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Kentucky bluegrass (Poa pratensis L.), a widely used cool-season turfgrass, shows a high sensitivity to soil salinity. Clarifying the adaptative mechanisms of Kentucky bluegrass that serve to improve its salt tolerance in saline environments is urgent for the application of this turfgrass in salt-affected regions. In this study, physiological responses of the Kentucky bluegrass cultivars "Explorer" and "Blue Best" to NaCl treatment, as well as gene expressions related to photosynthesis, ion transport, and ROS degradation, were analyzed. The results showed that the growth of "Explorer" was obviously better compared to "Blue Best" under 400 mM NaCl treatment. "Explorer" exhibited a much stronger photosynthetic capacity than "Blue Best" under NaCl treatment, and the expression of key genes involved in chlorophyll biosynthesis, photosystem II, and the Calvin cycle in "Explorer" was greatly induced by salt treatment. Compared with "Blue Best", "Explorer" could effectively maintain Na+/K+ homeostasis in its leaves under NaCl treatment, which can be attributed to upregulated expression of genes, such as HKT1;5, HAK5, and SKOR. The relative membrane permeability and contents of O2- and H2O2 in "Explorer" were significantly lower than those in "Blue Best" under NaCl treatment, and, correspondingly, the activities of SOD and POD in the former were significantly higher than in the latter. Moreover, the expression of genes involved in the biosynthesis of enzymes in the ROS-scavenging system of "Explorer" was immediately upregulated after NaCl treatment. Additionally, free proline and betaine are important organic osmolytes for maintaining hydration status in Kentucky bluegrass under NaCl treatment, as the contents of these metabolites in "Explorer" were significantly higher than in "Blue Best". This work lays a theoretical basis for the improvement of salt tolerance in Kentucky bluegrass.
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Na+ plays a vital role in numerous physiological processes across humans and animals, necessitating a comprehensive understanding of Na+ transmembrane transport. Among the various Na+ pumps and channels, light-driven Na+-pumping rhodopsin (NaR) has emerged as a noteworthy model in this field. This review offers a concise overview of the structural and functional studies conducted on NaR, encompassing ground/intermediate-state structures and photocycle kinetics. The primary focus lies in addressing key inquiries: (1) unraveling the translocation pathway of Na+; (2) examining the role of structural changes within the photocycle, particularly in the O state, in facilitating Na+ transport; and (3) investigating the timing of Na+ uptake/release. By delving into these unresolved issues and existing debates, this review aims to shed light on the future direction of Na+ pump research.
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Rodopsina , Animais , Humanos , Rodopsina/química , Transporte BiológicoRESUMO
Background: The basolateral potassium channels play an important role in maintaining the membrane transport in the renal proximal tubules (PT) and adenosine receptors have been shown to regulate the trans-epithelial Na+ absorption in the PT. The aim of the present study is to explore whether adenosine also regulates the basolateral K+ channel of the PT and to determine the adenosine receptor type and the signaling pathway which mediates the effect of adenosine on the K+ channel. Methods: We have used the single channel recording to examine the basolateral K+ channel activity in the proximal tubules of the mouse kidney. All experiments were performed in cell-attached patches. Results: Single channel recording has detected a 50 pS inwardly-rectifying K+ channel with high channel open probability and this 50 pS K+ channel is a predominant type K+ channel in the basolateral membrane of the mouse PT. Adding adenosine increased 50 pS K+ channel activity in cell-attached patches, defined by NPo (a product of channel Numbers and Open Probability). The adenosine-induced stimulation of the 50 pS K+ channel was absent in the PT pretreated with DPCPX, a selective inhibitor of adenosine A1 receptor. In contrast, adenosine was still able to stimulate the 50 pS K+ channel in the PT pretreated with CP-66713, a selective adenosine A2 receptor antagonist. This suggests that the stimulatory effect of adenosine on the 50 pS K+ channel of the PT was mediated by adenosine-A1 receptor. Moreover, the effect of adenosine on the 50 pS K+ channel was blocked in the PT pretreated with U-73122 or Calphostin C, suggesting that adenosine-induced stimulation of the 50 pS K+ channels of the PT was due to the activation of phospholipase C (PLC) and protein kinase C (PKC) pathway. In contrast, the inhibition of phospholipase A2 (PLA2) with AACOCF3 or inhibition of protein kinase A (PKA) with H8 failed to block the adenosine-induced stimulation of the 50 pS K+ channel of the PT. Conclusion: We conclude that adenosine activates the 50 pS K+ channels in the basolateral membrane of PT via adenosine-A1 receptor. Furthermore, the effect of adenosine on the 50 pS K+ channel is mediated by PLC-PKC signaling pathway.
