RESUMEN
BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.
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Sordera , Pez Cebra , Adolescente , Adulto , Animales , Niño , Preescolar , Sordera/genética , Endocitosis , Humanos , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Mutación , Proteinuria/metabolismo , Proteínas de Transporte Vesicular/genética , Adulto Joven , Pez Cebra/metabolismoRESUMEN
To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.
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Lesión Renal Aguda/tratamiento farmacológico , Albúminas/metabolismo , Dioxanos/farmacología , Galactosiltransferasas/antagonistas & inhibidores , Pirrolidinas/farmacología , Reabsorción Renal/efectos de los fármacos , Trihexosilceramidas/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Animales , Dioxanos/uso terapéutico , Modelos Animales de Enfermedad , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Gentamicinas/metabolismo , Gentamicinas/toxicidad , Humanos , Microscopía Intravital , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/ultraestructura , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía de Fluorescencia por Excitación Multifotónica , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Mioglobina/metabolismo , Mioglobina/toxicidad , Pirrolidinas/uso terapéutico , Receptores de Superficie Celular/metabolismo , Eliminación Renal/efectos de los fármacosRESUMEN
BACKGROUND: The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-ß (PDGFRß) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined. METHODS: In mice with fluorescent protein expression in renal tubular cells and PDGFRß-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration. RESULTS: Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRß-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRß-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRß-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRß impeded PDGFRß-positive cell migration to the tubular injury site and compromised the recovery of tubular function. CONCLUSIONS: Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRß-mediated communication between the renal interstitium and the tubular system.
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Desdiferenciación Celular , Células Epiteliales/fisiología , Túbulos Renales Proximales/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Regeneración , Urotelio/fisiología , Animales , Calcio/metabolismo , Comunicación Celular , Movimiento Celular/efectos de los fármacos , Femenino , Microscopía Intravital , Riñón/citología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/lesiones , Linfocinas/metabolismo , Masculino , Ratones , Inhibidores de Fosfodiesterasa/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Recuperación de la Función , Trapidil/farmacología , Urotelio/lesionesRESUMEN
Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.
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Albúminas/metabolismo , Angiotensina II/farmacología , Glomérulos Renales/fisiología , Podocitos/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Transcitosis/efectos de los fármacos , Vasoconstrictores/farmacología , Aminas , Animales , Femenino , Gentamicinas/farmacología , Microscopía Intravital , Glomérulos Renales/efectos de los fármacos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Microscopía Electrónica , Microscopía de Fluorescencia por Excitación Multifotónica , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Vesículas Transportadoras , OrinaRESUMEN
Intravital multiphoton microscopy is widely used to assess the structure and function of organs in live animals. Although different tissues vary in their accessibility for intravital multiphoton imaging, considerable progress has been made in the imaging quality of all tissues due to substantial technical improvements in the relevant imaging components, such as optics, excitation laser, detectors, and signal analysis software. In this review, we provide an overview of the technical background of intravital multiphoton microscopy. Then, we note a few seminal findings that were made through the use of multiphoton microscopy. Finally, we address the technical limitations of the method and provide an outlook for how these limitations may be overcome through future technical developments.
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Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Colorantes Fluorescentes/química , Humanos , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentaciónRESUMEN
Angiotensin-converting enzyme (ACE) inhibitors are commonly used antiproteinuric drugs. Here we assessed the effect of the ACE inhibitor enalapril on the glomerular sieving coefficient of albumin (GSCA) using intravital multiphoton microscopy. Munich Wistar Frömter (MWF) rats were used as a model of hypertension-related glomerular lesions. Young (9-week-old) MWF rats were nonproteinuric, similar to what was observed in control Wistar rats. However, urinary albumin excretion in the MWF rats gradually increased during aging, averaging 0.00062 ± 0.0001 at age 9 weeks and 0.0054 ± 0.0003 (mg/mOsmol per liter) at age 52 weeks (P < 0.0001). Albuminuria in aged MWF rats was accompanied by structural changes, which were indicative of glomerular lesions. The GSCA was low in young MWF rats but increased markedly during aging, averaging 0.00057 ± 4.7 × 10(-5) (n = 25) in young MWF rats and 0.0027 ± 0.00036 in 52-week-old MWF rats (n = 36; P < 0.0001). Treatment of proteinuric 12-month-old MWF rats with enalapril over a 4-week period reduced the GSCA from 0.0027 ± 0.00036 to 0.00139 ± 0.00013 (P = 0.0005). Similarly, urinary albumin excretion was reduced, averaging 0.0051 ± 0.0003 and 0.0036 ± 0.0005 mg/mOsmol per liter before and after enalapril administration, respectively (P = 0.0089). In parallel, enalapril treatment reduced the mean arterial blood pressure (144.6 ± 6.5 mm Hg in untreated versus 110.9 ± 0.6 mm Hg in enalapril-treated MWF rats) and increased the glomerular filtration rate from 1.64 ± 0.3 ml/min to 3.58 ± 0.3 ml/min (P = 0.0025 versus baseline). In summary, enalapril reduced the GSCA in proteinuric MWF rats, which was paralleled by a similar reduction in urinary albumin excretion. These data suggest that glomerular rather than tubular mechanisms account for the beneficial antiproteinuric effects of the ACE inhibitor.
