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Diet-induced iron deficiency in rats impacts small intestinal calcium and phosphate absorption
Abstract
Aims
Recent reports suggest that iron deficiency impacts both intestinal calcium and phosphate absorption, although the exact transport pathways and intestinal segment responsible have not been determined. Therefore, we aimed to systematically investigate the impact of iron deficiency on the cellular mechanisms of transcellular and paracellular calcium and phosphate transport in different regions of the rat small intestine.
Methods
Adult, male Sprague‐Dawley rats were maintained on a control or iron deficient diet for two weeks and changes in intestinal calcium and phosphate uptake were determined using the in situ intestinal loop technique. The circulating levels of the hormonal regulators of calcium and phosphate were determined by ELISA, while the expression of transcellular calcium and phosphate transporters, and intestinal claudins were determined using qPCR and western blotting.
Results
Diet‐induced iron deficiency significantly increased calcium absorption in the duodenum but had no impact in the jejunum and ileum. In contrast, phosphate absorption was significantly inhibited in the duodenum and to a lesser extent the jejunum, but remained unchanged in the ileum. The changes in duodenal calcium and phosphate absorption in the iron deficient animals were associated with increased claudin 2 and 3 mRNA and protein levels, while levels of parathyroid hormone, fibroblast growth factor‐23 and 1,25‐dihydroxy vitamin D3 were unchanged.
Conclusion
We propose that iron deficiency alters calcium and phosphate transport in the duodenum. This occurs via changes to the paracellular pathway, whereby upregulation of claudin 2 increases calcium absorption and upregulation of claudin 3 inhibits phosphate absorption.
... (4). Iron is mostly absorbed from the duodenum (16). In our theory, the lack of iron atoms that play the role of magnet in the neuroenteric dynamo mechanism also leads to a decrease in the energy produced by the neuroenteric dynamo. ...
Objective: Although the neurophysical mechanism of subthalamic nucleus (STN) stimulation is still unclear, STN with decreasing electric field strength may be recharged by battery. According to the law defined by Einstein, the unified electromagnetic field (UF) energy formed by the electrically charged Auerbach ganglia co-oscillating with bowel movements, can be transported by afferent nerves to charge the brain, like as battery. This study examines the rationality of this theory. Methods: In this study, 18 rats with 360±20 gr weighted were divided into 3 groups according to their intestinal pulsation ranges as: 10±3/GI; 7±2/GII and 3±1/GIII. Auerbach's ganglia density (n/AG/mm3), Auerbach ganglia neuron density (n/AGN/mm3) were estimated by taking 0.5 mm sections at 10 mm intervals from 5 different distances from the midline of the ascending colon; and STN neuron densities (n/STN/mm3) were estimated. The Auerbach ganglia neurons-accepted as vibrating particles-numbers (VPN/mm3) estimated with: VPN=nAxnAG;q the unified field strength (UFS) values formed by Auerbach's ganglia was estimated by UFS=fxVPN equation. UFS and n/STN values were compared Mann Whitney U test. Results: VPN/UFS/nSTN values were: (13.345±2.143)/(11.146±1.689)/132.863±12.654 in GI; (11.762±1.843)/(8.434±1.119)/121.371±9.872 in GII and (8.659±903)/(7.109±768) /118.127±6942 in GIII) Statistical results between UFS/nSTN were found as: p<0.005 of GI/GII; p<0.0005 of GII/GIII and p<0.00001 of GI/GIII. Conclusion: Electromagnetic energy emitted from the intestinal UFs which created by the Auerbach's ganglia have predestinative role on STN life with mechanisms such as batteries.
... При разработке экспериментальной модели важен адекватный выбор животного (44,49). Чаще всего биологическими моделями служат мыши (43,62) и крысы (15,18,25,39,44). В нашем опыте модельными животными были крысы, которые более схожи с пушными зверями, и в частности с норками, по физиолого-биохимическим особенностям. ...
... При разработке экспериментальной модели важен адекватный выбор животного (44,49). Чаще всего биологическими моделями служат мыши (43,62) и крысы (15,18,25,39,44). В нашем опыте модельными животными были крысы, которые более схожи с пушными зверями, и в частности с норками, по физиолого-биохимическим особенностям. ...
... Recent reports have described a correlation between claudin 3 and intestinal phosphate transport; in rat small intestine, this sealing claudin is highly expressed in the duodenum (Markov et al., 2010). Intestinal phosphate transport in the duodenum is significantly inhibited by diet-induced iron deficiency, an effect that is associated with the upregulation of claudin 3 mRNA and protein levels; in the jejunum, where diet-induced iron deficiency had no effect on phosphate absorption, claudin 3 remained unchanged (Asowata et al., 2021). In keeping with these findings, Hashimoto et al. (2020) have demonstrated that the VDR agonist lithocholic acid (LCA) suppresses claudin 3 levels in mouse ileum, resulting in increased phosphate permeability, and that in claudin 3 (CLDN3) KO mice, phosphate-but not calcium-absorption in the jejunum and ileum is significantly increased. ...
Phosphate homeostasis is dependent on the interaction and coordination of four main organ systems: thyroid/parathyroids, gastrointestinal tract, bone and kidneys, and three key hormonal regulators, 1,25-hydroxyvitamin D3, parathyroid hormone and FGF23 with its co- factor klotho. Phosphorus is a critical nutritional element for normal cellular function, but in excess can be toxic to tissues, particularly the vasculature. As phosphate, it also has an important interaction and inter-dependence with calcium and calcium homeostasis sharing some of the same controlling hormones, although this is not covered in our article. We have chosen to provide a current overview of phosphate homeostasis only, focusing on the role of two major organ systems, the gastrointestinal tract and kidneys, and their contribution to the control of phosphate balance. We describe in some detail the mechanisms of intestinal and renal phosphate transport, and compare and contrast their regulation. We also consider a significant example of phosphate imbalance, with phosphate retention, which is chronic kidney disease; why consequent hyperphosphatemia is important, and some of the newer means of managing it.
... Of the plethora of papers published in Acta Physiologica from 2006 to May 2022 (not considering Errata and conference abstracts) 26% feature "blotting," in any way shape or form. References are made to Western blotting, 7 immunoblotting, 8 electroblotting, 9 Northern blotting, 10 and so forth. 26% of articles features a single, adapted methodological principle. ...
