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Eicosanoids and Renal Function
Perhaps nothing underscores the special relationship between the kidney and the eicosanoids better than the profound clinical effects non-steroidal anti-inflammatory drugs (NSAIDS) have on kidney function. NSAIDs are widely used to treat pain and inflammatory diseases, and work by blocking the enzymatic synthesis of prostaglandins, a type of eicosanoid, from arachidonic acid. However, chronic NSAID use is often complicated by major side effects, including renal sodium retention, resulting in edema, hypertension, and congestive heart failure. Conversely, in the sodium depleted state, NSAIDs can reduce renal blood flow, glomerular filtration rate, and cause acute renal failure. These observations underscore the critical role cyclooxygenase-derived arachidonic acid metabolites play in maintaining normal kidney function – particularly in the setting of physiological stress.
Perhaps nothing underscores the special relationship between the kidney and the eicosanoids better than the profound clinical effects non-steroidal anti-inflammatory drugs (NSAIDS) have on kidney function. NSAIDs are widely used to treat pain and inflammatory diseases, and work by blocking the enzymatic synthesis of prostaglandins, a type of eicosanoid, from arachidonic acid. However, chronic NSAID use is often complicated by major side effects, including renal sodium retention, resulting in edema, hypertension, and congestive heart failure. Conversely, in the sodium depleted state, NSAIDs can reduce renal blood flow, glomerular filtration rate, and cause acute renal failure. These observations underscore the critical role cyclooxygenase-derived arachidonic acid metabolites play in maintaining normal kidney function – particularly in the setting of physiological stress.
Cellular Origin of Eicosanoids
Eicosanoids are a family of biologically active, oxygenated metabolites derived from arachidonic acid (AA). AA is comprised of 20 carbon atoms configured as a polyunsaturated fatty acid chain with four double bonds (C20:4). Mammals lack the enzymatic machinery to synthesize AA de novo , instead it must be formed from dietary linoleic acid (C18:2) by addition of two carbons and further desaturation. Essential fatty acid (EFA) deficiency occurs in the absence of dietary linoleic and other fatty acid AA precursors, depleting the hormone-responsive pool of AA metabolite products. Of the approximate 10 gm of linoleic acid ingested per day, only about 1 mg/day is eliminated as end products of AA metabolism. Following its formation, AA is esterified into cell membrane phospholipids, principally at the 2 position of the phosphatidylinositol fraction (i.e., sn-2 esterified AA). This source comprises the major hormone-sensitive pool of AA that is susceptible to release by phospholipases.
Phospholipase-Mediated Arachidonic Acid Release
Multiple stimuli lead to release of membrane-phospholipid esterified AA via activation of cellular phospholipases, principally phospholipase A2s (PLA 2 ). This cleavage step is rate-limiting in the production of arachidonate metabolites. Activation of phospholipase C or PLD, on the other hand, releases AA via the sequential action of the phospholipase C-mediated production of diacylglycerol (DAG), with subsequent release of AA from DAG by DAG lipase. The physiological significance of AA release by these other phospholipases remains uncertain since, at least in the setting of inflammation, phospholipase A 2 action appears to be essential for the generation of biologically active AA metabolites. Cellular levels of free arachidonic acid available for eicosanoid production are primarily controlled by phospholipase A2 (PLA2). So far, more than 30 enzymes with PLA 2 activity have been identified, and have been classified into four groups: secretory PLA 2 (sPLA2); cytosolic PLA2 (cPLA2); calcium-independent PLA2 (iPLA2); and PAF acetylhydrolases (PAF-AH). The activity of cPLA2 is regulated by diverse cell membrane receptors, including the EGF receptor, and transmembrane guanine-nucleotide protein coupled (GPCRs) including adrenergic receptors, angiotensin II receptors, and purinergic receptors. These receptors activate guanine nucleotide-binding (G) proteins, leading to PLA 2 -mediated release of AA from membrane phospholipids. Alternatively, these receptors may activate cPLA 2 via mitogen-activated protein kinases (MAPK), protein kinase C (PKC), and Ca 2+ -calmodulin-dependent kinases.
