Regulation of renal proximal tubule Na-K-ATPase by prostaglandins

Abstract

Prostaglandins (PGs) play a number of roles in the kidney, including regulation of salt and water reabsorption. In this report, evidence was obtained for stimulatory effects of PGs on Na-K-ATPase in primary cultures of rabbit renal proximal tubule (RPT) cells. The results of our real-time PCR studies indicate that in primary RPTs the effects of PGE(2), the major renal PG, are mediated by four classes of PGE (EP) receptors. The role of these EP receptors in the regulation of Na-K-ATPase was examined at the transcriptional level. Na-K-ATPase consists of a catalytic α-subunit encoded by the ATP1A1 gene, as well as a β-subunit encoded by the ATP1B1 gene. Transient transfection studies conducted with pHβ1-1141 Luc, a human ATP1B1 promoter/luciferase construct, indicate that both PGE(1) and PGE(2) are stimulatory. The evidence for the involvement of both the cAMP and Ca(2+) signaling pathways includes the inhibitory effects of the myristolylated PKA inhibitor PKI, the adenylate cyclase (AC) inhibitor SQ22536, and the PKC inhibitors Gö 6976 and Ro-32-0432 on the PGE(1) stimulation. Other effectors that similarly act through cAMP and PKC were also stimulatory to transcription, including norepinephrine and dopamine. In addition to its effects on transcription, a chronic incubation with PGE(1) was observed to result in an increase in Na-K-ATPase mRNA levels as well as an increase in Na-K-ATPase activity. An acute stimulatory effect of PGE(1) on Na-K-ATPase was observed and was associated with an increase in the level of Na-K-ATPase in the basolateral membrane.

Fig. 1.

Effects of prostaglandins on transcription. Primary renal proximal tubule (RPT) cells were transfected with pHβ1-1141 Luc and pSVβgal, as described in materials and methods. A: effect of PGE₁ and 8-Br-cAMP. Transfected primary RPT cells were incubated for 18 h in either DMEM/F12 supplemented with 5 μg/ml insulin and 5 μg/ml transferrin (control), control medium with 1.4 μM PGE₁, or control medium with 1 mM 8-Br-cAMP. B: effect of 1.4 μM PGE₁, PGE₂, and PGF_2α. Primary RPT cells were incubated for 18 h either in control medium or in control medium further supplemented with 0.14 μM PGE₁, PGE₂, or PGF_2α. C: effect of PGE₂ concentration. Transfected primary RPT cells were incubated for 18 h with PGE₂ at concentrations ranging from 0 to 1.4 μM. At the end of the 18-h incubation, the cultures were lysed. After the luciferase and β-galactosidase activity was determined in each cell lysate, the luciferase activity was standardized for transfection efficiency. In each condition, the fold-stimulation of luciferase activity was compared with the control. Values are averages ± SE of quadruplicate determinations. *P < 0.05 vs. control.

Fig. 2.

Expression of EP receptors in RPT Cells. A: expression of EP1, EP2, EP3, and EP4 receptor mRNA in primary RPT cells was compared with the levels in purified RPTs as described in materials and methods. B: expression of EP1, EP2, EP3, and EP4 receptor mRNA in primary RPT cells and purified RPTs was compared with the levels in the medulla. Values are averages ± SE of triplicate determinations.

Fig. 3.

Effects of EP2 and EP4 receptor agonists and antagonists on transcription. A: effects of butaprost and PGE₁-OH. B: effects of 0.28 μM PGE₁, PGE₂, and 16–16 dimethyl PGE₂. C: effect of L161, 982 and MF191. D: effect of PKI and SQ22536. Values are the averages ± SE of quadruplicate determinations. *P < 0.05.

Fig. 4.

Effects of EP1 agonists and antagonists on transcription. A: effect of 17-phenyltrinor PGE₂. B: effect of 1 μM AH 6809 and 1 μM SC51089 either in the presence or absence of 1.4 μM PGE₁. *P < 0.05 vs. control.

Fig. 5.

Effects of 12-O-tetradecatetradecanoyl phorbol 12-myristate-13 acetate (TPA) and PKC inhibitors on transcription. A: effect of TPA. Primary RPT cell cultures were incubated for 4 h with either 0, 10⁻⁹ M, 10⁻⁸ M, or 10⁻⁷ M TPA. B: effect of Gö 6976 on PGE₁ stimulation. Primary RPT cell cultures were incubated for 4 h with either 10⁻⁹ M TPA, 1.4 μM PGE₁, 1.4 μM PGE₁+Gö 6976, 10⁻⁹ M TPA+40 nM Gö 6976, or no further supplement (control). C: effect of Ro-32-0432 on PGE₁ Stimulation. Primary RPT cells were incubated for 4 h with either 1.4 μM PGE₁, 10⁻⁹ M TPA, 25 nM Ro-32-0432+1.4 μM PGE₁, 25 nM Ro-32–0432+10⁻⁹ M TPA, 25 nM Ro-32-0432, or no further supplement (control). *Differences were statistically significant when P < 0.05.

Fig. 6.

