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BACKGROUND: The KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the tubulopathy and potential therapeutic interventions. METHODS: We used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/- and KCNJ16-/-) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface. RESULTS: KCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16-/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16-/- kidney organoids. CONCLUSIONS: Mature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.
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Inhibidores de Hidroximetilglutaril-CoA Reductasas , Células Madre Pluripotentes Inducidas , Organoides , Canales de Potasio de Rectificación Interna , Humanos , Organoides/metabolismo , Organoides/efectos de los fármacos , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/metabolismo , Riñón/patología , Riñón/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacosRESUMEN
The rising prevalence of kidney diseases urges the need for novel therapies. Kidney organoids and tubuloids are advanced in vitro models and have recently been described as promising tools to study kidney (patho)physiology. Recent developments have shown their application in disease modeling, drug screening, and nephrotoxicity. These applications rely on their ability to mimic (dys)function in vitro including endocrine activity and drug, electrolyte, and water transport. This review provides an overview of these emerging kidney models and focuses on the most recent developments that utilize their functional capabilities. In addition, we cover current limitations and provide future perspectives for this rapidly evolving field, including what these functional properties mean for translational and personalized medicine now and in the future.
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Riñón , Organoides , HumanosRESUMEN
Kidney tubuloids are cell models that are derived from human or mouse renal epithelial cells and show high similarities with their in vivo counterparts. Tubuloids grow polarized in 3D, allow for long-term expansion, and represent multiple segments of the nephron, as shown by their gene expression pattern. In addition, human tubuloids form tight, functional barriers and have been succesfully used for drug testing. Our knowledge of mouse tubuloids, on the other hand, is only minimal. In this study, we further characterized mouse tubuloids and differentiated them towards the collecting duct, which led to a significant upregulation of collecting duct-specific mRNAs of genes and protein expression, including the water channel AQP2 and the sodium channel ENaC. Differentiation resulted in polarized expression of collecting duct water channels AQP2 and AQP3. Also, a physiological response to desmopressin and forskolin stimulation by translocation of AQP2 to the apical membrane was demonstrated. Furthermore, amiloride-sensitive ENaC-mediated sodium uptake was shown in differentiated tubuloids using radioactive tracer sodium. This study demonstrates that mouse tubuloids can be differentiated towards the collecting duct and exhibit collecting duct-specific function. This illustrates the potential use of mouse kidney tubuloids as novel in vitro models to study (patho)physiology of kidney diseases.
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BACKGROUND: A potential concern of formative testing using web-based applications ("apps") is provision of limited feedback. Adopting a randomised controlled trial in 463 first year (bio) medical students, we explored if providing immediate, detailed feedback during "app"-based formative testing can further improve study behaviour and study performance of (bio)medical students. METHODS: Students had access to a formative testing "app", which involved 7 formative test modules throughout the 4-week course. In a randomised order, subjects received the "app" with (n = 231, intervention) or without (n = 232, control) detailed feedback during the formative test modules. RESULTS: No differences in app-use was found between groups (P = 0.15), whereas the intervention group more frequently reviewed information compared to controls (P = 0.007). Exam scores differed between non-/moderate-/intensive- users of the "app" (P < 0.001). No differences in exam scores were found between intervention (6.6 ± 1.1) versus control (6.6 ± 1.1, P = 0.18). Time spent studying was significantly higher compared to previous courses in moderate- and intensive-users (P = 0.006 and < 0.001, respectively), but not in non-users (P = 0.55). Time spent studying did not differ between groups (P > 0.05). CONCLUSIONS: Providing detailed feedback did not further enhance the effect of a web-based application of formative testing on study behaviour or study performance in (bio)medical students, possibly because of a ceiling-effect.