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Albuminuria/tratamiento farmacológico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Enalapril/farmacología , Envejecimiento/efectos de los fármacos , Envejecimiento/orina , Albúminas/metabolismo , Albuminuria/metabolismo , Albuminuria/orina , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Enalapril/uso terapéutico , Barrera de Filtración Glomerular/efectos de los fármacos , Barrera de Filtración Glomerular/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Permeabilidad/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
PURPOSE OF REVIEW: Transepithelial salt transport in the thick ascending limb of Henle's loop (TAL) crucially depends on the activity of the Na/K/2Cl cotransporter NKCC2. The pharmacologic blockade of NKCC2 leads to pronounced natriuresis and diuresis, which indicate key roles for NKCC2 in renal salt retrieval. The inadequate regulation of NKCC2 and the loss of NKCC2 function are associated with the disruption of salt and water homoeostasis. This review provides a specific overview of our current knowledge with respect to the regulation of NKCC2 by differential splicing and phosphorylation. RECENT FINDINGS: Several mechanisms have evolved to adapt NKCC2 transport to reabsorptive needs. These mechanisms include the regulation of NKCC2 gene expression, the differential splicing of the NKCC2 pre-mRNA, the membrane trafficking, and the modulation of the specific transport activity. Substantial progress has been made over the past few years in deciphering the function of kinases in the regulatory network controlling NKCC2 activity and in elucidating the underlying mechanism and the functional consequences of the regulated differential splicing of the NKCC2 pre-mRNA. SUMMARY: NKCC2 differential splicing and phosphorylation are critically involved in the modulation of the thick ascending limb of Henle's loop reabsorptive capacity and, consequently, in salt homoeostasis, volume regulation, and blood pressure control.
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Transporte Biológico/fisiología , Asa de la Nefrona/metabolismo , Cloruro de Sodio Dietético/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Animales , Humanos , FosforilaciónRESUMEN
The Na-K-2Cl cotransporter (NKCC2; BSC1) is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates â¼20-25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport.
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Asa de la Nefrona/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/fisiología , Empalme Alternativo , Animales , Síndrome de Bartter/fisiopatología , Regulación de la Expresión Génica , Humanos , Riñón , Ratones , Isoformas de Proteínas/fisiología , Miembro 1 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
In this study, we assessed the acute effects of angiotensin II on the albumin glomerular sieving coefficient (GSC) using intravital microscopy. The experiments were performed on Munich Wistar Froemter (MWF) rats. Alexa-Fluor-594 albumin was injected intravenously, and the fluorescence intensity in the glomerular capillaries and Bowman's space was determined to calculate the albumin GSC. The GSC was measured before and during the constant infusion of angiotensin II (10 ng·min(-1)·kg(-1) body wt). Baseline mean arterial pressure (MAP) was 99 ± 5 mmHg and stabilized at 137 ± 5 mmHg during angiotensin II infusion. The baseline GSC averaged 0.00044 ± 4.8 × 10(-5) and increased by 286 ± 44% after angiotensin II infusion (P < 0.0001). The proximal tubular Alexa-Fluor-594 albumin uptake was enhanced during angiotensin II infusion (518% of the baseline value during angiotensin II vs. 218% in controls; P < 0.0001). No change in GSC was observed when the AT1 antagonist losartan was injected before the start of angiotensin II infusion. The AT2 antagonist PD123319 increased the baseline GSC from 0.00052 ± 3.6 × 10(-5) to 0.00074 ± 8.2 × 10(-5) (P = 0.02) without altering the MAP. During angiotensin II infusion with losartan, PD123319 increased the albumin GSC from 0.00037 ± 5.8 × 10(-5) to 0.00115 ± 0.00015 (P = 0.001). When the renal perfusion pressure was mechanically controlled, the GSC increased from 0.0007 ± 0.00019 to 0.0025 ± 0.00063 during angiotensin II infusion (P = 0.047), similar to what was observed when the renal perfusion pressure was allowed to increase. In summary, AT1 activation acutely increases the albumin GSC. This effect appears to be largely independent of changes in the renal perfusion pressure. The AT2 receptor partially attenuates the proteinuric effects of the AT1 receptor.
