Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells

Abstract

The Na(+)/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of P(i) in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary P(i) intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na(+)-transporting systems such as NHE3 and Na(+)-K(+)-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D(1)-like receptor agonists fenoldopam (10 microM) and SKF-38393 (10 microM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D(2)-like selective agonist quinpirole (1 microM) had no effect. The D(1) and D(2) agonists did not affect the renal Na(+)/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D(1)-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 microM chelerythrine) or ERK1/2 (20 microM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 microM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D(1)-like receptors, an effect that is mediated by PKA.

Fig. 1

Internalization of the NaPi-IIa cotransporter in mouse kidney slices after stimulation of D₁-like, but not D₂-like, receptors. A–C: immunohistochemical detection of the NaPi-IIa cotransporter in the renal cortex of control slices (A) and slices incubated with D₁-like agonists fenoldopam (10 µM; B) and SKF-38393 (10 µM; C). D: incubation with the D₂-like agonist quinpirole (1 µM) for 1 h had no effect on NaPi-IIa immunoreactivity. Original magnification: ×40. E–H: higher-magnification views of single proximal tubules stained for NaPi-IIa (green) and β-actin filaments (red). Scale bar, ~10 µm. E: a high degree of overlap (yellow) between NaPi-IIa and actin under control conditions, indicating localization in the brush border membrane (BBM). After treatment with fenoldopam (F) or SKF-38393 (G), NaPi-IIa immunoreactivity disappeared from the BBM and a distinct subapical signal was seen, demonstrating internalization. H: treatment with quinpirole did not alter BBM localization of NaPi-IIa (H).

Fig. 2

No effect of dopamine receptor agonists on the renal Na⁺/sulfate cotransporter NaSi. Immunodetection of NaSi in mouse proximal tubules under control conditions (top) and after treatment with fenoldopam (middle) or quinpirole (bottom). Localization of NaSi in the BBM was not affected by treatments with fenoldopam or quinpirole. Scale bar, ~10 µm.

Fig. 3

Activation of D₁, but not D₂, receptors reduces NaPi-IIa abundance in the BBM. Mouse kidney slices were incubated with control solution (Co), PTH-(1–34) (100 nM), the D₁-like agonist fenoldopam (Fen, 20 µM), or the D₂-like agonist quinpirole (Quin, 2 µM) for 1 h, and BBMs were prepared. Protein (10 µg/lane) was loaded, and membranes were stained for NaPi-IIa and actin to control for loading. Densitometry was performed, and values were normalized against actin, with control values set as 100%. n = 3 independent experiments. Values are means ± SD. Reduction in NaPi-IIa expression was significantly lower than under control conditions. There was no significant difference between the 3 treatments. Significant difference: *P < 0.05; **P < 0.001.

Fig. 4

Effect of basolateral and luminal application of D₁ and D₂ agonists on expression of the NaPi-IIa cotransporter in isolated and perfused mouse proximal tubules. Immunodetection of the NaPi-IIa cotransporter (green) and β-actin (red) in isolated proximal tubules. Tubules were perfused, and the D₁-like agonist SKF-38393 or the D₂-like agonist quinpirole was added to the lumen or bath. Luminal administration of SKF-38393 induced removal of cotransporters without affecting β-actin staining. Basolateral application of SKF-38393 did not influence NaPi-IIa immunofluorescence. All downregulatory effects were specific for the D₁-like receptor agonist, because application of the D₂-like receptor agonist quinpirole did not induce internalization of NaPi-IIa, applied apically or basolaterally. Scale bar, ~10 µm.

Fig. 5

D₁-like receptor agonist-induced NaPi-IIa internalization requires intact PKA. Freshly isolated mouse kidney slices were incubated with control solution or the dopamine D₁-like receptor agonist fenoldopam in the absence or presence of inhibitors of several signaling cascades for 1 h. Sections were stained for NaPi-IIa (green) and β-actin (red). Incubation with control solution (A) did not induce NaPi-IIa internalization, whereas incubation with fenoldopam did (B). Coincubation of fenoldopam with the PKA inhibitor H-89 (C) abolished internalization of NaPi-IIa. The PKC inhibitor chelerythrine (D) or the ERK1/2 MAPK kinase inhibitor PD-098089 (E) did not prevent fenoldopam-induced internalization. Scale bar, 10 µm.

Fig. 6

Dopamine-induced internalization of NaPi-IIa in opossum kidney (OK) cells. OK cells were transiently transfected with NaPi-IIa with a COOH-terminal enhanced green fluorescent protein (EGFP) tag. At 48 h after transfection, vehicle or dopamine was added for 30 min. Cells were fixed, labeled with rhodamine, and visualized with laser confocal microscopy (red = actin; green = NaPi-IIa/EGFP). Two representative cells from vehicle- or dopamine-treated cells are shown from 2 independent experiments.