... The interdependence of metal ions is not unidirectional. Not only do several metal ions influence iron utilization but also iron deficiency, for example, affects intestinal absorption of calcium [17]. ...
Humans require about 20 chemical elements. Half of them are essential metal ions. Many additional, non-essential metal ions are present in our bodies through environmental exposures, including in our diet, with functional consequences. Their accumulation is accelerated due to the increasing pollution of soil, air, water and manufacturing processes that employ chemical elements to which we have not been exposed in our evolutionary history. Yet other metal ions are essential for other forms of life, which calls on life scientists to consider the interactions of life processes with most of the chemical elements in the periodic table. Only in this century have attempts been made to integrate specialty disciplines into a science of bioelements called metallomics. Metallomics forms a fifth group when added to the traditional four building blocks of living cells and their areas of investigations, i.e., sugars (glycomics), fats (lipidomics), proteins (proteomics) and nucleic acids (genomics). Neither an understanding of all the essential metals and their interactions nor the functional impacts of the non-essential metals for life, except established toxic elements such as lead, are widely perceived as important in the basic science communities and in the applied sciences such as medicine and engineering. It is a remarkable oversight that this article attempts to address with representative examples.
... It is proposed that tight junctions, and in particular the claudin family of proteins, may determine phosphate permeability [48]. Although there is emerging evidence that changes in claudin 3 protein levels in response to the vitamin D receptor agonist lithocholic acid [48], and following dietary iron deficiency [49], impacts intestinal phosphate absorption in rodents, there is no data to confirm whether claudin expression impacts phosphate absorption in the human small intestine. As the main sites for intestinal phosphate significantly differ between species (duodenum and jejunum in humans and rats [50,51] and ileum in mice [51]), results from rodent models studying the processes of intestinal phosphate handling may not fully translate to humans. ...
Phosphate homeostasis is essential for health and is achieved via interaction between the bone, kidney, small intestine, and parathyroid glands and via intricate processes involving phosphate transporters, phosphate sensors, and circulating hormones. Numerous genetic and acquired disorders are associated with disruption in these processes and can lead to significant morbidity and mortality. The role of the kidney in phosphate homeostasis is well known, although it is recognized that the cellular mechanisms in murine models and humans are different. Intestinal phosphate transport also appears to differ in humans and rodents, with recent studies demonstrating a dominant role for the paracellular pathway. The existence of phosphate sensing has been acknowledged for decades; however, the underlying molecular mechanisms are poorly understood. At least three phosphate sensors have emerged. PiT2 and FGFR1c both act as phosphate sensors controlling Fibroblast Growth Factor 23 secretion in bone, whereas the calcium-sensing receptor controls parathyroid hormone secretion in response to extracellular phosphate. All three of the proposed sensors are expressed in the kidney and intestine but their exact function in these organs is unknown. Understanding organ interactions and the mechanisms involved in phosphate sensing requires significant research to develop novel approaches for the treatment of phosphate homeostasis disorders.
There has recently been significant interest in the concept of directly targeting intestinal phosphate transport to control hyperphosphatemia in patients with chronic kidney disease. However, we do not have a complete understanding of the cellular mechanisms that govern dietary phosphate absorption. Studies in the 1970s documented both active and passive pathways for intestinal phosphate absorption. However, following the cloning of the intestinal SLC34 cotransporter, NaPi-IIb, much of the research focused on the role of this protein in active transcellular phosphate absorption and the factors involved in its regulation. Generation of a conditional NaPi-IIb knockout mouse has demonstrated that this protein is critical for the maintenance of skeletal integrity during periods of phosphate restriction and that under normal physiological conditions, the passive sodium-independent pathway is likely be the more dominant pathway for intestinal phosphate absorption. The review aims to summarise the most recent developments in our understanding of the role of the intestine in phosphate homeostasis, including the acute and chronic renal adaptations that occur in response to dietary phosphate intake. Evidence regarding the overall contribution of the transcellular and paracellular pathways for phosphate absorption will be discussed, together with the clinical benefit of inhibiting these pathways for the treatment of hyperphosphatemia in chronic kidney disease.
The Na(+) -dependent phosphate-cotransporter NaPi-IIb (SLC34A2) is widely expressed, with intestine, lung, and testis among the organs with highest levels of mRNA abundance. In mice, the intestinal expression of NaPi-IIb is restricted to the ileum, where the cotransporter localizes specifically at the brush border membrane (BBM) and mediates the active transport of inorganic phosphate (Pi). Constitutive full ablation of NaPi-IIb is embryonically lethal whereas the global but inducible removal of the transporter in young mice leads to intestinal loss of Pi and lung calcifications. Here we report the generation of a constitutive but intestinal-specific NaPi-IIb/Slc34a2-deficient mouse model. Constitutive intestinal ablation of NaPi-IIb results in viable pups with normal growth. Homozygous mice are characterized by fecal wasting of Pi and complete absence of Na/Pi cotransport activity in BBM vesicles (BBMVs) isolated from ileum. In contrast, the urinary excretion of Pi is reduced in these animals. The plasma levels of Pi are similar in wild-type and NaPi-IIb-deficient mice. In females, the reduced phosphaturia associates with higher expression of NaPi-IIa and higher Na/Pi cotransport activity in renal BBMVs, as well as with reduced plasma levels of intact FGF-23. A similar trend is found in males. Thus, NaPi-IIb is the only luminal Na(+) -dependent Pi transporter in the murine ileum and its absence is fully compensated for in adult females by a mechanism involving the bone-kidney axis. The contribution of this mechanism to the adaptive response is less apparent in adult males. © 2015 American Society for Bone and Mineral Research.
Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe(2+) and H(+) cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na(+)/H(+) exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na(+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in β-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.
Iron is essential in oxygen transport and participates in many enzymatic systems in the body, with important roles in collagen synthesis and vitamin D metabolism. The relationship between iron and bone health comes from clinical observations in iron overload patients who suffered bone loss. The opposite scenario—whether iron deficiency, with or without anemia, affects bone metabolism—has not been fully addressed. This is of great interest, as this nutrient deficiency is a worldwide public health problem and at the same time osteoporosis and bone alterations are highly prevalent. This review presents current knowledge on nutritional iron deficiency and bone remodeling, the biomarkers to evaluate iron status and bone formation and resorption, and the link between iron and bone metabolism. Finally, it is hypothesized that chronic iron deficiency induces bone resorption and risk of osteoporosis, thus complete recovery from anemia and its prevention should be promoted in order to improve quality of life including bone health. Several mechanisms are suggested; hence, further investigation on the possible impact of chronic iron deficiency on the development of osteoporosis is needed.
