NDT Advance Access published March 31, 2015 Nephrol Dial Transplant (2015) 0: 1–8 doi: 10.1093/ndt/gfv028
Full Review Glutamate receptors in the kidney Stuart E. Dryer1,2 1
Department of Biology and Biochemistry, University of Houston, Houston, TX, USA and 2Division of Nephrology, Baylor College of Medicine,
Correspondence and offprint requests to: Stuart Dryer; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
L-Glutamate
The non-essential amino acid glutamate (L-Glu) plays many roles in cells. Besides being a component of proteins, L-Glu is a key intermediate in metabolic pathways related to energy production, nitrogen metabolism and responses to oxidative stress. Within the kidney, L-Glu contributes to ammonia secretion and regulation of acid-base balance. Significantly, L-Glu is also the most abundant excitatory neurotransmitter in the vertebrate central nervous system (CNS) [1]. L-Glu is packaged into specialized vesicles in presynaptic elements by means of specific transport systems. Calcium-dependent exocytosis mediates release of L-Glu, leading to an explosive increase in its concentration within 30–50 nm synaptic clefts. L-Glu diffuses to postsynaptic elements almost instantaneously, where it activates a variety of receptors [2]. Activation of L-Glu receptors triggers multiple responses that occur over a wide range of time scales, including transient membrane depolarization, as well as persistent or even permanent biochemical changes, including changes in gene expression [3]. L-Glu is removed from the synaptic cleft by transporter systems located within neurons and surrounding glial cells, but if sufficient amounts of L-Glu are released, it can diffuse to receptors in extrasynaptic regions of neurons and neighboring glial cells [3]. L-Glu and co-agonists of L-Glu receptors, such as glycine and D-serine, can also be released from surrounding glial cells and thereby modulate synaptic function, a process known as gliotransmission [4]. These observations, made over a period of four decades, have shed considerable light on plasticity in the nervous system, including phenomena implicated in learning and memory [5], and formation and refinement of neural circuits within the developing CNS [6]. Excessive activation of L-Glu receptors in the CNS can also induce a phenomenon known as excitotoxicity, which has been associated with several forms of neurodegeneration [7]. At the other extreme, hypo-function
(L-Glu) plays an essential role in the central nervous system (CNS) as an excitatory neurotransmitter, and exerts its effects by acting on a large number of ionotropic and metabotropic receptors. These receptors are also expressed in several peripheral tissues, including the kidney. This review summarizes the general properties of ionotropic and metabotropic L-Glu receptors, focusing on N-methyl-D-aspartate (NMDA) and Group 1 metabotropic glutamate receptors (mGluRs). NMDA receptors are expressed in the renal cortex and medulla, and appear to play a role in the regulation of renal blood flow, glomerular filtration, proximal tubule reabsorption and urine concentration within medullary collecting ducts. Sustained activation of NMDA receptors induces Ca2+ influx and oxidative stress, which can lead to glomerulosclerosis, for example in hyperhomocysteinemia. Group 1 mGluRs are expressed in podocytes and probably in other cell types. Mice in which these receptors are knocked out gradually develop albuminuria and glomerulosclerosis. Several endogenous agonists of L-Glu receptors, which include sulfur-containing amino acids derived from L-homocysteine, and quinolinic acid (QA), as well as the co-agonists glycine and D-serine, are present in the circulation at concentrations capable of robustly activating ionotropic and metabotropic L-Glu receptors. These endogenous agonists may also be secreted from renal parenchymal cells, or from cells that have migrated into the kidney, by exocytosis or by transporters such as system x(-)(c), or by transporters involved in ammonia secretion. L-Glu receptors may be useful targets for drug therapy, and many selective orally-active compounds exist for investigation of these receptors as potential drug targets for various kidney diseases. Keywords: excitotoxicity, glomerulosclerosis, glutamate, homocysteine, NMDA © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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of certain L-Glu receptors has been associated with severe cognitive disturbances, including schizophrenia [8]. As a result of decades of work on L-Glu receptor, a remarkably rich pharmacology has been developed. Many years ago, it was recognized that L-Glu receptors are also expressed in peripheral tissues [2]. It is now clear that these receptors are functional, although their role in normal physiology and pathophysiology is not as well understood as in the CNS. In the rest of this review, we will summarize current knowledge of L-Glu receptors expressed in the kidney. A recurring theme will be that L-Glu is not the only endogenous molecule capable of activating L-Glu receptors; indeed at least some renal receptors may be primarily responsive to dynamic fluctuations in the concentration of other endogenous ligands.
TYPES OF L-Glu RECEPTORS
G E N E R A L F E AT U R E S O F N M D A R E C E P T O R S NMDA receptors are robustly activated by the canonical agonist NMDA, and by several other structurally related diacidic molecules, including several sulfur-containing amino acids derived from metabolism of L-cysteine (L-Cys) and Lmethionine (L-Met) [11]. Several potent agonists [11], including L-aspartate (L-Asp), D-aspartate (D-Asp), L-homocysteic acid (L-HCA) and quinolinic acid (QA), occur within the
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G E N E R A L F E AT U R E S O F G R O U P 1 M E TA B O T R O P I C L - G l u R E C E P T O R S Group 1 metabotropic glutamate receptors (mGluRs) are homodimers of subunits that each contain seven membranespanning helical domains. The gene encoding mGluR1 is expressed in five different splice variants (a–e) whereas the gene
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There are two broad categories of receptors responsive to [2]. The first are the so-called ionotropic receptors. Based on sequence and pharmacology, these receptors can be divided into three groups of tetrameric proteins, including Nmethyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainate (KA) receptors. The names of these receptors are based historically on their canonical selective agonists, none of which occurs endogenously [1]. Ionotropic L-Glu receptors function primarily as chemically-gated cation channels that trigger changes in membrane potential in target cells, although it has been recently suggested that NMDA receptors can also evoke responses that are independent of ion flux [9]. The second broad class is comprised of metabotropic L-Glu receptors (mGluRs) classified within the Class C superfamily G protein-coupled receptors (GPCRs), along with GABAB receptors, calcium-sensing receptors and certain vomeronasal and taste receptors [2]. There are eight known mGluRs, each encoded by a distinct gene, which are further subdivided into Group 1 (mGluRs 1 and 5), Group 2 (mGluRs 2 and 3) and Group 3 (mGluRs 4, 6, 7 and 8). These receptors are coupled to number of standard G protein cascades, typically resulting in changes in cAMP, phospholipase C and/or Ca2+ dynamics. Transcripts encoding nearly all of the known L-Glu receptors and many of their interacting proteins have been detected in the kidney [10]. However, most of the functional studies in kidney have examined NMDA receptors and Group 1 mGluRs, and the rest of this review will focus on those receptors, their ligands, and their role in renal function. L-Glu
circulation and elsewhere. Functional NMDA receptors are always heteromeric, but two NR1 subunits (also known as GluN1 or Grin1) are always present and are obligatory in order to form a functional receptor [12]. Functional NMDA receptors also contain two NR2 (GluN2 or Grin2) subunits, or one NR2 and one NR3 (GluN3 or Grin3) subunit [12]. In heterologous expression systems, it is possible to form a functional receptor comprised of two NR1 and two NR3 subunits, but those receptors are activated by glycine and not by L-Glu or NMDA, and may not occur endogenously [13]. The four types of NR2 subunits (referred to as NR2A, NR2B, NR2C and NR2D) are each encoded by separate genes, as are the NR3A and NR3B subunits [12]. These features are summarized in Figure 1. NMDA and related di-acidic agonists activate these receptors by binding to sites located on NR2 subunits, which occurs at micromolar concentrations. Activation of NMDA receptors also requires binding of smaller monoacidic co-agonists, such as glycine or D-serine, to specific sites on NR1 subunits, often referred to as glycineB sites [14]. Recent studies suggest that some NMDA receptor glycineB sites are selective for different coagonists [14]. At central synapses, the local changes in L-Glu concentration are much larger and more rapid than those of glycine or D-serine. However, it is probably not justified to assume similar agonist dynamics in the vicinity of peripheral NMDA receptors. Other endogenous moieties, including Zn2+ and polyamines, can further modulate the function of NMDA receptors [15]. A crucial feature of NMDA receptors is their high Ca2+ permeability relative to that of monovalent cations (PCa/PM) [16]. Another is voltage-dependent blockade of the receptor pore by intracellular Mg2+ [17], which in neuronal receptors can introduce a zone of negative slope conductance into current-voltage relationships, leading to bi-stable membrane potential behavior. While these features generally apply to all NMDA receptors, quantitative details of ligand sensitivity, gating kinetics, unitary conductance, PCa/PM and sensitivity to Mg2+ block, depend on subunit composition [12]. Only a subset of the many possible subunit stoichiometries has been characterized [12], and the properties of NMDA receptors may be further altered by post-translational modifications. The pharmacology of NMDA receptors is richly developed, and now includes a large number of small molecules that inhibit these receptors by targeting sites on NR2 subunits [D-2-aminophosphonovaleric acid (D-APV)], the glycineB sites on NR1 subunits (kynurenic acid and many others) or the receptor pore (e.g. MK-801 and memantine). Many of these compounds are orally active, including the low-affinity pore blocker memantine, which is now widely used for treatment of neurodegenerative disorders [18].