Ambient physical conditions in the kidney including hypoxia, oxidative stress, and mechanical stretch can also activate PLA 2 activity. Dysregulated renal PLA 2 activity with attendant change in AA release results in altered substrate availability for the production of downstream metabolic products. This activation is believed to contribute to pathologic processes including acute kidney injury, diabetic nephropathy, and inflammatory glomerulonephritis. Some snake and bee venoms are imbued with high levels of secretory PLA 2 activity and, in part through this activity, can induce acute renal failure . A role for secretory PLA 2 in the pathogenesis of acute ischemic-reperfusion renal injury has also been supported by studies showing that sPLA 2 neutralizing antibodies protect rats from this form of injury.
Phospholipase A2 Receptors
Recently, an important role for a transmembrane cell surface secretory PLA 2 receptor (PLA 2 R) has been recognized in the pathogenesis of human idiopathic membranous nephropathy. Auto-antibodies to PLA 2 R are detected in ~70% of cases of human idiopathic membranous nephropathy. The antigen appears to be selectively expressed in podocytes ; however, the mechanism by which the auto-antibodies induce proteinuria and how these auto-antibodies arise remains to be determined. PLA 2 R is a type I transmembrane receptor and one of four mammalian members of the mannose-receptor family. PLA 2 R was initially identified as a binding protein for secreted phospholipase A 2 (PLA 2 ) that now has been expanded to a PLA 2 R family that exhibit different affinities for the secreted PLA 2 . New studies suggest these receptors could play additional transmembrane signaling roles, and may promote terminal cell differentiation and mitotic arrest.
Arachidonic Acid Metabolism
Following its release from membrane phospholipids, AA is usually rapidly re-esterified into the membrane or avidly bound by intracellular proteins, becoming unavailable for further metabolism. Should AA escape re-esterification and protein binding, it may be metabolized through one of three major enzymatic transformations, the common result of which is the incorporation of oxygen atoms at various sites of the fatty acid backbone, with accompanying changes in its molecular structure (such as ring formation). This results in the formation of biologically active molecules, collectively referred to as “eicosanoids.” The specific nature of the products generated is a function of the initial stimuli for AA release, as well as the metabolic enzymes available, as determined by the cell type involved.
Enyzmes capable of mediating AA metabolism through all three known pathways are present in the kidney, including cyclooxygenases 1 and 2, lipoxygenases, and cytochrome P450s ( Figure 17.1 ). Cyclooxygenase (COX, also called Prostaglandin H2 synthase or PGHS)-mediated AA metabolism comprises the first committed step in the formation of prostaglandins (PGs), prostacyclin, and thromboxane. The lipoxygenase pathway mediates the formation of mono-, di-, and trihydroxyeicosatetraenoic acids (HETEs), leukotrienes (LTs), and lipoxins (LXs), and the cytochrome P450-dependent oxygenation of AA mediates the formation of epoxyeicosatrienoic acids (EETs), their corresponding diols, HETEs, and monooxygenated AA derivatives. Fish oil diets, rich in n-3 polyunsaturated fatty acids ( n-3 fatty acids are those in which the double bond is three carbons from the terminal, i.e., n carbon, that is furthest from the carboxy-group atom, AA is thus an n-6 fatty acid) interfere with metabolism via all three pathways by competing with AA oxygenation, resulting in the formation of biologically inactive end-products. Interference with the production of pro-inflammatory lipids has been hypothesized to underlie the beneficial effects of fish-oil in IgA nephropathy, membranous nephropathy, and other cardiovascular diseases.
