RESUMO
Inflammation is an important component of fibrosis but immune processes that orchestrate kidney fibrosis are not well understood. Here we apply single-cell sequencing to a mouse model of kidney fibrosis. We identify a subset of kidney tubule cells with a profibrotic-inflammatory phenotype characterized by the expression of cytokines and chemokines associated with immune cell recruitment. Receptor-ligand interaction analysis and experimental validation indicate that CXCL1 secreted by profibrotic tubules recruits CXCR2+ basophils. In mice, these basophils are an important source of interleukin-6 and recruitment of the TH17 subset of helper T cells. Genetic deletion or antibody-based depletion of basophils results in reduced renal fibrosis. Human kidney single-cell, bulk gene expression and immunostaining validate a function for basophils in patients with kidney fibrosis. Collectively, these studies identify basophils as contributors to the development of renal fibrosis and suggest that targeting these cells might be a useful clinical strategy to manage chronic kidney disease.
Assuntos
Basófilos , Insuficiência Renal Crônica , Animais , Fibrose , Humanos , Rim/metabolismo , Túbulos Renais , Camundongos , Insuficiência Renal Crônica/metabolismo , Análise de Célula ÚnicaRESUMO
The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.
Assuntos
Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Queratinócitos/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Transcriptoma , Biópsia , Linhagem da Célula/genética , Biologia Computacional/métodos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Humanos , Nefrite Lúpica/patologia , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1ß. The adaptor ASC is necessary for NLRP3-dependent inflammasome function, but it is not known whether ASC is a sufficient partner and whether inflammasome formation occurs in the cytosol or in association with mitochondria is controversial. Here, we show that the mitochondria-associated adaptor molecule, MAVS, is required for optimal NLRP3 inflammasome activity. MAVS mediates recruitment of NLRP3 to mitochondria, promoting production of IL-1ß and the pathophysiologic activity of the NLRP3 inflammasome in vivo. Our data support a more complex model of NLRP3 inflammasome activation than previously appreciated, with at least two adapters required for maximal function. Because MAVS is a mitochondria-associated molecule previously considered to be uniquely involved in type 1 interferon production, these findings also reveal unexpected polygamous involvement of PYD/CARD-domain-containing adapters in innate immune signaling events.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/química , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Humanos , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Túbulos Renais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Monócitos/imunologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/patologia , Domínios e Motivos de Interação entre Proteínas , Alinhamento de SequênciaRESUMO
Mitochondria play a key role in kidney physiology and pathology. They produce ATP to fuel energy-demanding water and solute reabsorption processes along the nephron. Moreover, mitochondria contribute to cellular health by the regulation of autophagy, (oxidative) stress responses, and apoptosis. Mitochondrial abundance is particularly high in cortical segments, including proximal and distal convoluted tubules. Dysfunction of the mitochondria has been described for tubulopathies such as Fanconi, Gitelman, and Bartter-like syndromes and renal tubular acidosis. In addition, mitochondrial cytopathies often affect renal (tubular) tissues, such as in Kearns-Sayre and Leigh syndromes. Nevertheless, the mechanisms by which mitochondrial dysfunction results in renal tubular diseases are only scarcely being explored. This review provides an overview of mitochondrial dysfunction in the development and progression of kidney tubulopathies. Furthermore, it emphasizes the need for further mechanistic investigations to identify links between mitochondrial function and renal electrolyte reabsorption.
Assuntos
Síndrome de Bartter , Síndrome de Kearns-Sayre , Nefropatias , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Síndrome de Bartter/metabolismo , Síndrome de Bartter/patologia , Síndrome de Kearns-Sayre/metabolismo , Síndrome de Kearns-Sayre/patologia , Nefropatias/patologia , MitocôndriasRESUMO
Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist1-3. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood1,2,4. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.
Assuntos
Linhagem da Célula , Fibrose/patologia , Túbulos Renais/patologia , Miofibroblastos/patologia , Insuficiência Renal Crônica/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Masculino , Mesoderma/citologia , Mesoderma/patologia , Camundongos , Miofibroblastos/metabolismo , Pericitos/citologia , Pericitos/patologia , RNA-Seq , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Análise de Célula Única , TranscriptomaRESUMO
Drug-induced acute renal failure (ARF) is a public health concern that hinders optimal drug therapy. However, pathological mechanisms of drug-induced ARF remain to be elucidated. Here, we show that a pathological process of drug-induced ARF is mediated by proinflammatory cross-talk between kidney tubular cells and macrophages. Both polymyxin B and colistin, polypeptide antibiotics, frequently cause ARF, stimulated the ERK and NF-κB pathways in kidney tubular cells, and thereby upregulated M-CSF and MCP-1, leading to infiltration of macrophages into the kidneys. Thereafter, the kidney-infiltrated macrophages were exposed to polypeptide antibiotics, which initiated activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Interestingly, blockade of the NLRP3 activation clearly ameliorated the pathology of ARF induced by polypeptide antibiotics, suggesting that a combination of the distinct cellular responses to polypeptide antibiotics in kidney tubular cells and macrophages plays a key role in the pathogenesis of colistin-induced ARF. Thus, our results provide a concrete example of how drugs initiate ARF, which may give insight into the underlying pathological process of drug-induced ARF.
