Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Several phosphate transport processes are present in vascular smooth muscle cells.

We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.

Substrates and inhibitors of phosphate transporters: from experimental tools to pathophysiological relevance.

The control of inorganic phosphate homeostasis is mediated through the activity of sodium-coupled Pi transporters located in the intestine, kidneys, and bone. To study these transporters in either the native tissue or after heterologous expression, it is very important to use specific inhibitors of the studied transporter, in order to know the corresponding relevance in the total Pi uptake and to differentiate from the activity of other transporters. Inhibitors are also necessary as drugs for treating Pi homeostasis disorders. Under normal physiological conditions, the renal and intestinal excretion of Pi matches dietary intestinal absorption, but when the number of non-functional nephrons increase in chronic kidney disease and end-stage renal disease, the excretion of surplus Pi is progressively impaired, thereby increasing the risk of hyperphosphatemia and Pi toxicity. When the compensatory mechanisms that increase Pi excretion fail, Pi toxicity can only be prevented by reducing the intestinal absorption of Pi through phosphate binders that reduced the free Pi concentration in the lumen, and inhibitors of intestinal Pi transporters and of the paracellular absorption route. Although many potentially interesting inhibitors have been reported to date, only a few are available for experimental purposes, and even fewer have been used in independent clinical trials. In this review, we summarize the different groups of compounds reported to date as inhibitors of Pi transport. To help understand and characterize the inhibition mechanisms, we also summarize the kinetic analysis approaches and screening methods that could be applied.

Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line.

NEW FINDINGS: What is the central question of this study? The opossum kidney (OK) cell line is the main in vitro model of proximal tubular Pi transport, but it is incomplete because only the NaPiIIa Pi transporter has been identified. What is the main finding and its importance? We have cloned and characterized the Pi transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to Pi transport. All four transporters are involved in the upregulated Pi transport of cells incubated using a low-Pi medium, and only PiT1 is not involved in basal transport. ABSTRACT: The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary Pi excretion. The opossum kidney (OK) cell line is a good model for studying this function, but only NaPiIIa (NaPi4) has been identified to date as a Pi transporter in this cell line. In this work, we have identified three additional Pi transporters that are present in OK cells: NaPiIIc, PiT1 and PiT2. All three sequences are similar to the corresponding orthologues, but PiT1 is missing the first transmembrane domain. Confluent cells exhibit characteristics of type II Pi transport, which increases with alkalinity and is inhibited by phosphonoformic acid (PFA), and they mainly express NaPiIIa and NaPiIIc, with a low abundance of PiT1 and PiT2. Proliferating cells show a higher expression of PiT1 and PiT2 and a low expression of NaPiIIa and NaPiIIc. Adaptation to a low Pi concentration for 24 h induces the expression of RNA from NaPiIIa and NaPiIIc, which is not prevented by actinomycin D. Small interfering RNA transfections revealed that PiT1 is not necessary for Pi transport, but it is necessary for adaptation to a low Pi , similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four Pi transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between Pi transport and RNA levels.

Identifying early pathogenic events during vascular calcification in uremic rats.

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

Intestinal phosphate absorption is mediated by multiple transport systems in rats.

Apical inorganic phosphate (Pi) transport in the small intestine seems to be mainly mediated by the sodium/Pi cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum for all three times. Alkaline pH inhibited Pi transport, despite the increasing concentration of [Formula: see text] (NaPi2b substrate), whereas acidity increased transport when the concentration of the PiT1/PiT2 substrate, [Formula: see text], was at its highest. The effect of Pi deprivation was maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 h of Pi deprivation in the duodenum, jejunum, and ileum, whereas PiT1 required 5-10 days in the duodenum and jejunum. Therefore, whereas transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and phosphonoformic acid precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression were also inconsistent when feeding was limited to 4 h daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries [Formula: see text] and is inhibited by phosphonoformate.NEW & NOTEWORTHY The combined effects of dietary inorganic phosphate (Pi) content, pH, and phosphonoformate inhibition suggest that the resulting apical Pi transport in the small intestine cannot be fully explained by the presence of NaPi2b, PiT1, or PiT2. We provide evidence of the presence of a new sodium-coupled Pi transporter that uses [Formula: see text] as the preferred substrate and is inhibited by phosphonoformate, and its expression correlates with Pi transport in all assayed conditions.

