Type IIa Sodium-phosphate Cotransporter Research Articles

Dietary citric acid improves growth performance, feed utilization, phosphorus utilization, lateral scute hardness, and duodenal microstructure in juvenile hybrid sturgeon (Acipenser baerii ♀ × A. schrenckii ♂)

This study was performed to evaluate the feasibility of using citric acid (CA) as a feed supplement to improve phosphorus (P) utilization by juvenile hybrid sturgeon (19.75 ± 0.13 g). Four isophosphoric (1.00% total P) diets containing 0.5%, 1.0%, 2.0%, 4.0% CA were formulated (1.0% P-0.5% CA, 1.0% P-1% CA, 1.0% P-2% CA, 1.0% P-4% CA). A normal P (1.00% total P) diet and a high P (1.40% total P) diet without CA addition were designed as a negative control (1.0% P-0% CA) and a positive control (1.4% P-0% CA), respectively. A commercial diet (1.48% total P) was applied as a reference group (1.4% P-commercial). The seven diets were randomly allocated to 21 tanks with 15 fish per tank. The fish were hand fed to apparent satiation three meals daily for 8 weeks. The results manifested that significantly improved growth performance (final body weight, weight gain rate) and feed utilization (feed conversion ratio, protein efficiency ratio) were discovered in 1.0% P-0.5% CA group compared with two control groups. The P contents in dorsal muscle, vertebrae, and serum were significantly higher in 1.0% P-1% CA group compared with negative control. Additionally, the P retention rate in 1.0% P-0.5% CA and 1.0% P-1% CA groups were significantly higher than 1.4% P-0% CA group. Dietary addition of 0.5% and 1.0% CA significantly improved hardness of lateral scute compared with negative control. Compared with the negative control, the serum alkaline phosphatase activity was significantly improved by addition of 0.5% and 1.0% CA. Supplementation with 0.5% CA significantly elevated duodenal micromorphology (villi height, muscularis thickness, lamina propria width) compared with negative control. The duodenal type IIa sodium phosphate cotransporter (NaPi-IIa) mRNA level was significantly up-regulated by addition of 1.0% CA compared with two control groups. In a nutshell, CA could be a suitable feed supplement in sturgeon diet and dietary supplementation of 0.5% CA improved growth performance, feed utilization, P utilization efficiency, lateral scute hardness, and duodenal micromorphology.

High-fat diets provoke phosphorus absorption from the small intestine in rats.

The ratio of dietary carbohydrate to fat may affect phosphorus metabolism because both calcium and phosphorus are regulated by similar metabolic mechanisms, and a high-fat diet (HFD) induces deleterious effects on the absorption of dietary calcium. We hypothesized that an HFD induces an increase in phosphorus absorption. The aim of this study was to evaluate the effects of differences in the quantity and quality of dietary fat on phosphorus metabolism over the short- and long-term. Eighteen 8-wk-old Sprague-Dawley male rats were fed an isocaloric diet containing varied ratios of carbohydrates to fat energy and sources of fat (control diet, HFD, and high- saturated fat diet [HF-SFA]). At 3 d and 7 wk after the allocation and initiation of the test diets, feces and urine were collected and used for phosphorus and calcium measurement. The fecal phosphorous concentration (F-Pi) was lower in the HF-SFA group than in the other two groups; however, the urine phosphorus concentration (U-Pi) was significantly higher in the HF-SFA group than the other two groups when the rats were fed over the short- (P < 0.01) and long -term (P < 0.01 versus control, P < 0.05 versus HFD group). There were no significant differences in type-IIa sodium-phosphate cotransporter (NaPi-2 a) and type-IIc sodium-phosphate cotransporter (NaPi-2 c) mRNA expression, which are renal phosphate transport-related genes; however, the expression of type-IIb sodium-phosphate cotransporter (NaPi-2 b) and type-III sodium-phosphate cotransporter (Pit-1) mRNA in the duodenum was higher in the HFD and HF-SFA groups than in the control group (P < 0.05), although there were no significant differences in these in the jejunum. The present results indicated that an HFD, particularly HF-SFA, increases intestinal phosphate absorption compared with control.

Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1

Background/Aims: Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels. Methods: Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter. Results: Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation. Conclusion: NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.

Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.

