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Choline has important physiological functions as a precursor for essential cell components, signaling molecules, phospholipids, and the neurotransmitter acetylcholine. Choline is a water-soluble charged molecule requiring transport proteins to cross biological membranes. Although transporters continue to be identified, membrane transport of choline is incompletely understood and knowledge about choline transport into intracellular organelles such as mitochondria remains limited. Here we show that SLC25A48 imports choline into human mitochondria. Human loss-of-function mutations in SLC25A48 show impaired choline transport into mitochondria and are associated with elevated urine and plasma choline levels. Thus, our studies may have implications for understanding and treating conditions related to choline metabolism.
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Different cell channels and transporters tightly regulate cytoplasmic levels and the intraorganelle distribution of cations. Perturbations in these processes lead to human diseases that are frequently associated with kidney impairment. The family of melastatin-related transient receptor potential (TRPM) channels, which has eight members in mammals (TRPM1-TRPM8), includes ion channels that are highly permeable to divalent cations, such as Ca2+, Mg2+ and Zn2+ (TRPM1, TRPM3, TRPM6 and TRPM7), non-selective cation channels (TRPM2 and TRPM8) and monovalent cation-selective channels (TRPM4 and TRPM5). Three family members contain an enzymatic protein moiety: TRPM6 and TRPM7 are fused to α-kinase domains, whereas TRPM2 is linked to an ADP-ribose-binding NUDT9 homology domain. TRPM channels also function as crucial cellular sensors involved in many physiological processes, including mineral homeostasis, blood pressure, cardiac rhythm and immunity, as well as photoreception, taste reception and thermoreception. TRPM channels are abundantly expressed in the kidney. Mutations in TRPM genes cause several inherited human diseases, and preclinical studies in animal models of human disease have highlighted TRPM channels as promising new therapeutic targets. Here, we provide an overview of this rapidly evolving research area and delineate the emerging role of TRPM channels in kidney pathophysiology.
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Canais de Cátion TRPM , Canais de Potencial de Receptor Transitório , Animais , Humanos , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Mamíferos/metabolismo , Proteínas Serina-Treonina QuinasesRESUMO
The kidneys operate at the interface of plasma and urine by clearing molecular waste products while retaining valuable solutes. Genetic studies of paired plasma and urine metabolomes may identify underlying processes. We conducted genome-wide studies of 1,916 plasma and urine metabolites and detected 1,299 significant associations. Associations with 40% of implicated metabolites would have been missed by studying plasma alone. We detected urine-specific findings that provide information about metabolite reabsorption in the kidney, such as aquaporin (AQP)-7-mediated glycerol transport, and different metabolomic footprints of kidney-expressed proteins in plasma and urine that are consistent with their localization and function, including the transporters NaDC3 (SLC13A3) and ASBT (SLC10A2). Shared genetic determinants of 7,073 metabolite-disease combinations represent a resource to better understand metabolic diseases and revealed connections of dipeptidase 1 with circulating digestive enzymes and with hypertension. Extending genetic studies of the metabolome beyond plasma yields unique insights into processes at the interface of body compartments.
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Rim , Metaboloma , Rim/metabolismo , MetabolômicaRESUMO
Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.
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Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Reprodutibilidade dos Testes , Edição de Genes/métodos , Linhagem Celular , Células CultivadasRESUMO
The kidneys generate about 180 l of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here, we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.
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Drosophila , Podócitos , Animais , Endocitose/fisiologia , Junções Intercelulares/metabolismo , Camundongos , Podócitos/metabolismo , Proteinúria/metabolismoRESUMO
Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.
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Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.
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Células Epiteliais/patologia , Epitélio/patologia , Rim Policístico Autossômico Dominante/genética , Transcrição Gênica/genética , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Rim Policístico Autossômico Dominante/patologia , Transdução de Sinais/genética , Canais de Cátion TRPP/genéticaRESUMO
Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.
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Calpaína , Canais de Cátion TRPP , Animais , Autofagia , Calpaína/metabolismo , Lisossomos/metabolismo , Camundongos , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.
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Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica/fisiologia , Interferon gama/metabolismo , Camundongos TransgênicosRESUMO
The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite-locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research.
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Biotransformação/genética , Rim/metabolismo , Locos de Características Quantitativas , Insuficiência Renal Crônica/urina , Acetiltransferases/genética , Acetiltransferases/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/urina , Estudos de Coortes , Citocromo P-450 CYP2D6/genética , Estudo de Associação Genômica Ampla , Humanos , Inativação Metabólica , Rim/citologia , Metoprolol/farmacocinética , Polimorfismo de Nucleotídeo Único , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Urina/fisiologia , Xenobióticos/farmacocinética , Xenobióticos/urinaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes) and Brachyury, which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor ß (TGF-ß)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury.
