplasmic ATP occurs as a physiologically regulated mechanism; indeed, ATP, given that it is a highly charged molecule, does not cross the plasma membrane.^5,6 Only a small fraction of ATP is released from the cells into the extracellular space through a series of finely tuned processes. For instance, in nonexcitable cells, ATP release occurs through exocytosis, ion channels, gap junction hemichannels, nucleotide transporters, and the cystic fibrosis transmembrane conductance regulator.⁷ The extracellular release of ATP can be triggered by a wide range of stimuli such as mechanical stress, cell membrane damage, inflammation, hypoxia, and excitation of neural tissue, and by cell growth and death.⁷ Although the concentration of ATP extracellularly is 1000-fold lower compared with the intracellular space, it can still exert precise, albeit partially unknown, signaling function on the cell membrane, by binding and activating membrane-anchored ionotropic P2X (P2XRs) and metabotropic P2Y (P2YRs), purinergic receptors that are likely ubiquitous. The eight G protein–coupled P2YRs (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁–P2Y₁₄) are activated by a range of native agonists (e.g., ATP, ADP, UTP, and UDP). Aside from the crucial role exerted by P2Y₁₂ in platelet aggregation, the targeting of which has yielded successful therapeutic strategies,⁸ additional functions of P2Y₁₂ (e.g., influencing microglial motility and stimulating ciliary movement and secretion of epithelial cells) have been discovered.⁹ In addition to the activation of purinergic signaling, the effect of extracellular ATP (eATP) relies upon the activity of ectonucleotidases (e.g., CD39 and CD73), which degrade eATP to ADP, AMP, and adenosine, all of which are capable themselves of exerting P1R- and P2R-mediated functions.¹⁰ The P2XRs (ligand-gated ion channels), of which seven have been identified thus far (P2X₁–P2X₇), bind ATP as their principal ligand and are involved in a variety of biologic responses, mainly related to inflammation, tissue damage and cell proliferation, and the graft-versus-host response.¹⁰ Among these P2XRs, P2X₇R appears particularly intriguing due to its emerging role in NLRP3/ASC/caspase1 inflammasome activation^11,12 as well as its involvement in acute allograft rejection.¹³ eATP-P2Rs signaling modulates several aspects of normal kidney function, but it is also implicated in the development of renal damage during chronic diseases such as diabetes and hypertension, as well as in inherited conditions including polycystic kidney disease (PKD), although the precise mechanisms underlying the involvement of these receptors is not fully understood.¹⁴ The present review addresses the pathophysiologic roles exerted in the kidney by eATP and by P2 purinergic receptors.

The eATP/P2Rs Axis in the Kidney

eATP Production in the Kidney

Sources of eATP in the kidney include perivascular and peritubular nerve terminals, aggregating platelets, circulating erythrocytes, and resident endothelial and epithelial cells.¹⁵ In renal epithelial cells, derived from human nephron segments, ATP release occurs in both the apical and basolateral membrane, with apical release predominating.¹⁶ eATP has been also found in the interstitial fluid of canine renal cortex, possibly released by macula densa cells in response to BP-induced changes in renal vascular resistance.¹⁷ The effective amount of eATP found in the extracellular space could be influenced by several factors, including the activity of the hydrolyzing enzymes ectoapyrases¹⁸ (Figure 1).

Figure 1.

The purinergic system in the glomerular cells. eATP has a short life depending on the activity of ectonucleotidases that degrade eATP to generate ADP, AMP, and adenosine. The purinergic receptors P2YR, P2XR, and P1R bind extracellular nucleotides and nucleosides, and their activation causes a cascade of signaling. Such an effect may promote cellular proliferation or apoptosis of mesangial cells. Ado, adenosine; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; IP₃, inositol 1,4,5-triphosphate; AC, adenylate cyclase; PL, phospholipase.

Expression of Purinergic Receptors in the Kidney

eATP exerts effects on the kidney in both a paracrine and autocrine manner, via the activation of all P2Xs and some P2Ys receptors.¹⁵ These receptors are expressed in the cortical and in the medullary renal compartments^14,15; however, upon stringent analysis, some of these results appear controversial and mainly based on mRNA expression, which does not necessarily imply an increased functional activity of these receptors. The lack of availability of adequate antibodies as well as the complexity of the P2R structure may have caused uncertainty in their immunohistochemical evaluation. It can be also hypothesized that some of these receptors, particularly P2X₇R, are scarcely expressed in physiologic conditions but can be upregulated during inflammation. The localization of ionotropic and metabotropic receptors in the kidney is reported in Table 1.