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An increase in the size of single-crystal particles can effectively reduce the interfacial side reactions of layered oxides for sodium-ion batteries at high voltages but may result in sluggish Na+ transport. Herein, single-crystal Na0.66Ni0.26Zn0.07Mn0.67O2 with increased proportions of {010} planes is synthesized by adding low-cost NaCl as the molten salt. With the assistance of a NaCl molten salt, the median diameter (D50) of single-crystal Na0.66Ni0.26Zn0.07Mn0.67O2 increases to 10.46 µm relative to that of the comparison sample without NaCl (6.57 µm). Electrolyte decomposition on the surface of single-crystal Na0.66Ni0.26Zn0.07Mn0.67O2 is considerably suppressed, owing to a decrease in the specific surface area. Moreover, the increased exposure of {010} planes is favorable for improving the Na+ transport kinetics of single-crystal particles. Therefore, at 100 mA g-1, single-crystal Na0.66Ni0.26Zn0.07Mn0.67O2 exhibits a high-capacity retention of 96.6% after 100 cycles, which is considerably greater than that of the comparison sample (86.8%). Moreover, the rate performance of single-crystal Na0.66Ni0.26Zn0.07Mn0.67O2 (average discharge capacity of 81.2 mAh g-1) is superior to that of the comparison sample (average discharge capacity of 61.2 mAh g-1) at 2000 mA g-1. This work provides a new approach for promoting the single-crystal growth of layered oxides for highly stable interfaces at high voltages without compromising Na+ transport kinetics.
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The sodium cation (Na+ ) is the predominant cation with deleterious effects on crops in salt-affected agricultural areas. Salt tolerance of crop can be improved by increasing shoot Na+ exclusion. Therefore, it is crucial to identify and use genetic variants of various crops that promote shoot Na+ exclusion. Here, we show that a HKT1 family gene ZmNC3 (Zea mays L. Na+ Content 3; designated ZmHKT1;2) confers natural variability in shoot-Na+ accumulation and salt tolerance in maize. ZmHKT1;2 encodes a Na+ -preferential transporter localized in the plasma membrane, which mediates shoot Na+ exclusion, likely by withdrawing Na+ from the root xylem flow. A naturally occurring nonsynonymous SNP (SNP947-G) increases the Na+ transport activity of ZmHKT1;2, promoting shoot Na+ exclusion and salt tolerance in maize. SNP947-G first occurred in the wild grass teosinte (at a allele frequency of 43%) and has become a minor allele in the maize population (allele frequency 6.1%), suggesting that SNP947-G is derived from teosinte and that the genomic region flanking SNP947 likely has undergone selection during domestication or post-domestication dispersal of maize. Moreover, we demonstrate that introgression of the SNP947-G ZmHKT1;2 allele into elite maize germplasms reduces shoot Na+ content by up to 80% and promotes salt tolerance. Taken together, ZmNC3/ZmHKT1;2 was identified as an important QTL promoting shoot Na+ exclusion, and its favourable allele provides an effective tool for developing salt-tolerant maize varieties.
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Tolerância ao Sal , Zea mays , Tolerância ao Sal/genética , Zea mays/genética , Zea mays/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Alelos , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Renal ion channel transport and electrolyte disturbances play an important role in the process of functional impairment and fibrosis in the kidney. It is well known that there are limited effective drugs for the treatment of renal fibrosis, and since a large number of ion channels are involved in the renal fibrosis process, understanding the mechanisms of ion channel transport and the complex network of signaling cascades between them is essential to identify potential therapeutic approaches to slow down renal fibrosis. This review summarizes the current work of ion channels in renal fibrosis. We pay close attention to the effect of cystic fibrosis transmembrane conductance regulator (CFTR), transmembrane Member 16A (TMEM16A) and other Cl- channel mediated signaling pathways and ion concentrations on fibrosis, as well as the various complex mechanisms for the action of Ca2+ handling channels including Ca2+-release-activated Ca2+ channel (CRAC), purinergic receptor, and transient receptor potential (TRP) channels. Furthermore, we also focus on the contribution of Na+ transport such as epithelial sodium channel (ENaC), Na+, K+-ATPase, Na+-H+ exchangers, and K+ channels like Ca2+-activated K+ channels, voltage-dependent K+ channel, ATP-sensitive K+ channels on renal fibrosis. Proposed potential therapeutic approaches through further dissection of these mechanisms may provide new therapeutic opportunities to reduce the burden of chronic kidney disease.
