RESUMEN
Intestinal absorption of phosphate is bimodal, consisting of a transcellular pathway and a poorly characterized paracellular mode, even though the latter one contributes to the bulk of absorption under normal dietary conditions. Claudin-3 (Cldn3), a tight junction protein present along the whole intestine in mice, has been proposed to tighten the paracellular pathway for phosphate. The aim of this work was to characterize the phosphate-related phenotype of Cldn3-deficient mice. Cldn3-deficient mice and wildtype littermates were fed standard diet or challenged for 3 days with high dietary phosphate. Feces, urine, blood, intestinal segments and kidneys were collected. Measurements included fecal, urinary, and plasma concentrations of phosphate and calcium, plasma levels of phosphate-regulating hormones, evaluation of trans- and paracellular phosphate transport across jejunum and ileum, and analysis of intestinal phosphate and calcium permeabilities. Fecal and urinary excretion of phosphate as well as its plasma concentration was similar in both genotypes, under standard and high-phosphate diet. However, Cldn3-deficient mice challenged with high dietary phosphate had a reduced urinary calcium excretion and increased plasma levels of calcitriol. Intact FGF23 concentration was also similar in both groups, regardless of the dietary conditions. We found no differences either in intestinal phosphate transport (trans- or paracellular) and phosphate and calcium permeabilities between genotypes. The intestinal expression of claudin-7 remained unaltered in Cldn3-deficient mice. Our data do not provide evidence for a decisive role of Cldn3 for intestinal phosphate absorption and phosphate homeostasis. In addition, our data suggest a novel role of Cldn3 in regulating calcitriol levels.
Asunto(s)
Claudina-3 , Factor-23 de Crecimiento de Fibroblastos , Absorción Intestinal , Fosfatos , Animales , Fosfatos/metabolismo , Fosfatos/orina , Ratones , Claudina-3/metabolismo , Claudina-3/genética , Factor-23 de Crecimiento de Fibroblastos/metabolismo , Calcitriol/metabolismo , Calcitriol/sangre , Calcio/metabolismo , Ratones Endogámicos C57BL , Masculino , Ratones Noqueados , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Mucosa Intestinal/metabolismoRESUMEN
Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.
Asunto(s)
Biomineralización , Calcificación Fisiológica/fisiología , Homeostasis , Canales Iónicos/metabolismo , Larva/fisiología , Protones , Erizos de Mar/fisiología , Animales , Transporte Biológico , Concentración de Iones de Hidrógeno , Canales Iónicos/genética , Análisis de la Célula Individual , TranscriptomaRESUMEN
BACKGROUND: The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiologic role of claudin-10a in the kidney has been unclear. METHODS: To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice, confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining, and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. RESULTS: Mice deficient in claudin-10a were fertile and without overt phenotypes. On knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a result, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison with other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, and unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. CONCLUSIONS: Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyper-reabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.
Asunto(s)
Claudina-2 , Claudinas/metabolismo , Animales , Cationes/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Permeabilidad , Uniones Estrechas/fisiologíaRESUMEN
Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Claudinas/metabolismo , Túbulos Renales Distales/metabolismo , Canales Catiónicos TRPV/metabolismo , Uniones Estrechas/metabolismo , Transcitosis , Animales , Canales de Calcio/genética , Permeabilidad de la Membrana Celular , Claudinas/antagonistas & inhibidores , Claudinas/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Fosforilación , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genéticaRESUMEN
BACKGROUND: Single-cell transcriptomes from dissociated tissues provide insights into cell types and their gene expression and may harbor additional information on spatial position and the local microenvironment. The kidney's cells are embedded into a gradient of increasing tissue osmolality from the cortex to the medulla, which may alter their transcriptomes and provide cues for spatial reconstruction. METHODS: Single-cell or single-nuclei mRNA sequencing of dissociated mouse kidneys and of dissected cortex, outer, and inner medulla, to represent the corticomedullary axis, was performed. Computational approaches predicted the spatial ordering of cells along the corticomedullary axis and quantitated expression levels of osmo-responsive genes. In situ hybridization validated computational predictions of spatial gene-expression patterns. The strategy was used to compare single-cell transcriptomes from wild-type mice to those of mice with a collecting duct-specific knockout of the transcription factor grainyhead-like 2 (Grhl2CD-/-), which display reduced renal medullary osmolality. RESULTS: Single-cell transcriptomics from dissociated kidneys provided sufficient information to approximately reconstruct the spatial position of kidney tubule cells and to predict corticomedullary gene expression. Spatial gene expression in the kidney changes gradually and osmo-responsive genes follow the physiologic corticomedullary gradient of tissue osmolality. Single-nuclei transcriptomes from Grhl2CD-/- mice indicated a flattened expression gradient of osmo-responsive genes compared with control mice, consistent with their physiologic phenotype. CONCLUSIONS: Single-cell transcriptomics from dissociated kidneys facilitated the prediction of spatial gene expression along the corticomedullary axis and quantitation of osmotically regulated genes, allowing the prediction of a physiologic phenotype.