Despite the importance of extracellular phosphate in many essential biological processes, the mechanisms of phosphate transport across the epithelium of different intestinal segments remain unclear. We have used an in vitro method to investigate phosphate transport at the brush border membrane (BBM) of intact intestinal segments and an in vivo method to study transepithelial phosphate absorption. We have used micromolar phosphate concentrations known to favor NaPi-IIb-mediated transport, and millimolar concentrations that are representative of the levels we have measured in luminal contents, to compare the extent of Na+-dependent and Na+-independent phosphate transport along the rat duodenum, jejunum, ileum, and proximal and distal colon. Our findings confirm that overall the jejunum is the main site of phosphate absorption; however, at millimolar concentrations, absorption shows ~30% Na+-dependency, suggesting that transport is unlikely to be mediated exclusively by the Na+-dependent NaPi-IIb co-transporter. In the ileum, studies in vitro confirmed that relatively low levels of phosphate transport occur at the BBM of this segment, although significant Na+-dependent transport was detected using millimolar levels of phosphate in vivo. Since NaPi-IIb protein is not detectable at the rat ileal BBM, our data suggest the presence of an as yet unidentified Na+-dependent uptake pathway in this intestinal segment in vivo. In addition, we have confirmed that the colon has a significant capacity for phosphate absorption. Overall, this study highlights the complexities of intestinal phosphate absorption that can be revealed using different phosphate concentrations and experimental techniques.
Intestines are organs that not only digest food and absorb nutrients, but also provide a defense barrier against pathogens and noxious agents ingested. Tight junctions (TJs) are the most apical component of the junctional complex, providing one form of cell-cell adhesion in enterocytes and playing a critical role in regulating paracellular barrier permeability. Alteration of TJs leads to a number of pathophysiological diseases causing malabsorption of nutrition and intestinal structure disruption, which may even contribute to systemic organ failure. Claudins are the major structural and functional components of TJs with at least 24 members in mammals. Claudins have distinct charge-selectivity, either by tightening the paracellular pathway or functioning as paracellular channels, regulating ions and small molecules passing through the paracellular pathway. In this review, we have discussed the functions of claudin family members, their distribution and localization in the intestinal tract of mammals, their alterations in intestine-related diseases and chemicals/agents that regulate the expression and localization of claudins as well as the intestinal permeability, which provide a therapeutic view for treating intestinal diseases.
Calbindin-D9k (CaBP-9k) binds calcium with high affinity and regulates the distribution of free calcium in the cytoplasm. The expression of CaBP-9k is detected primarily in intestine that is vitamin D target tissue, and accumulates in the enterocytes of the duodenal villi. These enterocytes are the clearest example of vitamin D responsive cells, and the presence of CaBP-9k within them accentuates calcium absorption mediated by active transcellular calcium transport. It has been well established that the expression of CaBP-9k is mediated with vitamin D response element on its promoter and it regulates the amount of intracellular calcium in order to prevent cell death from reaching the toxicity of free calcium. There is now little doubt that glucocorticoid also decreases CaBP-9k expression in duodenal epithelial cells. In addition, it was reported that the level of CaBP-9k gene in enterocytes is increased in pregnancy when the plasma estradiol concentration is generally associated with a concomitant increase. Although calcium homeostasis was not disturbed in mice lacking the CaBP-9k gene, we found that CaBP-9k has a buffering role of free calcium in the cytosolic environment beyond that of calcium transfer. To expand our knowledge of the biological functions of CaBP-9k, our research has focused on defining the biological significance of intracellular CaBP-9k. Our findings suggest that the CaBP-9k gene is involved in compensatory induction of other calcium transporter genes in duodenal epithelial cells. This article summarizes the findings from recent studies on the expression and the functions of CaBP-9k in the small intestine.
Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally,
all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport
protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence
identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally
related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase
CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in
brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were
detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed inXenopus oocytes, CaT1 mediates saturable Ca2+uptake with a Michaelis constant of 0.44 mm. Transport of Ca2+ by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca2+uptake ratio close to 2:1, indicating that CaT1-mediated Ca2+ influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is
also permeant to Sr2+ and Ba2+ (but not Mg2+), although the currents evoked by Sr2+ and Ba2+ are much smaller than those evoked by Ca2+. The trivalent cations Gd3+ and La3+ and the divalent cations Cu2+, Pb2+, Cd2+, Co2+, and Ni2+ (each at 100 μm) do not evoke currents themselves, but inhibit CaT1-mediated Ca2+ transport. Fe3+, Fe2+, Mn2+, and Zn2+ have no significant effects at 100 μm on CaT1-mediated Ca2+ transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D3 administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca2+ entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.
Intestinal alkaline phosphatase (IAP) is a brush-border phosphomonoesterase. Its location suggests an involvement in the uptake of nutrients, but its role has not yet been defined. IAP expression parallels that of other proteins involved in Ca absorption under vitamin D stimulation. Experiments carried out in vitro with purified IAP have demonstrated an interaction between Ca and IAP. The gut is prepared to face different levels of Ca intake over time, but high Ca intake in a situation of a low-Ca diet over time would cause excessive entry of Ca into the enterocytes. The presence of a mechanism to block Ca entry and to avoid possible adverse effects is thus predictable. Thus, in the present study, Sprague-Dawley rats were fed with different amounts of Ca in the diet (0.2, 1 and 2 g%), and the percentage of Ca absorption (%Ca) in the presence and absence of L-phenylalanine (Phe) was calculated. The presence of Phe caused a significant increase in %Ca (52.3 (SEM 6.5) % in the presence of Phe v. 31.1 (sem 8.9) % in the absence of Phe, regardless of the amount of Ca intake; paired t test, P = 0.02). When data were analysed with respect to Ca intake, a significant difference was found only in the group with low Ca intake (paired t test, P = 0.03). Additionally, IAP activity increased significantly (ANOVA, P < 0.05) as Ca concentrations increased in the duodenal lumen. The present study provides in vivo evidence that luminal Ca concentration increases the activity of IAP and simultaneously decreases %Ca, acting as a minute-to-minute regulatory mechanism of Ca entry.