Cyclooxygenase Derived Prostanoids
Prostanoids, including the prostaglandins PGE 2 , PGF 2α , and PGD 2 , as well as the non-prostaglandin molecules thromboxane A 2 (TxA2) and prostacyclin (PGI 2 ), are derived from arachidonic acid via its di-oxygenation by cyclooxygenases 1 and 2 (COX1 and COX2). Cyclooxygenases exist as homodimers that are physically associated with, but do not pass through, the intracellular endoplasmic reticular membrane. Cyclooxygenases mediate a two-step reaction, initially converting free arachidonic acid to the unstable intermediate PGG 2 via a bis -oxygenase activity. PGG 2 is converted to PGH 2 via the peroxidase activity of COX. PGH 2 is subsequently metabolized to more stable primary biologically active prostanoids PGE 2 , PGF 2α , PGD 2 , PGI 2 , and TxA 2 by distinct enzymatic prostanoid synthases. These prostanoids exit the cell through uncharacterized mechanisms, where they exert paracrine or autocrine activity on specific and distinct cell surface G-protein coupled receptor(s). There is also less definitive evidence that prostanoids may provide physiologically relevant ligands for nuclear hormone receptors, including peroxisome proliferator activated receptors.
Two isoforms of COX have been identified, designated COX1 and COX2. Based on transcriptional elements in its 5′ upstream sequence, COX1 is believed to serve a constitutive housekeeping role, responsible for maintaining basic physiological function such as cytoprotection of the gastric mucosa, and control of platelet aggregation. Conversely, COX2 upstream promoter region has NF-κB, NFAT, and its expression is potently induced by inflammatory mediators and mitogens, consistent with its role in pathophysiologic processes including angiogenesis, inflammation, and tumorigenesis.
The major phenotype of COX2 knockout mice is renal dysgenesis, underscoring the special role COX2 plays in the kidney. This defect is characterized by a structurally normal medulla, but hypotrophic renal cortical development with small glomerular size, due to a defect occurring relatively late in partuition. The mechanism is undetermined, but may be related to the particular expression pattern of COX2 in the kidney since normally it is focally expressed adjacent to the glomerulus in the macula densa and the surrounding thick ascending limb cells. As in other organs, the housekeeping gene is COX1, which is also constitutively expressed at high levels in the kidney but in cellular compartments distinct from COX2, especially in the collecting duct and glomerular parietal epithelium. Low levels of COX1 are also detected in medullary interstitial cells, but these cells are also uniquely characterized by high endogenous levels of COX2.
Clinical pharmacologic studies are also consistent with a critical role of COX2 for maintaining cardiovascular homeostasis and normal renal function. Indeed, most of the clinically observed side effects associated with the use of non-selective NSAIDs, including edema, hypertension, increased congestive heart failure, hyperkalemia, and acute renal failure, have also been observed with COX2 selective inhibitors. COX2-dependent PGE 2 production is inversely related to luminal chloride concentration delivered to the macula densa, so that in volume depleted states high PGE 2 production rates may exert a vasodilator effect on the afferent arteriole, contributing to maintenance of glomerular blood flow. Impairment of renal function is presumed due to loss of specific prostanoids, derived from the metabolism of the common cyclooxygenase product PGH 2 .
Prostanoid Function
Once formed, the COX-derived arachidonate metabolite PGH 2 is further metabolized by prostanoid synthases into at least five primary biologically active prostanoids. Prostanoid synthases include PGE 2 synthase (PGES), prostacyclin synthase (PGIS), PGD synthase (PGDS), PGF synthase (PGFS), and thromboxane synthase, responsible for PGE2, PGI2, PGD2, PGF2α, and TxA2 biosynthesis respectively.
Most prostanoids are short lived, being highly susceptible to enzymatic inactivation, thereby limiting their effect to the immediate vicinity of their synthesis. The paracrine and autocrine biologic effects of COX-derived prostanoids are diverse and complex, depending on which prostanoid is produced and which receptor is available. Thus, the effects of prostanoids on kidney function rely on distinct enzymatic machinery that couples phospholipase and COX to specific prostanoid synthase in specific cells, yielding a specific prostanoid which acts locally through specific G-protein coupled receptors, exerting its particular effect.