Parathyroid hormone (PTH) is a key regulator of the expression and function of the type IIa sodium-phosphate cotransporter (Npt2a), the protein responsible for regulated renal phosphate reabsorption. We previously showed that PTH induces rapid decay of Npt2a mRNA through posttranscriptional mechanisms. We hypothesized that PTH-induced changes in RNA-binding protein (RBP) activity mediate the degradation of Npt2a mRNA. To address this aim, we treated opossum kidney (OK) cells, a PTH-sensitive proximal tubule cell culture model, with 100 nM PTH for 30 min and 2 h, followed by mass spectrometry characterization of the PTH-stimulated phosphoproteome. We identified 1,182 proteins differentially phosphorylated in response to PTH, including 68 RBPs. Preliminary analysis identified a phospho-RBP, hnRNPK-homology-type-splicing regulatory protein (KSRP), with predicted binding sites for the 3'-untranslated region (UTR) of Npt2a mRNA. Western blot analysis confirmed expression of KSRP in OK cells and showed PTH-dependent translocation to the nucleus. Immunoprecipitation of KSRP from control and PTH-treated cells followed by RNA isolation and RT-quantitative PCR analysis identified Npt2a mRNA from both control and PTH-treated KSRP pulldowns. Knockdown of KSRP followed by PTH treatment showed that KSRP is required for mediating PTH-stimulated reduction in sodium/hydrogen exchanger 3 mRNA, but not Npt2a mRNA. We conclude that 1) PTH is a major regulator of both transcription and translation, and 2) KSRP binds Npt2a mRNA but its role in PTH regulation of Npt2a mRNA is not clear.

Low Phosphate Stimulates Na‐H Exchanger Regulatory Factor I (NHERF1) Trafficking

NHERF1 anchors the type IIa sodium phosphate cotransporter (Npt2a) in the brush border membrane (BBM). We have shown that Npt2a associates with NHERF1 in the ER/Golgi and that low phosphate, a stimulus for Npt2a exocytosis, causes a transient decrease in BBM NHERF1 expression in OK cells, a model of proximal tubule. We hypothesized that low phosphate stimulates trafficking of NHERF1 between BBM and Golgi. To address this hypothesis, OK cells placed in low phosphate (0.1 mmol) medium for 4 or 8h were fractionated and blotted for NHERF1 and Npt2a. NHERF1 BBM expression progressively decreased while ER/Golgi expression increased at 4 and 8 hours. Npt2a expression increased at all times in the BBM. Using total internal reflection microscopy, plasma membrane (PM) expression of GFP-tagged Npt2a and mCherry-tagged NHERF1 showed progressive increase in Npt2a but cyclic variations in NHERF1 PM expression over 4h of low phosphate treatment. Particle tracking averages within the PM showed 0.02 NHERF1 trajectories and 0.034 Npt2a trajectories/min/μm2, corresponding to 0.84 NHERF1 and 1.28 Npt2a particles/min (filtered for 50 frame persistence of particle signal). We conclude that low phosphate exerts a differential effect on NHERF1 and Npt2a trafficking. NHERF1 traffics dynamically in and out of the PM and to the Golgi in response to low phosphate while Npt2a exhibits greater movement within PM and progressively accumulates in the PM. These findings are consistent with a chaperone function for NHERF1 in Npt2a exocytosis. Support: VA, University of Louisville

KSRP and Roquin‐2: Mediators of PTH‐Induced Npt2a mRNA Destabilization?

Parathyroid hormone (PTH) potently induces phosphaturia through the downregulation of the type IIa sodium-phosphate cotransporter (Npt2a), at the protein and the mRNA level. We previously identified 68 RNA-binding proteins (RBPs) whose phosphorylation status significantly changed in response to 30m or 2h PTH. Preliminary in silico analysis predicts that two of those RBPs – Roquin-2 and KSRP – are capable of interacting with the 3'UTR of Npt2a mRNA. We hypothesize that PTH stimulates Npt2a mRNA degradation through alterations in Roquin-2 and KSRP activity and/or expression. To address this hypothesis, we examined Roquin-2 and KSRP expression in opossum kidney (OK) cells, a proximal tubule cell culture model, treated with 100nM PTH for 15m to 2h. Western blot demonstrated a time-dependent increase in expression of both proteins in response to PTH and a decrease in expression in response to low phosphate media. Nuclear expression of KHSRP increased 10-fold following 2h PTH. Nuclear expression of Roquin-2 increased 207% after 2h PTH, and cytosolic expression increased 342%. Immunoprecipitation of either Roquin-2 or KSRP from control and PTH-treated cells was followed by RNA isolation. RT-qPCR analysis identified Npt2a mRNA from both Roquin-2 and KSRP pulldowns. We conclude that KSRP and Roquin-2 bind Npt2a mRNA and their expression is modulated by known regulators of Npt2a expression. These findings suggest that KSRP and Roquin-2 potentially mediate Npt2a mRNA destabilization in response to PTH. Funding for this research provided by VA to EDL.

