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Extracellular Nucleotides and Renal Function
Increasing evidence suggests that the ATP/P2 receptor system acts in an autocrine or paracrine fashion to affect various aspects of renal function. P2 receptors have been identified in most renal vessels and nephron segments; ATP is released from renal epithelial cells; and enzymes responsible for ATP breakdown are expressed in the vasculature and tubules. Stimulation of P2 receptors in the afferent arterioles by ATP released from renal nerve endings or from adjacent macula densa cells induces vasoconstriction and contributes to the control of renal haemodynamics. In the tubule, there is evidence for a variety of P2-mediated effects: inhibition of proximal tubular reabsorption; inhibition of Na + ,K + ,2Cl − cotransporter activity (through increased nitric oxide synthesis) in the thick ascending limb of the loop of Henle; inhibition of magnesium reabsorption in the distal tubule; and modulation of sodium and water reabsorption in the collecting duct. Finally, P2 receptors, particularly P2X subunits, appear to play an important role in renal pathology, specifically in cyst formation in polycystic kidney disease and in some forms of renal injury and inflammation.
Keywords
kidney, purinergic, purinoceptor, ATP, nitric oxide, nephron, ectonucleotidase, cyst, inflammation
Historically, the control of renal vascular and tubular function has been attributed solely to neural and endocrine regulation. However, in addition to these extrinsic factors, it is now recognized that several complex humoral control systems exist within the kidney that act in an autocrine and/or paracrine manner. One of these is the extracellular nucleotide/P2 receptor system.
Although physiological actions of extracellular adenine nucleotides were reported as long ago as 1929, it was not until many years later (1972) that the importance of ATP as a transmitter for non-adrenergic, non-cholinergic neurones of the autonomic nervous system was proposed by Geoffrey Burnstock. Since then it has become apparent that the function of extracellular nucleotides is not confined to neurones: rather, they are ubiquitous autocrine/paracrine agents regulating diverse physiological processes in almost every tissue in the body. Information on their roles in the kidneys has only really begun to emerge in the last decade.
P2 Receptors
Extracellular nucleotides exert their effects by binding to and activating cell surface located receptors; P2 receptors. These are subdivided into P2X receptors, of which seven mammalian subunits have been cloned (P2X 1–7 ), and P2Y receptors, of which eight mammalian subtypes are currently recognized, P2Y 1, 2, 4, 6, and P2Y 11–14 .
P2X Receptors
P2X receptor subunits are proteins with two transmembrane-spanning regions, the N- and C-termini being within the cell. Three P2X subunits assemble to form a P2X receptor ion channel that, when activated, is permeable to small cations (Na + , K + , Ca 2+ ). Each of the seven P2X subunits can make homomeric ion channels, and can also form heteromeric assemblies involving more than one type of subunit. Until recently it had been thought that P2X 7 subunits could only make homomeric assemblies, but a P2X 4/7 heteromer has now been described. As well as a non-selective ion channel, the P2X 7 receptor can form a larger membrane pore, and initiate cell death by necrosis or apoptosis.
The principal natural ligand for all P2X subunits is ATP. The P2X 1 subunit is the most sensitive (requiring sub-micromolar concentrations of ATP); P2X 2–6 subunits require micromolar concentrations, while the P2X 7 subunit is easily the least sensitive, requiring almost millimolar concentrations.
P2Y Receptors
P2Y receptors are G-protein-coupled receptors with seven transmembrane-spanning regions; the C-terminus is inside the cell and the N-terminus extracellular. In rodents, ATP is probably the principal natural ligand for P2Y 2, 4, and P2Y 11 subtypes and, at sufficiently high dose and/or receptor density, can activate P2Y 1, 12, and P2Y 13 subtypes. However, the natural ligand for P2Y 1, 12, and P2Y 13 subtypes is ADP. Although P2Y 6 receptors can also be activated by ADP, UDP is much more potent. In rodents, UTP activates P2Y 2 and P2Y 4 subtypes with similar potency to ATP, an observation often used in physiological studies as an initial pointer to receptor identity. Human P2Y 4 receptors, however, are activated primarily by UTP (50-fold more potent than ATP ). This is a particularly striking example of species differences, serving to highlight the need for caution before extrapolating from findings in one species to another. The P2Y 14 receptor is exceptional in that its natural ligand is UDP-glucose; although originally believed to be unaffected by unglycosylated purine- or pyrimidine-based nucleotides, it is now known that UDP is also a full agonist at rat and human P2Y 14 receptors.
P2Y receptors are coupled to either G q or G i signaling proteins. P2Y 1, 2, 4, 6, and P2Y 11 subtypes are coupled to G q /G 11 , resulting in PLC-β activation and increased [Ca 2+ ] i , while P2Y 12–14 are coupled to G i /G o , resulting in adenylyl cyclase inhibition and reduced cAMP levels. The P2Y 11 subtype is unusual, in that it can couple to both G q and G s , resulting in both PLC-β and adenylyl cyclase activation, causing increased cAMP levels.
