Abstract P245: Activation Of Protease-activated Receptors 1 Leads To Structural Changes In Immortalized Cultured Human Podocytes.

Previous studies have suggested that activation of the protease-activated receptors (PAR) exacerbates the development of diabetic nephropathy. PARs are transmembrane proteins activated by extracellular serine proteases, such as thrombin and trypsin, which are frequently elevated in disease states. We hypothesize that serine protease activation of the PAR1 signaling cascade upregulates calcium channel activity and promotes excessive intracellular Ca 2+ ([Ca 2+ ] i ), leading to podocyte foot retraction, apoptosis, and glomerular filtration barrier (GFB) damage. Immunofluorescent labeling revealed the co-localization of PAR1 and podocyte marker nephrin in freshly isolated human glomeruli. To explore the functional role of PAR1 mediated signaling, we used an immortalized cultured human podocyte (hPod) cell line. Live confocal imaging revealed rapid elevation of [Ca 2+ ] i in response to a PAR1 specific agonist (TFLLR-NH 2 ). Moreover, preincubation with a PAR1 antagonist (RWJ 56100) completely blocked this effect (1616±48 vs. 206±135 a.u., for TFLLR vs. TFLLR+RJW, n<100 cells, p<0.05). Furthermore, we tested potential downstream signaling proteins in hPod cells after PAR1 activation using Western blot analysis. Application of TFLLR-NH 2 led to a decrease in PAR1 expression due to internalization and lysosomal degradation, accompanied by an increase in TRPC6 expression and time-dependent changes in p-ERK1/2 and PLC-γ1 cascades. We further used scanning ion-conductance microscopy (SICM) to detect morphological changes in podocytes, which measures real-time surface topography in hPod cells. This imaging revealed normal protrusion of lamellipodium under control conditions. In contrast, PAR1 activation led to retraction of the lamellipodium, and a significant decrease in surface area (+51±46 vs. -15±8 μm 2 , 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05). Our studies suggested that PAR1 is involved in GPCR-induced calcium influx in podocytes. Furthermore, these data demonstrate that PAR1-mediated signaling is involved in podocyte structural alterations and may increase [Ca 2+ ] i , potentially leading to loss of GFB integrity and podocyte apoptosis.

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Switchable Lipid Provides pH-Sensitive Properties to Lipid and Hybrid Polymer/Lipid Membranes

Polymers ◽

10.3390/polym12030637 ◽

2020 ◽

Vol 12 (3) ◽

pp. 637 ◽

Cited By ~ 1

Author(s):

Victor Passos Gibson ◽

Martin Fauquignon ◽

Emmanuel Ibarboure ◽

Jeanne Leblond Chain ◽

Jean-François Le Meins

Keyword(s):

Structural Changes ◽

Morphological Changes ◽

Lipid Vesicles ◽

Ph Sensitive ◽

Confocal Imaging ◽

Amphiphilic Copolymers ◽

Hybrid Polymer ◽

Unilamellar Vesicles ◽

Triggered Release ◽

Submicron Scale

Blending amphiphilic copolymers and lipids constitutes a novel approach to combine the advantages of polymersomes and liposomes into a new single hybrid membrane. Efforts have been made to design stimuli-responsive vesicles, in which the membrane’s dynamic is modulated by specific triggers. In this investigation, we proposed the design of pH-responsive hybrid vesicles formulated with poly(dimethylsiloxane)-block-poly(ethylene oxide) backbone (PDMS36-b-PEO23) and cationic switchable lipid (CSL). The latter undergoes a pH-triggered conformational change and induces membrane destabilization. Using confocal imaging and DLS measurements, we interrogated the structural changes in CSL-doped lipid and hybrid polymer/lipid unilamellar vesicles at the micro- and nanometric scale, respectively. Both switchable giant unilamellar lipid vesicles (GUV) and hybrid polymer/lipid unilamellar vesicles (GHUV) presented dynamic morphological changes, including protrusions and fission upon acidification. At the submicron scale, scattered intensity decreased for both switchable large unilamellar vesicles (LUV) and hybrid vesicles (LHUV) under acidic pH. Finally, monitoring the fluorescence leakage of encapsulated calcein, we attested that CSL increased the permeability of GUV and GHUV in a pH-specific fashion. Altogether, these results show that switchable lipids provide a pH-sensitive behavior to hybrid polymer/lipid vesicles that could be exploited for the triggered release of drugs, cell biomimicry studies, or as bioinspired micro/nanoreactors.

