Intestinal Phosphate Research Articles

Hyperphosphataemia: Treatment Options

Hyperphosphataemia can be induced by three main conditions: a massive acute phosphate load, a primary increase in renal phosphate reabsorption, and an impaired renal phosphate excretion due to acute or chronic renal insufficiency. Renal excretion is so efficient in normal subjects that balance can be maintained with only a minimal rise in serum phosphorus concentration even for a large phosphorus load. Therefore, acute hyperphosphataemia usually resolves within few hours if renal function is intact. The most frequent cause of chronic hyperphosphataemia is chronic renal failure. Hyperphosphataemia in chronic kidney disease (CKD) is associated with increased cardiovascular morbidity and mortality. Lowering the phosphate load and maintaining serum phosphorus levels within the normal range are considered important therapeutic goals to improve clinical outcomes in CKD patients. Treatment consists of diminishing intestinal phosphate absorption by a low phosphate diet and phosphate binders. In CKD patients on dialysis an efficient dialysis removal of phosphate should be ensured. Dietary restriction of phosphorus while maintaining adequate protein intake is not sufficient to control serum phosphate levels in most CKD patients; therefore, the prescription of a phosphate binder is required. Aluminium-containing agents are efficient but no longer widely used because of their toxicity. Calcium-based salts are inexpensive, effective and most widely used, but there is now concern about their association with hypercalcaemia, parathyroid gland suppression, adynamic bone disease, and vascular and extraosseous calcification. The average daily dose of calcium acetate or carbonate prescribed in the randomised controlled trials to control hyperphosphataemia in dialysis patients ranges between 1.2 and 2.3g of elemental calcium. Such doses are greater than the recommended dietary calcium intake and can lead to a positive calcium balance. Although large amounts of calcium salts should probably be avoided, modest doses (<1g of elemental calcium) may represent a reasonable initial approach to reduced serum phosphorus levels. A non-calcium-based binder can then be added when large doses of binder are required. At present, there are three types of non-calcium-based phosphate binders available: sevelamer, lanthanum carbonate and magnesium salts. Each of these compounds is as effective as calcium salts in lowering serum phosphorus levels depending on an adequate prescribed dose and adherence of the patient to treatment. Sevelamer is the only non-calcium-containing phosphate binder that does not have potential for systemic accumulation and presents pleiotropic effects that may impact on cardiovascular disease. In contrast, lanthanum carbonate and magnesium salts are absorbed in the gut and their route of excretion is biliary for lanthanum and urinary for magnesium. There are insufficient data to establish the comparative superiority of non-calcium binding agents over calcium salts for such important patient-level outcomes as all-cause mortality and cardiovascular end points. Moreover, full adoption of sevelamer and lanthanum by government drug reimbursement agencies in place of calcium salts would lead to a large increase in health-care expenditure. Therefore, the choice of phosphate binder should be individualised, considering the clinical context, the costs, and the individual tolerability the concomitant effects on other parameters of mineral metabolism, such as serum calcium and parathyroid hormone, besides those on serum phosphorus.

Liver X Receptor activation inhibits intestinal phosphate transport

Renal Pi reabsorption has been considered the main mechanism controlling Pi homeostasis. However, recently small intestine Pi absorption has emerged as a putative pharmacological target for the control of hyperphosphatemia, especially important in the setting of chronic kidney disease. We have recently reported that activation of Liver X Receptor (LXR) with LXR agonists leads to a decreased serum Pi concentration and increased urinary Pi excretion. In parallel, these changes were associated with the decrease of renal and intestinal NaPi transporters expression. To investigate the potential mechanisms behind this regulation we used two transgenic mouse models expressing: 1) constitutively active form of the LXRα fused with the activation domain of VP16 under LXRα endogenous promoter (VP16‐LXRα Tg); 2) enterocyte‐specific LXRα under villin promoter (villin VP16‐LXRα Tg).VP16‐LXRα Tg and villin VP16‐LXRα Tg mice showed reduced sodium dependent Pi uptake in the brush border membrane (BBM) of enterocytes. This was accompanied by reduction of NaPi2b protein abundance in the BBM and also reduced ileum NaPi‐2b mRNA level.Our results show that the effects of enterocyte‐specific activation of LXR are independent of systemic influences and also highlight importance of the intestine in Pi homeostasis regulation. However, the putative mechanisms of LXR effects on Pi transport remain to be elucidatedSupported by NIH

Vascular calcification--is aldosterone a culprit?

