Dissertation > Excellent graduate degree dissertation topics show

RAS dysfunction induced by high glucose in renal tubular epithelial cells and its mechanism of action

Author: ZhouLi
Tutor: LuLiMin
School: Fudan University
Course: Physiology
Keywords: Diabetic nephropathy Epithelial cells Renin - angiotensin system MAPK TGF-β1 miR-181a
CLC: R692.9
Type: Master's thesis
Year: 2011
Downloads: 187
Quote: 0
Read: Download Dissertation

Abstract

Diabetic nephropathy (Diabetic nephropathy, DN) is one of the most serious complications of diabetes, but also the clinical ESRD (end-stage renal disease, ESRD) incidence main reason, research shows there are 40% Ⅰ diabetes and 15% Ⅱ diabetic patients may be complicated by kidney damage, and ultimately lead to renal failure. DN early pathological changes characteristic of glomerular hypertrophy, extracellular matrix (extracellular matrix, ECM) excessive deposition of basement membrane thickening and late development of glomerulosclerosis and tubulointerstitial diffuse fibrosis. Complex etiology of diabetic nephropathy, since its occurrence cellular and molecular mechanisms are not yet fully elucidated, but with further research, scholars generally believe that renal interstitial fibrosis, including diabetic nephropathy, including a variety of chronic kidney disease progress to end ESRD common pathological basis of tubulointerstitial fibrosis mechanisms for prevention and treatment of DN, delaying chronic renal damage provide new ideas and methods. Tubulointerstitial fibrosis mainly including kidney fibroblast activation and transdifferentiation of renal tubular epithelial cells (epithelial mesenchymal transition, EMT), and finally to the kidney increased synthesis of extracellular matrix degradation reduced, resulting in excessive deposition of intercellular matrix . Although mature tubular epithelial cells are terminally differentiated cells, but more and more studies show that in a variety of pathological conditions, renal tubular epithelial cells like fibroblasts to muscle transdifferentiation capacity; while after transdifferentiation muscle-like fibroblasts are able to interstitial infiltration area, migration, access to cell proliferation, extracellular matrix synthesis and secretion of enhanced participation in the process of renal interstitial fibrosis. EMT is considered to be the development of renal interstitial fibrosis is one of the initiating factor, revealing the mechanism of EMT is the current hot new kidney research, kidney damage caused by a variety of factors, including diabetes, are confirmed to be caused by EMT. Renin - angiotensin system (RAS) in the body cardiovascular system, water and salt balance and regulation of cell function plays an important role. The basis of a large number of experimental and clinical studies have confirmed that high glucose condition excessive activation of RAS in the pathogenesis of diabetic nephropathy play a very important role. In recent years, with several new members have been found RAS, RAS has been a major update in the concept, in the new RAS, in addition to the traditional ACE-Ang Ⅱ-AT1 pathway, there also exists with parallel ACE2-Ang- (1-7)-Mas pathway. In the cardiovascular study found, RAS two pathways are functionally antagonistic, Ang-(1-7) and Ang Ⅱ phase produces antagonistic biological effects. In this issue, we have high glucose changes under local kidney RAS, RAS changing relationship with EMT and high sugar causes changes in the mechanism of RAS and other issues have been studied, trying to elucidate the mechanism of diabetes EMT. Part I: high glucose condition tubular epithelial cells RAS ACE-Ang Ⅱ-AT1 pathway changes and EMT's role in research: experimental in vitro cultured rat renal tubular epithelial cells (NRK-52E) on using observed by immunofluorescence cytochemistry transdifferentiation of renal tubular epithelial marker protein by Western blot analysis of muscle-like cells quantitatively marker protein (α-SMA) and epithelial cell marker protein (E-cadherin) levels; method using Real-timePCR detecting AGT, ACE, AT1 mRNA levels by enzyme-linked immunosorbent assay (ELISA) to detect cell culture fluid concentrations of Ang Ⅱ. Experimental results show that: renal tubular epithelial cells to give 30 mmol / L D-glucose stimulation 48-72 hrs, cell morphology changed significantly by changing oval cobblestone growth spindle; Immunofluorescence staining showed that muscle-like cell markers protein α-SMA expression was significantly increased, while the epithelial cell marker protein E-cadherin expression was decreased; Western blot test results also confirmed that the tubular epithelial cells in high glucose increased expression of α-SMA, E-cadherin protein expression was decreased; addition , Real-time PCR results showed that cells were cultured under high glucose 24 and 48 hrs later, cells AG1, ACE, AT1 mRNA levels were significantly increased, culture medium Ang Ⅱ levels were significantly increased. These results suggest that: high glucose can cause renal tubular epithelial cells transdifferentiation, while the local RAS system cell dysfunction, RAS in the ACE-Ang Ⅱ-AT1 axis enhancements. On this basis, we further pathological state ACE-Ang Ⅱ-AT1 axis is involved in changing the transdifferentiation of renal tubular epithelial cells were studied. The results showed that: a high glucose condition ACE-Ang Ⅱ-AT1 axis changes in renal tubular epithelial cells experimental 10-5-10-7 mol / L concentration ATl receptor blocker Losartan pretreatment 30min, then give cells stimulated by high glucose 48 hrs, with immunofluorescence staining and Western Blot observed transdifferentiation marker protein E-cadherin and Vimentin expression. Experimental results show that high glucose 48 hrs can cause renal tubular epithelial cells in epithelial cell marker protein E-cadherin expression was decreased, while the muscle-like fibroblast marker protein Vimentin expression; Losartan can reverse the high glucose concentration-dependent manner caused transdifferentiation marker protein changes; given alone 10-5mol / L of Losartan on E-cadherin and Vimentin protein expression was not significantly affected. Second, the observation of Losartan high glucose induced inhibition of renal tubular epithelial cells increased synthesis of TGF-β1 cells pretreated with 10-5mol / L concentrations of Losartan, and then cultured in high glucose 48 hrs, the results show (30mM) glucose to make tubular epithelial cells TGF -β1 synthesis, Losartan can inhibit these effects, but can not completely block the high glucose induced TGF-β1 synthesis. Third, the observation Losartan inhibited high glucose induced renal tubular epithelial cells increased synthesis of extracellular matrix proteins cells were treated with 10-5-10-7mol / L concentrations of Losartan pretreatment 30 min, and then cultured in high glucose 48 hrs, observed by Western Blot method Fibronectin extracellular matrix protein and MMP-9 protein levels. Experimental results show that high glucose 48 hrs, the cells synthesize Fibronectin and MMP-9 increased, Losartan dose-dependently inhibited high glucose induced Fibronectin and MMP-9 synthesis, but even high concentrations of Losartan (10-5 mol / L) can not completely block the synthesis Fibronectin; cultured in normal cells, given alone 10-5mol / L concentrations of Losartan, the synthesis of cell Fibronectin and MMP-9 was not significantly affected. Part II: Because AT1 receptor blocker Losartan only partially reversed by high glucose induced EMT and cell transdifferentiation functional changes after this issue, we further the RAS in the newly discovered ACE2-Ang (1-7 )-Mas axis changes and is also involved in the high glucose induced EMT studied. The results showed that: a high glucose stimulation resulted in ACE2-Ang (1-7)-Mas pathway significantly reduced. Cells were treated with high glucose 24 or 48 hrs, Real-time PCR results showed that, ACE2 mRNA levels were significantly lower than the control group; Mas receptor mRNA levels in the high 24 hrs after glucose stimulation has decreased, but no statistically significant , while in high glucose 48 hrs later, there was a marked decrease. Two, ACE2-Ang (1-7)-Mas metabolic pathways involved in high glucose-induced changes in renal tubular epithelial cells induced transdifferentiation process. High glucose-induced renal tubular epithelial cells decreased expression of E-cadherin protein and Vimentin protein expression, given different concentrations of Ang-(1-7) (10 \stimulation of EMT marker proteins changed. Meanwhile, immunofluorescence experiments confirmed 10-5M concentrations of Ang-(1-7) significantly inhibited high glucose caused by increased expression of a-SMA. three, Ang-(1 - 7) can reverse high glucose stimulation pathway MAPKs ERK1 / 2 and p38 phosphorylation levels, but on high glucose induced JNK phosphorylation levels had no significant effect. cells cultured in high glucose at different times ( 15,30,60 min) after, MAPK family (ERK, p38, JJNK) phosphorylation levels were significantly increased in high glucose after 60min, the cells in the three MAPK pathway is still in high phosphorylation state; give Ang-(1 -7) (10-5 M) inhibited high glucose induced ERK1 / 2 and p38 phosphorylation levels, but on high glucose induced JNK phosphorylation levels had no significant effect; during normal cultured cells alone give Ang-(1-7) did not significantly alter the phosphorylation of MAPK pathway three level four, Ang-(1-7) inhibited high glucose induced renal tubular epithelial cells TGF-β1 synthesis and secretion increases. give 10 -5 M concentrations of Ang-(1-7) inhibits high glucose induced renal tubular epithelial cells TGF-β1 mRNA levels increased, and the cell culture medium elevated levels of TGF-β1, the inhibitory effect can be Ang-(1-7) receptor Mas receptor specific antagonist blocked by A-779. be administered alone Ang-(1-7) or the A-779 cell synthesis and secretion of TGF-β1 had no significant role. TGF-β1 generation and high