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Educación de Pregrado en Medicina , Retroalimentación Formativa , Aplicaciones Móviles/estadística & datos numéricos , Retención en Psicología/fisiología , Teléfono Inteligente/estadística & datos numéricos , Estudiantes de Medicina , Instrucción por Computador , Curriculum , Evaluación Educacional , Humanos , Aprendizaje , Evaluación de Programas y Proyectos de Salud , Estudiantes de Medicina/estadística & datos numéricos , Habilidades para Tomar ExámenesRESUMEN
The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Ingeniería de Tejidos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Línea Celular , Humanos , Fallo Renal Crónico/genética , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/patología , Túbulos Renales Proximales/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Proteína 1 de Transporte de Anión Orgánico/genéticaRESUMEN
BACKGROUND: Proton pump inhibitors (PPI) are among the most widely prescribed drugs to treat gastric acid-related disorders. PPI-induced hypomagnesaemia, a defect in intestinal absorption of Mg(2+) , can be a severe side effect of chronic PPI use. AIM: To restore serum Mg(2+) concentrations in PPI-induced hypomagnesaemia patients by dietary supplementation with inulin fibres. METHODS: Eleven patients with PPI-induced hypomagnesaemia and 10 controls were treated with inulin (20 g/day). Each trial consisted of two cycles of 14-day inulin treatment followed by a washout period of 14 days. Patients continued to use their PPI. Serum Mg(2+) levels served as the primary endpoint. RESULTS: Inulin significantly enhanced serum Mg(2+) levels from 0.60 to 0.68 mmol/L in PPI-induced hypomagnesaemia patients, and from 0.84 to 0.93 mmol/L in controls. As a consequence 24 h urinary Mg(2+) excretion was significantly increased in patients with PPI-induced hypomagnesaemia (0.3-2.2 mmol/day). Symptoms related to hypomagnesaemia, including muscle cramps and paraesthesia, were reduced during intervention with inulin. CONCLUSION: Inulin increases serum Mg(2+) concentrations under PPI maintenance in patients with PPI-induced hypomagnesaemia.
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Inulina/administración & dosificación , Magnesio/sangre , Inhibidores de la Bomba de Protones/efectos adversos , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Absorción Intestinal , Deficiencia de Magnesio/sangre , Masculino , Persona de Mediana Edad , Calambre Muscular/tratamiento farmacológico , Inhibidores de la Bomba de Protones/uso terapéutico , Adulto JovenRESUMEN
AIM: The molecular interactions between transient receptor potential vanilloid subtype 4 channels (TRPV4) and cell junction formation were investigated in the human and mouse urogenital tract. MATERIALS AND METHODS: A qualitative study was performed to investigate TRPV4 channels, adherence junctions (AJs) and tight junctions (TJs) in kidney, ureter and bladder tissues from humans and wild-type and transgenic TRPV4 knockout (-/-) mice with immunohistochemistry, Western blotting, immunoprecipitation and reverse trasnscription-PCR. Cell junction formation in the wild-type and TRPV4 knockout (-/-) mouse was evaluated with immunohistochemistry and transmission electron microscope (TEM) techniques. RESULTS: TRPV4 channels are predominantly located in membranes of epithelial cells of the bladder, ureter and the collecting ducts of the kidney. There is a molecular interaction between the TRPV4 channel and the AJ. TEM evaluation showed that AJ formation is disrupted in the TRPV4 -/- mouse resulting in deficient intercellular connections and integrity of the epithelium. CONCLUSIONS: TRPV4 is believed to be a mechanoreceptor in the bladder. This study demonstrates that TRPV4 is also involved in intercellular connectivity and structural integrity of the epithelium.
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Barrera Hematonerviosa/fisiología , Uniones Intercelulares/fisiología , Canales Catiónicos TRPV/fisiología , Sistema Urogenital/metabolismo , Animales , Barrera Hematonerviosa/ultraestructura , Humanos , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Riñón/fisiología , Riñón/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Canales Catiónicos TRPV/metabolismo , Vejiga Urinaria/fisiología , Vejiga Urinaria/ultraestructura , Sistema Urogenital/ultraestructura , Urotelio/fisiología , Urotelio/ultraestructuraRESUMEN
The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.
