scholarly journals Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eva Maria Pastor-Arroyo ◽  
Josep M. Monné Rodriguez ◽  
Giovanni Pellegrini ◽  
Carla Bettoni ◽  
Moshe Levi ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Intestinal Absorption ◽  
Intestinal Tract ◽  
Apical Membrane ◽  
Wild Type ◽  
Intestinal Epithelia ◽  
Dietary Phosphate ◽  
Perinatal Lethality ◽  
Paracellular Pathways ◽  
Inactivating Mutations

AbstractAbsorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na+/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

2002 ◽  
Vol 282 (5) ◽  
pp. G776-G784 ◽  
Author(s):  
Lara R. Gawenis ◽  
Xavier Stien ◽  
Gary E. Shull ◽  
Patrick J. Schultheis ◽  
Alison L. Woo ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Intestinal Tract ◽  
Apical Membrane ◽  
Intracellular Camp ◽  
Wild Type ◽  
Rt Pcr ◽  
Nacl Transport ◽  
Nacl Absorption ◽  
Small Intestinal ◽  
Absorptive Flux

Sodium/proton exchangers [Na+/H+ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+ and Cl− flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na+ (∼6 μeq/cm2 · h) and Cl− (∼3 μeq/cm2 · h) absorption. In contrast, NHE3(−) jejuna had reduced net Na+ absorption (∼2 μeq/cm2 · h) but absorbed Cl− at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-( N-ethyl- N-isopropyl) amiloride (EIPA) completely inhibited net Na+ and Cl−absorption in all genotypes. Studies of the Na+ absorptive flux ( J [Formula: see text]) indicated that J [Formula: see text] in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereas J [Formula: see text] in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl− secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+ absorption; 2) an amiloride-sensitive Na+ transporter partially compensates for Na+ absorption in NHE3(−) jejunum; 3) cAMPi stimulation abolishes net Na+ absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl− absorption is not directly dependent on either NHE2 or NHE3.

Cardiac expression of a gain-of-function α5-integrin results in perinatal lethality

2001 ◽  
Vol 280 (1) ◽  
pp. H361-H367 ◽  
Author(s):  
Maria L. Valencik ◽  
John A. McDonald
Keyword(s):  
Cardiac Development ◽  
Wild Type ◽  
Integrin Receptors ◽  
Gain Of Function ◽  
Transgenic Expression ◽  
Physiological Regulation ◽  
Myocyte Differentiation ◽  
Perinatal Lethality ◽  
And Function

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. α5β1-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of α5β1-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human α5-integrin in cardiomyocytes: the full-length wild-type α5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated α5-1-integrin. Overexpression of the wild-type α5-integrin has no detectable adverse effects in the mouse, whereas expression of α5-1-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.

Intestinal absorption of calcium: role of dietary phosphate and vitamin D

1981 ◽  
Vol 241 (1) ◽  
pp. G49-G53
Author(s):  
N. Brautbar ◽  
B. S. Levine ◽  
M. W. Walling ◽  
J. W. Coburn
Keyword(s):  
Vitamin D ◽  
Intestinal Absorption ◽  
Vitamin D3 ◽  
Control Groups ◽  
Dihydroxyvitamin D3 ◽  
Dietary Phosphorus ◽  
P Concentration ◽  
Dietary Phosphate ◽  
Intestinal Ca Absorption

The intestinal absorption of calcium (Ca) has been shown to depend on vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary phosphorus (P) concentration. This study was designed to evaluate the role of dietary P independent of vitamin D3 or 1,25(OH)2D3. Vitamin D-deficient rats were studied during dietary P restriction and were compared with control groups raised on a normal-phosphorus diet (NP). Balance studies were sued. Net intestinal Ca absorption was significantly lower with dietary P restriction compared with the NP group. This malabsorption of Ca was corrected by the administration of either D3 for 1,25(OH)2D3, despite hypophosphatemia. Everted gut sacs showed a marked reduction in the uptake of 45Ca in the duodenum, jejunum, and ileum during dietary P restriction. We concluded that dietary P concentration plays a major role in intestinal Ca absorption in the vitamin D-deficient rats. These findings suggest an effect of the low-phosphate diet on the vitamin D-dependent, Ca-transport mechanism.

Modern Eggs, Not Wild Type Eggs, Predispose Risk of Cardiovascular Disease, Diabetes and Cancer?

2019 ◽  
pp. 301-314
Author(s):  
Dominic Pella ◽  
Jan Fedacko ◽  
Daniel Pella ◽  
Viola Vargova ◽  
Vilium Mojto ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Wild Type

scholarly journals Attenuation of myocardial injury in mice with functional deletion of the circadian rhythm gene mPer2

2010 ◽  
Vol 298 (3) ◽  
pp. H1088-H1095 ◽  
Author(s):  
Jitka A. I. Virag ◽  
Jessica L. Dries ◽  
Peter R. Easton ◽  
Amy M. Friesland ◽  
Jon H. DeAntonio ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Disease ◽  
Circadian Rhythm ◽  
Contractile Function ◽  
Myocardial Infarct ◽  
Inflammatory Cells ◽  
Diurnal Rhythms ◽  
Cardiomyocyte Hypertrophy ◽  
Wild Type ◽  
Infarct Zone

