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BACKGROUND: The expression of long chain non-coding RNA PGM5-AS1 (lnc RNA PGM5-AS1) is reduced in peripheral blood of patients with acute immune rejection after renal transplantation. The effect of its expression on human glomerular endothelial cell (HRGEC) survival and macrophage chemotaxis remains to be studied. OBJECTIVE: To investigate the expression of lnc RNA PGM5-AS1 in serum of patients with acute rejection of renal transplantation and non-acute rejection patients and its effect on proliferation, cell cycle, apoptosis and macrophages chemotropism of HRGECs. METHODS: Forty-six patients with renal transplantation were divided into acute rejection group (n=17) and non-acute rejection group (n=29) according to whether or not acute rejection occurred within 1 month after operation. Peripheral blood sample was drawn from each patient at 1 day before operation, 1, 2, 3, and 4 weeks after operation. qRT-PCR was used to detect the expression of PGM5-AS1 in serum of patients with or without acute rejection. si-control and si-PGM5-AS1 were transfected into glomerular endothelial cells, and the expression of PGM5-AS1 in the transfected cells was detected by qPCR. MTT was used to detect cell proliferation, flow cytometry was applied to detect apoptosis and cell cycle, ELISA was adopted to detect cellular inflammatory factor secretion, and Transwell was used to detect chemotaxis of macrophages. Approval for this study was obtained from the Ethics Committee of Zhengzhou People’s Hospital, and all patients signed informed consent prior to the participation in the trial. RESULTS AND CONCLUSION: Compared with non-acute rejection patients, patients with acute rejection to renal transplantation had significantly lower PGM5-AS1 expression in serum at 1, 2, 3, and 4 weeks after transplantation (P <0.05). After PGM5-AS1 silencing, the expression of GM5-AS1 in HRGEC cells was significantly lower than that in the si-control group and normal control group (F=379.658, P < 0.05). MTT results showed that PGM5-AS1 silencing significantly inhibited HRGEC cell proliferation (P < 0.05). Flow cytometry results showed that PGM5-AS1 silencing induced HRGEC cell arrest in G0/G1 phase and increased apoptosis (P < 0.05). ELISA results showed that PGM5-AS1 silencing inhibited interleukin-13 expression and increased interleukin-6, interferon-γ and tumor necrosis factor-α expression (P < 0.05). Transwell results indicated that HRGEC cells silenced by PGM5-AS1 significantly increased the chemotaxis of macrophages (P < 0.05). All these findings indicate that PGM5-AS1 is lowly expressed in serum of patients with acute rejection of renal transplantation, and inhibition of PGM5-AS1 can promote HRGEC cell damage, which can be used as a peripheral blood diagnostic marker for early acute rejection, and may be a molecular target for the treatment of acute rejection.
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Objective: To explore the effects and mechanism of high expression of ARNO (ARF nucleotide-binding-site opener) on the permeability of human renal glomerular endothelial cells (HRGECs) under high glucose conditions. Methods: HRGECs were cultured in vitro, and ARNO expression and endothelial permeability were detected in high glucose time gradient (15, 30, 45 and 60 min) and concentration gradient (10, 20, 30 and 40 mmol/L) experiments, respectively. Cell lines of HRGECs with ARNO and Arf6 gene silencing were obtained by infecting HRGECs with ARNO siRNA and Arf6 siRNA recombinant lentivirus vectors, respectively. The effects of ARNO and Arf6 gene silencing on endothelial permeability were observed. Results: In the time gradient experiments, compared with control group, ARNO expressions increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (0.670±0.051, 0.960±0.106, 0.716±0.026, 0.531±0.030 vs. 0.242±0.029. P<0.05). Compared with high glucose 15 min group, ARNO expression increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, ARNO expression decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, ARNO expression decreased in high glucose 60 min group (P<0.05). Compared with control group, endothelial permeability increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (1.196±0.004, 1.399±0.012, 1.301±0.052, 1.184±0.030 vs. 1.000, P<0.05). Compared with high glucose 15 min group, endothelial permeability increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, endothelial permeability decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, endothelial