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Anionic charge concentration of rat kidney glomeruli and glomerular basement membrane.

ABSTRACT: Estimates of levels of glomerular and glomerular-basement-membrane anion charge should serve as useful quantitative markers for the integrity of the tissues in health and disease. We have developed a simple, rapid, technique to measure this charge through the use of ion exchange with radioisotopes 22Na+ and 36Cl- at low ionic strengths in phosphate buffer. When this technique is used, normal glomeruli isolated from rat have a measured net anion charge concentration of 17.4 +/- 3.7 p-equiv. per glomerulus (n = 20). Perfused rat kidneys that lose approximately half of their glomerular heparan [35S]sulphate content (owing to oxygen-radical damage) exhibited a lower anion charge, of 7.5 +/- 1.6 p-equiv. per glomerulus (n = 5). Glomerular basement membranes prepared from rat glomeruli by a sonication-centrifugation procedure in the presence of enzyme inhibitors had a charge concentration of 6.3 +/- 0.7 mu-equiv./g wet wt. of tissue (n = 4), whereas membranes prepared by sonication, centrifugation, DNAse and detergent treatment had a charge concentration of 7.1 +/- 1.6 mu-equiv./g wet wt. (n = 4). Isotope-dilution experiments with 3H2O on these detergent-prepared glomerular basement membranes demonstrated that they had a water content of approx. 93%, which would then give a net anion charge concentration of 7.6 +/- 1.7 m-equiv./l (n = 4). These values are in good agreement with those obtained by others using titration techniques [Bray and Robinson (1984) Kidney Int. 25, 527-533]. The relatively low magnitude of glomerular anion charge in normal kidneys is consistent with other recent findings that glomerular anion charge is too low to affect the glomerular transport of charged molecules in a direct, passive, biophysical manner through electrostatic interactions.

SUBMITTER: Comper WD

PROVIDER: S-EPMC1132224 | biostudies-other | 1993 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundCD151 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.MethodsNext-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.ResultsWe 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.ConclusionsOur 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. A higher resolution version of the Graphical abstract is available as Supplementary information.

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Anionic charge concentration of rat kidney glomeruli and glomerular basement membrane.

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