encoding mGluR5 occurs in two splice variants (a–b) [2, 19]. The extracellular amino-terminal portions of mGluRs are large, with more than 500 residues extending outside of the plasma membrane. These domains contain the recognition sites for amino acids, and have some structural homology to proteins involved in bacterial amino acid transport. The ligand-binding domain is a bi-lobed structure sometimes referred to as a Venus fly-trap (VFT) domain, which closes when an activating ligand is bound within its pocket. The VFT domain is linked to the transmembrane regions by a short cysteine-rich domain (CRD) that promotes stable dimerization of the receptors [19]. The CRD also translates conformational changes that occur upon ligand binding to the rest of the protein, and it is thought that the CRDs move closer together upon receptor activation [20]. The carboxy-terminals of Group 1 mGluRs contain a proline-rich Homer-binding motif that mediates interactions with a number of other proteins and anchors these receptors at specific sites on the cell surface, for example at postsynaptic densities [19]. As with most GPCRs, activation of Group 1 mGluRs activates transduction cascades that are highly pleiotropic and cell-type specific. Moreover, there is evidence that some of the effects of mGluRs are G protein-independent and entail activation of Src family tyrosine kinases [21]. Several selective agonists and antagonists have been discovered that act on Group 1 mGluRs.
Renal glutamate receptors
The most widely used selective Group 1 agonist is (S)-3,5dihydroxyphenylglycine (DHPG), whereas there are a number of competitive and non-competitive Group 1 antagonists, such as (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Group 1 receptors can also be activated by acidic L-homocysteine derivatives, including L-HCA, at physiologically relevant concentrations [22]. These features are summarized in Figure 2.
SOURCES OF ENDOGENOUS AGONISTS FOR RENAL mGluRs AND NMDA RECEPTORS In principle, endogenous agonists can be delivered to renal L-Glu receptors through the circulation, or released by neurons, renal parenchymal cells, or from other types of cells that have migrated into the kidney. Several potential agonists are continuously present at relatively high concentrations in the circulation. For example, plasma L-Glu concentration is normally 25–35 μM [23, 24], and can increase to above 100 µM after a highprotein meal [23]. Plasma L-Asp is 20–30 µM [24], while plasma glycine typically ranges from 200 to 500 µM [24]. The co-agonist D-serine is produced by amino acid racemases and can be detected in the circulation and in the kidney [25]. In fact, circulating L-Glu concentrations are already close to the top of the concentration-response curves reported for many NMDA
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receptors are tetrameric proteins comprised to two NR1 subunits (gray) and two NR2 subunits (blue), which may be the same or different. The active receptor catalyzes cation flux when the small monoacidic agonists glycine or D-serine bind to active sites on NR1 subunits, and when appropriate di-acidic molecules (e.g. NMDA, L-Glu or L-HCA) bind to sites on NR2 subunits. The open pores have a high Ca2+ permeability and are subjected to voltage-dependent block by Mg2+. (B) Some endogenous receptors have one NR2 and one NR3 subunit (green), along with two NR1 subunits. Those receptors are somewhat less sensitive to Mg2+ block, and have reduced Ca2+ permeability. (C) In heterologous expression systems it is possible to form glycine receptors from two NR1 subunits and two NR3 subunits. Those receptors have unusual pharmacological properties that do not correspond to any native receptors described to date. (D) A large number of inhibitors have been discovered (a few of which are shown in red). These small molecules target active sites on NR1 subunits, NR2 subunits or within the pore. In addition, there are several allosteric modulators of NMDA receptors, such as Zn2+ and polyamines, whose precise actions depend on subunit composition.
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F I G U R E 1 : General features of NMDA receptors. Detailed properties of the receptors depend on subunit composition. (A) Typical NMDA
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receptors [11, 26] and mGluRs [22]. This raises the question of why renal receptors for L-Glu are not continuously saturated, or alternatively, fully desensitized. In this regard, we will present data further below showing that NMDA receptors of podocytes are surprisingly resistant to activation by L-Glu and L-Asp [27]. There is evidence that L-Glu can be locally released from renal cells by exocytotic mechanisms similar to those found in neurons. For example, L-Glu-containing vesicles have been detected in podocyte foot processes, along with vesicular L-Glu transporters, and many of the other proteins needed to stimulate Ca2+-dependent exocytosis are also present in foot processes [10]. Moreover, application of toxins such as α-latrotoxin can evoke tetanus toxin-sensitive release of L-Glu from cultured podocytes [10], suggesting that exocytotic mechanisms are functional. L-Glu and related amino acids can also be released by non-exocytotic mechanisms. One of particular interest is the system x(-)(c) transporter that catalyzes exchange of L-cystine (L-Cys2) for L-Glu in a variety of cells [28]. This transporter, which is widely expressed, supplies cells with L-Cys, a limiting substrate in the biosynthesis of glutathione (GSH), since L-Cys2 is the dominant form in extracellular fluids. Through system x(-)(c), oxidative stressors that induce GSH depletion will stimulate release of L-Glu in order to regenerate GSH within the kidney, local extracellular L-Glu concentrations may change during acid-base regulatory pathways that mediate ammonia excretion in response to acidosis [29], hypertonicity [30] and possibly in podocyte diseases [31]. Finally, it is worth noting that L-Glu can also be released from gap junction hemichannels [32]. Locally released l-Glu and related amino acids may be removed by excitatory amino acid transporters (EAATs), some of which are expressed in proximal tubules [33]. Any down-regulation of EAATs could also result in increased local availability of endogenous agonists.
