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High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. (Cell Physics)
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 [en]
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: urn:nbn:se:kth:diva-249002ISBN: 978-91-7873-163-3 (print)OAI: oai:DiVA.org:kth-249002DiVA, id: diva2:1303756
Public defence
2019-05-03, Air & Fire, Tomtebodavägen 23A, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20190411

Available from: 2019-04-11 Created: 2019-04-10 Last updated: 2019-04-11Bibliographically approved
List of papers
1. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.
Open this publication in new window or tab >>Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.
2016 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 89, no 1, p. 243-247Article in journal (Refereed) Published
Abstract [en]

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labelled optically cleared intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
Keywords
glomerulus, podocyte, optical clearing, STED, super-resolution
National Category
Other Physics Topics Biological Sciences Cell Biology Biophysics
Identifiers
urn:nbn:se:kth:diva-182108 (URN)10.1038/ki.2015.308 (DOI)000368321300034 ()26444032 (PubMedID)2-s2.0-84943311622 (Scopus ID)
Funder
Swedish Research Council, 2013-6041Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160218

Available from: 2016-02-15 Created: 2016-02-15 Last updated: 2019-04-10Bibliographically approved
2. Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement
Open this publication in new window or tab >>Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement
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(English)Manuscript (preprint) (Other academic)
National Category
Urology and Nephrology
Identifiers
urn:nbn:se:kth:diva-248125 (URN)
Note

QC 20190425

Available from: 2019-04-03 Created: 2019-04-03 Last updated: 2019-04-25Bibliographically approved
3. Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion
Open this publication in new window or tab >>Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion
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2018 (English)In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 93, no 4, p. 1008-1013Article in journal (Refereed) Published
Abstract [en]

The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE INC, 2018
Keywords
glomerulus, imaging, podocyte, renal pathology
National Category
Atom and Molecular Physics and Optics Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:kth:diva-226201 (URN)10.1016/j.kint.2017.09.019 (DOI)000428169200029 ()29241621 (PubMedID)2-s2.0-85037568536 (Scopus ID)
Note

QC 20180518

Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2019-04-10Bibliographically approved
4. Mediator complex subunit 22 is necessary for glomerular homeostasis by regulation of podocyte vesicular trafficking
Open this publication in new window or tab >>Mediator complex subunit 22 is necessary for glomerular homeostasis by regulation of podocyte vesicular trafficking
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(English)Manuscript (preprint) (Other academic)
National Category
Urology and Nephrology
Identifiers
urn:nbn:se:kth:diva-248127 (URN)
Note

QC 20190521

Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-05-21Bibliographically approved
5. Renal diagnostics using a simplified optical clearing and swelling protocol
Open this publication in new window or tab >>Renal diagnostics using a simplified optical clearing and swelling protocol
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(English)Manuscript (preprint) (Other academic)
National Category
Urology and Nephrology
Identifiers
urn:nbn:se:kth:diva-248126 (URN)
Note

QC 20190404

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

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