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1.
J Am Soc Nephrol ; 33(4): 732-745, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35149593

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.


Asunto(s)
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/metabolismo
2.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-36077596

RESUMEN

Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate immune response converging to NF-ĸB pathway induction. Despite the clear role of NF-ĸB in inflammation, it has recently been acknowledged that NF-ĸB may impact other cell functions. To identify NF-ĸB function with respect to metabolism, vascular function and oxidative stress after I/R injury and to decipher in detail the underlying mechanism, we generated a transgenic mouse model with targeted deletion of IKKß along the tubule and applied I/R injury followed by its analysis after 2 and 14 days after I/R injury. Tubular IKKß deletion ameliorated renal function and reduced tissue damage. RNAseq data together with immunohistochemical, biochemical and morphometric analysis demonstrated an ameliorated vascular organization and mRNA expression profile for increased angiogenesis in mice with tubular IKKß deletion at 2 days after I/R injury. RNAseq and protein analysis indicate an ameliorated metabolism, oxidative species handling and timely-adapted cell proliferation and apoptosis as well as reduced fibrosis in mice with tubular IKKß deletion at 14 days after I/R injury. In conclusion, mice with tubular IKKß deletion upon I/R injury display improved renal function and reduced tissue damage and fibrosis in association with improved vascularization, metabolism, reactive species disposal and fine-tuned cell proliferation.


Asunto(s)
Lesión Renal Aguda , Daño por Reperfusión , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Animales , Apoptosis/genética , Fibrosis , Quinasa I-kappa B/genética , Isquemia , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Daño por Reperfusión/genética
3.
J Cell Mol Med ; 23(10): 6543-6553, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31368174

RESUMEN

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria-dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod ) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full-length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+ /K+ -ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.


Asunto(s)
Catepsina B/metabolismo , Canales Epiteliales de Sodio/metabolismo , Hipertensión/etiología , Hipertensión/metabolismo , Síndrome Nefrótico/complicaciones , Síndrome Nefrótico/metabolismo , Amilorida/farmacología , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Bloqueadores del Canal de Sodio Epitelial/farmacología , Glomeruloesclerosis Focal y Segmentaria/enzimología , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/orina , Hipertensión/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Túbulos Renales/citología , Túbulos Renales/metabolismo , Lisosomas/enzimología , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Síndrome Nefrótico/genética , Proteinuria/metabolismo , Proteolisis , Sodio/metabolismo
4.
J Am Soc Nephrol ; 28(1): 230-241, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27297946

RESUMEN

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR-dependent regulatory network for nutrient transport in renal proximal tubular cells.


Asunto(s)
Endocitosis/fisiología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Complejos Multiproteicos/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Transporte de Proteínas
5.
Biochem J ; 473(19): 3237-52, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27422782

RESUMEN

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.


Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Animales , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Corteza Renal/metabolismo , Ratones , Microscopía Confocal , Unión Proteica , Técnicas del Sistema de Dos Híbridos
6.
J Am Soc Nephrol ; 27(3): 731-44, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26116357

RESUMEN

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.


Asunto(s)
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 , Orina
7.
J Am Soc Nephrol ; 26(6): 1269-78, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25270072

RESUMEN

Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented >2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance.


Asunto(s)
Acuaporina 1/genética , Permeabilidad de la Membrana Celular/fisiología , Regulación de la Expresión Génica , Túbulos Renales Proximales/fisiopatología , Equilibrio Hidroelectrolítico/genética , Adaptación Fisiológica , Animales , Acuaporina 1/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Tasa de Filtración Glomerular/fisiología , Ratones , Ratones Transgénicos , Microvellosidades/metabolismo , Ósmosis , Transporte de Proteínas/fisiología , Distribución Aleatoria , Sensibilidad y Especificidad , Factores de Tiempo , Equilibrio Hidroelectrolítico/fisiología
8.
Am J Physiol Renal Physiol ; 308(10): F1047-55, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25651559

