Phosphate Cotransporter Research Articles

Co‐immunolocalizing proteins involved in fructose metabolism and calcitriol regulation in the rat kidney

In rodents chronically consuming high fructose diets, serum calcitriol levels decrease markedly and may be linked to increases in serum levels of FGF23 known to regulate expression of CYP27B1 and CYP24A1 that metabolize this active form of vitamin D. To determine the cell types involved in this complex interaction, we used immunohistochemistry of renal biomarkers of fructose metabolism (its transporter GLUT5 and fructokinase (FK)), of FGF23 effects (klotho, the obligate renal FGF23 coreceptor) as well as established biomarkers of proximal (the phosphate cotransporter NaPi2a) and distal (peanut agglutinin (PNA)) tubule cells. NaPi2a but not PNA colocalized with GLUT5 and FK, while GLUT5 colocalized also with FK. Compared to glucose or starch, fructose‐feeding markedly increased expression of GLUT5 and decreased that of NaPi2a. Thus dietary fructose regulates renal fructose transport and metabolism occurring primarily in proximal and not distal tubule cells. In contrast, PNA, but not NaPi2a, GLUT5 and FK, colocalized with klotho, suggesting that klotho and FGF23 regulation occurs mainly in the distal tubule, contrary to most findings, and that fructose metabolism in the proximal tubule may not directly interact with klotho‐regulated calcitriol metabolism. (NSF‐IOS1121049)

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate- and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics.

Antibody-Mediated Activation of FGFR1 Induces FGF23 Production and Hypophosphatemia

The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs) can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb) in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.

The phosphate-lowering effect of nicotinamide is offset by reduced Fgf23 levels in a murine model of familial tumoral calcinosis

Familial tumoral calcinosis is caused by mutations in the GALNT3 gene. Lack of GalNAc transferase 3, encoded by GALNT3, destabilizes FGF23, a key hormone that suppresses phosphate reabsorption and 1,25-dihydroxyvitamin D synthesis in the kidney. The destabilized FGF23 is more susceptible to proteolytic cleavage, thereby reducing the secretion of biologically active intact FGF23. Persistent hyperphosphatemia due to decreased FGF23 concentrations leads to often large, ectopic calcific masses in soft tissues, which are usually removed surgically. However, the calcific masses often recur, requiring a more permanent solution to the problem. Nicotinamide is reported to lower serum phosphate by decreasing type IIb sodium-dependent phosphate cotransporter in the gut. This effect of nicotinamide has promoted its use in treatment of limited cases of tumoral calcinosis, though its effectiveness remains largely unclear. Therefore, we investigated nicotinamide as a potential therapy for tumoral calcinosis, using a murine model of the disease – Galnt3 knockout mice. Initially, nicotinamide (doses 0, 2.5, 5, 7.5, and 10 mmol/kg/day) was given to normal mice for 2 weeks. Treatment had no effect on serum phosphate levels; however, Fgf23 was decreased in a dose-dependent manner. Subsequently, high-dose nicotinamide (10 mmol/kg/day) was tested for 4 weeks in Galnt3 knockout mice on a high-phosphate diet. The radiographic data pretreatment and posttreatments showed that the treatment did not eliminate the calcification, but retarded its growth, while in the untreated mice, calcifications increased in size. The therapy did not change serum phosphate levels despite moderately increased phosphate excretion, likely due to decreased serum intact Fgf23 levels. Quantification of calcium and phosphate contents in hearts and kidneys revealed that the treated mice had significantly high calcium in the heart. In summary, nicotinamide did not alter serum phosphate levels because the phosphate-lowering effect of nicotinamide was diminished by reduction in intact Fgf23 concentrations. The increased calcium in the heart suggests that nicotinamide therapy may also have an adverse effect.