Assuntos
Injúria Renal Aguda , Antibacterianos , Inflamassomos , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Camundongos , Inflamassomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Polimixina B/farmacologia , Camundongos Endogâmicos C57BL , Colistina/efeitos adversos , Colistina/farmacologia , Peptídeos/farmacologia , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/efeitos dos fármacos , Masculino , NF-kappa B/metabolismoRESUMO
Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Epitélio , Organoides/metabolismo , Túbulos Renais , Diferenciação CelularRESUMO
Classical physiological studies using electrophysiological, biophysical, biochemical, and molecular techniques have created a detailed picture of molecular transport, bioenergetics, contractility and movement, and growth, as well as the regulation of these processes by external stimuli in cells and organisms. Newer systems biology approaches are beginning to provide deeper and broader understanding of these complex biological processes and their dynamic responses to a variety of environmental cues. In the past decade, advances in mass spectrometry-based proteomic technologies have provided invaluable tools to further elucidate these complex cellular processes, thereby confirming, complementing, and advancing common views of physiology. As one notable example, the application of proteomics to study the regulation of kidney function has yielded novel insights into the chemical and physical processes that tightly control body fluids, electrolytes, and metabolites to provide optimal microenvironments for various cellular and organ functions. Here, we systematically review, summarize, and discuss the most significant key findings from functional proteomic studies in renal epithelial physiology. We also identify further improvements in technological and bioinformatics methods that will be essential to advance precision medicine in nephrology.
Assuntos
Túbulos Renais/metabolismo , Túbulos Renais/fisiologia , Animais , Biologia Computacional/métodos , Humanos , Espectrometria de Massas/métodos , Proteômica/métodosRESUMO
Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.
Assuntos
Metabolismo Energético , Túbulos Renais , Mitocôndrias , Animais , Camundongos , Mitocôndrias/metabolismo , Túbulos Renais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Biogênese de Organelas , Transporte Biológico , Glicólise/fisiologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Surgery-induced renal ischemia and reperfusion (I/R) injury and nephrotoxic drugs like cisplatin can cause acute kidney injury (AKI), for which there is no effective therapy. Lipid accumulation is evident following AKI in renal tubules although the mechanisms and pathological effects are unclear. Here, we report that Ehmt2-encoded histone methyltransferase G9a is upregulated in patients and mouse kidneys after AKI. Renal tubular specific knockout of G9a (Ehmt2Ksp ) or pharmacological inhibition of G9a alleviates lipid accumulation associated with AKI. Mechanistically, G9a suppresses transcription of the lipolytic enzyme Ces1; moreover, G9a and farnesoid X receptor (FXR) competitively bind to the same promoter regions of Ces1. Ces1 is consistently observed to be downregulated in the kidney of AKI patients. Pharmacological inhibition of Ces1 increases lipid accumulation, exacerbates renal I/R-injury and eliminates the beneficial effects on AKI observed in Ehmt2Ksp mice. Furthermore, lipid-lowering atorvastatin and an FXR agonist alleviate AKI by activating Ces1 and reducing renal lipid accumulation. Together, our results reveal a G9a/FXR-Ces1 axis that affects the AKI outcome via regulating renal lipid accumulation.
Assuntos
Injúria Renal Aguda , Túbulos Renais , Camundongos , Animais , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/induzido quimicamente , Lipídeos , Rim/patologia , Camundongos Endogâmicos C57BLRESUMO
The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout (dKO) of the upstream Hippo kinases Mst1 and Mst2 promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell overproliferation, tubular injury, and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistent with this, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell overproliferation, tubular injury, and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, where tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell overproliferation, tubular injury, and renal inflammation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Túbulos Renais , Fatores de Transcrição Kruppel-Like , Camundongos Knockout , Proteínas de Sinalização YAP , Animais , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Serina-Treonina Quinase 3 , Transdução de Sinais , Proliferação de Células , Regulação da Expressão Gênica , Modelos Animais de Doenças , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Cisplatino/farmacologiaRESUMO
Despite the recent discovery of tissue regeneration enhancers in highly regenerative animals, upstream and downstream genetic programs connected by these enhancers still remain unclear. Here, we performed a genome-wide analysis of enhancers and associated genes in regenerating nephric tubules of Xenopus laevis. Putative enhancers were identified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analyses. Their target genes were predicted based on their proximity to enhancers on genomic DNA and consistency of their transcriptome profiles to ATAC-seq/ChIP-seq profiles of the enhancers. Motif enrichment analysis identified the central role of Krüppel-like factors (Klf) in the enhancer. Klf15, a member of the Klf family, directly binds enhancers and stimulates expression of regenerative genes, including adrenoreceptor alpha 1A (adra1a), whereas inhibition of Klf15 activity results in failure of nephric tubule regeneration. Moreover, pharmacological inhibition of Adra1a-signaling suppresses nephric tubule regeneration, while its activation promotes nephric tubule regeneration and restores organ size. These results indicate that Klf15-dependent adrenergic receptor signaling through regeneration enhancers plays a central role in the genetic network for kidney regeneration.
