Membrane Nanoparticles Derived from ACE2-rich Cells Block SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Cheng Wang ◽  
Shaobo Wang ◽  
Yin Chen ◽  
Jianqi Zhao ◽  
Songling Han ◽  
...  
Keyword(s):  
Viral Entry ◽  
Inhibitory Concentration ◽  
Cell Metabolism ◽  
Host Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Dose Dependent

ABSTRACTThe ongoing COVID-19 epidemic worldwide necessitates the development of novel effective agents against SARS-CoV-2. ACE2 is the main receptor of SARS-CoV-2 S1 protein and mediates viral entry into host cells. Herein, the membrane nanoparticles prepared from ACE2-rich cells are discovered with potent capacity to block SARS-CoV-2 infection. The membrane of human embryonic kidney-239T cell highly expressing ACE2 is screened to prepare nanoparticles. The nanomaterial termed HEK-293T-hACE2 NPs contains 265.1 ng mg−1 of ACE2 on the surface and acts as a bait to trap SARS-CoV-2 S1 in a dose-dependent manner, resulting in reduced recruitment of the viral ligand to host cells. Interestingly, SARS-CoV-2 S1 can translocate to the cytoplasm and affect the cell metabolism, which is also inhibited by HEK-293T-hACE2 NPs. Further studies reveal that HEK-293T-hACE2 NPs can efficiently suppress SARS-CoV-2 S pseudovirions entry into human proximal tubular cells and block viral infection with a low half maximal inhibitory concentration. Additionally, this biocompatible membrane nanomaterial is sufficient to block the adherence of SARS-CoV-2 D614G-S1 mutant to sensitive cells. Our study demonstrates a easy-to-acheive memrbane nano-antagonist for curbing SARS-CoV-2, which enriches the existing antiviral arsenal and provides new possibilities to treat COVID-19. Graphical Table of Contents

ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop

1995 ◽  
Vol 268 (5) ◽  
pp. F940-F947 ◽  
Author(s):  
G. Wolf ◽  
F. N. Ziyadeh ◽  
U. Helmchen ◽  
G. Zahner ◽  
R. Schroeder ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Cell Line ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Ang Ii ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Proximal Tubular

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.

Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule

2000 ◽  
Vol 278 (6) ◽  
pp. F886-F893 ◽  
Author(s):  
Peter White ◽  
R. Brian Doctor ◽  
Rolf H. Dahl ◽  
Jing Chen
Keyword(s):  
Control Level ◽  
Dependent Manner ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Proximal Tubular Cells ◽  
Actin Bundle ◽  
Tubular Cells ◽  
Contrast Microscopy ◽  
Proximal Tubular ◽  
Interference Contrast Microscopy

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

scholarly journals Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

2020 ◽  
Author(s):  
Lamiaa El-Shennawy ◽  
Andrew D. Hoffmann ◽  
Nurmaa K. Dashzeveg ◽  
Paul J. Mehl ◽  
Zihao Yu ◽  
...  
Keyword(s):  
Viral Entry ◽  
Therapeutic Potential ◽  
Adaptive Immune Response ◽  
Host Cells ◽  
Dependent Manner ◽  
Angiotensin Converting Enzyme 2 ◽  
Healthy Donors ◽  
Dose Dependent ◽  
Entry Receptor ◽  
Antiviral Mechanism

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells

2003 ◽  
Vol 284 (5) ◽  
pp. F1037-F1045 ◽  
Author(s):  
Kiho Takaya ◽  
Daisuke Koya ◽  
Motohide Isono ◽  
Toshiro Sugimoto ◽  
Takeshi Sugaya ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Renal Diseases ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Reporter Activity ◽  
Proximal Tubular

Persistent proteinuria has been indicated to be a major risk factor for the development of tubulointerstitial damage through a process of proinflammatory molecule expression. Monocyte chemoattractant protein-1 (MCP-1) was shown to contribute to recruitment of immune cells into the renal interstitium in acute and chronic renal diseases. However, the molecular mechanisms by which proteinuria causes MCP-1 expression in proximal tubular cells have not been fully clarified. In this study, we examined whether albumin overload-induced MCP-1 expression was regulated by mitogen-activated protein kinase (MAPK) in mouse proximal tubular (mProx) cells. Exposure of mProx cells to delipidated bovine serum albumin (BSA) induced mRNA and protein expression of MCP-1 in a time- and dose-dependent manner. BSA activated extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. The MEK inhibitor U-0126 partially suppressed BSA-induced MCP-1 expression and MCP-1 promoter/luciferase reporter activity. U-0126 also inhibited an increase in nuclear factor-κB and activator protein-1 DNA-binding activity of MCP-1 promoter by protein overload in mProx cells. In addition, we found that U-0126 inhibited BSA-induced nuclear factor-κB reporter activity and inhibitory protein degradation in mProx cells. In conclusion, these findings indicate that ERK signaling is involved in BSA-induced MCP-1 expression in mProx cells.

