The impact of translationally controlled tumor protein on the podocyte hypertrophy under diabetic conditions

의학과/박사[한글]배경: 세포 비후 과정은 세포 주기 조절 단백 중 주로 cyclin-dependent kinase 억제제 (CKIs)의 발현 증가와 밀접하게 연관되어 있다. 최근의 연구에 의하면, CKIs 이외에도 mammalian target of rapamycin (mTOR)와 같은 mRNA 전사를 조절하는 세포 신호 전달계의 활성화도 세포 비후를 유발시키는 것으로 보고되고 있다. Translationally controlled tumor protein (TCTP)은 대부분의 진핵세포에 잘 보존된 상태 (conserve)로 존재하며, mRNA 전사 조절 과정을 통하여 세포 증식 및 비후 등 다양한 세포 내 현상과 관련이 있을 것으로 생각되어 왔다. 당뇨병성 신병증의 병리학적 특징 중 하나가 사구체 비후이기 때문에, TCTP와 당뇨병성 신병증의 발생 사이에 연관이 있을 가능성이 있으나, 이에 대한 연구는 전무한 실정이다. 이에 본 연구자는 고포도당으로 자극한 족세포와 실험적 당뇨 사구체에서 TCTP의 발현 변화를 관찰하였으며, 당뇨 조건 하에서 TCTP가 족세포의 비후에 미치는 영향을 규명하고자 하였다. 방법: 생체 내 실험으로는 불멸 생쥐 족세포를 정상 포도당군 (5.6 mM), 정상 포도당+만니톨군 (24.4 mM), 정상 포도당+안지오텐신 II 군 (10-6 M), 고포도당군 (30 mM), 그리고 고포도당+L-158,809 처치군 (10-7 M)으로 나누어 배양하였으며, TCTP 발현 억제를 위하여 TCTP shRNA를 포함한 lentivirus로 전처치한 실험도 시행하였다. 생체 내 실험으로는 32마리의 C57BL/6 생쥐를 사용하였으며, 16마리는 대조군, 그리고 16마리는 streptozotocin으로 당뇨를 유발시킨 당뇨군으로 나누었고, 각 군에서 8마리씩은 TCTP shRNA를 포함한 lentivirus로 처치하였다. 배양 족세포 및 분리한 사구체 내 TCTP mRNA의 발현은 real-time PCR으로, TCTP, phospho-4EBP1, 4EBP1, phospho-p70S6K, p70S6K, p27, p21, 그리고 β-actin의 단백 발현은 Western blot을 이용하여 분석하였다. 사구체 내 TCTP 단백의 발현 위치는 synaptopodin과 TCTP을 이용한 이중 면역형광염색으로 관찰하였다. 족세포 비후는 단백량/세포 수 및 유세포 분석으로, 그리고 사구체 비후는 morphometry를 이용하여 확인하였다. 결과: TCTP mRNA 및 단백의 발현은 정상 포도당군에 비하여 고포도당에서 의미있게 증가되었으며, 고포도당군에서의 TCTP 발현 증가는 L-158,809에 의하여 의의있게 억제되었다. 안지오텐신 II 역시 배양 족세포에서 TCTP의 발현을 유의하게 증가시켰다. 사구체 내 TCTP의 발현도 대조군에 비하여 당뇨군에서 의미있게 증가되었으며, 이중 면역형광염색상 당뇨 사구체 내 TCTP의 발현 증가는 주로 족세포의 발현 증가에 기인함을 확인하였다. 생체 내 및 생체 외 실험상 TCTP shRNA를 포함한 lentivirus로 처치한 경우 당뇨 조건 하에서 phospho-4EBP1, phospho-p70S6K, 그리고 p27의 단백 발현 증가뿐만 아니라 족세포 및 사구체 비후가 유의하게 억제되었다. 결론: 당뇨 조건 하에서 족세포 내 TCTP의 발현이 증가되었으며, TCTP 억제를 통하여 당뇨 조건 하에서 세포비후와 관련된 4EBP1과 p70S6K의 활성화 및 CKIs의 발현 증가와 함께 족세포 비후가 의미있게 억제되었다. 이상의 결과를 종합해 볼 때, TCTP는 mRNA의 전사 조절과 세포 주기의 진행 억제를 통하여 당뇨 조건 하에서의 족세포 비후에 관여할 것으로 생각된다. [영문]Background: Hypertrophic mechanism requires cell cycle arrest at the G1/S interphase, which is principally mediated by inhibitors of cyclin-dependent kinase (CDK), namely CDK-inhibitors (CKIs). In addition to CKIs, recent studies have shown that activation of mRNA translation regulating signaling pathways such as mammalian target of rapamycin (mTOR) is also implicated in cellular hypertrophy in various diseases. Translationally controlled tumor protein (TCTP) is highly conserved in eukaryotic cells and has been suggested to be involved in various intracellular processes including cell proliferation and growth by regulating mRNA translation pathway. Since glomerular cells hypertrophy is a characteristic finding in diabetic nephropathy, there is a possibility