In chronic kidney disease (CKD), increased plasma phosphate concentrations cause vascular calcification which substantially contribute to cardiovascular events and increased mortality of CKD patients. Similar to CKD patients, klotho-hypomorphic mice (kl/kl) also suffer from excessive vascular calcification leading to growth deficit, rapid ageing and early death. The hyperphosphataemia of kl/kl mice results from excessive formation of 1,25(OH)2D3 causing excessive intestinal phosphate absorption. Further, kl/kl mice further suffer from hyperaldosteronism and compelling evidence points to an active role of mineralocorticoids in triggering osteoinductive programmes in the vasculature, thus further contributing to the development of vascular calcification. Conversely, in kl/kl mice, the mineralocorticoid receptor antagonist spironolactone decreased the vascular osteoinductive processes and reversed the excessive expression of osteogenic programmes, i.e. type III sodium-dependent phosphate transporter Pit1, tumour necrosis factor α (Tnfα), transcription factors Msx2, Cbfa1/Runx2 and osterix as well as alkaline phosphatase (Alp). In human aortic vascular smooth muscle cells (HAoSMCs), aldosterone alone similarly triggered an 'osteogenic' programme, thus increasing PIT1, TNFα, MSX2, CBFA1/RUNX2 and ALP expression as well as ALP activity and potentiated the effects of phosphate treatment. These effects were again reversed by spironolactone and in addition by PIT1 silencing. The above observations reveal that the severe vascular calcification is not only the result of high plasma phosphate concentrations, but also promoted by aldosterone-driven osteoinductive signalling. Future studies in CKD patients will be required to define the role of aldosterone and the potential impact of its inhibition by spironolactone in the pathophysiology of vascular calcification.

Carrier systems for yerba mate extract (Ilex paraguariensis) to enrich instant soups. Release mechanisms under different pH conditions

The high antioxidant properties of yerba mate (Ilex paraguariensis) extract make it suitable as a natural food additive. Encapsulation of herbal extracts is often necessary to enhance handling and product stability. Release strategies depend on the encapsulation system and the intended use. Calcium alginate capsules, with and without a chitosan layer, containing the yerba mate extract were analyzed, in wet and dried state. Polyphenol release was quantified in gastric (HCl solution, pH = 2) and intestinal (phosphate buffer pH = 7.4) simulated fluids. Most of the encapsulated polyphenols were released in the acidic medium. However, wet capsules with chitosan maintained a higher amount of the active compound before reaching the intestinal medium. Several models were tested to fit kinetic release data; wet and dry capsules showed different release mechanisms in the acidic solution. The dry capsules hydrated, while the wet ones eroded before releasing their content mainly by diffusion. Carrier systems with yerba mate extract were added to instant vegetable soups. Capsules addition increased polyphenols content without modifying the flowability of the soups. The sensory evaluation carried out by a non-trained panel showed no significant differences between enriched and commercial soups.

Lanthanum Carbonate Inhibits Intestinal Oxalate Absorption and Prevents Nephrocalcinosis After Oxalate Loading in Rats

Increased intestinal oxalate absorption leads to increased urinary oxalate excretion (secondary hyperoxaluria) and calcium oxalate crystal formation, contributing to nephrocalcinosis/lithiasis. Lanthanum carbonate is an intestinal phosphate binder that is orally administered to patients on dialysis to treat hyperphosphatemia. It is hypothesized that lanthanum can also bind oxalate, in addition to phosphate. We evaluated this in vitro and in vivo. In vitro oxalate binding was evaluated by oxalate precipitation from a solution by lanthanum. In vivo oxalate absorption kinetics and the effect of lanthanum carbonate on nephrocalcinosis development were assessed in male Sprague-Dawley® rats that received 1) 1,000 mg lanthanum carbonate and oxalate, 2) carboxymethylcellulose and oxalate or 3) carboxymethylcellulose by gavage for up to 12 hours (kinetics) or 7 days (nephrocalcinosis). Plasma and urinary oxalate concentrations were measured at several time points after gavage. The degree of nephrocalcinosis was assessed histomorphometrically on von Kossa stained sections and by measuring total calcium content in renal tissue. In vitro lanthanum bound oxalate in a pH range comparable to the range of the intestine. In vivo oxalate administration in untreated animals resulted in a biphasic pattern of increased plasma oxalate levels, which was almost abolished in lanthanum treated rats. In the urine of treated rats oxaluria and calcium oxalate crystalluria were blunted. Moreover, significantly decreased nephrocalcinosis was observed compared with that in untreated rats. Lanthanum carbonate is a promising agent for the future prevention/treatment of secondary hyperoxaluria.