Medial vascular calcification revisited: review and perspectives.

Vascular calcifications (VCs) are actively regulated biological processes associated with crystallization of hydroxyapatite in the extracellular matrix and in cells of the media (VCm) or intima (VCi) of the arterial wall. Both patterns of VC often coincide and occur in patients with type II diabetes, chronic kidney disease, and other less frequent disorders; VCs are also typical in senile degeneration. In this article, we review the current state of knowledge about the pathology, molecular biology, and nosology of VCm, expand on potential mechanisms responsible for poor prognosis, and expose some of the directions for future research in this area.

Na+-independent phosphate transport in Caco2BBE cells.

Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved.

Prevention of vascular calcification by polyphosphates and nucleotides- role of ATP.

BACKGROUND: In recent decades, the prevention of vascular calcification (VC) by pyrophosphate (PPi), bisphosphonates, and polyphosphates has been extensively reported. However, the possibility of direct inhibition of calcium phosphate deposition (CPD) by nucleoside-associated polyphosphates has not been addressed. We analyzed the role of ATP as an inhibitor of calcification in 2 ways: by characterizing the extracellular hydrolysis of ATP as source of PPi in the aorta, and by demonstrating the ability of ATP to prevent CPD by acting as a polyphosphate. METHODS AND RESULTS: In our study, both PPi and ATP hydrolysis in the rat aorta was kinetically characterized, thereby resulting in apparent Michaelis-Menten constants of 179 and 435 µmol/l, respectively, with the corresponding maximal velocities of 55.1 and 6,177 nmol·g(-1)·min(-1). According to these kinetic parameters, the theoretical PPi concentration in the aortic wall was 0.4-3.5 µmol/L (for an ATP concentration range of 0.1-1.0 µmol/L). In addition, we showed that nonhydrolyzable molecules are more efficient as CPD inhibitors than endogenous compounds, in accordance with the IC50 values: 1.2-2.4 µmol/L for bisphosphonates vs. 8.8 µmol/L for PPi, and 0.5-1.5 µmol/L for nonhydrolyzable ATP analogs vs. 3.2 µmol/L for ATP. CONCLUSIONS: Extracellular ATP can play an important role in the prevention of VC, not only as the source of PPi but also as a direct inhibitor of CPD.

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification.

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca²(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM² but not with 2 × 1 or 1 × 4 Ca × P(i) mM². Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.

Liver X receptor-activating ligands modulate renal and intestinal sodium-phosphate transporters.

Cholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium-phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels.

Calcium phosphate deposition with normal phosphate concentration. -Role of pyrophosphate-.

BACKGROUND: Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation. METHODS AND RESULTS: We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC50 of ≍200 nmol/L. CONCLUSIONS: CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).

The Thermodynamics of Medial Vascular Calcification.

Medial vascular calcification (MVC) is a degenerative process that involves the deposition of calcium in the arteries, with a high prevalence in chronic kidney disease (CKD), diabetes, and aging. Calcification is the process of precipitation largely of calcium phosphate, governed by the laws of thermodynamics that should be acknowledged in studies of this disease. Amorphous calcium phosphate (ACP) is the key constituent of early calcifications, mainly composed of Ca2+ and PO4 3- ions, which over time transform into hydroxyapatite (HAP) crystals. The supersaturation of ACP related to Ca2+ and PO4 3- activities establishes the risk of MVC, which can be modulated by the presence of promoter and inhibitor biomolecules. According to the thermodynamic parameters, the process of MVC implies: (i) an increase in Ca2+ and PO4 3- activities (rather than concentrations) exceeding the solubility product at the precipitating sites in the media; (ii) focally impaired equilibrium between promoter and inhibitor biomolecules; and (iii) the progression of HAP crystallization associated with nominal irreversibility of the process, even when the levels of Ca2+ and PO4 3- ions return to normal. Thus, physical-chemical processes in the media are fundamental to understanding MVC and represent the most critical factor for treatments' considerations. Any pathogenetical proposal must therefore comply with the laws of thermodynamics and their expression within the medial layer.

Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit-2 (SLC20A2) during Pi deprivation and acidosis.

The role of four Pi transporters in the renal handling of Pi was analyzed using functional and molecular methods. The abundance of NaPi-IIa, NaPi-IIc, and Pit-2 was increased by 100% in kidney from rats on a 0.1% Pi diet, compared to a 0.6% Pi diet. Pit-1 was not modified. Type II-mediated Pi uptake in Xenopus oocytes increased as the pH of the uptake medium increased, and the opposite occurred with Pit-1 and Pit-2. At pH 6.0, Pi uptake mediated through type II was approximately 10% of the uptake at pH 7.5, but the uptake through Pit-2 was 250% of the activity at pH 7.5. Real brush-border membrane vesicles (BBMV) responded to pH changes following the same pattern as type II transporters. Adaptation to a 0.1% Pi diet was accompanied by a 65% increase in the V (max) of BBMV Pi transport at pH 7.5, compared to a 0.6% Pi diet. The increase was only 11% at pH 6.0. Metabolic acidosis increased the expression of NaPi-IIc and Pit-2 in animals adapted to a low Pi diet, and phosphaturia was only observed in control diet animals. The combination of the pH effect, Pi adaptation, and metabolic acidosis suggests very modest involvement of Pit-2 in renal Pi handling. Real-time PCR and mathematical analyses of transport findings suggest that NaPi-IIa RNA accounts for 95% of all Pi transporters and that type II handles 97% of Pi transport at pH 7.5 and 60% of Pi transport at pH 6.0, depending on the pH and the physiological conditions.

Arsenate transport by sodium/phosphate cotransporter type IIb.

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As(III) have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As(V) transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using (73)As(V) as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H(3)AsO(4) were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K(m) for arsenate that was >1mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As(V) in mouse (57 microM), rat (51 microM), and human (9.7 microM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As(V).

Phosphonoformic acid prevents vascular smooth muscle cell calcification by inhibiting calcium-phosphate deposition.

OBJECTIVE: The role of inorganic phosphate in the pathogenesis of vascular calcification (VC) has been studied extensively in recent years. Phosphonoformic acid (PFA), an inhibitor of type II Pi transporters, has been traditionally used to study the involvement of Pi transport in VC, because PFA also prevents calcium deposition in vitro. However, aortic vascular smooth muscle cells (VSMCs) only express PFA-resistant, type III transporters (Pit-1 and Pit-2). Therefore, in this article we have studied the mechanism of VC prevention by PFA. METHODS AND RESULTS: Radiotracer Pi uptake in rat VSMCs was not inhibited at the concentrations at which PFA prevents calcification. Alternative mechanisms whereby PFA could prevent calcification, such as cytotoxicity or phosphodiesterase inhibition, have also been excluded. The progression of calcification also took place in fixed cells. The kinetics of VC prevention by PFA, pyrophosphate, phosphonoacetate, and bisphosphonates was similar in live and fixed cells, showing mean effective concentrations in the micromolar range. CONCLUSIONS: PFA mainly prevents VC through a physicochemical mechanism that is independent of any cellular metabolic activity, including Pi transport. Conversely, PFA seems to act similarly to its chemical analogues, inorganic pyrophosphate, and bisphosphonates, as suggested decades ago.

Effects of oral exposure to arsenite on arsenic metabolism and transport in rat kidney.

