RESUMO
Jiang et al. show that zinc finger FYVE-type containing 21, a Rab5 effector in glomerular endothelial cells is involved in the maintenance of glomerular filtration barrier homeostasis through the stabilization of activated endothelial nitric oxide synthase on subcellular vesicles. The study demonstrates that zinc finger FYVE-type containing 21 could modulate the levels of caveolin-1 in glomerular endothelial cells using vesicle-based trafficking, thereby supporting endothelial nitric oxide synthase activity. The authors provide evidence that decreased zinc finger FYVE-type containing 21 expression in glomerular endothelial cells could play a role in aging-related glomerular filtration barrier dysfunction.
Assuntos
Envelhecimento , Caveolina 1 , Células Endoteliais , Óxido Nítrico Sintase Tipo III , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Caveolina 1/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Animais , Barreira de Filtração Glomerular/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Glomérulos Renais/metabolismo , Rim/fisiopatologia , Rim/metabolismo , Endotélio Vascular/fisiopatologia , Endotélio Vascular/metabolismoRESUMO
Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.
Assuntos
Glomerulonefrite , Glomérulos Renais , Dispositivos Lab-On-A-Chip , Podócitos , Humanos , Podócitos/metabolismo , Podócitos/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Glomerulonefrite/patologia , Barreira de Filtração Glomerular/metabolismo , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Glomerulonefrite Membranosa/fisiopatologia , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/fisiopatologia , Permeabilidade , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Nefrite Lúpica/fisiopatologia , Sobrevivência Celular , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Nefrose Lipoide/fisiopatologiaRESUMO
Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.
Assuntos
Adiponectina , Diabetes Mellitus Tipo 2 , Glicocálix , Glomérulos Renais , Animais , Glicocálix/metabolismo , Glicocálix/efeitos dos fármacos , Adiponectina/metabolismo , Adiponectina/genética , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/efeitos dos fármacos , Humanos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Masculino , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Sindecana-4/metabolismo , Sindecana-4/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Membranous nephropathy (MN) is a chronic kidney disease and a precursor to end-stage kidney disease. In this study, we evaluated the potential protective effects of acidic and neutral Stigma maydis polysaccharides (ASMP and NSMP, respectively) on cationized bovine serum albumin-induced MN in mice. Both polysaccharides (SMPs) provided effective protection from kidney injury by decreasing daily proteinuria, kidney dysfunction, and hyperlipidemia and minimizing structural changes and immune complex expression. Furthermore, SMPs improved intestinal barrier damage by increasing the expression of tight junction proteins in the intestinal tissue. They also maintained the integrity of the glomerular filtration barrier by promoting slit diaphragm proteins expression and PI3K/AKT signaling. However, ASMP offered better protection against podocyte injury than NSMP. The use of natural polysaccharides could thus be a new protective measure against podocyte injury and perhaps be utilized for the development of functional foods to protect against MN.
Assuntos
Glomerulonefrite Membranosa , Podócitos , Polissacarídeos , Animais , Camundongos , Barreira de Filtração Glomerular/metabolismo , Glomerulonefrite Membranosa/tratamento farmacológico , Glomerulonefrite Membranosa/prevenção & controle , Rim/metabolismo , Glomérulos Renais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Podócitos/metabolismo , Polissacarídeos/farmacologia , Proteinúria/metabolismoRESUMO
Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.
Assuntos
Podócitos , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Barreira de Filtração Glomerular/metabolismo , Camundongos , Podócitos/metabolismo , Via Secretória , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney's filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive. METHODS: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene (Parva) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo, we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and PARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns. RESULTS: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB-dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly. CONCLUSIONS: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease.
Assuntos
Barreira de Filtração Glomerular , Proteínas dos Microfilamentos , Podócitos , Animais , Barreira de Filtração Glomerular/metabolismo , Integrinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismoRESUMO
In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.
