RESUMEN
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.
Asunto(s)
Lesión Renal Aguda , Daño por Reperfusión , Humanos , Ratones , Animales , NAD/metabolismo , Restricción Calórica , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/metabolismo , HipoxiaRESUMEN
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.
Asunto(s)
Podocitos , Animales , Ratones , Capilares , Orientación Espacial , Glomérulos Renales , Arteria Renal , MamíferosRESUMEN
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.
Asunto(s)
Aprendizaje Profundo , Glomeruloesclerosis Focal y Segmentaria , Enfermedades Renales , Podocitos , Ratones , Animales , Podocitos/patología , Glomérulos Renales/patología , Riñón/diagnóstico por imagen , Riñón/patología , Glomeruloesclerosis Focal y Segmentaria/diagnóstico por imagen , Glomeruloesclerosis Focal y Segmentaria/patología , Enfermedades Renales/diagnóstico por imagen , Enfermedades Renales/patologíaRESUMEN
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.
Asunto(s)
Síndrome Nefrótico , Podocitos , Humanos , Animales , Ratones , Podocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Albuminuria/genética , Predisposición Genética a la Enfermedad/genética , Barrera de Filtración Glomerular/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedades Renales/genética , Proteínas de la Membrana/genética , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Podocitos/patologíaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Podocitos , Actinas/metabolismo , Animales , Polaridad Celular , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/genética , Ratones , Podocitos/metabolismo , Proteína Quinasa CRESUMEN
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.
Asunto(s)
Lesión Renal Aguda , Daño por Reperfusión , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Animales , Restricción Calórica , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacología , Riñón/metabolismo , Masculino , Ratones , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
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.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria , Enfermedades Renales , Podocitos , Animales , Glomérulos Renales , Ratones , Estrés MecánicoRESUMEN
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.
Asunto(s)
Glomérulos Renales , Riñón , Animales , Riñón/diagnóstico por imagen , Ratones , MicroscopíaRESUMEN
BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.
Asunto(s)
Células Epiteliales/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Hipoxia de la Célula/fisiología , Cilios/metabolismo , Células HEK293 , Humanos , Ratones , Unión ProteicaRESUMEN
Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.
Asunto(s)
Lesión Renal Aguda/prevención & control , Restricción Calórica , Hipoxia/metabolismo , Proteoma/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Animales , Cisplatino/toxicidad , Activación de Complemento/efectos de los fármacos , Biología Computacional , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Hipoxia/etiología , Masculino , Ratones , Prueba de Estudio Conceptual , Proteolisis/efectos de los fármacos , Índice de Severidad de la EnfermedadRESUMEN
Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.
Asunto(s)
Actinas/metabolismo , Cilios/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteoma/metabolismo , Actinas/química , Animales , Biología Computacional , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Membranas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Miosinas/deficiencia , Miosinas/genética , Miosinas/metabolismo , Proteómica , Transducción de SeñalRESUMEN
In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.
Asunto(s)
Glomerulonefritis/metabolismo , Nefronas/metabolismo , Proteinuria/metabolismo , Proteoma , Proteómica/métodos , Espectrometría de Masas en Tándem , Animales , Variación Biológica Individual , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Glomerulonefritis/genética , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Humanos , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Nefronas/patología , Nefronas/fisiopatología , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Síndrome Nefrótico/fisiopatología , Podocitos/metabolismo , Podocitos/patología , Proteinuria/genética , Proteinuria/patología , Proteinuria/fisiopatología , Proteostasis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Albúmina Sérica/metabolismo , Proteínas WT1RESUMEN
Genetic diseases constitute the most important cause for end-stage renal disease in children and adolescents. Mutations in the ACTN4 gene, encoding the actin-binding protein α-actinin-4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease-causing ACTN4 mutation (p.G195D, de novo) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry (MS) of patient-derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased when compared with healthy controls. By resolving the peptide bearing the mutated residue, we could proof that the mutant protein is less abundant when compared with the wild-type protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome, which was predominantly composed of cytoskeletal modulators and LIM domain-containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient-derived cells. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations of the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high-resolution proteomics analyses to study the pathogenicity of rare gene variants.