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Structural changes in uterine tissues after uterine artery embolization in patients with leiomyoma complicated by uterine bleeding

GYNECOLOGY ◽

10.26442/2079-5696_20.1.78-82 ◽

2018 ◽

Vol 20 (1) ◽

pp. 78-82

Author(s):

G P Titova ◽

M M Damirov ◽

L S Kokov ◽

O N Oleynikova ◽

G E Belozerov

Keyword(s):

Uterine Artery Embolization ◽

Uterine Artery ◽

Structural Changes ◽

Morphological Changes ◽

Uterine Bleeding ◽

Small Scale ◽

Artery Embolization ◽

Vessel Occlusion ◽

Uterine Arteries ◽

Uterine Tumors

Uterine leiomyoma (UL) is often complicated by the development of uterine bleeding. In urgent gynecology for the implementation of endovascular hemostasis, uterine artery embolization (UAE) is used. Performing UAE allows to stop and/or significantly reduce the intensity of bleeding and prepare a patient for surgical intervention. At the same time, the morphological changes that occur in uterine tissues in operated UL patients after performing the UAE are not studied. The aim was to study the peculiarities of pathomorphological changes in uterine tumors and tissues in operated UL patients complicated by uterine bleeding after performing UAE. Material and methods. The results of morphological changes appearing in tumors and tissues of the uterus in 39 operated UL patients, who were used for stopping uterine bleeding, were analyzed. Results. After applying different types of embolizing agents in macroscopic study of the uterus, signs of ischemia of its tissues were revealed, and the most pronounced disorders were detected in the UL nodes. Morphologically it was established that UAE microemboli resulted in vessel occlusion with increasing thrombosis in their distal sections. UAE was not accompanied by occlusal occlusion of the arteries and resulted in small-scale necrosis of the tumor with complete regeneration of the endometrium. Conclusions. The results of the morphological study showed that after the UAE was performed, the myomatous nodes underwent dystrophic, necrobiotic and necrotic changes. Depending on the nature of occlusion of the uterine arteries, various variants of necrosis (scale and completeness of the process) developed in the tumor tissue, which was aseptic in nature.

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Thermal characterization of ABS-Graphene blended three dimensional printed functional prototypes for electro chemical energy storage

International Journal of Computational Physics Series ◽

10.29167/a1i1p1-11 ◽

2018 ◽

Vol 1 (1) ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Kamaljit Singh Boparai ◽

Rupinder Singh

Keyword(s):

Energy Storage ◽

Structural Changes ◽

Fused Deposition Modeling ◽

Morphological Changes ◽

Three Dimensional ◽

Thermal Characterization ◽

Chemical Energy ◽

Flow Index ◽

Fused Deposition

This study highlights the thermal characterization of ABS-Graphene blended three dimensional (3D) printed functional prototypes by fused deposition modeling (FDM) process. These functional prototypes have some applications as electro-chemical energy storage devices (EESD). Initially, the suitability of ABS-Graphene composite material for FDM applications has been examined by melt flow index (MFI) test. After establishing MFI, the feedstock filament for FDM has been prepared by an extrusion process. The fabricated filament has been used for printing 3D functional prototypes for printing of in-house EESD. The differential scanning calorimeter (DSC) analysis was conducted to understand the effect on glass transition temperature with the inclusion of Graphene (Gr) particles. It has been observed that the reinforced Gr particles act as a thermal reservoir (sink) and enhances its thermal/electrical conductivity. Also, FT-IR spectra realized the structural changes with the inclusion of Gr in ABS matrix. The results are supported by scanning electron microscopy (SEM) based micrographs for understanding the morphological changes.

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Automated computational analysis reveals structural changes in the enteric nervous system of nNOS deficient mice

Scientific Reports ◽

10.1038/s41598-021-96677-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ben R. Cairns ◽

Benjamin Jevans ◽

Atchariya Chanpong ◽

Dale Moulding ◽

Conor J. McCann

Keyword(s):

Machine Learning ◽

Nervous System ◽

Enteric Nervous System ◽

Structural Changes ◽

Computational Analysis ◽

Confocal Imaging ◽

Learning Approaches ◽

Composition And Structure ◽

Oxide Synthase ◽

Computational Image Analysis

AbstractNeuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1−/− colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1−/− ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies.

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Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608763114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2395-2400 ◽

Cited By ~ 14

Author(s):

Umesh Vivekananda ◽

Pavel Novak ◽

Oscar D. Bello ◽

Yuri E. Korchev ◽

Shyam S. Krishnakumar ◽

...

Keyword(s):

Potassium Channels ◽

Calcium Influx ◽

Digital Transmission ◽

Ion Conductance ◽

Scanning Ion Conductance Microscopy ◽

Spike Shape ◽

Presynaptic Spike ◽

Synaptic Boutons ◽

Episodic Ataxia Type

Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog–digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of episodic ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels.

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Perspectives on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00378.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. L1-L8 ◽

Cited By ~ 202

Author(s):

Enrique Arciniegas ◽

Maria G. Frid ◽

Ivor S. Douglas ◽

Kurt R. Stenmark

Keyword(s):

Pulmonary Hypertension ◽

Smooth Muscle ◽

Vascular Remodeling ◽

Serine Proteases ◽

Structural Changes ◽

Transforming Growth Factor ◽

Pulmonary Arteries ◽

Potential Contribution ◽

Mesenchymal Transition ◽

Endothelial Mesenchymal Transition

All forms of pulmonary hypertension are characterized by structural changes in pulmonary arteries. Increased numbers of cells expressing α-smooth muscle (α-SM) actin is a nearly universal finding in the remodeled artery. Traditionally, it was assumed that resident smooth muscle cells were the exclusive source of these newly appearing α-SM actin-expressing cells. However, rapidly emerging experimental evidence suggests other, alternative cellular sources of these cells. One possibility is that endothelial cells can transition into mesenchymal cells expressing α-SM actin and that this process contributes to the accumulation of SM-like cells in vascular pathologies. We review the evidence that endothelial-mesenchymal transition is an important contributor to cardiac and vascular development as well as to pathophysiological vascular remodeling. Recent work has provided evidence for the role of transforming growth factor-β, Wnt, and Notch signaling in this process. The potential roles of matrix metalloproteinases and serine proteases are also discussed. Importantly, endothelial-mesenchymal transition may be reversible. Thus insights into the mechanisms controlling endothelial-mesenchymal transition are relevant to vascular remodeling and are important as we consider new therapies aimed at reversing pulmonary vascular remodeling.

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Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00381.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. C966-C976 ◽

Cited By ~ 22

Author(s):

Sunwoo Lee ◽

Joon-Chul Kim ◽

Yuhua Li ◽

Min-Jeong Son ◽

Sun-Hee Woo

Keyword(s):

Ventricular Myocytes ◽

Fluid Pressure ◽

Inhibitory Effect ◽

Cellular Mechanism ◽

Confocal Imaging ◽

Rat Ventricular Myocytes ◽

Whole Cell Patch Clamp ◽

Current Voltage ◽

Intracellular Ca ◽

High Concentrations

This study examines whether fluid pressure (FP) modulates the L-type Ca2+ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (∼16 dyn/cm2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca2+ current ( ICa) and cytosolic Ca2+ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed ICa (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of ICa. The level of ICa suppression by FP depended on the level and duration of pressure. The Ba2+ current through the Ca2+ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca2+ channel during FP stimulation. The cytosolic Ca2+ transients and the basal Ca2+ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the ICa and on the Ca2+ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca2+ buffer, eliminated the FP-induced acceleration of ICa inactivation and reduced the inhibitory effect of the FP on ICa by ≈80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca2+ release, eliminated the accelerating effect of FP on the ICa inactivation, and they reduced the inhibitory effect of FP on the ICa. These results suggest that the fluid pressure indirectly suppresses the Ca2+ channel by enhancing the Ca2+-induced intracellular Ca2+ release in rat ventricular myocytes.

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Staphylococcal infection and pathomurfological characteristic of the pituitary-adrenal-thyroid system

NATURE AND SCIENCE ◽

10.36719/2707-1146/04/69-73 ◽

2020 ◽

Vol 03 (04) ◽

pp. 69-73

Author(s):

Samira Mammadhasan Yagubova ◽

◽

Elchin Chingiz Akbarov ◽

Tarana Nadir Mirzayeva ◽

◽

...

Keyword(s):

Structural Changes ◽

Adrenal Glands ◽

Early Stage ◽

Morphological Changes ◽

Endocrine System ◽

Staphylococcal Infection ◽

The Body ◽

Glandular Cells ◽

Exogenous Factors ◽

Specific Symptoms

During the staphylococcal infection, changes in the interaction of glandular cells, dystrophic and disorganizing pathologies in tissues, especially acute structural and hemodynamic changes in the stroma of the glands in the pituitary-adrenal-thyroid system, develop from the first day of the experiment. At the end of the experiment, on the background of a decrease in exudative processes, fibroplastic reactions are significantly activated, resulting in signs of incomplete regeneration – mainly sclerotic processes and cystic-atrophic changes in the parenchyma. Structural changes in tissues in the early stages of staphylococcal infection and the dynamics of development are characterized by specific symptoms in each of the glands. Since the pituitary gland is exposed to endogenous and exogenous factors earlier and more often than the adrenal glands, and the adrenal glands are earlier than the thyroid gland, dystrophic and destructive changes in the pituitary and adrenal glands are more pronounced at the early stage of the experiment. These morphological changes can change the hormonal status of the body and lead to dysfunction of the endocrine system as a whole – a decrease in the functional activity of the glands to some extent, and even inhibition of adenohypophyseal cells. Key words: staphylococcal infection, peritonitis, pituitary, adrenal and thyroid glands

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Abstract 516: Knockdown of the Hypertension Associated Gene NOSTRIN Alters Glomerular Barrier Function in Zebrafish (Danio rerio)

Hypertension ◽

10.1161/hyp.60.suppl_1.a516 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Torsten Kirsch ◽

Jessica Kaufeld ◽

Ron Korstanje ◽

Dirk Hentschel ◽

Hermann Haller ◽

...

Keyword(s):

Glomerular Filtration ◽

Barrier Function ◽

Morphological Changes ◽

Glomerular Filtration Barrier ◽

Cell Dysfunction ◽

Larval Zebrafish ◽

Filtration Barrier ◽

Important Regulator ◽

Glomerular Barrier ◽

Prime Source

The bioavailability of nitric oxide (NO) has been associated with the development and progression of vascular and renal disease. NOSTRIN (for eNOS Traffic Inducer) has primarily been recognized as one important regulator of eNOS, the prime source of NO in the cardiovascular system, with a possible role in the pathogenesis of pre-eclampsia and the development of increased intrahepatic resistance in liver disease. Here, we identified NOSTRIN in the center of a QTL-overlap region in rat and human trait loci that are associated with hypertension. Glomerular NOSTRIN expression is detectable in podocytes in human and rat glomeruli and podocytic NOSTRIN expression is diminished in hypertensive kidney disease. We show that knockdown of NOSTRIN alters the glomerular filtration barrier function in larval zebrafish, inducing proteinuria and leading to ultrastructural morphological changes on the endothelial as well as epithelial side and the GBM of the glomerular capillary loop. We also demonstrate that NOSTRIN interacts with proteins associated with the podocyte slit membrane. We conclude that NOSTRIN expression is an important factor for the integrity of the glomerular filtration barrier. Disease related alteration of NOSTRIN expression may not only affect the vascular endothelium and therefore contribute to endothelial cell dysfunction but may also contribute to the development of podocyte disease and proteinuria.

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Three-dimensional Super-resolved Imaging of Paraffin-embedded Kidney Samples

10.1101/2021.09.02.458679 ◽

2021 ◽

Author(s):

David Unnersjoe-Jess ◽

Amer Ramdedovic ◽

Martin Hoehne ◽

Linus Butt ◽

Felix C Koehler ◽

...

Keyword(s):

New Technologies ◽

Three Dimensional ◽

Kidney Tissue ◽

Confocal Imaging ◽

Disease Assessment ◽

Gold Standard Method ◽

Filtration Barrier ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Monitoring Treatment

Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. New technologies, including super-resolved nanoscopy and expansion microscopy, as well as new sample preparation techniques, are starting to revolutionize imaging of biopsy specimens. However, our previous approaches for simple and fast three-dimensional imaging of optically cleared samples are to date not compatible with formalin fixed paraffin-embedded (FFPE) tissue, impeding application in clinical routine. Here we provide protocols that circumvent these limitations and allow for three dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. This would increase the feasibility to implement these protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

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