Washington University Kidney O’Brien Center for Chronic Kidney Disease Research
Summary
The Washington University Kidney O’Brien Center for Chronic Kidney Disease Research (WUCKD-NRC) primary goals are to develop and disseminate tools and new technologies for the investigation of chronic kidney disease and fibrosis. The WUCKD-NRC achieves these goals through two Biomedical Cores – the Metabolism Core (Meta-Core) and the Variant Validation Core.
Metabolism Core (Meta-Core)
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Description
The aim of the Metabolism Core (Meta-Core) is to facilitate the use of metabolic assays in pre-clinical models of chronic kidney disease (CKD) for the kidney community by providing the following services following consultation with core faculty:
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Services
- Assistance with manuscript development, grant or publication support
- BCA Assay for normalization of fatty acid oxidation data
- Bioinformatics and biostatistical support
- Cell quantification with Biotek Lionheart (to normalize Seahorse data)
- Choosing the right metabolic assay
- Discussion of the data generated and guidance for next steps
- Experimental planning and experimental design tailored to user's question
- Fatty acid oxidation in tissue ex vivo using 3H-palmitate
- Generation of figures from data
- Generation of primary cells for Seahorse or stable isotope studies
- Glucose oxidation in tissue ex vivo using 14C-pyruvate
- High resolution respirometry (Oroboros)
- Placement of jugular catheter for administration of stable isotopes
- Preparation of kidney tissue for substrate oxidation assays or stable isotope labeling
- Seahorse bioflux analyzer (XF24/XF96) to measure glucose oxidation in primary proximal tubule cells
- Seahorse bioflux analyzer (XF24/XF96) to measure OCR or ECAR
- Shipping of animals and/or biospecimens to core
- Stable isotope flux studies – proximal tubules in vivo
- Stable isotope flux studies – proximal tubules: 13C-glucose
- Stable isotope flux studies – tubules ex vivo
- Untargeted metabolomics – lipids
- Untargeted metabolomics – water soluble
- Other
Variant Validation Core
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Description
The widespread and increasing use of whole exome sequencing (WES) and whole genome sequencing (WGS) to investigate the genetic bases for CKD and other kidney disorders has led to a much better understanding of the etiology of monogenic diseases. The aim of the Variant Validation Core (VVC) is to investigate variants of uncertain significance (VUS) discovered in patients with kidney disease or a kidney developmental disorder for potential pathogenicity. The VVC will use experimental approaches in cultured cells and in mice. The experimental approach for each VUS will be tailored for 1) the particular gene and protein that are potentially involved; and 2) the particular disease or disorder that would likely develop if the VUS is indeed pathogenic. Immediate services and resources that are being offered include:
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Services
- Analysis of variant protein trafficking in cultured cells
- Analysis of next generation sequences from potential founder mice
- Analysis of variant UMOD protein trafficking in cultured cells
- Analysis of VUS in non-COL4A genes through design and implementation of tailored laboratory assays
- Analysis of VUS in non-COL4A genes through generation and analysis of gene edited mice
- Analysis of wild-type and variant protein trafficking in cultured cells by fluorescence microscopy
- Antigen retrieval and COL4A antibody staining of paraffin sections to assay the impact of COL4A VUS on GBM architecture
- Choosing an assay for COL4A3 VUS analysis
- Choosing an assay for COL4A4 VUS analysis
- Choosing an assay for COL4A5 VUS analysis
- Choosing an assay for non-COL4A gene VUS analysis
- COL4A gene VUS analysis by high resolution quantitative immunofluorescence imaging in paraffin sections of kidney biopsies
- COL4A3 intronic VUS analysis by minigene RNA splicing assays using transfected cells
- COL4A3 VUS (missense) analysis by N-terminal split nano-Luciferase collagen IV assembly and secretion assays in transfected cells
- COL4A3 VUS (missense) analysis by split C-terminal nano-Luciferase collagen IV assembly and secretion assays using transfected cells
- COL4A3 VUS analysis by high resolution quantitative immunofluorescence imaging in paraffin sections of kidney biopsies
- COL4A3/A4/A5 VUS analysis in vivo by mouse gene editing
- COL4A4 intronic VUS analysis by minigene RNA splicing assays using transfected cells
- COL4A4 VUS (missense) analysis by N-terminal split nano-Luciferase collagen IV assembly and secretion assays in transfected cells
- COL4A4 VUS (missense) analysis by split C-terminal nano-Luciferase collagen IV assembly and secretion assays using transfected cells
- COL4A5 intronic VUS analysis by minigene RNA splicing assays using transfected cells
- COL4A5 VUS (missense) analysis by N-terminal split nano-Luciferase collagen IV assembly and secretion assays using transfected cells
- COL4A5 VUS (missense) analysis by split C-terminal nano-Luciferase collagen IV assembly and secretion assays using transfected cells
- Data transfer to Core users
- Design and validation of CRISPR/Cas9 and homologous recombination reagents for gene editing in vivo
- Design of assays for other protein trafficking in cultured cells
- Design of minigene vectors for COL4A gene VUS analysis (intronic/splicing)
- Design of minigene vectors for non-COL4A gene VUS analysis (intronic/splicing)
- Design of tailored laboratory assays for analysis of VUS in non-COL4A genes
- Design of vectors for COL4A gene VUS analysis (missense/point mutant)
- Design of vectors for non-COL4A gene VUS analysis (missense/point mutant)
- Electroporation of CRISPR reagents into zygotes and implantation into pseudopregnant females
- Genotyping of founder mice by next generation sequencing
- In silico analysis of VUS for impact on RNA splicing
- Intronic non-COL4A gene VUS analysis by minigene RNA splicing assays using transfected cells
- Preliminary analysis of founder mice for VUS-induced phenotypes
- Preparation of RNA from cells transfected with mini-genes
- Production of vectors for COL4A gene VUS analysis
- Production of vectors for non-COL4A gene VUS analysis
- Provision of data and assembly of tables, graphs, and images to users for publishing data generated by the Core
- Provision of Materials and Methods and other text
- Purification and sequencing of wild-type and novel RT-PCR splice products from minigene assays
- RT-PCR and agarose gel analysis of splice products from transfected cells
- Sectioning and deparaffinization of kidney biopsy specimens
- Sequence analysis of splice products from minigene assays for potential splicing VUS
- Shipping of animal biospecimens to core users
- Shipping of cell lines to core users
- Shipping of gene-edited models to core users
- Shipping of nucleic acids or proteins to core users
- Training in the performance of collagen IV heterotrimerization assays using split nanoLuciferase-tagged COL4A chains
- Transfection of vectors encoding other variant proteins into cultured cells
- Transfection of vectors encoding variant UMOD proteins into cultured cells
- Other
Contact Information
If you would like more information regarding services provided by WUCKD-NRC, please contact:
Benjamin Humphreys
humphreysbd@wustl.edu
Website