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Circulating agonists can be derived from the metabolism of and tryptophan (L-Try) in the liver and elsewhere. Thus, L-Met is converted to L-homocysteine during the methionine cycle, which spontaneously oxidizes to form L-HCA, a potent agonist of NMDA receptors [11] and many types of mGluRs [22]. L-Met is normally regenerated from L-homocysteine during the folate cycle, a pathway catalyzed by a methionine synthetase, which uses methyl-tetrahydrofolate as a substrate and vitamin B12 as a cofactor. L-homocysteine can also be converted to L-Cys by transulfuration pathways that require cystathionine-β-synthetase, L-serine and S-adenosylmethionine [34]. Patients with hyperhomocysteinemia have considerably elevated concentrations of circulating agonists that are especially effective at NMDA receptors [11, 26]. The NMDA receptor agonist QA [11, 26] is produced by the kynurenine pathways of L-Try metabolism that also produce kynurenic acid, a competitive antagonist of the glycineB site on NMDA receptors [35]. It has been suggested that the ratio of local QA to kynurenic acid will determine the extent of NMDA receptor activation, and there is evidence for increased production of QA during immune activation, diabetes and as a consequence of renal insufficiency [35]. The activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD) in liver may be especially important in determining the ratio of QA to kynurenic acid [36]. It is not believed that QA is an effective agonist at mGluRs. It also should be noted that the uremic toxin guanidinosuccinate is also an effective NMDA agonist [37]. L-Met
EXPRESSION AND FUNCTION OF NMDA RECEPTORS AND mGluRs IN THE KIDNEY Microarray studies have shown that all known NMDA receptor transcripts can be detected in kidney [10], and there is now
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F I G U R E 2 : Features of Group 1 mGluRs. This group includes mGluR1 and mGluR5, which are expressed in podocytes. (A) These proteins are homodimeric GPCRs comprised of two heptahelical subunits connected to each other by covalent interactions between their CRD. The very large amino-terminal domains contain a structure known as a VFT domain that binds agonists, including the endogenous compounds L-Glu and certain acidic L-homocysteine derivatives, as well as various synthetic compounds. The CRD also transmits conformational changes from the VFT to other parts of the receptor, and may bind allosteric modulators. These receptors appear to interact with G proteins via their second intracellular loops, shown in red. The carboxy-terminals contain a Homer-binding motif that allows these receptors to form complexes with a wide range of other proteins, some of which may also include NMDA receptors. (B) Structures of the canonical agonist DHPG, and one of the most extensively studied antagonists, AIDA. Knocking out mGluR1 results in a gradually developing albuminuria and glomerulosclerosis in mice, whereas the agonist DHPG may be protective in certain mouse models of drug-induced glomerular disease.
a consensus that activation of these receptors affects renal function, and in some cases may induce renal dysfunction. Because NR1 subunits are obligatory for any functional receptor and are encoded by a single gene, many studies have focused on the distribution of these subunits. NR1 subunits are expressed at high levels in glomeruli, in podocytes and probably also in mesangial cells [10, 38, 39]. NR1 is also expressed in brush border membranes of the proximal tubule [40, 41] and possibly within the medulla [38]. There is strong evidence that NR2A subunits are expressed in normal glomeruli [38]. NR3 subunits have recently been detected in medullary collecting duct cells, as well as in cell lines derived from those structures [42]. The functional properties of NMDA receptors have been most extensively studied in podocytes [27]. Using whole-cell recordings, our group has observed that cationic currents are
robustly activated by NMDA (Figure 3), with half-maximal activation occurring around 50 μM [27]. There is a significant contribution of Ca2+ to these cationic currents (PCa/PM = 2.1), albeit considerably less than is commonly seen with neuronal NMDA receptors [16, 27]. These receptors are blocked by standard inhibitors acting at NR2 subunits (D-APV), at NR1 subunits (L689,560) and within the pore (MK-801 and Mg2+). They are robustly activated by L-HCA, QA and D-Asp, and their activation is markedly enhanced in the presence of the glycineB site agonist D-serine, although glycine is less effective in this respect [27], as recently reported for some synaptic receptors [14]. Notably, podocyte NMDA receptors are only weakly activated by L-Glu or L-Asp, and they do not desensitize even in the extended continuous presence of NMDA [27]. The weak responses to L-Glu and L-Asp are unusual, although this has been seen previously in at least
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F I G U R E 3 : Some characteristics of NMDA receptors revealed by whole-cell recordings. These particular examples are from an immortalized
mouse podocyte cell line, although functional NMDA receptors are also expressed in human podocyte cell lines and primary rat podocytes. (A) Schematic of experimental apparatus, showing recording electrode in contact with cell cytosol, and an adjacent micropipette used to deliver NMDA by means of pressure pulses. (B) Responses in a single cell to NMDA with this technique are highly reproducible, in this case consisting of inward cationic currents from a holding potential of −60 mV (top trace). This response can be maintained without desensitization even during a continuous 1-min application of 50 µm NMDA (middle trace). When membrane potential is allowed to fluctuate, we observe marked depolarization in response to NMDA, associated with a drop in input resistance (bottom trace). (C) Concentration-response relationship for NMDA in podocytes is quite similar to what is seen in neurons. (D) Responses to NMDA are markedly enhanced by adding D-serine to the bath, and the entire response is nearly abolished using one of the standard glycineB site inhibitors, L689,560, indicating that as with neuronal forms, both classes of active sites are required for activation of these receptors. For more details and additional data, see ref. [27].
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receptors that mediate these opposing actions [3, 14]. There is now considerable evidence that excessive NMDA receptor activation is also toxic for renal cells in vivo and in vitro. In cultured podocytes, we have observed that adding 50 µM NMDA or 50 µM L-HCA to media for 6–12 h markedly reduces steady-state surface expression of nephrin and podocin. Longer exposure to NMDA (24 h) markedly increased the accumulation of reactive oxygen species (ROS), which was accompanied by activation of RhoA, calcineurinNFATc pathways and increased expression of TRPC6 channels, especially on the cell surface ([46]; see also [47]), which probably is a secondary consequence of ROS generation [46, 47]. Induction of TRPC6 would tend to magnify Ca2+ overload evoked by NMDA receptors. After 72 h of continuous NMDA or L-HCA, there was a marked loss of viable cells associated with lactate dehydrogenase release into the medium, nuclear fragmentation and activation of several apoptotic markers, suggesting a combination of necrosis and apoptosis [46], as is seen with severe neuronal excitotoxicity [3]. All of these effects were blocked by MK-801. It is certainly possible that metabolic disturbances, especially alterations in energy metabolism, could make renal cells more susceptible to excitotoxic effects of NMDA agonists and co-agonists (Figure 4). Given this, it is not surprising that severe glomerulosclerosis and foot process effacement are observed in multiple rodent models of hyperhomocysteinemia, including high-methionine or low-folate diets in uni-nephrectomized rats [39, 47, 48], and cystathionine-β-synthestase knockout mice [49]. It is important to note that glomerulosclerosis and glomerular ROS generation in rats with hyperhomocysteinemia, due in part to activation of NADPH oxidases, is completely blocked by concurrent administration of MK-801, indicating an essential role for NMDA receptors in the resulting pathology [38]. A similar pattern is seen in cultured podocytes [46]. An increase in the expression or functional up-regulation of NMDA receptors could drive glomerulosclerosis, even without an increase in circulating ligands. In models of Alzheimer’s disease, it has been suggested that β-amyloid oligomers upregulate NMDA receptors, thereby inducing excitotoxicity [50]. Therefore, it is interesting that expression of renal NR1 subunits is increased following ischemia-reperfusion [41] and in the Akita mouse model of type-1 diabetes [51]. Moreover, there are reports that glycine infusion exacerbates, whereas glycineB inhibitors reduce kidney injuries evoked by ischemiareperfusion [52]. Finally we turn to possible functions of Group 1 mGluRs in kidney. It has been shown that mGluR1 and mGluR5 are expressed in podocyte foot processes [53, 54]. Rastaldi and co-workers have observed a progressive development of foot process effacement, glomerular basement membrane thickening, albuminuria and glomerulosclerosis in mice lacking mGluR1. The effect was not seen at birth but was robust by two months of age [53]. Conversely, application of the Group 1 agonist DHPG activated several transduction cascades in podocytes (including cAMP and CREB pathways) and is reported to protect Balb/c mice from nephrosis evoked by doxorubicin or puromycin aminonucleoside [54]. The mechanisms of these protective effects are not known.
