Differential Gene Expression According to the Size ofGlomeruli in Experimental Diabetic Nephropathy

Purpose: Although a few gene-profiling studies with whole renal tissue have been described in experimental diabetic nephropathy, there is only one microarray study using diabetic glomeruli. Furthermore, hypertrophic glomeruli have not been explored. The purpose of this study is to elucidate gene expression profiles of hypertrophic glomeruli in early diabetic nephropathy. Methods: Forty-male Sprague-Dawley rats were injected with diluent (N=20) or streptozotocin intraperitoneally (DM, N=20) and were sacrificed at 6- and 12-week. Glomeruli were isolated by sieving technique. Glomeruli from 125 and 75 m sieves were classified into large (hypertrophic, DM-LG) and small glomeruli (DM-SG), respectively. After RNA extraction, hybridization was performed on the Rat cDNA 5K chip in triplicate, and slides were analyzed. The significant genes were selected using significant analysis of microarray. Results: At 6-week, hierarchical clustering revealed that gene expression profiles of DM-LG were different from those of DM-SG, whereas DM-SG and C glomeruli showed similar gene expression pattern. In contrast, gene expression profiles at 12-week were similar between DM-LG and DM-SG, whereas C glomeruli showed different gene expression pattern from DM glomeruli. At 6-week, a total of 207 genes showed greater than 1.5-fold differential expression. 149 genes were upregulated, whereas 58 were downregulated in DM-LG. On the other hand, differential gene expression greater than 1.4 - fold was observed in 37 genes at 12-week, upregulated in 26 and downregulated in 11. Conclusion: These results suggest that the gene expression profiles of DM-LG are different from DM-SG, and the gene expression patterns change with the progression of diabetic nephropathy.ope

Gamma linolenic acid exerts anti-inflammatory and anti-fibrotic effects in diabetic nephropathy

PURPOSE: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 μM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. RESULTS: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. CONCLUSION: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.ope

Effects of an oral adsorbent on oxidative stress and fibronectin expression in experimental diabetic nephropathy

BACKGROUND: Previous studies have demonstrated that AST-120 (Kremezin((R))), a well-known oral adsorbent, inhibits the progression of diabetic (DM) and non-DM chronic kidney disease along with a decrease in oxidative stress. This study was undertaken to investigate whether AST-120 could reduce oxidative stress and ameliorate the development of nephropathy in experimental DM rats with normal renal function. METHODS: Rats were injected with diluent (C, n = 16) or 65 mg/kg streptozotocin intraperitoneally (DM, n = 16), and eight rats from each group were treated with chow containing 5% AST-120. After 3 months, plasma advanced oxidation protein products (AOPP) and total malondialdehyde (MDA) levels, 24-h urinary albumin excretion, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion were determined by ELISA. Glomerular endothelial nitric oxide synthase (eNOS), subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91phox, p47phox and p22phox), and fibronectin (FN) mRNA and protein expressions were determined by real-time PCR and western blot, respectively. In addition, dichlorodihydrofluorescein diacetate (DCF-DA) staining was performed to detect glomerular reactive oxygen species (ROS) production. RESULTS: Compared to the C group, 24-h urinary albumin excretion was significantly higher in the DM group (P < 0.01), and AST-120 treatment significantly reduced albuminuria in DM rats (P < 0.05). Glomerular eNOS, gp91phox, p47phox and FN expression were significantly increased in DM rats compared to C rats, and these increases in DM glomeruli were significantly abrogated by AST-120 treatment (P < 0.05). The increases in plasma AOPP and MDA levels as well as renal oxidative stress in DM rats, assessed by DCF-DA staining and urinary 8-OHdG excretion rates, were also significantly attenuated by AST-120 treatment (P < 0.05). CONCLUSION: In conclusion, the renoprotective effects of AST-120 in DM nephropathy seem to be associated with the amelioration of enhanced oxidative stress and FN expression under diabetic conditions.ope

Differential gene expression according to the size of glomeruli in experimental diabetic nephropathy

