Elsevier

Biochemical Pharmacology

Volume 67, Issue 1, 1 January 2004, Pages 191-199
Biochemical Pharmacology

Evidence that unsaturated fatty acids are potent inhibitors of renal UDP-glucuronosyltransferases (UGT): kinetic studies using human kidney cortical microsomes and recombinant UGT1A9 and UGT2B7

https://doi.org/10.1016/j.bcp.2003.08.025Get rights and content

Abstract

Renal ischaemia is associated with accumulation of fatty acids (FA) and mobilisation of arachidonic acid (AA). Given the capacity of UDP-glucuronosyltransferase (UGT) isoforms to metabolise both drugs and FA, we hypothesised that FA would inhibit renal drug glucuronidation. The effect of FA (C2:0–C20:5) on 4-methylumbelliferone (4-MU) glucuronidation was investigated using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7 as the enzyme sources. 4-MU glucuronidation exhibited Michaelis–Menten kinetics with HKCM (apparent Km (Kmapp) 20.3 μM), weak substrate inhibition with UGT1A9 (Kmapp 10.2 μM, Ksi 289.6 μM), and sigmoid kinetics with UGT2B7 (S50app440.6 μM) Similarly, biphasic UDP-glucuronic acid (UDPGA) kinetics were observed with HKCM (S50 354.3 μM) and UGT1A9 (S50 88.2 μM). In contrast, the Michaelis–Menten kinetics for UDPGA observed with UGT2B7 (Kmapp 493.2 μM) suggested that kinetic interactions with UGTs were specific to the xenobiotic substrate and the co-substrate (UDPGA). FA (C16:1–C20:5) significantly inhibited (25–93%) HKCM, UGT1A9 or UGT2B7 catalysed 4-MU glucuronidation. Although linoleic acid (LA) and AA were both competitive inhibitors of 4-MU glucuronidation by HKCM (Kiapp 6.34 and 0.15 μM, respectively), only LA was a competitive inhibitor of UGT1A9 (Kiapp 4.06 μM). In contrast, inhibition of UGT1A9 by AA exhibited atypical kinetics. These data indicate that LA and AA are potent inhibitors of 4-MU glucuronidation catalysed by human kidney UGTs and recombinant UGT1A9 and UGT2B7. It is conceivable therefore that during periods of renal ischaemia FA may impair renal drug glucuronidation thus compromising the protective capacity of the kidney against drug-induced nephrotoxicity.

Introduction

FA are predominantly straight-chain monocarboxylic acids ranging from 4 to 24 carbon atoms that may be either saturated or unsaturated. They are essential for integrated functioning of the human body, serving both as an energy source and as a unit for energy storage. Their importance, however, extends beyond their role in anabolic and catabolic pathways. FA per se act as regulatory molecules affecting enzyme activity (e.g. acetyl-CoA carboxylase) and gene transcription as endogenous ligands of peroxisome-proliferator-activated receptors [1]. In addition, FA are esterified in membrane phospholipids and are critical for the structure and hydrophobicity of mammalian cell membranes. While all FA contribute to membranous hydrophobicity, polyunsaturated FA additionally fulfil a unique role as precursors of potent signal molecules. Oxidative metabolism of FA produces prostaglandins, leukotrienes and hydroxy-fatty acids, collectively referred to as eicosanoids. These bioactive molecules are synthesised from 20-carbon polyunsaturated FA namely dihomo-γ-linolenic acid, eicosapentaenoic acid and AA. Eicosanoids synthesised from AA contribute to major pathophysiological conditions including chronic inflammatory diseases (e.g. rheumatoid arthritis, psoriasis) [2], [3], toxic and immune mediated nephropathies [4], formation of thrombi and atheroma, and cell proliferation [5].

AA (C20:4n−6) is the most abundant polyunsaturated FA in membrane phospholipids and is liberated as a result of the action of phospholipases and mechanical or mitogenic stimuli. Free AA is rapidly metabolised by three enzyme systems: cyclo-oxygenases (COX), lipo-oxygenases (LOX) and cytochromes P-450 (CYP). Oxygenation of released AA via the action of COX produces prostaglandins of the 2-series (e.g. PGE2) and thromboxanes; via the LOX pathway the 4-series leukotrienes (e.g. LTB4) and hydroxy-fatty acids; and via CYP the epoxyeicosatrienoic acids (EETs) and their corresponding diols and hydroxyeicosatetraenoic acids (HETEs).

