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1.
Cell Death Dis ; 14(8): 562, 2023 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-37626062

RESUMO

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.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Nefropatias Diabéticas , Animais , Camundongos , Nefropatias Diabéticas/genética , Rim , Túbulos Renais Proximais , Glomérulos Renais , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética
2.
J Extracell Vesicles ; 12(7): e12338, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37408115

RESUMO

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.


Assuntos
Antineoplásicos , Vesículas Extracelulares , Neoplasias Ovarianas , Humanos , Feminino , Proteínas ADAM/metabolismo , Receptores ErbB , Vesículas Extracelulares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Proteína ADAM17
3.
Sci Adv ; 9(13): eadf4055, 2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-37000885

RESUMO

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.


Assuntos
Galectina 3 , Metaloendopeptidases , Camundongos , Animais , Galectina 3/genética , Galectina 3/metabolismo , Metaloproteases/metabolismo , Proteólise , Camundongos Knockout , Homeostase
4.
Sci Signal ; 15(762): eabo7940, 2022 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-36445937

RESUMO

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.


Assuntos
COVID-19 , Glutamina , Humanos , Animais , Camundongos , SARS-CoV-2 , Rim , Nutrientes , Antivirais , Lipídeos
5.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077596

RESUMO

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.


Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Apoptose/genética , Fibrose , Quinase I-kappa B/genética , Isquemia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Traumatismo por Reperfusão/genética
6.
J Am Soc Nephrol ; 33(4): 732-745, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35149593

RESUMO

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.


Assuntos
Surdez , Peixe-Zebra , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Surdez/genética , Endocitose , Humanos , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Mutação , Proteinúria/metabolismo , Proteínas de Transporte Vesicular/genética , Adulto Jovem , Peixe-Zebra/metabolismo
7.
Biochim Biophys Acta Mol Cell Res ; 1869(4): 119190, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34968578

RESUMO

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.


Assuntos
Catepsina B/metabolismo , Rim/metabolismo , Animais , Catepsina B/química , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Humanos , Inflamassomos/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Lisossomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
8.
Acta Physiol (Oxf) ; 234(2): e13735, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34704357

RESUMO

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.


Assuntos
Injúria Renal Aguda , Fosfatase 3 de Especificidade Dupla , Traumatismo por Reperfusão , Injúria Renal Aguda/metabolismo , Animais , Fosfatase 3 de Especificidade Dupla/genética , Células Endoteliais/metabolismo , Inflamação/genética , Inflamação/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo
9.
Cells ; 12(1)2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36611847

RESUMO

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.


Assuntos
Endocitose , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Albuminas/metabolismo , Clatrina , Ligantes , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transporte Proteico
10.
FASEB Bioadv ; 3(2): 94-107, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33615154

RESUMO

The cJun N-terminal Kinases (JNK) emerged as a major link between obesity and insulin resistance, but their role in the loss of pancreatic ß-cell mass and function driving the progression from insulin resistance to type-2 diabetes and in the complications of diabetes was not investigated to the same extent. Furthermore, it was shown that pan-JNK inhibition exacerbates kidney damage in the db/db model of obesity-driven diabetes. Here we investigate the role of JNK1 in the db/db model of obesity-driven type-2 diabetes. Mice with systemic ablation of JNK1 (JNK1-/-) were backcrossed for more than 10 generations in db/+ C57BL/KS mice to generate db/db-JNK1-/- mice and db/db control mice. To define the role of JNK1 in the loss of ß-cell mass and function occurring during obesity-driven diabetes we performed comprehensive metabolic phenotyping, evaluated steatosis and metabolic inflammation, performed morphometric and cellular composition analysis of pancreatic islets, and evaluated kidney function in db/db-JNK1-/- mice and db/db controls. db/db-JNK1-/- mice and db/db control mice developed insulin resistance, fatty liver, and metabolic inflammation to a similar extent. However, db/db-JNK1-/- mice displayed better glucose tolerance and improved insulin levels during glucose tolerance test, higher pancreatic insulin content, and larger pancreatic islets with more ß-cells than db/db mice. Finally, albuminuria, kidney histopathology, kidney inflammation and oxidative stress in db/db-JNK1-/- mice and in db/db mice were similar. Our data indicate that selective JNK1 ablation improves glucose tolerance in db/db mice by reducing the loss of functional ß-cells occurring in the db/db mouse model of obesity-driven diabetes, without significantly affecting metabolic inflammation, steatosis, and insulin sensitivity. Furthermore, we have found that, differently from what previously reported for pan-JNK inhibitors, selective JNK1 ablation does not exacerbate kidney dysfunction in db/db mice. We conclude that selective JNK1 inactivation may have a superior therapeutic index than pan-JNK inhibition in obesity-driven diabetes.

