| 1. |
- Butt, Linus, et al.
(författare)
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A molecular mechanism explaining albuminuria in kidney disease
- 2020
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Ingår i: Nature Metabolism. - : Springer Nature. - 2522-5812. ; 2:5, s. 461-474
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.
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| 2. |
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| 3. |
- 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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| 4. |
- Charrin, Emmanuelle, et al.
(författare)
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Soluble Klotho protects against glomerular injury through regulation of ER stress response
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- alpha Klotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases. Transgenic overexpression of alpha Klotho in hepatocytes results in protection against renal insults, possibly through modulation of the ER stress response by circulating alpha Klotho. In contrast, alpha Klotho overexpressed in podocytes is not renoprotective.
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| 5. |
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| 6. |
- Fontana, Jacopo M., et al.
(författare)
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Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney
- 2019
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Ingår i: The FASEB Journal. - : FEDERATION AMER SOC EXP BIOL. - 0892-6638 .- 1530-6860. ; 33:3, s. 4089-4096
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Fontana, Jacopo, et al.
(författare)
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Temporal calcium activity in metanephric mesenchyme cells regulates kidney branching morphogenesis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 8. |
- 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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| 9. |
- Jess, David Unnersjö, et al.
(författare)
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Advanced optical imaging reveals preferred spatial orientation of podocyte processes along the axis of glomerular capillaries
- 2023
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 104:6, s. 1164-1169
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the hydrostatic pressure in glomerular capillaries, which is considerably higher than in most other tissues. Interdigitating cellular processes of podocytes form the slits for fluid filtration connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Several previous publications speculated that podocyte processes may display a preferable orientation on glomerular capillaries instead of a random distribution. However, for decades, the controversy over spatially oriented filtration slits could not be resolved due to technical limitations of imaging technologies. Here, we used advanced high-resolution, three-dimensional microscopy with high data throughput to assess spatial orientation of podocyte processes and filtration slits quantitatively. Filtration-slit-generating secondary processes preferentially align along the capillaries' longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. This preferential orientation required maturation in development of the mice but was lost in mice with kidney disease due to treatment with nephrotoxic serum or with underlying heterologous mutations in the podocyte foot process protein podocin. Thus, the observation that podocytes maintain a preferred spatial orientation of their processes on glomerular capillaries goes well in line with the role of podocyte foot processes as mechanical buttresses to counteract mechanical forces resulting from pressurized capillaries. Future studies are needed to establish how podocytes establish and maintain their orientation and why orientation is lost under pathological conditions.
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| 10. |
- Jess, David Unnersjö, et al.
(författare)
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Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion
- 2018
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Ingår i: Kidney International. - : ELSEVIER SCIENCE INC. - 0085-2538 .- 1523-1755. ; 93:4, s. 1008-1013
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 11. |
- Jess, David Unnersjö, et al.
(författare)
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Deep learning-based segmentation and quantification of podocyte foot process morphology suggests differential patterns of foot process effacement across kidney pathologies
- 2023
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 103:6, s. 1120-1130
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Tidskriftsartikel (refereegranskat)abstract
- Morphological alterations at the kidney filtration barrier increase intrinsic capillary wall permeability resulting in albuminuria. However, automated, quantitative assessment of these morphological changes has not been possible with electron or light microscopy. Here we present a deep learning-based approach for segmentation and quantitative analysis of foot processes in images acquired with confocal and super-resolution fluorescence microscopy. Our method, Automatic Morphological Analysis of Podocytes (AMAP), accurately segments podocyte foot processes and quantifies their morphology. AMAP applied to a set of kidney diseases in patient biopsies and a mouse model of focal segmental glomerulosclerosis allowed for accurate and comprehensive quantification of various morphometric features. With the use of AMAP, detailed morphology of podocyte foot process effacement was found to differ between categories of kidney pathologies, showed detailed variability between diverse patients with the same clinical diagnosis, and correlated with levels of proteinuria. AMAP could potentially complement other readouts such as various omics, standard histologic/electron microscopy and blood/urine assays for future personalized diagnosis and treatment of kidney disease. Thus, our novel finding could have implications to afford an understanding of early phases of kidney disease progression and may provide supplemental information in precision diagnostics.
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| 12. |
- Jess, David Unnersjö
(författare)
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High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 13. |
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| 14. |
- Kvedaraite, Egle, et al.
(författare)
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Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142−/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142−/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.
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| 15. |
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| 16. |
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| 17. |
- Rodriguez, Patricia Q., et al.
(författare)
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Inactivation of mediator complex protein 22 in podocytes results in intracellular vacuole formation, podocyte loss and premature death
- 2020
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Podocytes are critical for the maintenance of kidney ultrafiltration barrier and play a key role in the progression of glomerular diseases. Although mediator complex proteins have been shown to be important for many physiological and pathological processes, their role in kidney tissue has not been studied. In this study, we identified a mediator complex protein 22 (Med22) as a renal podocyte cell-enriched molecule. Podocyte-specific Med22 knockout mouse showed that Med22 was not needed for normal podocyte maturation. However, it was critical for the maintenance of podocyte health as the mice developed progressive glomerular disease and died due to renal failure. Detailed morphological analyses showed that Med22-deficiency in podocytes resulted in intracellular vacuole formation followed by podocyte loss. Moreover, Med22-deficiency in younger mice promoted the progression of glomerular disease, suggesting Med22-mediated processes may have a role in the development of glomerulopathies. This study shows for the first time that mediator complex has a critical role in kidney physiology.
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| 18. |
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| 19. |
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| 20. |
- Schwarz, Angelina, et al.
(författare)
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Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 21. |
- 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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| 22. |
- Unnersjö-Jess, David, 1985-, et al.
(författare)
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Confocal imaging of slit diaphragm proteins in expanded kidney tissue
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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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| 23. |
- Unnersjö-Jess, David, 1985-
(författare)
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High-resolution imaging of kidney tissue samples
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 24. |
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| 25. |
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| 26. |
- Unnersjö-Jess, David, et al.
(författare)
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Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.
- 2016
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Ingår i: Kidney International. - : Nature Publishing Group. - 0085-2538 .- 1523-1755. ; 89:1, s. 243-247
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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