permeability decreased in high glucose 60 min group (P<0.05). In the concentration gradient experiments, compared with normal concentration glucose group, ARNO expressions increased significantly in the groups with high glucose of 20, 30 and 40 mmol/L (0.632±0.031, 0.927±0.041, 1.183±0.098 vs. 0.169±0.033, P<0.05), and the ARNO expression level was increased along with the increase of glucose concentration (P<0.05). Compared with normal concentration glucose group, endothelial permeability increased significantly in the groups with high glucose of 10, 20, 30 and 40 mmol/L (1.147±0.015, 1.237±0.023, 1.351±0.015, 1.444±0.019 vs. 1.000, P<0.05), and the endothelial permeability increased along with the increase of glucose concentration (P<0.05). After transfection with lentivirus, compared with normal concentration glucose untransfected group and normal concentration glucose empty vector group, ARNO mRNA transcription reduced significantly (0.255±0.056 vs. 1.000, 1.183±0.297), and ARNO protein expression also reduced significantly (0.088±0.005 vs. 0.246±0.011, 0.237±0.009) in normal concentration glucose ARNO siRNA group (P<0.05). In addition, compared with those groups, Arf6 mRNA transcription was reduced significantly (0.314±0.090 vs. 1.000, 1.140±0.236), and Arf6 protein expression was also reduced significantly (0.690±0.012 vs. 0.917±0.009, 0.919±0.009) in the normal concentration glucose Arf6 siRNA group (P<0.05). After silencing ARNO, compared with high-glucose untransfected group and high-glucose empty vector group, the ARNO protein expression was reduced significantly (0.572±0.021 vs. 0.915±0.005, 0.916±0.012), Arf6 activity was reduced significantly (0.263±0.007 vs. 0.484±0.014, 0.490±0.008), and endothelial permeability was also decreased (0.718±0.017 vs. 1.000, 0.978±0.040) in high-glucose ARNO siRNA group (P<0.05). Besides, after silencing Arf6, compared with high-glucose untransfected group and high-glucose empty vector group, Arf6 protein expression was reduced significantly (0.673±0.015 vs. 0.932±0.020, 0.899±0.022), and endothelial permeability was also decreased (0.768±0.050 vs. 1.000, 0.978±0.040) in high-glucose Arf6 siRNA group (P<0.05). Conclusion: High-glucose induced high permeability of HRGECs is related to the activation of ARNO/Arf6 signal, and inhibition of ARNO expression and Arf6 activity may inhibit the glomerular endothelial hyperpermeability in diabetic nephropathy, and is expected to be a new direction for the treatment of glomerular endothelial dysfunction in diabetic nephropathy.
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AIM:To observe the expression of hypoxia-inducible factor-1α (HIF-1α) , vascular endothelial growth factor A (VEGFA) and vascular endothelial cadherin (VE-cadherin) in cultured rat renal glomerular endothelial cells (rRGECs) exposed to glucose at different concentrations in vitro, and to verify the hypothesis that salidroside attenuates high glucose (HG) -induced injury of rRGECs by boosting HIF-1αlevel.METHODS:rRGECs were divided into 4group:normal glucose (NG) group, HG groups, hypertonic group and salidroside+HG group.The viability of rRGECs was measured by MTT assay.The mRNA expression of VEGFA, VE-cadherin and HIF-1αwas detected by RT-qPCR.The protein expression of HIF-1αwas determined by Western blot.RESULTS:Compared with NG group, the mRNA and protein expression of HIF-1αwas increased when the rRGECs were treated with glucose at concentration of 20 mmol/L for 24h (P<0.01).Compared with NG group, the mRNA expression of HIF-1αwas decreased in HG groups for 120 h (P<0.05).Compared with NG group, the mRNA expression of VE-cadherin was significantly down-regulated in HG groups for24 h or 120 h (P<0.05).Compared with NG group, the mRNA expression of VEGFA was increased in HG groups at 24h (P<0.05) , while the mRNA expression of VEGFA was decreased at 120 h (P<0.05).Compared with NG group, no statistical difference in the mRNA expression levels of HIF-1α, VE-cadherin and VEGFA in DM group was observed.Compared with HG group, salidroside promoted the viability of rRGECs (P<0.01) , and up-regulated the mRNA expression of HIF-1αand VE-cadherin, and the protein expression of HIF-1α (P<0.05).CONCLUSION:High glucose regulates HIF-1αexpression in rRGECs in connection with cell viability, the concentration of glucose, the culture time and HIF/VEGF signaling.Salidroside promotes rRGEC growth against high glucose-induced cell apoptosis via up-regulating the expression of HIF-1α.
