DNA damage in human glomerular endothelial cells induces nodular glomerulosclerosis via an ATR and ANXA2 pathway

AbstractCollagen type VI (COL6) deposition occurs in various glomerular diseases, causing serious pathological damage like nodular lesions. However, the mechanisms underlying the deposition of COL6 remain unclear. In renal biopsy samples, immunohistochemical analyses revealed that COL6 and phosphorylated histone H2AX (γ-H2AX), a DNA damage marker, were detected mainly in diabetic nodular glomerulosclerosis, in which the γ-H2AX-positive area was identified as the independent factor significantly associated with the COL6-positive area (β: 0.539, t = 2.668). In in vitro studies, COL6 secretion from human renal glomerular endothelial cells (HRGECs) was assessed by measuring the decrease in the cytoplasmic COL6-positive cells and an increase in the amount of COL6 in the culture medium. Mitomycin C (MMc) treatment of HRGECs increased the number of γ-H2AX-positive cells and COL6 secretion, which were suppressed by a specific inhibitor of ataxia telangiectasia and Rad3-related (ATR). MMc-induced COL6 secretion was also suppressed by Annexin A2 (ANXA2) siRNA transfection. Moreover, the inhibition of ATR activity did not induce any extra suppression in the MMc-induced COL6 secretion by ANXA2 siRNA transfected cells. These results confirm that nodular glomerulosclerosis partially results from DNA damage in the glomerulus and that DNA damage-induced COL6 secretion from HRGECs occurs through an ATR and ANXA2-mediated pathway.

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The human glomerular endothelial cells are potent pro-inflammatory contributors in an in vitro model of lupus nephritis

Scientific Reports ◽

10.1038/s41598-019-44868-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Paraskevi Dimou ◽

Rachael D. Wright ◽

Kelly L. Budge ◽

Angela Midgley ◽

Simon C. Satchell ◽

...

Keyword(s):

Endothelial Cells ◽

Lupus Nephritis ◽

In Vitro Model ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells ◽

Vitro Model

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Isolation, characterization, and propagation in vitro of human glomerular endothelial cells.

Journal of Experimental Medicine ◽

10.1084/jem.160.1.323 ◽

1984 ◽

Vol 160 (1) ◽

pp. 323-328 ◽

Cited By ~ 53

Author(s):

G E Striker ◽

C Soderland ◽

D F Bowen-Pope ◽

A M Gown ◽

G Schmer ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Platelet Derived Growth Factor ◽

Continuous Growth ◽

Phase Microscopy ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells ◽

Von Willebrand’S Factor ◽

Umbilical Vein Endothelial Cells

Human glomerular endothelial cells have been isolated, cloned, and characterized. They appeared as the first outgrowth from human glomeruli in the presence of platelet-derived growth factor, which was also a requirement for continuous growth. By phase microscopy they appeared as monolayers of polygonal cells. Von Willebrand's factor (VWF) was detected in the cytoplasm of all clones. Their intermediate filaments differed antigenically from that present in human umbilical vein endothelial cells. Like other endothelial cells, they demonstrated high levels of membrane-associated angiotensin-converting enzyme (ACE).

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Uptake of atrial natriuretic peptide and production of cGMP in cultured human glomerular endothelial cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v541091 ◽

1994 ◽

Vol 5 (4) ◽

pp. 1091-1098

Author(s):

D F Green ◽

V A Duruibe ◽

G Blyden ◽

R E Laskey ◽

J J Bourgoignie

Keyword(s):