that TCTP may play an important role in the pathogenesis of diabetic nephropathy. However, the functional role of TCTP on cellular hypertrophy under diabetic conditions has never been explored. In this study, I examined not only the changes in TCTP expression in high glucose-stimulated podocytes and in experimental diabetic glomeruli but also the impact of TCTP on podocyte hypertrophy under diabetic conditions.Methods: In vitro, immortalized mouse podocytes ether with or without transfection of TCTP shRNA expressing lentivirus were serum restricted for 24 hr, after which the medium was changed to RPMI medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), NG+10-6 M angiotensin II (NG+ANG II), 30 mM glucose (HG), or HG+10-7 M L-158,809 (HG+ARB). In vivo, 32 C57BL/6 mice were injected either with diluent (n=16, C) or streptozotocin intraperitoneally for 5 consecutive days (n=16, DM). Eight mice from each group were treated with lentivirus containing TCTP shRNA (LV-shTCTP). Real-time PCR for TCTP mRNA expression and Western blotting for TCTP, phospho-4EBP1, 4EBP1, phospho-p70S6K, p70S6K, p27, p21 or β-actin protein expression were performed with cell lysates and sieved glomeruli. Double immunofluorescence (IF) staining with synaptopodin and TCTP was also performed with renal tissue. Podocyte hypertrophy was assessed by measurement of cellular protein/cell counts and by flow cytometry, and glomerular volume by morphometry.Results: Compared to NG cells, TCTP mRNA and protein expression were significantly increased in podocytes exposed to HG for 48 hrs, and this increase in TCTP expression was abrogated by ARB treatment. Similarly, ANG II induced TCTP mRNA and protein expression in cultured podocytes. Glomerular TCTP expression was also significantly higher in DM compared with C mice. Double IF staining for TCTP and synaptopodin revealed that podocytes were the main cells responsible for the increase in TCTP protein expression under diabetic conditions. TCTP inhibition using LV-shTCTP ameliorated the increase in phospho-4EBP1, phospho-p70S6K, and p27 protein expression in high glucose-stimulated podocytes and in diabetic glomeruli along with reduced podocyte and glomerular size.Conclusion: I demonstrate for the first time that TCTP expression is increased in podocytes under diabetic conditions and that inhibition of TCTP attenuates the activation of mTOR target molecules, 4EBP1 and p70S6K, and CKIs expression, along with reduced podocyte and glomerular size. These findings suggest that TCTP may play an important role in the process of podocyte hypertrophy under diabetic conditions via regulating mRNA translation and inducing cell cycle arrest at the G1/S interphase.ope

