Unmasking Sustainability in Early Childhood Education: Teachers' Voices from Bosnia and Herzegovina, Croatia, and Slovenia.

Education for sustainable development is one of the key components of the 2030 Agenda. Previous research emphasizes the importance of access to education as early as possible in accordance with the goals and contents of sustainable development. It is assumed that educational policies have incorporated education for sustainable development (ESD) into key documents regulating the early childhood education (ECE), although the existence of legislation is not a guarantee that the practice follows the educational policy. Preschool teachers are key persons who can integrate content that promotes and encourages acceptance of the concept of sustainability into the ECE setting. In order to determine ECE teachers' understanding of the concept of sustainability and contents that promote a sustainable lifestyle, an empirical study with a qualitative approach was conducted, where 91 ECE teachers from Bosnia and Herzegovina, Croatia and Slovenia participated. The participants presented their views on the socio-cultural, economic and environmental dimensions of sustainability, as well as gave many practical examples that, in their view, promote a sustainable lifestyle. They evaluated the efficacy of the researched segments of sustainability as well. Deductive content analysis was used for the analysis of the ECE teachers' answers. In order to get a real image of sustainability in ECE practices, responses were coded using the SOLO Taxonomy and the OMEP ESD Rating Scale. The empirical study showed that ECE teachers often understand sustainability differently from that described in educational policy documents but also pointed to creative approaches to implementation.

L'éducation au développement durable est l'un des éléments clés de l'Agenda 2030. Des recherches antérieures soulignent l'importance d'un accès à l'éducation le plus tôt possible conformément aux objectifs et contenus du développement durable. On suppose que les politiques éducatives ont intégré l'éducation pour le développement durable (EDD) dans les documents clés réglementant l'éducation de la petite enfance (EPE), bien que l'existence d'une législation ne garantisse pas que la pratique suive la politique éducative. Les enseignants du préscolaire sont les personnes clés qui peuvent intégrer un contenu qui promeut et encourage l'acceptation du concept de durabilité dans le cadre de l'EPE. Afin de conforter la compréhension des enseignants de l'EPE du concept de durabilité et des contenus qui promeuvent un mode de vie durable, une étude empirique avec une approche qualitative a été menée, à laquelle ont participé 91 enseignants de l'EPE de Bosnie-Herzégovine, de Croatie et de Slovénie. Les participants ont présenté leurs points de vue sur les dimensions socioculturelles, économiques et environnementales de la durabilité, ainsi que de nombreux exemples pratiques qui, à leur avis, favorisent un mode de vie durable. Ils ont également évalué l'efficacité des volets étudiés de la durabilité. L'analyse de contenu déductive a été utilisée pour l'analyse des réponses des enseignants de l'EPE. Afin d'obtenir une image réelle de la durabilité dans les pratiques d'EPE, les réponses ont été codées à l'aide de la taxonomie SOLO et de l'échelle d'évaluation ESD de l'OMEP. L'étude empirique a montré que les enseignants de l'EPE comprennent souvent la durabilité différemment de celle décrite dans les documents de politique éducative, mais a également souligné des approches créatives de la mise en oeuvre.