Role of PTH in the Renal Handling of Phosphate

Parathyroid hormone (PTH) is one of the primary phosphaturic hormones in the body. The type IIa sodium-phosphate cotransporter (Npt2a) is expressed in the apical membrane of the renal proximal tubule and is responsible for the reabsorption of the majority of the filtered load of phosphate. PTH acutely induces phosphaturia through the rapid stimulation of endocytosis of Npt2a and its subsequent lysosomal degradation. This review focuses on the homeostatic mechanisms underlying serum phosphate, with particular focus on the regulation of the phosphate transporter Npt2a by PTH within the renal proximal tubule. Additionally, the proximal tubular PTH-stimulated signaling events as they relate to PTH-induced phosphaturia are also highlighted. Lastly, we discuss recent findings by our lab concerning novel regulatory mechanisms of PTH-mediated reductions in Npt2a expression.

Aldosterone regulates Na+, K+ ATPase activity in human renal proximal tubule cells through mineralocorticoid receptor

The mechanisms by which aldosterone increases Na+, K+ ATPase and sodium channel activity in cortical collecting duct and distal nephron have been extensively studied. Recent investigations demonstrate that aldosterone increases Na–H exchanger-3 (NHE-3) activity, bicarbonate transport, and H+ ATPase in proximal tubules. However, the role of aldosterone in regulation of Na+, K+ ATPase in proximal tubules is unknown. We hypothesize that aldosterone increases Na+, K+ ATPase activity in proximal tubules through activation of the mineralocorticoid receptor (MR). Immunohistochemistry of kidney sections from human, rat, and mouse kidneys revealed that the MR is expressed in the cytosol of tubules staining positively for Lotus tetragonolobus agglutinin and type IIa sodium-phosphate cotransporter (NpT2a), confirming proximal tubule localization. Adrenalectomy in Sprague–Dawley rats decreased expression of MR, ENaC α, Na+, K+ ATPase α1, and NHE-1 in all tubules, while supplementation with aldosterone restored expression of above proteins. In human kidney proximal tubule (HKC11) cells, treatment with aldosterone resulted in translocation of MR to the nucleus and phosphorylation of SGK-1. Treatment with aldosterone also increased Na+, K+ ATPase-mediated 86Rb uptake and expression of Na+, K+ ATPase α1 subunits in HKC11 cells. The effects of aldosterone on Na+, K+ ATPase-mediated 86Rb uptake were prevented by spironolactone, a competitive inhibitor of aldosterone for the MR, and partially by Mifepristone, a glucocorticoid receptor (GR) inhibitor. These results suggest that aldosterone regulates Na+, K+ ATPase in renal proximal tubule cells through an MR-dependent mechanism.

Role of Vacuolar ATPase in the Trafficking of Renal Type IIa Sodium-phosphate Cotransporter

Background/Aims: Total body phosphate homeostasis is regulated by expression of type IIa sodium phosphate cotransporter (NpT2a) in the apical membrane (BBM) of renal proximal tubule cells. NpT2a expression is regulated by dietary phosphate and PTH but the mechanisms for trafficking of the protein are unknown. Based on 2D gel electrophoresis and mass spectroscopy data that changes in dietary phosphate stimulated changes in BBM expression of vacuolar H⁺-ATPase, we hypothesized that vacuolar H⁺-ATPase plays a significant role in regulation of NpT2a in opossum kidney (OK) cells, a model for renal proximal tubule transport. Methods: Role of vacuolar H⁺-ATPase was studied in opossum kidney (OK) cells by examining the effect of inhibition of vacuolar H⁺-ATPase on Pi uptake and NpT2a expression. Results: Pretreatment of OK cells with bafilomycin A₁ and concanamycin A, inhibitors of vacuolar H⁺-ATPases, blocked high phosphate- and PTH-induced degradation of NpT2a, but had no effect on high phosphate or PTH induced inhibition of sodium-dependent phosphate transport. Exposure of the cells to bafilomycin A₁ significantly decreased phosphate transport and apical membrane expression of NpT2a. Treatment with brefeldin A, an inhibitor of Golgi transport, decreased phosphate transport and apical membrane expression of NpT2a while treatment of cells with both brefeldin A and bafilomycin A₁ had no additive effect. Conclusion: We conclude that vacuolar H⁺-ATPase plays a significant role in exocytosis of NpT2a into the apical membrane and in degradation of NpT2a but has no role in endocytosis.