의과학과/석사[한글] 당뇨병성 신병증은 전 세계적으로 말기 신부전증의 원인 중 가장 많은 빈도를 차지하고 있는 질환으로, 병리학적으로는 사구체 및 세뇨관의 비후와 세포 외 기질의 축적, 그리고 임상적으로는 단백뇨가 특징적인 소견으로 되어있다. 당뇨병 신병증의 병태생리에 고혈압, 혈역동학적 변화, 그리고 각종 성장인자 등이 관여하는 것으로 알려져 있으나, 아직까지 분자생물학적 및 세포학적 기전은 명확히 정립되어 있지 않은 실정이다. 현재까지 당뇨병성 신병증에서 일부 유전자의 역할은 부분적으로 규명되어 있으나, 유전자 상호간의 관계에 대해서는 확실하게 밝혀져 있지 않다. 최근에는 microarray를 포함한 유전자 연구 방법의 발전으로 동시에 수천개의 RNA 발현을 검사하는 것이 가능해졌다. 실험적 당뇨병성 신병증 모델의 경우 신장 전체를 이용한 유전자 발현의 차이를 규명한 연구는 있었으나 당뇨 사구체만을 이용한 연구는 거의 없었으며, 더욱이 비후된 사구체에서의 전체 유전자 발현의 변화나 transcriptional profiling을 조사한 연구는 전무한 상태이다.이에 본 연구자는 초기 당뇨병성 신병증에서 사구체와 관련된 유전자를 알아보기 위하여 실험적 당뇨 백서로부터 분리한 사구체를 이용하여 microarray를 시행하였다. 당뇨는 streptozotocin (65mg/kg)을 복강 내로 주사하여 유발시켰으며, 당뇨군 20마리, 그리고 대조군 20마리를 대상으로 각각 10마리씩을 당뇨 유발 6주와 12주 후에 희생시켰다. 사구체는 체공이 250, 150, 125, 그리고 75 &#61549;m인 stainless sieve를 차례로 통과시켜 분리하였으며, 당뇨 사구체를 크기에 따라 125 &#61549;m 체공의 sieve에 걸린 사구체를 큰 사구체 (large DM glomeruli, LG), 75 &#61549;m 체공의 sieve에 걸린 사구체는 작은 사구체 (small DM glomeruli, SG)로 분류하였다. 사구체로부터 RNA를 추출한 후 Rat cDNA 5K chip을 이용한 microarray를 수행하였으며, 유의한 유전자는 significant analysis of microarray (SAM)을 이용하여 선별하였다. 또한 유의한 유전자의 기능은 National Institute of Health (NIH)와 Stanford 대학에서 제공하는 웹사이트를 검색하여 분류하였다. 이상의 과정을 통하여 다음과 같은 결과를 얻었다.1. 대조군과 당뇨군 백서 모두에서 실험 6주 및 12주 후에 체중이 증가되었으나, 대조군에서의 체중 증가가 통계적으로 유의하게 많았다 (p<0.01). 체중 당 신장 무게의 비는 대조군에 비하여 당뇨군에서 의의있게 높았다 (6주: 0.36 ± 0.01% vs. 0.65 ± 0.02%, 12주: 0.31 ± 0.01% vs. 0.61 ± 0.02%, p<0.01). 평균 혈당은 대조군에 비하여 당뇨군에서 의미있게 높았으며 (p<0.01), 24시간 뇨알부민 배설량도 대조군에 비하여 당뇨군에서 유의하게 높았다 (6주: 0.32 ± 0.02 mg/day vs. 1.28 ± 0.11 mg/day, 12주: 0.40 ± 0.06 mg/day vs. 1.99 ± 0.13 mg/day, p<0.05).2. Microarray 실험을 통한 전체 유전자의 발현 패턴을 hierarchical clustering을 수행하여 관찰한 결과, 실험 6주 후에는 작은 당뇨 사구체와 대조군 사구체의 유전자 발현 양상이 유사하였던 반면에, 큰 당뇨 사구체와 작은 당뇨 사구체의 유전자 발현 양상은 서로 상이하였다. 이와 반대로, 12주 후에는 큰 당뇨 사구체와 작은 당뇨 사구체의 유전자 발현 양상이 유사하였던 반면에, 대조군 사구체와 당뇨군 사구체의 유전자 발현 양상이 서로 상이하였다.3. 6주 당뇨 백서에서 분리한 큰 당뇨 사구체와 작은 당뇨 사구체 사이에 발현 차이를 보인 유전자 중 FDR을 기준으로하여 689개 (FDR 0.06%)의 유전자를 선별하였다. 이중 큰 사구체에서 유전자 발현이 1.5배 이상 증가된 유전자는 149개이었으며, 발현이 1.5배 이상 감소된 유전자는 58개이었다. 12주 당뇨 백서의 경우, 105개 (FDR 0.70%)의 유전자 중 큰 당뇨 사구체에서 유전자 발현이 1.4배 이상 증가된 유전자는 26개, 발현이 1.4배 이상 감소된 유전자는 11개이었다.이상의 결과로, 실험적 당뇨 백서에서 분리한 사구체의 크기에 따라 유전자 발현에 차이가 있으며, 당뇨병 유병 기간에 따라 크기에 따른 유전자 발현의 차이가 변할 것으로 생각된다. [영문]Diabetic nephropathy, the leading cause of end-stage renal disease in many countries, is pathologically characterized by cellular hypertrophy and increased extracellular matrix accumulation and clinically by proteinuria. While the diabetic milieu per se, hemodynamic changes, and local growth factors are considered mediators in the pathogenesis of diabetic nephropathy, the molecular and cellular mechanisms responsible for these remain incompletely resolved. The role of some genes in diabetic nephropathy has been described, but their interrelationship remains largely unclear. With the recent advances in genomic research including microarray technique, it is now possible to screen the RNA expression of thousands of genes in parallel. Although a few gene-profiling studies with whole