The kidney sodium–phosphate co-transporter alters bone quality in an age and gender specific manner

Mutations in the kidney NaPiIIa co-transporter are clinically associated with hypophosphatemia, hyperphosphaturia (phosphate wasting), hypercalcemia, nephrolithiasis and bone demineralization. The mouse lacking this co-transporter system was reported to recover its skeletal defects with age, but the “quality” of the bones was not considered. To assess changes in bone quality we examined both male and female NaPiIIa knockout (KO) mice at 1 and 7months of age using micro-computed tomography (micro-CT) and Fourier transform infrared imaging (FTIRI). KO cancellous bones at both ages had greater bone volume fraction, trabecular thickness and lesser structure model index based on micro-CT values relative to age- and sex-matched wildtype animals. There was a sexual-dimorphism in the micro-CT parameters, with differences at 7months seen principally in males. Cortical bone at 1month showed an increase in bone volume fraction, but this was not seen at 7months. Cortical thickness which was elevated in the male and female KO at 1month was lower in the male KO at 7months. FTIRI showed a reduced mineral and acid phosphate content in the male and female KO's bones at 1month with no change in acid phosphate content at 7months. Collagen maturity was reduced in KO cancellous bone at 1month. The observed sexual dimorphism in the micro-CT data may be related to altered phosphate homeostasis, differences in animal growth rates and other factors. These data indicate that the bone quality of the KO mice at both ages differs from the normal and suggests that these bone quality differences may contribute to skeletal phenotype in humans with mutations in this co-transporter.

Hypercholesterolemia and effects of high cholesterol diet in type IIa sodium-dependent phosphate co-transporter (Npt2a) deficient mice

The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol.

Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET

Fluorescence & Other Luminescence II 3496-Pos Board B651 Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET Luca Lanzano 1 , Hector Giral 2 , Moshe Levi 2 , Enrico Gratton 1 . University California, Irvine, CA, USA, 2 University Colorado, Denver, CO, USA. Phosphate (Pi) homeostasis is crucial for many cellular processes. Pi absorption in the kidney proximal tubule and in the intestine is mainly mediated by the type II family of the sodium-dependent phosphate co-transporters (NaPi) which are located in the apical membrane microvilli. Interaction with scaffolding PDZ domain containing proteins is thought to play a central role in the regulation of the abundance and activity of the NaPi transporters, in response to dietary and hormonal stimuli. We investigated protein-protein interactions between NaPi and PDZ proteins in cellular models of the proximal tubule and the intestine using the FLIM detection of FRET [1]. Also, we have recently introduced the nSPIRO method which generalizes the principles of Single Particle Tracking to the imaging of extended subcellular structures like the apical membrane microvilli [2]. using this method we can image the microvilli with high resolution even if they are moving. Here we combine the tracking of single microvilli with the phasor analysis of FLIM to quantify FRET interaction at the level of the single microvilli. We explore how the measurement of FRET on single microvilli compares to the measurement performed on the entire apical membrane, and discuss the advan- tages and limitations of this technique. Work supported in part by NIH RO1 DK066029-01A2 (ML, EG), 8P41GM103540 (EG and LL) and P50GM076516 (EG and LL). [1] Giral, Lanzano et al, J Biol Chem (2011) 286, 15032-15042; Giral, Cranston, Lanzano et al, J Biol Chem (2012) epub ahead of print [2] Lanzano L et al, J Biophotonics (2011) 4, 415-42; Lanzano L and Gratton E, Microsc Res Tech (2012) 75, 1253-1264

Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Directly Regulates Osteogenesis

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na(+)/H(+) exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na(+)/H(+) exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.