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Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [32 P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na+ , whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2 O with D2 O decreased the rates of pyrophosphate hydrolysis by both Na+ - and H+ -transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na+ -pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H+ -pumping and Na+ -pumping by mPPases.
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Difosfatos , Pirofosfatases , Difosfatos/metabolismo , Hidrólise , Isótopos , Cinética , Prótons , Pirofosfatases/metabolismo , Sódio/metabolismo , SolventesRESUMO
Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.
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Canais Epiteliais de Sódio , Fosfatidilinositol 3-Quinases , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
K+/Na+ homeostasis is important for land plants, particularly under salt stress. In this study, the structure and ion transport properties of the high-affinity K+ transporter (HKT) of the liverwort Marchantia polymorpha were investigated. Only one HKT gene, MpHKT1, was identified in the genome of M. polymorpha. Phylogenetic analysis of HKT proteins revealed that non-seed plants possess HKTs grouped into a clade independent of the other two clades including HKTs of angiosperms. A distinct long hydrophilic domain was found in the C-terminus of MpHKT1. Complementary DNA (cDNA) of truncated MpHKT1 (t-MpHKT1) encoding the MpHKT_Δ596-812 protein was used to examine the functions of the C-terminal domain. Both MpHKT1 transporters fused with enhanced green fluorescent protein at the N-terminus were localized to the plasma membrane when expressed in rice protoplasts. Two-electrode voltage clamp experiments using Xenopus laevis oocytes indicated that MpHKT1 mediated the transport of monovalent alkali cations with higher selectivity for Na+ and K+, but truncation of the C-terminal domain significantly reduced the transport activity with a decrease in the Na+ permeability. Overexpression of MpHKT1 or t-MpHKT1 in M. polymorpha conferred accumulation of higher Na+ levels and showed higher Na+ uptake rates, compared to those of wild-type plants; however, phenotypes with t-MpHKT1 were consistently weaker than those with MpHKT1. Together, these findings suggest that the hydrophilic C-terminal domain plays a unique role in the regulation of transport activity and ion selectivity of MpHKT1.
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Proteínas de Transporte de Cátions , Marchantia , Oryza , Proteínas de Transporte de Cátions/metabolismo , DNA Complementar/genética , Marchantia/genética , Marchantia/metabolismo , Oryza/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sódio/metabolismoRESUMO
Loss of nephron primary cilia due to disruption of the Ift88 gene results in sex- and age-specific phenotypes involving renal cystogenesis, blood pressure (BP) and urinary Na+ excretion. Previous studies demonstrated that male mice undergoing induction of nephron-specific Ift88 gene disruption at 2 months of age developed reduced BP and increased salt-induced natriuresis when pre-cystic (2 months post-induction) and became hypertensive associated with frankly cystic kidneys by 9 months post-induction; in contrast, female Ift88 KO mice manifested no unique phenotype 2 months post-induction and had mildly reduced BP 9 months post-induction. The current study utilized these Ift88 KO mice to investigate associated changes in renal Na+ transporter and channel protein expression. At 2 months post-induction, pre-cystic male Ift88 KO mice had reduced high salt diet associated total NKCC2 levels while female mice had no alterations in Na+ transporters or channels. At 9 months post-induction, cystic male Ift88 KO mice had increased total and phosphorylated NHE3 levels together with reduced NKCC2, phosphorylated and/or total NCC, and ENaC-α expression on normal and high salt diets. In contrast, female Ift88 KO mice at 9 months post-induction had no changes in Na+ transporters or channels beyond an increase in phosphorylated-NCC during high salt intake. Thus, reduced BP in pre-cystic, and elevated BP in renal cystic, male Ift88 KO mice are associated with unique sex-dependent changes in nephron Na+ transporter/channel expression.
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Cistos , Hipertensão , Animais , Pressão Sanguínea/fisiologia , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Néfrons/metabolismo , Sódio/metabolismo , Cloreto de Sódio na Dieta/metabolismoRESUMO
Within this review, sex-specific differences in alveolar epithelial functions are discussed with special focus on preterm infants and the respiratory disorders associated with premature birth. First, a short overview about fetal lung development, the challenges the lung faces during perinatal lung transition to air breathing and respiratory distress in preterm infants is given. Next, clinical observations concerning sex-specific differences in pulmonary morbidity of human preterm infants are noted. The second part discusses potential sex-specific causes of pulmonary complications, including pulmonary steroid receptors and local lung steroid metabolism. With regard to pulmonary steroid metabolism, it is important to highlight which steroidogenic enzymes are expressed at which stage during fetal lung development. Thereafter, we review the knowledge concerning sex-specific aspects of lung growth and maturation. Special focus is given to alveolar epithelial Na+ transport as a driver of perinatal lung transition and the sex differences that were noted in this process.
