Rajatsubhra Biswas

Cooperativity between the Phosphorylation of Thr95 and Ser77 of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport

The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser77 in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr95, another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser77 by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr95. These studies indicate a remarkable cooperativity between the phosphorylation of Thr95 and Ser77 of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr95 facilitates the phosphorylation of Ser77. This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.

Fibroblast Growth Factor-23-mediated Inhibition of Renal Phosphate Transport in Mice Requires Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1) and Synergizes with Parathyroid Hormone

Background: FGF-23 and PTH inhibit phosphate transport in renal proximal tubules. Results: FGF-23-mediated inhibition requires the PDZ-domain adaptor protein NHERF-1 and synergizes with PTH. Conclusion: FGF-23 and PTH induce modifications of NHERF-1 by different but complementary pathways. Significance: The interaction between FGF-23 and PTH on renal phosphate transport may provide a therapeutic rational for treatment of patients with FGF-23-associated hypophosphatemia. Fibroblast growth factor-23 (FGF-23) inhibits sodium-dependent phosphate transport in brush border membrane vesicles derived from hormone-treated kidney slices of the mouse and in mouse proximal tubule cells by processes involving mitogen-activated protein kinase (MAPK) but not protein kinase A (PKA) or protein kinase C (PKC). By contrast, phosphate transport in brush border membrane vesicles and proximal tubule cells from sodium-hydrogen exchanger regulatory factor-1 (NHERF-1)-null mice were resistant to the inhibitory effect of FGF-23 (10−9 m). Infection of NHERF-1-null proximal tubule cells with wild-type adenovirus-GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of FGF-23. Infection with adenovirus-GFP-NHERF-1 containing a S77A or T95D mutation also increased basal phosphate transport, but the cells remained resistant to FGF-23 (10−9 m). Low concentrations of FGF-23 (10−13 m) and PTH (10−11 m) individually did not inhibit phosphate transport or activate PKA, PKC, or MAPK. When combined, however, these hormones markedly inhibited phosphate transport associated with activation of PKC and PKA but not MAPK. These studies indicate that FGF-23 inhibits phosphate transport in the mouse kidney by processes that involve the scaffold protein NHERF-1. In addition, FGF-23 synergizes with PTH to inhibit phosphate transport by facilitating the activation of the PTH signal transduction pathway.

Sodium-Hydrogen Exchanger Regulatory Factor 1 (NHERF-1) Transduces Signals That Mediate Dopamine Inhibition of Sodium-Phosphate Co-transport in Mouse Kidney

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in 32P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

Increased renal dopamine and acute renal adaptation to a high-phosphate diet

The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.

Role of NHERF and scaffolding proteins in proximal tubule transport

Eukaryotic cells coordinate specific responses to hormones and growth factors by spatial and temporal organization of “signaling components.” Through the formation of multiprotein complexes, cells are able to generate “signaling components” that transduce hormone signals through proteins, such as PSD-95/Dlg/ZO-1(PDZ)-containing proteins that associate by stable and dynamic interactions. The PDZ homology domain is a common protein interaction domain in eukaryotes and with greater than 500 PDZ domains identified, it is the most abundant protein interaction domain in eukaryotic cells. The NHERF (sodium hydrogen exchanger regulatory factor) proteins are PDZ domain-containing proteins that play an important role in maintaining and regulating cell function. NHERF-1 was initially identified as a brush border membrane-associated phosphoprotein essential for the cAMP/PKA-induced inhibition of the sodium hydrogen exchanger isoform 3 (NHE3). Mouse, rabbit and human renal proximal tubules also express NHERF-2 (E3KARP), a structurally related protein, which in model cell systems also binds NHE3 and mediates its inhibition by cAMP. PDZK1 (NHERF-3) and IKEPP (NHERF-4) were later identified and found to have similar homology domains, leading to their recent reclassification. Although studies have revealed similar binding partners and overlapping functions for the NHERF proteins, it is clear that there is a significant amount of specificity between them. This review focuses primarily on NHERF-1, as the prototypical PDZ protein and will give a brief summary of its role in phosphate transport and the development of some forms of nephrolithiasis.

Sodium-Hydrogen Exchanger Regulatory Factor-1 Interacts with Mouse Urate Transporter 1 to Regulate Renal Proximal Tubule Uric Acid Transport

Sodium-hydrogen exchanger regulatory factor-1-deficient (NHERF-1(-/-)) mice demonstrate increases in the urinary excretion of phosphate, calcium, and uric acid associated with interstitial deposition of calcium in the papilla of the kidney. These studies examine the role of NHERF-1 in the tubular reabsorption of uric acid and regulation of mouse urate transporter 1 (mURAT1), a newly described transporter that is responsible for the renal tubular reabsorption of uric acid. In primary cultures of mouse renal proximal tubule cells, uric acid uptake was significantly lower in NHERF-1(-/-) cells compared with wild-type cells over a large range of uric acid concentrations in the media. Western immunoblotting revealed a 56 +/- 6% decrease in the brush border membrane (BBM) expression of mURAT1 in NHERF-1(-/-) compared with wild-type control kidneys (P < 0.05). Confocal microscopy confirmed the reduced apical membrane expression of mURAT1 in NHERF-1(-/-) kidneys and demonstrated mislocalization of mURAT1 to intracellular vesicular structures. Para-aminohippurate significantly inhibited uric acid uptake in wild-type cells (41 +/- 2%) compared with NHERF-1(-/-) cells (8.2 +/- 3%). Infection of NHERF-1(-/-) cells with adenovirus-green fluorescence protein-NHERF-1 resulted in significantly higher rates of uric acid transport (15.4 +/- 1.1 pmol/microg protein per 30 min) compared with null cells that were infected with control adenovirus-green fluorescence protein (7.9 +/- 0.3) and restoration of the inhibitory effect of para-aminohippurate (% inhibition 34 +/- 4%). These findings indicate that NHERF-1 exerts a significant effect on the renal tubular reabsorption of uric acid in the mouse by modulating the BBM abundance of mURAT1 and possibly other BBM uric acid transporters.

The ROMK potassium channel is present in mammalian urinary tract epithelia and muscle.

There is increasing evidence that mammalian urinary tract epithelial cells utilize membrane channels and transporters to transport solutes across their apical (luminal) and basalateral membranes to modify solute concentrations in both cell and urine. This study investigates the expression, localization, and regulation of the ROMK (K(ir) 1.1) potassium channels in rat and dog ureter and bladder tissues. Immunoblots of homogenates of whole ureter, whole bladder, bladder epithelial cells, and bladder smooth muscle tissues in both rat and dog identified approximately 45- to 50-kDa bands characteristic of ROMK in all tissues. RT-PCR identified ROMK mRNA in these same tissues in both animal species. ROMK protein localized by immunocytochemistry was strongly expressed in the apical membranes of the large umbrella cells lining the bladder lumen and to a lesser extent in the cytoplasm of epithelial cells and smooth muscle cells in the rat bladder. ROMK protein and mRNA were also discovered in cardiac, striated, and smooth muscle in diverse organs. There was no difference in immunoblot expression of ROMK abundance in bladder homogenates (whole bladder, epithelial cell, or muscle cell) or ureteral homogenates between groups of rats fed high- or low-potassium diets. Although the functional role of ROMK in urinary tract epithelia and smooth muscle is unknown, ROMK may participate in the regulation of epithelial and smooth muscle cell volume and osmolality, in the dissipation of potassium leaked or diffused from urine across the epithelial cell apical membranes or tight junctions, and in net or bidirectional potassium transport across urinary tract epithelia.