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AIM:To investigate the effect of angiotensin 1-7(Ang1-7)on the human glomerular endothelial cells(HGECs)injury induced by angiotensin Ⅱ(Ang Ⅱ)and its possible mechanism.METHODS: Cultured HGECs were divided into 6 groups randomly: control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779(an inhibitor of Mas receptor)group and A779 group.The apoptotic rate and reactive oxygen species (ROS)of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy.The levels of lactate de-hydrogenase(LDH),nitric oxide(NO),endothelin-1(ET-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α), transforming growth factor-β(TGF-β),monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)in the supernatant of cell cultures were measured.RESULTS:Compared with the control group,the apoptot-ic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group,IL-6,TNF-α,TGF-β,ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group(P<0.05).Compared with the Ang Ⅱ group,the apoptotic rate,ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group(P<0.05). Compared with the Ang Ⅱ +Ang1-7 group,adding A779 increased the cell apoptotic rate,ROS production and the releases of the above inflammatory factors in cell supernatants(P<0.05).Compared with the Ang Ⅱ group,adding Ang1-7 inhibi-ted the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner(P<0.05).CON-CLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.
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This study aimed to investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia conditions. The expression level of HIF-2α and tight junction proteins (occludin and ZO-1) in rGENCs were examined following 5% oxygen density exposure at different treatment times. HIF-2α lentivirus transfection was used to knockdown HIF-2α expression. Cells were divided into four groups: 1) control group (rGENCs were cultured under normal oxygen conditions), 2) hypoxia group (rGENCs were cultured under hypoxic conditions), 3) negative control group (rGENCs were infected with HIF-2α lentivirus negative control vectors and cultured under hypoxic conditions), and 4) Len group (rGENCs were transfected with HIF-2α lentivirus and cultured under hypoxic conditions). The hypoxia, negative control, and Len groups were kept in a hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 24 h and the total content of occludin and ZO-1, and the permeability of rGENCs were assessed. With increasing hypoxia time, the expression of HIF-2α gradually increased, while the expression of occludin decreased, with a significant difference between groups. ZO-1 expression gradually decreased under hypoxia conditions, but the difference between the 24 and 48 h groups was not significant. The permeability of cells increased following 24-h exposure to hypoxia compared to the control group (P<0.01). The knockdown of HIF-2α expression significantly increased occludin and ZO-1 content compared with hypoxia and negative control groups (P<0.01), while permeability was reduced (P<0.01). Hypoxia increased HIF-2α content, inducing permeability of rGENCs through the reduced expression of occludin and ZO-1.
