Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 μM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.
In primary hyperparathyroidism, chronically elevated levels of PTH produce low serum phosphorus through inhibition of proximal tubule expression of sodium‐phosphate cotransporters. We have previously shown that chronic PTH stimulation of proximal tubule cells is associated with decreased expression of NpT2a mRNA through destabilization, which is dependent on transcription. BLAST evaluation of the NpT2a promoter identified a CREB‐binding region. We hypothesize that PTH decreases NpT2a mRNA stability through activation of the PKA pathway. Opossum kidney (OK) cells were transfected with luciferase reporter gene constructs containing either full‐length or serially truncated NpT2a promoter regions. Luciferase reporter assays show no effect of PTH on the promoter function of the NpT2a gene. Direct activation of PKA with 8Br‐cAMP and PKC with PMA for 2h produced a decrease in NpT2a mRNA of 35.6 ± 12.2% and 28.9 ± 4.3%, respectively. We conclude that PTH decreases NpT2a mRNA expression through destabilization of NpT2a mRNA, and that both PKA and PKC pathways may contribute to this effect. Support for this project is provided by VA to EDL.
Under acute experimental conditions, PTH decreases expression of Npt2a (sodium phosphate cotransporter), NHE3 (sodium hydrogen exchanger isoform 3), and Na/K‐ATPase. In contrast, patients with primary hyperparathyroidism (HPTH) frequently have low serum phosphate but infrequently show metabolic acidosis or volume depletion. We hypothesize that acute and chronic PTH stimulation results in differential regulation of proximal tubule (PT) ion transporters. Using a transgenic mouse model that gradually develops HPTH due to parathyroid‐targeted cyclin D1 over‐expression, we examined the expression of Npt2a, Na/K‐ATPase, sodium‐hydrogen exchanger regulatory factor‐1 (NHERF‐1), and the PTH receptor (PTHr). In comparison with control mice, chronic HPTH resulted in sustained decreased expression of Npt2a, whereas expression of Na/K‐ATPase α subunit increased, contrary to what has been shown in acute conditions. The transgenic model showed no discernible difference in the expression of either NHERF‐1 or PTHr when compared to the control mice. We conclude that PTH differentially regulates PT ion transporters in acute versus chronic conditions through as yet unidentified mechanisms. We suggest that PTH regulation of transepithelial sodium transport undergoes desensitization while regulation of phosphate transport does not. Support for this project is provided by VA.
NpT2a, a proximal tubule apical membrane (AM) protein, plays a critical role in regulation of phosphate homeostasis. We and others have shown that 1) NpT2a is a glycosylated protein, 2) glycosylation is required for translocation to AM, and 3) NHERF1 deficiency causes aberrant AM expression. We hypothesized that NHERF1 associates with NpT2a prior to AM insertion and facilitates glycosylation. To test this hypothesis, we examined NpT2a‐NHERF1 association in Golgi (G) by density gradient centrifugation of opossum kidney cells. Cells transfected with GFP‐NpT2a at 16C showed retention of NpT2a in the G fractions while cells in 37C showed plasma membrane (PM) localization. NHERF1 was present in both G and PM fractions at both temperatures. NpT2a IP from G and PM fractions showed association with NHERF1. GFP‐NpT2a lacking the terminal – TRL motif lacked glycosylation and failed to associate with NHERF1 or traffic to the AM. Confocal imaging confirmed failure of the TRL deletion mutant to localize to the AM. We conclude that NpT2a associates with NHERF1 in the G through the C‐terminal PDZ binding domain and that this association is essential for NpT2a glycosylation and AM trafficking. Supported by VA
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