Table 1.

P2XR and P2YR localization in the kidney

Receptor	Species	RNA/Protein	Renal Cell	Reference
P2X1	Mouse	Yes/no	Medullary thick ascending limb, inner strip of outer medulla	72
	Rat	No/yes	Vascular networks	73
	Pig	Yes/no	Epithelial cell line (LLC-PK1)	74
	Human	Yes/no	Mesangial cell culture	36
P2X2	Mouse	Yes/no	Inner strip of outer medulla	72
	Human	Yes/no	Mesangial cell culture	36
P2X3	Mouse	Yes/no	Inner strip of outer medulla, inner medullary collecting duct cell line	72,75
P2X4	Mouse	Yes/no	Inner medullary collecting duct cell line, distal convoluted tubule immortalized cells, inner stripe of the outer medulla, isolated tubules in medullary thick ascending limb	72,75,76
	Human	Yes/no	Mesangial cell culture	36
P2X5	Mouse	Yes/no	Distal convoluted tubule immortalized cells, inner stripe of the outer medulla, isolated tubules in medullary thick ascending limb	72,76
	Human	Yes/no	Mesangial cell culture	36
P2X6	Human	Yes/no	Mesangial cell culture	36
P2X7	Mouse	Yes/no	Inner stripe of the outer medulla	72
	Rat	Yes/yes	Mesangial cells and glomeruli	77
	Madin-Darby canine	Yes/yes	Epithelial cells	78
	Human	Yes/no	Mesangial cell culture	36
P2Y1	Mouse	Yes/no	Inner medullary collecting duct cells	75
	Rat	Yes/yes	Podocytes, proximal convoluted tubule, descending and thin ascending limb of Henle’s loop, outer medullary collecting duct	31,79
	Madin-	Yes/no	Epithelial cells	80
	Darby canine
	Human	Yes/no	Mesangial cell culture, epithelial cell line (A498)	36,81
P2Y2	Mouse	Yes/no	Medullary thick ascending limb, inner stripe of the outer medulla, distal convoluted tubule cells, cortical collecting duct cells	72,76,82
	Rat	Yes/yes	Proximal convoluted tubule, descending and thin ascending limb of Henle’s loop, outer medullary collecting duct, mesangial cells and glomeruli, inner medulla collecting duct	68,77,79,83
	Human	Yes/no	Mesangial cell culture, epithelial cell line (A498)	36,81
P2Y4	Rat	Yes/no	Mesangial cells and glomeruli, proximal convoluted tubule, thin ascending limb of Henle’s loop, outer medullary collecting duct	77,79
	Human	Yes/no	Mesangial cell culture	36
P2Y6	Mouse	Yes/no	Medullary thick ascending limb, inner strip of the outer medulla	72
	Rat	Yes/no	Mesangial cells and glomeruli, proximal convoluted and straight tubule, thin descending limb of Henle’s loop, medullary and cortical thick ascending limb, outer medullary and cortical collecting duct	68,84
	Human	Yes/no	Mesangial cell culture	36
P2Y11	Madin-	Yes/no	Epithelial cells	80
	Darby canine
	Human	Yes/no	Mesangial cell culture, epithelial cell line (A498)	36,81
P2Y12	Mouse	Yes/no	Inner strip of the outer medulla	72
	Human	Yes/no	Mesangial cell culture	36
P2Y13, P2Y14	Mouse	Yes/no	Inner strip of the outer medulla	72

Effects of eATP Signaling via Purinergic Receptors

Effects on Renal Hemodynamics

eATP exerts a fundamental role in the regulation of renal hemodynamics and microcirculation¹⁹ (Figure 2). For instance, the intrarenal infusion of ATP in canine kidneys in vivo produces vasodilation through P2XR and P2YR activation and stimulation of endothelial release of nitric oxide.²⁰ However, some studies suggest that eATP-mediated vasoconstriction may occur in juxtamedullary nephron preparations in vitro and that eATP may induce preglomerular vasoconstriction of afferent arterioles.²¹ Another eATP-mediated effect on renal hemodynamics may stem from tubuloglomerular feedback, which is operated by the macula densa. Indeed, macula densa releases eATP in response to increased luminal NaCl concentration, thus regulating the GFR and renal blood flow²² (Figure 2).