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Cromatina/genética , Proteínas Fetais/genética , Camadas Germinativas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas com Domínio T/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Separação Celular/métodos , Endoderma/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Placa Neural/fisiologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Differential phosphorylation of proteins is a key regulatory mechanism in biology. Immunoprecipitation-coupled mass spectrometry facilitates the targeted analysis of transient receptor ion potential channel polycystin-2 (TRPP2) phosphorylation. However, empirical testing is required to optimize experimental conditions for immunoprecipitation and mass spectrometry. Here, we present a detailed workflow for the reliable analysis of endogenous TRPP2 phosphorylation in differentiated renal epithelial cells.
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Espectrometria de Massas/métodos , Canais de Cátion TRPP/metabolismo , Animais , Células Epiteliais/enzimologia , Rim/enzimologia , Camundongos , Fosforilação , Canais de Cátion TRPP/químicaRESUMO
Progressive cyst expansion in autosomal-dominant polycystic kidney disease has been shown to cause regional hypoxia in cystic and pericystic tissue. This results in up-regulation of hypoxia-inducible transcription factor (HIF) 1α in cyst epithelial cells. However, the functional impact of HIF-1α in cystic kidney disease was unknown. A new study has shown that HIF-1α promotes cyst progression in a mouse model of autosomal-dominant polycystic kidney disease.
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Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Animais , Cistos , Células Epiteliais , Subunidade alfa do Fator 1 Induzível por Hipóxia , CamundongosRESUMO
Cilia are organelles specialized in movement and signal transduction. The ciliary transient receptor potential ion channel polycystin-2 (TRPP2) controls elementary cilia-mediated physiological functions ranging from male fertility and kidney development to left-right patterning. However, the molecular components translating TRPP2 channel-mediated Ca2+ signals into respective physiological functions are unknown. Here, we show that the Ca2+-regulated mitochondrial ATP-Mg/Pi solute carrier 25 A 25 (SLC25A25) acts downstream of TRPP2 in an evolutionarily conserved metabolic signaling pathway. We identify SLC25A25 as an essential component in this cilia-dependent pathway using a genome-wide forward genetic screen in Drosophila melanogaster, followed by a targeted analysis of SLC25A25 function in zebrafish left-right patterning. Our data suggest that TRPP2 ion channels regulate mitochondrial SLC25A25 transporters via Ca2+ establishing an evolutionarily conserved molecular link between ciliary signaling and mitochondrial metabolism.
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Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Antiporters/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Drosophila melanogaster/metabolismo , Heterozigoto , Humanos , Canais Iônicos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais , Peixe-ZebraRESUMO
Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.
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Quimiocina CCL2/metabolismo , Cílios/patologia , Doenças Renais Císticas/congênito , Rim Policístico Autossômico Dominante/patologia , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto , Cães , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Doenças Renais Císticas/patologia , Túbulos Renais/citologia , Túbulos Renais/patologia , Macrófagos/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/fisiologia , Rim Policístico Autossômico Dominante/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Peixe-ZebraRESUMO
Ion channels control the membrane potential and mediate transport of ions across membranes. Archetypical physiological functions of ion channels include processes such as regulation of neuronal excitability, muscle contraction, or transepithelial ion transport. In that regard, transient receptor potential ion channel polycystin 2 (TRPP2) is remarkable, because it controls complex morphogenetic processes such as the establishment of properly shaped epithelial tubules and left-right-asymmetry of organs. The fascinating question of how an ion channel regulates morphogenesis has since captivated the attention of scientists in different disciplines. Four loosely connected key insights on different levels of biological complexity ranging from protein to whole organism have framed our understanding of TRPP2 physiology: 1) TRPP2 is a non-selective cation channel; 2) TRPP2 is part of a receptor-ion channel complex; 3) TRPP2 localizes to primary cilia; and 4) TRPP2 is required for organ morphogenesis. In this review, we will discuss the current knowledge in these key areas and highlight some of the challenges ahead.
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Canais de Cátion TRPP/metabolismo , Animais , Cílios/metabolismo , Humanos , Falência Renal Crônica/patologia , Rim Policístico Autossômico Dominante/patologia , Domínios Proteicos , Canais de Cátion TRPP/químicaRESUMO
Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.