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  • 1. Butt, Linus
    et al.
    Unnersjö-Jess, David
    Rinschen, Markus
    Höhne, Martin
    Edwards, Aurelie
    Ebert, Lena
    Castrop, Hayo
    Brismar, Hjalmar
    Blom, Hans
    Brinkkötter, Paul
    Schermer, Bernhard
    Benzing, Thomas
    Elucidating the pathogenesis of focal segmental glomerulosclerosis using CRISPR/Cas9 mediated genome editingManuscript (preprint) (Other academic)
  • 2.
    Fontana, Jacopo
    et al.
    Karolinska Institutet.
    Khodus, Georgiy
    Uppsala universitet.
    Unnersjö-Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Aperia, Anita
    Karolinska Institutet.
    Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesisManuscript (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.

  • 3.
    Fontana, Jacopo M.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden.;Sci Life Lab, Stockholm, Sweden..
    Jess, David Unnersjö
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden..
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden..
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden.;Karolinska Inst, Womens & Childrens Hlth, Stockholm, Sweden..
    Aperia, Anita
    Karolinska Inst, Womens & Childrens Hlth, Stockholm, Sweden..
    Role of calcium signaling for GDNF secretion, ureter branching and early nephron formation2016In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 30Article in journal (Other academic)
  • 4.
    Fontana, Jacopo M.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Khodus, Georgiy R.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Unnersjö Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Aperia, Anita
    Karolinska Inst, Sci Life Lab, Dept Womens & Childrens Hlth, Solna, Sweden..
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney2019In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 33, no 3, p. 4089-4096Article in journal (Refereed)
    Abstract [en]

    The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.Fontana, J. M., Khodus, G. R., Unnersjo-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

  • 5.
    Jess, David Unnersjö
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples2019Doctoral 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.

  • 6.
    Jess, David Unnersjö
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Dept Appl Phys, Sci Life Lab, Solna, Sweden..
    Scott, Lena
    Karolinska Inst, Dept Womens & Childrens Hlth, Sci Life Lab, Solna, Sweden..
    Sevilla, Sonia Zambrano
    Karolinska Univ Hosp Huddinge, Karolinska Inst, Dept Lab Med, KI AZ Integrated CardioMetab Ctr, Stockholm, Sweden..
    Patrakka, Jaakko
    Karolinska Univ Hosp Huddinge, Karolinska Inst, Dept Lab Med, KI AZ Integrated CardioMetab Ctr, Stockholm, Sweden..
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion2018In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 93, no 4, p. 1008-1013Article in journal (Refereed)
    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.