Hypoparathyroidism

This review provides an update on hypoparathyroidism (HPT), focusing on the major aspects of diagnosis, clinical manifestations and management of patients with hypocalcaemia due to HPT. Recent advances in the understanding of the physiologic actions of parathormone (PTH) and vitamin D, and the application of molecular genetics, have clarified certain aspects of the pathogenesis, classification, diagnosis and management of HPT. PTH promotes bone resorption, decreases urinary calcium excretion, enhances the conversion of 25-hydroxyvitamin D to 1, 25-dihydroxyvitamin D and increases intestinal calcium absorption and phosphate renal excretion. Understanding the molecular cause of the disease in patients and their families has the potential for proper tailoring of genetic counselling, family screening and treatment. Signs and symptoms may be associated not only with the severity, chronicity and therapeutic endpoints in HPT but also with the different causes of the disease. Hypocalcaemia may be an asymptomatic laboratory finding or a life-threatening metabolic disturbance. Although the therapy of acute hypocalcaemia is usually readily accomplished, chronic hypocalcaemia remains a very difficult treatment problem. Replacement therapy with PTH could be a therapeutic option for refractory HPT.

The roles of intestinal and renal sodium dependent phosphate transporters in phosphate homeostasis

Inorganic phosphate (Pi) is an essential nutrient for several biological functions, including intracellular signal transduction, the production and function of cell membranes, and energy exchange. To achieve these functions, a transport system is required to transfer Pi across hydrophobic cell membranes. Pi (re) absorption in the small intestine and renal proximal tubules is important for Pi homeostasis. Three types of NaPi transporters (types I - III ) have been identified : solute carrier series SLC17A1 (NPT1/NaPi- I /OATv1) , SLC34 (NaPi- II a, NaPi- II b, NaPi- II c) , and SLC20 (PiT1, PiT2) , respectively. In this review, we discuss the role of NaPi transporters in Pi homeostasis.

The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb

The vitamin D analog ED-71 [1α,25-dihydroxy-2β-(3-hydroxypropyloxy)vitamin D(3)] has been approved for treatment of osteoporosis in Japan, but its effects on mineral metabolism have not been fully explored. We investigated the actions of ED-71 on phosphate (Pi) absorption and induction of the intestinal sodium/phosphate cotransporters. Oral treatment of vitamin D-deficient rats with ED-71 (20 pmol every other day for 8 d) produced a maximal 8-fold increase in duodenal Pi absorption, measured by the in situ loop method, whereas 1,25-dihyroxyvitamin D(3) [1,25(OH)(2)D(3]), at doses up to 150 pmol, had no effect. This action of ED-71 was attributable to a dramatic 24-fold induction of sodium-dependent Pi transporter type IIb (NaPi-IIb) mRNA in the duodenum; Pit-1 and Pit-2 mRNA levels were not increased. In vitamin D-replete rats, ED-71 treatment (50 pmol) at 72 and 24 h before death increased NaPi-IIb mRNA in the duodenum and jejunum, but not the ileum, whereas 1,25(OH)(2)D(3) at 1000 pmol was ineffective in all segments. Single oral doses of ED-71 increased mouse intestinal NaPi-IIb mRNA and protein between 6 and 24 h. Surprisingly, rat lung NaPi-IIb was not increased by ED-71, despite its coexpression with the vitamin D receptor in alveolar type II cells. However, ED-71 did not induce intestinal NaPi-IIb in vitamin D receptor-ablated mice. The greater potency of ED-71 than 1,25(OH)(2)D(3) on NaPi-IIb appears to be due to much higher and more prolonged levels of ED-71 in the circulation. In summary, ED-71, due to its disparate pharmacokinetics, is a much more potent inducer of intestinal Pi absorption and NaPi-IIb than 1,25(OH)(2)D(3), suggesting a role for this analog in the treatment of Pi-wasting disorders.

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.

Molecular Mechanisms of Vitamin D Action

The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP(9k), and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.

