Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 141
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Nature ; 616(7958): 814-821, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37046086

RESUMO

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.


Assuntos
Envelhecimento , Longevidade , Elongação da Transcrição Genética , Animais , Humanos , Camundongos , Ratos , Envelhecimento/genética , Insulina/metabolismo , Longevidade/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais , Drosophila melanogaster/genética , Caenorhabditis elegans/genética , RNA Circular , Somatomedinas , Nucleossomos , Histonas , Divisão Celular , Restrição Calórica
2.
EMBO J ; 42(21): e113891, 2023 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-37743763

RESUMO

Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.


Assuntos
Cílios , Organelas , Cílios/metabolismo , Diferenciação Celular
3.
Cell ; 150(3): 533-48, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863007

RESUMO

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exoma , Doenças Renais Císticas/genética , Proteínas dos Microtúbulos/metabolismo , Animais , Cílios/metabolismo , Técnicas de Silenciamento de Genes , Genes Recessivos , Humanos , Proteína Homóloga a MRE11 , Camundongos , Proteínas , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
4.
Hum Mol Genet ; 32(22): 3153-3165, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37565816

RESUMO

Mutations in genes encoding nuclear pore proteins (NUPs) lead to the development of steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS). However, the precise molecular mechanisms by which NUP dysfunction contributes to podocyte injury preceding FSGS remain unclear. The tightly regulated activity of Yes-associated protein (YAP) and WW-domain-containing transcription regulator 1 (TAZ), the transcriptional effectors of the Hippo pathway, is crucial for podocytes and the maintenance of the glomerular filter. In this study, we investigate the impact of NUPs on the regulation of YAP/TAZ nuclear import and activity in podocytes. In unbiased interactome studies using quantitative label-free mass spectrometry, we identify the FSGS disease gene products NUP107, NUP133, NUP205, and Exportin-5 (XPO5) as components of YAP and TAZ protein complexes in podocytes. Moreover, we demonstrate that NUP205 is essential for YAP/TAZ nuclear import. Consistently, both the nuclear interaction of YAP/TAZ with TEA domain transcription factor 1 and their transcriptional activity were dependent on NUP205 expression. Additionally, we elucidate a regulatory feedback mechanism whereby YAP activity is modulated in response to TAZ-mediated NUP205 expression. In conclusion, this study establishes a connection between the FSGS disease protein NUP205 and the activity of the transcriptional regulators and Hippo effectors YAP and TAZ and it proposes a potential pathological role of YAP/TAZ dysregulation in podocytes of patients with pathogenic NUP205 variants.


Assuntos
Glomerulosclerose Segmentar e Focal , Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Carioferinas , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosfoproteínas/genética , RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP
5.
J Am Soc Nephrol ; 34(5): 772-792, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36758124

RESUMO

SIGNIFICANCE STATEMENT: AKI is a major clinical complication leading to high mortality, but intensive research over the past decades has not led to targeted preventive or therapeutic measures. In rodent models, caloric restriction (CR) and transient hypoxia significantly prevent AKI and a recent comparative transcriptome analysis of murine kidneys identified kynureninase (KYNU) as a shared downstream target. The present work shows that KYNU strongly contributes to CR-mediated protection as a key player in the de novo nicotinamide adenine dinucleotide biosynthesis pathway. Importantly, the link between CR and NAD+ biosynthesis could be recapitulated in a human cohort. BACKGROUND: Clinical practice lacks strategies to treat AKI. Interestingly, preconditioning by hypoxia and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. METHODS: Kynureninase (KYNU) knockout mice were generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and comparative transcriptome, proteome and metabolite analyses of murine kidneys pre- and post-ischemia-reperfusion injury in the context of CR or ad libitum diet were performed. In addition, acetyl-lysin enrichment and mass spectrometry were used to assess protein acetylation. RESULTS: We identified KYNU as a downstream target of CR and show that KYNU strongly contributes to the protective effect of CR. The KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway is necessary for CR-associated maintenance of NAD+ levels. This finding is associated with reduced protein acetylation in CR-treated animals, specifically affecting enzymes in energy metabolism. Importantly, the effect of CR on de novo NAD+ biosynthesis pathway metabolites can be recapitulated in humans. CONCLUSIONS: CR induces the de novo NAD+ synthesis pathway in the context of IRI and is essential for its full nephroprotective potential. Differential protein acetylation may be the molecular mechanism underlying the relationship of NAD+, CR, and nephroprotection.


Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Humanos , Camundongos , Animais , NAD/metabolismo , Restrição Calórica , Traumatismo por Reperfusão/prevenção & controle , Injúria Renal Aguda/metabolismo , Hipóxia
6.
J Am Soc Nephrol ; 34(8): 1366-1380, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37367205

RESUMO

SIGNIFICANCE STATEMENT: Treatment of acute, crescentic glomerulonephritis (GN) consists of unspecific and potentially toxic immunosuppression. T cells are central in the pathogenesis of GN, and various checkpoint molecules control their activation. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown potential for restraining inflammation in other T-cell-mediated disease models. To investigate its role in GN in a murine model of crescentic nephritis, the authors induced nephrotoxic nephritis in BTLA-deficient mice and wild-type mice. They found that BTLA has a renoprotective role through suppression of local Th1-driven inflammation and expansion of T regulatory cells and that administration of an agonistic anti-BTLA antibody attenuated experimental GN. These findings suggest that antibody-based modulation of BTLA may represent a treatment strategy in human glomerular disease. BACKGROUND: Modulating T-lymphocytes represents a promising targeted therapeutic option for glomerulonephritis (GN) because these cells mediate damage in various experimental and human GN types. The immune checkpoint molecule B and T-lymphocyte attenuator (BTLA) has shown its potential to restrain inflammation in other T-cell-mediated disease models. Its role in GN, however, has not been investigated. METHODS: We induced nephrotoxic nephritis (NTN), a mouse model of crescentic GN, in Btla -deficient ( BtlaKO ) mice and wild-type littermate controls and assessed disease severity using functional and histologic parameters at different time points after disease induction. Immunologic changes were comprehensively evaluated by flow cytometry, RNA sequencing, and in vitro assays for dendritic cell and T-cell function. Transfer experiments into Rag1KO mice confirmed the observed in vitro findings. In addition, we evaluated the potential of an agonistic anti-BTLA antibody to treat NTN in vivo . RESULTS: The BtlaKO mice developed aggravated NTN, driven by an increase of infiltrating renal Th1 cells. Single-cell RNA sequencing showed increased renal T-cell activation and positive regulation of the immune response. Although BTLA-deficient regulatory T cells (Tregs) exhibited preserved suppressive function in vitro and in vivo , BtlaKO T effector cells evaded Treg suppression. Administration of an agonistic anti-BTLA antibody robustly attenuated NTN by suppressing nephritogenic T effector cells and promoting Treg expansion. CONCLUSIONS: In a model of crescentic GN, BTLA signaling effectively restrained nephritogenic Th1 cells and promoted regulatory T cells. Suppression of T-cell-mediated inflammation by BTLA stimulation may prove relevant for a broad range of conditions involving acute GN.


Assuntos
Glomerulonefrite Membranoproliferativa , Glomerulonefrite , Nefrite , Camundongos , Humanos , Animais , Proteínas de Checkpoint Imunológico , Glomerulonefrite/patologia , Glomerulonefrite Membranoproliferativa/complicações , Inflamação/complicações , Camundongos Endogâmicos C57BL
7.
Kidney Int ; 104(6): 1164-1169, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37774923

RESUMO

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions.


