scholarly journals Overexpression of TGF-β Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans

2016 ◽  
Vol 40 (5) ◽  
pp. 819-830 ◽  
Author(s):  
Janina Müller-Deile ◽  
Finn Gellrich ◽  
Heiko Schenk ◽  
Patricia Schroder ◽  
Jenny Nyström ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Glomerular Filtration ◽  
Cell Types ◽  
Endothelial Damage ◽  
Micro Rna ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Filtration Barrier ◽  
Glomerular Endothelial Cells ◽  
Apoptotic Pathways

Background: TGF-β is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-β. Since miR-143-3p (miR-143) is described as a TGF-β inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. Methods: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-β. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. Results: We detected a time dependent, TGF-β inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. Conclusion: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

scholarly journals Progress in Pathogenesis of Proteinuria

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Aihua Zhang ◽  
Songming Huang
Keyword(s):  
Endothelial Cells ◽  
Glomerular Filtration ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Renal Diseases ◽  
Barrier Dysfunction ◽  
Glomerular Filtration Barrier ◽  
Glomerular Proteinuria ◽  
Filtration Barrier ◽  
Glomerular Endothelial Cells

Aims. Proteinuria not only is a sign of kidney damage, but also is involved in the progression of renal diseases as an independent pathologic factor. Clinically, glomerular proteinuria is most commonly observed, which relates to structural and functional anomalies in the glomerular filtration barrier. The aim of this paper was to describe the pathogenesis of glomerular proteinuria.Data Sources. Articles on glomerular proteinuria retrieved from Pubmed and MEDLINE in the recent 5 years were reviewed.Results. The new understanding of the roles of glomerular endothelial cells and the glomerular basement membrane (GBM) in the pathogenesis of glomerular proteinuria was gained. The close relationships of slit diaphragm (SD) molecules such as nephrin, podocin, CD2-associated protein (CD2AP), a-actinin-4, transient receptor potential cation channel 6 (TRPC6), Densin and membrane-associated guanylate kinase inverted 1 (MAGI-1),α3β1 integrin, WT1, phospholipase C epsilon-1 (PLCE1), Lmx1b, and MYH9, and mitochondrial disorders and circulating factors in the pathogenesis of glomerular proteinuria were also gradually discovered.Conclusion. Renal proteinuria is a manifestation of glomerular filtration barrier dysfunction. Not only glomerular endothelial cells and GBM, but also the glomerular podocytes and their SDs play an important role in the pathogenesis of glomerular proteinuria.

Glomerular Endothelium and its Impact on Glomerular Filtration Barrier in Diabetes: Are the Gaps Still Illusive?

2018 ◽  
Vol 25 (13) ◽  
pp. 1525-1529 ◽  
Author(s):  
Joseph Fomusi Ndisang
Keyword(s):  
Endothelial Cells ◽  
Basement Membrane ◽  
Glomerular Filtration ◽  
Glomerular Basement Membrane ◽  
Healthy Individuals ◽  
Kidney Dysfunction ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Glomerular Endothelial Cells ◽  
Fenestrated Endothelium

Background: Glomerular capillaries are lined with highly specialized fenestrated endothelium which are primarily responsible to regulate high flux filtration of fluid and small solutes. During filtration, plasma passes through the fenestrated endothelium and basement membrane before it reaches the slit diaphragm, a specialized type of intercellular junction that connects neighbouring podocytes. Methods: A PubMed search was done for recent articles on components of the glomerular filtration barrier such as glomerular endothelial cells, podocytes and glomerular basement membrane, and the effect of diabetes on these structures. Results and Conclusion: Generally, the onset of kidney dysfunction in many diabetic patients is characterized by albuminuria/proteinuria, a pathophysiological event triggered by several factors including; (i) endothelial activation and shading of glycocalyx, (ii) loss of endothelial cell function, (ii) re-uptake of albumin by podocyte through a scavenger receptors and (iv) rearrangement of podocyte cytoskeleton. Howeover, as podocyte effacement does not always lead to proteinuria, the dynamic interplay between all constituents of the glomerular filtration barrier including podocytes, endothelial cells and the basement membrane may be fundamental for the effective filtration in healthy individuals. Thus, a putative cross-talk amongst podocytes, endothelial cells and the basement membrane in the homeostasis of glomerular function is envisaged. Although, the exact nature of this cross-talk remains to be clearly elucidated, it is possible that the interaction between: (i) glomerular endothelial cells and podocytes, (ii) glomerular endothelial cells and glomerular basement membrane, (iii) podocytes and glomerular basement membrane, and (iv) the simultaneous interaction amongst the three components collectively underpin effective filtration in healthy individuals. A comprehensive understanding of these different interactions still remains elusive. The elucidation of these multifaceted interactions will set the stage for greater understanding of the pathophysiology of kidney dysfunction.