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some neuronal preparations [43]. The overall pattern derived from electrophysiological observations [27] raises the possibility that these receptors contain an NR3 subunit [12]. It is also quite likely that NMDA receptors in other parts of the kidney have different properties. The functional significance of NMDA receptors for normal kidney physiology is not well understood. The earliest studies suggested that activation of NMDA receptors by infusion of glycine caused an increased blood flow in denervated kidneys that could be measured using Doppler flow probes, as well as an increase in glomerular filtration rate (GFR) as assessed by inulin clearance [44]. These effects of glycine were blocked by the NMDA receptor pore inhibitor MK-801 and also by the glycineB site antagonist 5,7-dichlorokynurenic acid [44]. Thomson and his co-workers subsequently demonstrated that local glycine infusion stimulated net reabsorption by the proximal tubule, and independently increased the single nephron GFR [40]. Both effects were blocked by MK-801, and the experimental design suggested that the relevant receptors are located within nephrons. On the other hand, Yang and co-workers observed that NMDA infusion into the renal artery caused a fall in GFR and urinary Na+ and K+ excretion that was exacerbated after ischemia-reperfusion, and blocked stereospecifically by dAPV [41], a competitive inhibitor of NMDA that acts at NR2 subunits. The reasons for these seemingly contradictory observations are not known, although it should be noted that the studies from the initial studies primarily utilized glycine infusion into male rats [40, 44], whereas the study by Yang et al. infused NMDA into female rats [41]. The nature of the agonist may be important, and it should not be simply assumed that ligands acting on NR2 subunits are the ones most responsible for dynamic signaling in the periphery. More recently, it has been observed that activation of NMDA receptors may help to maintain the epithelial phenotypes of proximal tubule cells, in opposition to inflammatory mediators such as TGFβ [45]. In cells derived from inner medullary collecting duct (IMCD), Bell and his co-workers observed that NR3A subunits are increased following hyperosmotic stress or ischemia. Knocking down NR3A subunits also reduced intracellular and cell-surface expression of aquaporin-2 in IMCD cells [42]. In addition, mice with a constitutive deletion of NR3A subnits (NR3A−/− mice) had reduced aquaporin-2 expression throughout the medulla [42]. These mice also exhibit a >6 h delay in their ability to produce concentrated urine in response to water deprivation [42]. Therefore, medullary NMDA receptors therefore appear to play a significant role in renal function. However, the activating ligands that produce these responses are not known, and the other NMDA receptor subunits that must be present in IMCD cells have not been identified. The presence of NR3 subunits suggests that glycine could be particularly effective at activating NMDA receptors in the medullary collecting ducts. NMDA receptors have been strongly implicated in a variety of CNS disorders, and can induce excitotoxicity when receptors are excessively stimulated for extended periods [3, 7, 14, 18]. NMDA receptor activation also produces pro-survival effects on neurons, and there has been an extensive literature and some controversy on the location and composition of
CONCLUSIONS
REFERENCES
It has been known for many years that receptors for L-Glu originally identified in the CNS have a wide distribution in the periphery, including several parts of the kidney. A very rich pharmacology has been developed to manipulate these receptors, and there is now evidence that agents acting on these receptors or their activating ligands might be useful in kidney diseases. Nevertheless, many additional studies will be needed to elucidate how these receptors regulate normal renal physiology. This will require determining which of the several possible endogenous agonists exert the greatest dynamic control of these receptors under various physiological conditions.
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FUNDING Research in our laboratory is currently supported by a grant from the Juvenile Diabetes Research Foundation. Our past work has also been supported by a grant from Pfizer Inc. C O N F L I C T O F I N T E R E S T S TAT E M E N T The results presented in this paper have not been published previously in whole or part, except in abstract format.
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marked oxidative stress, with accumulations of ROS such as H2O2 in bulk cytosol apparent after 24 h of continuous exposure. Bear in mind that elevated ROS generation probably occurs in certain sensitive compartments long before that. NMDA treatment for 24 h also causes activation of RhoA (B), calcineurin-NFAT pathways that are sensitive to cyclosporine (CsA) (C) and increases steady-state surface expression of Ca2+permeable TRPC6 channels (D). NMDA treatment also causes reduced surface expression of essential slit diaphragm proteins including nephrin and podocin as assessed by cell-surface biotinylation assays (E). Longer exposures to NMDA, for 72 h, cause nuclear fragmentation and increases in various markers of apoptosis (F). The overall pattern is similar to excitotoxic cell death evoked by a variety of NMDA receptor agonists in neurons. However, these effects require substantially longer exposures in podocytes. For more details, see ref. [46].
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F I G U R E 4 : Deleterious effects of sustained NMDA receptor activation in mouse podocytes. (A) The selective agonist NMDA (50 µM) causes
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35. Schwarcz R, Bruno JP, Muchowski PJ et al. Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci 2012; 13: 465–477 36. Matsuda H, Sato M, Yakushiji M et al. Regulation of rat hepatic α-aminoβ-carboxymuconate-ε-semialdehyde decarboxylase, a key enzyme in the tryptophan-NAD pathway, by dietary cholesterol and sterol regulatory element-binding protein-2. Eur J Nutr 2014; 53: 469–477 37. D’Hooge R, Raes A, Lebrun P et al. N-methyl-d-aspartate receptor activation by guanidinosuccinate but not by methylguanidine: behavioural and electrophysiological evidence. Neuropharmacology 1996; 35: 433–440 38. Giardino L, Armelloni S, Corbelli A et al. Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier. J Am Soc Nephrol 2009; 20: 1929–1940 39. Zhang C, Yi F, Xia M et al. NMDA receptor-mediated activation of NADPH oxidase and glomerulosclerosis in hyperhomocysteinemic rats. Antioxid Redox Signal 2010; 13: 975–986 40. Deng A, Thomson SC. Renal NMDA receptors independently stimulate proximal reabsorption and glomerular filtration. Am J Physiol Renal Physiol 2009; 296: F976–F982 41. Yang CC, Chien CT, Wu MH et al. NMDA receptor blocker ameliorates ischemia-reperfusion-induced renal dysfunction in rat kidneys. Am J Physiol Renal Physiol 2008; 294: F1433–F1440 42. Sproul A, Steele SL, Thai TL et al. N-methyl-d-aspartate receptor subunit NR3A expression and function in principal cells of the collecting duct. Am J Physiol Renal Physiol 2011; 301: F44–F54 43. Näström J, Schneider SP, Perl ER. Differential L-glutamate responsiveness among superficial dorsal horn neurons. J Neurophysiol 1994; 72: 2956–2965 44. Deng A, Valdivielso JM, Munger KA et al. Vasodilatory N-methyl-daspartate receptors are constitutively expressed in rat kidney. J Am Soc Nephrol 2002; 13: 1381–1384 45. Bozic M, de Rooij J, Parisi E et al. Glutamatergic signaling maintains the epithelial phenotype of proximal tubular cells. J Am Soc Nephrol 2011; 22: 1099–1111 46. Kim EY, Anderson M, Dryer SE. Sustained activation of N-methyl-d-aspartate receptors in podoctyes leads to oxidative stress, mobilization of transient receptor potential canonical 6 channels, nuclear factor of activated T cells activation, and apoptotic cell death. Mol Pharmacol 2012; 82: 728–737 47. Han H, Wang Y, Li X et al. Novel role of NOD2 in mediating Ca2+ signaling: evidence from NOD2-regulated podocyte TRPC6 channels in hyperhomocysteinemia. Hypertension 2013; 62: 506–511 48. Yi F, dos Santos EA, Xia M et al. Podocyte injury and glomerulosclerosis in hyperhomocysteinemic rats. Am J Nephrol 2007; 27: 262–268 49. Sen U, Basu P, Abe OA et al. Hydrogen sulfide ameliorates hyperhomocysteinemia-associated chronic renal failure. Am J Physiol Renal Physiol 2009; 297: F410–F419 50. Danysz W, Parsons CG. Alzheimer’s disease, β-amyloid, glutamate, NMDA receptors and memantine─searching for the connections. Br J Pharmacol 2012; 167: 324–352 51. Kundu S, Pushpakumar SB, Tyagi A et al. Hydrogen sulfide deficiency and diabetic renal remodeling: role of matrix metalloproteinase-9. Am J Physiol Endocrinol Metab 2013; 304: E1365–E1378 52. Arora S, Kaur T, Kaur A et al. Glycine aggravates ischemia reperfusioninduced acute kidney injury through N-methyl-d-aspartate receptor activation in rats. Mol Cell Biochem 2014; 393: 123–131 53. Puliti A, Rossi PI, Caridi G et al. Albuminuria and glomerular damage in mice lacking the metabotropic glutamate receptor 1. Am J Pathol 2011; 178: 1257–1269 54. Gu L, Liang X, Wang L et al. Functional metabotropic glutamate receptors 1 and 5 are expressed in murine podocytes. Kidney Int 2012; 81: 458–468 Received for publication: 12.12.2014; Accepted in revised form: 8.1.2015
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12. Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 2013; 14: 383–400 13. Smothers CT, Woodward JJ. Pharmacological characterization of glycineactivated currents in HEK 293 cells expressing N-methyl-d-aspartate NR1 and NR3 subunits. J Pharmacol Exp Ther 2007; 322: 739–748 14. Papouin T, Ladépêche L, Ruel J et al. Synaptic and extrasynaptic NMDA receptors are gated by different endogenous co-agonists. Cell 2012; 150: 633–646 15. Zheng X, Zhang L, Durand GM et al. Mutagenesis rescues spermine and Zn2+ potentiation of recombinant NMDA receptors. Neuron 1994; 12: 811–818 16. Mayer ML, Westbrook GL. Permeation and block of N-methyl-d-aspartic acid receptor channels by divalent cations in mouse cultured central neurones. J Physiol 1987; 394: 501–527 17. Mayer ML, Westbrook GL, Guthrie PB. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 1984; 309: 261–263 18. Chen HS, Lipton SA. The chemical biology of clinically tolerated NMDA receptor antagonists. J Neurochem 2006; 97: 1611–1626 19. Hermans E, Challiss RA. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C Gprotein-coupled receptors. Biochem J 2001; 359: 465–484 20. Huang S, Cao J, Jiang M et al. Interdomain movements in metabotropic glutamate receptor activation. Proc Natl Acad Sci U S A 2011; 108: 15480–15485 21. Heuss C, Scanziani M, Gähwiler BH et al. G-protein-independent signaling mediated by metabotropic glutamate receptors. Nat Neurosci 1999; 2: 1070–1077 22. Shi Q, Savage JE, Hufeisen SJ et al. l-homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists. J Pharmacol Exp Ther 2003; 305: 131–142 23. Stegink LD, Filer LJ, Jr, Baker GL. Plasma glutamate concentrations in adult subjects ingesting monosodium L-glutamate in consomme. Am J Clin Nutr 1985; 42: 220–225 24. Zhou Y, Qiu L, Xiao Q et al. Obesity and diabetes related plasma amino acid alterations. Clin Biochem 2013; 46: 1447–1452 25. Horio M, Kohno M, Fujita Y et al. Levels of d-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice. Neurochem Int 2011; 59: 853–859 26. Vlachová V, Vyklický L, Vyklický L, Jr et al. The action of excitatory amino acids on chick spinal cord neurones in culture. J Physiol 1987; 386: 425–438 27. Anderson M, Suh JM, Kim EY et al. Functional NMDA receptors with atypical properties are expressed in podocytes. Am J Physiol Cell Physiol 2011; 300: C22–C32 28. Bridges R, Lutgen V, Lobner D et al. Thinking outside the cleft to understand synaptic activity: contribution of the cystine-glutamate antiporter (System xc-) to normal and pathological glutamatergic signaling. Pharmacol Rev 2012; 64: 780–802 29. Nissim I. Newer aspects of glutamine/glutamate metabolism: the role of acute pH changes. Am J Physiol 1999; 277: F493–F497 30. Klawitter J, Rivard CJ, Brown LM et al. A metabonomic and proteomic analysis of changes in IMCD3 cells chronically adapted to hypertonicity. Nephron Physiol 2008; 109: 1–10 31. Altintas MM, Moriwaki K, Wei C et al. Reduction of proteinuria through podocyte alkalinization. J Biol Chem 2014; 289: 17454–17467 32. Montero TD, Orellana JA. Hemichannels: New pathways for gliotransmitter release. Neuroscience. 2014; 286C: 45–59 33. Bailey CG, Ryan RM, Thoeng AD et al. Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria. J Clin Invest 2011; 121: 446–453 34. Finkelstein JD. Pathways and regulation of homocysteine metabolism in mammals. Semin Thromb Hemost 2000; 26: 219–225
Full Review Glutamate receptors in the kidney Stuart E. Dryer1,2 1
Department of Biology and Biochemistry, University of Houston, Houston, TX, USA and 2Division of Nephrology, Baylor College of Medicine,
Correspondence and offprint requests to: Stuart Dryer; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
L-Glutamate
The non-essential amino acid glutamate (L-Glu) plays many roles in cells. Besides being a component of proteins, L-Glu is a key intermediate in metabolic pathways related to energy production, nitrogen metabolism and responses to oxidative stress. Within the kidney, L-Glu contributes to ammonia secretion and regulation of acid-base balance. Significantly, L-Glu is also the most abundant excitatory neurotransmitter in the vertebrate central nervous system (CNS) [1]. L-Glu is packaged into specialized vesicles in presynaptic elements by means of specific transport systems. Calcium-dependent exocytosis mediates release of L-Glu, leading to an explosive increase in its concentration within 30–50 nm synaptic clefts. L-Glu diffuses to postsynaptic elements almost instantaneously, where it activates a variety of receptors [2]. Activation of L-Glu receptors triggers multiple responses that occur over a wide range of time scales, including transient membrane depolarization, as well as persistent or even permanent biochemical changes, including changes in gene expression [3]. L-Glu is removed from the synaptic cleft by transporter systems located within neurons and surrounding glial cells, but if sufficient amounts of L-Glu are released, it can diffuse to receptors in extrasynaptic regions of neurons and neighboring glial cells [3]. L-Glu and co-agonists of L-Glu receptors, such as glycine and D-serine, can also be released from surrounding glial cells and thereby modulate synaptic function, a process known as gliotransmission [4]. These observations, made over a period of four decades, have shed considerable light on plasticity in the nervous system, including phenomena implicated in learning and memory [5], and formation and refinement of neural circuits within the developing CNS [6]. Excessive activation of L-Glu receptors in the CNS can also induce a phenomenon known as excitotoxicity, which has been associated with several forms of neurodegeneration [7]. At the other extreme, hypo-function
(L-Glu) plays an essential role in the central nervous system (CNS) as an excitatory neurotransmitter, and exerts its effects by acting on a large number of ionotropic and metabotropic receptors. These receptors are also expressed in several peripheral tissues, including the kidney. This review summarizes the general properties of ionotropic and metabotropic L-Glu receptors, focusing on N-methyl-D-aspartate (NMDA) and Group 1 metabotropic glutamate receptors (mGluRs). NMDA receptors are expressed in the renal cortex and medulla, and appear to play a role in the regulation of renal blood flow, glomerular filtration, proximal tubule reabsorption and urine concentration within medullary collecting ducts. Sustained activation of NMDA receptors induces Ca2+ influx and oxidative stress, which can lead to glomerulosclerosis, for example in hyperhomocysteinemia. Group 1 mGluRs are expressed in podocytes and probably in other cell types. Mice in which these receptors are knocked out gradually develop albuminuria and glomerulosclerosis. Several endogenous agonists of L-Glu receptors, which include sulfur-containing amino acids derived from L-homocysteine, and quinolinic acid (QA), as well as the co-agonists glycine and D-serine, are present in the circulation at concentrations capable of robustly activating ionotropic and metabotropic L-Glu receptors. These endogenous agonists may also be secreted from renal parenchymal cells, or from cells that have migrated into the kidney, by exocytosis or by transporters such as system x(-)(c), or by transporters involved in ammonia secretion. L-Glu receptors may be useful targets for drug therapy, and many selective orally-active compounds exist for investigation of these receptors as potential drug targets for various kidney diseases. Keywords: excitotoxicity, glomerulosclerosis, glutamate, homocysteine, NMDA © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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of certain L-Glu receptors has been associated with severe cognitive disturbances, including schizophrenia [8]. As a result of decades of work on L-Glu receptor, a remarkably rich pharmacology has been developed. Many years ago, it was recognized that L-Glu receptors are also expressed in peripheral tissues [2]. It is now clear that these receptors are functional, although their role in normal physiology and pathophysiology is not as well understood as in the CNS. In the rest of this review, we will summarize current knowledge of L-Glu receptors expressed in the kidney. A recurring theme will be that L-Glu is not the only endogenous molecule capable of activating L-Glu receptors; indeed at least some renal receptors may be primarily responsive to dynamic fluctuations in the concentration of other endogenous ligands.