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Síndrome do Desconforto Respiratório do Recém-Nascido , Feminino , Feto/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Pulmão/metabolismo , Masculino , Gravidez , Síndrome do Desconforto Respiratório do Recém-Nascido/etiologia , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Caracteres SexuaisRESUMO
BACKGROUND: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. METHODS: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. RESULTS: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. CONCLUSION: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.
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DNA Mitocondrial/genética , Síndrome de Gitelman/genética , Mutação , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Pré-Escolar , Feminino , Genótipo , Síndrome de Gitelman/metabolismo , Síndrome de Gitelman/patologia , Células HEK293 , Humanos , Lactente , Rim/metabolismo , Rim/ultraestrutura , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Modelos Biológicos , Conformação de Ácido Nucleico , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/genética , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Adulto JovemRESUMO
In rice, the high-affinity K+ transporter, OsHKT1;3, functions as a Na+-selective transporter. mRNA variants of OsHKT1;3 have been reported previously, but their functions remain unknown. In this study, five OsHKT1;3 variants (V1-V5) were identified from japonica rice (Nipponbare) in addition to OsHKT1;3_FL. Absolute quantification qPCR analyses revealed that the transcript level of OsHKT1;3_FL was significantly higher than other variants in both the roots and shoots. Expression levels of OsHKT1;3_FL, and some variants, increased after 24 h of salt stress. Two electrode voltage clamp experiments in a heterologous expression system using Xenopus laevis oocytes revealed that oocytes expressing OsHKT1;3_FL and all of its variants exhibited smaller Na+ currents. The presented data, together with previous data, provide insights to understanding how OsHKT family members are involved in the mechanisms of ion homeostasis and salt tolerance in rice.
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Salt stress is one of the major environmental restricts for crop production and food safety. Barley (Hordeum vulgare L.) is the most salt-tolerant cereal crop, which could be the pioneer for shifting agricultural crop production to marginal saline lands. However, probably due to high genetic complexity of salinity tolerance trait, the progress in the identification of salt-tolerant locus or genes of barley roots moves slowly. Here, we determined physiological and ionic changes in mini-core barley accessions under salt conditions. Na+ content was lower in whole-plant but higher in roots of the salt tolerant genotypes than sensitive ones under salt stress. Genome-wide association study (GWAS) analysis identified 43 significant SNPs out of 12,564 SNPs and 215 candidate genes (P < 10-3) in the roots of worldwide barley accessions, highly associated with root relative dry weight (RDW) and Na+ content after hydroponic salinity in greenhouse and growth chamber. Meanwhile, transcriptomic analysis (RNA-Seq) identified 3217 differentially expression genes (DEGs) in barley roots induced by salt stress, mainly enriched in metabolism and transport processes. After GWAS and RNA-Seq integrating analysis, 39 DEGs were verified by qRT-PCR as salt-responding genes, including CYPs, LRR-KISS and CML genes, mostly related to the signal regulation. Taken together, current results provide genetic map-based genes or new locus useful for improving salt tolerance in crop and contributing to the utilization of saline soils.