Assuntos
Elementos Facilitadores Genéticos , Túbulos Renais , Fatores de Transcrição Kruppel-Like , Receptores Adrenérgicos , Regeneração , Animais , Cromatina/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Túbulos Renais/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores Adrenérgicos/metabolismo , Regeneração/genética , Xenopus laevisRESUMO
SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.
Assuntos
Túbulos Renais , Fosfotransferases (Aceptor do Grupo Fosfato) , Animais , Masculino , Camundongos , Rim/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Túbulos Renais/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismoRESUMO
AIMS: Lactate accumulation is reported to be a biomarker for diabetic nephropathy progression. Lactate drives lysine lactylation, a newly discovered post-translational modification that is involved in the pathogenesis of cancers and metabolic and inflammatory disease. Here, we aimed to determine whether lysine lactylation is involved in the pathogenesis of diabetic nephropathy. METHODS: Renal biopsy samples from individuals with diabetic nephropathy (n=22) and control samples from individuals without diabetes and kidney disease (n=9) were obtained from the First Affiliated Hospital of Zhengzhou University for immunohistochemical staining. In addition, we carried out global lactylome profiling of kidney tissues from db/m and db/db mice using LC-MS/MS. Furthermore, we assessed the role of lysine lactylation and acyl-CoA synthetase family member 2 (ACSF2) in mitochondrial function in human proximal tubular epithelial cells (HK-2). RESULTS: The expression level of lysine lactylation was significantly increased in the kidneys of individuals with diabetes as well as in kidneys from db/db mice. Integrative lactylome analysis of the kidneys of db/db and db/m mice identified 165 upregulated proteins and 17 downregulated proteins, with an increase in 356 lysine lactylation sites and a decrease in 22 lysine lactylation sites decreased. Subcellular localisation analysis revealed that most lactylated proteins were found in the mitochondria (115 proteins, 269 sites). We further found that lactylation of the K182 site in ACSF2 contributes to mitochondrial dysfunction. Finally, the expression of ACSF2 was notably increased in the kidneys of db/db mice and individuals with diabetic nephropathy. CONCLUSIONS: Our study strongly suggests that lysine lactylation and ACSF2 are mediators of mitochondrial dysfunction and may contribute to the progression of diabetic nephropathy. DATA AVAILABILITY: The LC-MS/MS proteomics data have been deposited in the ProteomeXchange Consortium database ( https://proteomecentral.proteomexchange.org ) via the iProX partner repository with the dataset identifier PXD050070.
Assuntos
Nefropatias Diabéticas , Túbulos Renais , Lisina , Animais , Camundongos , Humanos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Lisina/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Coenzima A Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Lipoilação , Camundongos Endogâmicos C57BL , FemininoRESUMO
The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.
Assuntos
Túbulos Renais , Proteínas dos Microfilamentos , Animais , Camundongos , Junções Aderentes/metabolismo , Túbulos Renais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Néfrons/metabolismoRESUMO
The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.