MO005GENERATION OF NOVEL 3D CO-CULTURE SYSTEMS TO STUDY PODOCYTOPATHIES EX VIVO IN A PERSONALIZED MANNER*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Victoria Rose ◽  
Nina Sopel ◽  
Alexandra Ohs ◽  
Christina Warnecke ◽  
Mario Schiffer ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Lines ◽  
Ex Vivo ◽  
Time Lapse ◽  
Cell Types ◽  
Glomerular Diseases ◽  
Glomerular Cell ◽  
Glomerular Endothelial Cells ◽  
Culture Model

Abstract Background and Aims The smallest filtration unit of the kidney, the glomerulus, is composed of capillaries formed by glomerular endothelial cells (GEC), glomerular basement membrane and podocytes. Furthermore, glomerular mesangial cells (GMC) between the capillary loops give structural support. It is known that loss of podocyte foot processes is leading to a dysfunctional glomerular filtration barrier and is seen in glomerular diseases like focal segmental glomerulosclerosis and other podocytopathies. Indeed, it is hard to investigate podocytes in culture because podocytes are terminally end-differentiated cells that do not proliferate, lack foot processes and cell type-specific markers. Although conditionally immortalized human podocytes regained the capacity of proliferation, marker expression and behaviour differ between cell lines. The overarching aims of this study are to generate a 3D glomerular co-culture model that better reflects the in vivo phenotype of glomerular cell types. We want to investigate cell-cell contact, interaction and communication and extracellular matrix production in 3D glomerular co-cultures. Furthermore, patient-derived hiPSC-podocytes will be used in the glomerular co-cultures to investigate podocyte disease in a personalized manner and to identify potential therapeutic targets. Method The hanging droplet method was used to produce 3D glomerular spheroids. Therefore, human differentiated immortalized podocytes, human GECs and human GMCs were inserted in a medium-droplet hanging from the lid of a petri dish and harvested at different time points. Fluorescent cell lines of the different glomerular cell types were tracked in a time-lapse experiment to study if cell attachment and spheroid formation undergoes a specific order and structure. Glomerular spheroids were further characterized regarding the expression of podocyte-specific markers and extracellular matrix synthesis by immunohistochemistry, electron microscopy and qPCR and were compared to human cells isolated from glomeruli. Furthermore, scRNA-sequencing analysis was performed in 2D mono-cultures of human GECs, GMCs and immortalized podocytes and on 3D co-cultures to see if this change in culture conditions leads to transcriptomic alterations. For the generation of patient-derived podocytes, skin fibroblast of patients with podocyte mutations (INF2 mutation and WT1 mutation) and from healthy controls were reprogramed in iPSCs and differentiated into podocytes that keep the patient’s mutation. Results First time-lapse experiments of glomerular co-cultures showed that human podocytes and human glomerular endothelial cells attach to each other (Fig. 1a) and histological sections revealed that the glomerular spheroids are encapsulated by a monolayer of cells (Fig. 1b). SEM allowed ultrastructural characterization of the 3D spheroid-like structures (Fig. 1c). TEM revealed cell protrusions of podocytes that were not seen in monocultures (Fig. 1d, e). We could also demonstrate production of extracellular matrix by the cells (Fig. 1f). Immunohistochemistry and qPCR showed expression of collagen-IV and laminin. During the reprogramming of patient-derived fibroblasts, size of the generated hiPSC decreased and the nuclei to cell body ratio increased. HiPSCs formed colonies with distinct boarders and the proliferation rate increased. Furthermore, generated hiPSC showed similar gene expression of pluripotency markers compared to a commercial hiPSC control cell line and podocytes derived from these hiPSC expressed synaptopodin (Fig. 2). Conclusion We generated a 3D co-culture model that better represents the complexity of the glomerulus ex vivo. It is indicated that this model provides better physiological conditions. By an insertion of patient-specific hiPSC-derived podocytes in the 3D co-culture we will investigate glomerular diseases in a personalized manner in the future.

