Review

10.1517/17425255.1.3.399 © 2005 Ashley Publications Ltd ISSN 1742-5255 399

Ashley Publicationswww.ashley-pub.com

Novel mechanisms of nonsteroidal anti-inflammatory drug-induced renal toxicityKathleen M Knights†, Paraskevi Tsoutsikos & John O Miners†Flinders University and Flinders Medical Centre, Department of Clinical Pharmacology, Bedford Park, Adelaide 5042, Australia

The exact mechanism(s) of NSAID-induced nephrotoxicity remains unclear,but most theories centre on the initial inhibition of COX and the subsequentperturbation of the numerous actions of COX in the kidney. Since the nine-teenth century no NSAIDs have been developed that are devoid of renaladverse effects, including the COX-2 selective inhibitors. Formation of renaleicosanoids from arachidonic acid is significantly increased in the presence ofvarious stimuli, and metabolic degradation of arachidonic acid and its biolog-ically active metabolites is crucial to the maintenance of renal homeostaticmechanisms. An important family of enzymes that function in this capacityare the uridine 5′-diphosphate glucuronosyltransferases (UGTs) that variouslymetabolise arachidonic acid and its metabolites. This review focuses on ara-chidonic acid and its biologically active metabolites and their respective fatessubsequent to COX inhibition by NSAIDs. The common involvement of UGT inthe metabolism of arachidonic acid, eicosanoids and NSAIDs is discussed inthe context of novel mechanisms of NSAID-induced nephrotoxicity.

Keywords: acyl glucuronides, arachidonic acid, cyclooxygenase, cytochrome P450, lipoxygenase, nephrotoxicity, NSAIDs, renal metabolism, uridine 5′-diphosphate-glucuronosyltransferases

Expert Opin. Drug Metab. Toxicol. (2005) 1(3):399-408

1. Introduction

NSAIDs comprise one of the most commonly used groups of therapeutic drugsworldwide, with annual estimates of > 100 million prescriptions and billions ofover-the-counter sales in Australia, the UK and the US. In the US alone, NSAIDsaccount for ∼ 70 million prescriptions and 30 billion over-the-counter sales [1].From March 2003 to March 2004 > 10 million prescriptions for NSAIDs wererequested through the Australian Pharmaceutical Benefits Scheme, representing anaverage of one in two Australians being treated with an NSAID. These statistics aresurprising because NSAIDs historically are known to cause serious adverse effects,including gastric ulceration and renal function abnormalities.

The commercial development of anti-inflammatory drugs dates back to the latenineteenth century, with the introduction of aspirin in 1899. By the early part of thetwentieth century aspirin was in widespread use principally for its anti-inflamma-tory, antipyretic and analgesic actions. However, within 20 years of clinical use,adverse effects of aspirin were reported, including gastrointestinal haemorrhage andimpaired renal function. Since that time other drugs with similar properties havecontinued to be developed and include the pyrazolon derivatives (e.g., phenyl-butazone; 1949), N-phenylanthranilic acids (fenamates; 1950s), indomethacin(1960s), arylpropionic acids (profens; 1970s), oxicams (1980s) and, more recently,the coxibs (late 1990s). Despite the plethora of NSAIDs, gastrointestinal and renaltoxicity remain characteristic clinical features of NSAID use. Although renal adverseeffects are frequently eclipsed by the severity of the gastrointestinal problems, which

1. Introduction

2. Arachidonic acid and its

bioactive metabolites

3. NSAIDs and COX

4. Renal metabolism of NSAIDs

5. Novel mechanisms of NSAID

nephrotoxicity

6. Expert opinion

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400 Expert Opin. Drug Metab. Toxicol. (2005) 1(3)

Figure 1. NSAID adverse renal reactions profile. Source data from the Adverse Drug Reactions Advisory Committee [101]. Total reports current as of August 2003 for NSAIDs dispensed through the Australian Pharmaceutical Benefits Scheme.

Celecoxib

Diclofenac

Diflunisal

Ibuprofen

Indomethacin

Ketoprofen

Mefenamic acid

Meloxicam

Naproxen

Piroxicam

Rofecoxib

Sulindac

Tiaprofenic acid

Renal impairment16.1%

Interstitialnephritis

3.8%

Nephrotic syndrome2.0%

Renal failure0.9%

Glomerulo-nephritis

0.6%

Acuterenalfailure22.4%

Renal tubulardisorder< 0.1%

Nephritis0.1%

Toxic nephropathy0.2%

Fluidretention

0.4%

Chronic renal failure 0.6%

Renal tubular necrosis 0.5%

decreasingfrequency ofoccurenceNSAIDs

include ulcer bleeding and perforation, recent events with thecoxibs have highlighted the fact that the kidney remains animportant target organ for the adverse effects of NSAIDs.