Transport of phosphate across intestinal and renal epithelia is essential for normal phosphate balance, yet we know less about the mechanisms and regulation of intestinal phosphate absorption than we do about phosphate handling by the kidney. Recent studies have provided strong evidence that the sodium-phosphate cotransporter NaPi-IIb is responsible for sodium-dependent phosphate absorption by the small intestine, and it might be that this protein can link changes in dietary phosphate to altered renal phosphate excretion to maintain phosphate balance. Evidence is also emerging that specific regions of the small intestine adapt differently to acute or chronic changes in dietary phosphate load and that phosphatonins inhibit both renal and intestinal phosphate transport. This review summarizes our current understanding of the mechanisms and control of intestinal phosphate absorption and how it may be related to renal phosphate reabsorption; it also considers the ways in which the gut could be targeted to prevent, or limit, hyperphosphatemia in chronic and end-stage renal failure.
In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R
epi). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R
epi, followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R
trans or TER) overestimated true R
epi by ~60%. In colon, strongest expression of “tightening” claudins 1, 3, 4, 5, and 8 was detected. In accordance with R
epi the most proximal of the small intestinal segments, duodenum exhibited highest expression of “tightening” claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.
Intestinal phosphate absorption occurs through both a paracellular mechanism involving tight junctions and an active transcellular mechanism involving the type II sodium-dependent phosphate cotransporter NPT2b (SLC34a2). To define the contribution of NPT2b to total intestinal phosphate absorption, we generated an inducible conditional knockout mouse, Npt2b(-/-) (Npt2b(fl/fl):Cre(+/-)). Npt2b(-/-) animals had increased fecal phosphate excretion and hypophosphaturia, but serum phosphate remained unchanged. Decreased urinary phosphate excretion correlated with reduced serum levels of the phosphaturic hormone FGF23 and increased protein expression of the renal phosphate transporter Npt2a. These results demonstrate that the absence of Npt2b triggers compensatory renal mechanisms to maintain phosphate homeostasis. In animals fed a low phosphate diet followed by acute administration of a phosphate bolus, Npt2b(-/-) animals absorbed approximately 50% less phosphate than wild-type animals, confirming a major role of this transporter in phosphate regulation. In vitro analysis of active phosphate transport in ileum segments isolated from wild-type or Npt2b(-/-) mice demonstrated that Npt2b contributes to >90% of total active phosphate absorption. In summary, Npt2b is largely responsible for intestinal phosphate absorption and contributes to the maintenance of systemic phosphate homeostasis.
Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.
Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.
The role of putative humoral factors, known as phosphatonins, in phosphate homeostasis and the relationship between phosphate handling by the kidney and gastrointestinal tract are incompletely understood. Matrix extracellular phosphoglycoprotein (MEPE), one of several candidate phosphatonins, promotes phosphaturia, but whether it also affects intestinal phosphate absorption is unknown. Here, using the in situ intestinal loop technique, we demonstrated that short-term infusion of MEPE inhibits phosphate absorption in the jejunum but not the duodenum. Simultaneous measurement of urinary phosphate excretion suggests that the phosphaturic action of MEPE correlates with a significant reduction in the protein levels of the renal sodium-phosphate co-transporter NaPi-IIa in the proximal convoluted tubules of the outer renal cortex, assessed by Western blotting and immunohistochemistry. This short-term inhibitory effect of MEPE on renal and intestinal phosphate handling occurred without any changes in circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D(3), or fibroblast growth factor 23. Taken together, these findings suggest that MEPE is a candidate phosphatonin involved in phosphate homeostasis, acting in both the kidney and the gastrointestinal tract.
The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis.
This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body.
An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter
(DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed
in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport
in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent
mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of
the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.
The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.
Phosphate/calcium homeostasis is crucial for health maintenance. Lithocholic acid, a bile acid produced by intestinal bacteria, is an agonist of vitamin D receptor. However, its effects on phosphate/calcium homeostasis remain unclear. Here, we demonstrated that lithocholic acid increases intestinal phosphate/calcium absorption in an enterocyte vitamin D receptor-dependent manner. Lithocholic acid was found to increase serum phosphate/calcium levels and thus to exacerbate vascular calcification in animals with chronic kidney disease. Lithocholic acid did not affect levels of intestinal sodium-dependent phosphate transport protein 2b, Pi transporter-1, -2, or transient receptor potential vanilloid subfamily member 6. Everted gut sac analyses demonstrated that lithocholic acid increased phosphate/calcium absorption in a transcellular pathway-independent manner. Lithocholic acid suppressed intestinal mucosal claudin 3 and occludin in wild-type mice, but not in vitamin D receptor knockout mice. Everted gut sacs of claudin 3 knockout mice showed an increased permeability for phosphate, but not calcium. In patients with chronic kidney disease serum 1,25(OH)2 vitamin D levels were decreased, probably as an intrinsic adjustment to reduce phosphate/calcium burden. In contrast, serum and fecal lithocholic acid levels and fecal levels of bile acid 7α-dehydratase, a rate-limiting enzyme involved in lithocholic acid production, were not downregulated. The effects of lithocholic acid were eliminated by bile acid adsorptive resin in mice. Thus, lithocholic acid and claudin 3 may represent novel therapeutic targets for reducing phosphate burden.
Besides the two canonical calciotropic hormones, namely parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH)2D3], there are several other endocrine and paracrine factors, such as prolactin, estrogen, and insulin-like growth factor that have been known to directly stimulate intestinal calcium absorption. Generally, to maintain an optimal plasma calcium level, these positive regulators enhance calcium absorption, which is indirectly counterbalanced by a long-loop negative feedback mechanism, i.e., through calcium-sensing receptor in the parathyroid chief cells. However, several lines of recent evidence have revealed the presence of calcium absorption inhibitors present in the intestinal lumen and extracellular fluid in close vicinity to enterocytes, which could also directly compromise calcium absorption. For example, luminal iron, circulating fibroblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive calcium uptake under certain conditions. Interestingly, the intestinal epithelial cells themselves could lower their rate of calcium uptake after exposure to high luminal calcium concentration, suggesting a presence of an ultra-short negative feedback loop independent of systemic hormones. The existence of neural regulation is also plausible but this requires more supporting evidence. In the present review, we elaborate on the physiological significance of these negative feedback regulators of calcium absorption, and provide evidence to show how our body can efficiently restrict a flood of calcium influx in order to maintain calcium homeostasis.