scholarly journals Major Pathways of Polymyxin-Induced Apoptosis in Rat Kidney Proximal Tubular Cells

2015 ◽  
Vol 59 (4) ◽  
pp. 2136-2143 ◽  
Author(s):  
Mohammad A. K. Azad ◽  
Jesmin Akter ◽  
Kelly L. Rogers ◽  
Roger L. Nation ◽  
Tony Velkov ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Polymyxin B ◽  
Time Dependent ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Induced Apoptosis ◽  
Kidney Proximal Tubular Cells

ABSTRACTIdentifying the pathways involved in the apoptotic cell death that is associated with polymyxin-induced nephrotoxicity is crucial for the development of strategies to ameliorate this dose-limiting side effect and for the development of novel safer polymyxins. The primary aim of this study was to identify the major pathways which lead to polymyxin-induced apoptosis in cultured rat kidney proximal tubular cells (NRK-52E). Caspase-3, -8, and -9 were activated by polymyxin B treatment in a concentration-dependent manner. Concentration- and time-dependent expression of FasL and deformation of mitochondrial morphology were revealed following polymyxin B treatment. The proportion of cells with filamentous mitochondria (regular morphology) following an 8-h treatment with 1.0 mM polymyxin B was 56.2% ± 9.7% (n= 3). This was decreased to 30.7% ± 7.5% when the polymyxin B concentration was increased to 2.0 mM. The mitochondrial membrane potential (Δψm) decreased to 14.1% ± 2.9% in the cells treated with 1.0 mM polymyxin B for 24 h (n= 3) compared to that in the untreated control group. Concomitantly, concentration- and time-dependent production of mitochondrial superoxide was also observed. This study is the first to have demonstrated that polymyxin-induced apoptosis is mediated through both the death receptor and mitochondrial pathways in cultured renal tubular cells. It provides key information not only for the amelioration of polymyxin-induced nephrotoxicity but also for the discovery of novel safer polymyxin-like antibiotics against Gram-negative “superbugs.”

Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells

2000 ◽  
Vol 98 (3) ◽  
pp. 295-301 ◽  
Author(s):  
Richard DIXON ◽  
Nigel J. BRUNSKILL
Keyword(s):  
Human Albumin ◽  
Mitogen Activated Protein Kinase ◽  
Proximal Tubular Cells ◽  
Opossum Kidney ◽  
Tubular Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Proximal Tubular ◽  
Dose Dependent ◽  
Recombinant Human Albumin

The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [3H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed.

scholarly journals Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

2021 ◽  
Author(s):  
Lamiaa El-Shennawy ◽  
Andrew Hoffmann ◽  
Nurmaa Dashzeveg ◽  
Paul Mehl ◽  
Zihao Yu ◽  
...  
Keyword(s):  
Viral Entry ◽  
Therapeutic Potential ◽  
Adaptive Immune Response ◽  
Host Cells ◽  
Dependent Manner ◽  
Angiotensin Converting Enzyme 2 ◽  
Healthy Donors ◽  
Negative Controls ◽  
Dose Dependent ◽  
Antiviral Mechanism

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes, but not the ACE2-negative controls, blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50–150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Furthermore, a dramatic increase in plasma ACE2+ exosome levels were detected in patients with severe COVID-19 pathogenesis. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

The Use of Porcine Proximal Tubular Cells for Studying Nephrotoxicity In Vitro: Validation of the Effects of Culturing and Cryopreservation

1994 ◽  
Vol 22 (6) ◽  
pp. 462-473
Author(s):  
Marieke Kruidering ◽  
Diedka H. Maasdam ◽  
Winfried R. Leeman ◽  
Emile de Heer ◽  
J. Fred Nagelkerke
Keyword(s):  
In Vitro Models ◽  
Halogenated Hydrocarbons ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Extracellular Matrix Components ◽  
Proximal Tubular ◽  
Dose Dependent Decrease ◽  
Controlled Freezing ◽  
Dose Dependent

The susceptibility to nephrotoxins of freshly isolated porcine proximal tubular cells (PPTC) and cultured PPTC in suspension were compared, with a view to using PPTC as in vitro models for studying nephrotoxicity. In a previous paper, we described how, in freshly isolated PPTC, well-known nephrotoxins such as mercury (II) chloride, cisplatin, p-aminophenol and halogenated hydrocarbons caused a dose-dependent decrease in the viability and mitochondrial membrane potential of the PPTC. In this paper, we show that suspensions of cultured PPTC, harvested by trypsinisation, are less susceptible to nephrotoxins, possibly due to the synthesis of extracellular matrix components. PPTC in primary culture are suitable for relatively long-term nephrotoxicity studies. This was demonstrated by incubation with mercury (II) chloride for 24 hours, resulting in a dose-dependent loss of viability. Freshly isolated PPTC can be cryopreserved by computer-controlled freezing. The cryopreserved PPTC displayed an increased susceptibility to mercury (II) chloride and a decreased susceptibility to cisplatin and 1,1-dichloro-2,2-difluoroethylene-cysteine, the toxicity of the latter indicating that the renal enzyme β-lyase remains active during cryopreservation.

scholarly journals Hormonal regulation of expression of the angiotensinogen gene in cultured opossum kidney proximal tubular cells.

1992 ◽  
Vol 2 (10) ◽  
pp. 1516-1522
Author(s):  
J S Chan ◽  
M Ming ◽  
Z R Nie ◽  
R Sikstrom ◽  
S Lachance ◽  
...  
Keyword(s):  
Hormonal Regulation ◽  
Messenger Rna ◽  
Fusion Gene ◽  
Dependent Manner ◽  
Fusion Genes ◽  
Proximal Tubular Cells ◽  
Opossum Kidney ◽  
Tubular Cells ◽  
Ok Cells ◽  
Proximal Tubular

Angiotensinogen (ANG) messenger RNA is expressed in cultured opossum kidney (OK) proximal tubular cells. The aim of these studies was to investigate whether steroid hormones (dexamethasone, estradiol, testosterone, and progesterone) could stimulate the expression of renal ANG gene in vitro. Fusion genes consisting of various lengths of the 5'-flanking region of the rat ANG gene linked to a chloramphenicol acetyl transferase (CAT) reporter gene were constructed and introduced into cultured OK cells. The level of expression of fusion genes was determined by the level of cellular CAT enzymatic activity. The addition of dexamethasone (10(-12) to 10(-6) M) stimulates the expression of the pOCAT (ANG N-1498/+18) fusion gene in OK cells in a dose-dependent manner with a maximum stimulation at 10(-6) M and a half-maximal stimulation at 10(-9) M. Combination of dexamethasone (10(-6) M) and thyroid hormone, L-T3 (10(-6) M), further enhanced the effect of the dexamethasone alone. Testosterone (10(-6) M), estradiol (10(-6) M), and progesterone (10(-6) M) did not have this effect. Moreover, dexamethasone also stimulates the expression of the pOCAT (ANG N-688/+18) but not pOCAT (ANG N-110/+18), pOCAT (ANG N-53/+18) and pOCAT (ANG N-35/+18). These studies demonstrate that the glucocorticoid hormone is effective at stimulating the transcription of the ANG gene in OK cells, but stimulation is not observed from testosterone, estradiol, or progesterone. Moreover, glucocorticoid and L-T3 act synergistically to stimulate the transcription of the ANG gene.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stanniocalcin 2 is positively and negatively controlled by 1,25(OH)2D3 and PTH in renal proximal tubular cells

2009 ◽  
Vol 42 (3) ◽  
pp. 261-268 ◽  
Author(s):  
Yuichiro Takei ◽  
Hironori Yamamoto ◽  
Masashi Masuda ◽  
Tadatoshi Sato ◽  
Yutaka Taketani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Ok Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular

We have previously identified a second mammalian stanniocalcin (STC2) in humans and demonstrated that STC2 inhibits phosphate uptake in an opossum renal proximal tubular cell line (opossum kidney (OK) cells). However, the regulation of Stc2 gene expression in OK cells is not well understood. In this study, we identified the opossum Stc2 cDNA sequence. The opossum STC2 amino acid sequence had 78.8% homology with human STC2, and has a conserved putative N-linked glycosylation site. Next, we investigated the regulation of Stc2 gene expression by the classical calcium and phosphate-regulating factors 1,25(OH)2D3 and PTH in OK cells. In western blot analysis using affinity-purified anti-STC2 antibody, the secretion of STC2 protein was stimulated by 1,25(OH)2D3 in a dose-dependent manner. By contrast, PTH suppressed the induction of STC2 protein secretion by 1,25(OH)2D3. Real-time PCR analysis revealed that Stc2 mRNA expression was increased by 1,25(OH)2D3 in a dose- and time-dependent manner. In addition, actinomycin D, an RNA synthesis inhibitor, prevented the effects of 1,25(OH)2D3 on Stc2 gene expression. On the other hand, PTH and phorbol 12,13-myristic acetate, a specific PKC activator, but not 8-bromo-cyclic AMP, a specific PKA activator, reduced the mRNA levels of Stc2. In addition, Gö6976, a specific PKC inhibitor, abolished the downregulation of Stc2 mRNA expression by PTH. Furthermore, we demonstrated that the renal Stc2 mRNA expression was increased by 1,25(OH)2D3 and decreased by PTH in vivo. These results suggest that STC2 is positively and negatively controlled by 1,25(OH)2D3 and PTH in renal proximal tubular cells.

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