Heterodimeric Receptors and Dinucleotide Receptors
A further layer of complexity has been added to the picture with the finding that adenosine A1 receptors can be co-expressed with P2Y 1 or P2Y 2 receptors (and possibly other P2Y subtypes) as a discrete receptor type, at least in non-renal cells. The chimeric nature of such receptors is reflected in their mixed pharmacological and signaling properties. The possible functional significance of these heterodimeric P1/P2Y receptors with regard to the kidneys is currently unknown.
Finally, a number of dinucleotides, in which the 5′-carbon positions of two nucleosides are linked by a polyphosphate chain, occur naturally in the body. These dinucleotides can be symmetrical (e.g., Ap 4 A, where two adenosine moieties are linked by a chain of four phosphates) or asymmetrical (e.g., Up 4 A, where a uridine moiety and an adenosine moiety are similarly linked). Dinucleotides can have both vascular and tubular effects within the kidneys ( vide infra ), but the receptors responsible are unknown; evidence for dinucleotide-specific receptors has been provided in other tissues, but several P2Y receptors (P2Y 1, 2, 4, and P2Y 6 ) and P2X receptors (P2X 1–5 ) are known to be dinucleotide-sensitive.
It is likely that both adenine-based and uracil-based nucleotides are released from most cells in the body (including renal cells); moreover, ecto-enzymes that metabolize nucleotides, either inactivating them or converting them to molecular forms that can stimulate different P2 receptor subtypes, are ubiquitous ( vide infra ). Figure 18.1 shows the molecular structures of some of the principal nucleotides involved, and Figure 18.2 provides a simplified overview of nucleotide release, degradation, and purinoceptor (i.e., P1 (adenosine) receptor and P2 receptor) activation.
Synthetic Agonists and Antagonists of P2 Receptors
Agonists
An ever-increasing range of synthetic nucleotide analogs and non-nucleotide agonists is being developed in an attempt to find agents that, unlike naturally occurring nucleotides, are not subject to degradation by ectonucleotidases, and can act as selective agonists for given receptor subtypes. Such exclusivity is rarely achieved, although substantial progress is now being made. Unfortunately, many of the initial observations on P2 receptor stimulation and renal function were made at a time when information on the selectivity of agonists was incomplete, and the agonists used were often more promiscuous than was appreciated, giving rise to misleading interpretations. Thus, although 2 meSADP, for example, has been used as an agonist for P2Y 1 receptors, it also activates P2Y 12 and P2Y 13 subtypes; the same applies to 2 meSATP which, additionally, can stimulate a number of P2X receptors, while ATPγS, originally used as a P2Y 2 and/or P2Y 4 agonist, is now known as a broad-spectrum agonist, being effective in a range of P2Y and P2X receptors. Another ATP analog, 2′3′- O -(4-benzoylbenzoyl)ATP (BzATP), has often been used as a “selective” P2X 7 agonist, given that it is more potent than ATP at this receptor subunit, but it is also effective at P2X 1, 3 and P2Y 5 subunits, so it is in reality only a non-selective P2X agonist. Furthermore, BzATP has been shown to act as an antagonist at P2Y 4 receptors.
As our knowledge of truly selective P2 agonists expands, future investigations should provide more precise information about the purinoceptor subtype(s) involved in a given physiological response. That knowledge, however, is still limited. The N -methanocarba-ADP derivative MRS2365 is selective for P2Y 1 receptors; MRS2698 and INS365 (Up 4 U or “diquafosol”) are selective P2Y 2 agonists; and UDPβS, INS48823, and MRS2693 are selective P2Y 6 agonists. At the time of writing, a selective agonist for P2Y 4 receptors has not been identified. Similarly, a truly selective agonist for any of the P2X subunits is still lacking.
Antagonists
As with agonists, nucleotide receptor-selective antagonists are something of a rarity. Probably the compound most commonly used to inhibit P2 receptors is suramin, although it also affects a variety of other cellular processes. In sufficient concentration, suramin antagonizes practically every P2 receptor subtype, be it P2Y or P2X. The same comment applies to PPADS (pyridoxal-5-phosphate-6-azophenyl 2′,4′-disulphonic acid) and, to a lesser extent, reactive blue 2 (RB-2). However, a clutch of selective and potent antagonists is now available. Thus, the ADP derivatives MRS2179, MRS2279, and MRS2500 are selective P2Y 1 antagonists; AR-C126313 and AR-C118925 are selective P2Y 2 antagonists; MRS2578 is a selective P2Y 6 antagonist; INS49266, INS50589, and AZD6140 are selective P2Y 12 antagonists; and MRS2211 is a selective P2Y 13 antagonist.
For P2X subunits, the list is shorter. Ip 5 I is a selective P2X 1 antagonist, and A-740003 and A-438079 are selective P2X 7 antagonists. Trinitrophenyl-ATP (TNP-ATP) “selectively” antagonizes P2X 1–5 subunits without affecting P2Y receptors.