Figure 2.

eATP signaling effects along the nephron. NO, nitric oxide; ENaC, epithelial sodium channel; TGF, tubuloglomerular feedback; TRPC6, transient receptor potential cation channel, subfamily C, member 6.

Effects on Tubular Transport Function

Mechanical stimulation of renal tubules, either by cell swelling or by an increase in tubular flow, promotes ATP release by renal epithelial cells, which in turn activates P2 receptors in the apical and basolateral membrane, thus modulating renal tubular transport.^23,24 In the absence of fluid flow, primary cilium, expressed by renal epithelial cells, protrudes perpendicularly into the tubular lumen, while fluid flow triggers cilium bending that elicits tubular release of eATP and inhibits the renal tubular transport of solute and water.²⁴ In the thick ascending limb, ATP release may activate basolateral (via P2XRs) and luminal (via P2YRs) membrane and stimulate nitric oxide production, thus regulating NaCl absorption²⁵ and potentially modulating the activity of the epithelial sodium channel in the collecting duct²⁶ (Figure 2). eATP may therefore control extracellular fluid volume status and BP,^26,27 and maintenance of these regulatory functions appears to be performed by the connexin 30 hemichannel (Cx30) in the collecting duct.²⁸ Cx30 knockout (KO) mice, with a salt retention phenotype in response to acute elevations in BP, have a reduced ability to excrete urinary salt and water.²⁸ Interestingly, Cx30 KO mice showed reduced luminal ATP release in vitro²⁸ and in vivo, suggesting that Cx30-dependent purinergic intracellular calcium signaling may control the regulation of salt and water reabsorption.²⁹ Consistent with the aforementioned observations, upon injection of ATP agonists (α,β-methylene ATP and β,γ-methylene ATP) into the femoral vein of rat, there is an increase in diuresis and sodium excretion,³⁰ while pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a nonselective P2 antagonist,³⁰ abolished this effect. In conclusion, taken together, these findings confirm that eATP has an important role in the regulation of renal hemodynamics, salt, and water reabsorption, thus contributing to the maintenance of normal BP, body fluid, and electrolyte balance (Figure 2).

Effects on Permeation

Recent relevant contributions have identified and characterized P2YRs in podocytes.^31,32 eATP activates podocyte TRPC6 Ca²⁺-permeable channels, thus influencing foot process effacement and subsequent changing in glomerular basement membrane permeability.³³ Mechanical-induced podocyte injuries appear to be mediated, at least in part, by P2Rs, including P2Y₂.³²

Effects on Cell Proliferation and Extracellular Matrix Deposition

Extracellular nucleotides may regulate renal fibroblast proliferation and activity, thus influencing fibroblast-to-myofibroblast transformation, confirming the existence of an interesting P2R-mediated cross-talk between epithelial cells and fibroblasts.^34,35 eATP also increases the proliferation of human mesangial cells, via P2YR-mediated activation of the Ras-Raf-MAPK^42/44 signal transduction pathway.³⁶

Pathologic Role of eATP in Kidney Diseases

Diabetic Nephropathy

Preclinical studies on the role of eATP signaling in diabetic nephropathy are limited by the lack of suitable murine models of the disease.^37,38 In the CD39 (the main vascular ectonucleotidase)–null diabetic mouse model, glomerulosclerosis is more severe than in age-matched diabetic wild-type animals, thus suggesting a protective role against inflammation of this eATP-hydrolyzing enzyme.³⁹ More recently, the genetic deletions of the adenosine A_2B receptor and of CD73 (a key enzyme that produces extracellular adenosine) in mice have been found to be associated with more severe diabetic nephropathy.⁴⁰ Increased eATP signaling via P2X₄R due to hyperglycemia induces activation of the NLRP3 inflammasome, thus stimulating IL-1β and IL-18 release, with development of tubulointerstitial inflammation^41,42 (Table 2). Two studies revealed that in individuals with type 2 diabetes and diabetic nephropathy, P2X₄R, P2X₇R, and NLPR3 are upregulated compared with controls, and that tubular P2X₄R expression colocalized in confocal analysis with NLRP3, IL-1β, and IL-18 expression.^12,41 This is in agreement with the results from in vitro studies in HK-2 cells, in which high-glucose challenge increased protein expression of NLRP3 and augmented the release of IL-1β and IL-18⁴¹ (Table 2). Thus, the activation of eATP signaling may play a crucial role in the development of diabetic nephropathy by enhancing inflammation.