Inorganic phosphate (Pi) is crucial for many biological functions such as energy metabolism, signal transduction and pH buffering. Efficient systems must exist to ensure sufficient supply of the body with Pi from diet. Previous experiments in humans and rodents suggest that two pathways for the absorption of Pi exist, an active transcellular Pi transport and a second paracellular pathway. Whereas the identity, role and regulation of active Pi transport has been extensively studied, much less is known about the properties of the paracellular pathway. In Ussing chamber experiments we characterized paracellular intestinal Pi permeabilities and fluxes. Dilution potential measurements in intestinal cell culture models demonstrated that the tight junction is permeable to Pi, with monovalent Pi having a higher permeability than divalent Pi. These findings were confirmed in rat and mouse intestinal segments using Ussing chambers and a combination of dilution potential measurements and fluxes of radiolabeled 32Pi. Both techniques yielded very similar results showing that paracellular Pi fluxes were bidirectional and that Pi permeability was about 50% of the permeability for Na+ or Cl-. Pi fluxes were a function of the concentration gradientand Pi species (mono- vs. divalent Pi). In mice lacking the active transcellular Pi transport component, NaPi-IIb, the paracellular pathway was not upregulated. In summary, the small and large intestine have a very high paracellular Pi permeability that may favor monovalent Pi fluxes and allows efficient uptake of Pi even in the absence of active transcellular Pi uptake.
Previously, β-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of β-globin gene, has been reported to induce osteopenia and impaired whole-body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how β-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous β-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermate, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice, which could be restored by 7-day 1,25(OH)2D3 treatment. Such the 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, PMCA1b, as well as vesicular transport inhibitor. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors, MG132 and bortezomib. In conclusion, 1,25(OH)2D3 impairment of calcium absorption. Our study has, therefore, shed light on the development of treatment strategy to rescue calcium dysregulation in β-thalassemia.
Control of serum phosphorus (PO4) has been long recognized as a goal in the nutritional and medical management of the patients with chronic kidney disease. Phosphate-binding compounds were introduced in the 1970s for the treatment of hyperphosphatemia in patients on dialysis after it was observed that oral administration of aluminum hydroxide as an antacid also reduced serum PO4 levels. Forty years later, aluminum is very seldom used as a phosphate binder as many other safer compounds are now available. This article is a comprehensive review, geared to the renal dietitian, of the most common binder categories. It will discuss pharmacokinetics, side effects, initial and optimal doses, phosphate affinity, and controversies of use. It will also review two novel approaches to serum PO4 management in chronic kidney disease patients receiving dialysis and provide a new calculation by which binders can be compared.
The Na(+) -dependent phosphate-cotransporter NaPi-IIb (SLC34A2) is widely expressed, with intestine, lung and testis among the organs with highest levels of mRNA abundance. In mice, the intestinal expression of NaPi-IIb is restricted to the ileum, where the cotransporter localizes specifically at brush border membrane (BBM) and mediates the active transport of inorganic phosphate (Pi). Constitutive full ablation of NaPi-IIb is embryonically lethal whereas the global but inducible removal of the transporter in young mice leads to intestinal loss of Pi and lung calcifications. Here we report the generation of a constitutive but intestinal-specific NaPi-IIb/Slc34a2 deficient mouse model. Constitutive intestinal ablation of NaPi-IIb results in viable pups with normal growth. Homozygous mice are characterized by fecal wasting of Pi and complete absence of Na/Pi cotransport activity in BBM vesicles (BBMV) isolated from ileum. In contrast, the urinary excretion of Pi is reduced in these animals. The plasma levels of Pi are similar in wild type and NaPi-IIb deficient mice. In females, the reduced phosphaturia associates with higher expression of NaPi-IIa and higher Na/Pi cotransport activity in renal BBMV, as well as with reduced plasma levels of intact FGF-23. A similar trend is found in males. Thus, NaPi-IIb is the only luminal Na(+) -dependent Pi transporter in the murine ileum and its absence is fully compensated in adult females by a mechanism involving the bone-kidney axis. The contribution of this mechanism to the adaptive response is less apparent in adult males. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Calcium (Ca(2+)) is a key constituent in a myriad of physiological processes from intracellular signalling to the mineralization of bone. As a consequence, Ca(2+) is maintained within narrow limits when circulating in plasma. This is accomplished via regulated interplay between intestinal absorption, renal tubular reabsorption, and exchange with bone. Many studies have focused on the highly regulated active transcellular transport pathways for Ca(2+) from the duodenum of the intestine and the distal nephron of the kidney. However, comparatively little work has examined the molecular constituents creating the paracellular shunt across intestinal and renal epithelium, the transport pathway responsible for the majority of transepithelial Ca(2+) flux. More specifically, passive paracellular Ca(2+) absorption occurs across the majority of the intestine in addition to the renal proximal tubule and thick ascending limb of Henle's loop. Importantly, recent studies demonstrated that Ca(2+) transport through the paracellular shunt is significantly regulated. Therefore, we have summarized the evidence for different modes of paracellular Ca(2+) flux across renal and intestinal epithelia and highlighted recent molecular insights into both the mechanism of secondarily active paracellular Ca(2+) movement and the identity of claudins that permit the passage of Ca(2+) through the tight junction of these epithelia.
The tight junction creates an intercellular barrier limiting paracellular movement of solutes and material across epithelia. Currently many proteins have been identified as components of the tight junction and understanding their architectural organization and interactions is critical to understanding the biology of the barrier. In general the architecture can be conceptualized into compartments with the transmembrane barrier proteins (claudins, occludin, JAM-A, etc.), linked to peripheral scaffolding proteins (such as ZO-1, afadin, MAGI1, etc.) which are in turned linked to actin and microtubules through numerous linkers (cingulin, myosins, protein 4.1, etc.). Within this complex network are associated many signaling proteins that affect the barrier and broader cell functions. The PDZ domain is a commonly used motif to specifically link individual junction protein pairs. Here we review some of the key proteins defining the tight junction and general themes of their organization with the perspective that much will be learned about function by characterizing the detailed architecture and subcompartments within the junction.
Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions.