11.
Cell Death Differ ; 28(5): 1610-1626, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33273695

RESUMO

The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans.


Assuntos
COVID-19/enzimologia , Primidona/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , SARS-CoV-2/metabolismo , Animais , COVID-19/patologia , Morte Celular/efeitos dos fármacos , Células HEK293 , Células HT29 , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Células Jurkat , Camundongos , Células NIH 3T3 , Células U937 , Tratamento Farmacológico da COVID-19
12.
Sci Rep ; 9(1): 12403, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455864

RESUMO

In proteinuric nephropathies of chronic kidney disease, the epithelial cells of the nephron including the collecting duct are exposed to high concentrations of luminal albumin. Albumin is taken up from collecting duct cells by endocytosis causing excessive reactive oxygen species (ROS) production and a proinflammatory response. Curcumin used in the traditional medicine possesses anti-inflammatory and antioxidant effects. ROS and ADP-ribose (ADPR) activate the cation channel TRPM2. We hypothesize, that albumin-induced cell stress and proinflammatory response are mediated by Ca2+ and can be reduced by curcumin. The cortical collecting duct (CCD) cells mpkCCDc14 exhibit spontaneous and inducible Ca2+ oscillations, which can be blocked by pre-treatment with curcumin. Curcumin accumulates in plasma membrane and intracellular vesicles, where it interferes with TRPM2 and decreases the influx of Ca2+. Albumin reduces cell viability and increases apoptosis, NF-κB activation, and mitochondrial membrane depolarization via Ca2+-dependent signaling, which results in increased ROS production. Albumin-induced cell stress is diminished by the inhibition of TRPM2 after administration of curcumin and ADPR (PARP1) inhibitors. Curcumin did not reduce the Ca2+ elevation induced by thapsigargin in Ca2+-free medium, but it reduced the function of store-operated Ca2+ channels and ATP-evoked Ca2+ response. In conclusion, albumin-induced oxidative stress is mediated by Ca2+-dependent signaling via TRPM2 and leads to cell damage and a proinflammatory response, strengthening the role of CCD cells in the progression of chronic kidney disease.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Curcumina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Soroalbumina Bovina/farmacologia , Canais de Cátion TRPM/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPM/genética , Tapsigargina/farmacologia
13.
J Cell Mol Med ; 23(10): 6543-6553, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31368174

RESUMO

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.


Assuntos
Catepsina B/metabolismo , Canais Epiteliais de Sódio/metabolismo , Hipertensão/etiologia , Hipertensão/metabolismo , Síndrome Nefrótica/complicações , Síndrome Nefrótica/metabolismo , Amilorida/farmacologia , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Glomerulosclerose Segmentar e Focal/enzimologia , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/urina , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Lisossomos/enzimologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Síndrome Nefrótica/genética , Proteinúria/metabolismo , Proteólise , Sódio/metabolismo
14.
J Am Heart Assoc ; 7(15): e008363, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30371243