NSAID-induced renal abnormalities range from fluidand electrolyte disturbances, which are most commonlyreported, through to the less commonly observed acuterenal failure, nephrotic syndrome with interstitial nephritisand, rarely, renal papillary necrosis. In humans, virtually allNSAIDs will to varying degrees lead to sodium and waterretention, but only in < 5% of patients will this electrolyteimbalance manifest as clinically detectable oedema [2].Although this proportion may seem low, current Australiandata suggests that of those individuals currently using pre-scription NSAIDs ∼ 525,000 people annually are likely todevelop some form of renal dysfunction. Comparable datafor the US indicates that 0.5 – 2.5 million US citizens are atrisk of one of a number of NSAID-induced renal abnormal-ities [3]. Recent surveillance data (August 2003) from theAdverse Drug Reactions Advisory Committee on currentlyused NSAIDs within Australia indicate that renal and uri-nary disorders consistently rank within the top 10 ofadverse effects for NSAIDs and account for 5.4% of allreports of NSAID-induced adverse reactions. Of the 5.4%

of reports, acute renal failure accounted for 22.4%,followed by the more serious, although less common, inter-stitial nephritis (3.8%), nephrotic syndrome (2%) andchronic renal failure (0.63%) (Figure 1). There were noreports in Australia up to 2003 of NSAID-induced renalpapillary necrosis. Overall, acute renal failure accounted for1.2% of the total adverse renal effects, similar to an earlierstudy reporting the occurrence of acute renal as 0.5 – 1.0%of all patients taking NSAIDs [2]. Clearly, data accumulatedover decades have established that acute renal failure andchanges in fluid and electrolyte balance are the major acuteeffects of NSAIDs on the kidney.

Historically, N-phenylanthranilic acid and substitutedN-phenylanthranilic acids, such as flufenamic acid,mefenamic acid and meclofenamic acid, were used for induc-ing renal papillary necrosis in rats during the 1960s [4], andthis continued throughout the 1970s [5]. During the 1980s, itbecame apparent that mefenamic acid was responsible formefenamic acid nephropathy: a renal disorder characterisedby acute reversible interstitial nephritis in elderly patients withpre-existing factors that contributed to mefenamic-inducedrenal failure [6]. Similarly, in the early 1980s, fenoprofen andnaproxen were the first arylpropionic acids reported to cause

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interstitial nephritis leading to acute renal failure, which wasaccompanied occasionally by nephrotic syndrome [7].

There is now a substantial body of evidence from bothexperimental and clinical studies that all NSAIDs variouslyaffect the kidney, and altered renal haemodynamics are a com-mon feature. Risk factors for NSAID-induced renal abnor-malities are also well recognised and include concomitantdrug use (diuretics and angiotensin-converting enzyme inhib-itors and/or angiotensin receptor antagonists), concurrent dis-eases (hypertension, diabetes, pre-existing renal failure andcardiac failure), older age and plasma volume contraction. Sofar, the exact mechanism(s) of NSAID-induced nephro-toxicity remain unclear. Although it is generally accepted thatNSAIDs perturb renal haemodynamics, does this account forall of the adverse renal effects? To address this question thisreview focuses on arachidonic acid, a molecule of majorbiological and physiological relevance.

2. Arachidonic acid and its bioactive metabolites

2.1 Synthesis and physiological role of prostaglandinsThe precursor molecule to the synthesis of prostaglandins(PGs) is arachidonic acid (20:4n-6), which is liberated frommembrane lipids by phospholipase A2 in response tophysiological, pathological and pharmacological stimuli.The concentration of free arachidonic acid in resting cells isuniversally described as ‘low’, but once released can be inthe range of 10 – 100 µM [8]. Most of the effects of arachi-donic acid are attributable to its metabolism to biologically

active products. Oxygenation of released arachidonic acidvia the action of COX produces the series-2 PGs andthromboxane (TX): PGD2, PGI2, PGE2, PGF2α and TXA2;via lipoxygenase (LOX), the series-4 leukotrienes (e.g.,LTB4) and hydroxyeicosatetraenoic acids (HETEs) are pro-duced; and via cytochrome P450s (CYP), the epoxyeicosa-trienoic acids (EETs), their corresponding diols, andHETEs are produced (Figure 2).

The synthesis of PGs from arachidonic acid is catalysed byone of two distinct COX isozymes: COX-1 or -2 (refer to ref-erences [9-10] for extensive reviews of COX). A significant fea-ture of the COX-2 gene is its induction by various stimuli(e.g., cytokines, forskolin, IL-1, endothelin), which results inincreased protein expression. This inducible response ofCOX-2 contrasts with COX-1, which has been termed a con-stitutive enzyme. This simplistic designation reflects the situa-tion in some tissues in which COX-1 predominates, but inhuman kidney COX-2 is constitutively expressed in renal vas-culature (afferent arterioles), medullary interstitial cells andthe macula densa of older adults [11], and can be upregulatedparticularly in the macula densa [12-13]. COX-2 expression hasalso been documented in endothelial and smooth muscle cellsof renal vasculature (arteries and veins) and intraglomerularlyin podocytes [12].