Assuntos
Injúria Renal Aguda , Cílios , Mitocôndrias , Triptofano , Animais , Cílios/metabolismo , Cílios/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Cães , Triptofano/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Células Madin Darby de Rim Canino , Espécies Reativas de Oxigênio/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/deficiência , Camundongos KnockoutRESUMO
T cells mediate organ injury and repair. A proportion of unconventional kidney T cells called double-negative (DN) T cells (TCR+ CD4- CD8-), with anti-inflammatory properties, were previously demonstrated to protect from early injury in moderate experimental acute kidney injury (AKI). However, their role in repair after AKI has not been studied. We hypothesized that DN T cells mediate repair after severe AKI. C57B6 mice underwent severe (40 min) unilateral ischemia-reperfusion injury (IRI). Kidney DN T cells were studied by flow cytometry and compared with gold-standard anti-inflammatory CD4+ regulatory T cells (Tregs). In vitro effects of DN T cells and Tregs on renal tubular epithelial cell (RTEC) repair after injury were quantified with live-cell analysis. DN T cells, Tregs, CD4, or vehicle were adoptively transferred after severe AKI. Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-sinistrin. Fibrosis was assessed with Masson's trichrome staining. Profibrotic genes were measured with qRT-PCR. Percentages and the numbers of DN T cells substantially decreased during repair phase after severe AKI, as well as their activation and proliferation. Both DN T cells and Tregs accelerated RTEC cell repair in vitro. Post-AKI transfer of DN T cells reduced kidney fibrosis and improved GFR, as did Treg transfer. DN T cell transfer lowered transforming growth factor (TGF)ß1 and α-smooth muscle actin (αSMA) expression. DN T cells reduced effector-memory CD4+ T cells and IL-17 expression. DN T cells undergo quantitative and phenotypical changes after severe AKI, accelerate RTEC repair in vitro as well as improve GFR and renal fibrosis in vivo. DN T cells have potential as immunotherapy to accelerate repair after AKI.NEW & NOTEWORTHY Double-negative (DN) T cells (CD4- CD8-) are unconventional kidney T cells with regulatory abilities. Their role in repair from acute kidney injury (AKI) is unknown. Kidney DN T cell population decreased during repair after ischemic AKI, in contrast to regulatory T cells (Tregs) which increased. DN T cell administration accelerated tubular repair in vitro, while after severe in vivo ischemic injury reduced kidney fibrosis and increased glomerular filtration rate (GFR). DN T cell infusion is a potential therapeutic agent to improve outcome from severe AKI.
Assuntos
Injúria Renal Aguda , Taxa de Filtração Glomerular , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão , Linfócitos T Reguladores , Animais , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Masculino , Modelos Animais de Doenças , Fibrose , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transferência Adotiva , Camundongos , Rim/patologia , Rim/imunologia , Rim/metabolismo , Fenótipo , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Regeneração , Células CultivadasRESUMO
Kidney epithelial cells have very high energy requirements, which are largely met by fatty acid oxidation. Complex changes in lipid metabolism are observed in patients with kidney disease. Defects in fatty acid oxidation and increased lipid uptake, especially in the context of hyperlipidemia and proteinuria, contribute to this excess lipid build-up and exacerbate kidney disease development. Recent studies have also highlighted the role of increased de novo lipogenesis in kidney fibrosis. The defect in fatty acid oxidation causes energy starvation. Increased lipid uptake, synthesis, and lower fatty acid oxidation can cause toxic lipid build-up, reactive oxygen species generation, and mitochondrial damage. A better understanding of these metabolic processes may open new treatment avenues for kidney diseases by targeting lipid metabolism.
Assuntos
Ácidos Graxos , Túbulos Renais , Metabolismo dos Lipídeos , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Animais , Ácidos Graxos/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Mitocôndrias/metabolismo , Lipogênese , Oxirredução , Fibrose , Espécies Reativas de Oxigênio/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Metabolismo EnergéticoRESUMO
Acute kidney injury is still associated with high morbidity and mortality. Reichardt et al. investigated DNA-binding protein-A (Ybx3) in acute kidney injury induced by ischemia-reperfusion injury and found that mice lacking Ybx3 have altered mitochondrial function and increased antioxidant activity, making them more resistant to ischemia-reperfusion injury-acute kidney injury. The study highlights a new role of the multifaceted protein DNA-binding protein-A, which could be potentially therapeutically exploited.
Assuntos
Injúria Renal Aguda , Células Epiteliais , Túbulos Renais , Traumatismo por Reperfusão , Animais , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/etiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Camundongos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/citologia , Humanos , Mitocôndrias/metabolismo , Estresse OxidativoRESUMO
BACKGROUND: Patients with type 2 diabetes often face early tubular injury, necessitating effective treatment strategies. This study aimed to evaluate the impact of the SGLT2 inhibitor empagliflozin on early tubular injury biomarkers in type 2 diabetes patients with normoalbuminuria. METHODS: A randomized controlled clinical study comprising 54 patients selected based on specific criteria was conducted. Patients were divided into an intervention group (empagliflozin, n = 27) and a control group (n = 27) and treated for 6 weeks. Tubular injury biomarkers KIM-1 and NGAL were assessed pre- and post-treatment. RESULTS: Both groups demonstrated comparable baseline characteristics. Post-treatment, fasting and postprandial blood glucose levels decreased similarly in both groups. The intervention group exhibited better improvements in total cholesterol, low-density lipoprotein, and blood uric acid levels. Renal function indicators, including UACR and eGFR, showed greater enhancements in the intervention group. Significant reductions in KIM-1 and NGAL were observed in the intervention group. CONCLUSION: Treatment with empagliflozin in type 2 diabetes patients with normoalbuminuria led to a notable decrease in tubular injury biomarkers KIM-1 and NGAL. These findings highlight the potential of SGLT2 inhibitors in early tubular protection, offering a new therapeutic approach.