La educación para el desarrollo sostenible es uno de los componentes clave de la Agenda 2030. Investigaciones anteriores enfatizan la importancia del acceso a la educación lo antes posible de acuerdo con las metas y contenidos del desarrollo sostenible. Se supone que las políticas educativas han incorporado la educación para el desarrollo sostenible (EDS) en documentos clave que regulan la educación inicial (EPI), aunque la existencia de legislación no es garantía de que las prácticas sigan la política educativa. Los maestros de preescolar son personas clave que pueden integrar contenidos que promuevan y fomenten la aceptación del concepto de sostenibilidad en el entorno de ECE. Con el fin de determinar la comprensión de los docentes de ECE sobre el concepto de sostenibilidad y contenidos que promueven un estilo de vida sostenible, se realizó un estudio empírico con un enfoque cualitativo, donde participaron 91 docentes de ECE de Bosnia y Herzegovina, Croacia y Eslovenia. Los participantes presentaron sus perspectivas sobre las dimensiones socioculturales, económicas y ambientales de la sostenibilidad, y dieron muchos ejemplos prácticos que, desde su punto de vista, promueven un estilo de vida sostenible. También evaluaron la eficacia de las dimensiones de la sostenibilidad investigados. Para el análisis de las respuestas de los docentes de ECE se utilizó el análisis de contenido deductivo. Para obtener una imagen real de la sostenibilidad en las prácticas de ECE, las respuestas se codificaron utilizando la Taxonomía SOLO y la Escala de Calificación OMEP ESD. El estudio empírico mostró que los maestros de ECE a menudo entienden la sostenibilidad de manera diferente a la descrita en los documentos de política educativa, pero también señalaron enfoques creativos para la implementación.

Hypermagnesemia disturbances in rats, NO-related: pentadecapeptide BPC 157 abrogates, L-NAME and L-arginine worsen.

AIM: Stable gastric pentadecapeptide BPC 157, administered before a high-dose magnesium injection in rats, might be a useful peptide therapy against magnesium toxicity and the magnesium-induced effect on cell depolarization. Moreover, this might be an NO-system-related effect. Previously, BPC 157 counteracts paralysis, arrhythmias and hyperkalaemia, extreme muscle weakness; parasympathetic and neuromuscular blockade; injured muscle healing and interacts with the NOS-blocker and NOS-substrate effects. MAIN METHODS: Assessment included magnesium sulfate (560 mg/kg intraperitoneally)-induced muscle weakness, muscle and brain lesions, hypermagnesemia, hyperkalaemia, increased serum enzyme values assessed in rats during and at the end of a 30-min period and medication (given intraperitoneally/kg at 15 min before magnesium) [BPC 157 (10 µg, 10 ng), L-NAME (5 mg), L-arginine (100 mg), alone and/or together]. In HEK293 cells, the increasing magnesium concentration from 1 to 5 mM could depolarize the cells at 1.75 ± 0.44 mV. KEY FINDINGS: L-NAME + magnesium-rats and L-arginine + magnesium-rats exhibited worsened severe muscle weakness and lesions, brain lesions, hypermagnesemia and serum enzymes values, with emerging hyperkalaemia. However, L-NAME + L-arginine + magnesium-rats exhibited all control values and normokalaemia. BPC 157 abrogated hypermagnesemia and counteracted all of the magnesium-induced disturbances (including those aggravated by L-NAME or L-arginine). Thus, cell depolarization due to increasing magnesium concentration was inhibited in the presence of BPC 157 (1 µM) in vitro. SIGNIFICANCE: BPC 157 likely counteracts the initial event leading to hypermagnesemia and the life-threatening actions after a magnesium overdose. In contrast, a worsened clinical course, higher hypermagnesemia, and emerging hyperkalaemia might cause both L-NAME and L-arginine to affect the same events adversely. These events were also opposed by BPC 157.

Stable gastric pentadecapeptide BPC 157 and bupivacaine.

