Figure 1.
Validation of Oatp4c1 expression and subcellular localization in MDCKII cells.
(A) Western blot analysis for Oatp4c1 expression. Protein lysates (50 µg per lane) from MDCKII cells transfected with pcDNA (empty vector) and Oatp4c1 were prepared from cells grown in monolayers. (B) Immunohistochemical analysis for Oatp4c1 expression. Paraformaldehyde-fixed paraffin-embedded cell sections were stained with PA1343. Color development with NovaRed signifies Oatp4c1 staining. All sections were counterstained with hemotoxylin. Rabbit IgG was used as a negative control. (C) Immunolocalization of Oatp4c1 in polarized MDCKII cells. Cells were double stained with Oatp4c1 (red) and ZO-1 (green). Nuclei were stained with DAPI (blue). Center image in the Oatp4c1 panel is a single optical section of the x–y plane while top and right images represent x–z and y–z planes, respectively, reconstructed from image stacks. The apical and basal sides can be demarcated by ZO-1 and the nuclei, respectively, in both x–z and y–z sections. (D) Western blot analysis of Oatp4c1 in proteins isolated from either the apical (AP) or basolateral (BL) membranes of polarized MDCKII cells. Surface proteins were isolated by biotin-streptavidin labeling on either the apical AP or BL compartment. Na+/K+-ATPase served as a BL marker to demonstrate the relative efficiency of AP and BL membrane separation. (Magnification (40×)).
Figure 2.
Oatp4c1 expression and localization in rat kidney.
(A) Western blot analysis for Oatp4c1 expression in rat tissues. Crude membrane fractions from rat liver and kidney were prepared. PNGaseF was used for deglycosylation. Oatp4c1 was detected by PA1343. β-actin was used as a loading control. (B) Immunohistochemical analysis for Oatp4c1 subcellular localization in rat tissues. Paraformaldehyde-fixed paraffin-embedded rat tissue sections were stained with PA1343. Color development with NovaRed signifies Oatp4c1 staining. (C) Double immunofluorescence staining for Oatp4c1 (green) and E-cadherin (red) in rat kidney. E-cadherin served as a basolateral marker (Magnification: 40×).
Figure 3.
Expression of Oatp4c1 in isolated kidney brush border membrane (BBM) and basolateral membrane (BLM) fractions.
(A) Western blot analysis for Oatp4c1 expression in BBM and BLM. Biochemical separation of membrane proteins (20 µg per lane) from rat kidney cortex was achieved by Percoll gradient. Na+/K+-ATPase alpha 1 was used as a basolateral marker. Abcg2 and Mrp4 served as apical markers. Oatp4c1 was detected by PA1343. (B) LC-MS/MS based proteomic analysis of ∼60–150 kD proteins isolated from BBM and BLM fractions. Spectral counts detected in protein digests obtained from ∼60–150 kD sections excised from the SDS-PAGE gel. Proteins with 3 or more spectral counts in one fraction are presented as “Apical” or “Basolateral” based on previously published data. Spectral counts indicate the number of unique peptides detected in each fraction. (AP, apical).
Figure 4.
Oatp4c1 localization in paraformaldehyde-fixed paraffin-embedded rat kidney tissue sections.
Paraformaldehyde-fixed paraffin-embedded kidney sections were stained with antibodies as indicated. Following staining with PA1343 antibody, color development with NovaRed signifies Oatp4c1 staining (A). Sections were double-stained with PA1343 (B, C, red) and the proximal tubule marker AQP1 (B, green) or the distal tubule marker calbindin (C, green). Nuclei (blue) were stained with DAPI (Magnification: 4× (A), 10× (B, C)).
Figure 5.
Oatp4c1 colocalization with major efflux transporters in the kidney cortex.
Paraformaldehyde-fixed paraffin-embedded rat kidney sections were stained for Oatp4c1 (red), Mrp4 (red), P-gp (green), and Abcg2 (red) (A). Sections were double stained for Mrp4 (red) and Oatp4c1 (green); (C) P-gp (green) and Oatp4c1 (red); and (D) Abcg2 (red) and Oatp4c1 (green) (B). Yellow/orange color in merged panels indicates colocalization. Nuclei (blue) were stained with DAPI. Images presented in panel 5A were stitched from six individual images using NIH Image J with Fiji plugin (Magnification: 4× (A), 10× (B–D)).
Figure 6.
Characteristics of Oatp4c1-mediated E3S transport.
Time dependent [3H]-E3S uptake at pH 5.5 by MDCKII-pcDNA and MDCKII-Oatp4c1 cells during incubation with 0.5 µM [3H]-E3S (A). Time dependent Oatp4c1 mediated uptake of [3H]-E3S at pH 4.5, 5.5 and 7.4 (B). Concentration dependent [3H]-E3S uptake in MDCKII-pcDNA and MDCKII-Oatp4c1 cells after incubation at pH 5.5 for 1 min (C). Concentration dependent Oatp4c1 mediated uptake (1 min) of [3H]-E3S at pH 4.5, 5.5 and 7.4 (D). GF: GF120918, MTX: methotrexate. Each point represents the mean ± S.D. of triplicates. *p<0.05, significant differences from control.
Table 1.
Transport kinetic parameters of Oatp4c1-mediated E3S transport at pH 4.5, 5.5 and 7.4 (mean ± S.E.).
Figure 7.
The effect of pH on uptake transport clearance.
The uptake clearance was estimated using the parameter estimates in Table 1. The solid lines show the best fit to the data using
obtained from [3H]-E3S uptake (Fig. 6).
Figure 8.
The effect of uremic toxins and anionic physiological substances on Oatp4c1-mediated E3S transport.
Inhibition of Oatp4c1-mediated [3H]-E3S uptake (1 min) was determined in the absence (control) and presence of various compounds (100 µM) at pH 5.5 (A) and 7.4 (B). Oatp4c1-mediated uptake was estimated after subtracting the nonspecific uptake measured in pcDNA cells treated under identical conditions. In all studies the [3H]-E3S concentration was 0.5 µM. GF: GF120918, MTX: methotrexate. Each point represents the mean ± S.D. of triplicates. *p<0.05, significant differences from control.