Endothelial Cells ◽

Atrial Natriuretic Peptide ◽

Natriuretic Peptide ◽

Mesangial Cells ◽

Specific Binding ◽

Specific Inhibitor ◽

Scatchard Analysis ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells ◽

Atrial Natriuretic

Interactions between human glomerular endothelial cells and atrial natriuretic peptide (ANP) were studied with 125I-alpha-human-ANP binding and intracellular accumulation of cGMP. Uptake for alpha-hANP (1-28 or 5-28) by homogeneous cultures of human glomerular endothelial cells was dose and time dependent with optimal uptake occurring after 30 min of incubation at 37 degrees C. Scatchard analysis of the specific binding data with a two-compartmental model identified both high (Kd = 0.3 nM)- and low (Kd = 10 nM)-affinity receptors, with a binding site density of 12,000 and 18,060 receptors per cell, respectively. alpha-hANP markedly stimulated glomerular endothelial cell-associated cGMP. After a 2-min incubation, cGMP increased 1.3-fold (from 17.88 +/- 1.29 to 23.33 +/- 3 pmol/mg of protein), in the presence of 1 nM ANP, to more than threefold (from 21 +/- .1 to 80.5 +/- 14.5 pmol/mg of protein) with 1 microM ANP (P < 0.05). In contrast, a 10 microM concentration of the clearance receptor C-ANP4-23 increased cGMP by 1.6 +/- 0.6 fold. ANP stimulation of intracellular cGMP was 100 times more sensitive in human glomerular endothelial than in mesangial cells. In comparison, higher doses of bradykinin were necessary to evoke similar responses in glomerular endothelial cells. In the presence of 10 microM bradykinin, cellular cGMP increased by 1.75 +/- 0.6-fold versus control cells. However, unlike ANP, bradykinin-stimulated cGMP synthesis was significantly inhibited by prior treatment with oxyhemoglobin (10(-5) M), an inhibitor of soluble guanylate cyclase, and NG-nitro-L-arginine (NO2Arg), a specific inhibitor of endothelial-derived relaxing factor (EDRF).(ABSTRACT TRUNCATED AT 250 WORDS)

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Activation of general control nonderepressible 2 kinase protects human glomerular endothelial cells from harmful high-glucose-induced molecular pathways

International Urology and Nephrology ◽

10.1007/s11255-016-1377-x ◽

2016 ◽

Vol 48 (10) ◽

pp. 1731-1739 ◽

Cited By ~ 10

Author(s):

Theodoros Eleftheriadis ◽

Konstantina Tsogka ◽

Georgios Pissas ◽

Georgia Antoniadi ◽

Vassilios Liakopoulos ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Molecular Pathways ◽

General Control ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells

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Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation

Frontiers in Immunology ◽

10.3389/fimmu.2021.789142 ◽

2021 ◽

Vol 12 ◽

Author(s):

Solvey Pollmann ◽

David Scharnetzki ◽

Dominique Manikowski ◽

Malte Lenders ◽

Eva Brand

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Fabry Disease ◽

Specific Inhibitor ◽

Small Vessel ◽

Endothelial Glycocalyx ◽

Enzyme Replacement ◽

Anti Inflammatory

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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Cytosolic Sensors of Viral RNA Are Involved in the Production of Interleukin-6 via Toll-Like Receptor 3 Signaling in Human Glomerular Endothelial Cells

Kidney & Blood Pressure Research ◽

10.1159/000498837 ◽

2019 ◽

Vol 44 (1) ◽

pp. 62-71 ◽

Cited By ~ 4

Author(s):

Qiang Liu ◽

Tadaatsu Imaizumi ◽

Tomomi Aizawa ◽

Koji Hirono ◽

Shogo Kawaguchi ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Interleukin 6 ◽

Viral Rna ◽

Pivotal Role ◽

Toll Like Receptor ◽

Polycytidylic Acid ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells ◽

Tlr3 Signaling

Background/Aims: Dysregulation of interleukin-6 (IL-6) production in residual renal cells may play a pivotal role in the development of glomerulonephritis (GN). Given that Toll-like receptor 3 (TLR3) signaling has been implicated in the pathogenesis of some forms of GN, we examined activated TLR3-mediated IL-6 signaling in cultured normal human glomerular endothelial cells (GECs). Methods: We treated GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of IL-6 and the cytosolic viral RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) using reverse transcription quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays. To further elucidate the effects of poly IC on this signaling pathway, we subjected the cells to small interfering RNA (siRNA) against TLR3, interferon (IFN)-β, RIG-I, and MDA5. Results: We found that poly IC induced the expression of RIG-I, MDA5 and IL-6 via TLR3/IFN-β signaling in GECs. siRNA experiments revealed that both MDA5 and RIG-I were involved in the poly IC-induced expression of IL-6, with MDA5 being upstream of RIG-I. Conclusion: Interestingly, cytosolic sensors of viral RNA were found to be involved in IL-6 production via TLR3 signaling in GECs. Regional activation of TLR3/IFN-β/ MDA5/RIG-I/IL-6 axis due to viral and “pseudoviral” infections is involved in innate immunity and inflammatory reactions in GECs. We believe this signaling pathway also plays a pivotal role in the development of some forms of GN.

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