Bupivacaine toxicity following accidental overdose still lacks therapeutic solution. However, there are major arguments for testing BPC 157 against bupivacaine toxicity in vivo in rats, in particular, and then finally, in vitro. These are: the lack of any known BPC 157 toxicity, a lifesaving effect via the mitigation of arrhythmias in rats underwent hyperkalemia or digitalis toxicity, the elimination of hyperkalemia and arrhythmias in rats underwent succinylcholine toxicity and finally, the reduction of potassium-induced depolarization in vitro (in HEK293 cells) in severe hyperkalemia. Most importantly, BPC 157 successfully prevents and counteracts bupivacaine cardiotoxicity; BPC 157 is effective even against the worst outcomes such as a severely prolonged QRS complex. Here, rats injected with bupivacaine (100mg/kg IP) exhibited bradycardia, AV-block, ventricular ectopies, ventricular tachycardia, T-wave elevation and asystole. All of the fatalities had developed T-wave elevation, high-degree AV-block, respiratory arrest and asystole. These were largely counteracted by BPC 157 administration (50µg/kg, 10µg/kg, 10ng/kg, or 10pg/kg IP) given 30min before or 1min after the bupivacaine injection. When BPC 157 was given 6min after bupivacaine administration, and after the development of prolonged QRS intervals (20ms), the fatal outcome was markedly postponed. Additionally, the effect of bupivacaine on cell membrane depolarization was explored by measuring membrane voltages (Vm) in HEK293 cells. Bupivacaine (1mM) alone caused depolarization of the cells, while in combination with BPC 157 (1µm), the bupivacaine-induced depolarization was inhibited. Together, these findings suggest that the stable gastric pentadecapeptide BPC 157 should be a potential antidote for bupivacaine cardiotoxicity.

Urodilatin reverses the detrimental influence of bradykinin in acute ischemic stroke.

Occlusion of cerebral arteries leads to ischemic stroke accompanied by subsequent brain edema. Bradykinin (BK) is involved in the formation of cerebral edema, and natriuretic peptides (NPs) potentially have beneficial effects on brain edema formation via a still unknown mechanism. The aim of this study was clarifying the mechanisms of action of NPs on BK signaling, and their interactive effects after ischemic brain injury. We used a mouse model for stroke, the middle cerebral artery (MCA) occlusion. Brain lesion and edema were measured by microcomputerized tomography volumetric measurements. To determine the effects of NPs on the BK signaling pathway in the MCAs we measured changes in vessel diameter and membrane potentials in endothelial cells. To determine the effects of NPs on BK signaling pathway in isolated astrocytes and neurons, membrane potentials and intercellular Ca2+ concentrations were measured. Urodilatin inhibited and when applied together with BK, reduced the formation of the ischemic lesion via activation of G-Protein-Signaling Protein Type 4 at the cellular (atrocities, neurons) and blood vessel (endothelial cells and isolated MCA) level as well as in in vivo experiments. The results of this study show the existence of a natural antagonist of BK in the brain, and the possible use of NPs in the treatment of stroke.

Involvement of bradykinin in brain edema development after ischemic stroke.

Stroke is the third leading cause of death in the Western world. Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in the blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain edema formation, contributes to further deterioration of the already impaired brain function. Bradykinin increases blood-brain barrier permeability and raises intracranial capillary blood pressure by arterial dilatation and venous constriction leading to brain edema formation. The aim of this paper is to summarize the recent research in the field of bradykinin function (structure, synthesis, signaling pathways, mechanism of action) followed by characterization of different types of brain edema development related to ischemic brain injury, together with the involvement of bradykinin in edema formation. Since there is currently no causal treatment addressing brain edema after ischemic stroke, specific bradykinin receptor antagonists are proposed as a possible new therapeutic approach.

The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3.

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 µM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.

Mortal hyperkalemia disturbances in rats are NO-system related. The life saving effect of pentadecapeptide BPC 157.