Nephrotoxicity is within the recognized toxic effects of arsenic. In this study we assessed the effect of arsenite on the renal capacity to metabolize and handle arsenicals in rats exposed to drinking water with 0, 1, 5, or 10 ppm sodium arsenite for ten days. Arsenite treatment did not affect the gene expression of the main enzyme catalyzing methylation of arsenite, As3mt, while it reduced the expression of GSTO1 mRNA and protein. Arsenite decreased the expression of Aqp3, Mrp1, Mrp4, and Mdr1b (i.e., transporters and channels used by arsenic), but not that of Aqp7, Glut1, Mrp2, and Mdr1a. The protein abundance of AQP3 was also reduced by arsenite. Arsenite increased urinary NGAL and FABP3 and decreased Klotho plasma levels, without alteration of creatinine, which evidenced early tubular damage. Renal Klotho mRNA and protein expressions were also downregulated, which may exacerbate renal damage. No effect was observed in selected miRNAs putatively associated with renal injury. Plasma PTH and FGF23 were similar between groups, but arsenite decreased the renal expression of Fgfr1 mRNA. In conclusion, exposure to arsenite alters the gene expression of proteins involved in the cellular handling of arsenical species and elicits tubular damage.

Diagnosis of genetic amyloidosis through the analysis of transthyretin gene mutation using high-resolution melting.

Transthyretin amyloidosis can be either the wild-type (ATTR-wt) or the hereditary form (ATTR-m) with autosomal dominant inheritance. ATTR seems to be an underdiagnosed disease, despite now being recognized as one of the most frequent causes of heart failure (HF) with preserved ejection fraction. The confirmation of diagnosis includes a genetic analysis as a critical step to distinguish between ATTR-wt and hereditary amyloidosis. The present study aimed to evaluate the potential application of High-Resolution Melting (HRM) analysis for identifying gene mutations in patients with suspected ATTR-m. We have adapted and validated the use of HRM for TTR mutations. We, therefore, sequenced the TTR gene and used HRM in a group of 134 patients suspected of suffering from amyloidosis. Seven patients were diagnosed with mutations in the TTR gene (p.Glu74Gln, heterozygous p.Val142Ile, and homozygous p.Val142Ile). HRM is capable of clearly detecting these TTR mutations, including the heterozygous and homozygous variants. The results show a 100% correlation between the HRM study and TTR sequencing. These results support future studies of applying HRM analysis as a diagnostic approach for ATTR-m, mainly for epidemiological studies.

Different effects of arsenate and phosphonoformate on P(i) transport adaptation in opossum kidney cells.

The main nonhormonal mechanism for controlling inorganic phosphate (P(i)) homeostasis is renal adaptation of the proximal tubular P(i) transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular P(i) concentration. We have studied how two competitive inhibitors of P(i) transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high P(i) concentrations. OK cells show very high affinity for As(V) (inhibitory constant, K(i) 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low P(i) (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM P(i). Adaptation with 2 mM P(i) or As(V) was characterized by decreases in the maximal velocity (V(max)) of P(i) transport and an abundance of the NaPi-IIa P(i) transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM P(i) increased the V(max) and transporter abundance. Changes in the V(max) were limited to a 50% variation, which was not paralleled by changes in the concentration of P(i) or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high P(i) concentration. PFA blocks the uptake of P(i) but is not transported, and it therefore simulates a low P(i) concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in P(i) transport V(max).

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-alpha, and Pi.

Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study, we have determined the correlation between calcification induced by Pi, platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor-alpha and Pi transport activity in primary cultures of rat aortic VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 osteogene messenger RNA in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100%, while DNA content and number of cells were not modified. PDGF increased the expression of the Pi transporter, Pit-1, but membrane protein biotinylation showed that Pit-1 abundance was not modified in the cell surface. Immunofluorescence revealed that, under basal conditions, Pit-1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell. The intracellular signal colocalizes with endoplasmic reticulum (ER) markers, and PDGF increases Pit-1 expression in the ER but not the cell membrane. In conclusion, Pi transport across the plasma membrane does not correlate directly with calcification, but the expression of Pit-1 in the ER opens new possibilities for the study of the pathogenesis of vascular calcification.