Assuntos
Podócitos , Animais , Camundongos , Capilares , Orientação Espacial , Glomérulos Renais , Artéria Renal , Mamíferos
8.
Kidney Int ; 103(6): 1120-1130, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36990215

RESUMO

Morphological alterations at the kidney filtration barrier increase intrinsic capillary wall permeability resulting in albuminuria. However, automated, quantitative assessment of these morphological changes has not been possible with electron or light microscopy. Here we present a deep learning-based approach for segmentation and quantitative analysis of foot processes in images acquired with confocal and super-resolution fluorescence microscopy. Our method, Automatic Morphological Analysis of Podocytes (AMAP), accurately segments podocyte foot processes and quantifies their morphology. AMAP applied to a set of kidney diseases in patient biopsies and a mouse model of focal segmental glomerulosclerosis allowed for accurate and comprehensive quantification of various morphometric features. With the use of AMAP, detailed morphology of podocyte foot process effacement was found to differ between categories of kidney pathologies, showed detailed variability between diverse patients with the same clinical diagnosis, and correlated with levels of proteinuria. AMAP could potentially complement other readouts such as various omics, standard histologic/electron microscopy and blood/urine assays for future personalized diagnosis and treatment of kidney disease. Thus, our novel finding could have implications to afford an understanding of early phases of kidney disease progression and may provide supplemental information in precision diagnostics.


Assuntos
Aprendizado Profundo , Glomerulosclerose Segmentar e Focal , Nefropatias , Podócitos , Camundongos , Animais , Podócitos/patologia , Glomérulos Renais/patologia , Rim/diagnóstico por imagem , Rim/patologia , Glomerulosclerose Segmentar e Focal/diagnóstico por imagem , Glomerulosclerose Segmentar e Focal/patologia , Nefropatias/diagnóstico por imagem , Nefropatias/patologia
9.
Nat Immunol ; 12(9): 898-907, 2011 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-21841785

RESUMO

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.


Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Proteínas de Ligação à Região de Interação com a Matriz/imunologia , Tolerância a Antígenos Próprios , Linfócitos T Reguladores/imunologia , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lentivirus , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Interferência de RNA , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância a Antígenos Próprios/efeitos dos fármacos , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Transdução Genética
10.
BMC Nephrol ; 24(1): 378, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38114895

RESUMO

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.


Assuntos
Síndrome Nefrótica , Podócitos , Humanos , Animais , Camundongos , Podócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , RNA Mensageiro/metabolismo
11.
J Am Soc Nephrol ; 33(1): 138-154, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853150

RESUMO

BACKGROUND: Diseases of the kidney's glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results. METHODS: To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q ). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments. RESULTS: Heterozygous PodR231Q/wild-type mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wild-type mice showed a more severe course of disease compared with Podwild-type/wild-type mice. Podocin protein levels were decreased in PodR231Q/wild-type and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation. CONCLUSIONS: Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.


Assuntos
Albuminúria/genética , Predisposição Genética para Doença/genética , Barreira de Filtração Glomerular/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Nefropatias/genética , Proteínas de Membrana/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/patologia
12.
Int J Mol Sci ; 24(3)2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36768831

RESUMO

Chronic Kidney Disease (CKD), a global health burden, is strongly associated with age-related renal function decline, hypertension, and diabetes, which are all frequent consequences of obesity. Despite extensive studies, the mechanisms determining susceptibility to CKD remain insufficiently understood. Clinical evidence together with prior studies from our group showed that perinatal metabolic disorders after intrauterine growth restriction or maternal obesity adversely affect kidney structure and function throughout life. Since obesity and aging processes converge in similar pathways we tested if perinatal obesity caused by high-fat diet (HFD)-fed dams sensitizes aging-associated mechanisms in kidneys of newborn mice. The results showed a marked increase of γH2AX-positive cells with elevated 8-Oxo-dG (RNA/DNA damage), both indicative of DNA damage response and oxidative stress. Using unbiased comprehensive transcriptomics we identified compartment-specific differentially-regulated signaling pathways in kidneys after perinatal obesity. Comparison of these data to transcriptomic data of naturally aged kidneys and prematurely aged kidneys of genetic modified mice with a hypomorphic allele of Ercc1, revealed similar signatures, e.g., inflammatory signaling. In a biochemical approach we validated pathways of inflammaging in the kidneys after perinatal obesity. Collectively, our initial findings demonstrate premature aging-associated processes as a consequence of perinatal obesity that could determine the susceptibility for CKD early in life.