Tracking of single microvilli to study regulation of the intestinal phosphate transporters

Intestinal phosphate (Pi) uptake is a key mechanism in systemic Pi homeostasis. Modulation of intestinal Pi transport has been recently recognized as an important target in prevention of hyperphosphatemia and the associated cardiovascular complications in Chronic Kidney Disease. Intestinal Pi absorption is mainly mediated by the sodium‐phosphate co‐transporter NaPi2b expressed in the intestinal apical microvilli. Recent evidences show that agonists of the LXR nuclear hormone receptor inhibit intestinal NaPi transport activity, however, the detailed mechanisms of how LXR decreases intestinal NaPi transport activity, including the role of changes in membrane cholesterol or PDZ interacting proteins, remain to be determined.We study these processes at the molecular level applying the Modulation Tracking nanoimaging method to microvilli of live cells expressing fluorescently tagged NaPi2b. Tracking single microvilli we measure their motion, image NaPi2b distribution on the membrane and perform Fluorescence Correlation Spectroscopy techniques (RICS, N&amp;B) in steady state, even if the microvilli are moving. Motility of the microvilli, protein diffusion and clustering, are monitored under stimulation to get insights into the process of intestinal transporters regulation.Work supported in part by NIH R01 DK066029‐01A2 (ML, EG and LL), NIH‐P41‐RR03155 (EG and LL) and P50‐GM076516 (EG, MD and LL).

The Effects of 24R, 25‐Dihydroxyvitamin D3 and 24S, 25Dihydroxyvitamin D3 on Phosphate Transport in Vivo

The steroid hormone 1, 25‐dihydroxyvitamin D3 [1,25D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while 24,25‐dihydroxyvitamin D3 [24,25D3] inhibits the rapid stimulation by 1,25D3. in isolated intestinal cells and perfused duodenal loops. Studies were undertaken to determine if 24,25D3 had a similar effect in vivo. 24,25D3 has two isomers which are 24R, 25dihydroxyvitamin D3 [24R,25D3] and 24S, 25dihydroxyvitamin D3 [24S,25D3]. We studied these two isomers separately and tested them over a time course of 1, 5, 10, 15 and 18 h after steroid using chicks on regular diet, but fasted, and chicks on a lower vitamin D diet. All chicks were anesthetized prior to surgical exposure of the duodenal loop and injection of H3 32PO4 in buffer into the lumen. An initial study determined that 3 to 9 min of absorption in vivo was in a linear range, as judged by serum levels of radioactivity. Chicks were then injected IM with either 200 μg 24R,25D3, 20 μg 24S,25D3 or vehicle (1–18 h prior to experimentation) within the same time course studies. The isomers had different effects on phosphate absorption: 24R,25D3 had a hypophosphatemic effect in vivo in which serum levels at 5, 15 and 18 h had decreased to 42%, 39% and 43%, respectively, (P&lt; 0.05) compared with controls. In contrast, 24S, 25(OH)2D3 stimulated intestinal phosphate absorption at the 5h time point by 64%, but had no effect at the other time points tested. Supported by Agriculture and Food Research Initiative Competitive Grant no. 2010‐65206‐20652 from the USDA National Institute of Food and Agriculture.

Intestinal Cell Phosphate Uptake and the Targeted Knockout of the 1,25D3-MARRS Receptor/PDIA3/ERp57

We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Enterocytes isolated from 3- to 4-wk-old littermate (LM) male mice responded to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with enhanced phosphate uptake relative to corresponding controls within 1 min of addition, whereas in cells from targeted knockout (KO) mice, the response was severely blunted. Unlike chick enterocytes, mouse enterocytes did not respond to phorbol ester with enhanced phosphate uptake. However, forskolin, which does not stimulate phosphate uptake in chick intestinal cells, did so in enterocytes isolated from either young male LM or KO mice. Intestinal cells isolated from young female LM mice also responded to 1,25(OH)₂D₃ with enhanced phosphate uptake within 5 min of hormone addition, whereas cells from KO mice did not. Forskolin also stimulated phosphate uptake in enterocytes from young female KO or LM mice. As with intestinal cells from adult male chickens or rats, cells from adult (8 wk) male LM mice lost the ability to respond to 1,25(OH)₂D₃) with enhanced phosphate uptake. In contrast, intestinal cells from adult female LM mice did respond with enhanced phosphate uptake within 1 min of steroid hormone addition relative to corresponding controls, and the magnitude of the effect was greater than that observed in enterocytes of young females. Cells isolated from young or adult male or female LM mice failed to respond to 1,25(OH)₂D₃ with enhanced protein kinase C activity. Finally, we have previously reported that mouse enterocytes have cell surface vitamin D receptor; however preincubation of such cells with anti-vitamin D receptor antibodies demonstrated that the classical receptor is not involved in the rapid 1,25(OH)₂D₃-stimulated uptake of phosphate.

