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Mediator complex subunit 22 is necessary for glomerular homeostasis by regulation of podocyte vesicular trafficking
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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(English)Manuscript (preprint) (Other academic)
National Category
Urology and Nephrology
Identifiers
URN: urn:nbn:se:kth:diva-248127OAI: oai:DiVA.org:kth-248127DiVA, id: diva2:1302242
Note

QC 20190521

Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-05-21Bibliographically approved
In thesis
1. High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
Open this publication in new window or tab >>High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The kidney is one of the most important and complex organs in the humanbody with the task of filtering hundreds of litres of blood daily. It is responsiblefor the salt and acid/base balance in the body, as well as secretinghormones important for red blood cell production and blood pressure regulation. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understandingthe function of the kidney in both health and disease. Kidney failure or end stage renal disease (ESRD) is irreversible and requires treatment with dialysisor transplantation. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (below 200 nanometers), and thus electron microscopy has previously been the only method with high enough resolution to study the morphology and topology of these minute structures. In three studies included in this thesis, we show that the finest elements of the kidney can now be resolved using different light microscopy techniques. In study 1, we show that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney can be resolved, with widths around 75 nanometers. In study 3, a novel sample preparation method, expansion microscopy, is utilized to isotropically expand kidney tissue samples in space. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragmusing diffraction-limited confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved even further (<20 nm). In our most recent work, study 5, we apply a simplified, moderate tissue swelling protocol which together with optimization of the confocal imaging provides sufficient resolution to resolve foot processes and parts of the filtration barrier. This new protocol is fast and technically simple, making it ideal for routine use, such as for future clinical pathology. In collaboration with kidney researchers, we have applied both STED microscopy and expansion microscopy to various disease models, showing that these tools can be used to both visualize and quantify pathologies occurring in different parts of the glomerular filtration barrier (GFB). In study 2, STED microscopy in combination with optical clearing is used to study the localization of Coro2b in secondary foot processes in both mouse and human tissue. In two ongoing studies with preliminary results presented in the thesis, we use STED microscopy and optical clearing to study the pathogenesis of focal segmental glomerulosclerosis (FSGS) by the use of genetic mouse models. Based on STED images, we extract different morphological parameters from foot processes and the glomerular filtration barrier (GFB) at different stages of the disease. In study 4, we apply a tissue expansion protocol to answer questions about the phenotype seen in podocytes where the mediator complex subunit 22 (Med22) is inactivated. By inactivating Med22 in a transgenic mouse line with cytosolic expression of tdTomato in podocytes, we saw strong indications that the vesicle-like structures seen in EM micrographs were indeed intracellular vesicles and not dilated sub-podocyte space. In summary, the work presented in this thesis has contributed to the development of a new toolbox for imaging renal ultra-structure using light microscopy, a field previously reserved for electron microscopy.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 85
Series
TRITA-SCI-FOU ; 2019:18
Keywords
Kidney, Renal Pathology, Microscopy, Super-resolution microscopy, Tissue expansion, Foot processes, Glomerular filtration, Podocytes
National Category
Biophysics Cell Biology Cell and Molecular Biology Urology and Nephrology Medical Laboratory and Measurements Technologies
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-249002 (URN)978-91-7873-163-3 (ISBN)
Public defence
2019-05-03, Air & Fire, Tomtebodavägen 23A, Solna, 09:00 (English)
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Supervisors
Note

QC 20190411

Available from: 2019-04-11 Created: 2019-04-10 Last updated: 2019-04-11Bibliographically approved

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Jess, David Unnersjö

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