In healthy volume-replete individuals, basal PG synthesisvia COX-1 activity is low, and hence PGs are not principallyinvolved in regulating renal function, but instead serve tomoderate the effect of vasoconstrictor molecules such asangiotensin II or noradrenaline. However, in situations whererenal blood flow is perturbed, for example, congestive heart

Figure 2. Major pathways of eicosanoid synthesis. CYP: Cytochrome P450; EET: Epoxyeicosatrienoic acids; HETE: Hydroxyeicosatetraenoic acids; LOX: Lipoxygenase; LT: Leukotriene; PG: Prostaglandin; TXA2: Thromboxane A2.

Arachidonic acid

Phospholipase A2

+

Membrane phospholipid

Re-esterifiedin membrane

Renal vasodilation Renal vasoconstriction Renal vasodilation

COX

LOX CYP

PGs(PGE2)

TXAs(TXA2)

LTs(LTB4)

5-HETE8-HETE

12-HETE15-HETE

19-HETE20-HETE

14-EET15-EET

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failure, intravascular volume depletion or hepato–renal syn-drome, PG synthesis increases to preserve renal function. Thisoccurs primarily via PG mediated vasodilation of renal vessels,renin secretion and enhanced sodium excretion. Thus, thekidney becomes dependent on continual synthesis of PGs tomaintain the balance of vasoconstrictor–vasodilator forces.

The PGs of greatest importance in maintaining renal func-tion are PGI2 and PGE2. Both are synthesised within discreteanatomic regions of the kidney, which reflects their principalsites of action. PGI2 acts as a vasodilator of cortical arterioles,maintains glomerular filtration, inhibits sodium reabsorptionin the Loop of Henle and stimulates renin secretion via anaction in the juxtaglomerular apparatus. PGE2 is produced to agreater extent in the renal medulla in comparison with thecortex, but functionally it acts in the same manner as PGI2 [10].

In summary, following synthesis, PGs act on any of anumber of PG receptors, thereby modulating cell function[10]. In general, interest in PGs per se diminishes at this pointand little is written about their subsequent metabolism. Thequestion is what terminates the biological reactivity of PGsderived from arachidonic acid?

2.2 Metabolism of prostaglandinsCarboxylic acids are common substrates for glucuronidationcatalysed by endoplasmic reticulum-bound uridine 5′-diphos-phate glucuronosyltransferases (UGT). Of the 27 knownhuman UGT genes, 15 (UGT1A1, -1A3, -1A4, -1A6, -1A7,-1A8, -1A9, -1A10, -2A1, -2B4, -2B7, -2B10, -2B15, -2B17

and -2B28) encode proteins that are catalytically activetowards a myriad of endogenous and exogenous compounds[14]. The glucuronidation of medium chain fatty acids and theunsaturated fatty acids linoleic acid, α-linolenic acid and ara-chidonic acid by rat UGT2B1 was reported in 1994 [15].Recently, glucuronidation of linoleic acid, the immediate pre-cursor of arachidonic acid, by human liver microsomes andglucuronidation of the two naturally occurring metabolites oflinoleic acid, 13-hydroxyoctadecadienoic acid and13-oxooctadecadienoic acid by human UGT2B7, wasreported [16-18]. A subsequent study established that arachi-donic acid and PGE2 were substrates for human hepatic andintestinal UGTs and recombinant UGT2B7 [19]. Clearly,glucuronidation provides a clearance pathway for botharachidonic acid and PGE2 (Figure 3).

2.3 Synthesis and metabolism of leukotrienesArachidonic acid oxygenation by LOX produces 5-, 8-, 12- and15-HETE, and the series-4 leukotrienes (e.g., LTB4).12-HETE is the predominant LOX metabolite and althoughmuch remains to be learned about the actions of LOX meta-bolites on renal haemodynamics, it is noteworthy that 12- and15-HETE are potent glomerular and renal vascular vasocon-strictors, and that LOX metabolites play a key role in glomeru-lar nephritis [20]. Similar to PGs, the biological half-lifes of theHETEs and LTB4 (a potent chemotactic for neutrophils and aninflammatory mediator) is determined by metabolic degrada-tion. Although LTB4 metabolism via oxidative pathways has

Figure 3. Glucuronidation of arachidonic acid-derived metabolites. Eicosanoid glucuronides identified so far using human renaltissue and recombinant UGT.AA: Arachidonic acid; CYP: Cytochrome P450; EET: Epoxyeicosatrienoic acids; HETE: Hydroxyeicosatetraenoic acids; LOX: Lipoxygenase; LT: Leukotriene; PG: Prostaglandin; TXA2: Thromboxane A2; UGT: Uridine 5′-diphosphate glucuronosyltransferases.