Asunto(s)
Corteza Renal/metabolismo , Corteza Renal/patología , Médula Renal/metabolismo , Médula Renal/patología , Transcriptoma , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hibridación in Situ , Túbulos Renales/metabolismo , Túbulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Concentración OsmolarRESUMEN
AIM: Dimethyl fumarate and nicotinic acid activate the hydroxy-carboxylic acid receptor 2 (HCA2 ) and induce flushing. It is not known whether HCA2 mediates other adverse drug reactions (ADRs) to these two substances. This study aims to compare ADRs associated with dimethyl fumarate and nicotinic acid, and to discuss whether they are HCA2 -mediated. METHODS: We identified spontaneous reports of suspected ADRs to dimethyl fumarate and nicotinic acid in the European Adverse Drug Reaction Database (EudraVigilance). These reports were analysed at different hierarchical levels of the Medical Dictionary for Regulatory Activities (MedDRA). In addition, we screened murine organs for HCA2 expression. RESULTS: Similarities in the ADR profile of dimethyl fumarate and nicotinic acid included "gastrointestinal signs and symptoms" (odds ratio [OR] 0.8 [0.6-1.1]), "hepatobiliary investigations" (OR 1.3 [0.7-2.5]) and "anxiety disorders and symptoms" (OR 0.9 [0.3-2.2]) in High Level Group Terms; "diarrhoea (excluding infective)" (OR 1.2 [0.7-1.8]) and "liver function analyses" (OR 1.3 [0.7-2.6]) in High Level Terms; and "diarrhoea" (OR 1.2 [0.7-2.0]) and "vomiting" (OR 0.9 [0.4-1.7]) in Preferred Terms. In analogy, HCA2 was expressed in the gastrointestinal tract, liver and central nervous system (CNS) of murine organs. A discrepant ADR profile was seen for "lymphopenia" (n = 777) at the preferred term level (only reported for dimethyl fumarate) and "blood glucose increased" (more often reported for nicotinic acid; OR 0.1 [0.0-0.5]). CONCLUSION: The gastrointestinal ADRs common to both substances may be mediated by HCA2 . Other ADRs not common to both substances are compound or indication-specific reactions and likely do not involve HCA2 .
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Niacina , Sistemas de Registro de Reacción Adversa a Medicamentos , Animales , Bases de Datos Factuales , Dimetilfumarato/efectos adversos , Humanos , Ratones , Niacina/efectos adversosRESUMEN
Whether the tight junction is permeable to water remains highly controversial. Here, we provide evidence that the tricellular tight junction is important for paracellular water permeation and that Ig-like domain containing receptor 1 (ILDR1) regulates its permeability. In the mouse kidney, ILDR1 is localized to tricellular tight junctions of the distal tubules. Genetic knockout of Ildr1 in the mouse kidney causes polyuria and polydipsia due to renal concentrating defects. Microperfusion of live renal distal tubules reveals that they are impermeable to water in normal animals but become highly permeable to water in Ildr1 knockout animals whereas paracellular ionic permeabilities in the Ildr1 knockout mouse renal tubules are not affected. Vasopressin cannot correct paracellular water loss in Ildr1 knockout animals despite normal effects on the transcellular aquaporin-2-dependent pathway. In cultured renal epithelial cells normally lacking the expression of Ildr1, overexpression of Ildr1 significantly reduces the paracellular water permeability. Together, our study provides a mechanism of how cells transport water and shows how such a mechanism may be exploited as a therapeutic approach to maintain water homeostasis.