TYPES OF L-Glu RECEPTORS
G E N E R A L F E AT U R E S O F N M D A R E C E P T O R S NMDA receptors are robustly activated by the canonical agonist NMDA, and by several other structurally related diacidic molecules, including several sulfur-containing amino acids derived from metabolism of L-cysteine (L-Cys) and Lmethionine (L-Met) [11]. Several potent agonists [11], including L-aspartate (L-Asp), D-aspartate (D-Asp), L-homocysteic acid (L-HCA) and quinolinic acid (QA), occur within the
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G E N E R A L F E AT U R E S O F G R O U P 1 M E TA B O T R O P I C L - G l u R E C E P T O R S Group 1 metabotropic glutamate receptors (mGluRs) are homodimers of subunits that each contain seven membranespanning helical domains. The gene encoding mGluR1 is expressed in five different splice variants (a–e) whereas the gene
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There are two broad categories of receptors responsive to [2]. The first are the so-called ionotropic receptors. Based on sequence and pharmacology, these receptors can be divided into three groups of tetrameric proteins, including Nmethyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainate (KA) receptors. The names of these receptors are based historically on their canonical selective agonists, none of which occurs endogenously [1]. Ionotropic L-Glu receptors function primarily as chemically-gated cation channels that trigger changes in membrane potential in target cells, although it has been recently suggested that NMDA receptors can also evoke responses that are independent of ion flux [9]. The second broad class is comprised of metabotropic L-Glu receptors (mGluRs) classified within the Class C superfamily G protein-coupled receptors (GPCRs), along with GABAB receptors, calcium-sensing receptors and certain vomeronasal and taste receptors [2]. There are eight known mGluRs, each encoded by a distinct gene, which are further subdivided into Group 1 (mGluRs 1 and 5), Group 2 (mGluRs 2 and 3) and Group 3 (mGluRs 4, 6, 7 and 8). These receptors are coupled to number of standard G protein cascades, typically resulting in changes in cAMP, phospholipase C and/or Ca2+ dynamics. Transcripts encoding nearly all of the known L-Glu receptors and many of their interacting proteins have been detected in the kidney [10]. However, most of the functional studies in kidney have examined NMDA receptors and Group 1 mGluRs, and the rest of this review will focus on those receptors, their ligands, and their role in renal function. L-Glu
circulation and elsewhere. Functional NMDA receptors are always heteromeric, but two NR1 subunits (also known as GluN1 or Grin1) are always present and are obligatory in order to form a functional receptor [12]. Functional NMDA receptors also contain two NR2 (GluN2 or Grin2) subunits, or one NR2 and one NR3 (GluN3 or Grin3) subunit [12]. In heterologous expression systems, it is possible to form a functional receptor comprised of two NR1 and two NR3 subunits, but those receptors are activated by glycine and not by L-Glu or NMDA, and may not occur endogenously [13]. The four types of NR2 subunits (referred to as NR2A, NR2B, NR2C and NR2D) are each encoded by separate genes, as are the NR3A and NR3B subunits [12]. These features are summarized in Figure 1. NMDA and related di-acidic agonists activate these receptors by binding to sites located on NR2 subunits, which occurs at micromolar concentrations. Activation of NMDA receptors also requires binding of smaller monoacidic co-agonists, such as glycine or D-serine, to specific sites on NR1 subunits, often referred to as glycineB sites [14]. Recent studies suggest that some NMDA receptor glycineB sites are selective for different coagonists [14]. At central synapses, the local changes in L-Glu concentration are much larger and more rapid than those of glycine or D-serine. However, it is probably not justified to assume similar agonist dynamics in the vicinity of peripheral NMDA receptors. Other endogenous moieties, including Zn2+ and polyamines, can further modulate the function of NMDA receptors [15]. A crucial feature of NMDA receptors is their high Ca2+ permeability relative to that of monovalent cations (PCa/PM) [16]. Another is voltage-dependent blockade of the receptor pore by intracellular Mg2+ [17], which in neuronal receptors can introduce a zone of negative slope conductance into current-voltage relationships, leading to bi-stable membrane potential behavior. While these features generally apply to all NMDA receptors, quantitative details of ligand sensitivity, gating kinetics, unitary conductance, PCa/PM and sensitivity to Mg2+ block, depend on subunit composition [12]. Only a subset of the many possible subunit stoichiometries has been characterized [12], and the properties of NMDA receptors may be further altered by post-translational modifications. The pharmacology of NMDA receptors is richly developed, and now includes a large number of small molecules that inhibit these receptors by targeting sites on NR2 subunits [D-2-aminophosphonovaleric acid (D-APV)], the glycineB sites on NR1 subunits (kynurenic acid and many others) or the receptor pore (e.g. MK-801 and memantine). Many of these compounds are orally active, including the low-affinity pore blocker memantine, which is now widely used for treatment of neurodegenerative disorders [18].
encoding mGluR5 occurs in two splice variants (a–b) [2, 19]. The extracellular amino-terminal portions of mGluRs are large, with more than 500 residues extending outside of the plasma membrane. These domains contain the recognition sites for amino acids, and have some structural homology to proteins involved in bacterial amino acid transport. The ligand-binding domain is a bi-lobed structure sometimes referred to as a Venus fly-trap (VFT) domain, which closes when an activating ligand is bound within its pocket. The VFT domain is linked to the transmembrane regions by a short cysteine-rich domain (CRD) that promotes stable dimerization of the receptors [19]. The CRD also translates conformational changes that occur upon ligand binding to the rest of the protein, and it is thought that the CRDs move closer together upon receptor activation [20]. The carboxy-terminals of Group 1 mGluRs contain a proline-rich Homer-binding motif that mediates interactions with a number of other proteins and anchors these receptors at specific sites on the cell surface, for example at postsynaptic densities [19]. As with most GPCRs, activation of Group 1 mGluRs activates transduction cascades that are highly pleiotropic and cell-type specific. Moreover, there is evidence that some of the effects of mGluRs are G protein-independent and entail activation of Src family tyrosine kinases [21]. Several selective agonists and antagonists have been discovered that act on Group 1 mGluRs.
Renal glutamate receptors
The most widely used selective Group 1 agonist is (S)-3,5dihydroxyphenylglycine (DHPG), whereas there are a number of competitive and non-competitive Group 1 antagonists, such as (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Group 1 receptors can also be activated by acidic L-homocysteine derivatives, including L-HCA, at physiologically relevant concentrations [22]. These features are summarized in Figure 2.
SOURCES OF ENDOGENOUS AGONISTS FOR RENAL mGluRs AND NMDA RECEPTORS In principle, endogenous agonists can be delivered to renal L-Glu receptors through the circulation, or released by neurons, renal parenchymal cells, or from other types of cells that have migrated into the kidney. Several potential agonists are continuously present at relatively high concentrations in the circulation. For example, plasma L-Glu concentration is normally 25–35 μM [23, 24], and can increase to above 100 µM after a highprotein meal [23]. Plasma L-Asp is 20–30 µM [24], while plasma glycine typically ranges from 200 to 500 µM [24]. The co-agonist D-serine is produced by amino acid racemases and can be detected in the circulation and in the kidney [25]. In fact, circulating L-Glu concentrations are already close to the top of the concentration-response curves reported for many NMDA
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receptors are tetrameric proteins comprised to two NR1 subunits (gray) and two NR2 subunits (blue), which may be the same or different. The active receptor catalyzes cation flux when the small monoacidic agonists glycine or D-serine bind to active sites on NR1 subunits, and when appropriate di-acidic molecules (e.g. NMDA, L-Glu or L-HCA) bind to sites on NR2 subunits. The open pores have a high Ca2+ permeability and are subjected to voltage-dependent block by Mg2+. (B) Some endogenous receptors have one NR2 and one NR3 subunit (green), along with two NR1 subunits. Those receptors are somewhat less sensitive to Mg2+ block, and have reduced Ca2+ permeability. (C) In heterologous expression systems it is possible to form glycine receptors from two NR1 subunits and two NR3 subunits. Those receptors have unusual pharmacological properties that do not correspond to any native receptors described to date. (D) A large number of inhibitors have been discovered (a few of which are shown in red). These small molecules target active sites on NR1 subunits, NR2 subunits or within the pore. In addition, there are several allosteric modulators of NMDA receptors, such as Zn2+ and polyamines, whose precise actions depend on subunit composition.