renal tissue have been described in experimental diabetic nephropathy, there is only one microarray study performed with diabetic glomeruli. Furthermore, global gene expression or transcriptional profiling specific to hypertrophic glomeruli has not been explored.The purpose of this study is to elucidate gene expression profiles of hypertrophic glomeruli in early diabetic nephropathy. Forty male Sprague-Dawley rats were injected with diluent (N=20) or streptozotocin intraperitoneally (DM, N=20) and were sacrificed at 6-week and at 12-week. Body weight, kidney weight, blood glucose, and 24-hour urinary albumin excretion were determined at the time of sacrifice. Glomeruli were isolated by sieving technique using sieves with pore size of 250 &#61549;m, 150 &#61549;m, 125 &#61549;m, and 75 &#61549;m. Diabetic glomeruli from 125 &#61549;m and 75 &#61549;m sieves were classified into large (hypertrophic) glomeruli (DM-LG) and small glomeruli (DM-SG), respectively. After RNA extraction, RNAs were pooled, hybridization was performed on the Rat cDNA 5K chip in triplicate, and slides were scanned and analyzed. The significant genes were selected using significant analysis of microarray, and functional annotation of the genes was based on the website of National Institute of Heath or Stanford University. The results were as follows;1. All animals gained weight over the 12-week experimental period, but weight gain was significantly higher in C compared to DM rats (p<0.01). The ratios of kidney weight to body weight at 6-week and at 12-week in DM (0.65 ± 0.02%, 0.61 ± 0.02%, respectively) were significantly higher than those in C rats (0.36 ± 0.01%, 0.31 ± 0.01%, respectively) (p<0.01). The mean blood glucose levels of DM were significantly higher compared to C throughout the study period (p<0.01). Compared to the C, 24-hour urinary albumin excretion was significantly higher in the DM group at 6-week (0.32 ± 0.02 vs. 1.28 ± 0.11 mg, p<0.05) and at 12-week (0.40 ± 0.06 vs. 1.99 ± 0.13 mg, p<0.05).2. At 6-week, hierarchical clustering revealed that gene expression profiles of DM-LG were different from those of DM-SG, whereas DM-SG and C glomeruli showed similar gene expression pattern. In contrast, gene expression profiles at 12-week were similar between DM-LG and DM-SG, whereas C glomeruli showed different gene expression pattern from DM glomeruli.3. To identify the differential genes expressed in DM-LG compared to DM-SG, 689 genes (FDR 0.06%) at 6-week and 105 genes (FDR 0.70%) at 12-week were selected based on the FDR. At 6-week, a total of 207 genes showed greater than 1.5-fold differential expression. 149 genes were upregulated, whereas 58 were downregulated in DM-LG. On the other hand, differential gene expression greater than 1.4-fold was observed in 37 genes at 12-week, upregulated in 26 and downregulated in 11.In conclusion, these results suggest that the gene expression profiles of DM-LG are different from DM-SG, and the gene expression patterns change with the progression of diabetic nephropathy.ope