Effects of Mosapride on Upper Gastrointestinal Symptoms and Gastric Emptying in Patients with Functional Dyspepsia

Background/Aims: Mosapride is a new, prokinetic, 5-HT4 agonistic agent free of dopamine D2 receptor antagonist properties. The aim of this study was to evaluate the effects of mosapride on upper GI symptoms and gastric emptying in patients with functional dyspepsia. Methods: Eighty-one patients were randomly allocated to treatment with mosapride (5 mg t.i.d) (n=42) or domperidone (10 mg t.i.d) (n=39) in a single-blind, prospective, multicenter study. The changes in gastric emptying and symptom severity score after 2-week treatment were evaluated for the treatment efficacy. Results: The proportion of patients with symptom improvement more than 50% after medications was 84.6% in the mosapride-treated group and 80.6% in the domperidone-treated group, and this difference was not significant ( p=0.6426). In both groups, gastric emptying measured at 120 minutes after test meal was significantly accelerated after 2-week treatment compared with that of baseline. However, there was no significant difference between te two groups after 2-week treatment. Mosapride was well tolerated, and no serious adverse events were observed. Conclusions: Mosapride is a safe and useful drug for the treatment of patients with functional dyspepsia.ope

Interobserver Variation in the Endoscopic Diagnosis of Gastroesophageal Reflux Disease

Background/Aims: A diagnosis of gastroesophageal reflux disease (GERD) is based on the typical symptoms, such as acid regurgitation and heartburn. However, there is a very high inter-observer variation in the evaluation of GERD patients. Methods: The endoscopic images of forty-two cases with reflux symptoms (2 still images and 15-second video images per case) were analyzed by 18 experienced endoscopists and 22 trainees. The findings were classified into the following: (1) 6 groups (modified LA classification: 4 LA groups, minimal, and normal), (2) erosinve and non-erosive, and (3) confluent erosive and others. The level of inter-observer variation is expressed as a kappa value. Results: The level of inter-observer agreement of the 18 experienced endoscopists for classifying the patients into 6 groups was fairly low (kappa=0.364). However, when the findings were classified into the 2 groups suggested in the Genval workshop (NERD, A, or B versus C or D), the level of inter- observer agreement increased substantially (kappa=0.710). The kappa value of the 22 trainees for classifying the patients into 6 groups was 0.402. Conclusions: Modified LA classification with minimal change lesions showed a fairly low level of agreement. The problem caused by inter-observer variations decreased significantly when the findings were classified into two groups.ope

Genetic Association Analysis of Lipid Profiles Using Linear Mixed Model

Background and Objectives: Analyzing the association between multiple SNPs and the disease outcomes will provide new insight into the disease’s etiology. However, this presents an analytic difficulty due to the large number of SNPs and the complex relationships among them. We proposed using the mixed model approach to identify the significant multi-locus genotypes and the high-order gene-to-gene interactions. Subjects and Methods: We described the mixed effects model and applied this approach to real world data. For the purpose of these analyses, we examine the association of four types of SNPs (AGT5, APOB, CETP3 and ACE6) with the lipid profiles and the measures related with cardiovascular disease. We used data from 672 healthy individuals (283 males and 389 females) who were without cardiovascular diseases. Results: The results of our analysis suggested that there were significant random genotype patterns and genotype groups according to the gender effect on the lipid profiles. In other words, there was significant variability across the genotype groups because of the effect of gender on the lipid profiles. Conclusion: The mixed model approach provided a flexible statistical framework for controlling potential confounding variables and for identifying a significant genetic contributions that may come about through the effects of multi-locus genotypes or through an interaction between the genotype and environmental variables (e.g. gender) with the variations in quantitative traits (e.g. lipid profiles). There were significant genetic contributions to the variability in the lipid profiles, and these were explained by the 4 SNPs described in our real data.ope