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F I G U R E 1 : General features of NMDA receptors. Detailed properties of the receptors depend on subunit composition. (A) Typical NMDA
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receptors [11, 26] and mGluRs [22]. This raises the question of why renal receptors for L-Glu are not continuously saturated, or alternatively, fully desensitized. In this regard, we will present data further below showing that NMDA receptors of podocytes are surprisingly resistant to activation by L-Glu and L-Asp [27]. There is evidence that L-Glu can be locally released from renal cells by exocytotic mechanisms similar to those found in neurons. For example, L-Glu-containing vesicles have been detected in podocyte foot processes, along with vesicular L-Glu transporters, and many of the other proteins needed to stimulate Ca2+-dependent exocytosis are also present in foot processes [10]. Moreover, application of toxins such as α-latrotoxin can evoke tetanus toxin-sensitive release of L-Glu from cultured podocytes [10], suggesting that exocytotic mechanisms are functional. L-Glu and related amino acids can also be released by non-exocytotic mechanisms. One of particular interest is the system x(-)(c) transporter that catalyzes exchange of L-cystine (L-Cys2) for L-Glu in a variety of cells [28]. This transporter, which is widely expressed, supplies cells with L-Cys, a limiting substrate in the biosynthesis of glutathione (GSH), since L-Cys2 is the dominant form in extracellular fluids. Through system x(-)(c), oxidative stressors that induce GSH depletion will stimulate release of L-Glu in order to regenerate GSH within the kidney, local extracellular L-Glu concentrations may change during acid-base regulatory pathways that mediate ammonia excretion in response to acidosis [29], hypertonicity [30] and possibly in podocyte diseases [31]. Finally, it is worth noting that L-Glu can also be released from gap junction hemichannels [32]. Locally released l-Glu and related amino acids may be removed by excitatory amino acid transporters (EAATs), some of which are expressed in proximal tubules [33]. Any down-regulation of EAATs could also result in increased local availability of endogenous agonists.
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Circulating agonists can be derived from the metabolism of and tryptophan (L-Try) in the liver and elsewhere. Thus, L-Met is converted to L-homocysteine during the methionine cycle, which spontaneously oxidizes to form L-HCA, a potent agonist of NMDA receptors [11] and many types of mGluRs [22]. L-Met is normally regenerated from L-homocysteine during the folate cycle, a pathway catalyzed by a methionine synthetase, which uses methyl-tetrahydrofolate as a substrate and vitamin B12 as a cofactor. L-homocysteine can also be converted to L-Cys by transulfuration pathways that require cystathionine-β-synthetase, L-serine and S-adenosylmethionine [34]. Patients with hyperhomocysteinemia have considerably elevated concentrations of circulating agonists that are especially effective at NMDA receptors [11, 26]. The NMDA receptor agonist QA [11, 26] is produced by the kynurenine pathways of L-Try metabolism that also produce kynurenic acid, a competitive antagonist of the glycineB site on NMDA receptors [35]. It has been suggested that the ratio of local QA to kynurenic acid will determine the extent of NMDA receptor activation, and there is evidence for increased production of QA during immune activation, diabetes and as a consequence of renal insufficiency [35]. The activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD) in liver may be especially important in determining the ratio of QA to kynurenic acid [36]. It is not believed that QA is an effective agonist at mGluRs. It also should be noted that the uremic toxin guanidinosuccinate is also an effective NMDA agonist [37]. L-Met
EXPRESSION AND FUNCTION OF NMDA RECEPTORS AND mGluRs IN THE KIDNEY Microarray studies have shown that all known NMDA receptor transcripts can be detected in kidney [10], and there is now
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F I G U R E 2 : Features of Group 1 mGluRs. This group includes mGluR1 and mGluR5, which are expressed in podocytes. (A) These proteins are homodimeric GPCRs comprised of two heptahelical subunits connected to each other by covalent interactions between their CRD. The very large amino-terminal domains contain a structure known as a VFT domain that binds agonists, including the endogenous compounds L-Glu and certain acidic L-homocysteine derivatives, as well as various synthetic compounds. The CRD also transmits conformational changes from the VFT to other parts of the receptor, and may bind allosteric modulators. These receptors appear to interact with G proteins via their second intracellular loops, shown in red. The carboxy-terminals contain a Homer-binding motif that allows these receptors to form complexes with a wide range of other proteins, some of which may also include NMDA receptors. (B) Structures of the canonical agonist DHPG, and one of the most extensively studied antagonists, AIDA. Knocking out mGluR1 results in a gradually developing albuminuria and glomerulosclerosis in mice, whereas the agonist DHPG may be protective in certain mouse models of drug-induced glomerular disease.
a consensus that activation of these receptors affects renal function, and in some cases may induce renal dysfunction. Because NR1 subunits are obligatory for any functional receptor and are encoded by a single gene, many studies have focused on the distribution of these subunits. NR1 subunits are expressed at high levels in glomeruli, in podocytes and probably also in mesangial cells [10, 38, 39]. NR1 is also expressed in brush border membranes of the proximal tubule [40, 41] and possibly within the medulla [38]. There is strong evidence that NR2A subunits are expressed in normal glomeruli [38]. NR3 subunits have recently been detected in medullary collecting duct cells, as well as in cell lines derived from those structures [42]. The functional properties of NMDA receptors have been most extensively studied in podocytes [27]. Using whole-cell recordings, our group has observed that cationic currents are
robustly activated by NMDA (Figure 3), with half-maximal activation occurring around 50 μM [27]. There is a significant contribution of Ca2+ to these cationic currents (PCa/PM = 2.1), albeit considerably less than is commonly seen with neuronal NMDA receptors [16, 27]. These receptors are blocked by standard inhibitors acting at NR2 subunits (D-APV), at NR1 subunits (L689,560) and within the pore (MK-801 and Mg2+). They are robustly activated by L-HCA, QA and D-Asp, and their activation is markedly enhanced in the presence of the glycineB site agonist D-serine, although glycine is less effective in this respect [27], as recently reported for some synaptic receptors [14]. Notably, podocyte NMDA receptors are only weakly activated by L-Glu or L-Asp, and they do not desensitize even in the extended continuous presence of NMDA [27]. The weak responses to L-Glu and L-Asp are unusual, although this has been seen previously in at least
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F I G U R E 3 : Some characteristics of NMDA receptors revealed by whole-cell recordings. These particular examples are from an immortalized
mouse podocyte cell line, although functional NMDA receptors are also expressed in human podocyte cell lines and primary rat podocytes. (A) Schematic of experimental apparatus, showing recording electrode in contact with cell cytosol, and an adjacent micropipette used to deliver NMDA by means of pressure pulses. (B) Responses in a single cell to NMDA with this technique are highly reproducible, in this case consisting of inward cationic currents from a holding potential of −60 mV (top trace). This response can be maintained without desensitization even during a continuous 1-min application of 50 µm NMDA (middle trace). When membrane potential is allowed to fluctuate, we observe marked depolarization in response to NMDA, associated with a drop in input resistance (bottom trace). (C) Concentration-response relationship for NMDA in podocytes is quite similar to what is seen in neurons. (D) Responses to NMDA are markedly enhanced by adding D-serine to the bath, and the entire response is nearly abolished using one of the standard glycineB site inhibitors, L689,560, indicating that as with neuronal forms, both classes of active sites are required for activation of these receptors. For more details and additional data, see ref. [27].