Membrane phospholipid

Re-esterifiedin membrane Phospholipase A2

COX

LOX CYPUGT

Arachidonic acid

AA-glucuronidePGs

(PGE2)TXAs

(TXA2)LTs

(LTB4)5-HETE8-HETE

12-HETE15-HETE

19-HETE20-HETE

14-EET15-EET

+

PGE2-glucuronide LTB4-glucuronide20-HETE-glucuronide

5,12,15-HETE-glucuronide

UGT UGT

UGT

UGT

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been characterised, the first report of LTB4 glucuronidation waspublished in 1999. Using primary cultures of human hepato-cytes, Wheelan et al. [21] identified glucuronide conjugates ofLTB4, 20-COOH-LTB4 and 10,11-dihydro-LTB4. Subse-quently, using urine from subjects injected with LTB4 an addi-tional number of conjugates were identified. These includedthe glucuronides of LTB4, 17-, 18-, 19- and 20-hydroxy-LTB4,10-hydroxy-4,6,12-octadecatrienoic acid and 10,11-dihydro-LTB4 [22]. Using recombinant UGT proteins, it was establishedthat LTB4 was glucuronidated by UGT1A3; UGT2B7 and 5-,12- and 15-HETE were glucuronidated by UGT1A9, -2B4, -2B7 and -2B10; and 20-COOH-LTB4 was glucuronidated byUGT1A9 [23]. These data demonstrate that UGT1A3, -1A9,-2B4 and -2B7 play a role in the metabolic degradation of ara-chidonic acid, PGE2, LTB4 and its metabolites, and 5-, 12- and15-HETE (Figure 3). So far, nine human UGTs have beenidentified in the kidney: UGT1A3, -1A6, -1A9, -2B4, -2B7,-2B10, -2B11, -2B15 and -2B17 [24-26].

2.4 Synthesis and metabolism of EETs and HETEsFocus on COX and LOX overshadowed for many years therole of CYP in the metabolism of arachidonic acid and therole of CYP-derived metabolites in the control of renal func-tion and haemodynamics. Renal arachidonic acid metabolismby CYP is well established, and CYP activity in the kidney iscomparably one of the highest of any organ [20]. There are tworenal CYP pathways: CYP epoxygenase and CYP hydroxylase.Enzymes of the CYP2C family are the major contributors tothe formation of EETs, whereas the CYP4A family catalysesformation of HETEs [27]. 20-HETE is the principal meta-bolite generated by CYP, and is a potent vasoconstrictor witha central role in regulating renal function and vascular tone. Italso serves as a second messenger for endothelin-1, mediatesselective renal effects of angiotensin II and is the subject ofintense investigation because of a link with the developmentof hypertension [27]. In addition to regulation of the renal cir-culation, EETs and 20-HETE regulate sodium/potassiumATPase activity, and sodium/potassium/chloride transport inthe nephron [27]. Interestingly, 20-HETE glucuronide hasbeen identified in human urine [28] and the first UGTreported to catalyse 20-HETE glucuronidation was UGT2B7[19]. A subsequent study confirmed the involvement ofUGT2B7, but further established that UGT1A1, -1A3 and-1A4 catalyse 20-HETE glucuronidation [29].

2.5 PerspectiveClassically, renal arachidonic acid metabolism is consideredonly in terms COX, LOX and CYP, but this should now beextended to include UGT. Fatty acids accumulate intra-cellularly during acute renal ischaemia, and it is known thathigh concentrations of fatty acids and their metabolites areinherently cytotoxic. For example, accumulation of arachi-donic acid, through inhibition of COX-2 by NSAIDs, report-edly induced apoptosis in stable cell lines [30]. Analogous toarachidonic acid, linoleic acid and some linoleic acid

metabolites (i.e., linoleic acid-diols) are toxic to renal proxi-mal tubular cells, although the exact mechanism of toxicity isunknown [31-32]. Similarly, linoleic acid and linoleic acid-diolsare glucuronidated, and it has been reported that the latter areprimarily glucuronidated by UGT2B7 [17]. Given the affinityof arachidonic acid for UGT (apparent Km ∼ 45 µM) [29], andin light of potential intracellular concentrations of arachi-donic acid ≤ 100 µM [8], it appears that metabolism via UGTprovides a local intrarenal detoxification pathway. Thus, glu-curonidation is a pivotal pathway in the kidney for limitingthe biological reactivity of arachidonic acid and its bioactivemetabolites, particularly PGE2, LTB4, EETs and HETEs.

