Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical ...research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.
Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell ...populations remains unknown.
We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.
Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.
Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.
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.
Synopsis
Primary cilia are important mediators of human disease and cell signaling. A local proximity labeling strategy to identify the ciliary membrane‐associated proteome (cmAPEX) reveals the close association of actin binding proteins (ABPs) with the ciliary membrane.
cmAPEX and complementary affinity purification analyses identified association of actin‐binding proteins (ABPs) with cilia.
cmAPEX resolves dynamic relocalization of ciliary proteins including ABPs upon inhibition of actin polymerization.
Myo5a is a novel and essential ciliary protein that relocates into the cilium upon induction of ciliary disassembly.
Primary cilia are important mediators of human disease and cell signaling. A local proximity labeling strategy to identify the ciliary membrane‐associated proteome (cmAPEX) reveals the close association of actin binding proteins with the ciliary membrane.
Cystic transformation of kidney tissue is a key feature of various disorders including autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and ...disorders of the nephronophthisis spectrum (NPH). While ARPKD and NPH typically affect children and adolescents, pediatric onset of ADPKD is less frequently found. While both ADPKD and ARPKD are characterized by formation of hundreds of cysts accompanied by hyperproliferation of tubular epithelia with massive renal enlargement, NPH patients usually show kidneys of normal or reduced size with cysts limited to the corticomedullary border. Recent results suggest the hippo pathway to be a central regulator at the crossroads of the renal phenotype in both diseases. Hippo signaling regulates organ size and proliferation by keeping the oncogenic transcriptional co-activators Yes associated protein 1 (YAP) and WW domain containing transcription regulator 1 (TAZ) in check. Once this inhibition is released, nuclear YAP/TAZ interacts with TEAD family transcription factors and the consecutive transcriptional activation of TEA domain family members (TEAD) target genes mediates an increase in proliferation. Here, we review the current knowledge on the impact of NPHP and ADPKD mutations on Hippo signaling networks. Furthermore, we provide an outlook towards potential future therapeutic strategies targeting Hippo signaling to alleviate cystic kidney disease.
Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for ...electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively ...investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).
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•Anaerobic glycolysis represents the predominant energy source of podocytes•Neither mitochondrial turnover nor mtDNA transcription impairs podocyte function•These findings elucidate podocyte metabolism, function, and glomerular integrity
Glomerular podocytes form the third and most outer layer of the kidney filtration barrier responsible for restricting the passage of proteins into the urine. Brinkkoetter et al. show that podocyte metabolism primarily relies on anaerobic glycolysis and the fermentation of glucose to lactate.
Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and ...decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose.
To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK).
Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK.
In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive ...analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.
Podocytes are visceral epithelial cells supporting the function of the glomerular filter. Interdigitating foot processes of podocytes enwrap the glomerular capillaries and are connected by a highly ...specialized cell junction, the slit diaphragm. Signal transduction at the slit diaphragm is essential for the proper function of the kidney filtration barrier. The slit diaphragm constitutes a dynamic multiprotein signaling complex that contains structural proteins, receptors, signaling adaptors, ion channels, and scaffolding proteins. Function of some of these proteins requires cholesterol attached to the multiprotein complex. Recruitment of cholesterol is achieved through the PHB domain protein podocin, a member of a novel family of lipid-binding proteins that are conserved through evolution. The finding that cholesterol interaction regulates the activity of ion channels at the glomerular filtration barrier has important implications for renal physiology and pathophysiology.
MicroRNAs (miRNAs) regulate gene expression by binding the 3' untranslated region of mRNAs. To define their role in glomerular function, miRNA biogenesis was disrupted in mouse podocytes using a ...conditional Dicer allele. Mutant mice developed proteinuria by 3 wk after birth and progressed rapidly to end-stage kidney disease. Podocyte pathology included effacement, vacuolization, and hypertrophy with crescent formation. Despite normal expression of WT1, podocytes underwent dedifferentiation, exemplified by cytoskeletal disruption with early transcriptional downregulation of synaptopodin. These abnormalities differed from Cd2ap(-/-) mice, indicating they were not a general consequence of glomerular disease. Glomerular labeling of ezrin, moesin, and gelsolin was altered at 3 wk, but expression of nestin and alpha-actinin was unchanged. Abnormal cell proliferation or apoptosis was not responsible for the glomerular injury. Mutant podocytes were incapable of synthesizing mature miRNA, as revealed by their loss of miR-30a. In contrast, expression of glomerular endothelial and mesangial cell miRNAs (miR-126 and miR-145, respectively) was unchanged. These findings demonstrate a critical role for miRNA in glomerular function and suggest a pathway that may participate in the pathogenesis of kidney diseases of podocyte origin. The unique architecture of podocytes may make them especially susceptible to cytoskeletal alterations initiated by aberrant miRNA dynamics.
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