Table 2.

Kidney diseases in which a putative role for eATP signaling is claimed

Disease	Model	eATP Signaling	Reference
Diabetic nephropathy	In vitro	Hyperglycemia induces an increase in eATP signaling via P2X4R with NLRP3 activation that stimulates IL-1β and IL-18 release and development of tubulointerstitial inflammation	41,42
	In vitro	P2X4R, P2X7R, and NLRP3 are upregulated in individuals with T2D and diabetic nephropathy, tubular P2X4R expression colocalizes with NLRP3, IL-1β, and IL-18	12,41
	In vitro	In HK-2 cells, high-glucose challenge increases expression of NLRP3, cleaves caspase-1, and IL-1β and the release of IL-1β and IL-18	41
Hypertension	In vivo	In a murine model of hypertension, P2X7R KO mice show reduced BP, renal injury, urinary albumin excretion, and renal interstitial fibrosis	43
	In vivo	There is an association between BP and the rs591874 polymorphism of the P2X7R gene	46
GN	In vivo	In rat and murine models of GN and in renal biopsies of individuals with lupus nephritis, an increase in glomerular P2X7R expression is observed	48
	In vivo	In a model of GN using P2X7R KO mice, P2X7R deficiency is renoprotective	49
PKDs	In vivo	In Han:SPRD rats, renal P2Y2R, P2Y6R, and P2X7R expression is increased	53
	In vitro	Epithelial cells generated from individuals with PKD release a higher amount of eATP compared with healthy controls as well as in a murine model of PKD	16,54
Kidney allograft rejection	In vivo	No direct evidence of eATP signaling in kidney transplantation
		In murine models of heart, islet, and lung transplantation P2X1R expression increases in syngenic and allogeneic graft, P2X7R upregulation is during allograft rejection	56
Other kidney diseases	In vitro	P2X7R is implicated in interstitial inflammation and fibrosis, tubular atrophy, and renal cell apoptosis	59

T2D, type 2 diabetes; NLRP3, NOD-like receptor-family protein 3.

Hypertension

Some studies have suggested that P2X₇Rs may be relevant in the onset of hypertension by inducing a proinflammatory setting, in addition to influencing NaCl and water reabsorption.^18,43,44 P2X₇R KO mice were used to investigate the role of P2X₇R in a deoxycorticosterone acetate high-salt diet–induced hypertension murine model.⁴³ Although P2X₇R mRNA and protein expression increased after treatment in the kidney of wild-type mice, P2X₇R KO mice showed reduced BP, renal injury, urinary albumin excretion, and renal interstitial fibrosis compared with controls⁴³ (Table 2). Furthermore, a recent article suggested that CD73 and adenosine A_2B receptor mRNA/protein and mRNA expression, respectively, were augmented in kidneys of angiotensin II–infused mice.⁴⁵ Despite these interesting observations, very few human studies, mostly genetic, have thus far explored the link between purinergic signaling and BP control. An association between BP and the single nucleotide polymorphism rs591874 in the first intron of the P2X₇R gene was found⁴⁶ (Table 2). However, another study found no relationship between two single nucleotide polymorphisms of the P2X₇R gene, Glu496Ala and His155Tyr, and endothelial function or arterial stiffness in essential hypertensive patients.⁴⁷ Taken together, these studies suggest that altered eATP signaling may contribute to hypertension by controlling vascular tone and by favoring Na⁺ retention (Table 2).