At steady-state PGE 2 is the most abundant prostanoid in the mouse kidney, followed by PGI 2 , PGF 2α , and TxA 2 . Under basal conditions, both COX1 and COX2 pathways are responsible for the biosynthesis of these prostanoids. Similarly, PGE2 is the most abundant prostanoid in human urine, and under basal, non-stressed conditions is produced by both COX1 and COX. In contrast, COX2 primarily contributes to angiotensin II-induced PGE 2 and PGI2 generation in the kidney, and under conditions of low-sodium diet in humans. The intrarenal cellular sites where COX1 and COX2 prostanoids are synthesized remain to be fully defined.
Following their synthesis, these prostanoids become available to exert their biological effects via a diverse family of membrane spanning G-protein coupled prostanoid receptors. These include the DP, EP, FP, IP, and TP receptors, each of which is selectively activated by a specific ligand – PGD 2 , PGE 2 , PGF 2α , PGI 2 or TXA 2 , respectively. PGE2 receptors, designated EP receptors, are unique in that they are encoded by four distinct genes encoding the proteins for EP1, EP2, EP3, and EP4 receptors. Each prostanoid receptor activates a distinct G-protein coupled signaling pathway. The IP, DP1, EP2, and EP4 receptors are coupled to the stimulatory G-protein (Gs) and signal by increasing intracellular cAMP levels, whereas the TP, FP, and EP1 receptors induce calcium mobilization. The FP, DP2, and EP3 receptors can couple to an inhibitory G-protein (Gi) and reduce cAMP synthesis.
Restricted cellular expression of prostanoid receptors provides an important mechanism by which a COX-derived prostanoid can exert differential actions in physiological and pathophysiological processes. In the kidney the EP receptors map to distinct segments of the nephron. Similarly, all four EP receptors have been described in major inflammatory cells including T-lymphocytes, B-lymphocytes, macrophage, and mast cells ; however, whether these receptors are simultaneously expressed in individual cells is uncertain. It has been proposed that activation of different receptors on different cells at different stages of inflammation may account for the pro- or anti-inflammatory action of PGE 2 .
Prostaglandin E2
PGE 2 is synthesized by at least three forms of PGE synthases, including microsomal PGE synthase 1 (mPGES1), microsomal PGE synthase 2 (mPGES2), and cytosolic PGE synthase (cPGES1). The two membrane associated PGE 2 synthases are 33 kDa and 16 kDa enzymes designated mPGES1 and mPGES2, respectively. Microsomal PGES1 displays a higher catalytic activity relative to other PGE synthases and, like COX2, its expression can be induced by cytokines and inflammatory stimuli. In contrast, the expression of cPGES and mPGES2 do not seem to be inducible and may play housekeeping functions.
Genetic disruption confirms that mPGES1 −/− mice exhibit a marked reduction in inflammatory responses compared with mPGES1 +/+ mice, and indicates that mPGES1 is critical for the induction of inflammatory fever. It has been proposed that mPGES1 couples primarily to the inducible COX2 in inflammatory cells. In contrast, intrarenal expression of mPGES1 maps to cells of the collecting duct that primarily express COX1 with lower expression in medullary interstitial cells and macula densa that express COX2 ( Figure 17.2 ). Thus, in the kidney mPGE1 co-localizes with both cyclooxygenase 1 and 2. The renal phenotype of the mPGE1 knockout mouse is relatively subtle, and is characterized by increased blood pressure sensitivity to high-sodium diet and mineralocorticoids, as well as increased vascular reactivity to angiotensin-II, although not all investigators have seen these effects. These results are consistent with a role for mPGES1-derived PGE 2 in buffering physiologic stresses that tend to increase blood pressure. Notably, the kidneys of mPGES1 −/− mice are normal and do not exhibit the renal dysgenesis observed in COX2 −/− mice. Nor do these mice exhibit perinatal death from patent ductus arteriosus observed with the prostaglandin EP4 receptor knockout mouse, suggesting other sources of PGE2 production are sufficient to provide adequate receptor activation. These sources could include cPGES and mPGES2. Both cPGES and mPGES2 are expressed in the kidney ; however, their intrarenal role(s) have not yet been elucidated. In addition, several cytosolic glutathione-S-transferases have the capacity to convert PGH 2 to PGE 2 ; however, their physiologic role in this process remains uncertain.
