scholarly journals NMDA Receptor-mediated CaMKII/ERK Activation Contributes to Renal Fibrosis

2019 ◽  
Author(s):  
Jingyi Zhou ◽  
Shuaihui Liu ◽  
Luying Guo ◽  
Rending Wang ◽  
Jianghua Chen ◽  
...  
Keyword(s):  
Western Blot ◽  
Renal Fibrosis ◽  
Type I Collagen ◽  
Ischemia Reperfusion ◽  
In Vivo Studies ◽  
Type I ◽  
Erk Activation ◽  
Expression Levels

Abstract Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered. Methods: RF was induced in vivo by unilateral ureteral obstruction (UUO) using 8-week-old C57BL/6 mice. The expression levels of the NMDAR’s functional subunit, NR1, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed using Masson’s trichrome staining. Expression of NR1, fibrotic markers (α-smooth muscle actin (α-SMA), type I collagen (COL1A4), S100A4 and fibronectin), and EMT markers (snail and E-cadherin) were measured using immunohistochemistry and western blot analysis. RF was induced after TGF-β-treatment in HK-2 cells in vitro. NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were included in this study for pathway determination. Expression of NR1, total and phosphorylation of CaMKII (p-CaMKII), total and p-ERK were measured using western blot and immunofluorescent assays. Results from in vitro studies were confirmed using in vivo studies for NR1, CaMKII and ERK expression levels. In addition, ischemia-reperfusion injury (IRI) mouse model was used to determine whether oral NMDAR inhibitor dextromethorphan (DXM) could inhibit chronic fibrosis. Results: Increased NR1 expression was observed in both UUO-injured kidneys and TGF-β-treated tubular cells. NR1 knockdown and MK801 administration downregulated CaMKII/ERK activation. In vitro administered CaMKII antagonist KN93 reduced ERK phosphorylation and was not affected by NR1 expression levels. DXM protected IRI-injured kidneys from atrophy and fibrosis. Conclusions: NMDAR participates in renal fibrogenesis by activating the CaMKII/ERK pathway. NMDAR could be a potential therapeutic target for renal fibrosis.

scholarly journals NMDA Receptor-mediated CaMKII/ERK Activation Contributes to Renal Fibrosis

2020 ◽  
Author(s):  
Jingyi Zhou ◽  
Shuaihui Liu ◽  
Luying Guo ◽  
Rending Wang ◽  
Jianghua Chen ◽  
...  
Keyword(s):  
Western Blot ◽  
Renal Fibrosis ◽  
Ischemia Reperfusion Injury ◽  
Lentiviral Vector ◽  
Ischemia Reperfusion ◽  
Erk Activation ◽  
Function Impairment ◽  
Dependent Protein

Abstract Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered. Methods: Acute RF was induced in mice by unilateral ureteral obstruction (UUO). NR1, which is the functional subunit of NMDAR, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed by Masson’s trichrome staining. Expression of NR1, fibrotic and EMT markers were measured by immunohistochemistry and western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 administration were further included in this study for pathway determination. Expression of NR1, total and phosphorylated CaMKII, total and phosphorylated ERK were measured using western blot and immunofluorescent assays. Chronic renal fibrosis was introduced by sublethal ischemia-reperfusion injury in mice, and oral NMDAR inhibitor dextromethorphan (DXM) administration was performed. Results: NR1 expressions were upregulated in both obstructed kidneys and TGF-β treated HK-2 cells. NR1 knockdown, MK801 and KN93 reduced the fibrotic morphology in vivo and in vitro respectively, and companied with the downregulated ERK activation, while KN93 administration had no effect on NR1 and CaMKII levels. Mice in the DXM group had better preservation of kidney structures and corticomedullary volumes. Conclusions: NMDAR participates in both acute and chronic renal fibrogenesis via CaMKII/ERK activation, and is a potential therapeutic target for renal fibrosis.

scholarly journals NMDA Receptor-mediated CaMKII/ERK Activation Contributes to Renal Fibrosis