Experimental Induction of Calcium Oxalate Nephrolithiasis in Mice

The availability of various transgenic and knockout mice provides an excellent opportunity to better understand the pathophysiology of calcium oxalate stone disease. However, attempts to produce calcium oxalate nephrolithiasis in mice have not been successful. We hypothesized that calcium oxalate nephrolithiasis in mice requires increasing urine calcium and oxalate excretion, and experimentally induced hyperoxaluria alone is not sufficient. To provide evidence we induced hyperoxaluria by administering hyperoxaluria inducing agents in normocalciuric and hypercalciuric mice, and investigating various aspects of nephrolithiasis. We administered ethylene glycol, glyoxylate or hydroxyl proline via diet in male and female normocalciuric B6 mice, and in hypercalciuric sodium phosphate co-transporter type 2 a -/- mice for 4 weeks. We collected 24-hour urine samples on days 0, 3, 7, 14, 21 and 28, and analyzed them for pH, creatinine, lactate dehydrogenase calcium and oxalate. Kidneys were examined using light microscopy. Urine was examined for crystals using light and scanning electron microscopy. Hypercalciuric mice on hydroxyl proline did not tolerate treatment and were sacrificed before 28 days. All mice on ethylene glycol, glyoxylate or hydroxyl proline became hyperoxaluric and showed calcium oxalate crystalluria. No female, normocalciuric or hypercalciuric mice showed renal calcium oxalate crystal deposits. Calcium oxalate nephrolithiasis developed in all mice on glyoxylate and in some on ethylene glycol. In all mice the kidneys showed epithelial injury. Male mice particularly on glyoxylate had more renal injury and inflammatory cell migration into the interstitium around the crystal deposits. Results confirm that hyperoxaluria induction alone is not sufficient to create calcium oxalate nephrolithiasis in mice. Hypercalciuria is also required. Kidneys in male mice are more prone to injury than those in female mice and are susceptible to calcium oxalate crystal deposition. Perhaps epithelial injury promotes crystal retention. Thus, calcium oxalate nephrolithiasis in mice is gender dependent, and requires hypercalciuria and hyperoxaluria.

Common Genetic Variants Associate with Serum Phosphorus Concentration

Phosphorus is an essential mineral that maintains cellular energy and mineralizes the skeleton. Because complex actions of ion transporters and regulatory hormones regulate serum phosphorus concentrations, genetic variation may determine interindividual variation in phosphorus metabolism. Here, we report a comprehensive genome-wide association study of serum phosphorus concentration. We evaluated 16,264 participants of European ancestry from the Cardiovascular Heath Study, Atherosclerosis Risk in Communities Study, Framingham Offspring Study, and the Rotterdam Study. We excluded participants with an estimated GFR <45 ml/min per 1.73 m(2) to focus on phosphorus metabolism under normal conditions. We imputed genotypes to approximately 2.5 million single-nucleotide polymorphisms in the HapMap and combined study-specific findings using meta-analysis. We tested top polymorphisms from discovery cohorts in a 5444-person replication sample. Polymorphisms in seven loci with minor allele frequencies 0.08 to 0.49 associate with serum phosphorus concentration (P = 3.5 x 10(-16) to 3.6 x 10(-7)). Three loci were near genes encoding the kidney-specific type IIa sodium phosphate co-transporter (SLC34A1), the calcium-sensing receptor (CASR), and fibroblast growth factor 23 (FGF23), proteins that contribute to phosphorus metabolism. We also identified genes encoding phosphatases, kinases, and phosphodiesterases that have yet-undetermined roles in phosphorus homeostasis. In the replication sample, five of seven top polymorphisms associate with serum phosphorous concentrations (P < 0.05 for each). In conclusion, common genetic variants associate with serum phosphorus in the general population. Further study of the loci identified in this study may help elucidate mechanisms of phosphorus regulation.

