Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity

Abstract

Plasma membrane monoamine transporter (PMAT) is a novel polyspecific organic cation transporter that transports organic cations and the purine nucleoside, adenosine. PMAT is expressed in the kidney, but the specific localization and function of this transporter in renal cells are unclear. In this study, we developed a polyclonal antibody toward a 14-amino acid sequence in the last intracellular loop of PMAT and determined the precise cellular localization of PMAT in human and rat kidneys. Surprisingly, we found that the PMAT protein was predominantly expressed in the glomerulus with minimal expression in tubular cells. Within the glomerulus, dual-color immunofluorescence labeling showed that the PMAT protein was specifically localized to the visceral glomerular epithelial cells, i.e., podocytes. There was no significant PMAT immunoreactivity in mesangial or glomerular endothelial cells. We further showed that puromycin aminonucleoside (PAN), a classic podocyte toxin that induces massive proteinuria and severe glomerulopathy, is transported by PMAT. Expression of PMAT in Madin-Darby canine kidney cells significantly increased cell sensitivity to PAN. Decynium 22, a potent PMAT inhibitor, abolished PAN toxicity in PMAT-expressing cells. Together, our data suggest that PMAT is specifically expressed in podocytes and may play an important role in PAN-induced kidney injury.

Fig. 1.

Characterization of a purified polyclonal anti-plasma membrane monoamine transporter (PMAT) antibody. A: membrane topology of human PMAT and the amino acid sequence (shown in dark) used to generate P469 polyclonal antibody. B: Western analysis of PMAT- and vector-transfected Madin-Darby canine kidney (MDCK) cells using P469 antibody at 1:1,600 dilution. C: Western analysis of PMAT expression in human kidney using P469 antibody at 1:1,000 dilution. D-F: immunocytochemistry in MDCK cells. Cells transfected with empty vector pcDNA3 (D) and human PMAT (E) were stained with P469 antibody. F: PMAT-transfected MDCK cells were stained with prebleed sera. All cells were permeabilized with 0.2% Triton X-100 before staining, and the antibody was used at 1:200 dilution. Cell nuclei were counterstained with TO-PRO-3.

Fig. 2.

Immunolocalization of PMAT in the glomerulus in normal rat and human kidneys. Immunofluorescence analysis was performed on cryosections of normal rat (A-C) or human kidneys (D-F). Sections incubated with prebleed sera were served as controls (C and F). Incubation of sections with PMAT-specific antibody revealed strong fluorescent signals (green) in the glomerulus (A, B, D, E).

Fig. 3.

Podocyte-specific localization of PMAT in the human glomerulus. Human kidney cryosection was costained with P469 antibody (green) and markers of mesangial cells (α-smooth muscle actin, red; A), glomerular endothelial cells (CD31, blue; B), and podocytes (WT1, red; C and D). D: high magnification showed membrane staining of PMAT and nucleus staining of WT1 of the same cell in the glomerulus.

Fig. 4.

Effect of PMAT expression or inhibition on cell sensitivity to puromycin aminonucleoside (PAN) toxicity. A: PAN dose-response curves in vector (○)- and PMAT (•)-transfected MDCK cells. Cells were incubated with graded concentrations of PAN for 72 h at 37°C. B: effect of a PMAT inhibitor on cell sensitivity to PAN. Vector-transfected (open bar) and PMAT-transfected (filled bar) cells were incubated with 250 μM PAN in the absence or presence of 2 μM decynium 22 (Dy22) for 72 h at 37°C. Cytotoxicity was determined by MTT assay described in methods. *Significantly different from vector control (P < 0.0001). #Significantly different from PMAT-expressing cells treated with PAN without Dy22 (P < 0.0001).

Fig. 5.

Uptake of PAN in PMAT-expressing MDCK cells. Vector (open bar)- and PMAT (filled bar)-expressing MDCK cells were incubated at 37°C with 100 μM PAN for 1 min at pH 6.6 or 7.4. Cellular concentration of PAN was determined by liquid chromatography/mass spectrometry assay described in methods. *Significantly different from vector control (P < 0.01). #Significantly different from PMAT-expressing cells at pH 7.4 (P < 0.001).