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Objective:To investigate the protective effect and potential mechanisms of microRNA-26a-5p (miR-26a-5p) on podocyte injury in diabetic kidney disease (DKD).Methods:(1) In vivo experiment: Four-week-old db/db mice were divided into db/db group, db/db+agomir-NC group and db/db+miR-26a-5p agomir group according to random number table method, with 10 mice in each group, and 10 db/m mice of the same week-old were set as normal control group. At the age of 10 weeks, pathological changes were observed through light and electron microscopy. Kidney weight/body weight (KW/BW), urinary albumin to creatinine ratio (ACR), fasting blood glucose (FBG) and other biochemical indicators were also detected. The position and expression of miR-26a-5p in kidney tissue were determined through fluorescence in situ hybridization and quantitative real-time PCR, while the expressions of transient receptor potential cation channel-6 (TRPC6) and Nephrin in kidney tissue were determined by Western blotting and immunohistochemistry. (2) In vitro experiment: The immortalized mouse podocytes (MPC5) were divided into 5 groups: normal glucose group, high mannitol group, high glucose group, high glucose+miR-26a-5p mimic group, and high glucose+mimic-NC group. The expressions of miR-26a-5p, TRPC6 and Nephrin were detected. Luciferase reporter assay was conducted to research the relationship of miR-26a-5p and TRPC6. Results:(1) In vivo experiment: Compared with db/m group, db/db mice exhibited lower KW/BW and disrupted conditions of ACR, FBG, total cholesterol, triglycerides and low density lipoprotein cholesterol (all P<0.01). Increased glomeruli volume, more extracellular matrix deposition, thicker basement membrane and more foot process fusion were observed by light and electron microscope. Increased expression of TRPC6 protein as well as decreased expression of Nephrin protein and miR-26a-5p were detected in kidney tissues of db/db mice ( P<0.05). Compared with db/db+agomir-NC group, db/db mice transfected by miR-26a-5p agomir exhibited less albuminuria, with less protein expression of TRPC6 and more Nephrin in kidney tissue (all P<0.05). (2) In vitro experiment: Compared with normal glucose group, high glucose-treated podocytes exhibited increased expression of TRPC6 ( P<0.05), as well as decreased expression of Nephrin ( P<0.05) and miR-26a-5p ( P<0.01). Compared with high glucose+mimic-NC group, lower expression of TRPC6 and higher expression of Nephrin were detected in podocytes transfected by miR-26a-5p mimic (both P<0.05). Luciferase reporter assay confirmed that miR-26a-5p could regulate the expression of TRPC6 precisely. Conclusions:The expression of miR-26a-5p in podocytes is down-regulated in the context of high glucose and miR-26a-5p protects podocytes from injury via inhibiting the expression of TRPC6 in DKD.
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ABSTRACT Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.
RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).