Asunto(s)
Acuaporinas/fisiología , Capacidad de Concentración Renal/fisiología , Receptores de Superficie Celular/fisiología , Animales , Acuaporina 2/metabolismo , Acuaporinas/metabolismo , Transporte Biológico , Permeabilidad de la Membrana Celular/fisiología , Células Epiteliales/metabolismo , Riñón/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales Distales/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptores de Superficie Celular/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/fisiología , Vasopresinas/metabolismoRESUMEN
The thick ascending limb (TAL) of Henle's loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2.
Asunto(s)
Claudinas/metabolismo , Asa de la Nefrona/metabolismo , Magnesio/metabolismo , Sodio/metabolismo , Animales , Claudinas/genética , Células HEK293 , Humanos , Asa de la Nefrona/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Sprague-Dawley , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND: Arginine-vasopressin (AVP) binding to vasopressin V2 receptors promotes redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. This pathway fine-tunes renal water reabsorption and urinary concentration, and its perturbation is associated with diabetes insipidus. Previously, we identified the antimycotic drug fluconazole as a potential modulator of AQP2 localization. METHODS: We assessed the influence of fluconazole on AQP2 localization in vitro and in vivo as well as the drug's effects on AQP2 phosphorylation and RhoA (a small GTPase, which under resting conditions, maintains F-actin to block AQP2-bearing vesicles from reaching the plasma membrane). We also tested fluconazole's effects on water flow across epithelia of isolated mouse collecting ducts and on urine output in mice treated with tolvaptan, a VR2 blocker that causes a nephrogenic diabetes insipidus-like excessive loss of hypotonic urine. RESULTS: Fluconazole increased plasma membrane localization of AQP2 in principal cells independent of AVP. It also led to an increased AQP2 abundance associated with alterations in phosphorylation status and ubiquitination as well as inhibition of RhoA. In isolated mouse collecting ducts, fluconazole increased transepithelial water reabsorption. In mice, fluconazole increased collecting duct AQP2 plasma membrane localization and reduced urinary output. Fluconazole also reduced urinary output in tolvaptan-treated mice. CONCLUSIONS: Fluconazole promotes collecting duct AQP2 plasma membrane localization in the absence of AVP. Therefore, it might have utility in treating forms of diabetes insipidus (e.g., X-linked nephrogenic diabetes insipidus) in which the kidney responds inappropriately to AVP.
Asunto(s)
Acuaporina 2/metabolismo , Transporte Biológico/genética , Colforsina/farmacología , Diabetes Insípida Nefrogénica/tratamiento farmacológico , Fluconazol/farmacología , Proteína de Unión al GTP rhoA/efectos de los fármacos , Análisis de Varianza , Animales , Membrana Celular/metabolismo , Células Cultivadas , Diabetes Insípida Nefrogénica/metabolismo , Modelos Animales de Enfermedad , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación/genética , Distribución Aleatoria , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.
Asunto(s)
Equilibrio Ácido-Base/efectos de los fármacos , Receptores de Vasopresinas/efectos de los fármacos , Vasopresinas/farmacología , Equilibrio Ácido-Base/genética , Animales , Células Cultivadas/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Inmunohistoquímica , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratas Brattleboro , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Vasopresinas/genética , Sensibilidad y Especificidad , Urinálisis/métodosRESUMEN
Inorganic phosphate (Pi) is crucial for many biological functions, such as energy metabolism, signal transduction, and pH buffering. Efficient systems must exist to ensure sufficient supply for the body of Pi from diet. Previous experiments in humans and rodents suggest that two pathways for the absorption of Pi exist, an active transcellular Pi transport and a second paracellular pathway. Whereas the identity, role, and regulation of active Pi transport have been extensively studied, much less is known about the properties of the paracellular pathway. In Ussing chamber experiments, we characterized paracellular intestinal Pi permeabilities and fluxes. Dilution potential measurements in intestinal cell culture models demonstrated that the tight junction is permeable to Pi, with monovalent Pi having a higher permeability than divalent Pi. These findings were confirmed in rat and mouse intestinal segments by use of Ussing chambers and a combination of dilution potential measurements and fluxes of radiolabeled 32Pi. Both techniques yielded very similar results, showing that paracellular Pi fluxes were bidirectional and that Pi permeability was ~50% of the permeability for Na+ or Cl-. Pi fluxes were a function of the concentration gradient and Pi species (mono- vs. divalent Pi). In mice lacking the active transcellular Pi transport component sodium-dependent Pi transporter NaPi-IIb, the paracellular pathway was not upregulated. In summary, the small and large intestines have a very high paracellular Pi permeability, which may favor monovalent Pi fluxes and allow efficient uptake of Pi even in the absence of active transcellular Pi uptake.NEW & NOTEWORTHY The paracellular permeability for phosphate is high along the entire axis of the small and large intestine. There is a slight preference for monovalent phosphate. Paracellular phosphate fluxes do not increase when transcellular phosphate transport is genetically abolished. Paracellular phosphate transport may be an important target for therapies aiming to reduce intestinal phosphate absorption.