Clinical Research Design and Biostatistical Methods

Purpose: To proceed effectively with clinical research requires an understanding of the fundamental principles of study design and biostatistical methods. In this article, we identified and summarized basic clinical research designs and some of the key biostatistical methods that have been commonly used in clinical research. Materials and Methods: In an observational study, cross-sectional, case- control and Cohort designs were illustrated and compared. In a clinical trial study, parallel group design and cross-over designs were described according to their characteristics. Also, the biostatistical methods for their usages classified and summarized. Results: Understanding and evaluating research design are part of the process researchers must use to determine both the quality and usefulness of their research. Adequate applications to biostatistical methods are need; i.e., descriptive statistics, Student´s t-test, ANOVA, nonparametrics, categorical data analysis, correlation and regression, and survival analysis. Conclusions: Research findings are used by clinical researcher to guide their practice and reduce their uncertainty in clinical decision making. However, to understand how to interpret research results, it is important to be able to understand basic statistical concepts and types of study design. Clinicians should also appropriately choose the biostatistical methods to suit their purposes.ope

Prevalence of Metabolic Syndrome in obese Children

Purpose : Obesity has been known to be a cause of insulin resistance and dyslipidemia, and along with coronary artery disease and diabetes, is associated with metabolic syndrome(MS). This study aimed to ascertain the cause and prevalence of MS in obese children and adolescents. Methods : Two hundred and seventy-seven school children, who showed more than a 95th percentile of body mass index(BMI) for age and sex, underwent oral glucose tolerance tests; fasting plasma lipid profiles, leptin, and CRP were measured; and ultrasonography was done. Results : Out of 277 obese children, the prevalence of MS was 37.5%, with 38.7% occurring in males and 35.2% in females. The prevalence was 20.8% in primary school children and 50.3% in middle school children. MS was present in 25.2% of mildly obese children, 43.9% of moderate and 71.4% of highly obese children, showing increased occurrence among the severer degrees of obese groups. Increased prevalence was observed in males with high blood pressure, and females with high triglyceride levels. The ratio of children satisfying more than one, more than two, more than three, more than four and all of the five criteria for diagnosis of MS were 90.6%, 63.5%, 37.5%, 8.3%, and 0.4%, respectively. Aside from diagnostic criteria for MS, a statistically significant difference was present between obese patients with or without the syndrome in such items as weight, BMI, degree of obesity, visceral fat thickness, ratio of body fat, leptin, and adiponectin, fasting and 2 hour postprandial insulin concentration. Conclusion : The prevalence of MS is currently on the rise among children, due to the rapidly increasing rate of obesity, westernized diet, higher calorie intake and reduced exercise. The prevalence of MS in obese children was 37.5% - higher than US results - after implementation of modified pediatric criteria established by ATP III Asia-Pacific standard in 2003. It would therefore be necessary to establish an acceptable universal standard for the diagnosis of MS, and extend the study to the general pediatric population in order to acquire more accurate data on the prevalence of the disease.ope

Analysis of the Relative Effects of SNPs within a Gene to Serum Lipid ProfilesUsing Stepwise Linear Regression

BACKGROUND AND OBJECTIVES: It is very important to distinguish between the primary and secondary genetic effects at different sites within a small genetic region. Therefore, we evaluated the relative effects of single nucleotide polymorphisms (SNPs) within a gene on the serum lipid profiles by using individual data. SUBJECTS AND METHODS: To evaluate the contributions of SNPs in a region to the serum lipid profiles (total cholesterol, triglyceride, low density lipoprotein, high density lipoprotein), we used data that consisted of 808 individuals (327 males and 481 females) who did not have cardiovascular disease. In this study, we used a stepwise regression procedure to analyze the relative effects of four single nucleotide polymorphisms (ACE6, ACE7, ACE8, ACE10) in a gene region on the development of the serum lipid profiles in each gender group. RESULTS: In the males, there were epistatic interaction effects between two loci (ACE6xACE7, ACE6xACE8, ACE6xACE10, ACE8xACE10 and ACE7xACE8) and among three loci (ACE6xACE7xACE8, ACE6xACE7xACE10 and ACE6xACE8xACE10). Also, there are interaction effects between two loci (ACE6xACE7, ACE6xACE8, ACE6xACE10, ACE7xACE10 and ACE8xACE10) and among three loci (ACE6xACE7xACE8, ACE6xACE7xACE10, ACE6xACE8xACE10 and ACE7xACE8xACE10) in the females. CONCLUSION: The results suggested that each of these loci is important in causing a relative change of the serum lipid profiles, even with simultaneously accounting for the effects at the other loci. In the results of the analysis, there existed the effects of individual loci and significant interaction between the loci on the serum lipid profiles in each gender group. It was confirmed that this stepwise regression method can be suitable for evaluating the relative effects of SNPs and it is easily performed.ope