Carboxylic acids are common substrates for UGT (EC 2.4.1.17). Of the 27 known human UGT genes, 14 (UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B15, 2B17, and 2B28) encode proteins that are catalytically active towards a myriad of xeno- and endo-biotics. The glucuronidation of medium chain FA and the unsaturated FA, LA, α-linolenic and AA by rat UGT2B1 was reported in 1994 [6]. Later studies demonstrated that monkey UGT2B9 (89% identical to human UGT2B7) and human UGT1A3 glucuronidated C2–C12, and C10 and C12 straight-chain saturated FA, respectively [7], [8]. More recently, glucuronidation of LA, the immediate precursor of AA, by human liver microsomes and glucuronidation of the two naturally occurring metabolites of LA, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-oxooctadecadienoic acid (13-OXO) by human UGT2B7, was reported [9], [10], [11]. Subsequent studies also established that AA, 20-HETE and PGE2 were substrates for human hepatic and intestinal UGTs and recombinant UGT2B7 [12].

Although knowledge of xenobiotic metabolism is often based on studies using predominantly liver, significant glucuronidation activity towards endogenous and exogenous compounds has been reported for human kidney [13]. Substrates for renal UGTs include paracetamol, morphine, 4-MU, 1-naphthol, 4-nitrophenol, propofol, mycophenolic acid, hyodeoxycholic acid, estradiol and testosterone amongst others [14], [15], [16]. UGT activity is primarily localised to the cortical region and the isoforms identified in human renal tissue include 1A3, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15 and 2B17 [17], [18], [19].

The physiological functions of the kidney are varied and numerous, and the metabolism of AA plays an important role in integrating renal dynamics in particular renal vascular tone, glomerular function and fluid and electrolyte balance. Although the concentration of free AA in resting cells is universally described as ‘low’, once released the concentration can range from 10 to 100 μM [20]. Progression to acute renal failure is also accompanied by increased intracellular accumulation of FA. Since most UGT isoforms are capable of metabolising both drugs and endobiotics, we hypothesised that FA would inhibit renal drug and chemical glucuronidation. In order to assess the potential effects of FA on renal glucuronidation, we utilised 4-MU as the probe UGT substrate and HKCM and recombinant UGT1A9 and UGT2B7 as the enzyme sources. UGT1A9 and UGT2B7 were selected for investigation as both are known to be expressed in human kidney [17], [18] and both have the capacity to glucuronidate substrates containing a carboxylic acid function, a characteristic chemical feature of FA [17]. Accordingly, the inhibitory effect of a range of saturated (C2–C20) and unsaturated (C14:1–C20:5) FA on 4-MU glucuronidation by HKCM and recombinant UGT1A9 and UGT2B7 was determined.

Section snippets

Materials

The following compounds were purchased from Sigma-Aldrich: UDPGA, 4-MU, 4-MU–β-d-glucuronide and the FA (either as the free acid or the sodium salt) acetic (C2:0), butyric (C4:0), n-caproic (C6:0), caprylic (C8:0), capric (C10:0), myristic (C14:0), myristoleic (C14:1), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), α-linolenic (C18:3), arachidic (C20:0), gondoic (C20:1), eicosadienoic (C20:2n6), dihomo-γ-linolenic (C20:3), arachidonic (C20:4),

UDPGA kinetics

The kinetic parameters for the co-substrate UDPGA and for glucuronidation of 4-MU were studied using HKCM and recombinant UGT1A9 and UGT2B7. In the presence of a fixed concentration of 4-MU (0.5 mM) apparent biphasic Eadie–Hofstee plots were observed for UDPGA when using HKCM (Fig. 1A) and UGT1A9 (Fig. 1B). The data for HKCM were best fitted to the Hill equation with h<1, indicative of negative co-operativity (Table 2). The apparent S50 for individual kidneys ranged from 259.1 to 416.9 μM, with a

Discussion

This study has demonstrated that 4-MU glucuronidation activity in HKCM (CLint 428.5 μL/min/mg) is directly comparable to that reported previously for 4-MU glucuronidation by human liver microsomes (CLint 413.8 μL/min/mg) [24]. Recent studies have also reported comparable activity between human liver and human kidney microsomes for the glucuronidation of mycophenolic acid [16] and propofol [27]. Although extrapolation of in vitro kinetic parameters (using appropriate scaling factors) indicates

Acknowledgements

Thanks are due to Virginia Papangelis for coordinating renal tissue collection and Xiao Hui Guo for expression of recombinant UGT proteins. The research was supported by grants from the Flinders Medical Research Institute (K.M.K.) and the National Health and Medical Research Council of Australia (J.O.M.).

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