Kinetics of aldosterone glucuronidation by human liver and human kidney cortical microsomes and recombinant UGTs

Kathleen Knights, Leanne Winner, David Elliot & John O Miners, Department of Clinical Pharmacology, Flinders University and Flinders Medical Centre, Bedford Park, Adelaide, Australia, 5042.

Aldosterone is an important mineralocorticoid that acts in the kidney to promote sodium and water reabsorption and enhance potassium excretion. It similarly mediates sodium and water transport in the colon. Less than 3% of aldosterone is excreted unchanged in urine and the 18β-glucuronide of aldosterone, which is principally formed in the kidney accounts for 5-15% of the daily excretion of aldosterone. Aldosterone is a substrate for UDP-glucuronosyltransferases (UGT), in particular UGT2B7 (Girard et al., 2003). NSAIDs also promote sodium and water retention and given that many NSAIDs are substrates for UGT2B7, there is the potential for an intrarenal aldosterone-NSAID interaction. The aims of the present study were to define the kinetics of aldosterone glucuronidation using human liver and kidney cortical microsomes and identify specific UGTs that catalyse aldosterone glucuronidation. Approval for collection and use of human liver and kidney tissue was obtained from the Research and Ethics Committee, Repatriation General Hospital and the Flinders Clinical Research Ethics Committee.

Human liver (0.5mg/mL, HLM), or kidney cortical (0.2mg/mL, HKCM) microsomes or recombinant UGT protein (UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17, 2B28; 0.5-1mg/mL, HEK293 cell lysate ) was incubated at 37° for 45, 20 and 60min, respectively in the presence of 10-2000µM aldosterone, UDP-glucuronic acid (5mM) and 4mM MgCl2 in 0.1M phosphate buffer (pH7.4). Reactions were terminated and an aliquot (35µL) of the supernatant fraction was injected onto a Waters Nova-Pak C18 column, which was eluted with 95% water- 5% acetonitrile (containing 0.002% v/v acetic acid, mobile phase A) and 100% acetonitrile (mobile phase B) at a flow rate of 1.0mL/min. The proportion of mobile phase B was increased to 65% over 9 min. Analytes were detected at 241nm. The retention times for aldosterone glucuronide and aldosterone were 4.68 and 6.36 min, respectively.

Aldosterone 18β- glucuronidation by human liver and kidney cortical microsomes and recombinant UGTs exhibited Michaelis-Menten kinetics. Of the recombinant proteins, only UGT1A10 and UGT2B7 catalysed aldosterone glucuronidation (Table 1). Data are tabulated as mean ± SD.

The results indicate that the intrinsic clearance for aldosterone via 18β-glucuronidation is comparable between human liver and kidney. UGT2B7 is expressed in both liver and kidney whereas UGT1A10 is expressed only in colon. Thus, the data suggest that UGT2B7 is responsible for the hepatic and renal glucuronidation of aldosterone. As NSAIDs are substrates for UGT2B7, inhibition of renal aldosterone glucuronidation may contribute to the onset of NSAID induced hypertension observed in older adults. Interestingly the involvement of UGT1A10 in aldosterone glucuronidation suggests a role for this enzyme in the metabolism of aldosterone in the colon.

Girard et al., (2003) Endocrinology, 144(6):2659-2668.