Cunningham R, Biswas R, Brazie M, Steplock D, Shenolikar S, Weinman EJ. Signaling pathways utilized by PTH and dopamine to inhibit phosphate transport in mouse renal proximal tubule cells. Am J Physiol Renal Physiol 296: F355-F361, 2009. First published November 5, 2008 doi:10.1152/ajprenal.90426.2008.-The present experiments were designed to detail factors regulating phosphate transport in cultured mouse proximal tubule cells by determining the response to parathyroid hormone (PTH), dopamine, and second messenger agonists and inhibitors. Both PTH and dopamine inhibited phosphate transport by over 30%. The inhibitory effect of PTH was completely abolished in the presence of chelerythrine, a PKC inhibitor, but not by Rp-cAMP, a PKA inhibitor. By contrast, both chelerythrine and Rp-cAMP blocked the inhibitory effect of dopamine. Chelerythrine inhibited PTH-mediated cAMP accumulation but also blocked the inhibitory effect of 8-bromo-cAMP on phosphate transport. On the other hand, Rp-cAMP had no effect on the ability of DOG, a PKC activator, to inhibit phosphate transport. PD98059, an inhibitor of MAPK, had no effect on PTH-or dopamine-mediated inhibition of sodium-phosphate cotransport. Finally, compared with 8-bromo-cAMP, 8-pCPT-2Ј-O-Me-cAMP, an activator of EPAC, had no effect on phosphate transport. These results outline significant differences in the signaling pathways utilized by PTH and dopamine to inhibit renal phosphate transport. Our results also suggest that activation of MAPK is not critically involved in PTH-or dopaminemediated inhibition of phosphate transport in mouse renal proximal tubule cells in culture. parathyroid hormone; PKA; PKC IN RECENT EXPERIMENTS from this laboratory, we used primary cultures of mouse proximal tubule cells to study phosphate transport with emphasis on elucidating the role of adaptor proteins such as 15,30,33). From such studies, we provided evidence that parathyroid hormone (PTH) inhibits phosphate transport utilizing downstream signaling pathways involving activation of protein kinase C (PKC) and protein kinase A (PKA) (7, 8). We have not, however, detailed the role of these pathways in this tissue. This is important since there may be differences in second messenger signaling pathways between model cell systems (3,22). Moreover, hormones such as PTH have been demonstrated to interact with PTH1 receptors on both the apical and basolateral sides of renal proximal tubule cells and although the receptors are the same, signaling following the binding of PTH differs (5,19,26,31). Accordingly, in the present experiments, we employ inhibitors and activators of specific protein kinases to determine the role of PKC, PKA, and MAPK on both PTH-and dopaminemediated inhibition of phosphate transport in primary cultures of mouse proximal tubule cells. In addition, we contrasted the effect of cAMP with a specific activator of exchange protein activated by cAMP (EPAC) (10,16,27). METHODS Animals and preparation of renal proximal tubule cells.Male C57BL/6 mice 12 to 16 wk were used in the...

T he sodium-hydrogen exchanger regulatory factor (NHERF) proteins represent a family of proteins that are abundantly expressed in the apical membranes of transporting epithelia, such as the renal proximal convoluted tubule and small intestine. These adaptor proteins have multiple PSD-95/Dlg/ZO-1 (PDZ) protein interactive domains and, in the case of NHERF-1 and NHERF-2, a C-terminal domain that binds to ezrin, radixin, moesin, and merlin (1-4). In mice, inactivation of the NHERF-1 gene, the founding member of this family, results in increased urinary excretion of phosphate as a result of decreased membrane abundance of the sodium-dependent phosphate co-transporter 2a (Npt2a), hypercalciuria as a result of the reciprocating changes in the plasma concentrations of 1,25 (OH) 2 vitamin D, and the interstitial deposition of calcium primarily in the papilla of the kidney (5,6). We also observed that both male and female NHERF-1 Ϫ/Ϫ mice have an increase in the urinary excretion of uric acid, the mechanism of which is unknown (6). Accordingly, these experiments were designed to study the relation between NHERF-1 and the renal transport of uric acid. In cultured proximal tubule cells, we demonstrate a decrease in the uptake of uric acid NHERF-1 null cells. This was associated with a decrease in the brush border membrane (BBM) abundance of mURAT1 (7,8). Although mouse URAT1 (mURAT1) does not transport para-aminohippurate (PAH), our studies indicate that uric acid transport was inhibited by PAH in cultured wild-type proximal tubule cells but not in NHERF-1 null cells. Rescue of NHERF-1 null cells using adenovirus-mediated gene transfer significantly increased uric acid transport and restored the inhibitory effect of PAH. When considered together, these findings indicate an important role for NHERF-1 in regulating uric acid transport in renal proximal tubule cells and provide evidence that NHERF-1 interacts with mURAT1 as well as other uric acid transporters. Materials and MethodsMale NHERF-1 Ϫ/Ϫ mice (F.129-Slc9a3r1 tmSsl /Ssl) that were bred into a C57BL/6 background for six generations and parental wild-type

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 (Kir 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 ϳ45-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. epithelial sodium channel; calponin MAMMALIAN URINARY TRACT EPITHELIA (urothelia) has long been held to serve solely as a storage conduct for urine made by the kidney and, in particular, to prevent modification of urine by inhibiting net flux of water and solutes across the epithelial cell luminal membrane (7). The barrier(s) preventing transepithelial flux of urine constituents have been shown to include: the lumenal membrane (containing unique uroplakin proteins) of the large ("umbrella") cells lining the urinary tract lumen (12), tight junctions adjoining the umbrella cells, and a glycosaminoglycans layer overlying and adjacent to the lumenal membrane (13, reviewed in Ref. 17). Evidence for the effectiveness of these barriers, which have been thought to be passive in nature and not to utilize or require membrane transporters, comes from Ussing chamber studies documenting very low urothelial permeability to water, ions, and nonelectrolytes and very high transepithelial cell resistance in stretched bladders and apical membrane endosomes obtained from rabbits under basal conditions (2, 24).However, although the permeabilities of urothelia for water and various urinary solutes may be low under basal conditions, urothelial permeabilities in animals subjected to dietary, physiological (e.g., water loading/water restricted), or other stress could be altered. Furthermore, urothelial cells are...