E-Prostanoid Receptors
All four E-Prostanoid receptors (EP receptors) are expressed in the kidney ( Figure 17.3 ). Each exhibits a distinct mRNA expression profile along the nephron. The EP4 receptor predominates in the glomerulus, while the EP3 and EP1 receptors are primarily detected in the thick limb and collecting duct. The EP2 receptor is expressed at lower levels in the renal vaculature and stroma. Each receptor plays a distinct role in these regions, mediating many of the well-defined physiologic actions of PGE 2 that have been identified over the past several decades.
EP 1 Receptor
The EP1 receptor was originally identified pharmacologically via its smooth muscle constrictor activity in guinea pig ileum, and its unique profile of response to a series of prostanoid analogs. The EP1 receptor cDNA has been cloned from numerous species, including human, dog, mouse, rat, and rabbit. The human EP1 receptor cDNA encodes a 402 amino acid polypeptide with a predicted molecular mass of 41,858 kDa. This receptor signals via a mechanism linked to increased cell Ca + , and is accompanied by modest increases in IP 3 generation.
Studies of EP1 receptors have taken advantage of several relatively selective antagonists that block their activation, including SC-19220, SC-53122, and ONO-8130. A significant impetus behind the development of clinically active EP1 receptor antagonists derives from evidence that the EP 1 receptor plays an important role in prostaglandin-mediated pain, and that EP1 receptor antagonists have EP1 properties. These antagonists provide useful tools to study EP 1 receptor physiology in vivo .
The EP1 receptor is highly expressed in the kidney, where it primarily localizes to the collecting duct with an increasing mRNA expression gradient from the cortical to the medullary collecting duct. In the collecting duct, activation of the EP1 receptor inhibits Na + and water reabsorption via a Ca 2+ -coupled mechanism. These results suggest that renal EP 1 receptor activation contributes to PGE 2 -dependent natriuresis by inhibiting Na + transport in the collecting duct. Despite this in vitro demonstration, these natriuretic effects have been difficult to demonstrate in vivo .
Genetic disruption of the EP1 receptor does not lead to a significant impairment of sodium excretion; however, EP1 knockout mice do exhibit increased renin and aldosterone levels, consistent with maintenance of normotension at the expense of activation of the renin–angiotensin system. EP1 receptor knockout mice not only exhibit reduced blood pressure on normal chow, but also impaired pressor response to angiotensin II. These studies identified EP1 mRNA expression in small resistance vessels of mice including the afferent arterioles of the glomerulus, and are consistent with more recent studies suggesting Ang II-stimulated vasoconstriction may in part be mediated by activation of vascular EP1 receptors. EP1 receptors have also been identified in glomerular mesangial cells, where they may contribute to mesangial contraction. Inhibition of the EP1 receptor slows the progression of mesangial expansion in experimental models of diabetic nephropathy. EP1 receptor knockout mice are resistant to the pressor effects of angiotensin II, and EP1 receptor antagonists can also block the Ang II pressor activity. It is instructive to consider the role of PGE 2 as a vasoconstrictor through its actions on the EP1 receptor, as opposed to its classically characterized role as a vasodilator/vasodepressor. This underscores the capacity of PGE 2 to serve as a physiological buffer of blood pressure, either in support or reduction of blood pressure (see below).
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