Buyer beware! Doctor be aware!—Answers

Acute hyperphosphatemia with hypocalcemic tetany from exogenous phosphate load was diagnosed. The serum phosphorous level was 45.2 mg/dl, total calcium 5.1 mg/dl and ionized calcium 1.83mg/dl (normal range 4.40–5.40 mg/dl). An adult Fleet® enema (C.B. Fleet Co., Lynchburg, VA) has a volume of 118 ml and contains 19 g of monobasic sodium phosphate monohydrate, 7 g of dibasic sodium phosphate heptahydrate and 4.4 g of sodium [1]. Hypocalcemia is induced by calcium complexing with phosphate, and the magnitude of hypocalcemia is proportional to the degree by which the solubility product of calcium phosphate is exceeded. Although the exact threshold is impossible to determine, a product as low as 60 mg/dl has been associated with increased visceral calcification [2, 3]. Phosphorous is the sixth most abundant element in the human body, and under normal conditions most total body phosphorous exists in the bone in the divalent form (HPO4 ). Of the remaining phosphorous 10 % exists intracellularly, and only 1 % is found in the extracellular fluid of which 15 % is protein bound and another 5 % is complexed as salts with cations such as sodium, calcium and magnesium [4, 5]. Active absorption of dietary phosphorous occurs in the small intestine (duodenum and jejunum) via sodium phosphate cotransporter 2b (NaPi2b). Passive absorption takes place throughout the intestine, including the colon. As a reference, the typical adult diet contains 1 g of phosphorous daily. The recommended elemental phosphorous intake for a 6-year-old child is 500 mg per day. Our patient received 15.8 g (488.5 mmol) of elemental phosphate within 12 h. Control of plasma phosphate is normally maintained by altering renal excretion or redistribution within the body compartments. Phosphate is freely filtered at the glomerulus, and 85 % is reabsorbed into the blood at the level of the proximal tubule. Phosphate enters the proximal tubule cell against an electrical gradient via the NaPi2a and NaPi2c transporters, both of which are located along the brush border membrane [5]. Inhibition of proximal tubule reabsorption produces phosphaturia, which not uncommonly can cause hypophosphatemia. In our case, the large exogenous phosphorous load overwhelmed the limited ability of the gut and kidney to regulate phosphorous absorption and excretion. Hyperphosphatemia occurs from exogenous sources, as in our case, or from endogenous sources, such as tumor lysis syndrome or rhabdomyolysis. In clinical medicine, chronic kidney disease is the most common cause. Severe This article refers to the article that can be found at http://dx/doi.org/ 10.1007/s00467-012-2338-y E. Anyaegbu : T. Keefe Davis (*) :K. Hruska :A. Beck (*) Division of Nephrology, Department of Pediatrics, Washington University School of Medicine, Box 8208, 660 S. Euclid Ave, St. Louis, MO 63110, USA e-mail: davis_tk@kids.wustl.edu e-mail: beck_a@kids.wustl.edu

Molecular cloning and functional characterization of swine sodium dependent phosphate cotransporter type II b (NaPi-IIb) gene

A sodium-dependent phosphate transporter gene, NaPi-IIb, was isolated from swine small intestine using cDNA library screening method. Sequencing analysis revealed that the NaPi-IIb cDNA sequences was 2,016 bp in length and encoded an open-reading frame consisting of 671 amino acids. The cDNA showed 83.1 % sequences identity to the human NaPi-IIb and 78.7 % sequences identity to the chicken NaPi-IIb. Prediction of membrane spanning domains based on the hydrophilic and hydrophobic properties of the amino acids suggested that a putative protein had nine transmembrane domains, with both the NH(2) and COOH terminal being intracellular. By northern blot, a ~4.2 kb transcript was found to be abundantly expressed in mall intestine, lung, ovary, mammary glands, liver, kidney, salivary glands, placenta and thymus. Microinjection of swine NaPi-IIb cRNA into Xenopus oocytes demonstrated that the NaPi-IIb showed sodium-dependent Pi cotransport activity, and an approximate 31-fold increase of Pi uptake was seen in cRNA injected oocytes. The swine NaPi-IIb transporter expressed in Xenopus oocytes had a Km for Pi of ~79.35 ± 7.2 μM. Furthermore, the pH dependency characterization of swine NaPi-IIb transporter showed activation at extracellular alkaline-pH.

NHERF1 regulation of PTH-dependent bimodal Pi transport in osteoblasts

Control of systemic inorganic phosphate (Pi) levels is crucial for osteoid mineralization. Parathyroid hormone (PTH) mediates actions on phosphate homeostasis mostly by regulating the activity of the type 2 sodium–phosphate cotransporter (Npt2), and this action requires the PDZ protein NHERF1. Osteoblasts express Npt2 and in response to PTH enhance osteogenesis by increasing mineralized matrix. The regulation of Pi transport in osteoblasts is poorly understood. To address this gap we characterized PTH-dependent Pi transport and the role of NHERF1 in primary mouse calvarial osteoblasts. Under proliferating conditions osteoblasts express Npt2a, Npt2b, PTH receptor, and NHERF1. Npt2a mRNA expression was lower in calvarial osteoblasts from NHERF1-null mice. Under basal conditions Pi uptake in osteoblasts from wild-type mice was greater than that of knockout mice. PTH inhibited Pi uptake in proliferating osteoblasts from wild-type mice, but not in cells from knockout mice. In vitro induction of mineralization enhanced osteoblast differentiation and increased osterix and osteocalcin expression. Contrary to the results with proliferating osteoblasts, PTH increased Pi uptake and ATP secretion in differentiated osteoblasts from wild-type mice. PTH had no effect on Pi uptake or ATP release in differentiated osteoblasts from knockout mice. NHERF1 regulation of PTH-sensitive Pi uptake in proliferating osteoblasts is mediated by cAMP/PKA and PLC/PKC, while modulation of Pi uptake in differentiated osteoblasts depends only on cAMP/PKA signaling. The results suggest that NHERF1 cooperates with PTH in differentiated osteoblasts to increase matrix mineralization. We conclude that NHERF1 regulates PTH that differentially affects Na-dependent Pi transport at distinct stages of osteoblast proliferation and maturation.