Subject(s)
Animals , Rats , Endothelial Cells/chemistry , Basic Helix-Loop-Helix Transcription Factors/metabolism , Occludin/metabolism , Zonula Occludens-1 Protein/metabolism , Kidney Glomerulus/cytology , Permeability , Time Factors , Cell Hypoxia/physiology , Endothelial Cells/metabolism , Cell ProliferationABSTRACT
Objective To observe the role and related mechanism of chemerin and its receptor ChemR23 in glomerular endothelial cells (GEnCs) stimulated by high glucose.Methods Mouse GEnCs were cultured and divided into control group,20.0 mmol/L high glucose group,40.0 mmol/L high glucose group and mannitol control group.Then the expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supematant as well as the expressions of intracellular protein and mRNA of chemerin,ChemR23,IL-6 and TNF-α were detected.Lentiviral transfection targeting ChemR23 was applied before high glucose-or Chemerin-stimulated,and expressions of supernatant and intracellular mRNA of IL-6 and TNF-α were measured.Meanwhile whether p38 mitogen-activated protein kinase (p38 MAPK) pathway was activated by high glucose was detected.The specific inhibitor of p38 MAPK was added prior to high glucose-stimulated,then supernatant and intracellular mRNA expressions of IL-6 and TNF-α was detected.The supernatant expressions of IL-6 and TNF-α were measured by ELISA.The intracellular protein expression and p38 MAPK phosphorylation activity were detected by Western blotting.The mRNA expression was detected by real time PCR.Results Compared with those in the control group,in high glucose groups the expressions of IL-6,TNF-α and chemerin were significantly increased (all P ＜ 0.05),however,the expressions of ChemR23 did not change (all P ＞ 0.05);the supernatant and mRNA expressions of IL-6 and TNF-α were also elevated in the chemerin group (all P ＜ 0.05).Lentivirus baring shRNA could efficiently suppress ChemR23 expression,and the Chemerin-or high glucose-induced expressions of IL-6 and TNF-α were reduced (all P ＜ 0.05).Also it could significantly reduce the expression of phosphorylated-p38 MAPK (p-p38 MAPK) induced by high glucose (P ＜ 0.05),as high glucose group had higher p-p38 MAPK than control group (P ＜ 0.05).While the high glucose-elevated expressions of IL-6 and TNF-α were significantly attenuated by p38 MAPK inhibitor (all P ＜ 0.05).Conclusions High glucose stimulation can induce the expression of chemerin in GEnCs.By binding to ChemR23,chemerin activates p38 MAPK signaling pathway,and then promotes the expressions of IL-6 and TNF-α.These inflammatory cytokines aggravate inflammation of GEnCs.
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Objective To observe the effect of TGF‐β1 in the endothelial‐mesenchymal transition EndM T of glomeruli ,and to explore the effect of TGF‐β/Smad signaling pathway mediated by integrin linked kinase(ILK ) in the progress of renal fibrosis . Methods Human glomerular endothelial cells (HGEnC) incubated in vitro were divided into blank control group ,TGF‐β1 (12 .5 , 25 .0 ,50 .0 ng/mL) group .TGF‐β1 50 .0 ng/mL receptor type one antagonist (LY364749)5 .0 μmol/L group ,ILK (QLT‐0267)5 .0μmol/L antagonist group .The mRNA level of P‐Smad 2/3 and ILK was determined by RT‐PCR ,and the protein level of P‐Smad 2/3 ,ILK ,E‐cadherin ,CD31 ,α‐SMA and FSP‐1 was determined by Western blot after 48 h and 72 h after incubation in each group un‐der the above‐mentioned condition .Results (1)TGF‐β1 could significantly increased the mRNA level of P‐Smad2/3 and ILK (P0 .05) .Conclusion TGF‐β1 as the effector molecule in downstream can promote endothelial‐mesenchymal transition of HGEnC ,and TGF‐β/Smad signaling pathway mediated integrin linked kinase participate in this process ,which probably play important role in the progress of renal fibrosis .
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Objective To investigate the effect of advanced glycation end products (AGEs) on the disruption of tight junctions in rat glomerular endothelial cells (rGEnCs) and the role of renin-angiotensin system (RAS) in this pathological procedure.Methods Primary cultured rGEnCs were incubated with AGEs at concentrations of 20 mg/L,40 mg/L and 80 mg/L,for 6 h,12 h and 24 h respectively.The cells were treated with captopril (1 mmol/L) or valsartan (10 μ mol/L)to block RAS.The endothelial permeability was investigated by transendothelial electrical resistance and the flux of fluorescein isothiocyanate-conjugated bovine serum albumin.The expression of AGEs receptor (RAGE),tight junction proteins [occludin,claudin-5,junctional adhesion molecules A (JAM-A) and zona occludens-1 (ZO-1)]and RAS components [angiotensinogen,renin and angiotensin Ⅱ type 1 receptor (AT1)]were detected by Western blotting.Immunofluorescence was used to demonstrate the disruptions of the tight junction proteins.The activity of angiotensin converting enzyme (ACE) was evaluated by UV spectrophotometry.Angiotensin Ⅱ (Ang Ⅱ ) was measured by enzyme immunoassay.Results The monolayer permeability,the expression of RAGE,the activity of ACE,the concentration of Ang Ⅱ and the expression of AT1 of rGEnCs were increased after induced by AGEs.Meanwhile,AGEs decreased the expression of occludin,claudin5 and JAM-A and induced disruption of tight junction proteins.Pretreatment with anti-RAGE antibody (100 mg/L),captopril or valsartan could attenuate the detrimental effect of AGEs.Conclusion The changes of permeability induced by AGEs in glomerular endothelial cells are partly mediated by RAS through RAGE.