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Membranas Artificiales , Proteínas de Transporte de Catión Orgánico/metabolismo , Cationes/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Cimetidina/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Antagonistas de los Receptores H2 de la Histamina/farmacología , Humanos , Inmunohistoquímica , Transporte Iónico/efectos de los fármacos , Túbulos Renales Proximales/citología , Metilaminas/química , Metilaminas/metabolismo , Transportador 2 de Cátion Orgánico , Permeabilidad/efectos de los fármacos , Compuestos de Piridinio/química , Compuestos de Piridinio/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.
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Bioprótesis , Biotecnología/métodos , Técnicas de Cocultivo/métodos , Riñones Artificiales , Ingeniería de Tejidos/métodos , Animales , Línea Celular , Células Epiteliales , Matriz Extracelular , Humanos , Ratones , Modelos Biológicos , Células Madre , PorcinosRESUMEN
The ability of cells to incorporate azidosugars metabolically is a useful tool for extracellular glycan labelling. The exposed azide moiety can covalently react with alkynes, such as bicyclo[6.1.0]nonyne (BCN), by strain-promoted alkyne-azide cycloaddition (SPAAC). However, the use of SPAAC can be hampered by low specificity of the cycloalkyne. In this article we describe the synthesis of more polar BCN derivatives and their properties for selective cellular glycan labelling. The new polar derivatives [amino-BCN, glutarylamino-BCN and bis(hydroxymethyl)-BCN] display reaction rates similar to those of BCN and are less cell-permeable. The labelling specificity in HEK293 cells is greater than that of BCN, as determined by confocal microscopy and flow cytometry. Interestingly, amino-BCN appears to be highly specific for the Golgi apparatus. In addition, the polar BCN derivatives label the N-glycan of the membrane calcium channel TRPV5 in HEK293 cells with significantly enhanced signal-to-noise ratios.
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Alquinos/química , Azidas/química , Compuestos Bicíclicos con Puentes/síntesis química , Colorantes Fluorescentes/síntesis química , Polisacáridos/análisis , Compuestos Bicíclicos con Puentes/análisis , Química Clic , Reacción de Cicloadición , Citometría de Flujo , Colorantes Fluorescentes/análisis , Glicosilación , Células HEK293 , Humanos , Microscopía Confocal , Imagen Óptica , Polisacáridos/químicaRESUMEN
Promising renal replacement therapies include the development of a bioartificial kidney using functional human kidney cell models. In this study, human conditionally immortalized proximal tubular epithelial cell (ciPTEC) lines originating from kidney tissue (ciPTEC-T1 and ciPTEC-T2) were compared to ciPTEC previously isolated from urine (ciPTEC-U). Subclones of all ciPTEC isolates formed tight cell layers on Transwell inserts as determined by transepithelial resistance, inulin diffusion, E-cadherin expression and immunocytochemisty. Extracellular matrix genes collagen I and -IV α1 were highly present in both kidney tissue derived matured cell lines (p<0.001) compared to matured ciPTEC-U, whereas matured ciPTEC-U showed a more pronounced fibronectin I and laminin 5 gene expression (p<0.01 and p<0.05, respectively). Expression of the influx carrier Organic Cation Transporter 2 (OCT-2), and the efflux pumps P-glycoprotein (P-gp), Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) were confirmed in the three cell lines using real-time PCR and Western blotting. The activities of OCT-2 and P-gp were sensitive to specific inhibition in all models (p<0.001). The highest activity of MRP4 and BCRP was demonstrated in ciPTEC-U (p<0.05). Finally, active albumin reabsorption was highest in ciPTEC-T2 (p<0.001), while Na(+)-dependent phosphate reabsorption was most abundant in ciPTEC-U (p<0.01). In conclusion, ciPTEC established from human urine or kidney tissue display comparable functional PTEC specific transporters and physiological characteristics, providing ideal human tools for bioartificial kidney development.