GN

An increase in glomerular P2X₇R mRNA and protein expression was observed in rat and murine models of GN compared with controls, as well as in renal biopsies obtained from individuals with lupus nephritis.⁴⁸ P2X₇R deficiency was shown to be renoprotective in an experimental model of antibody-mediated GN, with P2X₇R KO mice displaying a reduction in glomerular thrombosis by 60%, in proteinuria by 52% and in serum creatinine by 38% compared with wild-type⁴⁹ (Table 2). Renal expression of the P2X₇/NLRP3 inflammasome pathway and IL-1β are significantly increased in a mouse model of lupus nephritis, and P2X₇ inhibition reduces immune complex deposition in the kidneys as well as the levels of circulating anti–double-stranded DNA antibodies.⁵⁰ In a rat model of GN, the activation of adenosine A_2A receptors prevents renal infiltration of macrophages and arrests the progression of kidney disease, by inhibiting incoming fibrosis.⁵¹ Therefore, eATP signaling is likely involved in the pathogenesis of GN; however, the underlying mechanisms and its functional importance need to be further elucidated.

PKD

Accumulating evidence suggests that eATP signaling via P2XRs and P2YRs could be detrimental with regard to progression of PKD.⁵² In Han:SPRD rats which developed polycystic kidneys, increased P2Y₂R, P2Y₆R, and P2X₇R renal mRNA expression was evident compared with control animals,⁵³ suggesting a possible role of these receptors in the disease (Table 2). Renal epithelial cells from individuals with PKD release higher amounts of ATP (from 0.5 μM to 2 μM) compared with those obtained from healthy controls, which then may be trapped in the lumen of the cyst.¹⁶ Interestingly, cyst fluid from PKD kidneys contained large amounts of ATP (up to 10 μM),¹⁶ and eATP may augment PKD cyst widening through stimulation of salt and water secretion across PKD cystic epithelia.¹⁶ It is possible that a defect in the degradation of eATP in epithelial cells, due to lack and/or mislocalization of ecto-ATPases, ectoapyrases, and ectonucleotidases, occurs in individuals with PKD; the eATP signal may therefore last longer in the PKD microenvironment.⁵⁴ Indeed, although normal renal epithelial cells degraded 50% of the total ATP in the medium in 20 minutes, 50% of the total ATP was degraded by 3 hours in PKD epithelial cells.⁵⁴ As far as the involvement of the adenosine receptor in polycystic kidney disease, PKD1-mutated cystic cells express increased levels of adenosine A_3A receptors.⁵⁵ Thus, cyst growth and expansion in polycystic kidney disease may be worsened by eATP and adenosine-mediated signaling, but it remains unclear whether this may lead to a clinically relevant therapeutic strategy.

Kidney Allograft Rejection

Although the literature is lacking in direct studies on the role of eATP in kidney transplantation, preclinical and clinical studies are available for other models of allotransplantation.¹⁰ An increased release of ATP is likely to occur in allograft transplantation, possibly due to ischemia/reperfusion or upon the activation of immune cells, thus acting as a danger signal that may modulate the alloimmune response.¹⁰ In models of murine heart, islet, and lung transplantation, P2X₁R expression was increased in both syngeneic and allogeneic grafts, whereas intragraft P2X₇R upregulation was observed only during allograft rejection⁵⁶ (Table 2). This may suggest that P2X₇R upregulation is specifically associated with the alloimmune response, whereas P2X₁R expression is more related to a peritransplant inflammatory response and possibly ischemia/reperfusion events.^13,57 Interestingly, adenosine A_2A receptor signaling may attenuate ischemia-reperfusion injury and allogeneic T-cell recognition, thus delaying allograft rejection.⁵⁸ The aforementioned data suggested that eATP is involved in the regulation of cellular and immunologic process that occurs during allograft organ rejection.

Other Kidney Diseases

P2X₇R is implicated in the onset of interstitial inflammation, tubular fibrosis, and atrophy that rapidly occur after unilateral ureteral obstruction,⁵⁹ whereas P2X₄R has recently unveiled protective properties in the above-described setting.⁶⁰ Extracellular adenosine has recently been regarded as an intriguing novel modulator of AKI through its renal signaling. The A_1A, A_2A, A_3A adenosine receptors have different beneficial effects against ischemia-reperfusion injury and AKI, by modulating metabolic demand and leukocyte-mediated renal inflammation, as well as by decreasing necrosis, apoptosis, and tissue damage.⁶¹ The activation of adenosine receptors is needed for efficient kidney protection, and the clinical availability of adenosine may suggest a fast-track for this compound to be translated into the clinical setting when supplementation is needed.