The Effects of 24R, 25-dihydroxyvitamin D3 and 24S, 25-dihydroxyvitamin D3 on Phosphate Transport in Chicks In Vivo

Previous work in this laboratory has indicated that 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits rapid, 1,25-dihuydroxyvitamin D3 [1,25(OH)2D3] stimulated phosphate uptake in isolated intestinal cells and perfused duodenal loops. Studies were undertaken to determine if 24,25(OH)2D3 had a similar effect in vivo. 24,25(OH)2D3 has two isomers which are 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] and 24S,25-dihydroxyvitamin D3 [24S,25(OH)2D3]., which we studied separately and tested over a time course of 1, 5, 10, 15 and 18 hours after steroid using chicks on regular diet, but fasted, and chicks on a lower vitamin D diet. All chicks were anesthetized prior to surgical exposure of the duodenal loop and injection of a solution containing H3 32PO4 into the lumen. Blood was then collected and serum prepared to determine transport of the radionuclide by liquid scintillation counting. An initial time course study of phosphate transport determined that 3 to 9 min of absorption in vivo was in a linear range, as judged by serum levels of radioactivity. Chicks were then injected with either 200 μg 24R,25(OH)2D3, 20 μg 24S,25(OH)2D3 or vehicle for control groups within the same time course studies. We found that the isomers had different effects on phosphate absorption. 24R,25(OH)2D3 had a hypophosphatemic effect in vivo. The serum levels of radionuclide revealed hypophosphatemic effects at 1, 5, 15 and 18 hours time points with a decrease of 56%, 42%, 39% and 43%, respectively, (P< 0.05) compared with controls. In contrast, 24S,25(OH)2D3 stimulated intestinal phosphate absorption at the five hour time point by 64%, but had no other effects at the other time points tested. We conclude that 24R,25(OH)2D3, which is made when dietary vitamin D levels are high, inhibits phosphate transport from the intestine, while 24S,25(OH)2D3 lacks activity in vivo. Keywords: Chick, hypocalcem, hypophosphatemia, intestine, in vivo, phosphate absorption, phosphate transport, serum phosphate, vitamin D, 24S, 25-dihydroxyvitamin D, 24R, 25-dihydroxyvitamin D, phosphate transport, 25-dihydroxyvitamin D3.

Candida albicans Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient.

Tracking of Single Microvilli to Study Regulation of the Intestinal Phosphate Transporters

Intestinal phosphate (Pi) uptake is one of the key mechanisms of systemic Pi homeostasis. Modulation of intestinal Pi transport has been recently recognized as an important target in prevention of hyperphosphatemia and the associated cardiovascular complications in Chronic Kidney Disease. Intestinal Pi absorption is mainly mediated by the type IIb sodium-phosphate co-transporter NaPi2b which is expressed in the intestinal apical microvilli. Recent evidences show that agonists of the LXR nuclear hormone receptor inhibit intestinal NaPi transport activity, NaPi2b protein and mRNA abundance, and decrease serum Pi level [1]. However, the detailed mechanisms of how LXR decreases intestinal NaPi transport activity, including the role of changes in membrane cholesterol or PDZ interacting proteins, remain to be determined.In order to study these processes at the molecular level we apply the Modulation Tracking nanoimaging method [2] to microvilli of live cells expressing fluorescently tagged NaPi2b. This technique generalizes the principles of Single Particle Tracking to allow the imaging of extended subcellular structures. Tracking the single microvilli we directly measure their motion, while we also image NaPi2b distribution along the membrane and perform Fluorescence Correlation Spectroscopy techniques (RICS, N&B) in steady state, even if the microvilli are moving. Different dynamic properties, including motility of the microvilli and protein diffusion and clustering, are monitored under stimulation to get insights into the process of intestinal transporters regulation.Work supported in part by NIH R01 DK066029-01A2 (ML, EG and LL), NIH-P41-RR03155 (EG and LL) and P50-GM076516 (EG, MD and LL).