Intestinal iron absorption is a critical process for maintaining body iron levels within the optimal physiological range. Iron in the diet is found in a wide variety of forms, but the absorption of non-heme iron is best understood. Most of this iron is moved across the enterocyte brush border membrane by the iron transporter divalent metal-ion transporter 1, a process enhanced by the prior reduction of the iron by duodenal cytochrome B and possibly other reductases. Enterocyte iron is exported to the blood via ferroportin 1 on the basolateral membrane. This transporter acts in partnership with the ferroxidase hephaestin that oxidizes exported ferrous iron to facilitate its binding to plasma transferrin. Iron absorption is controlled by a complex network of systemic and local influences. The liver-derived peptide hepcidin binds to ferroportin, leading to its internalization and a reduction in absorption. Hepcidin expression in turn responds to body iron demands and the BMP-SMAD signaling pathway plays a key role in this process. The levels of iron and oxygen in the enterocyte also exert important influences on iron absorption. Disturbances in the regulation of iron absorption are responsible for both iron loading and iron deficiency disorders in humans.
The acquisition and maintenance of bone mass and strength are influenced by environmental factors, including physical activity and nutrition. Among micronutrients, calcium (Ca) and inorganic (i) phosphate (P) are the two main constituents of hydroxyapatite, the bone mineral that strengthens the mechanical resistance of the organic matrix. Bone contains about 99% and 80% of the body's entire supply of Ca and P, respectively. The Ca/P mass ratio in bone is 2.2, which is similar to that measured in human milk. The initial step of Ca-Pi crystal nucleation takes place within matrix vesicles that bud from the plasma membrane of osteogenic cells and migrate into the extracellular skeletal compartment. They are endowed with a transport system that accumulates Pi inside the matrix vesicles, followed by the influx of Ca ions. This process leads to the formation of hydroxyapatite crystal and its subsequent association with the organic matrix collagen fibrils. In addition to this structural role, both Ca and Pi positively influence the activity of bone-forming and bone-resorbing cells. Pi plays a role in the maturation of osteocytes, the most abundant cells in bone. Osteocytes are implicated in bone mineralization and systemic Pi homeostasis. They produce fibroblast growth factor-23, a hormonal regulator of renal Pi reabsorption and 1,25-dihydroxy vitamin D production. This relationship is in keeping with the concept proposed several decades ago of a bone-kidney link in Pi homeostasis. In contrast to their tight association in bone formation and resorption, Ca and Pi renal reabsorption processes are independent from each other, driven by distinct molecular machineries. The distinct renal control is related to the different extraskeletal functions that Ca and Pi play in cellular metabolism. At both the renal and the intestinal levels, interactions of Ca and Pi have been documented that have important implications in the acquisition and maintenance of bone health, as well as in osteoporosis management. In the kidney, increased Pi intake enhances Ca reabsorption and Ca balance. During growth and adulthood, administration of Ca-Pi in a ratio close to that of dairy products leads to positive effects on bone health. In contrast, when separately ingested as pharmaceutical salt supplements, thus inducing large differences between Ca and Pi concentrations in the intestinal lumen, they might have adverse effects on bone health. In osteoporotic patients treated with anabolic agents, a Ca-Pi supplement appears to be preferable to carbonate or citrate Ca salt. In conclusion, Ca and Pi constitute a key duo for appropriate bone mineral acquisition and maintenance throughout life. Outside the skeleton, their essential but distinct physiological functions are controlled by specific transporters and hormonal systems that also serve to secure the appropriate supply of Ca and Pi for bone health. Key teaching points: Bone contains about 99% and 80% of the body's supply of Ca and P, respectively, as hydroxyapatite and has a Ca/P mass ratio of about 2.2, close to that measured in human milk. The first step of Ca-Pi crystal nucleation takes place within matrix vesicles that bud from the plasma membrane of osteogenic cells. In addition to their structural role, both Ca and Pi influence bone-forming and bone-resorbing cells. There is a bone-kidney link in Pi homeostasis in which fibroblast growth factor-23, a molecule produced by osteocytes, appears to play a pivotal role. In contrast to their tight association during bone formation and resorption, both intestinal and renal Ca and Pi processes are independent of each other. Observational and interventional studies suggest that Ca-Pi salt or dairy products can exert positive effects on bone acquisition and maintenance.
Hepcidin is a peptide hormone secreted by the liver in response to iron loading and inflammation. Decreased hepcidin leads
to tissue iron overload, whereas hepcidin overproduction leads to hypoferremia and the anemia of inflammation. Ferroportin
is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells. Here
we report that hepcidin bound to ferroportin in tissue culture cells. After binding, ferroportin was internalized and degraded,
leading to decreased export of cellular iron. The posttranslational regulation of ferroportin by hepcidin may thus complete
a homeostatic loop: Iron regulates the secretion of hepcidin, which in turn controls the concentration of ferroportin on the
cell surface.
The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.
Phosphate is absorbed in the small intestine by a minimum of 2 distinct mechanisms: paracellular phosphate transport which is dependent on passive diffusion, and active transport which occurs through the sodium-dependent phosphate cotransporters. Despite evidence emerging for other ions, regulation of the phosphate-specific paracellular pathways remains largely unexplored. In contrast, there is a growing body of evidence that active transport through the sodium-dependent phosphate cotransporter, Npt2b, is highly regulated by a diverse set of hormones and dietary conditions. Furthermore, conditional knockout of Npt2b suggests that it plays an important role in maintenance of phosphate homeostasis by coordinating intestinal phosphate absorption with renal phosphate reabsorption. The knockout mouse also suggests that Npt2b is responsible for the majority of sodium-dependent phosphate uptake. The type-III sodium-dependent phosphate transporters, Pit1 and Pit2, contribute to a minor role in total phosphate uptake. Despite coexpression along the apical membrane, differential responses of Pit1 and Npt2b regulation to chronic versus dietary changes illustrates another layer of phosphate transport control. Finally, a major problem in patients with CKD is management of hyperphosphatemia. The present evidence suggests that targeting key regulatory pathways of intestinal phosphate transport may provide novel therapeutic approaches for patients with CKD.
The mechanism by which hepcidin regulates iron export from macrophages has been well established and is believed to involve degradation of ferroportin. However, in the small intestine, hepcidin's mechanisms of action are not known. We studied human polarized intestinal (Caco-2/TC7) cells and mouse duodenal segments, ex vivo, to investigate the molecular mechanisms by which hepcidin down-regulates intestinal transepithelial iron transport.