Different phosphate transport in the duodenum and jejunum of chicken response to dietary phosphate adaptation.

Intestinal phosphate (Pi) absorption across the apical membrane of small intestinal epithelial cells is mainly mediated by the type IIb Na-coupled phosphate co-transporter (NaPi-IIb), but its expression and regulation in the chicken remain unclear. In the present study, we investigated the mRNA and protein levels of NaPi-IIb in three regions of chicken small intestine, and related their expression levels to the rate of net phosphate absorption. Our results showed that maximal phosphate absorption occurs in the jejunum, however the highest expression levels of NaPi-IIb mRNA and protein occurs in the duodenum. In response to a low-Pi diet (TP 0.2%), there is an adaptive response restricted to the duodenum, with increased brush border membrane (BBM) Na-Pi transport activity and NaPi-IIb protein and mRNA abundance. However, when switched from a low- (TP 0.2%) to a normal diet (TP 0.6%) for 4 h, there is an increase in BBM NaPi-IIb protein abundance in the jejunum, but no changes in BBM NaPi-IIb mRNA. Therefore, our study indicates that Na-Pi transport activity and NaPi-IIb protein expression are differentially regulated in the duodenum vs the jejunum in the chicken.

Solute carrier family 17 sodium-dependent inorganic phosphate cotransporter member 8 (SLC17A8; VGLUT3)

Solute carrier family 17 sodium-dependent inorganic phosphate cotransporter member 8 (SLC17A8; VGLUT3)

Sirolimus Induced Phosphaturia is Not Caused by Inhibition of Renal Apical Sodium Phosphate Cotransporters

The vast majority of glomerular filtrated phosphate is reabsorbed in the proximal tubule. Posttransplant phosphaturia is common and aggravated by sirolimus immunosuppression. The cause of sirolimus induced phosphaturia however remains elusive. Male Wistar rats received sirolimus or vehicle for 2 or 7 days (1.5mg/kg). The urine phosphate/creatinine ratio was higher and serum phosphate was lower in sirolimus treated rats, fractional excretion of phosphate was elevated and renal tubular phosphate reabsorption was reduced suggesting a renal cause for hypophosphatemia. PTH was lower in sirolimus treated rats. FGF 23 levels were unchanged at day 2 but lower in sirolimus treated rats after 7 days. Brush border membrane vesicle phosphate uptake was not altered in sirolimus treated groups or by direct incubation with sirolimus. mRNA, protein abundance, and subcellular transporter distribution of NaPi-IIa, Pit-2 and NHE3 were not different between groups but NaPi-IIc mRNA expression was lower at day 7. Transcriptome analyses revealed candidate genes that could be involved in the phosphaturic response. Sirolimus caused a selective renal phosphate leakage, which was not mediated by NaPi-IIa or NaPi-IIc regulation or localization. We hypothesize that another mechanism such as a basolateral phosphate transporter may be responsible for the sirolimus induced phosphaturia.

Hereditary hypophosphatemic rickets with hypercalciuria: case report.