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The mechanism of injury on the human glomerular endothelial cells (ciGENC) induced by preeclampsia serum was investigated.Concentration of maternal serum sFlt-1 protein was detected by ELISA.Fluorescently-labeled bovine serum albumin infiltrating through lower chamber of Transwell was measured by multifunction microplate reader.Morphologic change of ciGENC was observed under inverted phase contrast microscope.The concentration of sflt-1 in preeclampsia groups was significantly increased as compared with control group (P＜0.01).Permeability in preeclampsia groups was significantly increased as compared with control group (P＜0.01).By contrast with severe preeclampsia group,the permeability of ciGENC monolayer in mild preeclampsia group was decreased significantly (P＜0.05).Intervention of exogenous VEGF significantly decreased permeability of ciGENC in preeclampsia groups.It was concluded that sFlt-1 increased ciGENC permeability by damaging integrity of endothelial barrier function.
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Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.
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Animals , Rats , Angiotensin II/pharmacology , /biosynthesis , Endothelial Cells/metabolism , Kidney Glomerulus/cytology , NADPH Oxidases/antagonists & inhibitors , NF-kappa B/metabolism , Angiotensin II Type 1 Receptor Blockers/pharmacology , Blotting, Western , Benzimidazoles/pharmacology , Benzoates/pharmacology , /drug effects , Endothelial Cells/drug effects , Enzyme Inhibitors/pharmacology , Inflammation/metabolism , Onium Compounds/pharmacology , Oxidative Stress/physiology , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND: Glomerular endothelial cells should participate in the process of glomerular disease by expressions of HLA antigens and adhesion molecules. However, few have been known about the regulation of the expression of these molecules in human glomerular endothelial cells(HGEC). The aim of this study was to evaluate the expressions of VCAM-1, ICAM-1 and HLA-DR to see if there are any cytokine-dependent or time-dependent differences. METHODS: HGEC were isolated and cultured from the normal portion of the kidneys removed due to renal cell carcinoma, which was confirmed by factor VIII and fluorescent-labeled acetylated LDL. The effects of cytokine on the cell surface expression of VCAM-1, ICAM-1 and HLA-DR were measured by ELISA. RESULTS: ICAM-1 was increased by IL-1 beta, TNF-alpha and IFN-gamma. VCAM-1 was increased by IL-1 beta and TNF-alpha, not by IFN-gamma. IFN-gamma only increased expression of HLA-DR. Basal expression of ICAM-1 was higher than VCAM-1 and HLA-DR. The time course of expression was different according to adhesion molecule. CONCLUSIONS: These data showed that the expression of adhesion molecules and HLA-DR in HGEC were regulated differentially by inflammatory and immune-regulatory cytokines.