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Hordeum , Tolerância ao Sal , Sódio , Estudos de Associação Genética , Hordeum/genética , Raízes de Plantas/genética , Tolerância ao Sal/genética , TranscriptomaRESUMO
Na+/H+ exchangers (NHE) mediate at least part of Na+ entry into gill epithelia via Na+/NH4+ exchange. For homeostasis, Na+ entry into and exit via Na+/K+ ATPase from gill epithelia must balance. Na+/K+ ATPase activity is reduced in cold- compared to warm-acclimated freshwater temperate fish. We hypothesized gill NHE activity is greater in warm- than cold-acclimated fish when measured at acclimation temperatures, and NHE activity displays a temperature dependence similar to Na+/K+ ATPase. Since NHE mRNA expression does not differ, we measured the Na+-dependence of pH-induced Na+ fluxes in gill vesicles from warm- and cold-acclimated fathead minnows at 20o and 7 °C, and calculated maximum transport rates (Vmax) and Na+ K1/2s. We also measured NH4+-induced Na+ fluxes and Na+-induced H+ fluxes. In vesicles from warm-acclimated fish, NHE Vmaxs were 278 ± 33 and 149 ± 23 arbitrary unit/s (au/s) and Na+ K1/2s were 12 ± 4 and 6 ± 4 mmol/l when assayed at 20o and 7 °C (p < 0.004), respectively. In vesicles from cold-acclimated fish, Vmaxs were 288 ± 35 and 141 ± 13 au/s and Na+ K1/2s 17 ± 5 and 7 ± 2 mmol/l when assayed at 20o and 7 °C (p < 0.002), respectively. Na+-induced H+ fluxes were 98 ± 8 and 104 ± 26 au/s in warm- and cold-acclimated fish assayed at 20 °C, respectively. Na+/NH4+ exchange was 120 ± 11 and 158 ± 13 au/s in warm- and cold-acclimated fish, respectively. Conclusions: Gill NHE activity was greater in warm- than cold-acclimated fish assayed at acclimation temperatures. The temperature dependence of NHE activity was similar in both groups, but differed from that reported for Na+/K+ ATPase suggesting complex mechanisms to maintain Na+ homeostasis.
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Aclimatação/fisiologia , Cyprinidae/fisiologia , Brânquias/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Compostos de Amônio/química , Animais , Temperatura Baixa , Cyprinidae/metabolismo , Água Doce , Homeostase , Cinética , Concentração Osmolar , Potássio/química , RNA Mensageiro/metabolismo , Sódio/química , TemperaturaRESUMO
The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development-related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.
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Polaridade Celular/fisiologia , Células Epiteliais/citologia , Túbulos Renais Proximais/citologia , Túbulos Renais/metabolismo , Transporte Biológico , Linhagem Celular , Matriz Extracelular/metabolismo , Humanos , Rim/metabolismo , Sódio/metabolismoRESUMO
High-dietary K+ (HK) intake inhibits basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT), and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of NaCl cotransporter (NCC). Here, we examined whether neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single-channel recording and whole cell recording showed that neither HK decreased nor low-dietary K+ (LK) increased basolateral Kir4.1/Kir5.1 activity of the DCT in kidney tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in control mice [neural precursor cell expressed developmentally downregulated 4-like (Nedd4l)flox/flox]. Also, HK intake decreased the negativity of K+ current reversal potential in the DCT (depolarization) only in control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK intake decreased, whereas LK intake increased, hydrochlorothiazide-induced renal Na+ excretion only in control mice, but this effect was absent in Ks-Nedd4-2 KO mice. Western blot analysis also demonstrated that HK-induced inhibition of phosphorylated NCC (Thr53) and total NCC was observed only in control mice but not in Ks-Nedd4-2 KO mice. Furthermore, expression of all three subunits of the epithelial Na+ channel in Ks-Nedd4-2 KO mice on HK was higher than in control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.NEW & NOTEWORTHY Basolateral Kir4.1/Kir5.1 in the distal convoluted tubule plays an important role as a "K+ sensor" in the regulation of renal K+ excretion after high K+ intake. We found that neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) a role in mediating the effect of K+ diet on Kir4.1/Kir5.1 and NaCl cotransporter because high K+ intake failed to inhibit basolateral Kir4.1/Kir5.1 and NaCl cotransporter in kidney tubule-specific Nedd4-2 knockout mice.
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Túbulos Renais Distais/metabolismo , Ubiquitina-Proteína Ligases Nedd4/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Transporte Biológico/fisiologia , Transporte de Íons/fisiologia , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp/métodos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Membro 3 da Família 12 de Carreador de Soluto/genéticaRESUMO
This study was aimed at the genome-wide identification, a comprehensive in silico characterization of NHX genes from soybean (Glycine max L.) and their tissue-specific expression under varied levels (0-200 mM NaCl) of salinity stress. A total of nine putative NHX genes were identified from soybean. The phylogenetic analysis confirmed a total of five sub-groups and GmNHXs were distributed in three of them. Bioinformatics analyses confirmed all GmNHXs as ion transporters in nature, and all were localized on the vacuolar membrane. Several cis-acting regulatory elements involved in hormonal signal-responsiveness and abiotic stress including salinity responses were identified in the promoter regions of GmNHXs. Amiloride, which is a known Na+/H+ exchanger activity inhibitor, binding motifs were observed in all the GmNHXs. Furthermore, the identified GmNHXs were predicted-targets of 75 different miRNA candidates. To gain an insight into the functional divergence of GmNHX transporters, qRT-PCR based gene expression analysis was done in control and salt-treated root, stem and leaf tissues of two contrasting Indian soybean varieties MAUS-47 (tolerant) and Gujosoya-2 (sensitive). The gene up-regulation was tissue-specific and varied amongst the soybean varieties, with higher induction in tolerant variety. Maximum induction was observed in GmNHX2 in root tissues of MAUS-47 at 200 mM NaCl stress. Overall, identified GmNHXs may be explored further as potential gene candidates for soybean improvement.
