A novel nanoluciferase transgenic reporter to measure proteinuria in zebrafish

Naylor, Richard W.; Lemarie, Emmanuel; Jackson-Crawford, Anthony; Davenport, J Bernard; Mironov, Aleksandr; Lowe, Martin; Lennon, Rachel

doi:10.1101/2021.07.19.452884

Cited by 3 publications

(7 citation statements)

References 54 publications

(62 reference statements)

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“…In addition, we examined kidney physiology, which depends on BMs for filtration function ( 32 ). Using the NL:D3 excretion assay ( 33 ), proteinuria was observed in adamts3 and robo1 crispants, suggesting defects in BM (fig. S8H).…”

Section: Resultsmentioning

confidence: 99%

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A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations

et al. 2022

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Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFβ. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5 , MPZL2 , and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

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Section: Resultsmentioning

confidence: 99%

“…The NL:D3 transgenic zebrafish ( 33 ) were injected at the one-cell stage with gRNAs targeting adamts3 or robo1 (table S20) and Cas9 protein. Crispant NL:D3 embryos were grown to 4 days postfertilization (dpf) and then placed in wells of a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations

et al. 2022

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“…In addition, we examined kidney physiology, which depends on BMs for filtration function 9 . Using the NL:D3 excretion assay 42 , proteinuria was observed in adamts3 and robo1 crispants (Supplementary Fig. 6f), suggesting defects in BM.…”

Section: Functional In Vivo Studies Uncover New Regulators Of Bm Compositionmentioning

confidence: 99%

“…The NL:D3 transgenic zebrafish 78 were injected at the one-cell stage with gRNAs targeting adamts3 or robo1 (Supplementary table 19) and Cas9 protein. Crispant NL:D3 embryos were grown to 4 days post-fertilization (dpf) and then placed in wells of a 96-well plate.…”

Section: Kidney Filtration Assay In Zebrafishmentioning

confidence: 99%

A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

Jayadev

Morais

Ellingford

et al. 2021

Preprint

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Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins localized to BMs. Network analysis and screening in C. elegans and zebrafish identified new BM regulators, including ADAMTS, ROBO, and TGFβ. More than 100 BM-network genes associate with human phenotypes and by screening 63,039 genomes from families with rare disorders, we discovered loss-of-function variants in LAMA5, MPZL2, and MATN2, and show they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity and role of BMs in human health.

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“…Zebrafish were maintained and staged according to established protocols [ 12 ] and in accordance with the personal project license of Professor Rachel Lennon (P1AE9A736) and under the current guidelines of the UK Animals Act 1986. Embryos were collected from group-wise matings of nphs2:egfp or NL-D3 reporter fish [ 13 ]. Progeny from these crossings were injected with cd151 crRNAs whose design was based on the algorithm described by Wu et al [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

Basement membrane defects in CD151-associated glomerular disease

et al. 2022

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Background CD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease. Methods Next-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9. Results We report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence. Conclusions Our results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants. Graphical abstract A higher resolution version of the Graphical abstract is available as Supplementary information

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A novel nanoluciferase transgenic reporter to measure proteinuria in zebrafish

Cited by 3 publications

References 54 publications

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations

A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

Basement membrane defects in CD151-associated glomerular disease

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