Expression studies and functional characterization of renal human organic anion transporter 1 isoforms.

The human organic anion transporter 1 (hOAT1) facilitates the basolateral entry of organic anions such as endogenous metabolites, xenobiotics, and drugs into the proximal tubule cells. In the present study we investigated the general occurrence of hOAT1 isoforms in the kidneys and performed functional characterizations. Kidney specimens of 10 patients were analyzed by reverse transcription-polymerase chain reaction. We detected hOAT1-2 as the main transcript in almost all patients, and weak transcripts of hOAT1-1, hOAT1-3, and hOAT1-4 in many of them. An evaluation of the renal distribution showed all four mRNAs mostly restricted to the cortex. Western blot analysis of membrane fractions from two kidney specimens yielded two bands corresponding to the observed mRNA expression, suggesting hOAT1-3 and hOAT1-4 to be expressed on the protein level in vivo. This observation is further supported by immunofluorescence analyses of all four cloned hOAT1 isoforms transiently transfected in COS 7 cells. Functional characterizations did not show any transport activity of hOAT1-3 and hOAT1-4 for the tested substrates. Cotransfection studies of each of them with hOAT1-1 did not alter fluorescein uptake indicating no regulatory impact of these isoforms. Further functional comparisons of hOAT1-1 and hOAT1-2 in fluorescein uptake studies exhibited almost identical affinities for fluorescein with Michaelis constants of 11.6 +/- 3.7 microM (hOAT1-1) and 11.9 +/- 6.4 microM (hOAT1-2), and similar sensitivities to inhibition by p-aminohippurate [IC(50): 16 microM (hOAT1-1), 10 microM (hOAT1-2)], urate [IC(50): 440 microM (hOAT1-1), 385 microM (hOAT1-2)], and furosemide (IC(50): 14 microM (hOAT1-1), 20 microM (hOAT1-2)], implying functional equivalence.

RT-PCR-based evidence for the in vivo stimulation of renal tubularp-aminohippurate (PAH) transport by triiodothyronine (T3) or dexamethasone (DEXA) in kidney tissue of immature and adult rats.

Our previous studies have shown that a pre-treatment of rats with triiodothyronine (T3) or dexamethasone (DEXA) increases renal PAH excretion significantly. This stimulation was accompanied by an enhanced protein synthesis within the renal cortex. To explore the molecular basis for this sub-chronic induction process, we investigated the stimulation of PAH accumulation in renal cortical slices as well as the expression level of organic anion transporter 1 (OAT1), the recently cloned renal basolateral PAH-transporter, using RT-PCR techniques under the applied conditions. 10- and 55-day-old Han:WIST rats were treated in vivo with T3 (20 microg/100 g b.wt.) or DEXA (60 microg/100 g b.wt.), both for 3 days, once daily. Renal cortical slices were incubated for 2 hours in Cross-Taggart medium and PAH uptake into kidney tissue was measured time dependently (slice to medium ratio, QS/M). The accumulation capacity is comparable between immature and mature rats (control-QS/M: 6.7 +/- 0.1 vs. 6.9 +/- 0.2, respectively). Both age groups showed a significant increase of PAH accumulation capacity after T3 treatment (10-day-old rats: 15.0 +/- 0.2; 55-day-old rats: 11.7 +/- 1.3). After DEXA pre-treatment, PAH accumulation was only slightly changed (10-day-old rats: 5.9 +/- 0.2; 55-day-old rats: 8.2 +/- 1.3). Semi-quantitative measurements of OAT1 mRNA expression level showed a significant increase of OAT1 mRNA after pre-treatment with both T3 and DEXA in the two age groups. Thus, this is the first evidence that T3 and DEXA pre-treatment induces the expression of OAT1.