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Órganos Bioartificiales , Túbulos Renales Proximales/citología , Riñones Artificiales , Orina/citología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/metabolismo , Cadherinas/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Técnicas de Cultivo de Célula , Línea Celular , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/metabolismo , Fibronectinas/biosíntesis , Humanos , Inulina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Factor 2 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 2 de Transcripción de Unión a Octámeros/biosíntesis , Factor 2 de Transcripción de Unión a Octámeros/metabolismo , Ingeniería de Tejidos , Migración Transendotelial y Transepitelial/fisiología , KalininaRESUMEN
We explored the role of transient receptor potential vanilloid 4 (TRPV4) in murine bone metabolism and association of TRPV4 gene variants with fractures in humans. Urinary and histomorphometrical analyses demonstrated reduced osteoclast activity and numbers in male Trpv4(-/-) mice, which was confirmed in bone marrow-derived osteoclast cultures. Osteoblasts and bone formation as shown by serum procollagen type 1 amino-terminal propeptide and histomorphometry, including osteoid surface, osteoblast and osteocyte numbers were not affected in vivo. Nevertheless, osteoblast differentiation was enhanced in Trpv4(-/-) bone marrow cultures. Cortical and trabecular bone mass was 20% increased in male Trpv4(-/-) mice, compared to sex-matched wild type (Trpv4(+/+)) mice. However, at the same time intracortical porosity was increased and bone matrix mineralization was reduced. Together, these lead to a maximum load, stiffness and work to failure of the femoral bone, which were not different compared to Trpv4(+/+) mice, while the bone material was less resistant to stress and less elastic. The differential impacts on these determinants of bone strength were likely responsible for the lack of any changes in whole bone strength in the Trpv4(-/-) mice. None of these skeletal parameters were affected in female Trpv4(-/-) mice. The T-allele of rs1861809 SNP in the TRPV4 locus was associated with a 30% increased risk (95% CI: 1.1-1.6; p=0.013) for non-vertebral fracture risk in men, but not in women, in the Rotterdam Study. Meta-analyses with the population-based LASA study confirmed the association with non-vertebral fractures in men. This was lost when the non-population-based studies Mr. OS and UFO were included. In conclusion, TRPV4 is a male-specific regulator of bone metabolism, a determinant of bone strength, and a potential risk predictor for fractures through regulation of bone matrix mineralization and intra-cortical porosity. This identifies TRPV4 as a unique sexually dimorphic therapeutic and/or diagnostic candidate for osteoporosis.
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Huesos/patología , Fracturas Osteoporóticas/epidemiología , Caracteres Sexuales , Canales Catiónicos TRPV/deficiencia , Animales , Huesos/metabolismo , Módulo de Elasticidad , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Países Bajos/epidemiología , Osteoblastos/patología , Osteoclastos/patología , Fracturas Osteoporóticas/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Estrés Mecánico , Canales Catiónicos TRPV/genéticaRESUMEN