3. NSAIDs and COX

It is widely accepted and rarely challenged that NSAIDs act (irre-versibly in the case of aspirin and reversibly for all other NSAIDs)by inhibiting COX and hence PG synthesis. This is well recog-nised as the central mechanism that explains not only theanti-inflammatory effects, but also the adverse effects of NSAIDson the gastrointestinal tract and the kidney. During the period1972 – 1991, the existence of a second form of COX was at firstpostulated then established, and the concepts of ‘constitutiveCOX-1 activity’ and ‘inducible COX-2 activity’ pervaded theliterature. The identification of COX-2 provided new impetus torefining the COX theory of NSAID action by ascribing the anti-inflammatory, analgesic and antipyretic effects of NSAIDs toinhibition of COX-2 and the adverse effects (gastrointestinal andrenal) to inhibition of COX-1 [33]. With the development of cell-based and human whole-blood assay systems, relative ranking ofexisting NSAIDs in terms of selectivity for COX-1 and/or -2rapidly proceeded [34-36]. Despite the considerable variability inthe estimates of COX selectivity, considerable enthusiasm andeffort was expended on developing drugs that were COX-2 selec-tive. The first selective COX-2 inhibitors approved by the FDAwere celecoxib (December 1998) and rofecoxib (May 1999). Atthe time, differential expression of COX-1 and -2 in the kidney[12] led many to speculate that selective COX-2 inhibitors might‘spare’ the kidney as compared with nonselective NSAIDs. Thisperception was short lived, as it became apparent that COX-2was constitutively expressed in kidney and intimately involved inmaintaining renal homeostasis [37]. Within a few years of market-ing COX-2 inhibitors, it was established that COX-2-derivedprostanoids were essential for preserving renal blood flow andglomerular filtration, particularly during periods of low sodiumintake [38]. Cases of nephrotoxicity due to use of selective COX-2inhibitors soon appeared in the literature [39-40]. It is now gener-ally accepted that selective COX-2 inhibitors provide no clinicaladvantage over the older nonselective COX inhibitors withregard to renal adverse effects [41-42].

4. Renal metabolism of NSAIDs

Although knowledge of xenobiotic metabolism is predomi-nantly based on studies using liver, significant glucuronidation

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activity towards endogenous and exogenous compounds hasbeen reported for human kidney [43-44]. Of the 15 catalyticallyactive human UGT isoforms found in liver and kidney,UGT1A3, -1A9 and -2B7 are the predominant isoformsinvolved in glucuronidation of carboxylic acids, includingmany NSAIDs [45-46]. UGT2B7 is the major isoform and isranked highest of the NSAID glucuronidating UGTs(UGT2B7 > UGT1A9 ∼ UGT1A3) [47]. Studies using humankidney cortical microsomes (HKCM) and recombinantUGT1A3, -1A6, -1A9 and -2B7 have established glucuroni-dation of flufenamic, meclofenamic, mefenamic and niflumicacid at pharmacologically relevant concentrations, with S50

values (sigmoidal glucuronidation kinetics) in the range of∼ 56 – 250 µM ([48] and unpublished data). In addition,S-naproxen glucuronidation by HKCM and UGT2B7 exhibitsMichaelis–Menten kinetics (apparent Km = 72 µM) [49]. Otherhuman UGTs involved in NSAID glucuronidation includeUGT1A6 and -2B15, which have been shown to glucuroni-date diclofenac [46]. It is noteworthy that UGT1A3, -1A9 and-2B7, which are found in human kidney, are also the isoformsinvolved in eicosanoid glucuronidation. This raises the ques-tion of a link between the renal metabolism of NSAIDs, ara-chidonic acid and arachidonic acid-derived metabolites, andNSAID-induced nephrotoxicity.

5. Novel mechanisms of NSAID nephrotoxicity

5.1 NSAIDs, arachidonic acid and inhibition of renal glucuronidationThe kidney epitomises an array of unique functional networksand, undoubtedly, initial inhibition (∼ 75 – 90%) of COX-1and -2 activity by NSAIDs (and hence decreased synthesis of‘renal-protective’ vasodilatory PGs) plays a pivotal role inNSAID-induced nephrotoxicity. However, focusing solely onCOX inhibition by NSAIDs fails to address the complexity ofthe metabolism of arachidonic acid and its bioactive meta-bolites, and the role this may play in NSAID-induced nephro-toxicity. It is conceivable that with inhibition of COX byNSAIDs and with the rate-limiting nature of the arachidonicacid re-esterification pathway (via arachidonyl-coenzyme Aligase), greater amounts of nonesterified arachidonic acidwould be available for the LOX and CYP arms of the arachi-donic acid cascade (Figure 2). Termed the ‘shunting hypo-thesis’, it has been proposed that increased flux of arachidonicacid via LOX and the resulting increased synthesis of leuko-trienes contributes to the development of the nephrotic syn-drome that occurs with interstitial nephritis [1]. However,‘shunting’ may not occur exclusively through LOX, but mayalso involve CYP. Arachidonic acid metabolites arising fromCYP are thought to eclipse the effects of LOX and COXmetabolites with respect to regulation of renal tubular and vas-cular function [27]. Importantly, in the presence of an NSAID,inhibition of COX results not only in diminished synthesis ofPGs, but the CYP metabolite 20-HETE is not metabolised byCOX to the vasodilatory PG analogues 20-OH-PGF2α and

20-OH-PGE2 [50]. Clearly, the downstream effect of COXinhibition in this situation may shift the balance in favour ofglucuronidation as a local detoxification pathway for thepre-eminent vasoconstrictor, 20-HETE (Figure 3).