Assuntos
Senilidade Prematura , Insuficiência Renal Crônica , Feminino , Camundongos , Animais , Gravidez , Humanos , Senilidade Prematura/metabolismo , Obesidade/metabolismo , Rim/metabolismo , Insuficiência Renal Crônica/metabolismo , Dieta Hiperlipídica/efeitos adversos , Envelhecimento/genética
13.
Kidney Int ; 101(4): 733-751, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34929254

RESUMO

Glomerular diseases are a major cause for chronic kidney disorders. In most cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and loss of podocytes. Here, we establish a link between the Par3 polarity complex and actin regulators necessary to establish and maintain podocyte architecture by utilizing mouse and Drosophila models to characterize the functional role of Par3A and Par3B and its fly homologue Bazooka in vivo. Only simultaneous inactivation of both Par3 proteins caused a severe disease phenotype. Rescue experiments in Drosophila nephrocytes revealed atypical protein kinase C (aPKC)-Par6 dependent and independent effects. While Par3A primarily acts via aPKC-Par6, Par3B function was independent of Par6. Actin-associated synaptopodin protein levels were found to be significantly upregulated upon loss of Par3A/B in mouse podocytes. Tropomyosin2, which shares functional similarities with synaptopodin, was also elevated in Bazooka depleted nephrocytes. The simultaneous depletion of Bazooka and Tropomyosin2 resulted in a partial rescue of the Bazooka knockdown phenotype and prevented increased Rho1-GTP, a member of a GTPase protein family regulating the cytoskeleton. The latter contribute to the nephrocyte phenotype observed upon loss of Bazooka. Thus, we demonstrate that Par3 proteins share a high functional redundancy but also have specific functions. Par3A acts in an aPKC-Par6 dependent way and regulates RhoA-GTP levels, while Par3B exploits Par6 independent functions influencing synaptopodin localization. Hence, Par3A and Par3B link elements of polarity signaling and actin regulators to maintain podocyte architecture.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Podócitos , Actinas/metabolismo , Animais , Polaridade Celular , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/genética , Camundongos , Podócitos/metabolismo , Proteína Quinase C
14.
Kidney Int ; 102(3): 560-576, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35654224

RESUMO

Acute kidney injury is a frequent complication in the clinical setting and associated with significant morbidity and mortality. Preconditioning with short-term caloric restriction is highly protective against kidney injury in rodent ischemia reperfusion injury models. However, the underlying mechanisms are unknown hampering clinical translation. Here, we examined the molecular basis of caloric restriction-mediated protection to elucidate the principles of kidney stress resistance. Analysis of an RNAseq dataset after caloric restriction identified Cyp4a12a, a cytochrome exclusively expressed in male mice, to be strongly downregulated after caloric restriction. Kidney ischemia reperfusion injury robustly induced acute kidney injury in male mice and this damage could be markedly attenuated by pretreatment with caloric restriction. In females, damage was significantly less pronounced and preconditioning with caloric restriction had only little effect. Tissue concentrations of the metabolic product of Cyp4a12a, 20-hydroxyeicosatetraenoic acid (20-HETE), were found to be significantly reduced by caloric restriction. Conversely, intraperitoneal supplementation of 20-HETE in preconditioned males partly abrogated the protective potential of caloric restriction. Interestingly, this effect was accompanied by a partial reversal of caloric restriction--induced changes in protein but not RNA expression pointing towards inflammation, endoplasmic reticulum stress and lipid metabolism. Thus, our findings provide an insight into the mechanisms underlying kidney protection by caloric restriction. Hence, understanding the mediators of preconditioning is an important prerequisite for moving towards translation to the clinical setting.


Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Restrição Calórica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Rim/metabolismo , Masculino , Camundongos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle
15.
J Am Soc Nephrol ; 32(11): 2885-2899, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34607910

RESUMO

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.


Assuntos
Cardiomiopatia Dilatada/genética , Hipercalciúria/genética , Nefropatias/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Nefrocalcinose/genética , Erros Inatos do Transporte Tubular Renal/genética , Serina-Treonina Quinases TOR/metabolismo , Cardiomiopatia Dilatada/metabolismo , Feminino , Células HEK293 , Humanos , Hipercalciúria/metabolismo , Nefropatias/metabolismo , Túbulos Renais Distais/metabolismo , Masculino , Modelos Moleculares , Natriurese/genética , Nefrocalcinose/metabolismo , Linhagem , Conformação Proteica , Erros Inatos do Transporte Tubular Renal/metabolismo , Convulsões/genética , Convulsões/metabolismo , Transdução de Sinais , Sequenciamento do Exoma , Sequenciamento Completo do Genoma
16.
Kidney Int ; 100(5): 1054-1062, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34332959

RESUMO

Loss of podocytes, possibly through the detachment of viable cells, is a hallmark of progressive glomerular disease. Podocytes are exposed to considerable physical forces due to pressure and flow resulting in circumferential wall stress and tangential shear stress exerted on the podocyte cell body, which have been proposed to contribute to podocyte depletion. However, estimations of in vivo alterations of physical forces in glomerular disease have been hampered by a lack of quantitative functional and morphological data. Here, we used ultra-resolution data and computational analyses in a mouse model of human disease, hereditary late-onset focal segmental glomerular sclerosis, to calculate increased mechanical stress upon podocyte injury. Transversal shear stress on the lateral walls of the foot processes was prominently increased during the initial stages of podocyte detachment. Thus, our study highlights the importance of targeting glomerular hemodynamics to treat glomerular disease.


Assuntos
Glomerulosclerose Segmentar e Focal , Nefropatias , Podócitos , Animais , Glomérulos Renais , Camundongos , Estresse Mecânico
17.
Kidney Int ; 99(4): 1010-1020, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33285146

RESUMO

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.


Assuntos
Glomérulos Renais , Rim , Animais , Rim/diagnóstico por imagem , Camundongos , Microscopia
18.
J Am Soc Nephrol ; 31(3): 532-542, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924670

RESUMO

BACKGROUND: Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo. METHODS: To study the effects of AngII on podocyte calcium levels in vivo, we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3. RESULTS: In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2, the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease. CONCLUSIONS: Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Losartan/farmacologia , Proteínas de Membrana/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Masculino , Camundongos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Distribuição Aleatória , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Valores de Referência
19.
J Am Soc Nephrol ; 31(3): 544-559, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32047005

RESUMO

BACKGROUND: Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS: We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS: Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS: We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.


Assuntos
Filaminas/genética , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/patologia , Proteômica/métodos , Estresse Fisiológico/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/genética , Humanos , Camundongos , Podócitos/metabolismo , Proteinúria/genética , Proteinúria/fisiopatologia , Distribuição Aleatória , Ratos
20.
J Am Soc Nephrol ; 31(4): 716-730, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32111728

RESUMO

BACKGROUND: Although AKI lacks effective therapeutic approaches, preventive strategies using preconditioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. METHODS: To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. RESULTS: The gene expression signatures induced by both preconditioning strategies involve distinct common genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. CONCLUSIONS: This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny.cecad.uni-koeln.de:3838/IRaP/).


Assuntos
Injúria Renal Aguda/genética , Injúria Renal Aguda/prevenção & controle , Restrição Calórica , Hipóxia , Precondicionamento Isquêmico/métodos , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controle , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA