Transporters involved in renal excretion of N-carbamoylglutamate, an orphan drug to treat inborn n-acetylglutamate synthase deficiency

Abstract

Inborn defects in N-acetylglutamate (NAG) synthase (NAGS) cause a reduction of NAG, an essential cofactor for the initiation of the urea cycle. As a consequence, blood ammonium concentrations are elevated, leading to severe neurological disorders. The orphan drug N-carbamoylglutamate (NCG; Carbaglu), efficiently overcomes NAGS deficiency. However, not much is known about the transporters involved in the uptake, distribution, and elimination of the divalent organic anion NCG. Organic anion-transporting polypeptides (OATPs) as well as organic anion transporters (OATs) working in cooperation with sodium dicarboxylate cotransporter 3 (NaDC3) accept a wide variety of structurally unrelated drugs. To test for possible interactions with OATPs and OATs, the impact of NCG on these transporters in stably transfected human embryonic kidney-293 cells was measured. The two-electrode voltage-clamp technique was used to monitor NCG-mediated currents in Xenopus laevis oocytes that expressed NaDC3. Neither OATPs nor OAT2 and OAT3 interacted with NCG, but OAT1 transported NCG. In addition, NCG was identified as a high-affinity substrate of NaDC3. Preincubation of OAT4-transfected human embryonic kidney-293 cells with NCG showed an increased uptake of estrone sulfate, the reference substrate of OAT4, indicating efflux of NCG by OAT4. In summary, NaDC3 and, to a lesser extent, OAT1 are likely to be responsible for the uptake of NCG from the blood. Efflux of NCG across the luminal membrane into the tubular lumen probably occurs by OAT4 completing renal secretion of this drug.

Interaction of Excitatory Amino Acid Transporters 1 – 3 (EAAT1, EAAT2, EAAT3) with N-Carbamoylglutamate and N-Acetylglutamate

“…4A). For NAG and NCG, we recently determined in NaDC3-expressing oocytes K m values of 125 ± 10 and 25.7 ± 2.0 µM, respectively [14]. Using similar experimental conditions, a K m for OAA of 465 ± 59 µM was calculated (Fig.…”

“…The efficiency of transport was 2.38 and 0.53 nA/µM, indicating that the turnover rates for NCG and αKG, one of the reference substrates of NaDC3 (2.88 nA/µM; Table 2, [14]), are similar. At least in the rat, NaDC3 mRNA appears to be restricted to perivenous hepatocytes that are not primarily involved in urea synthesis, but utilize ammonia mainly for glutamine synthesis [15].…”

“…In a recent paper [14], we demonstrated transport of N -carbamoylglutamate and N -acetylglutamate by the sodium-dependent dicarboxylate transporter 3 (NaDC3) with K m values of 25.7 ± 2.0 and 125 ± 10 µM (c.f Table 2), respectively. The efficiency of transport was 2.38 and 0.53 nA/µM, indicating that the turnover rates for NCG and αKG, one of the reference substrates of NaDC3 (2.88 nA/µM; Table 2, [14]), are similar.…”

“…Using Xenopus laevis oocytes expressing the human sodium-dependent dicarboxylate transporter 3 (NaDC3), we recently demonstrated transport of N -acetylglutamate and N -carbamoylglutamate by NaDC3 [14]. In the rat, in-situ hybridization localized NaDC3 to perivenous hepatocytes [15], suggesting NaDC3 as a possible entry pathway for N -carbamoylglutamate at least in a subset of hepatocytes.…”

The Isoquinolone Derived Prolyl Hydroxylase Inhibitor ICA Is a Potent Substrate of the Organic Anion Transporters 1 and 3

“…OAT1-, OAT2-, OAT3-, OAT4- and vector-transfected HEK293 cells are used routinely in our laboratory and were prepared and treated as described previously [5,13]. For transport measurements, cells were incubated at room temperature in Mammalian Ringer (MRi) that contained the [ 3 H]nuclide at the concentration indicated in the respective figure legend and various experimental conditions.…”