Assuntos
Barreira de Filtração Glomerular , Glicocálix , N-Acetilglucosaminiltransferases , Barreira de Filtração Glomerular/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Barreira de Filtração Glomerular/metabolismo , Imunoglobulina G/imunologia , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Feminino , Barreira de Filtração Glomerular/efeitos dos fármacos , Humanos , Imunoglobulina G/classificação , Camundongos SCIDRESUMO
BACKGROUND: Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. METHODS: We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. RESULTS: We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome-associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. CONCLUSIONS: Key components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Barreira de Filtração Glomerular/química , Guanilato Quinases/química , Proteínas de Membrana/química , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Fenômenos Biofísicos , Moléculas de Adesão Celular/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/fisiologia , Proteínas de Fluorescência Verde , Guanilato Quinases/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Estrutura Molecular , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Transição de Fase , Domínios e Motivos de Interação entre ProteínasRESUMO
Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E2 (PGE2). Treating the endothelial specific Dgke-knockout mice with a stable PGE2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE2-dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE2, that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Diacilglicerol Quinase/genética , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Comunicação Autócrina , Ciclo-Oxigenase 2/metabolismo , Diacilglicerol Quinase/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Técnicas de Silenciamento de Genes , Barreira de Filtração Glomerular/efeitos dos fármacos , Barreira de Filtração Glomerular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores CXCR4/metabolismo , Microangiopatias Trombóticas/genética , Microangiopatias Trombóticas/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Hypoxia-inducible factor1 (HIF1) plays a pivotal role in ensuring cells adapt to low-oxygen conditions. Depletion of oxygen, a co-substrate during hydroxylation of prolyl (P402 and P564) residues of HIF1âº, evades HIF1⺠ubiquitination and enables its dimerization with HIF1ß to mediate global transcriptional response to hypoxia. Though HIF1 is largely considered eliciting a protective role during physiological or pathological hypoxia or ischemia, elevated HIF1 during chronic hypoxia contributes to glomerular diseases' pathology and proteinuria. The glomerulus is responsible for renal permselectivity and excretion of ultra-filtrated urine. Podocytes are the glomerulus' major cell types and are instrumental for glomerular filtration, permselectivity, and glomerular basement membrane maintenance. Podocyte injury is expected to impair the efficiency of glomerular filtration and manifestation of glomerulosclerosis and proteinuria. Accumulated evidence suggests that podocytes are susceptible to various insults during chronic hypoxia, including podocyte EMT, slit-diaphragm dysfunction, foot process effacement, and cytoskeletal derangement due to accumulation of HIF1. This review discusses how hypoxia/HIF1 signaling regulates various features and function of podocytes during exposure to chronic hypoxia or inducing HIF1 by various chemical modulators.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Glomerulonefrite/genética , Glomerulosclerose Segmentar e Focal/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Podócitos/metabolismo , Proteinúria/genética , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Doença Crônica , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/patologia , Glomerulonefrite/etiologia , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Hipóxia/complicações , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Podócitos/patologia , Proteinúria/etiologia , Proteinúria/metabolismo , Proteinúria/patologia , Transdução de Sinais , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismoRESUMO
RATIONALE: Glomerular capillaries are lined with a highly specialized fenestrated endothelium and contribute to the glomerular filtration barrier. The Notch signaling pathway is involved in regulation of glomerular filtration barrier, but its role in glomerular endothelium has not been investigated due to the embryonic lethality of animal models with genetic modification of Notch pathway components in the endothelium. OBJECTIVE: To determine the effects of aberrant activation of the Notch signaling in glomerular endothelium and the underlying molecular mechanisms. METHODS AND RESULTS: We established the ZEG-NICD1 (notch1 intracellular domain)/Tie2-tTA/Tet-O-Cre transgenic mouse model to constitutively activate Notch1 signaling in endothelial cells of adult mice. The triple transgenic mice developed severe albuminuria with significantly decreased VE-cadherin (vascular endothelial cadherin) expression in the glomerular endothelium. In vitro studies showed that either NICD1 (Notch1 intracellular domain) lentiviral infection or treatment with Notch ligand DLL4 (delta-like ligand 4) markedly reduced VE-cadherin expression and increased monolayer permeability of human renal glomerular endothelial cells. In addition, Notch1 activation or gene knockdown of VE-cadherin reduced the glomerular endothelial glycocalyx. Further investigation demonstrated that activated Notch1 suppression of VE-cadherin was through the transcription factors SNAI1 (snail family transcriptional repressor 1) and ERG (Ets related gene), which bind to the -373 E-box and the -134/-118 ETS (E26 transformation-specific) element of the VE-cadherin promoter, respectively. CONCLUSIONS: Our results reveal novel regulatory mechanisms whereby endothelial Notch1 signaling dictates the level of VE-cadherin through the transcription factors SNAI1 and ERG, leading to dysfunction of glomerular filtration barrier and induction of albuminuria. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Albuminúria/metabolismo , Barreira de Filtração Glomerular/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Caderinas/genética , Caderinas/metabolismo , Células Endoteliais/metabolismo , Barreira de Filtração Glomerular/citologia , Glicocálix/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Regulador Transcricional ERG/metabolismoRESUMO
Gangliosides constitute a subgroup of glycosphingolipids characterized by the presence of sialic acid residues in their structure. As constituents of cellular membranes, in particular of raft microdomains, they exert multiple functions, some of them capital in cell homeostasis. Their presence in cells is tightly regulated by a balanced expression and function of the enzymes responsible for their biosynthesis, ganglioside synthases, and their degradation, glycosidases. The dysregulation of their abundance results in rare and common diseases. In this review, we make a point on the relevance of gangliosides and some of their metabolic precursors, such as ceramides, in the function of podocytes, the main cellular component of the glomerular filtration barrier, as well as their implications in podocytopathies. The results presented in this review suggest the pertinence of clinical lipidomic studies targeting these metabolites.