The situation is further complicated, however, duringNSAID-induced renal ischaemia, as fatty acids, including ara-chidonic acid, accumulate. In a recent study using the non-selective UGT substrate 4-methylumbelliferone with humankidney cortical microsomes and recombinant UGT1A9 and-2B7 as the enzyme sources, arachidonic and linoleic acid,were described as the ‘most potent endogenous inhibitors ofUGT activity demonstrated to date’ [51]. PGE2, LTB4 and itsmetabolites, 5-, 12- and 15-HETE, and 20-HETE are all sub-strates for human renal UGTs. Thus, depending on the rela-tive affinities of the fatty acids and the selectivity of the UGTisoforms involved, the potential exists for inhibition of glu-curonidation of arachidonic acid-derived metabolites by fattyacids during renal ischaemia. In the presence of NSAIDs‘shunting’ may occur in the primary synthetic pathways, butequally, inhibition of enzymes in the degradative pathways(e.g., UGT) would exacerbate the renal insult of NSAIDsthrough imbalances in the prevailing vasodilator–vasocon-strictor forces (a chaotic combination of increased synthesisand decreased metabolism occurring in multiple pathways).

A comparison of the respective affinities (apparent Km) ofarachidonic acid for LOX, CYP and UGT suggests that in thewake of COX inhibition, CYP and UGT represent likelyroutes for arachidonic acid metabolism. Kinetic parametersfor arachidonic acid-derived 20-HETE formation viaCYP4F2 (Km = 24 µM) and CYP4A11 (Km = 228 µM) [52],are comparable with arachidonic acid glucuronide formationvia UGT1A9 (Km = 46 µM) and UGT2B7 (Km = 161 µM)[29]. Arachidonic acid is a proapoptotic molecule [8,30] and incontrast with metabolism via COX, LOX and CYP, meta-bolism via UGT limits the biological reactivity of arachidonicacid and its bioactive metabolites.

Recent studies have reported comparable activity betweenhuman liver and kidney microsomes for the glucuronidationof a number of xenobiotics. However, in the context of thefate of arachidonic acid, the renal metabolism of NSAIDsalso needs to be considered. The commonality of UGT1A3,-1A9 and -2B7 involvement in renal metabolism of NSAIDs,arachidonic acid, PGE2, LTB4 and its metabolites, and 5-,12- and 15-HETE, and 20-HETE suggests that competitionfor clearance via glucuronidation intrarenally is likely. In sup-port of this hypothesis, recent studies by the authors havedemonstrated significant inhibition of S-naproxen glucuroni-dation by arachidonic acid using human kidney corticalmicrosomes (50% inhibitory concentration [IC50] 17 µM)and recombinant UGT2B7 (IC50 = 16 µM) (unpublisheddata). As discussed in the preceding sections, the selectiveCOX-2 inhibitors elicited virtually the same spectrum ofadverse renal effects as the older nonselective NSAIDs. Inter-estingly, the carboxylic acid metabolite of hydroxymethyl-celecoxib [53] and 5-hydroxymethylvaldecoxib [54] and

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5-hydroxyrofecoxib [55] are all substrates for UGT, formingO-glucuronides in several species including humans.5-Hydroxyrofecoxib is principally metabolised by UGT2B15and -2B7 [55], which are UGT isoforms located in the kidney,with the latter playing a significant role in the metabolism ofarachidonic acid metabolites. Although glucuronidation ofcoxib metabolites has not been demonstrated using humanrenal tissue, the common involvement of UGT2B7 wouldpredict that similar competitive interactions would occurwith arachidonic acid and its metabolites.

5.1.1 PerspectiveFocusing on the central tenet that NSAID nephrotoxicity issynonymous with inhibition of COX fails to address thebiological reactivity of arachidonic acid per se and its meta-bolic fate subsequent to COX inhibition. Renal cells rapidlyamplify synthesis of eicosanoids in response to stimuli, butequally these compounds are rapidly metabolised. It follows,therefore, that subsequent to inhibition of COX byNSAIDs, further inhibition of glucuronidation of the CYP-and LOX-derived metabolites of arachidonic acid will resultin prolongation of their biological actions, which will con-tribute further to the spectrum of adverse renal effectsobserved with NSAIDs.