RESUMEN

The balance between vasoconstrictor/sodium-retaining and vasodilator/natriuretic systems is essential for maintaining body fluid and electrolyte homeostasis. Natriuretic peptides, such as atrial natriuretic peptide (ANP), belong to the vasodilator/natriuretic system. ANP is produced by the conversion of pro-ANP into ANP, which is achieved by a proteolytical cleavage executed by corin. In the kidney, ANP binds to the natriuretic peptide receptor-A (NPR-A) and enhances its guanylyl cyclase activity, thereby increasing intracellular cyclic guanosine monophosphate production to promote natriuretic and renoprotective responses. In the glomerulus, ANP increases glomerular permeability and filtration rate and antagonizes the deleterious effects of the renin-angiotensin-aldosterone system activation. Along the nephron, natriuretic and diuretic actions of ANP are mediated by inhibiting the basolaterally expressed Na(+)-K(+)-ATPase, reducing apical sodium, potassium, and protein organic cation transporter in the proximal tubule, and decreasing Na(+)-K(+)-2Cl(-) cotransporter activity and renal concentration efficiency in the thick ascending limb. In the medullary collecting duct, ANP reduces sodium reabsorption by inhibiting the cyclic nucleotide-gated cation channels, the epithelial sodium channel, and the heteromeric channel transient receptor potential-vanilloid 4 and -polycystin 2 and diminishes vasopressin-induced water reabsorption. Long-term ANP treatment may lead to NPR-A desensitization and ANP resistance, resulting in augmented sodium and water reabsorption. In mice, corin deficiency impairs sodium excretion and causes salt-sensitive hypertension. Characteristics of ANP resistance and corin deficiency are also encountered in patients with edema-associated diseases, highlighting the importance of ANP signaling in salt-water balance and renal pathophysiology.


Asunto(s)
Factor Natriurético Atrial/fisiología , Riñón/fisiopatología , Transducción de Señal/fisiología , Animales , Tasa de Filtración Glomerular/fisiología , Humanos , Ratones , Modelos Animales , Serina Endopeptidasas/fisiología , Equilibrio Hidroelectrolítico/fisiología
9.
J Extracell Vesicles ; 12(7): e12338, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37408115

RESUMEN

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.


Asunto(s)
Antineoplásicos , Vesículas Extracelulares , Neoplasias Ováricas , Humanos , Femenino , Proteínas ADAM/metabolismo , Receptores ErbB , Vesículas Extracelulares/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Proteína ADAM17
10.
Cell Death Dis ; 14(8): 562, 2023 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-37626062

RESUMEN

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.


Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Nefropatías Diabéticas , Animales , Ratones , Nefropatías Diabéticas/genética , Riñón , Túbulos Renales Proximales , Glomérulos Renales , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética
11.
Sci Adv ; 9(13): eadf4055, 2023 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-37000885

RESUMEN

The metalloproteases meprin α and meprin ß are highly expressed in the healthy gut but significantly decreased in inflammatory bowel disease, implicating a protective role in mucosal homeostasis. In the colon, meprin α and meprin ß form covalently linked heterodimers tethering meprin α to the plasma membrane, therefore presenting dual proteolytic activity in a unique enzyme complex. To unravel its function, we applied N-terminomics and identified galectin-3 as the major intestinal substrate for meprin α/ß heterodimers. Galectin-3-deficient and meprin α/ß double knockout mice show similar alterations in their microbiome in comparison to wild-type mice. We further demonstrate that meprin α/ß heterodimers differentially process galectin-3 upon bacterial infection, in germ-free, conventionally housed (specific pathogen-free), or wildling mice, which in turn regulates the bacterial agglutination properties of galectin-3. Thus, the constitutive cleavage of galectin-3 by meprin α/ß heterodimers may play a key role in colon host-microbiome homeostasis.


Asunto(s)
Galectina 3 , Metaloendopeptidasas , Ratones , Animales , Galectina 3/genética , Galectina 3/metabolismo , Metaloproteasas/metabolismo , Proteolisis , Ratones Noqueados , Homeostasis
12.
Kidney Int ; 82(1): 53-9, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22437417

RESUMEN

Severe sepsis is often accompanied by acute renal failure with renal tubular dysfunction. Albuminuria is a common finding in septic patients and we studied whether it was due to an impairment of proximal tubular endocytosis of filtered albumin. We studied the regulation of megalin and cubilin, the two critical multiligand receptors responsible for albumin absorption, during severe experimental endotoxemia. Lipopolysaccharide (LPS) caused a time- and dose-dependent suppression of megalin and cubilin expression that was paralleled by a decrease in plasma albumin levels and an increase in the urine concentration of albumin in mice. Incubation of rat renal cortical slices with LPS also reduced the mRNA expression of megalin and cubilin. Further, LPS suppressed megalin and cubilin mRNA expression in murine primary proximal tubule cells and decreased the uptake of FITC albumin in these cells. In addition, the increase in urine levels of albumin in response to ischemia/reperfusion-induced acute renal failure was paralleled by a decrease in the expression of megalin and cubilin. Thus, our data indicate that the expression of megalin and cubilin is decreased during experimental endotoxemia and in response to renal ischemia/reperfusion injury. This downregulation may contribute, in part, to an increase in urine levels of albumin during acute renal failure.