Targeting eATP and Its Receptors in the Kidney

Diabetic Nephropathy

The enzyme apyrase, the P2 receptor antagonist suramin, the P2X₄R selective antagonist 5-BDBD, and silencing of the P2X₄R gene are all able to nearly normalize NLRP3 expression and reduce the release of IL-1β and IL-18, which is increased by high glucose in HK-2 cells in vitro⁴¹ (Table 3). In another study, the increased expression of NLRP3 and IL-18 release were attenuated by P2X₇R silencing in murine podocytes.¹² Furthermore, the P2X₇R inhibitor periodate-oxidized ATP reduced TGF-β and mesangial extracellular matrix production by rat mesangial cell culture under high-glucose conditions⁴² (Table 3). Targeting of eATP signaling may reduce the inflammatory component of diabetic nephropathy and thus may be considered a future novel therapeutic tool for diabetic nephropathy.⁴¹

Table 3.

Preclinical targeting of eATP signaling in different kidney diseases

Target	Antagonist	Preclinical Effect	Reference
P2Xs/P2Ys	PPADS/Suramin	Reduces cyst size in Madin-Darby canine kidney-derived cysts	70
P2Xs/P2Ys	PPADS	Reduces glomerular mesangial cell proliferation in the anti-Thy1 rat model of GN	68
P2Xs/P2Ys	PPADS	Prevents afferent arteriolar thickening development in angiotensin II infusion in rat	62
		Reduces necrosis-associated inflammation	35
P2Xs/P2Ys	Suramin	Normalizes NLRP3 expression, reducing the release of IL-1β and IL-18 increased by high glucose in HK-2 cells	41
		Promotes renal repair after injury when administered with adult renal progenitor cells	71
P2Xs	NF279	Prevents vasoconstriction of afferent arteriolar diameter, driving an increase in renal perfusion pressure in rodents	63
P2Xs	TNP-ATP	Normalizes NLRP3 expression, reducing the release of IL-1β and IL-18 increased by high glucose in HK-2 cells	41
P2X1	PPADS/IP5I	Inhibits autoregulatory control of whole-kidney blood flow	64
P2X4	5-BDBD gene silencing	Normalizes NLRP3 expression, reducing the release of IL-1β and IL-18 increased by high glucose in HK-2 cells	41
P2X7	A-438079	Decreases glomerular thrombosis, glomerular macrophage infiltration and proteinuria in a rat model of GN	49
P2X7	Brilliant Blue G	Causes a reduction in arterial BP and a decrease in renal vascular resistance in the Fischer rat	65,66
P2X7	A-438079	Diminishes renal fibroblast death	35
P2X7	A-438079/oATP	Decreases the frequency of cystic phenotype	69
P2X7	oATP	Reduces the mesangial extracellular matrix and TGF-β upregulation in rat mesangial cell culture under high-glucose conditions	42
P2X7	oATP	Prolongs heart and islet graft survival in mouse	13,57
P2X7	P2X7R silencing	In murine podocytes, attenuates upregulated expression of NLRP3, pro-caspase 1, and release of IL-18	12
		Diminishes renal fibroblast death	35
eATP	apyrase	Normalizes NLRP3 expression, reducing the release of IL-1β and IL-18 increased by high glucose in HK-2 cells	41
		Reduces necrosis-associated inflammation	35
eATP	CD39-CD73 genetic deletion	Enhances the hypoxic injury in kidney transplantation murine models and reduces cardiac allograft survival	10

NF279, 8,8′-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphthalenetrisulfonic acid); TNP-ATP, 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate; IP5I, P1,P5-di(-inosine-5-pentaphosphate) pentasodium salt; 5-BDBD, 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one); A-438079, 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine; oATP, periodate-oxidized ATP; NLRP3, NOD-like receptor-family protein 3.

