| 1. |
- Butt, Linus, et al.
(författare)
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Super-Resolution Imaging of the Filtration Barrier Suggests a Role for Podocin R229Q in Genetic Predisposition to Glomerular Disease
- 2021
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Ingår i: Journal of the American Society of Nephrology. - : Wolters Kluwer. - 1046-6673 .- 1533-3450. ; 33:1, s. 138-154
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Tidskriftsartikel (refereegranskat)abstract
- Significance Statement Podocin R229Q results from the most frequent missense variant in NPHS2, and its association with FSGS when podocin R229Q is transassociated with a second mutation in NPHS2 is well recognized. However, because results from observational studies are ambiguous and appropriate animal studies are lacking, its isolated pathogenic potency is not entirely clear. In this study, the authors introduced this genetic alteration in mice and assessed the phenotype using super-resolution microscopy and albuminuria measurements. They demonstrated a deleterious effect of the variant on podocyte morphology and on the integrity of the glomerular filtration barrier under basal conditions and after external glomerular injury. Because this finding suggests that this mutation confers a genetic predisposition to glomerular disease, it has implications for a large number of carriers worldwide.Background Diseases of the kidney’s glomerular filtration barrier are a leading cause of end stage renal failure. Despite a growing understanding of genes involved in glomerular disorders in children, the vast majority of adult patients lack a clear genetic diagnosis. The protein podocin p.R229Q, which results from the most common missense variant in NPHS2, is enriched in cohorts of patients with FSGS. However, p.R229Q has been proposed to cause disease only when transassociated with specific additional genetic alterations, and population-based epidemiologic studies on its association with albuminuria yielded ambiguous results.Methods To test whether podocin p.R229Q may also predispose to the complex disease pathogenesis in adults, we introduced the exact genetic alteration in mice using CRISPR/Cas9-based genome editing (PodR231Q). We assessed the phenotype using super-resolution microscopy and albuminuria measurements and evaluated the stability of the mutant protein in cell culture experiments.Results Heterozygous PodR231Q/wild-type mice did not present any overt kidney disease or proteinuria. However, homozygous PodR231Q/R231Q mice developed increased levels of albuminuria with age, and super-resolution microscopy revealed preceding ultrastructural morphologic alterations that were recently linked to disease predisposition. When injected with nephrotoxic serum to induce glomerular injury, heterozygous PodR231Q/wild-type mice showed a more severe course of disease compared with Podwild-type/wild-type mice. Podocin protein levels were decreased in PodR231Q/wild-type and PodR231Q/R231Q mice as well as in human cultured podocytes expressing the podocinR231Q variant. Our in vitro experiments indicate an underlying increased proteasomal degradation.Conclusions Our findings demonstrate that podocin R231Q exerts a pathogenic effect on its own, supporting the concept of podocin R229Q contributing to genetic predisposition in adult patients.
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| 2. |
- He, Bing, et al.
(författare)
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Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies. The molecular identity of renal glomerular cells is poorly characterized and rodent glomerulopathy models translate poorly to humans. Here, the authors show molecular signatures of glomerulus-associated cells using single cell RNA sequencing and highlight differences between mouse and human cells.
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| 3. |
- Unnersjö Jess, David, et al.
(författare)
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A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales
- 2021
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 99:4, s. 1010-1020
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.
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