Proton pump inhibitors (PPIs) are potent blockers of gastric acid secretion, used by millions of patients suffering from gastric acid-related complaints. Although PPIs have an excellent safety profile, an increasing number of case reports describe patients with severe hypomagnesemia due to long-term PPI use. As there is no evidence of a renal Mg²âº leak, PPI-induced hypomagnesemia is hypothesized to result from intestinal malabsorption of Mg²âº. The aim of this study was to investigate the effect of PPIs on Mg ²âºhomeostasis in an in vivo mouse model. To this end, C57BL/6J mice were treated with omeprazole, under normal and low dietary Mg²âº availability. Omeprazole did not induce changes in serum Mg²âº levels (1.48 ± 0.05 and 1.54 ± 0.05 mmol/L in omeprazole-treated and control mice, respectively), urinary Mg²âº excretion (35 ± 3 µmol/24 h and 30 ± 4 µmol/24 h in omeprazole-treated and control mice, respectively), or fecal Mg²âº excretion (84 ± 4 µmol/24 h and 76 ± 4 µmol/24 h in omeprazole-treated and control mice, respectively) under any of the tested experimental conditions. However, omeprazole treatment did increase the mRNA expression level of the transient receptor potential melastatin 6 (TRPM6), the predominant intestinal Mg²âº channel, in the colon (167 ± 15 and 100 ± 7 % in omeprazole-treated and control mice, respectively, P < 0.05). In addition, the expression of the colonic Hâº,Kâº-ATPase (cHK-α), a homolog of the gastric Hâº,Kâº-ATPase that is the primary target of omeprazole, was also significantly increased (354 ± 43 and 100 ± 24 % in omeprazole-treated and control mice, respectively, P < 0.05). The expression levels of other magnesiotropic genes remained unchanged. Based on these findings, we hypothesize that omeprazole inhibits cHK-α activity, resulting in reduced extrusion of protons into the large intestine. Since TRPM6-mediated Mg²âºabsorption is stimulated by extracellular protons, this would diminish the rate of intestinal Mg²âº absorption. The increase of TRPM6 expression in the colon may compensate for the reduced TRPM6 currents, thereby normalizing intestinal Mg²âº absorption during omeprazole treatment in C57BL/6J mice, explaining unchanged serum, urine, and fecal Mg²âº levels.
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Colon/metabolismo , Omeprazol/farmacología , Inhibidores de la Bomba de Protones/farmacología , Canales Catiónicos TRPM/metabolismo , Animales , Colon/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Homeostasis , Absorción Intestinal/efectos de los fármacos , Magnesio/sangre , Magnesio/metabolismo , Magnesio/orina , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canales Catiónicos TRPM/genética , Transcripción GenéticaRESUMEN
In the past 8 years, there has been renewed interest in the role of iron in both acute kidney injury (AKI) and chronic kidney disease (CKD). In patients with kidney diseases, renal tubules are exposed to a high concentration of iron owing to increased glomerular filtration of iron and iron-containing proteins, including haemoglobin, transferrin and neutrophil gelatinase-associated lipocalin (NGAL). Levels of intracellular catalytic iron may increase when glomerular and renal tubular cells are injured. Reducing the excessive luminal or intracellular levels of iron in the kidney could be a promising approach to treat AKI and CKD. Understanding the role of iron in kidney injury and as a therapeutic target requires insight into the mechanisms of iron metabolism in the kidney, the role of endogenous proteins involved in iron chelation and transport, including hepcidin, NGAL, the NGAL receptor and divalent metal transporter 1, and iron-induced toxic effects. This Review summarizes emerging knowledge, which suggests that complex mechanisms of iron metabolism exist in the kidney, modulated directly or indirectly by cellular iron content, inflammation, ischaemia and oxidative stress. The potential exists for prevention and treatment of iron-induced kidney injury by customized iron removal or relocation, aided by detailed insight into the underlying pathological mechanisms.