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receptors that mediate these opposing actions [3, 14]. There is now considerable evidence that excessive NMDA receptor activation is also toxic for renal cells in vivo and in vitro. In cultured podocytes, we have observed that adding 50 µM NMDA or 50 µM L-HCA to media for 6–12 h markedly reduces steady-state surface expression of nephrin and podocin. Longer exposure to NMDA (24 h) markedly increased the accumulation of reactive oxygen species (ROS), which was accompanied by activation of RhoA, calcineurinNFATc pathways and increased expression of TRPC6 channels, especially on the cell surface ([46]; see also [47]), which probably is a secondary consequence of ROS generation [46, 47]. Induction of TRPC6 would tend to magnify Ca2+ overload evoked by NMDA receptors. After 72 h of continuous NMDA or L-HCA, there was a marked loss of viable cells associated with lactate dehydrogenase release into the medium, nuclear fragmentation and activation of several apoptotic markers, suggesting a combination of necrosis and apoptosis [46], as is seen with severe neuronal excitotoxicity [3]. All of these effects were blocked by MK-801. It is certainly possible that metabolic disturbances, especially alterations in energy metabolism, could make renal cells more susceptible to excitotoxic effects of NMDA agonists and co-agonists (Figure 4). Given this, it is not surprising that severe glomerulosclerosis and foot process effacement are observed in multiple rodent models of hyperhomocysteinemia, including high-methionine or low-folate diets in uni-nephrectomized rats [39, 47, 48], and cystathionine-β-synthestase knockout mice [49]. It is important to note that glomerulosclerosis and glomerular ROS generation in rats with hyperhomocysteinemia, due in part to activation of NADPH oxidases, is completely blocked by concurrent administration of MK-801, indicating an essential role for NMDA receptors in the resulting pathology [38]. A similar pattern is seen in cultured podocytes [46]. An increase in the expression or functional up-regulation of NMDA receptors could drive glomerulosclerosis, even without an increase in circulating ligands. In models of Alzheimer’s disease, it has been suggested that β-amyloid oligomers upregulate NMDA receptors, thereby inducing excitotoxicity [50]. Therefore, it is interesting that expression of renal NR1 subunits is increased following ischemia-reperfusion [41] and in the Akita mouse model of type-1 diabetes [51]. Moreover, there are reports that glycine infusion exacerbates, whereas glycineB inhibitors reduce kidney injuries evoked by ischemiareperfusion [52]. Finally we turn to possible functions of Group 1 mGluRs in kidney. It has been shown that mGluR1 and mGluR5 are expressed in podocyte foot processes [53, 54]. Rastaldi and co-workers have observed a progressive development of foot process effacement, glomerular basement membrane thickening, albuminuria and glomerulosclerosis in mice lacking mGluR1. The effect was not seen at birth but was robust by two months of age [53]. Conversely, application of the Group 1 agonist DHPG activated several transduction cascades in podocytes (including cAMP and CREB pathways) and is reported to protect Balb/c mice from nephrosis evoked by doxorubicin or puromycin aminonucleoside [54]. The mechanisms of these protective effects are not known.
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some neuronal preparations [43]. The overall pattern derived from electrophysiological observations [27] raises the possibility that these receptors contain an NR3 subunit [12]. It is also quite likely that NMDA receptors in other parts of the kidney have different properties. The functional significance of NMDA receptors for normal kidney physiology is not well understood. The earliest studies suggested that activation of NMDA receptors by infusion of glycine caused an increased blood flow in denervated kidneys that could be measured using Doppler flow probes, as well as an increase in glomerular filtration rate (GFR) as assessed by inulin clearance [44]. These effects of glycine were blocked by the NMDA receptor pore inhibitor MK-801 and also by the glycineB site antagonist 5,7-dichlorokynurenic acid [44]. Thomson and his co-workers subsequently demonstrated that local glycine infusion stimulated net reabsorption by the proximal tubule, and independently increased the single nephron GFR [40]. Both effects were blocked by MK-801, and the experimental design suggested that the relevant receptors are located within nephrons. On the other hand, Yang and co-workers observed that NMDA infusion into the renal artery caused a fall in GFR and urinary Na+ and K+ excretion that was exacerbated after ischemia-reperfusion, and blocked stereospecifically by dAPV [41], a competitive inhibitor of NMDA that acts at NR2 subunits. The reasons for these seemingly contradictory observations are not known, although it should be noted that the studies from the initial studies primarily utilized glycine infusion into male rats [40, 44], whereas the study by Yang et al. infused NMDA into female rats [41]. The nature of the agonist may be important, and it should not be simply assumed that ligands acting on NR2 subunits are the ones most responsible for dynamic signaling in the periphery. More recently, it has been observed that activation of NMDA receptors may help to maintain the epithelial phenotypes of proximal tubule cells, in opposition to inflammatory mediators such as TGFβ [45]. In cells derived from inner medullary collecting duct (IMCD), Bell and his co-workers observed that NR3A subunits are increased following hyperosmotic stress or ischemia. Knocking down NR3A subunits also reduced intracellular and cell-surface expression of aquaporin-2 in IMCD cells [42]. In addition, mice with a constitutive deletion of NR3A subnits (NR3A−/− mice) had reduced aquaporin-2 expression throughout the medulla [42]. These mice also exhibit a >6 h delay in their ability to produce concentrated urine in response to water deprivation [42]. Therefore, medullary NMDA receptors therefore appear to play a significant role in renal function. However, the activating ligands that produce these responses are not known, and the other NMDA receptor subunits that must be present in IMCD cells have not been identified. The presence of NR3 subunits suggests that glycine could be particularly effective at activating NMDA receptors in the medullary collecting ducts. NMDA receptors have been strongly implicated in a variety of CNS disorders, and can induce excitotoxicity when receptors are excessively stimulated for extended periods [3, 7, 14, 18]. NMDA receptor activation also produces pro-survival effects on neurons, and there has been an extensive literature and some controversy on the location and composition of
CONCLUSIONS
REFERENCES
It has been known for many years that receptors for L-Glu originally identified in the CNS have a wide distribution in the periphery, including several parts of the kidney. A very rich pharmacology has been developed to manipulate these receptors, and there is now evidence that agents acting on these receptors or their activating ligands might be useful in kidney diseases. Nevertheless, many additional studies will be needed to elucidate how these receptors regulate normal renal physiology. This will require determining which of the several possible endogenous agonists exert the greatest dynamic control of these receptors under various physiological conditions.
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FUNDING Research in our laboratory is currently supported by a grant from the Juvenile Diabetes Research Foundation. Our past work has also been supported by a grant from Pfizer Inc. C O N F L I C T O F I N T E R E S T S TAT E M E N T The results presented in this paper have not been published previously in whole or part, except in abstract format.
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marked oxidative stress, with accumulations of ROS such as H2O2 in bulk cytosol apparent after 24 h of continuous exposure. Bear in mind that elevated ROS generation probably occurs in certain sensitive compartments long before that. NMDA treatment for 24 h also causes activation of RhoA (B), calcineurin-NFAT pathways that are sensitive to cyclosporine (CsA) (C) and increases steady-state surface expression of Ca2+permeable TRPC6 channels (D). NMDA treatment also causes reduced surface expression of essential slit diaphragm proteins including nephrin and podocin as assessed by cell-surface biotinylation assays (E). Longer exposures to NMDA, for 72 h, cause nuclear fragmentation and increases in various markers of apoptosis (F). The overall pattern is similar to excitotoxic cell death evoked by a variety of NMDA receptor agonists in neurons. However, these effects require substantially longer exposures in podocytes. For more details, see ref. [46].
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F I G U R E 4 : Deleterious effects of sustained NMDA receptor activation in mouse podocytes. (A) The selective agonist NMDA (50 µM) causes
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