We demonstrate the full counteracting ability of stable gastric pentadecapeptide BPC 157 against KCl-overdose (intraperitoneal (i), intragastric (ii), in vitro (iii)), NO-system related. (i) We demonstrated potential (/kg) of: BPC 157 (10ng, 10µg ip, complete counteraction), l-arginine (100mg ip, attenuation) vs. L-NAME (5mg ip, deadly aggravation), given alone and/or combined, before or after intraperitoneal KCl-solution application (9mEq/kg). Therapy was confronted with promptly unrelenting hyperkalemia (>12mmol/L), arrhythmias (and muscular weakness, hypertension, low pressure in lower esophageal and pyloric sphincter) with an ultimate and a regularly inevitable lethal outcome within 30min. Previously, we established BPC 157-NO-system interaction; now, a huge life-saving potential. Given 30min before KCl, all BPC 157 regimens regained sinus rhythm, had less prolongation of QRS, and had no asystolic pause. BPC 157 therapy, given 10min after KCl-application, starts the rescue within 5-10min, completely restoring normal sinus rhythm at 1h. Likewise, other hyperkalemia-disturbances (muscular weakness, hypertension, low sphincteric pressure) were also counteracted. Accordingly with NO-system relation, deadly aggravation by L-NAME: l-arginine brings the values to the control levels while BPC 157 always completely nullified lesions, markedly below those of controls. Combined with l-arginine, BPC 157 exhibited no additive effect. (ii) Intragastric KCl-solution application (27mEq/kg) - (hyperkalemia 7mmol/L): severe stomach mucosal lesions, sphincter failure and peaked T waves were fully counteracted by intragastric BPC 157 (10ng, 10µg) application, given 30min before or 10min after KCl. (iii). In HEK293 cells, hyperkalemic conditions (18.6mM potassium concentrations), BPC 157 directly affects potassium conductance, counteracting the effect on membrane potential and depolarizations caused by hyperkalemic conditions.

Current understanding of guanylin peptides actions.

Guanylin peptides (GPs) family includes guanylin (GN), uroguanylin (UGN), lymphoguanylin, and recently discovered renoguanylin. This growing family is proposed to be intestinal natriuretic peptides. After ingestion of a salty meal, GN and UGN are secreted into the intestinal lumen, where they inhibit sodium absorption and induce anion and water secretion. At the same conditions, those hormones stimulate renal electrolyte excretion by inducing natriuresis, kaliuresis, and diuresis and therefore prevent hypernatremia and hypervolemia after salty meals. In the intestine, a well-known receptor for GPs is guanylate cyclase C (GC-C) whose activation increases intracellular concentration of cGMP. However, in the kidney of GC-C-deficient mice, effects of GPs are unaltered, which could be by new cGMP-independent signaling pathway (G-protein-coupled receptor). This is not unusual as atrial natriuretic peptide also activates two different types of receptors: guanylate cylcase A and clearance receptor which is also G-protein coupled receptor. Physiological role of GPs in other organs (liver, pancreas, lung, sweat glands, and male reproductive system) needs to be discovered. However, it is known that they are involved in pathological conditions like cystic fibrosis, asthma, intestinal tumors, kidney and heart failure, obesity, and metabolic syndrome.

Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells.

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 µM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1ß and IL-6, which all decreased the expression of hMATE1-mRNA and protein. CONCLUSION/SIGNIFICANCE: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.

Primers on molecular pathways: bicarbonate transport by the pancreas.

The pancreas has both endocrine and exocrine functions. As an endocrine organ, stimulation of the pancreatic ß-cells results in insulin secretion to control systemic glucose levels. The exocrine function of the pancreas and the need for alkaline pancreatic secretion (pH 8.0-8.5) have been appreciated for more than 40 years. Yet, our knowledge of the cellular mechanisms (signaling, transporters and channels) which accomplish these critical functions has evolved greatly. In the mid-1990s, basolateral Na-bicarbonate (HCO(3)(-)) uptake by NBCe1 (Slc4a4) was shown to be critical for the generation of approximately 75% of stimulated HCO(3)(-) secretion. In the last 10 years, several new HCO(3)(-) transporters in the Slc26 family and their interaction with the cystic fibrosis transmembrane conductance regulator-chloride channel have elucidated the HCO(3)(-) exit step at the ductal lumen. Most recently, both IRBIT (inositol 1,4,5-trisphosphate receptor-binding protein) and WNK [with no lysine (K)] kinase have been implicated as additional HCO(3)(-) secretory controllers. and IAP.

Slc26a9 is inhibited by the R-region of the cystic fibrosis transmembrane conductance regulator via the STAS domain.