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Lesión Renal Aguda/etiología , Sobrecarga de Hierro/complicaciones , Hierro/metabolismo , Riñón/metabolismo , Insuficiencia Renal Crónica/etiología , Lesión Renal Aguda/metabolismo , Humanos , Sobrecarga de Hierro/metabolismo , Insuficiencia Renal Crónica/metabolismoRESUMEN
During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Riñón/metabolismo , Mitocondrias/metabolismo , Uremia/metabolismo , Línea Celular , Cresoles/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/enzimología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Transporte de Electrón , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Riñón/enzimología , Mitocondrias/enzimología , Mitocondrias/genética , Preparaciones Farmacéuticas/metabolismo , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Umbeliferonas/metabolismo , Uremia/enzimología , Uremia/genéticaRESUMEN
BACKGROUND: Proton pump inhibitors (PPIs) are a mainstay therapy for all gastric acid-related diseases. Clinical concerns arise from a small but growing number of case reports presenting PPI-induced hypomagnesaemia (PPIH) as a consequence of long-term PPI use. Current opinion is that reduced intestinal magnesium absorption might be involved, but nothing is known on the molecular mechanism underlying PPIH. AIM: To investigate whether or not PPIH is a true, long-term drug-class effect of all PPIs and to scrutinise a possible role of comorbidity in its aetiology. Therefore, the primary objective in particular was to investigate serum magnesium dynamics in trials drug withdrawal and re-challenge. The secondary objective was to profile the 'patient at risk'. METHODS: We reviewed systematically all currently available case reports on the subject and performed a statistical analysis on extracted data. RESULTS: Proton pump inhibitor-induced hypomagnesaemia PPIH is a drug-class effect and occurred after 5.5 years (median) of PPI use, onset was broad and ranged from 14 days to 13 years. Discontinuation of PPIs resulted in fast recovery from PPIH in 4 days and re-challenge led to reoccurrence within 4 days. Histamine-2-receptor antagonists were the preferable replacement therapy in PPIH and prevented reoccurrence of hypomagnesaemia. In PPIH no specific risk profile was identified that was linked to the hypomagnesaemia. CONCLUSIONS: The cases of PPIH show severe symptoms of magnesium depletion and identification of its causation was only possible through withdrawal of the PPI. Clinical awareness of PPIH is key to avoid putting patients at risk.
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Enfermedades Gastrointestinales/tratamiento farmacológico , Deficiencia de Magnesio/inducido químicamente , Magnesio/sangre , Inhibidores de la Bomba de Protones/efectos adversos , Ensayos Clínicos como Asunto , Humanos , Magnesio/metabolismo , Deficiencia de Magnesio/sangre , Factores de RiesgoRESUMEN
Ca2+ and Mg2+ are essential ions in a wide variety of cellular processes and form a major constituent of bone. It is, therefore, essential that the balance of these ions is strictly maintained. In the last decade, major breakthrough discoveries have vastly expanded our knowledge of the mechanisms underlying epithelial Ca2+ and Mg2+ transport. The genetic defects underlying various disorders with altered Ca2+ and/or Mg2+ handling have been determined. Recently, this yielded the molecular identification of TRPM6 as the gatekeeper of epithelial Mg2+ transport. Furthermore, expression cloning strategies have elucidated two novel members of the transient receptor potential family, TRPV5 and TRPV6, as pivotal ion channels determining transcellular Ca2+ transport. These two channels are regulated by a variety of factors, some historically strongly linked to Ca2+ homeostasis, others identified in a more serendipitous manner. Herein we review the processes of epithelial Ca2+ and Mg2+ transport, the molecular mechanisms involved, and the various forms of regulation.
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Calcio/metabolismo , Células Epiteliales/fisiología , Magnesio/metabolismo , Animales , Transporte Biológico , HumanosRESUMEN
Ca(2+) is an essential ion in all organisms and many physiological functions in the body rely on the exact maintenance of the Ca(2+) balance. The epithelial Ca(2+) channels TRPV5 [TRP (transient receptor potential) vanilloid 5] and TRPV6 are the most Ca(2+)-selective members of the TRP superfamily and are generally considered as the gatekeepers of Ca(2+) entry across epithelia. TRPV5 is involved in Ca(2+) reabsorption from pro-urine, while TRPV6 has an essential role in intestinal Ca(2+) uptake. These channels are the prime targets of calciotropic hormonal regulation, including vitamin D and parathyroid hormone. In addition, extra- and intra-cellular signalling by associated proteins and Ca(2+) itself play key roles in TRPV5 and TRPV6 regulation. In this paper, we describe the present understanding of the concerted action of calbindin-D(28k), klotho and BSPRY (B-box and SPRY-domain-containing protein) at different levels throughout the epithelial cell to control Ca(2+) influx at the luminal entry gate.