Effect of agents that modulate Ca²⁺ metabolism on transcription. Primary RPT cell cultures were transiently transfected as described in materials and methods. Subsequently, effects of agents that modulate Ca²⁺ metabolism were examined. A: effect of ionomycin and BAPTA-AM. Primary RPT cell cultures were incubated for 4 h either with DMEM/F12 supplemented with 5 μg/ml insulin and 5 μg/ml transferrin (control) or in the control condition further supplemented with either 1 μM ionomycin, 2.5 × 10⁻⁵ M BAPTA-AM individually, or ionomycin and BAPTA in combination. B: effects of TMB-8 and ionomycin. Primary RPT cell cultures were incubated for 19 h with either 10⁻⁶ M TMB-8 or 1 μM ionomycin. *Significantly different from the control, P < 0.05.

Fig. 7.

Effects of arachidonic acid (AA) and its metabolites on transcription. A: effect of AA concentration. Primary RPT cells were transiently transfected with pHβ1-1141 Luc and pSVβgal. Subsequently, the transiently transfected primary RPT cells were incubated for 18 h with AA at concentrations ranging from 10⁻¹⁰ to 10⁻⁷ M. B: effect of indomethacin. Primary RPT cells were preincubated for 30 min in either the presence or absence of 10⁻⁶ M indomethacin. Subsequently, the cultures were incubated for 18 h with either 10⁻⁹ M AA, 10⁻⁹ M AA with 10 ⁻⁶ M indomethacin, 10⁻⁶ M indomethacin alone, 1.4 μM PGE₂, or with no further supplement (control). Cultures were lysed, and luciferase determinations were made as described in the legend to Fig. 1. *Differences were statistically significant when P < 0.05.

Fig. 8.

Effect of 20-HETE and 5,6-EET on transcription. A: effect of 20-HETE. Primary RPT cells were incubated for 4 h with either 1 μM 20-HETE, 1.4 μM PGE₁, 1 mM 8-Br-cAMP, or control (no further supplement). B: effect of 5,6-EET. Primary RPT cells were treated with either 1 μM 5,6 EET, 0.070 μM PGE₁, or 0.70 μM PGE₁ for 4 h. Values were significantly higher than control when *P < 0.05 or lower than the control when **P < 0.05 vs. AA.

Fig. 9.

Effect of norepinephrine and dopamine on transcription. Primary RPT cells were transiently transfected with pHβ1-1141 Luc and pSVβgal, after either 3 or 8 days in culture, as described in materials and methods. Subsequently, the primary cultures were incubated for 4 h in control medium supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 10⁻⁶ M indomethacin, and either 10⁻⁶ M norepinephrine, 10 μM dopamine, or no further supplement. Cells were lysed, and luciferase activity was determined as described in the legend to Fig. 1. Values are the averages ± SE of quadruplicate determinations. Four independent experiments were conducted.

Fig. 10.

Chronic and acute effects of PGE₁ on the Na-K-ATPase. A: chronic effect of PGE₁ on the level of the Na-K-ATPase in primary RPT cell cultures. Primary RPT cell cultures were grown to confluence in Medium RK-1 in the presence of 10 ⁻⁶ M indomethacin. Subsequently, the cultures were maintained for 4 days in Medium RK-1 containing 10⁻⁶ M indomethacin further supplemented in either the presence or absence of 0.7 μM PGE₁. The medium was changed daily. At the end of the 4-day incubation, the level of the α- and β-subunits of the Na-K-ATPase was determined by Western blot analysis. The level of α- and β-subunit mRNA was determined by real-time PCR. A: blots of the α- and β-subunits as well as β-actin are illustrated. B: results of scanning densitometry of α- and β-blots. Values are averages of duplicate determinations. C: results of determinations of α- and β-subunit mRNA are shown. Values are averages ± SE of triplicate determinations. D: initial rate of Rb⁺ uptake was determined in both the presence and absence of 1 mM ouabain. Before the uptake study, the primary RPT monolayers were maintained for 4 days in DMEM/F12 supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 10⁻⁶ M indomethacin, and either 0.7 μM PGE₁ or no further supplement (control). The difference between total Rb⁺ uptake, and ouabain-insensitive Rb⁺ uptake (the ouabain-sensitive component) was then calculated. Values are averages ± SE of triplicate determinations. E: activity of the Na-K-ATPase in cell lysates was determined in primary RPTs monolayers which were maintained for 4 days in DMEM/F12 supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 10⁻⁶ M indomethacin, and either 0.35 μM PGE₁ and 0.125 mM IBMX, 0.125 mM IBMX alone, or no further supplement (control). Values are averages ± SE of triplicate determinations.

Fig. 11.

Acute effect of PGE₁ on the α-subunit. A: primary RPT cells on Transwell membranes were cultured in Medium RK-1 containing 1 μM indomethacin. Following a 30-min incubation with either 1.4 μM PGE₁, 50 μM butaprost, or 50 μM 17-phenyltrinor PGE₂, the basolateral surface of the primary RPT cells was biotinylated. Samples were precipitated using streptavidin beads, separated by SDS-PAGE, and transferred to nitrocellulose. The level of the α-subunit of the Na-K-ATPase was determined by Western blot analysis, as described in materials and methods. B: primary RPT cells in 35-mm dishes were cultured in Medium RK-1 containing 1 μM indomethacin. The monolayers were incubated for 30 min at 23°C with either 1.4 μM PGE₁, 0.125 mM IBMX, 1.4 μM PGE₁+0.125 mM IBMX, or no further supplement (control), followed by a 15-min ⁸⁶Rb⁺ uptake period, as described in materials and methods. Values are averages of triplicate determinations ± SE.