βPix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

Therapeutic Effects of Anti-FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

X-linked hypophosphatemia (XLH), characterized by renal phosphate wasting, is the most common cause of vitamin D-resistant rickets. It has been postulated that some phosphaturic factor plays a causative role in XLH and its murine homolog, the Hyp mouse. Fibroblast growth factor 23 (FGF23) is a physiological phosphaturic factor; its circulatory level is known to be high in most patients with XLH and Hyp mice, suggesting its pathophysiological role in this disease. To test this hypothesis, we treated Hyp mice with anti-FGF23 antibodies to inhibit endogenous FGF23 action. A single injection of the antibodies corrected the hypophosphatemia and inappropriately normal serum 1,25-dihydroxyvitamin D. These effects were accompanied by increased expressions of type IIa sodium-phosphate cotransporter and 25-hydroxyvitamin-D-1alpha-hydroxylase and a suppressed expression of 24-hydroxylase in the kidney. Repeated injections during the growth period ameliorated the rachitic bone phenotypes typically observed in Hyp mice, such as impaired longitudinal elongation, defective mineralization, and abnormal cartilage development. Thus, these results indicate that excess actions of FGF23 underlie hypophosphatemic rickets in Hyp mice and suggest a novel therapeutic potential of the FGF23 antibodies for XLH.

Anti-FGF23 Neutralizing Antibodies Show the Physiological Role and Structural Features of FGF23

Fibroblast growth factor (FGF)23 is proposed to play a physiological role in the regulation of phosphate and vitamin D metabolism; deranged circulatory levels of FGF23 cause several diseases with abnormal mineral metabolism. This paper presents a novel approach to analyze the mechanism of action of FGF23 using anti-FGF23 monoclonal antibodies that can neutralize FGF23 activities both in vitro and in vivo. We developed two antibodies (FN1 and FC1) that recognize the N- and C-terminal regions of FGF23, respectively. Both FN1 and FC1 inhibited FGF23 activity in a cell-based Klotho-dependent reporter assay. Their administration caused marked increases in serum phosphate and 1,25D levels in normal mice. These changes were accompanied by altered expression in the kidney of type IIa sodium-phosphate cotransporter, 25-hydroxyvitamin-D-1alpha-hydroxylase, and 24-hydroxylase. Thus, this study using neutralizing antibodies confirms that FGF23 is a physiological regulator of phosphate and vitamin D metabolism. We addressed the mechanism of action for these neutralizing antibodies. Structural analysis of the FGF23/FN1-Fab complex showed that FN1 masked putative FGF receptor-binding sites in the N-terminal domain of FGF23, whereas biochemical analyses showed that FC1 interfered with the association between FGF23 and Klotho by binding to the C-terminal domain of FGF23. Taken together, our results suggest that the N- and C-terminal domains of FGF23 are responsible for association with cognate FGF receptors and Klotho, respectively, and that these interactions are indispensable for FGF23 activity.

Estrogen downregulates the proximal tubule type IIa sodium phosphate cotransporter causing phosphate wasting and hypophosphatemia

Estrogen treatment causes significant hypophosphatemia in patients. To determine the mechanisms responsible for this effect, we injected ovariectomized rats with either 17beta-estradiol or vehicle for three days. Significant renal phosphate wasting and hypophosphatemia occurred in estrogen-treated rats despite a decrease in their food intake. The mRNA and protein levels of the renal proximal tubule sodium phosphate cotransporter (NaPi-IIa) were significantly decreased in estradiol-treated ad-libitum or pair-fed groups. Estrogen did not affect NaPi-III or NaPi-IIc expression. In ovariectomized and parathyroidectomized rats, 17beta-estradiol caused a significant decrease in NaPi-IIa mRNA and protein expression compared to vehicle. Estrogen receptor alpha isoform blocker significantly blunted the anorexic effect of 17beta-estradiol but did not affect the downregulation of NaPi-IIa. Our studies show that renal phosphate wasting and hypophosphatemia induced by estrogen are secondary to downregulation of NaPi-IIa in the proximal tubule. These effects are independent of food intake or parathyroid hormone levels and likely not mediated through the activation of estrogen receptor alpha subtype.