Affecting Factors of Insulin Resistance in Obese Children and Adolescents

Purpose: Insulin resistance is the most important risk factor linked to the development of impaired glucose tolerance(IGT), diabetes mellitus and cardiovascular diseases in childhood and adolescent obesity, The purpose of this study was to see whether insulin resistance of obese adolescent is higher than that of obese children. and to analyze gender difference and affecting factors of insulin resistance. Methods: Of the 9,837 school children from 5 to 16 tears old, 92 obese children and 187 adolescent, underwent a two-hour oral glucose tolerance test and plasma glucose, insulin, lipid profiles, leptin and high sensitive C-reactive protein(hs-CRP) were measure. Results: Plasma insulin levels of female were higher compared to those of males during oral glucose tolerance test(P<0.05). Four(4.3%) in obese children and twenty five(13.3%) in obese adolescents met the criteria of IGT. Female, leptin, adiponectin and triglyceride concentrations were strongly correlated with homeostatic model assessment insulin-resistance(HOMA-IR) by multiple linear regression analysis(P<0.05). Conclusion: Obese adolescents might have higher insulin concentrations compared to obese children and obese girls higher insulin concentrations than obese boys. Obese boys and children with impaired glucose tolerance have higher insulin concentrations than those with normal glucose tolerance. HOMA-IR was significantly correlated with female, plasma leptin, adiponectin and triglyceride concentrations.ope

Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions

Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10(-7) M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.ope

Comparison of tramadol/acetaminophen and odeine/acetaminophen/ibuprofen in onset of analgesia and analgesic efficacy for postoperative acute pain

Background: Some clinical trials have reported that a new analgesic combination of tramadol and acetaminophen provides good efficacy in various pain models. For the more clinical uses of this agent, comparisons about the onset of analgesia and analgesic efficacy in the acute state of pain with the other drugs known as strong analgesics were needed. Purpose: The goal of this study was to compare the times to onset of analgesia and the other analgesic efficacy of 75 mg tramadol/650 mg acetaminophen and 20 mg codeine/500 mg acetaminophen/400 mg ibuprofen in the treatment of acute pain after oral surgery. Patients and Methods: Using a randomized, single-dose, parallel-group, single-center, and active-controlled test design, this clinical study compared the times to onset of analgesia using a two-stopwatch technique and the other analgesic efficacy of the single-dose tramadol/ acetaminophen and odeine/acetaminophen/ibuprofen. These were assessed in 128 healthy subjects with pain from oral surgical procedures involving extraction of one or more impacted third molars requiring bone removal. From the time of pain development, the times to onset of perceptible and meaningful pain relief, pain intensity, pain relief, an overall assessment, and adverse events of the study medications were recorded for 6 hours. Results: The demographic distribution and baseline pain data in the two groups were statistically similar. The median times to onset of perceptible pain relief were 21.0 and 24.4 minutes in the tramadol/acetaminophen and codeine/acetaminophen/ibuprofen groups respectively and those to onset of meaningful pain relief were 56.4 and 57.3 minutes, which were statistically similar. The other efficacy variables such as mean total pain relief (TOTPAR) and the sum of pain intensity differences (SPID) were also similar in the early period after pain development and drug dosing. The safety of tramadol/acetaminophen was well tolerated and very comparable to that of codeine/acetaminophen/ibuprofen. Conclusions: In this acute dental pain model, the onset of analgesia and analgesic efficacy of tramadol/acetaminophen was comparable to that of codeine/acetaminophen/ibuprofen. These results showed that tramadol/acetaminophen was recommendable for fast and effective treatment in the management of postoperative acute pain.ope