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 or publication support
- BCA Assay for normalization of fatty acid oxidation data
- Bioinformatics and biostatistical support
- Branched chain amino acid oxidation in tissue ex vivo using 14C-KIVA
- Cell quantification with Biotek Lionheart (to normalize Seahorse data)
- Choosing the right metabolic assay
- Fatty acid oxidation in tissue ex vivo using 3H-palmitate
- Generation of primary cells for Seahorse or stable isotope studies
- Glucose oxidation in tissue ex vivo using 14C-pyruvate
- High resolution respirometry (Oroboros)
- Metabolomics and stable isotope flux study- Zoom discussion with Patti lab over experimental design
- Placement of jugular catheter for administration of stable isotopes
- Seahorse bioflux analyzer (XF24/XF96) to measure glucose oxidation in primary proximal tubule cells
- Seahorse bioflux analyzer (XF24/XF96) to measure glutamine oxidation in primary proximal tubule cells
- Seahorse bioflux analyzer (XF24/XF96) to measure glycolysis (i.e. extracellular acidification rate) in primary proximal tubule cells
- Seahorse bioflux analyzer (XF24/XF96) to measure mitochondrial oxidative respiration in primary proximal tubule cells
- Seahorse bioflux analyzer (XF24/XF96) to measure palmitate (fatty acid) oxidation in primary proximal tubule cells
- Stable isotope flux studies- proximal tubules: 13C-glucose
- Stable isotope flux studies- proximal tubules: 13C-glutamine
- Stable isotope flux studies- proximal tubules: 13C-palmitate
- Stable isotope flux studies- proximal tubules: 15N-glutamine: data provisioning
- Stable isotope tracing studies in vivo: 13C-glucose
- Stable isotope tracing studies in vivo: 13C-glucose: data provisioning
- Stable isotope tracing studies in vivo: 13C-glutamine
- Stable isotope tracing studies in vivo: 13C-glutamine: data provisioning
- Stable isotope tracing studies in vivo: 13C-palmitate and 13C-oleate
- Stable isotope tracing studies in vivo: 13C-palmitate and 13C-oleate: data provisioning
- Stable isotope tracing studies in vivo: 15N-glutamine
- Stable isotope tracing studies in vivo: 15N-glutamine: data provisioning
- Stable isotope tracing studies in vivo: branched chain amino acids
- Stable isotope tracing studies in vivo: branched chain amino acids: data provisioning
- Untargeted metabolomics - lipids
- Untargeted metabolomics - water soluble
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 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 models
- 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
- 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
- COL4A4 VUS analysis by high resolution quantitative immunofluorescence imaging in paraffin sections of kidney biopsies
- 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 in transfected cells
- COL4A5 VUS (missense) analysis by split C-terminal nano-Luciferase collagen IV assembly and secretion assays using transfected cells
- COL4A5 VUS analysis by high resolution quantitative immunofluorescence imaging in paraffin sections of kidney biopsies
- Design of assays for other protein trafficking in cultured cells
- Intronic non-COL4A gene VUS analysis by minigene RNA splicing assays using transfected cells
- Training in the performance of collagen IV heterotrimerization assays using split nanlLuciferase tagged COL4A chains
Contact Information
If you would like more information regarding services provided by WUCKD-NRC, please contact:
Benjamin Humphreys
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