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BACKGROUND: The absorption, translocation, accumulation and excretion of substances are fundamental processes in all organisms including plants, and have been successfully studied using radiotracers labelled with 11C, 13N, 14C and 22Na since 1939. Sodium is one of the most damaging ions to the growth and productivity of crops. Due to the significance of understanding sodium transport in plants, a significant number of studies have been carried out to examine sodium influx, compartmentation, and efflux using 22Na- or 24Na-labeled salts. Notably, however, most of these studies employed destructive methods, which has limited our understanding of sodium flux and distribution characteristics in real time, in live plants. Positron emission tomography (PET) has been used successfully in medical research and diagnosis for decades. Due to its ability to visualise and assess physiological and metabolic function, PET imaging has also begun to be employed in plant research. Here, we report the use of a clinical PET scanner with a 22Na tracer to examine 22Na-influx dynamics in barley plants (Hordeum vulgare L. spp. Vulgare-cultivar Bass) under variable nutrient levels, alterations in the day/night light cycle, and the presence of sodium channel inhibitors. RESULTS: 3D dynamic PET images of whole plants show readily visible 22Na translocation from roots to shoots in each examined plant, with rates influenced by both nutrient status and channel inhibition. PET images show that plants cultivated in low-nutrient media transport more 22Na than plants cultivated in high-nutrient media, and that 22Na uptake is suppressed in the presence of a cation-channel inhibitor. A distinct diurnal pattern of 22Na influx was discernible in curves displaying rates of change of relative radioactivity. Plants were found to absorb more 22Na during the light period, and anticipate the change in the light/dark cycle by adjusting the sodium influx rate downward in the dark period, an effect not previously described experimentally. CONCLUSIONS: We demonstrate the utility of clinical PET/CT scanners for real-time monitoring of the temporal dynamics of sodium transport in plants. The effects of nutrient deprivation and of ion channel inhibition on sodium influx into barley plants are shown in two proof-of-concept experiments, along with the first-ever 3D-imaging of the light and dark sodium uptake cycles in plants. This method carries significant potential for plant biology research and, in particular, in the context of genetic and treatment effects on sodium acquisition and toxicity in plants.
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Dexamethasone augments mitochondrial protein abundance. The translocase of the outer membrane (Tom) of mitochondria plays a major role in importing largely cytosolically synthesized proteins into mitochondria. We hypothesize that dexamethasone upregulates the Tom transport system, leading to increase of mitochondrial protein localization. Tom20 and Tom70 are the two major subunits. Dexamethasone increased Tom20 and Tom70 mRNA levels by 53 ± 11% and 25 ± 9% and mitochondrial protein abundance by 27 ± 7% and 25 ± 4% (p < 0.05 for all), respectively, in HEK293 cells. In parallel, dexamethasone elevated the SGK1 mRNA by 79 ± 17% and activity by 190 ± 42%, and mitochondrial protein level by 41 ± 2% (all p < 0.05) without significantly affecting the cytosol counterpart. The discovery of the effect of dexamethasone on SGK1 protein restricted in the mitochondria attracted us to examine the effect of the hormone on MnSOD, an enzyme with known mitochondrial localization and function. Similarly, dexamethasone significantly increased MnSOD transcripts by 67 ± 15% and protein level only in the mitochondria dose-dependently. Inhibition of SGK1 by GSK650394 and RNAi significantly attenuated the effects of the hormone on Tom20, Tom70, and MnSOD, indicating that SGK1 relays the effects of dexamethasone. Catalase inhibited the effects of dexamethasone on SGK1 and the subsequent effects of SGK1 on Tom20, Tom70, and MnSOD. Finally, knock-down of Tom20 and Tom70 by their siRNAs reduced dexamethasone-induced increases in the mitochondrial localization of SGK1 and MnSOD proteins. In conclusion, dexamethasone upregulates Tom20, Tom70, and MnSOD, and these effects are dependent on reactive oxygen species and SGK1. Dexamethasone-induced increases of SGK1 and MnSOD mitochondrial localization requires Tom20 and Tom70.