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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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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
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.
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Colágeno Tipo IV , Estresse do Retículo Endoplasmático , Mutação , Nefrite Hereditária , Ácido Tauroquenodesoxicólico , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Humanos , Masculino , Camundongos , Apoptose/efeitos dos fármacos , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Nefrite Hereditária/genética , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/uso terapêuticoRESUMO
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and embedding conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
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Congenital nephrotic syndrome is an autosomal recessive inherited disorder that manifests as steroid-resistant massive proteinuria in the first three months of life. Defects in the glomerular filtration mechanism are the primary etiology. We present a child who developed severe nephrotic syndrome at two weeks of age and eventually required a bilateral nephrectomy. Genetic testing revealed compound heterozygous variants in NPHS1 including a known pathogenic variant and a missense variant of uncertain significance. Light microscopy revealed crescent formation-an atypical finding in congenital nephrotic syndrome caused by nephrin variants-in addition to focal segmental and global glomerulosclerosis. Electron microscopy showed diffuse podocyte foot process effacement. Confocal and Airyscan immunofluorescence microcopy showed aggregation of nephrin in the podocyte cell body that is not a result of diffuse podocyte foot process effacement as seen in minimal change disease. These findings confirm the novel variant as pathogenic.
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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
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.
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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
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
RESUMO
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 caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype 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 suggested 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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Familial hematuria is a clinical sign of a genetically heterogeneous group of conditions, accompanied by broad inter- and intrafamilial variable expressivity. The most frequent condition is caused by pathogenic (or likely pathogenic) variants in the collagen-IV genes, COL4A3/A4/A5. Pathogenic variants in COL4A5 are responsible for the severe X-linked glomerulopathy, Alport syndrome (AS), while homozygous or compound heterozygous variants in the COL4A3 or the COL4A4 gene cause autosomal recessive AS. AS usually leads to progressive kidney failure before the age of 40-years when left untreated. People who inherit heterozygous COL4A3/A4 variants are at-risk of a slowly progressive form of the disease, starting with microscopic hematuria in early childhood, developing Alport spectrum nephropathy. Sometimes, they are diagnosed with benign familial hematuria, and sometimes with autosomal dominant AS. At diagnosis, they often show thin basement membrane nephropathy, reflecting the uniform thin glomerular basement membrane lesion, inherited as an autosomal dominant condition. On a long follow-up, most patients will retain normal or mildly affected kidney function, while a substantial proportion will develop chronic kidney disease (CKD), even kidney failure at an average age of 55-years. A question that remains unanswered is how to distinguish those patients with AS or with heterozygous COL4A3/A4 variants who will manifest a more aggressive kidney function decline, requiring prompt medical intervention. The hypothesis that a subgroup of patients coinherit additional genetic modifiers that exacerbate their clinical course has been investigated by several researchers. Here, we review all publications that describe the potential role of candidate genetic modifiers in patients and include a summary of studies in AS mouse models.
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Nefrite Hereditária , Insuficiência Renal , Pré-Escolar , Humanos , Animais , Camundongos , Pessoa de Meia-Idade , Adulto , Hematúria/genética , Nefrite Hereditária/genética , Colágeno Tipo IV/genéticaRESUMO
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
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: Missense variants in COL4A genes are often found in patients with an Alport syndrome-like presentation, but their pathogenicity is not always clear. We encountered a woman with microscopic hematuria and proteinuria at 33 years of age with a diagnosis of thin basement membrane disease who was approaching end stage kidney disease at 59 years of age. We hypothesized that this patient's kidney disease was within the spectrum of Alport syndrome. Methods: We used histologic, genetic, and biochemical approaches to investigate the mechanisms of kidney disease. By immunofluorescence, we investigated collagen IV chain composition of the glomerular basement membrane (GBM). We employed targeted sequencing to search for pathogenic variants in COL4A and other relevant genes. We utilized N- and C-terminal split NanoLuciferase assays to determine the effect of a novel COL4A4 variant of uncertain significance (VUS) on collagen IV heterotrimer formation in vitro. We transfected COL4A4 expression constructs with split NanoLuciferase fragment-fused COL4A3 and COL4A5 constructs into human embryonic kidney 293T cells. To assay for α3α4α5(IV) heterotrimer formation and secretion, we measured luminescence in cell lysates and culture supernatants from transfected cells. Results: Immunostaining suggested that the collagen α3α4α5(IV) network was present throughout the patient's GBMs. DNA sequencing revealed a novel homozygous VUS: COL4A4 c.1180G>A (p. Gly394Ser). In the C-terminal split luciferase-based α3α4α5(IV) heterotrimer formation assays, luminescence levels for G394S were comparable to WT, but in the N-terminal tag assays, the extracellular luminescence levels for G394S were decreased by approximately 50% compared with WT. Conclusions: Our cell-based assay provides a platform to test COL4 VUS and shows that G394S impairs assembly of the α3α4α5(IV) N-terminus and subsequent trimer secretion. These data suggest that the COL4A4-G394S variant is pathogenic and causes an atypical mild form of autosomal recessive Alport syndrome.
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Nefrite Hereditária , Feminino , Humanos , Pessoa de Meia-Idade , Autoantígenos/genética , Colágeno Tipo IV/genética , Membrana Basal Glomerular/metabolismo , Hematúria/genética , Nefrite Hereditária/genéticaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.
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Cistos , Rim Policístico Autossômico Dominante , Humanos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Análise de Célula Única , Rim/metabolismo , Túbulos Renais/metabolismo , Células Epiteliais/metabolismo , Cistos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process-like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.
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Nefropatias , Podócitos , Células Epiteliais , Humanos , Rim , SarcômerosRESUMO
CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on the VP64 transcriptional activator have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced basal dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.
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Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , RNA Guia de Cinetoplastídeos/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Alport syndrome, a disease of kidney, ear, and eye, is caused by pathogenic variants in the COL4A3, COL4A4, or COL4A5 genes encoding collagen α3α4α5(IV) of basement membranes. Collagen IV chains that are truncated due to nonsense variants/premature termination codons (PTCs) cannot assemble into heterotrimers or incorporate into basement membranes. To investigate the feasibility of PTC readthrough therapy for Alport syndrome, we utilized two NanoLuc reporters in transfected cells: full-length for monitoring translation, and a split version for assessing readthrough product function. Full-length assays of 49 COL4A5 nonsense variants identified eleven as susceptible to PTC readthrough using various readthrough drugs. In split-NanoLuc assays, the predicted missense α5(IV) readthrough products of five nonsense mutations could heterotrimerize with α3(IV) and α4(IV). Readthrough was also observed in kidney cells from an engineered Col4a5 PTC mouse model. These results suggest that readthrough therapy is a feasible approach for a fraction of patients with Alport syndrome.
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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.