Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology

Pereira, Ester Miranda; Labilloy, Anatalia; Eshbach, Megan L.; Roy, Ankita; Subramanya, Arohan R.; Monte, Semíramis Jamil Hadad do; Labilloy, Guillaume; Weisz, Ora A.

doi:10.1152/ajprenal.00283.2016

Cited by 23 publications

(27 citation statements)

References 52 publications

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“…Unlike siRNA, which is transient and often presents different knockdown levels, the created GLA-null cells were stable, heritable, and a useful model to screen molecules that could improve rhα-GLA stability in a human cell without the interferences of endogenous GLA expression. In addition, the CRISPR/Cas9 genome-editing technique has also been used to develop human kidney cell model of FD in human immortalized podocytes [32]. These results, including our present report, elaborated the application of CRISPR/Cas9 in studying inherited diseases, e.g., FD.…”

Section: Discussionsupporting

confidence: 64%

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

Song¹,

Chiang

Tseng

et al. 2016

IJMS

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The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

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Section: Discussionsupporting

confidence: 64%

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

Song¹,

Chiang

Tseng

et al. 2016

IJMS

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“…A significant fraction of disorders affect the podocytes resulting in an altered actinbased cytoskeleton and disturbed cellular polarity (1). Podocyte cell culture models represent widely used tools in glomerular research (2,3) as environmental cues and gene and protein expression can be easily manipulated in vitro for mechanistic analyses (4,5).…”

mentioning

confidence: 99%

Protein half‐life determines expression of proteostatic networks in podocyte differentiation

et al. 2018

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Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

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“…Podocyte cell culture models were the first models to study podocyte biology and are still widely used ( 16 , 17 ) as gene and protein expression as well as environmental cues can be easily manipulated in vitro for mechanistic analyses ( 18 , 19 ). Multiple human, mouse, and rat podocyte cell lines have been generated in the past ( 16 , 17 , 20 – 22 ).…”

Section: Podocyte Cell Culturementioning

confidence: 99%

Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research

Hagmann

Brinkkoetter

2018

Front. Pediatr.

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Diseases affecting the glomeruli of the kidney, the renal filtration units, are a leading cause of chronic kidney disease and end-stage renal failure. Despite recent advances in the understanding of glomerular biology, treatment of these disorders has remained extraordinarily challenging in many cases. The use of experimental models has proven invaluable to study renal, and in particular, glomerular biology and disease. Over the past 15 years, studies identified different and very distinct pathogenic mechanisms that result in damage, loss of glomerular visceral epithelial cells (podocytes) and progressive renal disease. However, animal studies and, in particular, mouse studies are often protracted and cumbersome due to the long reproductive cycle and high keeping costs. Transgenic and heterologous expression models have been speeded-up by novel gene editing techniques, yet they still take months. In addition, given the complex cellular biology of the filtration barrier, certain questions may not be directly addressed using mouse models due to the limited accessibility of podocytes for analysis and imaging. In this review, we will describe alternative models to study podocyte biology experimentally. We specifically discuss current podocyte cell culture models, their role in experimental strategies to analyze pathophysiologic mechanisms as well as limitations with regard to transferability of results. We introduce current models in Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio that allow for analysis of protein interactions, and principle signaling pathways in functional biological structures, and enable high-throughput transgenic expression or compound screens in multicellular organisms.

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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology

Cited by 23 publications

References 52 publications

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease

Protein half‐life determines expression of proteostatic networks in podocyte differentiation

Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research

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