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl(-)-HCO(3)(-) exchanger and Cl(-) channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl(-)-HCO(3)(-) exchange and Cl(-) currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-DeltaSTAS) virtually eliminated the Cl(-) currents with only a modest affect on nCl(-)-HCO(3)(-) exchange activity. Co-expression of a9-DeltaSTAS and the (R)CFTR fragment did not alter the residual a9-DeltaSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.

Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells.

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.

MUPP1 complexes renal K+ channels to alter cell surface expression and whole cell currents.

We previously found that the Ca(2+)-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K(+) channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2DeltaPDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K(+) as well as other ions.

Slc26a9--anion exchanger, channel and Na+ transporter.

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl(-)-HCO(3)(-) exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO(3)(-) secretion; Na(+) electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl(-)-HCO(3)(-) exchanger, Cl(-) channel, and Na(+)-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

Renal physiology of SLC26 anion exchangers.

PURPOSE OF REVIEW: The multifunctional anion exchanger family (Slc26) encompasses 11 identified genes, but only 10 encode real proteins (Slc26a10 is a pseudogene). Most of the Slc26 proteins function primarily as anion exchangers, exchanging sulfate, iodide, formate, oxalate, hydroxyl ion, and bicarbonate anions, whereas other Slc26 proteins function as chloride ion channels or anion-gated molecular motors. The aim of this review is to present recent studies on the molecular function of the Slc26 family and its role in renal physiology and pathophysiology. RECENT FINDINGS: In proximal tubules, Slc26a1 (Sat-1) mediates sulfate and oxalate transport across the basolateral membrane, while Slc26a6 (CFEX, Pat-1) mediates a variety of anion exchange at the apical membrane to facilitate transcellular sodium chloride absorption. Targeted deletion of murine Slc26a6 leads to intestinal hyperabsorption of oxalate, hyperoxaluria, and kidney stones. Slc26a4 (pendrin) and Slc26a7 are expressed in intercalated cells, and are involved in acid-base homeostasis and blood pressure regulation. Messenger RNA for Slc26a2, Slc26a9, and Slc26a11 is also present in the kidney, yet the roles of these family members in renal physiology or pathophysiology are not clear. SUMMARY: Members of this multifunctional anion transporter family play evolving roles in the etiology of nephrolithiasis (Slc26a6) and hypertension (Slc26a4 and Slc26a6). Other Slc26 family members (Slc26a2, Slc26a9, Slc26a11) express mRNA in the kidney but their roles in renal physiology are not yet known.

Ligands and signaling of the G-protein-coupled receptor GPR14, expressed in human kidney cells.

Activation of the urotensin II (U-II) receptor, GPR14, leads to an increase in Ca(2+), activation of phospholipase A(2) (PLA(2)) and an increase in arachidonic acid. The signaling pathway for guanylin peptides in the kidney involves an unknown G-protein coupled receptor which activates PLA(2) and increases arachidonic acid as well. To test if guanylin peptides could be, as U-II, agonists for the GPR14 receptor in the kidney, we used HEK293 and CHO cells transfected with hGPR14 (HEK293+hGPR14, CHO+hGPR14, respectively). Effects of guanylin peptides and U-II were studied by slow-whole-cell patch-clamp analysis and microfluorimetric measurements of intracellular Ca(2+). Guanylin peptides and U-II depolarized HEK293+hGPR14 significantly more than wild type cells. These effects were inhibited in the presence of Ba(2+) or PLA(2) inhibition (AACOCF(3)), suggesting that guanylin peptides and U-II increase arachidonic acid and inhibit ROMK channels in these cells. However, only U-II was capable to increase the cellular Ca(2+), suggesting different mechanism of GPR14 activation by guanylin peptides and U-II. This signaling pathway of U-II involves PKC, because U-II effects in HEK293+hGPR14 cells were inhibited by calphostin C. Guanylin peptides activate PLA(2) and inhibit ROMK channels in HEK293 cells transfected with the human GPR14 receptor. Since GPR14 is present in mouse and human CCD it is a candidate for the guanylate cyclase independent receptor for guanylin peptides.