Asunto(s)
Actinina/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Fallo Renal Crónico/genética , Actinina/metabolismo , Actinas/metabolismo , Adolescente , Secuencia de Aminoácidos , Citoesqueleto/metabolismo , Femenino , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Fallo Renal Crónico/metabolismo , Proteínas de Microfilamentos/metabolismo , ProteómicaRESUMEN
The PHB-domain protein podocin maintains the renal filtration barrier and its mutation is an important cause of hereditary nephrotic syndrome. Podocin and its Caenorhabditis elegans orthologue MEC-2 have emerged as key components of mechanosensitive membrane protein signalling complexes. Whereas podocin resides at a specialized cell junction at the podocyte slit diaphragm, MEC-2 is found in neurons required for touch sensitivity. Here, we show that the ubiquitin ligase Ubr4 is a key component of the podocin interactome purified both from cultured podocytes and native glomeruli. It colocalizes with podocin and regulates its stability. In C. elegans, this process is conserved. Here, Ubr4 is responsible for the degradation of mislocalized MEC-2 multimers. Ubiquitylomic analysis of mouse glomeruli revealed that podocin is ubiquitylated at two lysine residues. These sites were Ubr4-dependent and were conserved across species. Molecular dynamics simulations revealed that ubiquitylation of one site, K301, do not only target podocin/MEC-2 for proteasomal degradation, but may also affect stability and disassembly of the multimeric complex. We suggest that Ubr4 is a key regulator of podocyte foot process proteostasis.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Humanos , Masculino , Ratones , Simulación de Dinámica Molecular , Síndrome Nefrótico/metabolismo , Prohibitinas , UbiquitinaciónRESUMEN
Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Enfermedades Renales/metabolismo , Podocitos/metabolismo , Proteolisis , Animales , Células Cultivadas , Humanos , Masculino , Ratones Noqueados , Proteoma , Proteómica/métodos , RatasRESUMEN
Polycystic kidney disease (PKD) is among the leading causes of end-stage renal disease. Increasing evidence exists that molecular therapeutic strategies targeted to cyst formation and growth might be more efficacious in early disease stages, highlighting the growing need for sensitive biomarkers. Here we apply quantitative magnetic resonance imaging techniques of T2 mapping and diffusion-weighted imaging in the jck mouse model for PKD using a clinical 3.0 T scanner. We tested whether kidney T2 values and the apparent diffusion coefficient (ADC) are superior to anatomical imaging parameters in the detection of early cystogenesis, as shown on macro- and histopathology. We also tested whether kidney T2 values and ADC have the potential to monitor early treatment effects of therapy with the V2 receptor antagonist Mozavaptane. Kidney T2 values and to a lesser degree ADC were found to be highly sensitive markers of early cystogenesis and superior to anatomical-based imaging parameters. Furthermore, kidney T2 values exhibited a nearly perfect correlation to the histological cystic index, allowing a clear separation of the two mouse genotypes. Additionally, kidney T2 values and ADC were able to monitor early treatment effects in the jck mouse model in a proof-of-principle experiment. Thus, given the superiority of kidney T2 values and ADC over anatomical-based imaging in mice, further studies are needed to evaluate the translational impact of these techniques in patients with PKD.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas/uso terapéutico , Benzazepinas/uso terapéutico , Quistes/diagnóstico por imagen , Riñón/diagnóstico por imagen , Enfermedades Renales Poliquísticas/diagnóstico por imagen , Adulto , Animales , Quistes/tratamiento farmacológico , Quistes/genética , Quistes/patología , Imagen de Difusión por Resonancia Magnética/métodos , Modelos Animales de Enfermedad , Diagnóstico Precoz , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Riñón/patología , Estudios Longitudinales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Mutación , Quinasas Relacionadas con NIMA/genética , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Prueba de Estudio Conceptual , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.
Asunto(s)
Técnicas de Sustitución del Gen , Genes Reporteros , Integrasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Péptidos/genética , Podocitos/metabolismo , Proteínas Virales/genética , Animales , Separación Celular/métodos , Codón , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas Luminiscentes/biosíntesis , Proteínas de la Membrana/biosíntesis , Ratones Endogámicos C57BL , Ratones Transgénicos , Prohibitinas , Regiones Promotoras Genéticas , Factores de Tiempo , Proteína Fluorescente RojaRESUMEN
Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.
Asunto(s)
Mitocondrias/fisiología , Podocitos/fisiología , Proteínas Represoras/deficiencia , Animales , Proteínas de Caenorhabditis elegans , Células Cultivadas , Células HEK293 , Humanos , Uniones Intercelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/fisiología , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/fisiopatología , Membranas Mitocondriales/fisiología , Membranas Mitocondriales/ultraestructura , Podocitos/ultraestructura , Prohibitinas , Proteinuria/etiología , Proteinuria/fisiopatología , Tacto/fisiologíaRESUMEN
Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.