Asunto(s)
Albuminuria/etiología , Endotoxemia/complicaciones , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores de Superficie Celular/metabolismo , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Albuminuria/sangre , Albuminuria/genética , Albuminuria/fisiopatología , Albuminuria/orina , Animales , Biomarcadores/sangre , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Endocitosis , Endotoxemia/sangre , Endotoxemia/inducido químicamente , Endotoxemia/genética , Endotoxemia/fisiopatología , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Capacidad de Concentración Renal , Túbulos Renales Proximales/fisiopatología , Lipopolisacáridos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Daño por Reperfusión/complicaciones , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Albúmina Sérica/metabolismo , Factores de Tiempo
13.
Am J Pathol ; 179(5): 2177-88, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21925138

RESUMEN

In many kidney diseases, the original insult primarily involves the glomerulus and may then pass onto the tubulointerstitium. Several hypotheses link glomerular disease to tubular injury; perhaps the foremost hypothesis involves chronic tubular hypoxia. The reported effects of hypoxia and consecutive stabilization of hypoxia-inducible factors (HIFs), however, are controversial. Hypoxia induces interstitial fibrosis but also has beneficial effects on renal disease progression when HIF is activated pharmacologically. To analyze the impact of HIF on tubulointerstitial disease development in primary glomerular disease, transgenic von Hippel Lindau (VHL)-knockout mice were generated and null expression was induced before the onset of autoimmune IgG-mediated anti-glomerular basement membrane glomerulonephritis (GN). Tubular VHL knockout and, thus, local HIF-α stabilization increased renal production of vascular endothelial growth factor, tumor growth factor-ß(1), and platelet-derived growth factor-B, resulting in augmented formation of capillaries and interstitial matrix, and conversion of fibroblasts to myofibroblasts. Within the glomerular disease, VHL knockout reduced the glomerular damage and attenuated tubulointerstitial injury. Likewise, proteinuria, plasma urea concentration, and tubulointerstitial matrix were decreased in VHL knockout with GN. These findings shown that tubular HIF-α stabilization in glomerular disease is beneficial for disease outcome. In comparison with VHL knockout alone, GN is a much stronger activator of fibrosis such that stimuli other than hypoxia may be considered important for renal disease progression.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/fisiopatología , Animales , Autoanticuerpos/administración & dosificación , Autoanticuerpos/farmacología , Capilares/citología , Proliferación Celular , Progresión de la Enfermedad , Inmunohistoquímica , Glomérulos Renales/irrigación sanguínea , Masculino , Ratones , Ratones Noqueados , Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas c-sis/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
14.
Biochim Biophys Acta Mol Cell Res ; 1869(4): 119190, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34968578

RESUMEN

Cathepsin B (CatB) is a very abundant lysosomal protease with endo- and carboxydipeptidase activities and even ligase features. In this review, we will provide a general characterization of CatB and describe structure, structure-derived properties and location-dependent proteolytic actions. We depict CatB action within lysosome and its important roles in lysosomal biogenesis, lysosomal homeostasis and autophagy rendering this protease a key player in orchestrating lysosomal functions. Lysosomal leakage and subsequent escape of CatB into the cytosol lead to harmful actions, e.g. the role in activating the NLPR3 inflammasome, affecting immune responses and cell death. The second focus of this review addresses CatB functions in the kidney, i.e. the glomerulus, the proximal tubule and collecting duct with strong emphasis of its role in pathology of the respective segment. Finally, observations regarding CatB functions that need to be considered in cell culture will be discussed. In conclusion, CatB a physiologically important molecule may, upon aberrant expression in different cellular context, become a harmful player effectively showing its teeth behind its smile.


Asunto(s)
Catepsina B/metabolismo , Riñón/metabolismo , Animales , Catepsina B/química , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Humanos , Inflamasomas/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Lisosomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo
15.
Cells ; 12(1)2022 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-36611847

RESUMEN

The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis.


Asunto(s)
Endocitosis , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Albúminas/metabolismo , Clatrina , Ligandos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Transporte de Proteínas
16.
Acta Physiol (Oxf) ; 234(2): e13735, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34704357

RESUMEN

AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.