5.2 NSAIDs, glucuronidation and renal dysfunctionIt is established that reduced renal function leads to a reduc-tion in renal excretion of drugs and/or their metabolites.NSAIDs are structurally diverse, and include salicylates, aryl-propionates, oxicams, fenamates, indoles, phenylacetic acidsand pyrazoles. They are all metabolised to varying degreesin vivo and, for most NSAIDs, only a small fraction of thedose is excreted unchanged in urine. Studies using humankidney microsomes have demonstrated formation of the acylglucuronides of diflunisal, mefenamic acid, naproxen, ibupro-fen, ketoprofen and flurbiprofen [43,56]. All of the NSAIDslisted in Figure 1 are glucuronidated in vivo, either as the par-ent carboxylic acid or as oxidative metabolites, or are sub-strates for glucuronidation using recombinant UGTs(unpublished data). In the case of the NSAID ketoprofen,acyl glucuronides excreted in urine account for ∼ 80% of theoral dose [57]. In haemodialysis-dependent individuals withend stage renal disease, hydrolysis in vivo of S-ketoprofen glu-curonide back to the parent drug led to an accumulation ofthe pharmacologically active S-enantiomer of ketoprofen [58].Thus, repetitive dosing in these individuals would lead to pro-longed exposure to S-ketoprofen and persistent inhibition ofCOX with ensuing suppression of homeostatic PG synthesis.In situations of reduced renal perfusion acyl (ester) glucuro-nides of NSAIDs are rapidly hydrolysed, and hence clearanceof the glucuronides is diminished, resulting in higher intra-renal concentration of the parent compound, the concept of‘futile-cycling’. In addition to futile-cycling, reactive NSAIDacyl glucuronides are more unstable than ether-linked glu-curonides and undergo acyl migration and covalent binding

to proteins – predominantly via the acyl-migrated isomers[59-60]. Indeed, one of the theories of NSAID-induced inter-stitial nephritis involves covalent binding of the drug and/ormetabolite to sites within renal tissue, eliciting an immunehypersensitivity reaction. NSAIDs metabolised to glucu-ronides and implicated in the formation of protein adductsinclude aspirin, benoxaprofen, carprofen, diclofenac, feno-profen, indomethacin, naproxen, suprofen, sulindac,zomepirac, diflunisal, ibuprofen, tolmetin and mefenamicacid [60-62]. Human UGTs have been shown to be targets forreactive acyl glucuronides and, in particular, ketoprofen acylglucuronide binds irreversibly to the cofactor (uridine5′-diphosphate-glucuronic acid) binding site on the UGTprotein, thereby inhibiting UGT activity nonspecifically [63].

5.2.1 PerspectiveIt is generally accepted that a link between formation of adrug–protein adduct and organ toxicity is often casual ratherthan causal. In the case of reactive NSAID acyl glucuronides,evidence suggests that in situations of renal function impair-ment hydrolysis may occur, leading to the release of the parentNSAID. This provides an opportunity for continued suppres-sion of PG synthesis through persistent inhibition of COX,thus further exacerbating a decline in renal function in at-riskindividuals. In addition, in the presence of reduced renalexcretion of glucuronides, reactivity of NSAID acyl glucu-ronides is such that the likelihood of binding to macromole-cules within the kidney is high. The latter may provide anexplanation for the rare cases of NSAID-induced irreversiblerenal papillary necrosis. Although the mechanism is unclear, ithas been suggested that renal papillary necrosis may resultfrom a combination of NSAID-induced decreased renal papil-lary perfusion and excessive concentrations of NSAID and/orNSAID-metabolites [64].

6. Expert opinion

Nephrotoxicity is a well-recognised clinical outcome thatarises from the use of NSAIDs. It is estimated that ≤ 5% ofNSAID-users will develop one of a number of renal functionabnormalities: an overwhelming statistic when considered inthe context of NSAID use worldwide. Emphasis on COXselectivity (COX-2 > COX-1) did not produce the predictedrenal-sparing NSAIDs, but merely reaffirmed ‘more of thesame’ in terms of renal adverse effects. The search for a saferaspirin continues, and the notion of dual COX–LOX inhibi-tors as the anti-inflammatory drugs of the future has beenproposed [65-68]. Selective (and nonselective) COX inhibitorshave not provided the answer, and from experience neitherhas a predominantly LOX inhibitor (e.g., benoxaprofen – thespectacularly short-lived NSAID – metabolised extensively toglucuronides). As succinctly stated by Gambaro and Perazella[41] ‘the complexity of and the fundamental physiological roleof the prostanoid network in the kidney make it extremelydifficult to safely interfere with the various enzyme pathways’.

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Will a combination of a COX–LOX inhibitor simply be a‘double blow’ to the kidney?

Complex synthetic and metabolic networks function in anintegrated manner to maintain renal homeostasis. Based on theconcept of ‘shunting’, inhibition of two pathways for arachi-donic acid metabolism will simply produce increased flux ofarachidonic acid via an alternative route, most probably CYPand the production of EETs, di-HETEs and the pre-eminentrenal vasoconstrictor 20-HETE. Dual COX–LOX inhibitorsmay be efficacious in terms of their anti-inflammatory,analgesic and antipyretic activities, but it is questionable thatthey will be free of adverse renal effects.