Hypertension

The involvement of the P2 receptorial system in the modulation of vascular tone and arterial pressure is complex and still under investigation. Administration of PPADS, a nonselective P2 antagonist, significantly prevented the development of afferent arteriolar thickening during angiotensin II infusion in rats, as well as reduced the increase in α-actin expression in mesangial cells⁶² (Table 3). In vitro exposure to a P2XR antagonist (NF279) prevented vasoconstriction of afferent arteriolar diameter, increasing renal perfusion pressure in rodents.⁶³ In vivo inhibition of P2X₁R with PPADS or IP5I inhibits autoregulatory control of whole-kidney blood flow,⁶⁴ whereas the infusion of Brilliant Blue G, a P2X₇R antagonist, to Fischer rats and Dahl salt-sensitive rats reduced arterial BP and decreased renal vascular resistance.^65,66 On the other hand, P2Y₂R modulates, via paracrine signaling, renal sodium excretion in the distal nephron in mice, thus playing a pivotal role in maintaining arterial BP within a normal range.⁶⁷ In conclusion, although it is difficult to anticipate the net effect of modulating eATP signaling on arterial BP, a reduction in vasoconstriction tone and in renal salt and water homeostasis suggests a potential benefit in hypertension.

GN

PPADS, a nonselective P2 antagonist, injected intravenously and intraperitoneally in vivo in the anti-Thy1 model of rat mesangial proliferative GN reduced glomerular mesangial cell proliferation, without affecting the proliferative activity of non-mesangial cells (i.e., mainly endothelial cells and monocytes/macrophages).⁶⁸ In a rat model of antibody-mediated GN, administration of A-438079 decreased glomerular thrombosis and reduced glomerular macrophage infiltration and proteinuria compared with the vehicle-treated group⁴⁹ (Table 3). The robust proinflammatory role of eATP signaling suggests a potential effective therapeutic option for its targeting in GN.

PKD

In a zebrafish model of PKD, oxidized ATP and A-438079, P2X₇R antagonists, decreased the frequency of the cystic phenotype.⁶⁹ In the growth of Madin-Darby canine kidney-derived cysts, the use of P2XR/P2YR inhibitors (suramin and PPADS) significantly reduced cyst size.⁷⁰ When ATP was removed from the culture medium using higher concentrations of apyrase, cyst growth was also reduced significantly⁷⁰ (Table 3). eATP signaling may thus promote cyst growth and its targeting may slow the progression of the disease.

Kidney Allograft Rejection

In heart, islet, and lung transplantation models, targeting of eATP/P2X₇R signaling has been associated with long-term graft function, thus potentially representing an intriguing therapeutic target in kidney transplantation.^10,56 Heart grafts of periodate-oxidized ATP–treated mice showed mild graft lymphocyte infiltration and reduced coronaropathy compared with untreated animals.¹³ Analogous results were obtained in a murine model of islet transplantation, wherein treatment with eATP targeting showed a significant synergism with rapamycin in reducing the alloimmune response⁵⁷ (Table 3). CD39 and CD73 ectoenzymes may represent future therapeutic targets as well.¹⁰ Genetic ablation of CD39 and CD73 enhanced hypoxic injury in kidney and liver transplantation murine models and reduced cardiac allograft survival¹⁰ (Table 3). Clinical studies in kidney transplant recipients are underway and will clarify whether eATP can serve as a therapeutic target.

Other Kidney Diseases

Depletion of eATP with apyrase or inhibition of the P2XR with PPADS has been shown to reduce the extent of necrosis-associated inflammation.³⁵ Furthermore, treatment with A438079, a highly selective P2X₇R inhibitor, or knockdown of the P2X₇R with small interference RNA diminished renal fibroblast death induced by supernatant collected from necrotic cells.³⁵ Mice treated with suramin and murine adult renal progenital cells underwent extensive renal repair after injury.⁷¹ Both studies suggested that blocking eATP may be a promising therapeutic treatment for AKI.

In conclusion, knowledge and understanding of eATP function in the renal system has grown in recent years. eATP signaling may modulate renal hemodynamics, microcirculation, BP, and tubular transport of solute and fluid. Furthermore, eATP signaling may play an important role in increasing kidney damage and inflammation in diabetic nephropathy, as well as in other systemic diseases characterized by a relevant degree of renal inflammatory response. It is also involved in the pathogenesis of hypertension, GN, and PKD, as well as in the regulation of cellular and immunologic processes that occur during allograft organ rejection.^10,52 P2XR inhibitors (e.g., CE224,535, AZD9056, and GSK1482160) are available for clinical use and are under evaluation as immunomodulatory agents.¹⁰ Thus, eATP signaling may be targeted in a wide range of pathologic renal conditions and may lead to the discovery of clinically relevant therapeutic strategies.

Disclosures

None.