Variations in circadian rhythms are evident in the incidence of cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. The suprachiasmatic nucleus is the central circadian pacemaker that regulates the daily rhythm of peripheral organs. Diurnal rhythms have more recently been shown to exist in myocardial tissue and are involved in metabolism and contractile function. Thus we sought to determine whether the functional deletion of the circadian rhythm mouse periodic gene 2 (mPer2) would protect the heart against ischemic injury. Nonreperfused myocardial infarction was induced in anesthetized, ventilated C57 ( n = 17) and mPer2 mutant (mPer2-M; n = 15) mice via permanent ligation of the left anterior descending coronary artery. At 4 days post-myocardial infarction, we observed a 43% reduction of infarct area in mPer2-M mice compared with wild-type mice. This is coincident with 25% less macrophage infiltration, 43% higher capillary density, 17% increase in hypertrophy, and 15% less cardiomyocyte apoptosis in the infarct zone. Also, matrix metalloproteinase-9 was expressed in inflammatory cells in both groups, but total protein was 40% higher in wild-type mice, whereas it was not elevated in mPer2-M mice in response to injury. The functional deletion of the mPer2 gene reduces the severity of myocardial infarct injury by limiting the inflammatory response, reducing apoptosis, and inducing cardiomyocyte hypertrophy, thus preserving cardiac function. These findings collectively imply that the disruption of the circadian clock gene mPer2 is protective. Understanding the interactions between circadian rhythm genes and cardiovascular disease may provide insights into potential preventative and therapeutic strategies for susceptible populations.

scholarly journals PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP

2009 ◽  
Vol 297 (6) ◽  
pp. F1560-F1565 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
Boyoung Cha ◽  
Olga Kovbasnjuk ◽  
Nicholas A. Frost ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Binding Proteins ◽  
Apical Membrane ◽  
Wild Type ◽  
Lateral Mobility ◽  
Proximal Tubule Cells ◽  
Ok Cells ◽  
Mobile Fraction ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ∼17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary Pi

1999 ◽  
Vol 277 (5) ◽  
pp. F676-F684 ◽  
Author(s):  
Heini Murer ◽  
Ian Forster ◽  
Nati Hernando ◽  
Georg Lambert ◽  
Martin Traebert ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Proximal Tubule ◽  
Posttranscriptional Regulation ◽  
Apical Membrane ◽  
Tubular Reabsorption ◽  
Transport Capacity ◽  
Phosphate Intake ◽  
Proximal Tubular ◽  
Dietary Phosphate ◽  
Proximal Tubular Reabsorption

The rate of proximal tubular reabsorption of phosphate (Pi) is a major determinant of Pi homeostasis. Deviations of the extracellular concentration of Piare corrected by many factors that control the activity of Na-Pi cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule Pi reabsorption via one particular Na-Pi cotransporter (the type IIa cotransporter) by parathyroid hormone (PTH) and dietary phosphate intake. Available data indicate that both factors determine the net amount of type IIa protein residing in the apical membrane. The resulting change in transport capacity is a function of both the rate of cotransporter insertion and internalization. The latter process is most likely regulated by PTH and dietary Pi and is considered irreversible since internalized type IIa Na-Picotransporters are subsequently routed to the lysosomes for degradation.

Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice

1999 ◽  
Vol 277 (2) ◽  
pp. F298-F302 ◽  
Author(s):  
Tong Wang ◽  
Chao-Ling Yang ◽  
Thecla Abbiati ◽  
Patrick J. Schultheis ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Significant Inhibition ◽  
Fluid Absorption ◽  
Wild Type ◽  
Significant Component ◽  
Nhe Isoforms

NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3−/−mouse with greatly reduced proximal tubule[Formula: see text] absorption compared with NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282–285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule [Formula: see text] reabsorption in NHE3−/− mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of[Formula: see text] ( J HCO3) and fluid absorption ( J v) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 μM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J HCO3 and J v in NHE3+/+ mice but failed to inhibit J HCO3 or J v in NHE3−/− mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 μM bafilomycin caused a similar absolute decrement in J HCO3 in wild-type and NHE3 null mice, indicating equivalent rates of[Formula: see text] absorption mediated by H+-ATPase. Addition of 10 μM Sch-28080 did not reduce J HCO3 in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating [Formula: see text] reabsorption in the proximal tubule. A significant component of[Formula: see text] reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

scholarly journals Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid

2018 ◽  
Vol 314 (1) ◽  
pp. G81-G90 ◽  
Author(s):  
Leela Rani Avula ◽  
Tiane Chen ◽  
Olga Kovbasnjuk ◽  
Mark Donowitz
Keyword(s):  
Brush Border ◽  
Apical Membrane ◽  
Cell Model ◽  
Pdz Domain ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Dual Emission ◽  
Two Photon Microscopy ◽  
Two Photon

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

2007 ◽  
Vol 292 (4) ◽  
pp. G1079-G1088 ◽  
Author(s):  
Janet E. Simpson ◽  
Clifford W. Schweinfest ◽  
Gary E. Shull ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Wild Type ◽  
Rt Pcr ◽  
Targeted Deletion ◽  
Anion Transporter ◽  
Dependent Transport

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

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