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Albúminas/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Glomérulos Renales/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Animales , Presión Sanguínea/fisiología , Femenino , Tasa de Filtración Glomerular/fisiología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Proteinuria/tratamiento farmacológico , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/fisiología , Receptor de Angiotensina Tipo 2/fisiologíaRESUMEN
Both sodium reabsorption in the thick ascending limb of the loop of Henle (TAL) and macula densa salt sensing crucially depend on the function of the Na/K/2Cl cotransporter NKCC2. The NKCC2 gene gives rise to at least three different full-length NKCC2 isoforms derived from differential splicing. In the present study, we addressed the influence of dietary salt intake on the differential splicing of NKCC2. Mice were subjected to diets with low-salt, standard salt, and high-salt content for 7 days, and NKCC2 isoform mRNA abundance was determined. With decreasing salt intake, we found a reduced abundance of the low-affinity isoform NKCC2A and an increase in the high-affinity isoform NKCC2B in the renal cortex and the outer stripe of the outer medulla. This shift from NKCC2A to NKCC2B during a low-salt diet could be mimicked by furosemide in vivo and in cultured kidney slices. Furthermore, the changes in NKCC2 isoform abundance during a salt-restricted diet were partly mediated by the actions of angiotensin II on AT1 receptors, as determined using chronic angiotensin II infusion. In contrast to changes in oral salt intake, water restriction (48 h) and water loading (8% sucrose solution) increased and suppressed the expression of all NKCC2 isoforms, without changing the distribution pattern of the single isoforms. In summary, the differential splicing of NKCC2 pre-mRNA is modulated by dietary salt intake, which may be mediated by changes in intracellular ion composition. Differential splicing of NKCC2 appears to contribute to the adaptive capacity of the kidney to cope with changes in reabsorptive needs.
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Empalme Alternativo/genética , Cloruro de Sodio Dietético/administración & dosificación , Cloruro de Sodio Dietético/farmacología , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Agua/administración & dosificaciónRESUMEN
Renal endothelial cells demonstrate an impressive remodeling potential during angiogenic sprouting, vessel repair or while transitioning into mesenchymal cells. These different processes may play important roles in both renal disease progression or regeneration while underlying signaling pathways of different endothelial cell plasticity routes partly overlap. Angiogenesis contributes to wound healing after kidney injury and pharmaceutical modulation of angiogenesis may home a great therapeutic potential. Yet, it is not clear whether any differentiated endothelial cell can proliferate or whether regenerative processes are largely controlled by resident or circulating endothelial progenitor cells. In the glomerular compartment for example, a distinct endothelial progenitor cell population may remodel the glomerular endothelium after injury. Endothelial-to-mesenchymal transition (EndoMT) in the kidney is vastly documented and often associated with endothelial dysfunction, fibrosis, and kidney disease progression. Especially the role of EndoMT in renal fibrosis is controversial. Studies on EndoMT in vivo determined possible conclusions on the pathophysiological role of EndoMT in the kidney, but whether endothelial cells really contribute to kidney fibrosis and if not what other cellular and functional outcomes derive from EndoMT in kidney disease is unclear. Sequencing data, however, suggest no participation of endothelial cells in extracellular matrix deposition. Thus, more in-depth classification of cellular markers and the fate of EndoMT cells in the kidney is needed. In this review, we describe different signaling pathways of endothelial plasticity, outline methodological approaches and evidence for functional and structural implications of angiogenesis and EndoMT in the kidney, and eventually discuss controversial aspects in the literature.
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Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.
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Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.
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Lesión Renal Aguda , Insuficiencia Renal Crónica , Humanos , Nefronas , Riñón , Atrofia , NecrosisRESUMEN
Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.
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Cadenas Ligeras de Clatrina , Clatrina , Animales , Clatrina/metabolismo , Cadenas Ligeras de Clatrina/genética , Endocitosis , Lípidos , Ratones , Microscopía Fluorescente/métodosRESUMEN
Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.