glucose induced activation of MAPK pathway, experiment, we further observe three MAPK pathway in high glucose induced TGF-β1 synthesis in the role of experimental results show that given alone PD98059 (ERK1 / 2 inhibitor), SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor) can partially inhibit the high glucose-induced TGF-β1 production, three kinds of blocking agents used in combination can be completely reversed high glucose induced increased production of TGF-β1 V. , Ang-(1-7) inhibited high glucose induced renal tubular epithelial cells increased synthesis of ECM Western blot results showed that high glucose for 48 hours, Fibronectin extracellular matrix synthesis increased significantly, giving 10-5M concentrations of Ang-( 1-7) partially inhibited high glucose-induced cell Fibronectin increased protein synthesis, and 10. M concentrations of Ang-(1-7) does not affect the synthesis of cell Fibronectin Part III: In vivo RAS dysfunction in diabetic nephropathy occurrence has received extensive attention, our study confirms the above experiment, the cells under high glucose conditions local RAS abnormalities, including ACE-Ang Ⅱ-AT1 pathway is activated, ACE2-Ang (1-7)-Mas pathway inhibition were associated with the occurrence of EMT However, high glucose causes dysfunction of the RAS specific mechanism is unclear. nicroRNAs involved in regulation of gene expression is important in vivo transcriptional regulation mechanisms we passed on the high glucose before and after the screening of differentially expressed microRNAs to study high Sugar causes tubule epithelial cells express an important member of the RAS change the mechanism. a high glucose-stimulated cells differentially expressed microRNAs screening: The experiment is divided into three groups: normal glucose group, mannitol control group and high glucose group, kidney tubule epithelial cells (NRK-52E) cultured 24-48 hrs, extraction cell RNA, followed by microarray analysis showed that microRNA-181a under high glucose conditions significantly upregulated by bioinformatic analysis, display Mas receptors may miR-181a is regulated target genes in vivo one two, with the Real-time PCR method validation of differentially expressed microRNA-181a: Cells cultured in high glucose 24 hrs, Real-time PCR results showed that, miR-181a levels compared with the normal group (NG) significantly increased, the results were consistent with the chip screening, while in STZ-induced diabetes three weeks of renal tissue, also observed that miR-181a levels were significantly increased compared with normal rats. three, microRNA-181a on the Mas receptor expression and regulation of authentication: Design miR-181a knockdown probe by liposome-mediated transfection of cultured normal tubular epithelial cells showed that transfection of miR-181a knockdown probe after 24 hrs , cells, miR-181a levels were significantly reduced, while the random sequence scramble transfected probe did not affect cell 1miR-181a levels, suggesting that miR-181a knockdown probes that specifically interfere with miR-181a expression further observation transfected with miR- 181a knockdown probe Mas receptor expression, the experimental results show that the normal glucose cultured cells transfected with miR-181a knockdown probe after, Mas receptor mRNA levels significantly higher than the control group; high glucose condition , cell Mas receptor mRNA levels decreased, while transfected miR-181a knockdown probe after, Mas receptor mRNA levels were significantly increased, confirming that miR-181a Mas receptor in vivo regulation of target genes. summary, The results of this research confirmed: 1, high glucose can induce in vitro transdifferentiation of renal tubular epithelial cells, the cells secrete ECM transdifferentiation ability to enhance the role of the kidneys in the body can promote fibrosis. partial ACE-Ang Ⅱ-AT1 pathway involved in the process, Ang Ⅱ ATl by acting on receptors, stimulating synthesis and secretion of TGF-β1, causing EMT occurs, however, ATl receptor blocker Losartan only partially reversed by high glucose induced EMT. 2, high sugar state, with ACE-Ang Ⅱ-AT1 pathway function photogenic antagonism ACE2-Ang-(1-7)-Mas axis is suppressed metabolic, ACE2-Ang-(1-7)-Mas pathway function is reduced also involved high glucose induced EMT, giving Mas receptor agonist Ang-(1-7) to some extent inhibited high glucose induced EMT, Ang-(1-7) Mas receptor activation can inhibit high glucose induced ERK and p38 phosphorylation. 3, in the early onset of STZ-induced diabetic animal models and high glucose stimulated tubule epithelial cells were observed in miR-181a levels were significantly elevated in this topic, use microRNAs interference confirmed, Mas receptor Is miR-181a target genes in vivo, one of high glucose levels under miR-181a and ACE2-Ang-(1-7)-Mas pathway function was reduced about.

Related Dissertations

CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Other diseases
© 2012 www.DissertationTopic.Net Mobile