We report a case of a male aged 50 years who consulted for renal disease recurrent lithiasis and nephrocalcinosis. The clinical examination showed external signs of rickets/osteomalacia and biochemical data as well as a severe loss of renal phosphate with hypophosphatemia, normal 25 OH vitamin D, high 1,25 OH vitamin D and hypercalciuria. Parathyroid hormone was low and renal ultrasound confirmed the existence of severe bilateral medullary nephrocalcinosis. They also found incipient chronic renal failure and incomplete renal tubular acidosis, both secondary to nephrocalcinosis and unrelated to the underlying disease. The molecular study found a change in homozygosity in intron 5 of gene SLC34A3 (NM_080877.2:c[ 448 +5G>A] + [ 448 +5G>A] ). His three children were carriers of the same variant in heterozygosis and although they were clinically asymptomatic two of them had hypercalciuria. All these data suggest that the patient had hereditary hypophosphataemic rickets with hypercalciuria (HHRH) secondary to an alteration in the sodium dependent phosphate cotransporter located in proximal tubule (NaPi-IIc). The HHRH is transmitted by autosomal recessive inheritance and is an extremely rare form of hypophosphatemic rickets. The diagnosis and treatment are essential to prevent bone sequelae of rickets and nephrocalcinosis. A correct differential diagnosis with other forms of hypophosphatemic rickets has implications on the treatment, as the management based only on phosphorus supplementation usually corrects all clinical and biochemical abnormalities, except for the loss of phosphorus in the urine. The exogenous supply of calcitriol, as advised in other hypophosphatemic rickets, may induce renal calcium deposits and nephrocalcinosis and worsens the prognosis.

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.

Clinical Consequences of Mutations in Sodium Phosphate Cotransporters

Three families of sodium phosphate cotransporters have been described. Their specific roles in human health and disease have not been defined. Review of the literature reveals that the type II sodium phosphate cotransporters play a significant role in transepithelial transport in a number of tissues including kidney, intestine, salivary gland, mammary gland, and lung. The type I transporters seem to play a major role in renal urate handling and mutations in these proteins have been implicated in susceptibility to gout. The ubiquitously expressed type III transporters play a lesser role in phosphate homeostasis but contribute to cellular phosphate uptake, mineralization, and inflammation. The recognition of species differences in the expression, regulation, and function of these transport proteins suggests an urgent need to find ways to study them in humans.

BuMPy road of delayed graft function

BuMPy road of delayed graft function

Secondary hyperparathyroidism and impaired renal phosphate excretion in mice lacking adenylyl cyclase 6

Parathyroid hormone (PTH) activates an unidentified isoform of adenylyl cyclase (AC) thereby retrieving Na+/phosphate cotransporters from the proximal tubule membrane and increasing phosphate (Pi) excretion. Fibroblast growth factor (FGF) 23 is a novel AC‐independent phosphaturic hormone. To test for a role of AC6 we studied wild‐type (WT, n=10) and AC6 knockout (AC6−/−, n=9) mice under different Pi intake (low: <0.02% Pi; control: 0.6% Pi; both 0.8% Ca2+). Plasma Pi was similar on control diet (both 1.6±0.1 mM) but AC6−/− had lower urinary Pi/creatinine (Crea) (7±1 vs 17±1 mM/mM, P<0.05) despite of higher plasma PTH and FGF23 vs WT (1057±223 vs 140±23 and 923±124 vs. 629±119 pg/ml, P<0.05). Low Pi intake (7 days) suppressed PTH (11±5 and 7±7 pg/ml); despite of higher FGF23 (629±119 vs 302±78 pg/ml, P<0.05) AC6−/− had lower urinary Pi/Crea (0.09±0.01 vs 0.13±0.01 mM/mM, P<0.05) and maintained higher plasma Pi vs WT (1.3±0.1 vs 0.9±0.1 mM, P<0.05). Acute oral phosphate loading (0.5 M NaH2PO4, 1% of bw) under low Pi diet induced higher plasma Pi in AC6−/− vs WT (Δ change 30 min: 1.3±0.2 vs 0.8±0.1 mM; 60 min: 2.1±0.3 vs 1.4±0.2 mM, P<0.05) but urinary Pi/Crea tended to be lower in AC6−/− (60 min: 35±14 vs 63±16 mM/mM). Dissociation of PTH and urinary Pi excretion in AC6−/− is consistent with AC6 mediating PTH‐induced inhibition of renal Pi reabsorption, while upregulation of FGF23 may compensate.

Renal proximal tubule ion transporters are discrepantly regulated by parathyroid hormone in acute versus chronic hyperparathyroidism

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.