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Endotelio Vascular , Microscopía Intravital/métodos , Glomérulos Renales , Análisis de la Célula Individual/métodos , Animales , Endotelio Vascular/citología , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/fisiología , Glomérulos Renales/citología , Glomérulos Renales/diagnóstico por imagen , Glomérulos Renales/fisiología , Ratones , Ratones TransgénicosRESUMEN
Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (µm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.
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Epidermis/anatomía & histología , Epidermis/metabolismo , Folículo Piloso/metabolismo , Morfogénesis/fisiología , Regeneración/fisiología , Proteína 1 Relacionada con Twist/metabolismo , Cicatrización de Heridas/fisiología , Animales , Epidermis/fisiología , Perfilación de la Expresión Génica , Folículo Piloso/anatomía & histología , Folículo Piloso/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis por Micromatrices , Microscopía de Fuerza Atómica , Modelos Psicológicos , Morfogénesis/genética , Murinae , RNA-Seq , Regeneración/genética , Medicina Regenerativa , Transducción de Señal/genética , Transducción de Señal/fisiología , Análisis Espacio-Temporal , Proteína 1 Relacionada con Twist/genética , Cicatrización de Heridas/genéticaRESUMEN
Renal epithelial cells show remarkable regenerative capacity to recover from acute injury, which involves specific phenotypic changes, but also significant profibrotic tubule-interstitial crosstalk. Tubule-derived profibrotic stimuli and subsequent myofibroblast activation and extracellular matrix deposition have been linked closely with decline of renal function and nephron loss. However, recent data have questioned the view of purely detrimental effects of myofibroblast activation in the injured kidney and even suggested its beneficial role for epithelial regeneration. This article reviews the current understanding of the underlying mechanisms of tubular cell turnover, new suggested pathways of proregenerative tubular-interstitial crosstalk, and relevant insights of proliferation-enhancing effects of myofibroblasts on epithelial cells in nonrenal tissues.
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Lesión Renal Aguda/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Túbulos Renales/metabolismo , Miofibroblastos/metabolismo , Regeneración/fisiología , Lesión Renal Aguda/fisiopatología , Animales , Proliferación Celular , Transición Epitelial-Mesenquimal , Matriz Extracelular/patología , Fibrosis , Humanos , Riñón/metabolismo , Riñón/patología , Riñón/fisiología , Túbulos Renales/patología , Túbulos Renales/fisiología , Transducción de SeñalRESUMEN
Acute kidney injury (AKI) is associated with an increased risk of CKD. Injury-induced multifaceted renal cell-to-cell crosstalk can either lead to successful self-repair or chronic fibrosis and inflammation. In this mini-review, we will discuss critical renal cell types acting as victims or executioners in AKI pathology and introduce intravital imaging as a powerful technique to further dissect these cell-to-cell interactions.
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Lesión Renal Aguda/patología , Lesión Renal Aguda/diagnóstico por imagen , Humanos , Riñón/patología , Macrófagos/patologíaRESUMEN
Intravital multiphoton microscopy of the kidney is a powerful technique to study alterations in tissue morphology and function simultaneously in the living animal and represents a dynamic and developing research tool in the field. Recent technological advances include serial intravital multiphoton microscopy of the same kidney regions over several weeks and combined with ex vivo histology for cellular biomarker expression of the same cells, which had been subject to serial imaging before. Thus, serial intravital multiphoton microscopy followed by ex vivo histology provides unique tools to perform long-term cell fate tracing of the same renal cells during physiological and pathophysiological conditions, thereby allowing the detection of structural changes of the same renal cells over time. Examples include renal cell migration and proliferation while linking these events to local functional alterations and eventually to the expression of distinct cellular biomarkers. Here, we provide a detailed step-by-step protocol to facilitate serial intravital multiphoton microscopy for long-term in vivo tracking of renal cells and subsequent ex vivo histology for immunohistological staining of the same cells in the fixed tissue.
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Rastreo Celular/métodos , Microscopía Intravital/métodos , Riñón/citología , Riñón/diagnóstico por imagen , Abdomen/diagnóstico por imagen , Animales , Colorantes Fluorescentes/química , Inyecciones , Riñón/cirugía , RatonesRESUMEN
The original version of this chapter was inadvertently published without a proper acknowledgement. The authors informed to insert the following acknowledgement in this chapter.