Zebrafish Slc5a12 encodes an electroneutral sodium monocarboxylate transporter (SMCTn). A comparison with the electrogenic SMCT (SMCTe/Slc5a8).

We have identified and characterized two different sodium-coupled monocarboxylate cotransporters (SMCT) from zebrafish (Danio rerio), electrogenic (zSMCTe) and electroneutral (zSMCTn). zSMCTn is the 12th member of the zebrafish Slc5 gene family (zSlc5a12). Both zSMCT sequences have approximately 50% homology to human SLC5A8 (hSMCT). Transport function and kinetics were measured in Xenopus oocytes injected with zSMCT cRNAs by measurement of intracellular Na(+) concentration ([Na(+)](i)) and membrane potential. Both zSMCTs oocytes increased [Na(+)](i) with addition of monocarboxylates (MC) such as lactate, pyruvate, nicotinate, and butyrate. By using two electrode voltage clamp experiments, we measured currents elicited from zSMCTe after MC addition. MC-elicited currents from zSMCTe were similar to hSMCT currents. In contrast, we found no significant MC-elicited current in either zSMCTn or control oocytes. Kinetic data show that zSMCTe has a higher affinity for lactate, nicotinate, and pyruvate (K(m)(L-lactate) = 0.17 +/- 0.02 mM, K(m)(nicotinate) = 0.54 +/- 0.12 mM at -150 mV) than zSMCTn (K(m)(L-lactate) = 1.81 +/- 0.19 mM, K(m)(nicotinate) = 23.68 +/- 4.88 mM). In situ hybridization showed that 1-, 3-, and 5-day-old zebrafish embryos abundantly express both zSMCTs in the brain, eyes, intestine, and kidney. Within the kidney, zSMCTn mRNA is expressed in pronephric tubules, whereas zSMCTe mRNA is more distal in pronephric ducts. zSMCTn is expressed in exocrine pancreas, but zSMCTe is not. Roles for Na(+)-coupled monocarboxylate cotransporters have not been described for the brain or eye. In summary, zSMCTe is the zebrafish SLC5A8 ortholog, and zSMCTn is a novel, electroneutral SMCT (zSlc5a12). Slc5a12 in higher vertebrates is likely responsible for the electroneutral Na(+)/lactate cotransport reported in mammalian and amphibian kidneys.

Renal electrolyte effects of guanylin and uroguanylin.

PURPOSE OF REVIEW: Guanylin peptides are secreted from the intestine and influence electrolyte and water transport in intestine and kidney, suggesting that these peptides act as intestinal natriuretic peptides. This review presents recent research on renal guanylin and uroguanylin effects. RECENT FINDINGS: After salty meals guanylin peptides are produced in the intestine activating anion secretion and inhibiting sodium absorption. In the kidney guanylin peptides induce saluresis and diuresis. The signaling of guanylin peptides in the intestine is well known, involving guanylate cyclase C and increases in cellular cGMP concentrations. As in the intestine in proximal tubule cells a cGMP and guanylate cyclase C-dependent signaling pathway exists. In guanylate cyclase C-deficient mice, renal effects are unaltered, which could be by explained by recently described new cGMP-independent signaling pathways. In proximal tubules, Uroguanylin activates a pertussis toxin-sensitive receptor. Another cGMP-independent signaling pathway of guanylin peptides involving phospholipase A2 and arachidonic acid is shown for principal cells of human and mouse cortical collecting ducts. SUMMARY: Mechanisms and sites of renal actions of guanylin peptides are still not completely understood. Renal receptors for guanylin peptides are probably G-protein-coupled. The influences of guanylin peptides on natriuresis, kaliuresis, and diuresis are complex and only further detailed studies will allow a complete understanding of the function of these peptides.

Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function.

The Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca(2+) metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for approximately 125 aa from the COOH terminus of the inwardly rectifying K(+) channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaR(R796W), and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K(+) for the basolateral Na(+)-K(+)-ATPase and thereby contribute to inhibition of Na(+) reabsorption.