Subject(s)
Animals , Male , Acetylcholine/pharmacology , Calcium/pharmacology , Thapsigargin/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium-Dependent Relaxing Factors/metabolism , Nitric Oxide/metabolism , Aorta, Thoracic/drug effects , Time Factors , Vasodilator Agents/pharmacology , Rats, Wistar , TRPC Cation Channels/metabolism , TRPV Cation Channels/drug effects , TRPV Cation Channels/metabolism , Calcium Release Activated Calcium Channels/metabolismABSTRACT
Objective To observe the expressions and distribution of transient receptor potential cation channel 6 (TRPC6) and integrin-linked kinase (ILK) in the glomeruli of renal biopsytissue of patients with proteinuric kidney diseases,and to investigate the effect of TRPC6 over-expression on ILK in vitro.Methods The archival histological specimens of patients admitted to Tangdu hospital from 2012 to 2013,with 24-hour urinary protein over 1 g,were collected.The expressions and distribution of TRPC6 and ILK in the glomeruli of renal biopsy tissue were observed by immunohistochemistry.MPC5 podocytes were cultured in vitro and they were stimulated with 10-7 mol/L ADR for 12,24 and 36 h.The pcDNA3.1(+)-TRPC6 plasmid and pcDNA3.1(+) were transfected into MPC5 podocytes by liposome 2000 reagent to establish the TRPC6 overexpression group and the negative control group respectively.Western blotting was used to detect the expressions of TRPC6 and ILK protein.Results There were 14 cases of membranous nephropathy,13 cases of focal segmental glomerulosclerosis (FSGS),15 cases of membranoproliferative glomerulonephritis,12 cases of mesangial proliferative glomerulonephritis,10 cases of hyperplastic sclerosis nephritis,15 cases of IgA nephropathy,13 cases of purpura nephritis,15 cases of lupus nephritis,13 cases of hypertensive renal injury,14 cases of diabetic nephropathy and 9 cases of normal renal tissue included.In glomerulus,TRPC6 was expressed mainly in podocytes,and the expressions of TRPC6 in these renal tissues were higher than that in normal renal tissues (all P ＜ 0.05),except for hypertensive nephropathy.ILK was expressed in podocytes and the mesangial areas.The expressions of ILK in FSGS,lupus nephritis and diabetic nephropathy were higher than that in normal kidney tissue (all P ＜ 0.05),while the other renal tissues was high but showed no statistical difference with normal kidney tissue (all P ＞ 0.05).The expressions of TRPC6 and ILK were positively correlated in renal tissues of FSGS and diabetic nephropathy (r=0.906,P ＜ 0.001;r=0.783,P=0.001 respectively).The expressions of TRPC6 and ILK protein in 24 and 36 h stimulating with ADR were significantly higher than that in the control group (all P ＜ 0.05).The expression of ILK in the TRPC6 overexpression group was significantly higher than that in the normal control group (P ＜ 0.05).Conclusions The expressions of TRPC6 and ILK increase in the glomeruli of patients with kidney diseases with proteinuria being the main manifestation,especially in FSGS and diabetic nephropathy.The up-regulation of TRPC6 can increase the expression of ILK protein,which may be involved in podocyte injury.
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Transient receptor potential canonical 6 (TRPC6) is a non-selective cation channel, which is involved in various physiological processes, including neuronal axonal growth cone guidance, promotion of dendritic growth and excitatory synaptic formation.Recent studies have shown that TRPC6 participates in many pathological process of central nervous system (CNS) diseases. Therefore, this review focuses on physiological roles of TRPC6,and its pathological roles in CNS,i.e.stroke,Alzheimer disease and epilepsy. We go on to discuss the research development of TRPC6 as drug targets.Finally,we overview and discuss the problems to be solved in further research, and the prospect of TRPC6 as a promising target for drug development.
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BACKGROUND:Transient receptor potential channel C6 (TRPC6) is a new and important slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Glomerular TRPC6 expression is closely associated with proteinuria in diabetic kidney disease. OBJECTIVE: To investigate the expression of canonical TRPC6 in mouse podocytes induced by high glucose, and to explore the possible mechanism of diabetic kidney disease. METHODS:Mouse podocyte cels were cultured and divided into normal glucose group (5.6 mmol/L D-glucose), normal control group (5.6 mmol/L D-glucose+25 mmol/L mannitol) and experimental groups which were in the environment of high glucose (30 mmol/L). The experimental groups included high glucose group, valsartan treatment groups (10-5 mol/L) and U73122 control group (10μmol/L U73122). After 48 hours, the expressions of mRNA and proteins of TRPC6, nephrin and angiotensin II (AngII) were detected respectively by real-time quantitative PCR and western blot analysis. RESULTS AND CONCLUSION:Compared with the normal control group, the expressions of mRNA and proteins of TRPC6 and angiotensin II were markedly elevated in the high glucose group (P 0.05). Angiotensin II-TRPC6 signaling pathway may mediate high glucose-induced podocyte injury, meanwhile it provides a new theoretical basis for the treatment of diabetic kidney disease, by which the angiotensin receptor blockers can protect podocytes in diabetic kidney disease.