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Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.
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Proteínas de Transporte , Cílios , Proteína 1 Homóloga a Discs-Large , Canais de Cátion TRPP , Animais , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genética , Camundongos , Proteína 1 Homóloga a Discs-Large/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Humanos , Transporte Proteico , Camundongos Knockout , Rim/metabolismo , Células Epiteliais/metabolismo , Ligação Proteica , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Anormalidades UrogenitaisRESUMO
Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.
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Rim , Multiômica , Humanos , Diferenciação Celular/genética , Células Cultivadas , Organoides/metabolismo , Análise de Célula ÚnicaRESUMO
BACKGROUND: Hematuria is an established sign of glomerular disease and can be associated with kidney failure, but there has been limited scientific study of this trait. METHODS: Here, we combined genetic data from the UK Biobank with predicted gene expression and splicing from GTEx kidney cortex samples (n = 65) in a transcriptome-wide association study (TWAS) to identify additional potential biological mechanisms influencing hematuria. RESULTS: The TWAS using kidney cortex identified significant associations for 5 genes in terms of expression and 3 significant splicing events. Notably, we identified an association between the skipping of COL4A4 exon 27, which is genetically predicted by intronic rs11898094 (minor allele frequency 13%), and hematuria. Association between this variant was also found with urinary albumin excretion. We found independent evidence supporting the same variant predicting this skipping event in glomeruli-derived mRNA transcriptomics data (n = 245) from NEPTUNE. The functional significance of loss of exon 27 was demonstrated using the split NanoLuc-based α3α4α5(IV) heterotrimer assay, in which type IV collagen heterotrimer formation was quantified by luminescence. The causal splicing variant for this skipping event is yet to be identified. CONCLUSIONS: In summary, by integrating multiple data types, we identify a potential splicing event associated with hematuria and albuminuria.
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Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α3α4α5(IV) and α1α1α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV):α1/2(IV) mean fluorescence intensities (α5:α1/2 intensity ratios) and thicknesses (α5:α1/2 thickness ratios) were calculated to represent the levels of collagen α3α4α5(IV) relative to α1α1α2(IV). The α5:α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.
Assuntos
Glomerulosclerose Segmentar e Focal , Nefrite Hereditária , Humanos , Membrana Basal Glomerular/patologia , Colágeno Tipo IV/genética , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Parafina , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Membrana Basal/patologiaRESUMO
COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress in vivo and in vitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function in vivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.
Assuntos
Apoptose , Autoantígenos , Colágeno Tipo IV , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Camundongos Transgênicos , Mutação , Nefrite Hereditária , Ácido Tauroquenodesoxicólico , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nefrite Hereditária/genética , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/patologia , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Apoptose/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Masculino , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismoRESUMO
PURPOSE OF REVIEW: With the latest classification, variants in three collagen IV genes, COL4A3 , COL4A4 , and COL4A5 , represent the most prevalent genetic kidney disease in humans, exhibiting diverse, complex, and inconsistent clinical manifestations. This review breaks down the disease spectrum and genotype-phenotype correlations of kidney diseases linked to genetic variants in these genes and distinguishes "classic" Alport syndrome (AS) from the less severe nonsyndromic genetically related nephropathies that we suggest be called "Alport kidney diseases". RECENT FINDINGS: Several research studies have focused on the genotype-phenotype correlation under the latest classification scheme of AS. The historic diagnoses of "benign familial hematuria" and "thin basement membrane nephropathy" linked to heterozygous variants in COL4A3 or COL4A4 are suggested to be obsolete, but instead classified as autosomal AS by recent expert consensus due to a significant risk of disease progression. SUMMARY: The concept of Alport kidney disease extends beyond classic AS. Patients carrying pathogenic variants in any one of the COL4A3/A4/A5 genes can have variable phenotypes ranging from completely normal/clinically unrecognizable, hematuria without or with proteinuria, or progression to chronic kidney disease and kidney failure, depending on sex, genotype, and interplays of other genetic as well as environmental factors.
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Nefrite Hereditária , Humanos , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Hematúria/genética , Rim/patologia , Colágeno Tipo IV/genética , MutaçãoRESUMO
SIGNIFICANCE STATEMENT: HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development. BACKGROUND: The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. METHODS: We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. RESULTS: Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation. CONCLUSIONS: This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.
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Redes Reguladoras de Genes , Rim , Humanos , Camundongos , Animais , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Regulação da Expressão Gênica , Organoides/metabolismo , Fator 4 Nuclear de Hepatócito/genéticaRESUMO
Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by â¼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.
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Membrana Basal/metabolismo , Bromo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Peroxidase/metabolismo , Animais , Biópsia , Bromatos/metabolismo , Brometos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Iminas/metabolismo , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica , PeroxidasinaRESUMO
BACKGROUND: Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. METHODS: We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail, while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion-beam scanning electron microscopy, and machine learning image segmentation. RESULTS: Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures, located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. CONCLUSIONS: Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.
