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High-resolution imaging of kidney tissue samples
KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
2017 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The kidney is one of the most important and complex organs in the human body, filtering hundreds of litres of blood daily. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understanding the function of the kidney in health and disease. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (on the sub-200 nm scale), and thus electron microscopy has been the only method of choice to visualize these minute structures. In one study, we show for the first time that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney, a structure around 75 nanometers in width, can now be resolved using light microscopy. In a second study, a novel sample preparation method, expansion microscopy, is utilized to physically expand kidney tissue samples. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragm using confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved further. In a third study, influences on the development of the kidney were studied. There is substantial knowledge regarding what genes (growth factors, receptors etc.) are important for the normal morphogenesis of the kidney. Less is known regarding the physiology behind how paracrine factors are secreted and delivered in the developing kidney. By depleting calcium transients in explanted rat kidneys, we show that calcium is important for the branching morphogenesis of the ureteric tree. Further, the study shows that the calcium-dependent initiator of exocytosis, synaptotagmin, is expressed in the metanephric mesenchyme of the developing kidney, indicating that it could have a role in the secretion of paracrine growth factors, such as GDNF, to drive the branching.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , 34 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2017:28
Keyword [en]
Super Resolution Microscopy, Kidney, Imaging, Fluorescence, Kidney development, Calcium
National Category
Biophysics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-207577ISBN: 978-91-7729-456-6 (print)OAI: oai:DiVA.org:kth-207577DiVA: diva2:1097792
Presentation
2017-06-15, Air-Fire, Science for Life Laboratories, Tomtebodavägen 23A, Solna, 09:30 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research , RIF14-0091Swedish Research Council, 2013-6041
Note

QC 20170523

Available from: 2017-05-23 Created: 2017-05-22 Last updated: 2017-05-23Bibliographically 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, 243-247 p.Article 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
Keyword
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: 2017-05-23Bibliographically approved
2. Confocal imaging of slit diaphragm proteins in expanded kidney tissue
Open this publication in new window or tab >>Confocal imaging of slit diaphragm proteins in expanded kidney tissue
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The subtlest element of the kidney, such as the slit diaphragm, 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 (STED) microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different molecular species (i.e. epitopes), and also the amount of volumetric data that can be extracted from a thick sample. However, STED microscopy is still a costly technique commonly not available to all life science researchers. An image technique with which the slit diaphragm proteins in the kidney can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded while optically cleared, 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 under both healthy and diseased conditions using confocal microscopy. This finding opens up the possibility for any researcher with access to a confocal microscope to study foot process protein distributions on the effective nanometer-scale. We also show that expansion microscopy can be combined with STED microscopy to further increase the effective spatial resolution down to below 20 nm.

Keyword
Expansion microscopy, kidney, slit diaphragm, Super-resolution microscopy
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-207579 (URN)
Funder
Swedish Foundation for Strategic Research , RFI14-0091Swedish Research Council, 2013-6041
Note

QC 20170523

Available from: 2017-05-22 Created: 2017-05-22 Last updated: 2017-05-23Bibliographically approved
3. Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesis
Open this publication in new window or tab >>Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesis
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The role of calcium signaling for development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used explanted embryonic rat kidneys to study the role of calcium for branching morphogenesis, a process that depends on reciprocal interaction between mesenchymal and epithelial ureteric bud cells. We recorded a spontaneous calcium activity characterized by stochastic and irregular calcium spikes, in the mesenchymal cells. This activity is due to calcium release from the endoplasmic reticulum (ER). Depletion of ER calcium stores results in down-regulation of the calcium activity, retardation of branching morphogenesis and formation of primitive nephrons, but has no effect on cell proliferation. We propose that the excretion of morphogenic factors that mediate the interaction between 26 the mesenchymal and epithelial cells, which initiate branching morphogenesis, is calcium dependent. In support of this we demonstrate expression of the calcium dependent excretory protein synaptotagmin1.

Keyword
Calcium signaling, kidney development, embryonic kidney, synaptotagmin
National Category
Developmental Biology
Identifiers
urn:nbn:se:kth:diva-207581 (URN)
Funder
Swedish Research Council, 2013-6041Swedish Research Council, 2015-04198
Note

QC 20170523

Available from: 2017-05-22 Created: 2017-05-22 Last updated: 2017-05-23Bibliographically approved

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