2020 ◽  
Author(s):  
Jingyi Zhou ◽  
Shuaihui Liu ◽  
Luying Guo ◽  
Rending Wang ◽  
Jianghua Chen ◽  
...  
Keyword(s):  
Western Blot ◽  
Renal Fibrosis ◽  
Ischemia Reperfusion Injury ◽  
Lentiviral Vector ◽  
Ischemia Reperfusion ◽  
Erk Activation ◽  
Function Impairment ◽  
Dependent Protein

Abstract Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered. Methods: Acute RF was induced in mice by unilateral ureteral obstruction (UUO). NR1, which is the functional subunit of NMDAR, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed by Masson’s trichrome staining. Expression of NR1, fibrotic and EMT markers were measured by immunohistochemistry and western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 administration were further included in this study for pathway determination. Expression of NR1, total and phosphorylated CaMKII, total and phosphorylated ERK were measured using western blot and immunofluorescent assays. Chronic renal fibrosis was introduced by sublethal ischemia-reperfusion injury in mice, and oral NMDAR inhibitor dextromethorphan (DXM) administration was performed. Results: NR1 expressions were upregulated in both obstructed kidneys and TGF-β treated HK-2 cells. NR1 knockdown, DXM, MK801, and KN93 reduced the fibrotic morphology in vivo and in vitro respectively, and companied with the downregulated ERK activation, while KN93 administration had no effect on NR1 and CaMKII levels. Conclusions: NMDAR participates in both acute and chronic renal fibrogenesis via CaMKII/ERK activation, and is a potential therapeutic target for renal fibrosis.

scholarly journals Fusion Potential of Human Osteoclasts In Vitro Reflects Age, Menopause, and In Vivo Bone Resorption Levels of Their Donors—A Possible Involvement of DC-STAMP

2020 ◽  
Vol 21 (17) ◽  
pp. 6368
Author(s):  
Anaïs M. J. Møller ◽  
Jean-Marie Delaissé ◽  
Jacob B. Olesen ◽  
Luisa M. Canto ◽  
Silvia R. Rogatto ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Type I Collagen ◽  
Transmembrane Protein ◽  
Type I ◽  
Contributing Factors ◽  
Expression Levels ◽  
Procollagen Type ◽  
Age Related

It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14+ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.

Cellular and humoral responses to collagen–polyvinylpyrrolidone administered during short and long periods in humans

2003 ◽  
Vol 81 (11) ◽  
pp. 1029-1035 ◽  
Author(s):  
Janette Furuzawa-Carballeda ◽  
Emilio Rojas ◽  
Mahara Valverde ◽  
Irma Castillo ◽  
Lino Diaz de León ◽  
...  
Keyword(s):  
Dna Damage ◽  
Type I Collagen ◽  
Cellular Damage ◽  
In Vivo Studies ◽  
Type I ◽  
Healthy Donors ◽  
Collagen Antibodies ◽  
Humoral Responses

Collagen, particularly type I, and its related derivatives have been extensively employed in many areas of pharmacology. The present study was performed to determine the safety of collagen–polyvinylpyrrolidone (collagen–PVP) by in vitro and in vivo studies. Sera and peripheral blood cells from healthy donors without treatment and patients treated with collagen–PVP were evaluated. We observed that the biodrug does not stimulate lymphoproliferation or DNA damage in vitro, nor does it induce human anti-porcine type I collagen or anti-collagen–PVP antibodies in vivo. Furthermore, no hepatic or renal metabolic dysfunctions were observed when collagen–PVP was administered by intradermal or intramuscular routes in short- or long-term treatments. In conclusion, the present work shows that no cellular damage or immunological adverse effects (cellular and humoral) occurred during collagen–PVP treatment, even after more than 400 weeks of consecutive administrations.Key words: collagen–polyvinylpyrrolidone, DNA damage, collagen antibodies, hypertrophic scar.

scholarly journals Type I Collagen Suspension Induces Neocollagenesis and Myodifferentiation in Fibroblasts In Vitro

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Francesca Lombardi ◽  
Paola Palumbo ◽  
Francesca Rosaria Augello ◽  
Ilaria Giusti ◽  
Vincenza Dolo ◽  
...  
Keyword(s):  
Western Blot ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Aqueous Suspension ◽  
Mouse Fibroblast ◽  
Type I ◽  
High Concentration ◽  
Collagen Suspension

The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, maturation, and secretion, prolyl 4-hydroxylase (P4H) and heat shock protein 47 (HSP47), as demonstrated by western blot experiments. The treatment led, also, to an increase of α-smooth muscle actin (α-SMA) expression, evaluated through western blot analysis, and cytoskeletal reorganization, as assessed by phalloidin staining. Moreover, scanning electron microscopy analysis highlighted the appearance of plasma membrane extensions and blebbing of extracellular vesicles. Altogether, these results strongly suggest that an aqueous collagen type I suspension is able to induce fibroblast myodifferentiation. Moreover, our findings also support in vitro models as a useful tool to evaluate the effects of a collagen suspension and understand the molecular signaling pathways possibly involved in the effects observed following collagen treatment in vivo.

scholarly journals The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 450-457 ◽  
Author(s):  
DG Moon ◽  
JE Kaplan ◽  
JE Mazurkewicz
Keyword(s):  
Platelet Aggregation ◽  
Type I Collagen ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
In Vivo Model ◽  
Type I ◽  
Plasma Fibronectin ◽  
Antithrombotic Effect

Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.

scholarly journals The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 450-457 ◽  
Author(s):  
DG Moon ◽  
JE Kaplan ◽  
JE Mazurkewicz
Keyword(s):  
Platelet Aggregation ◽  
Type I Collagen ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
In Vivo Model ◽  
Type I ◽  
Plasma Fibronectin ◽  
Antithrombotic Effect

Abstract Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.

scholarly journals NMDA receptor-mediated CaMKII/ERK activation contributes to renal fibrosis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Jingyi Zhou ◽  
Shuaihui Liu ◽  
Luying Guo ◽  
Rending Wang ◽  
Jianghua Chen ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Renal Fibrosis ◽  
Epithelial To Mesenchymal Transition ◽  
Ischemia Reperfusion Injury ◽  
Smooth Muscle Actin ◽  
Ischemia Reperfusion ◽  
Mesenchymal Transition ◽  
Erk Activation

Abstract Background This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-β administration. Methods Acute renal fibrosis (RF) was induced in mice by unilateral ureteral obstruction (UUO). Histological changes were observed using Masson’s trichrome staining. The expression levels of NR1, which is the functional subunit of NMDAR, and fibrotic and epithelial-to-mesenchymal transition markers were measured by immunohistochemical and Western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were administered for pathway determination. Chronic RF was introduced by sublethal ischemia–reperfusion injury in mice, and NMDAR inhibitor dextromethorphan hydrobromide (DXM) was administered orally. Results The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-β1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal–regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic–reperfusion injury. Conclusions NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.

Develoapmet of a Tissue Analog for Cartilage Repair

1991 ◽  
Vol 252 ◽  
Author(s):  
J. M. Pachence ◽  
S. R. Frenkel ◽  
H. Lin
Keyword(s):  
Type I Collagen ◽  
Collagen Matrix ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Type I ◽  
Collagen Matrices ◽  
Pore Sizes ◽  
The Matrix

ABSTRACTPurified type I collagen was formed into matrices whose pore sizes were defined on the basis of previous results. The first series of in vitro studies measured the metabolism of chondrocytes grown in matrices with various pore sizes; results revealed that the growth rate was independent of the average matrix pore size, but that ckmdrocyte infiltration throughout the matrix was optimal for pore sizes of 100 to 150 un. In a second series of studies, type I collagen was combined with hyaluranic acid; the HyA/collagen matrices had little effect on chcrdrocyte cell growth versus the collagen matrices. A third set of in vitro studies used collagen matrices incorporating varying cornentrations of insulin-like growth factor. It was found that the IGF-1/collagen matrices can significantly effect the growth and metabolism of the clxrihrocytes. These experiments were vital in establishing the collagen matrix parameters which will be used in subsequent in vivo studies.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

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