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Citoesqueleto de Actina/ultraestrutura , Imageamento Tridimensional/métodos , Aprendizado de Máquina , Microscopia Eletrônica , Podócitos/ultraestrutura , Animais , Camundongos , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
BACKGROUND: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. METHODS: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. RESULTS: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. CONCLUSIONS: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.
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Synaptopodin (Synpo) is an actin-associated protein in podocyte foot processes. By generating mice that completely lack Synpo, we previously showed that Synpo is dispensable for normal kidney function. However, lack of Synpo worsened adriamycin-induced nephropathy, indicating a protective role for Synpo in injured podocytes. Here, we investigated whether lack of Synpo directly impacts a genetic disease, Alport syndrome (AS), because Synpo is reduced in podocytes of affected humans and mice; whether this is merely an association or pathogenic is unknown. We used collagen type IV-α5 (Col4a5) mutant mice, which model X-linked AS, showing glomerular basement membrane (GBM) abnormalities, eventual foot process effacement, and progression to end-stage kidney disease. We intercrossed mice carrying mutations in Synpo and Col4a5 to produce double-mutant mice. Urine and tissue were taken at select time points to evaluate albuminuria, histopathology, and glomerular capillary wall composition and ultrastructure. Lack of Synpo in Col4a5-/Y, Col4a5-/-, or Col4a5+/- Alport mice led to the acceleration of disease progression, including more severe proteinuria and glomerulosclerosis. Absence of Synpo attenuated the shift of myosin IIA from the podocyte cell body and major processes to actin cables near the GBM in the areas of effacement. We speculate that this is mechanistically associated with enhanced loss of podocytes due to easier detachment from the GBM. We conclude that Synpo deletion exacerbates the disease phenotype in Alport mice, revealing the podocyte actin cytoskeleton as a target for therapy in patients with AS.NEW & NOTEWORTHY Alport syndrome (AS) is a hereditary disease of the glomerular basement with hematuria and proteinuria. Podocytes eventually exhibit foot process effacement, indicating actin cytoskeletal changes. To investigate how cytoskeletal changes impact podocytes, we generated Alport mice lacking synaptopodin, an actin-binding protein in foot processes. Analysis showed a more rapid disease progression, demonstrating that synaptopodin is protective. This suggests that the actin cytoskeleton is a target for therapy in AS and perhaps other glomerular diseases.
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Nefropatias/genética , Proteínas dos Microfilamentos/deficiência , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Modelos Animais de Doenças , Membrana Basal Glomerular/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismoRESUMO
Aminopeptidase A is one of the most potent enzymes within the renin-angiotensin system in terms of angiotensin II degradation. Here, we examined whether there is a kidney phenotype and any compensatory changes in other renin angiotensin system enzymes involved in the metabolism of angiotensin II associated with aminopeptidase A deficiency. Kidneys harvested from aminopeptidase A knockout mice were examined by light and electron microscopy, immunohistochemistry and immunofluorescence. Kidney angiotensin II levels and the ability of renin angiotensin system enzymes in the glomerulus to degrade angiotensin II ex vivo, their activities, protein and mRNA levels in kidney lysates were evaluated. Knockout mice had increased blood pressure and mild glomerular mesangial expansion without significant albuminuria. By electron microscopy, knockout mice exhibited a mild increase of the mesangial matrix, moderate thickening of the glomerular basement membrane but a striking appearance of knob-like structures. These knobs were seen in both male and female mice and persisted after the treatment of hypertension. In isolated glomeruli from knockout mice, the level of angiotensin II was more than three-fold higher as compared to wild type control mice. In kidney lysates from knockout mice angiotensin converting enzyme activity, protein and mRNA levels were markedly decreased possibly as a compensatory mechanism to reduce angiotensin II formation. Thus, our findings support a role for aminopeptidase A in the maintenance of glomerular structure and intra-kidney homeostasis of angiotensin peptides.
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Membrana Basal Glomerular , Glutamil Aminopeptidase , Rim , Angiotensina II/metabolismo , Animais , Feminino , Membrana Basal Glomerular/metabolismo , Glutamil Aminopeptidase/genética , Glutamil Aminopeptidase/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Sistema Renina-Angiotensina/genéticaRESUMO
Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.
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Fator 3 Ativador da Transcrição/metabolismo , Albuminúria/metabolismo , Regulação para Baixo , Estresse do Retículo Endoplasmático , Glucuronidase/biossíntese , Túbulos Renais/metabolismo , Transcrição Gênica , Fator 3 Ativador da Transcrição/genética , Albuminúria/genética , Albuminúria/patologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Glucuronidase/genética , Humanos , Túbulos Renais/patologia , Proteínas Klotho , Camundongos , Camundongos KnockoutRESUMO
Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.