Asunto(s)
Espacio Extracelular/fisiología , Mucosa Intestinal/metabolismo , Transporte Iónico/fisiología , Fosfatos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo , Uniones Estrechas/fisiología , Animales , Células Cultivadas , Absorción Intestinal , Ratones , Permeabilidad , Fosfatos/química , Fosfatos/metabolismo , RatasRESUMEN
Light has been demonstrated to enhance calcification rates in hermatypic coral species. To date, it remains unresolved whether calcifying epithelia change their ion transport activity during illumination, and whether such a process is mediated by the endosymbiotic algae or can be controlled by the coral host itself. Using a modified Ussing chamber in combination with H+ sensitive microelectrode measurements, the present work demonstrates that light triggers the generation of a skeleton positive potential of up to 0.9 mV in the hermatypic coral Stylophora pistillata. This potential is generated by a net flux of cations towards the skeleton and reaches its maximum at blue (450 nm) light. The effects of pharmacological inhibitors targeting photosynthesis 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and anion transport 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) were investigated by pH microelectrode measurements in coral tissues demonstrating a rapid decrease in tissue pH under illumination. However, these inhibitors showed no effect on the electrophysiological light response of the coral host. By contrast, metabolic inhibition by cyanide and deoxyglucose reversibly inhibited the light-induced cation flux towards the skeleton. These results suggest that ion transport across coral epithelia is directly triggered by blue light, independent of photosynthetic activity of algal endosymbionts. Measurements of this very specific and quantifiable physiological response can provide parameters to identify photoreception mechanisms and will help to broaden our understanding of the mechanistic link between light stimulation and epithelial ion transport, potentially relevant for calcification in hermatypic corals.
Asunto(s)
Antozoos/efectos de la radiación , Cationes/metabolismo , Transporte Iónico/efectos de la radiación , Luz , Animales , Antozoos/crecimiento & desarrollo , Antozoos/metabolismo , Calcificación Fisiológica , Cationes/efectos de la radiación , Fenómenos ElectrofisiológicosRESUMEN
Collecting ducts make up the distal-most tubular segments of the kidney, extending from the cortex, where they connect to the nephron proper, into the medulla, where they release urine into the renal pelvis. During water deprivation, body water preservation is ensured by the selective transepithelial reabsorption of water into the hypertonic medullary interstitium mediated by collecting ducts. The collecting duct epithelium forms tight junctions composed of barrier-enforcing claudins and exhibits a higher transepithelial resistance than other segments of the renal tubule exhibit. However, the functional relevance of this strong collecting duct epithelial barrier is unresolved. Here, we report that collecting duct-specific deletion of an epithelial transcription factor, grainyhead-like 2 (GRHL2), in mice led to reduced expression of tight junction-associated barrier components, reduced collecting duct transepithelial resistance, and defective renal medullary accumulation of sodium and other osmolytes. In vitro, Grhl2-deficient collecting duct cells displayed increased paracellular flux of sodium, chloride, and urea. Consistent with these effects, Grhl2-deficient mice had diabetes insipidus, produced dilute urine, and failed to adequately concentrate their urine after water restriction, resulting in susceptibility to prerenal azotemia. These data indicate a direct functional link between collecting duct epithelial barrier characteristics, which appear to prevent leakage of interstitial osmolytes into urine, and body water homeostasis.
Asunto(s)
Epitelio/fisiología , Túbulos Renales Colectores/fisiología , Osmorregulación/genética , Uniones Estrechas/genética , Uniones Estrechas/fisiología , Factores de Transcripción/genética , Animales , Acuaporina 2/metabolismo , Acuaporina 4/metabolismo , Arginina Vasopresina/metabolismo , Azotemia/etiología , Transporte Biológico/genética , Creatinina/orina , Perfilación de la Expresión Génica , Masculino , Ratones , Concentración Osmolar , Transducción de Señal , Urea/metabolismo , Orina , Agua/metabolismo , Privación de Agua/fisiologíaRESUMEN
The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.