Calcium oxalate crystal deposition in kidneys of hypercalciuric mice with disrupted type IIa sodium-phosphate cotransporter

The most common theories about the pathogenesis of idiopathic kidney stones consider precipitation of calcium phosphate (CaP) within the kidneys critical for the development of the disease. We decided to test the hypothesis that a CaP substrate can promote the deposition of calcium oxalate (CaOx) in the kidneys. Experimental hyperoxaluria was induced by feeding glyoxylate to male mice with knockout (KO) of NaP(i) IIa (Npt2a), a sodium-phosphate cotransporter. Npt2a KO mice are hypercalciuric and produce CaP deposits in their renal tubules. Experimental hyperoxaluria led to CaOx crystalluria in both the hypercalciuric KO mice and the normocalciuric control B6 mice. Only the KO mice produced CaOx crystal deposits in their kidneys, but the CaOx crystals deposited separately from the CaP deposits. Perhaps CaP deposits were not available for a CaOx overgrowth. These results also validate earlier animal model observations that showed that CaP substrate is not required for renal deposition of CaOx and that other factors, such as local supersaturation, may be involved. The absence of CaOx deposition in the B6 mice despite extreme hyperoxaluria also signifies the importance of both calcium and oxalate in the development of CaOx nephrolithiasis.

The role of ubiquitination of the type IIa sodium phosphate cotransporter (Npt2a) in protein trafficking

In the apical membrane of OK cells, Npt2a is associated with the PTH receptor, PKA, AKAP 79/150, NHERF, ezrin, and protein phosphatase 2a. PTH causes phosphorylation of several apical membrane proteins and ubiquitination of Npt2a We hypothesized that PTH‐stimulated phosphorylation of associated proteins was required for dissociation of Npt2a from the protein complex and that ubiquitination was required for Npt2a trafficking to the endosome. To address this hypothesis, we treated OK cells with vehicle or PTH, compared Npt2a‐associated proteins by immunoprecipitation (IP), protein phosphorylation, and Npt2a trafficking. PTH resulted in a rapid (within 15 min) expression of Npt2a in late endosomes (rab7, mannose6phosphate receptor positive), but not early endosomes (rab5 positive). Removal of PTH resulted in insertion of new proteins, not recycled Npt2a, into the apical membrane. Gel filtration of vehicle and PTH‐treated OK cell lysates demonstrated a shift in the fractions expressing NHERF and Npt2a. IP studies showed decreased association of Npt2a with NHERF, ezrin, RII, and the PTH receptor and decreased association of the PTH receptor with NHERF. In contrast, Npt2a continued to co‐associate with AKAP. PTH stimulated ubiquitination of Npt2a and association with VCP, a chaperone for ubiquitinated proteins. Under basal conditions, Npt2a degradation was blocked by inhibitors of either lysosomal or proteasomal degradation, while PTH‐stimulated Npt2a degradation was resistant to proteasomal inhibitors. We conclude that PTH stimulates endocytosis of Npt2a by ubiquitination of Npt2a, resulting in release of Npt2a from its anchoring complex in the apical membrane and targeting to the late endosomal fraction for degradation.

Aberrant Phex function in osteoblasts and osteocytes alone underlies murine X-linked hypophosphatemia

Patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a model of XLH characterized by a deletion in the Phex gene, manifest hypophosphatemia, renal phosphate wasting, and rickets/osteomalacia. Cloning of the PHEX/Phex gene and mutations in affected patients and hyp-mice established that alterations in PHEX/Phex expression underlie XLH. Although PHEX/Phex expression occurs primarily in osteoblast lineage cells, transgenic Phex expression in hyp-mouse osteoblasts fails to rescue the phenotype, suggesting that Phex expression at other sites underlies XLH. To establish whether abnormal Phex in osteoblasts and/or osteocytes alone generates the HYP phenotype, we created mice with a global Phex knockout (Cre-PhexDeltaflox/y mice) and conditional osteocalcin-promoted (OC-promoted) Phex inactivation in osteoblasts and osteocytes (OC-Cre-PhexDeltaflox/y). Serum phosphorus levels in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice were lower than those in normal mice. Kidney cell membrane phosphate transport in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice was likewise reduced compared with that in normal mice. Abnormal renal phosphate transport in Cre-PhexDeltaflox/y and OC-Cre-PhexDeltaflox/y mice was associated with increased bone production and serum FGF-23 levels and decreased kidney membrane type IIa sodium phosphate cotransporter protein, as was the case in hyp-mice. In addition, Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice manifested comparable osteomalacia. These data provide evidence that aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to underlie the hyp-mouse phenotype.

Shank2 Associates with and Regulates Na+/H+ Exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.

Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa.