Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion

Abstract Background: Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion. Methods: To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs. Results: The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability. Conclusion: Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.

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Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

Scientific Reports ◽

10.1038/s41598-021-93607-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shojiro Katoh ◽

Atsuki Fujimaru ◽

Masaru Iwasaki ◽

Hiroshi Yoshioka ◽

Rajappa Senthilkumar ◽

...

Keyword(s):

Regenerative Medicine ◽

Replicative Senescence ◽

Cultured Cells ◽

Three Dimensional ◽

Human Chondrocytes ◽

Cartilage Tissue ◽

Optimal Method ◽

Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

Scientific Reports ◽

10.1038/s41598-019-53196-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sun Young Lee ◽

Sung Bum Park ◽

Young Eun Kim ◽

Hee Min Yoo ◽

Jongki Hong ◽

...

Keyword(s):

Metabolic Disorders ◽

Cultured Cells ◽

Three Dimensional ◽

3D Cell Culture ◽

Proteomic Profiling ◽

Esi Ms ◽

Mitochondrial Fatty Acid ◽

2D And 3D

AbstractThe demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

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P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0053 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Anna Iervolino ◽

Tim Lange ◽

Florian Siegerist ◽

Maximilian Schindler ◽

Giovambattista Capasso ◽

...

Keyword(s):

Glomerular Filtration ◽

Fluorescent Protein ◽

Transgenic Zebrafish ◽

Renal Tubules ◽

Glomerular Filtration Barrier ◽

Podocyte Injury ◽

Zebrafish Larvae ◽

Filtration Barrier ◽

Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

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A Targeted Computational Screen of the SWEETLEAD Database Reveals FDA-Approved Compounds with Anti-Dengue Viral Activity

mBio ◽

10.1128/mbio.02839-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Jasmine Moshiri ◽

David A. Constant ◽

Bowen Liu ◽

Roberto Mateo ◽

Steven Kearnes ◽

...

Keyword(s):

Dengue Virus ◽

Cultured Cells ◽

Three Dimensional ◽

Basic Research ◽

Structural Similarity ◽

Virus Infections ◽

Chemical Structures ◽

Antiviral Compounds ◽

Computational Screening

ABSTRACT Affordable and effective antiviral therapies are needed worldwide, especially against agents such as dengue virus that are endemic in underserved regions. Many antiviral compounds have been studied in cultured cells but are unsuitable for clinical applications due to pharmacokinetic profiles, side effects, or inconsistent efficacy across dengue serotypes. Such tool compounds can, however, aid in identifying clinically useful treatments. Here, computational screening (Rapid Overlay of Chemical Structures) was used to identify entries in an in silico database of safe-in-human compounds (SWEETLEAD) that display high chemical similarities to known inhibitors of dengue virus. Inhibitors of the dengue proteinase NS2B/3, the dengue capsid, and the host autophagy pathway were used as query compounds. Three FDA-approved compounds that resemble the tool molecules structurally, cause little toxicity, and display strong antiviral activity in cultured cells were selected for further analysis. Pyrimethamine (50% inhibitory concentration [IC50] = 1.2 μM), like the dengue proteinase inhibitor ARDP0006 to which it shows structural similarity, inhibited intramolecular NS2B/3 cleavage. Lack of toxicity early in infection allowed testing in mice, in which pyrimethamine also reduced viral loads. Niclosamide (IC50 = 0.28 μM), like dengue core inhibitor ST-148, affected structural components of the virion and inhibited early processes during infection. Vandetanib (IC50 = 1.6 μM), like cellular autophagy inhibitor spautin-1, blocked viral exit from cells and could be shown to extend survival in vivo. Thus, three FDA-approved compounds with promising utility for repurposing to treat dengue virus infections and their potential mechanisms were identified using computational tools and minimal phenotypic screening. IMPORTANCE No antiviral therapeutics are currently available for dengue virus infections. By computationally overlaying the three-dimensional (3D) chemical structures of compounds known to inhibit dengue virus over those of compounds known to be safe in humans, we identified three FDA-approved compounds that are attractive candidates for repurposing as antivirals. We identified targets for two previously identified antiviral compounds and revealed a previously unknown potential anti-dengue compound, vandetanib. This computational approach to analyze a highly curated library of structures has the benefits of speed and cost efficiency. It also leverages mechanistic work with query compounds used in biomedical research to provide strong hypotheses for the antiviral mechanisms of the safer hit compounds. This workflow to identify compounds with known safety profiles can be expanded to any biological activity for which a small-molecule query compound has been identified, potentially expediting the translation of basic research to clinical interventions.

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Construction of Lung Tumor Model for Drug Screening Based on 3D Bio-Printing Technology

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2706 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1213-1226

Author(s):

Yadong Yang ◽

Geng Yang ◽

Xingzhu Liu ◽

Yimeng Xu ◽

Siyu Zhao ◽

...

Keyword(s):

Lung Cancer ◽

3D Model ◽

Cultured Cells ◽

Lung Tumor ◽

Three Dimensional ◽

Tumor Model ◽

Traditional Chinese Medicines ◽

Chinese Medicines

As is known to all, the biological characteristics of two-dimensional (2D) cultured cells are quite different from those in vivo, so the 2D screening model can no longer meet people’s needs. With the development of tissue engineering, people are committed to developing 3D tissue models that can better reflect the biology in vivo, and tend to be mass and miniaturized. In this study, three-dimensional (3D) bio-printing was used to develop an appropriate 3D model for screening sensitive anti-lung cancer drugs in vitro. A549 lung cancer cells were mixed with 8% sodium alginate and 5% gelatin as bio-printing ink to fabricate a cell-laden hydrogel grid scaffold structure. The sensitivity of the printed 3D model to drugs was evaluated with eight anti-tumor traditional Chinese medicines. A fluorescent live/dead staining was carried out at different time to assess the cell survival rate in the 3D scaffolds. MTT assay was used to determine the inhibitory rate of eight antitumor traditional Chinese medicines on A549 cell proliferation in 3D-printed lung tumor models and conventional 2D culture models.

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Shock-induced stress induces loss of microvascular endothelial Tie2 in the kidney which is not associated with reduced glomerular barrier function

AJP Renal Physiology ◽

10.1152/ajprenal.00137.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. F272-F281 ◽

Cited By ~ 36

Author(s):

Matijs van Meurs ◽

Neng F. Kurniati ◽

Francis M. Wulfert ◽

Sigridur A. Asgeirsdottir ◽

Inge A. de Graaf ◽

...

Keyword(s):

Endothelial Cell ◽

Barrier Function ◽

Human Kidney ◽

Mrna Levels ◽

Barrier Dysfunction ◽

Molecular Control ◽

Filtration Barrier ◽

Glomerular Barrier

Both hemorrhagic shock and endotoxemia induce a pronounced vascular activation in the kidney which coincides with albuminuria and glomerular barrier dysfunction. We hypothesized that changes in Tie2, a vascular restricted receptor tyrosine kinase shown to control microvascular integrity and endothelial inflammation, underlie this loss of glomerular barrier function. In healthy murine and human kidney, Tie2 is heterogeneously expressed in all microvascular beds, although to different extents. In mice subjected to hemorrhagic and septic shock, Tie2 mRNA and protein were rapidly, and temporarily, lost from the renal microvasculature, and normalized within 24 h after initiation of the shock insult. The loss of Tie2 protein could not be attributed to shedding as both in mice and healthy volunteers subjected to endotoxemia, sTie2 levels in the systemic circulation did not change. In an attempt to identify the molecular control of Tie2, we activated glomerular endothelial cell cultures and human kidney slices in vitro with LPS or TNF-α, but did not observe a change in Tie2 mRNA levels. In parallel to the loss of Tie2 in vivo, an overt influx of neutrophils in the glomerular compartment, which coincided with proteinuria, was seen. As neutrophil-endothelial cell interactions may play a role in endothelial adaptation to shock, and these effects cannot be mimicked in vitro, we depleted neutrophils before shock induction. While this neutrophil depletion abolished proteinuria, Tie2 was not rescued, implying that Tie2 may not be a major factor controlling maintenance of the glomerular filtration barrier in this model.

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The Role of Palladin in Podocytes

Journal of the American Society of Nephrology ◽

10.1681/asn.2017091039 ◽

2018 ◽

Vol 29 (6) ◽

pp. 1662-1678 ◽

Cited By ~ 8

Author(s):

Nadine Artelt ◽

Tim A. Ludwig ◽

Henrik Rogge ◽

Panagiotis Kavvadas ◽

Florian Siegerist ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Kidney Biopsy ◽

Three Dimensional ◽

Focal Adhesions ◽

Early Developmental Stage ◽

Zebrafish Embryos ◽

Filtration Barrier ◽

Biopsy Specimens

Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld−/− mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld−/− mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld−/− mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.

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Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses

Biochemical Society Transactions ◽

10.1042/bst0381072 ◽

2010 ◽

Vol 38 (4) ◽

pp. 1072-1075 ◽

Cited By ~ 68

Author(s):

Daniel J. Maltman ◽

Stefan A. Przyborski

Keyword(s):

Cell Culture ◽

Cell Growth ◽

Cultured Cells ◽

Ex Vivo ◽

Three Dimensional ◽

3D Cell Culture ◽

Proliferation And Differentiation ◽

In Vitro Systems

Drug discovery programmes require accurate in vitro systems for drug screening and testing. Traditional cell culture makes use of 2D (two-dimensional) surfaces for ex vivo cell growth. In such environments, cells are forced to adopt unnatural characteristics, including aberrant flattened morphologies. Therefore there is a strong demand for new cell culture platforms which allow cells to grow and respond to their environment in a more realistic manner. The development of 3D (three-dimensional) alternative substrates for in vitro cell growth has received much attention, and it is widely acknowledged that 3D cell growth is likely to more accurately reflect the in vivo tissue environments from which cultured cells are derived. 3D cell growth techniques promise numerous advantages over 2D culture, including enhanced proliferation and differentiation of stem cells. The present review focuses on the development of scaffold technologies for 3D cell culture.

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Three-Dimensional Visualization of the Podocyte Actin Network Using Integrated Membrane Extraction, Electron Microscopy, and Machine Learning

Journal of the American Society of Nephrology ◽

10.1681/asn.2021020182 ◽

2021 ◽

pp. ASN.2021020182

Author(s):

Chengqing Qu ◽

Robyn Roth ◽

Pongpratch Puapatanakul ◽

Charles Loitman ◽

Dina Hammad ◽

...

Keyword(s):

Electron Microscopy ◽

Machine Learning ◽

Ion Beam ◽

Three Dimensional ◽

Membrane Extraction ◽

Stress Fibers ◽

Filtration Barrier ◽

Actin Cables ◽

Actin Stress Fibers

Background Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. Methods We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion beam scanning electron microscopy, and machine learning image segmentation. Results Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. Conclusions Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.

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Measuring glomerular number and size in perfused kidneys using MRI

AJP Renal Physiology ◽

10.1152/ajprenal.00044.2011 ◽

2011 ◽

Vol 300 (6) ◽

pp. F1454-F1457 ◽

Cited By ~ 68

Author(s):

Scott C. Beeman ◽

Min Zhang ◽

Lina Gubhaju ◽

Teresa Wu ◽

John F. Bertram ◽

...

Keyword(s):

Three Dimensional ◽

Renal Diseases ◽

Resonance Imaging ◽

Mr Images ◽

Image Processing Algorithm ◽

Glomerular Volume ◽

Glomerular Number ◽

Magnetic Resonance Imaging Mri ◽

Intrarenal Distribution

The goal of this work was to nondestructively measure glomerular (and thereby nephron) number in the whole kidney. Variations in the number and size of glomeruli have been linked to many renal and systemic diseases. Here, we develop a robust magnetic resonance imaging (MRI) technique based on injection of cationic ferritin (CF) to produce an accurate measurement of number and size of individual glomeruli. High-field (19 Tesla) gradient-echo MR images of perfused rat kidneys after in vivo intravenous injection of CF showed specific labeling of individual glomeruli with CF throughout the kidney. We developed a three-dimensional image-processing algorithm to count every labeled glomerulus. MRI-based counts yielded 33,786 ± 3,753 labeled glomeruli ( n = 5 kidneys). Acid maceration counting of contralateral kidneys yielded an estimate of 30,585 ± 2,053 glomeruli ( n = 6 kidneys). Disector/fractionator stereology counting yielded an estimate of 34,963 glomeruli ( n = 2). MRI-based measurement of apparent glomerular volume of labeled glomeruli was 4.89 × 10−4mm3( n = 5) compared with the average stereological measurement of 4.99 × 10−4mm3( n = 2). The MRI-based technique also yielded the intrarenal distribution of apparent glomerular volume, a measurement previously unobtainable in histology. This work makes it possible to nondestructively measure whole-kidney glomerular number and apparent glomerular volumes to study susceptibility to renal diseases and opens the door to similar in vivo measurements in animals and humans.

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