Asunto(s)
Lesión Renal Aguda , Fosfatasa 3 de Especificidad Dual , Daño por Reperfusión , Lesión Renal Aguda/metabolismo , Animales , Fosfatasa 3 de Especificidad Dual/genética , Células Endoteliales/metabolismo , Inflamación/genética , Inflamación/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo
17.
Sci Signal ; 15(762): eabo7940, 2022 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-36445937

RESUMEN

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased ß-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.


Asunto(s)
COVID-19 , Glutamina , Humanos , Animales , Ratones , SARS-CoV-2 , Riñón , Nutrientes , Antivirales , Lípidos
18.
J Biol Chem ; 285(53): 41935-46, 2010 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-20966072

RESUMEN

The existence of a local renin angiotensin system (RAS) of the kidney has been established. Angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), angiotensin receptors, and high concentrations of luminal angiotensin II have been found in the proximal tubule. Although functional data have documented the relevance of a local RAS, the dualism between biosynthesis and endocytotic uptake of its components and their cellular processing has been incompletely understood. To resolve this, we have selectively analyzed their distribution, endocytosis, transcytosis, and biosynthesis in the proximal tubule. The presence of immunoreactive AGT, restricted to the early proximal tubule, was due to its retrieval from the ultrafiltrate and storage in endosomal and lysosomal compartments. Cellular uptake was demonstrated by autoradiography of radiolabeled AGT and depended on intact endocytosis. AGT was identified as a ligand of the multiple ligand-binding repeats of megalin. AGT biosynthesis was restricted to the proximal straight tubule, revealing substantial AGT mRNA expression. Transgenic AGT overexpression under the control of an endogenous promoter was also restricted to the late proximal tubule. Proximal handling of renin largely followed the patterns of AGT, whereas its local biosynthesis was not significant. Transcytotic transport of AGT in a proximal cell line revealed a 5% recovery rate after 1 h. ACE was expressed along late proximal brush-border membrane, whereas ACE2 was present along the entire segment. Surface expression of ACE and ACE2 differed as a function of endocytosis. Our data on the localization and cellular processing of RAS components provide new aspects of the functional concept of a "self-contained" renal RAS.


Asunto(s)
Endocitosis , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Nefronas/metabolismo , Sistema Renina-Angiotensina/fisiología , Animales , Animales Modificados Genéticamente , Endosomas/metabolismo , Humanos , Riñón/metabolismo , Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Sprague-Dawley
19.
Am J Pathol ; 177(2): 632-43, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20616344

RESUMEN

We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-beta1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-beta1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-beta1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-beta1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.


Asunto(s)
Autofagia/fisiología , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , Túbulos Renales , Riñón , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Epiteliales/ultraestructura , Fibrosis/metabolismo , Fibrosis/patología , Riñón/citología , Riñón/metabolismo , Riñón/patología , Túbulos Renales/metabolismo , Túbulos Renales/ultraestructura , Ratones , Ratones Endogámicos , Ratones Transgénicos
20.
Biochem J ; 431(1): 103-11, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20653565

RESUMEN

Distribution of selenium (Se) within the mammalian body is mediated by SePP (selenoprotein P), an Se-rich glycoprotein secreted by hepatocytes. Genetic and biochemical evidence indicate that the endocytic receptors ApoER2 (apolipoprotein E receptor 2) and megalin mediate tissue-specific SePP uptake. In the present study megalin-mutant mice were fed on diets containing adequate (0.15 p.p.m.) or low (0.08 p.p.m.) Se content and were analysed for tissue and plasma Se levels, cellular GPx (glutathione peroxidase) activities and protein expression patterns. Megalin-mutant mice displayed increased urinary Se loss, which correlated with SePP excretion in their urine. Accordingly, serum Se and SePP levels were significantly reduced in megalin-mutant mice, reaching marginal levels on the low-Se diet. Moreover, kidney Se content and expression of renal selenoproteins were accordingly reduced, as was SePP internalization along the proximal tubule epithelium. Although GPx4 expression was not altered in testis, Se and GPx activity in liver and brain were significantly reduced. When fed on a low-Se diet, megalin-mutant mice developed impaired movement co-ordination, but no astrogliosis. These findings suggest that megalin prevents urinary SePP loss and participates in brain Se/SePP uptake.


Asunto(s)
Encéfalo/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Mutación , Selenio/metabolismo , Selenoproteína P/metabolismo , Animales , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratas , Selenio/sangre , Selenio/orina , Selenoproteína P/sangre , Selenoproteína P/orina
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