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Na+/H+ exchange regulatory factor 1 (NHERF1), a member of a PDZ scaffolding protein family, was first identified as an organizer of membrane-bound protein complexes composed of hormone receptors, signal transduction pathways, and electrolyte and mineral transporters and channels. NHERF1 is involved in the regulation of Na+/H+ exchanger 3, Na+-dependent phosphate transporter 2a, and Na+-K+-ATPase through its ability to scaffold these transporters to the plasma membrane, allowing regulation of these protein complexes with their associated hormone receptors. Recently, NHERF1 has received increased interest in its involvement in a variety of functions, including cell structure and trafficking, tumorigenesis and tumor behavior, inflammatory responses, and tissue injury. In this review, we highlight the evidence for the expansive role of NHERF1 in cell biology and speculate on the implications for renal physiology and pathophysiology.

Na+/H+exchanger regulatory factor (NHERF1) plays a critical role in the renal transport of phosphate by binding to Na+-Picotransporter (NpT2a) in the proximal tubule. While the association between NpT2a and NHERF1 in the apical membrane is known, the role of NHERF1 to regulate the trafficking of NpT2a has not been studied. To address this question, we performed cell fractionation by sucrose gradient centrifugation in opossum kidney (OK) cells placed in low-Pimedium to stimulate forward trafficking of NpT2a. Immunoblot analysis demonstrated expression of NpT2a and NHERF1 in the endoplasmic reticulum (ER)/Golgi. Coimmunoprecipitation demonstrated a NpT2a-NHERF1 interaction in the ER/Golgi. Low-Pimedium for 4 and 8 h triggered a decrease in NHERF1 in the plasma membrane with a corresponding increase in the ER/Golgi. Time-lapse total internal reflection fluorescence imaging of OK cells placed in low-Pimedium, paired with particle tracking and mean square displacement analysis, indicated active directed movement of NHERF1 at early and late time points, whereas NpT2a showed active movement only at later times. Silence of NHERF1 in OK cells expressing green fluorescent protein (GFP)-NpT2a resulted in an intracellular accumulation of GFP-NpT2a. Transfection with GFP-labeled COOH-terminal (TRL) PDZ-binding motif deleted or wild-type NpT2a in OK cells followed by cell fractionation and immunoprecipitation confirmed that the interaction between NpT2a and NHERF1 was dependent on the TRL motif of NpT2a. We conclude that appropriate trafficking of NpT2a to the plasma membrane is dependent on the initial association between NpT2a and NHERF1 through the COOH-terminal TRL motif of NpT2a in the ER/Golgi and requires redistribution of NHERF1 to the ER/Golgi.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na+-K+-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-Picotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.

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 86Rb 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 86Rb 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.

Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.

We previously showed an inverse correlation between membrane cholesterol content and Na-Picotransport activity during the aging process and adaptation to alterations in dietary Piin the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-Picotransport activity and apical membrane Na-Piprotein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with β-methyl cyclodextrin (β-MCD) resulted in a significant increase in Na-Picotransport activity accompanied by a moderate increase in apical membrane Na-Piprotein abundance and no alteration of total cellular Na-Piprotein abundance. Conversely, acute cholesterol enrichment achieved with β-MCD/cholesterol resulted in a significant decrease in Na-Picotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-Piprotein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-Picotransport activity and apical membrane and total cellular Na-Piprotein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-Picotransport activity and apical membrane and total cellular Na-Piprotein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-Picotransport activity by diverse mechanisms that also include significant interactions of Na-Piprotein with lipid microdomains.

Parathyroid hormone (PTH) and dopamine (DA) inhibit Na-K ATPase activity and sodium-phosphate cotransport in proximal tubular cells. We previously showed that PTH and DA inhibit phosphate transport in opossum kidney (OK) cells through different signaling pathways. Therefore, we hypothesized that PTH and DA also inhibit Na-K ATPase through divergent pathways. We measured PTH and DA inhibition of Na-K ATPase activity in the presence of inhibitors of signaling pathways. PTH and DA inhibited Na-K ATPase in a biphasic manner, the early inhibition through protein kinase C (PKC)- and phospholipase A2(PLA2)-dependent pathways and the late inhibition through protein kinase A- and PLA2-dependent pathways. Inhibition of extracellular signal-regulated kinase (ERK) activation blocked early and late inhibition of Na-K ATPase by PTH but not by DA. Pertussis toxin blocked early and late inhibition by DA but not by PTH. Treatment with DA, but not PTH, resulted in an early downregulation of basolateral membrane expression of the α-subunit, whereas total cellular expression remained constant for both agonists. We conclude that PTH and DA regulate Na-K ATPase by different mechanisms through activation of divergent pathways.

Purinergic P2 receptors are present on proximal renal tubules, but their function is unknown. Because P2 agonists antagonize vasopressin-stimulated water transport in the distal tubule by inhibiting activation of adenylyl cyclase, we postulated that P2 receptor activation blocks parathyroid hormone (PTH) inhibition of phosphate uptake in proximal tubule by preventing PTH-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation. PTH inhibition of sodium-dependent phosphate uptake was attenuated by alpha,beta-methylene-ATP (AMP-CPP), a P2x receptor agonist, but not by 2-methyl-thio-ATP, a P2y receptor agonist, in a dose-dependent manner. AMP-CPP did not attenuate inhibition of phosphate uptake produced by direct activation of adenylyl cyclase with forskolin, by addition of the cAMP analogue 8-bromo-cAMP, or by inhibition of cAMP phosphodiesterase with RO-20-1724. Additionally, AMP-CPP had no effect on basal or PTH-stimulated cAMP production. As PTH also stimulates protein kinase C activation, the effect of AMP-CPP on inhibition of phosphate uptake stimulated by phorbol 12-myristate 13-acetate (PMA) was tested. AMP-CPP had no effect on PMA-induced inhibition of phosphate uptake. Pretreatment with pertussis toxin abolished the attenuating effect of AMP-CPP on PTH inhibition of sodium-dependent phosphate uptake. We conclude that activation of purinergic P2 receptors attenuates the inhibitory effect of PTH on sodium-dependent phosphate uptake by a G protein-dependent mechanism that is independent of cAMP generation protein kinase A activation, or protein kinase C activation.

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H2S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H2S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H2S was measured by colorimetric chemical method. To enhance endogenous production of H2S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47phoxwas measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H2S was attenuated. H2S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47phoxwas attenuated by H2S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H2S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H2S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electronmicroscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the α1-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of previous studies, we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA2 did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA2 also prevented PTH-mediated phosphorylation of Na+-K+-ATPase α1-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na+-K+-ATPase activity by the PLA2 metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na+-K+-ATPase α1-subunit and PKCα, which was prevented by PLA2 inhibition. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA2, and PKCα.

Pages F309'F316: E. D. Lederer and K. R. McLeish. “P2 purinoceptor stimulation attenuates PTH inhibition of phosphate uptake by a G protein-dependent mechanism.” The American Heart Association of Kentucky should have been acknowledged for their support of E. D. Lederer.