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Humans , Carcinoma, Renal Cell , Cytokines , Endothelial Cells , Enzyme-Linked Immunosorbent Assay , Factor VIII , HLA Antigens , HLA-DR Antigens , Intercellular Adhesion Molecule-1 , Interleukin-1beta , Kidney , Tumor Necrosis Factor-alpha , Vascular Cell Adhesion Molecule-1ABSTRACT
Cyclosporine(CsA) is a modulator of the immune system used therapeutically to prevent organ transplant rejection. However, it is nephrotoxic and causes thrombotic phenomena after renal and bone marrow transplantation. CsA nephrotoxicity in vivo is associated with elevated levels of von Willebrand factor(vWf), which is a multimeric plasm glycoprotein secreted by endothelial cells and platelets. CsA is not soluble in water and the intravenous form given to patients is dissolved in a vehicle called cremophor EL. The vehicle has been implicated in anaphylatic reactions and associated with the release of histamine in vivo. We hypothesized that CsA or cremophor might affect the release of vWf from human glomerular endothelial cells. vWf was measured in culture supernant using ELISA kit after speed vaccum for 3 hour. The expression of vWf in cultured human glomerular endothelial cells was relatively low compared to human plasm in vivo. CsA alone did not increase vWf release(100%, 99.9+/-0.7%, 99.1+/-2.9%, 106+/-21.5%, CsA 0, 0.1, 1, 10microM mean S.E., n=2), but cremophor EL increased vWf release (100%, 104.0+/-8.6%, 117.6+/-9.5%, 121.3+/-12.2%, mean+/-S.E., n=3). These results were same as the results of experiments under thrombin(1IU/ml) and histamine(10(-4)M). The increased expression of vWf in human glomerular endothelial cells in response to CsA seems to be related to cremophor, the solvent, rather than CsA itself in vitro culture experiments.
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Humans , Bone Marrow Transplantation , Cyclosporine , Endothelial Cells , Enzyme-Linked Immunosorbent Assay , Glycoproteins , Histamine , Immune System , Transplants , von Willebrand FactorABSTRACT
The administration of erythropoietin for renal failure patients is frequently associated with a mild-to-marked rise in arterial blood pressure. The erythropoietin-induced hypertension has been variably attributed to the rise in erythrocyte concentration and/or a direct or indirect pressor action of erythropoietin on vascular smooth muscle. Especially erythropoietin-induced hypertension may be mediated by increased production of the potent vasoactive peptide endothelin. This study was designed to determine the effect of erythropoietin to the production of endothelin in human glomerular endothelial cells and human umbilical vein endothelial cells. ELISA method was used to measure the endothelin- 1, after 4 X 10(4) endothelial cells were grown to confluency on 24-well plates, in which erythropoietin 25u/ml was incubated for 24 hours. The results showed that the endothelin production in human glomerular endothelial cells was insignificant (116.0+/-14.0%, P>0.05, mean+/-S.E., n=5, each n means the mean of duplicate experiments), but the endothelin production in human umbilical endothelial cells was increased after 25u/ml erythropoietin treatment(126.5+/-15.2%, P<0.05, mean+/-S.E., n=4). In glomerular endothelial cells, TGF-beta(0.5ng/ml) increased the production of endothelin(218.8+/-57.2%, P<0.01, mean+/-S.E., n=5), also it looks like that TNF-alpha and thrombin might increase the production of endothelin according to the concentration. In conclusion, the responsiveness of endothelial cells to erythropoietin may be different according to the cell type, and glomerular endothelial cells could increase the production of endothelin, if appropriate stimuli were given.
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Humans , Arterial Pressure , Endothelial Cells , Endothelins , Enzyme-Linked Immunosorbent Assay , Erythrocytes , Erythropoietin , Human Umbilical Vein Endothelial Cells , Hypertension , Muscle, Smooth, Vascular , Renal Insufficiency , Thrombin , Transforming Growth Factor beta , Tumor Necrosis Factor-alphaABSTRACT
Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been major difficulties in developing homogenous cultures of human glomerular endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogenous monolayers of human glomerular endothelial cells based on the method of Green DF et al published in 1992. Using the selective media and the sieving method, fibronectin was required as a surface matrix after adequate collagenase treatment, and endothelial cell growth factor and heparin was needed for the continuous growth of endothelial cells. The endothelial cell growth factor was isolated from the bovine hypothalamic extracts. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence and stained homogenously with von Willebrand factor(factor VIII). The cytokeratin and the actin were not stained. This study might be helpful for in vitro study to know the biological characteristics of human glomerular endothelial cells under the predetermined condition.