Iron transport was analyzed using ⁵⁵FeNTA. Expression of Divalent Metal Transporter 1 (DMT1) and ferroportin was evaluated by reverse-transcription quantitative polymerase chain reaction and immunoblotting. Videomicroscopy analysis was performed on live cells that expressed either DMT1 or ferroportin fused to green fluorescent protein.
In Caco-2/TC7 cells, physiologic doses of hepcidin (50-1000 nmol/L) inhibited transport of ⁵⁵Fe in a dose-dependent manner; a half-maximum effect was observed at 75-100 nmol/L. However, 200 nmol/L hepcidin induced a significant decrease in DMT1 protein expression but no change in ferroportin protein levels, unlike macrophages. This result was confirmed ex vivo in isolated duodenal segments: 200 nmol/L hepcidin induced a significant reduction in iron transport and DMT1 protein levels but no change in ferroportin levels. In Caco-2/TC7 cells, the effect of hepcidin on the DMT1 protein level was completely abolished in the presence of a proteasome inhibitor (MG-132); DMT1 ubiquitination was induced by the addition of hepcidin.
An acute increase in hepcidin concentration reduces intestinal iron absorption through ubiquitin-dependent proteasome degradation of DMT1.
A prerequisite of epithelial transport is a paracellular barrier function, which seals the tissue against an uncontrolled leak flux. Moreover, selective paracellular permeability has been shown to be crucial for physiological epithelial transport function. Claudins are tetraspan tight junction proteins which play a major role in paracellular ion permeability across epithelia. The multigene family consists of 24 members and several splice variants which show distinct tissue-specific expression profiles. Moreover, in diseases associated with a loss of barrier function such as forms of inflammatory bowel disease, the expression of claudins is altered. Functional characterization of single claudins revealed specific contribution to barrier properties in epithelia. This review gives an overview on the exploration of molecular structure and barrier function along the intestine and nephron, which not only share mechanisms of selective restriction of the paracellular pathway but also exhibit distinct organ-specific characteristics.
The purpose of this study was to clarify the manner in which dietary iron deficiency decreased bone mineral density (BMD) in rats. Eighteen 3-wk-old male Wistar rats were divided into 3 groups of 6 rats each. The rats in 2 of the 3 groups had free access to a control diet (C group) or an iron-deficient diet (ID group) for 4 wk. The rats in the third group (PF group) were pair-fed the control diet to the mean intake of the ID group. Compared with the C and PF groups, hematocrit and hemoglobin concentrations were significantly reduced and bone mineral content and BMD of the femur were significantly lower in the ID group. Bone histomorphometric parameters showed that the bone formation rate and osteoclast surface in the lumbar vertebra were significantly reduced in the ID group compared with the C and PF groups. Furthermore, dietary iron deficiency decreased serum 1,25-dihydroxycholecalciferol, insulin-like growth factor-I, and osteocalcin concentrations and urinary excretion of deoxypyridinoline. These results suggest that severe iron deficiency decreases not only bone formation but also bone resorption.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Kinetic analysis of transmural calcium transport, as evaluated by in situ intestinal loops, has confirmed the existence of two transport processes, a saturable, transcellular one that is regulated by vitamin D and predominates in the proximal intestine and a nonsaturable process similar in intensity throughout the intestine. Transport data obtained from everted sac experiments are kinetically consistent with events in the in situ loop. Analysis of the three component steps making up the saturable process, i.e., entry across the brush-border membrane, intracellular diffusion, and extrusion across the basolateral membrane, indicates that intracellular diffusion is likely to be the limiting step. Active calcium transport varies directly and proportionately with the content of calcium-binding protein (CaBP), a specific molecular expression of the action of vitamin D. Since CaBP is a cytosolic protein, it may act to facilitate calcium diffusion, a proposition advanced by Kretsinger, Mann, and Simmons and supported here quantitatively. We calculate that the rate of intracellular calcium diffusion in the absence of CaBP is only approximately 1/70 of what is found in the vitamin D-replete cell. Similar considerations have led to the proposal that calcium moved by the nonsaturable process travels largely via the paracellular route. The kinetic parameters derived here, i.e., Vm = 22 mumol X h-1 X g (wt wt-1, Km = 3.9 mM, and a nonsaturable rate of 0.16/h, can be used to predict calcium absorption data as determined in previously published balance experiments.
The electrical parameters and the unidirectional fluxes of 45Ca and 3H-mannitol were measured in preparations of rat colon descendens freed from the muscularis externa and mounted in a modified Ussing-chamber. Two criteria were used to differentiate between changes in the trans- and the paracellular calcium transport after treatment with 1,25(OH)2D3: the fluxes of the simultaneously measured 3H-mannitol as a paracellular marker; the 45Ca fluxes in preparations with clamped potentials. After a short-time (6 h) pretreatment by s.c. administration of 1,25(OH)2D3 (250 ng kg-1) in normal rats the mucosa (m) to serosa (s) 45Ca flux under short circuit conditions increased about 65%, whereas the electrical parameters and the 3H-mannitol fluxes remained unchanged. In clamped epithelia the PD-independent m to s 45Ca flux was increased, whereas the PD-dependent flux remained unchanged. In contrast, after long-time (4 days) induction by 1,25(OH)2D3 the m to s 45Ca flux increased under short circuit conditions by about 100% and the m to s 3H-mannitol flux increased by 50%, PD and Isc decreased by more than 60%, whereas tissue resistance was the same, in clamped epithelia the calculated PD-independent, transcellular m to s 45Ca flux was 2.4 times and the PD-dependent, paracellular 45Ca-flux was 1.9 times higher than in controls, whereas the s to m 45Ca flux remained unchanged. On the basis of the relevant references the following conclusions were drawn: after short-time exposure to 1,25(OH)2D3 only the PD-dependent, transcellular m to s calcium transport is increased; this is probably due to a liponomic effect of 1,25(OH)2D3 at the brush border membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Concentration dependence of unidirectional calcium fluxes across the rat colon ascendens were measured in a modified Ussing chamber. Both the mucosa (m) to serosa (s) and the s to m calcium flux exhibited saturation kinetics. The maximum transport rates and the affinity to the transporter of calcium was higher in the m to s direction than that from s to m, resulting in a remarkable net calcium absorption. The results obtained from measurements of unidirectional calcium fluxes in dependence on clamped transepithelial potentials showed that: (i) calcium transport in both direction had a voltage-independent component; (ii) the voltage-independent, i.e. non diffusive fraction of the m to s calcium flux was 3.2 times greater than that in the opposite direction; (iii) the voltage dependent, i.e. diffusional fraction of the m to s calcium flux, was about two times greater than the voltage-dependent fraction of the calcium flux in the s to m direction; and (iv) in the m to s direction 62%, and the s to m direction 73%, of the total unidirectional flux was voltage-dependent. Dexamethasone, known to enhance sodium and water absorption in the colon, had no significant influence on net calcium absorption but increased the unidirectional calcium fluxes in both directions. The increase in unidirectional calcium fluxes parallel to that of the extracellular marker mannitol suggests that dexamethasone has no influence on the transcellular calcium transport but increases the calcium flux along the paracellular way. Amiloride had no influence on the dexamethasone-induced changes of the epithelial electrical parameters as distinguished from the colon descendens.(ABSTRACT TRUNCATED AT 250 WORDS)