Tolerability and efficacy of the intestinal phosphate binder Lantharenol® in cats

BackgroundTolerability and efficacy of the intestinal phosphate binder Lantharenol® (lanthanum carbonate octahydrate) were tested in two prospective, randomized and negative controlled laboratory studies with healthy adult cats fed commercial maintenance diets non-restricted in phosphorus. In the first study, the maximal tolerated dose was determined. Starting from a dose of 0.125 g/kg body weight mixed with the daily feed ration, the dose of Lantharenol® was doubled every other week until signs of intolerability were observed (N = 10 cats compared to 5 untreated controls). In the second study, the effects of feed supplementation for two weeks with approximately 2, 6, and 20% of the maximal tolerated dose on phosphorus excretion patterns and balance were assessed (N = 8 cats per group).ResultsLantharenol® was found to be safe and well tolerated up to the dose of 1 g/kg bodyweight, corresponding to a concentration of 84 g Lantharenol®/kg complete feed, defined as dry matter with a standard moisture content of 12%. Feed supplementation for two weeks with approximately 2-20% of this dosage (i.e., 1.6, 4.8, and 16 g/kg complete feed) resulted in a shift from urinary to faecal phosphorus excretion. Apparent phosphorus digestibility was dose-dependently reduced compared to the control group fed with diet only (N = 8).ConclusionsThe feed additive was well accepted and tolerated by all cats. Therefore, Lantharenol® presents a well tolerated and efficacious option to individually tailor restriction of dietary phosphorus as indicated, for instance, in feline chronic kidney disease.

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca 2 + ) and inorganic phosphate (P i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca 2 + and P i transport are limited. The aim of the present study was to characterize the intestinal Ca 2 + and P i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca 2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca 2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca 2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D 9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca 2 + transport. Regarding the P i transport, the results indicate significant active P i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na +/K +-ATPase on the serosal-to-mucosal P i transport suggests a pivotal role of Na + in jejunal P i transport in the horse.

Frontiers in vitamin D; basic research and clinical application. Vitamin D and secondary hyperparathyroidism

In chronic kidney disease (CKD) , phosphate retention and low 1, 25-dihydroxyvitamin D [1, 25 (OH) ₂D] due to a decrease in the number of functioning nephrons had long been considered to contribute to the pathogenesis of secondary hyperparathyroidism (SHPT) . The discovery of fibroblast growth factor 23 (FGF23) drastically changed our understanding and provided new implication of the development of SHPT. In addition, CKD impairs the maintenance of normal 25-hydoxyvitamin D [25 (OH) D] levels which can act directly on the parathyroid glands to repress PTH synthesis. Active vitamin D therapy is widely used for the treatment of SHPT. However, the active form of vitamin D, 1, 25 (OH) ₂D and its analogs increase the calcium and phosphate levels by increasing the intestinal calcium and phosphate absorption. To suppress PTH secretion without these potential complications, several vitamin D analogs including paricalcitol, maxacalcitol and doxercalciferol are already in clinical use. A novel therapeutic concept in new vitamin D therapies are expected to improve patient outcome.

Treatment of Cardiovascular Calcification in Uremia

Uremic subjects, in addition to accelerated atherosclerosis, develop diffuse media vascular calcification (VC) which, in turn, is associated with increased vascular stiffness and mortality risk. Two sets of risk factors for VC can be considered: passive deposition of calcium-phosphate and active transformation of vascular smooth muscle cells into osteoblastic- like cells. The former is linked with the metabolic imbalance in divalent ions that affects renal patients at any stage of the disease; the latter is secondary to a recently discovered mechanism of cellular trans-differentiation caused by deranged local concentration of divalent ions. Also, the role of inhibitors of calcification is under investigation. These include circulating or local, substances like fetuin, matrix GLA protein or osteoprotegerin. Their biologic importance is supported by the occurrence of calcification in transgenic animals lacking these factors. Accordingly, VC is a complex biologic and incompletely understood process that deserves further research, in order to develop specific therapeutic strategies. In general, once established, these calcifications are not considered to be reversible; therefore, prevention is the main treatment option. With this aim, in uremic patients it is now recommended to adopt restricted ranges of serum concentrations for calcium, phosphate and parathyroid hormone which are associated with a lower rate of calcification. To target these recommended ranges, new drugs, like selective vitamin D receptor activators, calcium sensing receptor modulators and calcium free intestinal phosphate binders, have been introduced. Moreover new possible pathogenetic pathways are considered (e.g. vitamin K deficiency). Further, in patients with calciphylaxis, the most severe form of VC, experimental therapies are suggested, with drugs like sodium thiosulphate or bisphosphonates. Drugs capable of reversing the process of trans-differentiation from osteoblast-like to vascular smooth muscle cells could be developed in the future.