Assuntos
Membrana Celular/metabolismo , Gangliosídeos/metabolismo , Barreira de Filtração Glomerular/metabolismo , Podócitos/patologia , Animais , Humanos , Podócitos/metabolismoRESUMO
Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.
Assuntos
Doxorrubicina/farmacologia , Podócitos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Modelos Animais de Doenças , Barreira de Filtração Glomerular/efeitos dos fármacos , Barreira de Filtração Glomerular/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Rim/patologia , Glomérulos Renais/patologia , Larva/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVES: Islet transplantation has been proved to be effective in delaying early stage of DN. This study was established to observe the mechanism of islet transplantation on early diabetic nephropathy (DN). METHOD: The diabetes mellitus (DM) rat model was established by an injection of a single-dose streptozotocin. According to the treatment, the rats were randomly divided into 4 groups: the untreated DN rats (DN group); the C-peptide treated rats (CP group); the islet transplanted rats (IT group); the normal control rats (NC group). Renal function and structure of glomerular filtration barrier (GFB) were evaluated by urinalysis and histopathological examination, respectively. The renal fibrotic factors, TGF- ß1 and CTGF, as well as the anti-renal fibrosis factor HGF were assessed by immunohistochemical staining and western blotting methods. RESULTS: After C-peptide treatment and islet transplantation, the GFB structure was obviously improved. The blood glucose significantly decreased in the IT group. The 24h urine protein and glomerular basement membrane thickness decreased, the pathological changes of podocytes improved, TGF- ß1 and CTGF decreased and HGF increased in the CP group and the IT group compared with that in the DN group (P < 0.05), especially in the IT group. CONCLUSION: Islet transplantation could ameliorate the structure of GFB of early DN in a rat model, and the treatment effect was partly attributed to the restoration of C-peptide concentration. Suppressing the fibrosis system can be the potential mechanism of islet transplantation, which is independent of blood glucose control.
Assuntos
Peptídeo C/metabolismo , Diabetes Mellitus/terapia , Nefropatias Diabéticas/terapia , Barreira de Filtração Glomerular/metabolismo , Barreira de Filtração Glomerular/patologia , Transplante das Ilhotas Pancreáticas , Animais , Modelos Animais de Doenças , Fibrose , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Estreptozocina , Fator de Crescimento Transformador beta1/metabolismo , UrináliseRESUMO
Diabetic nephropathy as a deleterious complication of diabetes mellitus and an important cause of end-stage renal failure is characterized by changes in the molecular and cellular levels. Cell-cell communication via the gap and tight junctions are involved in the pathogenesis of diseases such as diabetes and kidney failure. Studying cell junctions including gap junctions, tight junctions, and anchoring junctions within the nephron can be used as an early sign of diabetic nephropathy. Furthermore, cell junctions may be an upcoming target by pharmacological methods to improve treatments of diabetic nephropathy and pave the way to introduce promising therapeutic strategies based on cell-cell communications effects and its translation into clinical studies for the treatment of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Barreira de Filtração Glomerular/metabolismo , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Nefropatias Diabéticas/patologia , Suscetibilidade a Doenças , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Humanos , Podócitos/metabolismo , Podócitos/patologiaRESUMO
Podocytes have foot processes that comprise an important cellular layer of the glomerular barrier involved in regulating glomerular permeability. The disturbance of podocyte function plays a central role in the development of proteinuria in diabetic nephropathy. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes, and these channels are involved in disturbing the glomerular filtration barrier in diabetes. Metformin is an anti-diabetic drug widely used for treating patients with type 2 diabetes. Recent studies have suggested that the therapeutic effect of metformin might be mediated by AMPK. The precise function of metformin on cellular function and intracellular signaling in podocytes under diabetic conditions is not fully understood. In this study, we demonstrated that metformin normalized TRPC6 expression via AMPKα1 activation in podocytes exposed to high glucose concentrations. A quantitative analysis showed that metformin increased the colocalization of TRPC6 and AMPKα1 subunits from 42% to 61% in standard glucose (SG) medium and from 29% to 52% in high glucose (HG) medium. AMPK activation was also necessary for maintaining appropriate levels of Rho-family small GTPase activity in HG conditions. Moreover, metformin through AMPK activation remodeled cytoskeleton dynamics, and consequently, reduced filtration barrier permeability in diabetic conditions.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Citoesqueleto/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Metformina/farmacologia , Podócitos/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Animais , Citoesqueleto/metabolismo , Nefropatias Diabéticas/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Barreira de Filtração Glomerular/efeitos dos fármacos , Barreira de Filtração Glomerular/metabolismo , Glucose/metabolismo , Masculino , Podócitos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
The endothelial glycocalyx (eGlx) constitutes the first barrier to protein in all blood vessels. This is particularly noteworthy in the renal glomerulus, an ultrafiltration barrier. Leakage of protein, such as albumin, across glomerular capillaries results in albumin in the urine (albuminuria). This is a hall mark of kidney disease and can reflect loss of blood vessel integrity in microvascular beds elsewhere. We discuss evidence demonstrating that targeted damage to the glomerular eGlx results in increased glomerular albumin permeability. EGlx is lost in diabetes and experimental models demonstrate loss from glomerular endothelial cells. Vascular endothelial growth factor (VEGF)A is upregulated in early diabetes, which is associated with albuminuria. Treatment with paracrine growth factors such as VEGFC, VEGF165b and angiopoietin-1 can modify VEGFA signalling, rescue albumin permeability and restore glomerular eGlx in models of diabetes. Manipulation of VEGF receptor 2 signalling, or a common eGlx biosynthesis pathway by these growth factors, may protect and restore the eGlx layer. This would help to direct future therapeutics in diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/terapia , Células Endoteliais/metabolismo , Glicocálix/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Células Endoteliais/efeitos dos fármacos , Barreira de Filtração Glomerular/efeitos dos fármacos , Barreira de Filtração Glomerular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagemRESUMO
Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson's trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease.
Assuntos
Colite/etiologia , Colite/metabolismo , Colágeno/metabolismo , Barreira de Filtração Glomerular/metabolismo , Glomérulos Renais/metabolismo , Animais , Biomarcadores , Biópsia , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Imuno-Histoquímica , Camundongos , Modelos BiológicosRESUMO
Acute cardiorenal syndrome (CRS-1) is a morbid complication of acute cardiovascular disease. Heart-to-kidney signals transmitted by "cardiorenal connectors" have been postulated, but investigation into CRS-1 has been limited by technical limitations and a paucity of models. To address these limitations, we developed a translational model of CRS-1, cardiac arrest and cardiopulmonary resuscitation (CA/CPR), and now report findings from nanoscale mass spectrometry proteomic exploration of glomerular filtrate 2 hours after CA/CPR or sham procedure. Filtrate acquisition was confirmed by imaging, molecular weight and charge distribution, and exclusion of protein specific to surrounding cells. Filtration of proteins specific to the heart was detected following CA/CPR and confirmed with mass spectrometry performed using urine collections from mice with deficient tubular endocytosis. Cardiac LIM protein was a CA/CPR-specific filtrate component. Cardiac arrest induced plasma release of cardiac LIM protein in mice and critically ill human cardiac arrest survivors, and administration of recombinant cardiac LIM protein to mice altered renal function. These findings demonstrate that glomerular filtrate is accessible to nanoscale proteomics and elucidate the population of proteins filtered 2 hours after CA/CPR. The identification of cardiac-specific proteins in renal filtrate suggests a novel signaling mechanism in CRS-1. We expect these findings to advance understanding of CRS-1.