  • 7. Kvedaraite, Egle
    et al.
    Jess, David Unnersjö
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Gavhed, Desiree
    Idestrom, Maja
    Svensson, Mattias
    Lourda, Magda
    Henter, Jan-Inge
    When Langerhans Met Crohn: A Clinical Case Report and a First Three-Dimentional Reconstruction of Human Gut2017In: Pediatric Blood & Cancer, ISSN 1545-5009, E-ISSN 1545-5017, Vol. 64, p. S20-S20Article in journal (Refereed)
  • 8. Kvedaraite, Egle
    et al.
    Lourda, Magdalini
    Van Acker, Aline
    Jess, David Unnersjö
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Gavhed, Desiree
    Mjosberg, Jenny
    Idestrom, Maja
    Henter, Jan-Inge
    Svensson, Mattias
    Stromal cell - innate immune cell interactions in inflammatory bowel disease patients2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 253-253Article in journal (Other academic)
  • 9. Rodriguez, Patricia Q.
    et al.
    Chen, Ping
    Jess, David Unnersjö
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zambrano, Sonia S.
    Guo, Jing
    Schwarz, Angelina
    Möller-Hackbarth, Katja
    Axelsson, Jonas
    Blom, Hans
    Sandberg, Rickard
    Jahnukainen, Timo
    Ebarasi, Lwaki
    Patrakka, Jaakko
    Mediator complex subunit 22 is necessary for glomerular homeostasis by regulation of podocyte vesicular traffickingManuscript (preprint) (Other academic)
  • 10. Schwarz, Angelina
    et al.
    Katja, Möller-Hackbarth
    Ebarasi, Lwaki
    Unnersjö-Jess, David
    Zambrano, Sonia
    Blom, Hans
    Wernerson, Annika
    Lal, Mark
    Patrakka, Jaakko
    Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacementManuscript (preprint) (Other academic)
  • 11.
    Schwarz, Angelina
    et al.
    Karolinska Univ Hosp Huddinge, Karolinska Inst, AstraZeneca Integrated Cardio Metab Ctr, Dept Lab Med, Stockholm, Sweden..
    Moller-Hackbarth, Katja
    Karolinska Univ Hosp Huddinge, Karolinska Inst, AstraZeneca Integrated Cardio Metab Ctr, Dept Lab Med, Stockholm, Sweden..
    Ebarasi, Lwaki
    Karolinska Univ Hosp Huddinge, Karolinska Inst, AstraZeneca Integrated Cardio Metab Ctr, Dept Lab Med, Stockholm, Sweden..
    Jess, David Unnersjö
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Dept Appl Phys, Sci Life Lab, Solna, Sweden..
    Zambrano, Sonia
    Karolinska Univ Hosp Huddinge, Karolinska Inst, AstraZeneca Integrated Cardio Metab Ctr, Dept Lab Med, Stockholm, Sweden..
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Wernerson, Annika
    Karolinska Inst, Dept Clin Sci Intervent & Technol, Div Renal Med, Stockholm, Sweden..
    Lel, Mark
    AstraZeneca, Innovat Med Biotech Unit, Biosci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Patrakka, Jaakko
    Karolinska Univ Hosp Huddinge, Karolinska Inst, AstraZeneca Integrated Cardio Metab Ctr, Dept Lab Med, Stockholm, Sweden..
    Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 8888Article in journal (Refereed)
    Abstract [en]

    Podocytes have an important role in the pathogenesis of diabetic nephropathy (DN). Podocyte foot process effacement, mediated largely by the actin-based cytoskeleton of foot processes, is commonly detected in DN and is believed to be a key pathogenic event in the development of proteinuria. In this study, we identified coronin 2b (Coro2b), a member of known actin-regulating proteins, the coronins, as a highly podocyte-enriched molecule located at the cytoplasmic side of the apical plasma membrane. Studies in human renal biopsies show that glomerular Coro2b expression is significantly down-regulated in patients with DN. Studies in knockout mice indicate that Coro2b is not required for the development or maintenance of the glomerular filtration barrier. Moreover, inactivation of Coro2b specifically in podocytes does not affect the outcome of nephropathy in a streptozotocin-induced diabetes model. However, Coro2b seems to modulate the reorganization of foot processes under pathological conditions as Coro2b knockout podocytes are partially protected from protamine sulfate perfusion-induced foot process effacement. Taken together, our study suggests a role for Coro2b in the pathogenesis of glomerulopathies. Further studies regarding the involvement of Coro2b in podocyte health and diseases are warranted.

  • 12.
    Unnersjö-Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    High-resolution imaging of kidney tissue samples2017Licentiate 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.

  • 13.
    Unnersjö-Jess, David
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Butt, Linus
    Höhne, Martin
    Patrakka, Jaakko
    Schermer, Bernhard
    Benzing, Thomas
    Scott, Lena
    Blom, Hans
    Brismar, Hjalmar
    Renal diagnostics using a simplified optical clearing and swelling protocolManuscript (preprint) (Other academic)
  • 14.
    Unnersjö-Jess, David
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Scott, Lena
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Optical Clearing Methods for Large Scale Studies of Renal Morphology2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29Article in journal (Other academic)
  • 15.
    Unnersjö-Jess, David
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Scott, Lena
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Institutet, Sweden.
    Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.2016In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 89, no 1, p. 243-247Article in journal (Refereed)
    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.

  • 16.
    Unnersjö-Jess, David
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Scott, Lena
    Karolinska Institutet.
    Zambrano Sevilla, Sonia
    Karolinska Institutet.
    Patrakka, Jakko
    Karolinska Institutet.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cellular Biophysics.
    Confocal imaging of slit diaphragm proteins in expanded kidney tissueManuscript (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.

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