Assignment of Physiological Responses to Specific P2 Receptor Subtypes
The plasma membranes of any renal cell, be it vascular or tubular, can contain a variety of P2 receptor subtypes. Moreover, epithelial cells can have different (as well as the same) subtypes on their apical and basolateral membranes. This raises the question of how to attribute a given functional response to a particular subtype. A number of approaches can be used. First, it is useful to identify immunologically the subtypes present in the region of interest (although this, of course, depends on the availability of suitable antibodies) and, if possible, to localize the receptor to apical and/or basolateral membrane. In some cases, instead of the immunohistochemical approach, determination of mRNA has been used, although this obviously does not guarantee the presence of the receptor protein itself. Second, it is possible to try to mimic the effect of the naturally occurring nucleotide using “selective” agonists and antagonists. However, as indicated above, only a few of these are truly selective (although the situation is improving). Consequently, it is usually necessary to compare the individual responses to a variety of agonists (both natural and synthetic) to provide a pharmacological profile from which tentative conclusions can be drawn, but even then their effects will depend not only on agonist/antagonist concentration, but also on the number and distribution of receptor subtypes. Moreover, naturally occurring agonists are degraded by ectonucleotidases, making it difficult to control their absolute concentrations at the receptor site. A further limitation is the use of intracellular Ca 2+ transients to assess responses to direct application of agonists, since these are not invariably associated with recognizable functional changes.
A completely different, and superficially more attractive, approach is to use “knockout” mice in which the gene encoding the receptor of interest has been deleted. However, this is not without its own potential problems. Life-long, global deletion of a receptor subtype that performs a vital function is likely to lead to compensatory changes in several organ systems. The P2 receptor profile within the kidney may then change in order to restore overall excretion rates, which could then lead to misleading conclusions about the role of the receptor. The Cre-loxP system adds a degree of refinement to the gene-targeting approach, permitting tissue- or cell-type-specific deletion. Nevertheless, compensatory changes in up- or downstream nephron segments cannot be excluded, and this approach is further complicated by incomplete (knockdown rather than knockout) and off-target deletion. Furthermore, genetically-engineered deletions have so far been restricted to mice, where P2Y 2 receptors seem to predominate in the renal tubule. There are important differences in the distributions of receptor subtypes between mice and rats – and presumably between mice and other species. Thus, for the foreseeable future it seems that we will need to continue to rely on a combination of approaches; as yet, there is no “silver bullet” when it comes to defining P2 receptor function.
Finally, to obviate the need for working with complex renal tubules, many investigators have made use of simpler systems: immortalized cell lines originally derived from renal-like tissue (e.g., Madin–Darby canine kidney (MDCK) cells). Unfortunately, these cell lines often express membrane proteins that differ from those found in native tissue. Consequently, in this chapter we will avoid deductions based solely on observations concerning P2 receptors in non-native renal tissue.
P2 Receptors and Renal Function
The Renal Vasculature
Figure 18.3 summarizes current knowledge about the distribution of P2 receptors in renal vascular and tubular structures. P2 receptors are expressed widely in the renal vasculature, in the glomerulus, and in the extraglomerular mesangium. Immunohistochemical and Western analyses indicate that P2X 1 receptors are present in the vascular smooth muscle of the rat renal artery, arcuate and interlobular arteries, and the afferent arteriole, but not in the efferent arteriole. Functional approaches have confirmed the expression of a P2X 1 -like receptor in the afferent arteriole. P2X 2 subunits have been immunolocalized in the smooth muscle of larger arteries and veins within the kidney, and molecular evidence has recently been provided for P2X 4 subunits, at least in arcuate and interlobular arteries. Of the P2Y receptors, P2Y 1 has an extensive distribution, being expressed in the endothelium of the large arteries, and both afferent and efferent arterioles.
Most information concerning P2 receptor expression in the glomerulus comes from cell culture systems. On the basis of mRNA detection and/or agonist profiling, P2Y 1,2,4, and P2Y 6 subtypes and P2X 2,3,4,5, and P2Y 7 subunits have been identified in glomerular mesangial cells ; P2Y 1,2 and P2Y 6 subtypes in podocytes ; and P2Y 1 and P2Y 2 subtypes in glomerular endothelial cells. Studies performed on RNA extracted from pools of intact glomeruli from rats found messages encoding P2Y 1,2,4 and P2Y 6 subtypes ; the expression of other P2Y receptors was not assessed. Immunohistochemical analysis and measurements of agonist-induced phosphoinositide production confirmed the presence P2Y 1 and P2Y 2 subtypes in the rat glomerulus. On the basis of co-localization with cell-specific markers, P2Y 1 receptors were localized in mesangial cells and P2Y 2 receptors in podocytes ; expression of P2Y 4 and P2Y 6 receptor protein could not be confirmed, either functionally or immunologically. Of the P2X subtypes, only a low and variable expression of P2X 7 immunoreactivity was found in the rat glomerulus.
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