Asunto(s)
Claudinas/metabolismo , Túbulos Renales Colectores/citología , Uniones Estrechas/metabolismo , Vasopresinas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Ratones , Concentración Osmolar , Ratas , Migración Transendotelial y TransepitelialRESUMEN
The nephron segments in the inner medulla are part of the urine concentrating mechanism. Depending on the diuretic state, they are facing a large range of extracellular osmolality. We investigated whether water homeostasis affects tubular transport and permeability properties in inner medullary descending thin limb (IMdTL) and ascending thin limb (IMaTL). Three experimental groups of rats under different diuretic states were investigated on metabolic cages: waterload, furosemide-induced diuresis, and control (antidiuresis). Urine production and osmolalities reflected the 3-day treatment. To functionally investigate tubular epithelial properties, we performed experiments in freshly isolated inner medullary thin limbs from these animals. Tubular segments were acutely dissected and investigated for trans- and paracellular properties by in vitro perfusion and electrophysiological analysis. IMdTL and IMaTL were distinguished by morphological criteria. We confirmed absence of transepithelial electrogenic transport in thin limbs. Although diffusion potential measurements showed no differences between treatments in IMdTLs, we observed increased paracellular cation selectivity under waterload in IMaTLs. NaCl diffusion potential was -5.64 ± 1.93 mV under waterload, -1.99 ± 1.72 mV under furosemide-induced diuresis, and 0.27 ± 0.40 mV under control. The corresponding permeability ratio PNa/Cl was 1.53 ± 0.21 (waterload), 1.22 ± 0.18 (furosemide-induced diuresis), and 0.99 ± 0.02 (control), respectively. Claudins are main constituents of the tight junction responsible for paracellular selectivity; however, immunofluorescence did not show qualitative differences in claudin 4, 10, and 16 localization. Our results show that IMaTLs change tight junction properties in response to diuretic state to allow adaptation of NaCl reabsorption.
Asunto(s)
Diuresis/efectos de los fármacos , Diuréticos/farmacología , Ingestión de Líquidos , Células Epiteliales/efectos de los fármacos , Furosemida/farmacología , Asa de la Nefrona/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Agua/metabolismo , Animales , Claudinas/metabolismo , Difusión , Células Epiteliales/metabolismo , Femenino , Asa de la Nefrona/metabolismo , Masculino , Concentración Osmolar , Permeabilidad , Ratas Sprague-Dawley , Reabsorción Renal/efectos de los fármacos , Cloruro de Sodio/orina , Uniones Estrechas/metabolismoRESUMEN
The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.
Asunto(s)
Claudinas/deficiencia , Hipercalciuria/prevención & control , Túbulos Renales Distales/metabolismo , Asa de la Nefrona/metabolismo , Deficiencia de Magnesio/prevención & control , Animales , Calcio/metabolismo , Claudinas/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Predisposición Genética a la Enfermedad , Hipercalciuria/genética , Hipercalciuria/metabolismo , Hipercalciuria/fisiopatología , Túbulos Renales Distales/patología , Túbulos Renales Distales/fisiopatología , Asa de la Nefrona/patología , Asa de la Nefrona/fisiopatología , Magnesio/metabolismo , Deficiencia de Magnesio/genética , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Nefrocalcinosis/genética , Nefrocalcinosis/metabolismo , Nefrocalcinosis/fisiopatología , Nefrocalcinosis/prevención & control , Fenotipo , Sodio/metabolismoRESUMEN
Mice lacking distal tubular expression of CLDN10, the gene encoding the tight junction protein Claudin-10, show enhanced paracellular magnesium and calcium permeability and reduced sodium permeability in the thick ascending limb (TAL), leading to a urine concentrating defect. However, the function of renal Claudin-10 in humans remains undetermined. We identified and characterized CLDN10 mutations in two patients with a hypokalemic-alkalotic salt-losing nephropathy. The first patient was diagnosed with Bartter syndrome (BS) >30 years ago. At re-evaluation, we observed hypocalciuria and hypercalcemia, suggesting Gitelman syndrome (GS). However, serum magnesium was in the upper normal to hypermagnesemic range, thiazide responsiveness was not blunted, and genetic analyses did not show mutations in genes associated with GS or BS. Whole-exome sequencing revealed compound heterozygous CLDN10 sequence variants [c.446C>G (p.Pro149Arg) and c.465-1G>A (p.Glu157_Tyr192del)]. The patient had reduced urinary concentrating ability, with a preserved aquaporin-2 response to desmopressin and an intact response to furosemide. These findings were not in line with any other known salt-losing nephropathy. Subsequently, we identified a second unrelated patient showing a similar phenotype, in whom we detected compound heterozygous CLDN10 sequence variants [c.446C>G (p.(Pro149Arg) and c.217G>A (p.Asp73Asn)]. Cell surface biotinylation and immunofluorescence experiments in cells expressing the encoded mutants showed that only one mutation caused significant differences in Claudin-10 membrane localization and tight junction strand formation, indicating that these alterations do not fully explain the phenotype. These data suggest that pathogenic CLDN10 mutations affect TAL paracellular ion transport and cause a novel tight junction disease characterized by a non-BS, non-GS autosomal recessive hypokalemic-alkalotic salt-losing phenotype.