scholarly journals Understanding the Mechanisms of Proteinuria: Therapeutic Implications

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Jorge E. Toblli ◽  
P. Bevione ◽  
F. Di Gennaro ◽  
L. Madalena ◽  
G. Cao ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Glomerular Filtration ◽  
Strong Predictor ◽  
Large Body ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Filtration Barrier ◽  
Wide Range

A large body of evidence indicates that proteinuria is a strong predictor of morbidity, a cause of inflammation, oxidative stress and progression of chronic kidney disease, and development of cardiovascular disease. The processes that lead to proteinuria are complex and involve factors such as glomerular hemodynamic, tubular absorption, and diffusion gradients. Alterations in various different molecular pathways and interactions may lead to the identical clinical end points of proteinuria and chronic kidney disease. Glomerular diseases include a wide range of immune and nonimmune insults that may target and thus damage some components of the glomerular filtration barrier. In many of these conditions, the renal visceral epithelial cell (podocyte) responds to injury along defined pathways, which may explain the resultant clinical and histological changes. The recent discovery of the molecular components of the slit diaphragm, specialized structure of podocyte-podocyte interaction, has been a major breakthrough in understanding the crucial role of the epithelial layer of the glomerular barrier and the pathogenesis of proteinuria. Thispaper provides an overview and update on the structure and function of the glomerular filtration barrier and the pathogenesis of proteinuria, highlighting the role of the podocyte in this setting. In addition, current antiproteinuric therapeutic approaches are briefly commented.

scholarly journals Podocyte Autophagy in Homeostasis and Disease

2021 ◽  
Vol 10 (6) ◽  
pp. 1184
Author(s):  
Qisheng Lin ◽  
Khadija Banu ◽  
Zhaohui Ni ◽  
Jeremy S. Leventhal ◽  
Madhav C. Menon
Keyword(s):  
Glomerular Filtration ◽  
Functional Role ◽  
Specific Reference ◽  
Protective Mechanism ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Filtration Barrier ◽  
Cellular Autophagy ◽  
Survival Mechanisms

Autophagy is a protective mechanism that removes dysfunctional components and provides nutrition for cells. Podocytes are terminally differentiated specialized epithelial cells that wrap around the capillaries of the glomerular filtration barrier and show high autophagy level at the baseline. Here, we provide an overview of cellular autophagy and its regulation in homeostasis with specific reference to podocytes. We discuss recent data that have focused on the functional role and regulation of autophagy during podocyte injury in experimental and clinical glomerular diseases. A thorough understanding of podocyte autophagy could shed novel insights into podocyte survival mechanisms with injury and offer potential targets for novel therapeutics for glomerular disease.

Cell Biology of the Glomerular Podocyte

2003 ◽  
Vol 83 (1) ◽  
pp. 253-307 ◽  
Author(s):  
Hermann Pavenstädt ◽  
Wilhelm Kriz ◽  
Matthias Kretzler
Keyword(s):  
Glomerular Filtration ◽  
Cell Biology ◽  
Considerable Increase ◽  
Gene Products ◽  
Common Theme ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Selective Permeability ◽  
Filtration Barrier

Glomerular podocytes are highly specialized cells with a complex cytoarchitecture. Their most prominent features are interdigitated foot processes with filtration slits in between. These are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier. Injury to podocytes leads to proteinuria, a hallmark of most glomerular diseases. New technical approaches have led to a considerable increase in our understanding of podocyte biology including protein inventory, composition and arrangement of the cytoskeleton, receptor equipment, and signaling pathways involved in the control of ultrafiltration. Moreover, disturbances of podocyte architecture resulting in the retraction of foot processes and proteinuria appear to be a common theme in the progression of acquired glomerular disease. In hereditary nephrotic syndromes identified over the last 2 years, all mutated gene products were localized in podocytes. This review integrates our recent physiological and molecular understanding of the role of podocytes during the maintenance and failure of the glomerular filtration barrier.

The podocyte in health and disease: insights from the mouse

2007 ◽  
Vol 112 (6) ◽  
pp. 325-335 ◽  
Author(s):  
Jean-Louis R. Michaud ◽  
Chris R. J. Kennedy
Keyword(s):  
Glomerular Filtration ◽  
Genetic Manipulation ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Form And Function ◽  
Filtration Barrier ◽  
Scientific Disciplines ◽  
Fenestrated Endothelium ◽  
Health And Disease ◽  
And Function

The glomerular filtration barrier consists of the fenestrated endothelium, the glomerular basement membrane and the terminally differentiated visceral epithelial cells known as podocytes. It is now widely accepted that damage to, or originating within, the podocytes is a key event that initiates progression towards sclerosis in many glomerular diseases. A wide variety of strategies have been employed by investigators from many scientific disciplines to study the podocyte. Although invaluable insights have accrued from conventional approaches, including cell culture and biochemical-based methods, many renal researchers continue to rely upon the mouse to address the form and function of the podocyte. This review summarizes how genetic manipulation in the mouse has advanced our understanding of the podocyte in relation to the maintenance of the glomerular filtration barrier in health and disease.

scholarly journals Enzymatic processing of angiotensin peptides by human glomerular endothelial cells

2012 ◽  
Vol 302 (12) ◽  
pp. F1583-F1594 ◽  
Author(s):  
Juan Carlos Q. Velez ◽  
Jessalyn L. Ierardi ◽  
Alison M. Bland ◽  
Thomas A. Morinelli ◽  
John M. Arthur ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Messenger Rna ◽  
Cell Types ◽  
Specific Cell ◽  
Ang Ii ◽  
Glomerular Diseases ◽  
Glomerular Cell ◽  
Glomerular Endothelial Cells ◽  
Neprilysin Inhibitor ◽  
Aspartyl Aminopeptidase

The intraglomerular renin-angiotensin system (RAS) is linked to the pathogenesis of progressive glomerular diseases. Glomerular podocytes and mesangial cells play distinct roles in the metabolism of angiotensin (ANG) peptides. However, our understanding of the RAS enzymatic capacity of glomerular endothelial cells (GEnCs) remains incomplete. We explored the mechanisms of endogenous cleavage of ANG substrates in cultured human GEnCs (hGEnCs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and isotope-labeled peptide quantification. Overall, hGEnCs metabolized ANG II at a significantly slower rate compared with podocytes, whereas the ANG I processing rate was comparable between glomerular cell types. ANG II was the most abundant fragment of ANG I, with lesser amount of ANG-(1–7) detected. Formation of ANG II from ANG I was largely abolished by an ANG-converting enzyme (ACE) inhibitor, whereas ANG-(1–7) formation was decreased by a prolylendopeptidase (PEP) inhibitor, but not by a neprilysin inhibitor. Cleavage of ANG II resulted in partial conversion to ANG-(1–7), a process that was attenuated by an ACE2 inhibitor, as well as by an inhibitor of PEP and prolylcarboxypeptidase. Further fragmentation of ANG-(1–7) to ANG-(1–5) was mediated by ACE. In addition, evidence of aminopeptidase N activity (APN) was demonstrated by detecting amelioration of conversion of ANG III to ANG IV by an APN inhibitor. While we failed to find expression or activity of aminopeptidase A, a modest activity attributable to aspartyl aminopeptidase was detected. Messenger RNA and gene expression of the implicated enzymes were confirmed. These results indicate that hGEnCs possess prominent ACE activity, but modest ANG II-metabolizing activity compared with that of podocytes. PEP, ACE2, prolylcarboxypeptidase, APN, and aspartyl aminopeptidase are also enzymes contained in hGEnCs that participate in membrane-bound ANG peptide cleavage. Injury to specific cell types within the glomeruli may alter the intrarenal RAS balance.

scholarly journals Adriamycin does not damage podocytes of zebrafish larvae

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242436
Author(s):  
Maximilian Schindler ◽  
Antje Blumenthal ◽  
Marcus Johannes Moeller ◽  
Karlhans Endlich ◽  
Nicole Endlich
Keyword(s):  
Glomerular Filtration ◽  
Rapid Development ◽  
Glomerular Injury ◽  
Glomerular Filtration Barrier ◽  
Glomerular Diseases ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Suitable Animal Model ◽  
Similar Phenotype

Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.

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