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Polaridade Celular/fisiologia , Citocinese/fisiologia , Proteína 1 Homóloga a Discs-Large/metabolismo , Anáfase , Animais , Cartilagem/metabolismo , Cartilagem/fisiologia , Ciclo Celular , Embrião de Galinha , Condrócitos/metabolismo , Proteína 1 Homóloga a Discs-Large/fisiologia , Desenvolvimento Embrionário , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Metáfase , Camundongos , Camundongos Knockout , Microscopia de Fluorescência/métodos , Mitose/fisiologia , Morfogênese/fisiologia , Vertebrados/metabolismoRESUMO
BACKGROUND: Synaptopodin (Synpo) is an actin-associated protein in podocytes and dendritic spines. Many functions in regulating the actin cytoskeleton via RhoA and other pathways have been ascribed to Synpo, yet no pathogenic mutations in the SYNPO gene have been discovered in patients. Naturally occurring Synpo isoforms are known (Synpo-short and -long), and a novel truncated version (Synpo-T) is upregulated in podocytes from Synpo mutant mice. Synpo-T maintains some Synpo functions, which may prevent a podocyte phenotype from emerging in unchallenged mutant mice. METHODS: Novel mouse models were generated to further investigate the functions of Synpo. In one, CRISPR/Cas9 deleted most of the Synpo gene, preventing production of any detectable Synpo protein. Two other mutant strains made truncated versions of the protein. Adriamycin injections were used to challenge the mice, and Synpo functions were investigated in primary cultured podocytes. RESULTS: Mice that could not make detectable Synpo (Synpo-/- ) did not develop any kidney abnormalities up to 12 months of age. However, Synpo-/- mice were more susceptible to Adriamycin nephropathy. In cultured primary podocytes from mutant mice, the absence of Synpo caused loss of stress fibers, increased the number and size of focal adhesions, and impaired cell migration. Furthermore, loss of Synpo led to decreased RhoA activity and increased Rac1 activation. CONCLUSIONS: In contrast to previous findings, podocytes can function normally in vivo in the absence of any Synpo isoform. Synpo plays a protective role in the context of podocyte injury through its involvement in actin reorganization and focal adhesion dynamics.
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Homeostase/fisiologia , Nefropatias/etiologia , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Animais , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Movimento Celular/fisiologia , Modelos Animais de Doenças , Nefropatias/metabolismo , Nefropatias/patologia , CamundongosRESUMO
BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals. RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency. CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.
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Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Animais , Membrana Basal/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas da Matriz Extracelular/genética , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Esclera/citologia , Esclera/metabolismoRESUMO
The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin ß2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.
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Síndrome Nefrótica , Distúrbios Pupilares , Animais , Laminina/genética , Camundongos , Camundongos Knockout , Síndrome Nefrótica/genética , Distúrbios Pupilares/genéticaRESUMO
Alport syndrome is experiencing a remarkable increase in preclinical investigations. To proactively address the needs of the Alport syndrome community, as well as offer clarity for future clinical research sponsors, the Alport Syndrome Foundation hosted a workshop to generate consensus recommendations for prospective trials for conventional drugs. Opinions of key stakeholders were carefully considered, including those of the biopharmaceutical industry representatives, academic researchers, clinicians, regulatory agency representatives, and-most critically-patients with Alport syndrome. Recommendations were established for preclinical researchers, the use and selection of biomarkers, standards of care, clinical trial designs, trial eligibility criteria and outcomes, pediatric trial considerations, and considerations for patient engagement, recruitment, and treatment. This paper outlines their recommendations.
Assuntos
Transplante de Rim , Nefrite Hereditária , Biomarcadores , Criança , Humanos , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/tratamento farmacológico , Estudos ProspectivosRESUMO
Alport syndrome is caused by mutations in the genes COL4A3, COL4A4 or COL4A5 and is characterised by progressive glomerular disease, sensorineural hearing loss and ocular defects. Occurring in less than 1:5000, Alport syndrome is a rare genetic disorder but still accounts for > 1% of the prevalent population receiving renal replacement therapy. There is also increasing awareness about the risk of chronic kidney disease in individuals with heterozygous mutations in Alport syndrome genes. The mainstay of current therapy is the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, yet potential new therapies are now entering clinical trials. The 2017 International Workshop on Alport Syndrome in Glasgow was a pre-conference workshop ahead of the 50th anniversary meeting of the European Society for Pediatric Nephrology. It focussed on updates in clinical practice, genetics and basic science and also incorporated patient perspectives. More than 80 international experts including clinicians, geneticists, researchers from academia and industry, and patient representatives took part in panel discussions and breakout groups. This report summarises the workshop proceedings and the relevant contemporary literature. It highlights the unique clinician, patient and researcher collaborations achieved by regular engagement between the groups.
Assuntos
Pesquisa Biomédica/organização & administração , Colaboração Intersetorial , Nefrite Hereditária/terapia , Participação do Paciente , Doenças Raras/terapia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Autoantígenos/genética , Pesquisa Biomédica/normas , Criança , Ensaios Clínicos como Assunto , Colágeno Tipo IV/genética , Congressos como Assunto , Humanos , Mutação , Nefrite Hereditária/complicações , Nefrite Hereditária/genética , Nefrologia/métodos , Nefrologia/organização & administração , Nefrologia/normas , Pediatria/métodos , Pediatria/organização & administração , Pediatria/normas , Guias de Prática Clínica como Assunto , Doenças Raras/complicações , Doenças Raras/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/prevenção & controle , Terapia de Substituição Renal , Sociedades Médicas , Terapias em EstudoRESUMO
How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.