Asunto(s)
Alcalosis/genética , Claudinas/genética , Hipopotasemia/genética , Defectos Congénitos del Transporte Tubular Renal/genética , Adolescente , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The medullary thick ascending limb of Henle's loop (mTAL) is crucial for urine-concentrating ability of the kidney. It is water tight and able to dilute the luminal fluid by active transcellular NaCl transport, fueling the counter current mechanism by increasing interstitial osmolality. While chloride is exclusively transported transcellularly, approx. 50% of sodium transport occurs via the paracellular route, driven by the lumen-positive transepithelial potential. Antidiuretic hormone (AVP) is known to increase active NaCl transport to support collecting duct water reabsorption. Here, we investigated the concomitant effects of AVP on the paracellular properties of mTAL. Freshly isolated mouse mTALs were perfused and electrophysiological transcellular and paracelluar properties were assessed in a paired fashion before and after AVP stimulation. In addition, the same parameters were measured in mice on a water-restricted (WR) or water-loaded (WL) diet for 5 days. Acute ex vivo stimulation as well as long-term in vivo water restriction increased equivalent short circuit current as a measure of active transcellular NaCl transport. Intriguingly, in both experimental approaches, this was accompanied by markedly increased paracellular Na+ selectivity. Thus, AVP is able to acutely regulate paracellular cation selectivity in parallel to transcellular NaCl transport, allowing balanced paracellular Na+ absorption under an increased transepithelial driving force.
Asunto(s)
Transporte Biológico/fisiología , Médula Renal/metabolismo , Túbulos Renales/metabolismo , Cloruro de Sodio/metabolismo , Sodio/metabolismo , Vasopresinas/metabolismo , Animales , Humanos , PermeabilidadRESUMEN
Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia-reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.
Asunto(s)
Apoptosis , Túbulos Renales/citología , Animales , Peso Corporal , Caspasa 8/genética , Caspasa 8/fisiología , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/fisiología , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Daño por Reperfusión/prevención & controlRESUMEN
Tacrolimus is a widely used immunosuppressive drug that inhibits the phosphatase calcineurin when bound to the 12 kDa FK506-binding protein (FKBP12). When this binding occurs in T cells, it leads to immunosuppression. Tacrolimus also causes side effects, however, such as hypertension and hyperkalemia. Previously, we reported that tacrolimus stimulates the renal thiazide-sensitive sodium chloride cotransporter (NCC), which is necessary for the development of hypertension. However, it was unclear if tacrolimus-induced hypertension resulted from tacrolimus effects in renal epithelial cells directly or in extrarenal tissues, and whether inhibition of calcineurin was required. To address these questions, we developed a mouse model in which FKBP12 could be deleted along the nephron. FKBP12 disruption alone did not cause phenotypic effects. When treated with tacrolimus, however, BP and the renal abundance of phosphorylated NCC were lower in mice lacking FKBP12 along the nephron than in control mice. Mice lacking FKBP12 along the nephron also maintained a normal relationship between plasma potassium levels and the abundance of phosphorylated NCC with tacrolimus treatment. In cultured cells, tacrolimus inhibited dephosphorylation of NCC. Together, these results suggest that tacrolimus causes hypertension predominantly by inhibiting calcineurin directly in cells expressing NCC, indicating thiazide diuretics may be particularly effective for lowering BP in tacrolimus-treated patients with hypertension.