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ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2
I Leier1, J Hummel-Eisenbeiss, Y Cui
1Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany. i.leier@dkfz-heidelberg.de
Background:
Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2). This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney.
Methods:
Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of [3H]PAH, and the net ATP-dependent transport into inside-out vesicles was determined. Comparative studies were performed with membrane vesicles containing recombinant human MRP1.
Results:
Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively. The Km value for PAH was 880 micromol/L. The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively. The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L. Ochratoxin A was itself a substrate for MRP2.
Conclusions:
PAH is a good substrate for the ATP-dependent export pump MRP2. The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.
Insights
Para-aminohippurate (PAH) is effectively transported by the multidrug resistance-associated protein 2 (MRP2), an ATP-dependent export pump found in kidney proximal tubules. This finding highlights MRP2's role in secreting PAH and other anionic compounds.
Area of Science:
- Nephrology
- Molecular Biology
- Pharmacology
Background:
- Para-aminohippurate (PAH) is a key substrate for studying organic anion transport in kidney proximal tubule epithelia.
- The apical multidrug resistance protein MRP2 (ABCC2) is an ATP-dependent export pump localized to the apical membrane of proximal tubules.
Purpose of the Study:
- To investigate if para-aminohippurate (PAH) is a substrate for the multidrug resistance-associated protein 2 (MRP2).
- To determine the functional role of MRP2 in the renal secretion of PAH.
Main Methods:
- Transport assays using membrane vesicles from HEK cells expressing recombinant human MRP2 and control cells.
- Incubation with varying concentrations of [3H]PAH to measure net ATP-dependent transport.
- Comparative studies with membrane vesicles expressing MRP1.
Main Results:
- MRP2-mediated PAH transport was significantly higher than in control vesicles (21.9 vs. 1.6 pmol x mg protein-1 x min-1 at 10 µmol/L PAH).
- The Michaelis constant (Km) for PAH was determined to be 880 µmol/L.
- Transport was inhibited by leukotriene C4 and MK571, and ochratoxin A, which is also an MRP2 substrate.
Conclusions:
- Para-aminohippurate (PAH) is confirmed as a good substrate for the ATP-dependent export pump MRP2.
- MRP2's localization and function in proximal tubules suggest its contribution to the secretion of PAH and other amphiphilic anions into the kidney tubule lumen.