Parathyroid hormone (PTH) regulates the sodium chloride cotransporter via Ras guanyl releasing protein 1 (Ras-GRP1) and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway

Abstract

The sodium chloride cotransporter (NCC) is the principal salt absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of thiazide diuretics. Using a mammalian cell model system to assess NCC function, we demonstrated previously that Ras guanyl releasing protein 1 (Ras-GRP1) mediates phorbol ester-induced suppression of the function and surface expression of NCC in a protein kinase C (PKC)-independent and extracellular signal-regulated kinase (ERK)1/2-dependent manner. Given that phorbol esters are functional analogs of diacylglycerol (DAG), this finding suggested a potential physiologic regulation of NCC by DAG. The parathyroid hormone (PTH) receptor is a G-protein-coupled receptor that is expressed in the DCT and activates PLC resulting in the generation of DAG. In this article, we demonstrate that PTH suppresses NCC function via a PLC/Ras-GRP1/ERK pathway. A functional assessment of NCC measuring thiazide-sensitive (22)Na(+) flux revealed that PTH suppresses NCC function. The inhibition of PLC prevented the suppression of NCC, indicating that PLC was necessary for this effect. Inhibitors of PKC and protein kinase A (PKA) had no effect on this suppression, but mitogen-activated protein kinase (MAPK) inhibitors prevented the PTH effect completely. Ras-GRP1 activates the MAPK pathway though activation of the small G-protein Ras. Gene silencing of Ras-GRP1 prevented the PTH-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of Ras-GRP1 in mediating the PTH-induced suppression of NCC activity through stimulation of the MAPK pathway.

Figure 1. Effect of PTH on NCC Activity

A. mDCT cells were grown to confluence and then treated with 100nM PTH or vehicle (DMSO) for the indicated times. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01. B. mDCT cells grown as above were treated with the indicated concentrations of PTH for 15 minutes. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01.

Figure 2. Effect of PLC Inhibition on the PTH Effect on NCC Activity

mDCT cells were grown to confluence and then treated with 100nM PTH or vehicle (DMSO, Control) in the presence or absence of a PLC inhibitor, U73122 (PLCi), for 15 minutes. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01.

Figure 3. Effect of RasGRP1 gene silencing on PTH-mediated effects on NCC Activity

mDCT cells transfected as above were treated with 100nM PTH or vehicle (DMSO, Control) for 15 minutes. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01. Inset: mDCT cells were transfected with either non-targeting (NT) or siRNA specific to RasGRP1 (siRG). 48 hours after transfection, cells were lysed and immunoblotted for RasGRP1. Blot shown is representative of 4 experiments.

Figure 4. Effect of ERK1/2, PKC, and PKA Inhibition on PTH-medaited effects on NCC Activity

A. mDCT cells were grown to confluence and then treated with 100nM PTH or vehicle (DMSO, Control) in the presence or absence of an MEK1/2 inhibitor, U0126, for 15 minutes. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01. B. mDCT cells were grown to confluence and then treated with 100nM PTH or vehicle (DMSO, Control) in the presence or absence of either the PKC inhibitors BIM (B, B+T) and Gö6976 (G, G+T), the PKA inhibitor H89 (H, H+T) or control (DMSO vehicle, C) for 15 minutes. Cells were subsequently incubated in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. n=6, * p<0.01.

Figure 5. Ras Activation in PTH-Treated mDCT Cells After Gene-Silencing of RasGRP1

mDCT cells transfected with siRNA for RasGRP1 (siRG) or non-targeting siRNA (NT) were treated for 15 minutes with 100nM PTH or vehicle (DMSO). Cells were then lysed and a Ras-GTP pulldown using the Raf binding domain was performed, followed by immunoblotting for total Ras or H-Ras (21kD). Shown is a representative immunoblot and densitometry (normalized to total Ras). n=4 *p<0.05 as compared to NT.

Figure 6. ERK Phosphorylation in mDCT Cells Treated with PTH

A. mDCT cells grown as above were treated with 100nM PTH or vehicle (DMSO) for 15 minutes in the presence or absence of a MEK inhibitor (U), PKC inhibitor (B), or PKA inhibitor (H). Cells were then lysed and immunoblotting for phosphorylated or total ERK1/2 was performed. ERK1 appears at 44kD. ERK2 appears at 42kD. n=4, * p<0.05 as compared to control. B. mDCT cells transfected with siRNA for RasGRP1 (siRG) or non-targeting siRNA (NT) were treated for 15 minutes with 100nM PTH or vehicle (DMSO). Cells were then lysed and immunoblotting for phosphorylated or total ERK1/2 was performed. ERK1 appears at 44kD. ERK2 appears at 42kD. n=4, * p<0.05 as compared to NT.