The direct effects of verapamil, diltiazem and nifedipine on duodenal calcium transport were assessed in rats by the in vivo ligated loop technique, using luminal calcium concentrations at which active and passive transport mechanisms predominate (2 and 50 mM Ca, respectively). At 2 mM Ca, addition of verapamil (0.3-10 mM) to the luminal solution caused a concentration-dependent decrease in calcium lumen-to-plasma uptake and an increase in calcium plasma-to-lumen translocation, such that at 10 mM verapamil there was a net secretion of calcium into the duodenal lumen. In contrast, nifedipine (0.3-3 mM) was without effect on calcium transport, and diltiazem reduced calcium lumen-to-plasma uptake and net calcium absorption only at 10 mM, without influencing plasma-to-lumen translocation. The verapamil-induced increase in calcium plasma-to-lumen translocation was abolished by bile duct ligation. Calcium transport was unaffected by any calcium channel blocker at 50 mM luminal calcium. Thus, verapamil can directly influence active calcium translocation in the intestine, in vivo, and may affect calcium homeostasis during chronic oral treatment with this drug.
1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.
An in situ ligated loop procedure was applied to dissect transmural calcium transport in the intestine into two components, a saturable and a nonsaturable process. The existence of two such processes was confirmed in the duodenum, but ileal calcium transport was devoid of the saturable component. There was a small saturable component in the upper jejunum. The level of CaBP, the vitamin D-dependent cytosolic calcium-binding protein (Mr, approximately or equal to 9,000), corresponded to the magnitude of the saturable component. No CaBP was detected in the ileum. Vitamin D dependence of the saturable component was established by inducing it in the duodenum of vitamin D-deficient animals following intraperitoneal injection of 1,25-dihydroxyvitamin D3. In these same animals, conversely, the ileum did not respond to exogenous 1,25-dihydroxyvitamin D3. This confirms the absence in the ileum of the saturable component of transmural calcium movement and the fact that the nonsaturable component is not vitamin D dependent. Everted sac experiments also showed that duodenal sacs from vitamin D-replete or -repleted animals transported calcium against a chemical gradient, whereas ileal sacs did not. Vitamin D regulation of intestinal calcium absorption thus occurs only in the proximal intestine, even though calcium is absorbed down its chemical gradient all along the small intestine.
Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5 mM MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 microgram/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na +K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 microgram/ml oliomycin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal lateral membranes possess two distinct mechanisms for Ca-transport: a) ATP-driven Ca-transport and b) Na/Ca-exchange.
We studied the development of nutritional iron deficiency 0, 10, 20, 30 and 40 days after the intake of a semisynthetic diet lacking iron (diet 0) and the possible interactions with calcium, phosphorus and magnesium in both control rats and rats after 40 days of iron deficiency. During this period, iron deficiency was found to produce stress in the rats, as evidenced by high levels of cortisol in the serum. High levels of parathyroid hormone (PTH) were also found. There was a considerable increase in the absorption of calcium, phosphorus and magnesium, but the phosphorus and magnesium balance decreased and that of calcium remained practically unchanged, although there was an increase in calcium urinary elimination. Despite the noticeable degree of bone demineralization, which was evident in the femur, serum levels of calcium, phosphorus and magnesium remained constant. The present study shows that severe nutritional ferropenic anaemia provokes significant alterations in the metabolism of calcium, phosphorus and magnesium. We conclude that these alterations should be taken into account in the treatment of this pathology, given its prevalence and the fact that it may exacerbate other pathologies, particularly those related to the metabolism of calcium and phosphorus.
Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are differentially expressed consistent with a potential role in creating these variable properties.
Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine.
All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis.
Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.
Calcium is absorbed in the mammalian small intestine by two general mechanisms: a transcellular active transport process, located largely in the duodenum and upper jejunum; and a paracellular, passive process that functions throughout the length of the intestine. The transcellular process involves three major steps: entry across the brush border, mediated by a molecular structure termed CaT1, intracellular diffusion, mediated largely by the cytosolic calcium-binding protein (calbindinD(9k) or CaBP); and extrusion, mediated largely by the CaATPase. Chyme travels down the intestinal lumen in approximately 3 h, spending only minutes in the duodenum, but over 2 h in the distal half of the small intestine. When calcium intake is low, transcellular calcium transport accounts for a substantial fraction of the absorbed calcium. When calcium intake is high, transcellular transport accounts for only a minor portion of the absorbed calcium, because of the short sojourn time and because CaT1 and CaBP, both rate-limiting, are downregulated when calcium intake is high. Biosynthesis of CaBP is fully and CaT1 function is approximately 90% vitamin D-dependent. At high calcium intakes CaT1 and CaBP are downregulated because 1,25(OH)(2)D(3), the active vitamin D metabolite, is downregulated.
Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.
We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D(9k) expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] injection strongly regulated duodenal calbindin D(9k) and CaT1 mRNA. Following a single dose of 1,25(OH)(2)D(3), induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 +/- 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)(2)D(3) injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)(2)D(3) in kidney and intestine and that there may be a specialized role for CaT1 in kidney in fetal and neonatal development. The rapid induction of intestinal CaT1 mRNA expression by 1,25(OH)(2)D(3), and the marked induction at weaning, suggest that CaT1 is critical for 1,25(OH)(2)D(3)-mediated intestinal Ca absorption.