| 1. |
- Bernhem, Kristoffer, et al.
(författare)
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AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
- 2017
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Ingår i: BMC Cardiovascular Disorders. - Stockholm : BioMed Central. - 1471-2261 .- 1471-2261. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Blockers of angiotensin II type 1 receptor (AT 1 R) and the voltage gated calcium channel 1.2 (Ca V 1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT 1 R signaling and whether there is a reciprocal regulation of AT 1 R signaling by Ca V 1.2.Methods: To elucidate these questions, we have studied the Ca 2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca 2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of Ca V 1.2 blockers. Semi- quantitative imaging methods were used to assess the plasma membrane expression of AT 1 R and G-protein activation.Results: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca 2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca 2+ response. The up-regulation of the Ca 2+ response to repeated 1 nM AngII doses and the down- egulation of the Ca 2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT 1 R plasma membrane expression. The Ca 2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the Ca V 1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT 1 R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT 1 R is sensitive to calcium influx.Conclusions: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca 2+ channel blockers as mono-therapy in hypertension.
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| 2. |
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| 3. |
- Bernhem, Kristoffer
(författare)
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Quantitative bioimaging in single cell signaling
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.
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| 4. |
- Bernhem, Kristoffer, et al.
(författare)
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Super resolution imaging reveals details in hyperglycemic induced apoptosis in kidney cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The role of the Bcl-family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods in studies of isolated mitochondria and transfected cell lines. There is however little knowledge about the mechanisms for Bcl protein interaction leading to apoptosis in intact cells. In particular, the time sequence and location for Bcl protein interaction has so far only been described in hypothetical models.Here we have used Stimulated Emission Depletion (STED) microscopy and Single Molecule Localization Microscopy (SMLM) to study the apoptotic process in immune-stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl-2 proteins, we used the nearest-neighbor algorithm. The anti-apoptotic protein Bcl-xLl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis-inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl-xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl-xL interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol1. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl-xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non-specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return.
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| 5. |
- Bernhem, Kristoffer, et al.
(författare)
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Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad
- 2021
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Ingår i: Cell Death and Disease. - : Springer Nature. - 2041-4889 .- 2041-4889. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.
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| 6. |
- Blom, Hans, et al.
(författare)
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Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy
- 2012
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Ingår i: Microscopy research and technique (Print). - : Wiley. - 1059-910X .- 1097-0029. ; 75:2, s. 220-228
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Tidskriftsartikel (refereegranskat)abstract
- Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.
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| 7. |
- Blom, Hans, et al.
(författare)
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Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy
- 2011
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 12, s. 16-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.
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| 8. |
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| 9. |
- Burlaka, Ievgeniia, et al.
(författare)
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Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease
- 2016
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Ingår i: Kidney International. - : Nature Publishing Group. - 0085-2538 .- 1523-1755. ; 90:1, s. 135-148
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Tidskriftsartikel (refereegranskat)abstract
- There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time-and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.
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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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