Asunto(s)
Canales de Calcio/fisiología , Regulación de la Expresión Génica , Canales Catiónicos TRPV/fisiología , Animales , Calbindinas , Calcio/metabolismo , Membrana Celular/metabolismo , Glucuronidasa/metabolismo , Humanos , Iones , Proteínas Klotho , Modelos Biológicos , Hormona Paratiroidea/metabolismo , Estructura Terciaria de Proteína , Proteína G de Unión al Calcio S100/química , Transducción de Señal , Vitamina D/químicaRESUMEN
Ca2+ homeostasis in the body is tightly controlled, and is a balance between absorption in the intestine, excretion via the urine, and exchange from bone. Recently, the epithelial Ca2+ channel (TRPV5) has been identified as the gene responsible for the Ca2+ influx in epithelial cells of the renal distal convoluted tubule. TRPV5 is unique within the family of transient receptor potential (TRP) channels due to its high Ca2+ selectivity. Ca2+ flux through TRPV5 is controlled in three ways. First, TRPV5 gene expression is regulated by calciotropic hormones such as vitamin D3 and parathyroid hormone. Second, Ca2+ transport through TRPV5 is controlled by modulating channel activity. Intracellular Ca2+, for example, regulates channel activity by feedback inhibition. Third, TRPV5 is controlled by mobilization of the channel through trafficking toward the plasma membrane. The newly identified anti-aging hormone Klotho regulates TRPV5 by cleaving off sugar residues from the extracellular domain of the protein, resulting in a prolonged expression of TRPV5 at the plasma membrane. Inactivation of TRPV5 in mice leads to severe hypercalciuria, which is compensated by increased intestinal Ca2+ absorption due to augmented vitamin D3 levels. Furthermore, TRPV5 deficiency in mice is associated with polyuria, urine acidification, and reduced bone thickness. Some pharmaceutical compounds, such as the immunosuppressant FK506, affect the Ca2+ balance by modulating TRPV5 gene expression. This underlines the importance of elucidating the role of TRPV5 in Ca(2+)-related disorders, thereby enhancing the possibilities for pharmacological intervention. This chapter describes a unique TRP channel and highlights its regulation and function in renal Ca2+ reabsorption and overall Ca2+ homeostasis.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/fisiología , Calcio/fisiología , Homeostasis/fisiología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiología , Animales , Biotransformación/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Electrofisiología , Humanos , Canales Catiónicos TRPV/efectos de los fármacosRESUMEN
Ca2+ homeostasis is an important factor, which is underlined by the numerous clinical symptoms that involve Ca2+ deficiencies. The overall Ca2+ balance is maintained by the concerted action of Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone, which are all under the control of the calciotropic hormones that are released upon a demand for Ca2+. In the kidney, these calciotropic hormones affect active Ca2+ reabsorption, which consists of TRPV5 as the apical entry gate for Ca2+ influx, calbindin-D28K as an intracellular ferry for Ca2+ and, NCX1 and PMCA1b for extrusion of Ca2+ across the basolateral membrane. This review highlights the action of hormones on renal Ca2+ handling and focuses on the coordinated control of the renal Ca2+ transport proteins. Parathyroid hormone stimulates renal Ca2+ handling by regulating active Ca2+ reabsorption on both the genomic and non-genomic level. Estrogens harbor calciotropic hormone characteristics positively regulating the expression of TRPV5, independently of vitamin D. Besides having a strong regulatory effect on the expression of the intestinal Ca2+ transport proteins, vitamin D contributes to the overall Ca2+ balance by enhancing the expression of the Ca2+ transport machinery in the kidney. Dietary Ca2+ is involved in regulating its own handling by controlling the expression of the renal Ca2+ transport proteins. Thus, the magnitude of Ca2+ entry via TRPV5 controls the expression of the other Ca2+ transport proteins underlining the gatekeeper function of this Ca2+ channel in the renal Ca2+ handling.