RESUMO

Background In pregnancy, a high plasma volume maintains uteroplacental perfusion and prevents placental ischemia, a condition linked to elevated maternal blood pressure ( BP ). Reducing BP by increasing Na+ intake via plasma volume expansion appears contra-intuitive. We hypothesize that an appropriate Na+ intake in pregnancy reduces maternal BP and adapts the renin-angiotensin system in a pregnancy-specific manner. Methods and Results BP was measured by implanted telemetry in Sprague-Dawley rats before and throughout pregnancy. Pregnant and nonpregnant animals received either a normal-salt (0.4%; NS ), high-salt (8%; HS ), or low-salt (0.01%; LS ) diet, or HS (days 1-14) followed by LS (days 14-20) diet ( HS / LS ). Before delivery (day 20), animals were euthanized and organs collected. Food, water, and Na+ intake were monitored in metabolic cages, and urinary creatinine and Na+ were analyzed. Na+ intake and retention increased in pregnancy ( NS , LS ), leading to a positive Na+ balance ( NS , LS ). BP was stable during LS , but reduced in HS conditions in pregnancy. The renin-angiotensin system was adapted as expected. Activating cleavage of α- and γ-subunits of the renal epithelial Na+ channel and expression of-full length medullary ß-subunits, accentuated further in all LS conditions, were upregulated in pregnancy. Conclusions Pregnancy led to Na+ retention adapted to dietary changes. HS exposure paradoxically reduced BP . Na+ uptake while only modestly linked to the renin-angiotensin system is enhanced in the presence of posttranslational renal epithelial Na+ channel modifications. This suggests (1) storage of Na+ in pregnancy upon HS exposure, bridging periods of LS availability; and (2) that potentially non-renin-angiotensin-related mechanisms participate in EN aC activation and consecutive Na+ retention.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Sódio na Dieta/farmacologia , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Angiotensinas/efeitos dos fármacos , Angiotensinas/genética , Animais , Dieta Hipossódica , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/genética , Gravidez , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/genética , Sistema Renina-Angiotensina/genética , Telemetria , Água
15.
Sci Rep ; 7(1): 4149, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28646163

RESUMO

Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption.


Assuntos
Aldosterona/farmacologia , Canais Epiteliais de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Linhagem Celular , Canais Epiteliais de Sódio/ultraestrutura , Córtex Renal/metabolismo , Córtex Renal/ultraestrutura , Camundongos , Subunidades Proteicas/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/ultraestrutura
16.
J Am Soc Nephrol ; 28(1): 230-241, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27297946

RESUMO

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.


Assuntos
Endocitose/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Complexos Multiproteicos/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Transporte Proteico
17.
Biochem J ; 473(19): 3237-52, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27422782

RESUMO

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.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Córtex Renal/metabolismo , Camundongos , Microscopia Confocal , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
18.
J Am Soc Nephrol ; 27(3): 731-44, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26116357

RESUMO

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.


Assuntos
Albuminas/metabolismo , Angiotensina II/farmacologia , Glomérulos Renais/fisiologia , Podócitos/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transcitose/efeitos dos fármacos , Vasoconstritores/farmacologia , Aminas , Animais , Feminino , Gentamicinas/farmacologia , Microscopia Intravital , Glomérulos Renais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência por Excitação Multifotônica , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Vesículas Transportadoras , Urina
19.
Am J Physiol Renal Physiol ; 308(10): F1047-55, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25651559

RESUMO

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.


Assuntos
Fator Natriurético Atrial/fisiologia , Rim/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Taxa de Filtração Glomerular/fisiologia , Humanos , Camundongos , Modelos Animais , Serina Endopeptidases/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia
20.
J Am Soc Nephrol ; 26(6): 1269-78, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25270072

RESUMO

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.


Assuntos
Aquaporina 1/genética , Permeabilidade da Membrana Celular/fisiologia , Regulação da Expressão Gênica , Túbulos Renais Proximais/fisiopatologia , Equilíbrio Hidroeletrolítico/genética , Adaptação Fisiológica , Animais , Aquaporina 1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Taxa de Filtração Glomerular/fisiologia , Camundongos , Camundongos Transgênicos , Microvilosidades/metabolismo , Osmose , Transporte Proteico/fisiologia , Distribuição Aleatória , Sensibilidade e Especificidade , Fatores de Tempo , Equilíbrio Hidroeletrolítico/fisiologia
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