The reactivity of arachidonic acid per se and its metabolitesinfers a significant role for renal UGTs in limiting the durationof action of a multitude of molecules that contribute to theregulation of renal and cardiovascular function. The kidneymay be small in mass in comparison with the liver, but the cata-lytic capacity for glucuronidation of NSAIDs is similar. Hence,the potential arises for intrarenal endobiotic–xenobiotic interac-tions resulting from common involvement of UGT enzymes inthe metabolism of eicosanoids and NSAIDs. It is time to lookbeyond COX, LOX and CYP and expand our horizon toinclude the UGTs when searching for an answer to the questionas to why no NSAID is ‘renal-sparing’.

BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

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20. IMIG JD: Eicosanoid regulation of renal vasculature. Am. J. Physiol. (2000) 279:F965-F981.

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•• Excellent review on human UGTs.

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• Review on COX-2 inhibitors and renal effects.

38. KRAMER BK, KAMMERL MC, KOMHOFF M: Renal cyclooxygenase-2 (COX-2) physiological, pathophysiological, and clinical implications. Kidney Blood Press. Res. (2004) 27:43-62.

• A general review of the area.

39. PERAZELLA MA, TRAY K: Selective COX-2 inhibitors: a pattern of nephrotoxicity similar to traditional nonsteroidal anti-inflammatory drugs. Am. J. Med. (2001) 111:64-67.

•• Key paper reporting cases of nephrotoxicity due to COX-2 inhibitors.

40. AHMAD SR, KORTEPETER C, BRINKER A, CHEN M, BEITZ J: Renal failure associated with the use of celecoxib and rofecoxib. Drug Safety (2002) 25(7):537-544.

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43. MCGURK KA, BRIERLY CH, BURCHELL B: Drug glucuronidation by human renal UDP-glucuronosyltransferases. Biochem. Pharmacol. (1998) 55(7):1005-1012.

44. BOWALGAHA K, MINERS JO: The glucuronidation of mycophenolic acid by human liver, kidney and jejunum microsomes. Br. J. Clin. Pharmacol. (2001) 52:605-609.

45. JIN C, MINERS JO, LILLYWHITE KJ, MACKENZIE PI: Complementary deoxyribonucleic acid cloning and expression of human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs. J. Phamacol. Exp. Ther. (1993) 264(1):475-479.

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47. SAKAGUCHI K, GREEN M, STOCK N, REGER TS, ZUNIC J, KING C: Glucuronidation of carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms. Arch. Biochem. Biophys. (2004) 424(2):219-225.

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•• Key paper reporting potency of linoleic and arachidonic acid as inhibitors of renal glucuronidation.

52. LASKER JM, CHEN WB, WOLF I, BLOSWICK BP, WILSON PD, POWELL PK: Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of CYP4F2 and CYP4A11. J. Biol. Chem. (2000) 275(6):4118-4126.

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55. ZHANG JY, ZHAN J, COOK CS, INGS RM, BREAU AP: Involvement of human UGT2B7 and 2B15 in rofecoxib metabolism. DMD (2003) 31:652-658.

56. MURRAY MD, BRATER DC: Renal toxicity of the nonsteroidal anti-inflammatory drugs. Ann. Rev. Pharmacol. Toxicol. (1993) 32:435-465.

57. FOSTER RT, JAMALI F, RUSSELL AS, ALBALLA SR: Pharmacokinetics of ketoprofen enantiomers in young and elderly arthritic patients following single and multiple doses. J. Pharm. Sci. (1988) 77(3):191-195.

58. GRUBB NG, RUDY DW, BRATER DC, HALL SD: Stereoselective pharmacokinetics of ketoprofen and ketoprofen glucuronide in end-stage renal disease: evidence for a ‘futile cycle’ of elimination. Br. J. Clin. Pharmacol. (1999) 48:494-500.

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59. DICKINSON RG, KING AR Studies on the reactivity of acyl glucuronides – II. Interaction of diflunisal acyl glucuronide and its isomers with human serum albumin in vitro. Biochem. Pharmacol. (1991) 42:2301-2306.

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63. TERRIER N, BENOIT E, SENAY C et al.: Human and rat liver UDP-glucuronosyltransferase are targets of ketoprofen acylglucuronide. Mol. Pharmacol. (1999) 56:226-234.

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64. WHELTON A, STURMER T, PORTER GA: Non-steroidal anti-

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Website

101. http:// www.tga.gov.au

AffiliationKathleen M Knights† PhD, Paraskevi Tsoutsikos & John O Miners†Author for correspondenceFlinders University and Flinders Medical Centre, Department of Clinical Pharmacology, Bedford Park, Adelaide 5042, AustraliaTel: +61 (8) 8204 4331; Fax: +61 (8) 8204 5114;E-mail: Kathie.knights@flinders.edu.au

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