당뇨 조건 하에서 kallikrein-kinin 계가 족세포의 세포사멸에 미치는 영향

의학과/박사[한글] [영문]Background: Recent studies have shown that podocyte injury plays an important role in the pathogenesis of various proteinuric glomerular diseases, including diabetic nephropathy. The number of podocytes is decreased in diabetic glomeruli and angiotensin II (AII)-mediated apoptosis is known to be involved in the process of podocyte loss under diabetic conditions. The kallikrein-kinin system (KKS) is known to closely interact with the renin-angiotensin system (RAS) and to serve as the physiologic counterbalance to the RAS. Since the RAS is activated in diabetic glomeruli and is considered to play an important role in glomerular injury, the KKS is supposed to have a protective effect on the pathogenesis of diabetic nephropathy. However, the results of recent studies, which investigated the role of the KKS on diabetic nephropathy, were absolutely contrary. Moreover, the presence of a local KKS in podocytes and the changes of its components under diabetic conditions have never yet been explored. In this study, I examined whether a local KKS existed in podocytes and whether the expression of the components of the KKS and bradykinin (BK) production were changed in diabetic glomeruli and in cultured podocytes exposed to high glucose medium. I also elucidated the functional role of BK in podocyte apoptosis, which is implicated as a potential mechanism of podocyte loss characterized in diabetic nephropathy.Methods: In vivo, 32 Sprague-Dawley rats were injected either with diluent (n=16, C) or with streptozotocin intraperitoneally (IP) (n=16, DM), and 8 rats from each group were treated with BK (0.5 μg/hour) via subcutaneously implanted osmotic minipumps for 6 weeks. In vitro, immortalized mouse podocytes were cultured in media containing normal glucose (5.6 mM, NG), NG+24.4 mM mannitol (NG+M), NG+10-7 M AII (NG+AII), high glucose (30 mM, HG) with or without 6-hour pretreatment of 10-8 M BK. BK levels in sieved glomeruli and cell lysates were measured by ELISA. Real-time PCR and Western blot for kallikrein, kininogen, BK B1-receptor (B1R), and B2-receptor (B2R) mRNA and protein expression, respectively, were performed with sieved glomeruli and cell lysates. For the assessment of apoptosis, Western blot for Bax, Bcl-2, and active fragments of caspase-3 were performed. TUNEL assay and Hoechst 33342 staining were also performed with renal tissue and cultured podocytes.Results: 24-hour urinary albumin excretion was significantly higher in DM compared to C rats, and this increment was ameliorated by BK treatment in DM rats. Not only kininogen, kallikrein, B1R, and B2R mRNA and protein expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG medium. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli and HG- and AII-stimulated podocytes were significantly abrogated by BK treatment. The antiapoptotic effect of BK in experimental diabetic glomeruli and in cultured podocytes under diabetic conditions seemed to be mediated through the proapoptotic p38 mitogen-activated protein kinase pathway.Conclusion: I demonstrate for the first time that the expression of all components of the KSS is decreased in diabetic glomeruli and in cultured podocytes exposed to high glucose, and this suppressed KKS is associated with podocyte apoptosis. In addition, BK treatment ameliorated podocyte apoptosis under diabetic conditions. These findings suggest that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.ope

수수료가 있는 이항옵션 가격의 정규근사

MasterWe consider an option pricing model with transaction costs in a discrete-time framework. European options are replicated as in the Cox-Ross-Rubinstein binomial option pricing model, reserving extra amount to cover proportional transaction costs at each trading. We construct a simple Black-Scholes type approximate pricing formula based on a higher-order approximation of the value of replicating portfolio. We compare our numerical results to Boyle and Vorst's which is based on a first-order approximation

Induction of heme oxygenase-1 protects against podocyte apoptosis under diabetic conditions.

Heme oxygenase-1 (HO-1) is an anti-oxidant enzyme normally upregulated in response to oxidant injury. Here we determined the role of HO-1 in podocyte apoptosis in glomeruli of streptozotocin-treated rats and in immortalized mouse podocytes cultured in media containing normal or high glucose. HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, and active caspase-3 fragments were all significantly higher in isolated glomeruli of diabetic rats and in high glucose-treated podocytes. These increases were inhibited by zinc protoporphyrin treatment of the rats or by HO-1 siRNA treatment of the podocytes in culture. The number of apoptotic cells was also significantly increased in the glomeruli of diabetic rats and in high glucose-treated podocytes. Inhibition of HO-1 accentuated the increase in apoptotic cells both in vivo and in vitro. Our findings suggest that HO-1 expression protects against podocyte apoptosis under diabetic conditionsope

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30 mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-β1 with or without anti-TGF-β1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20 db/m and 20 db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-β1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-β1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-β1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db + RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-β1 may contribute to podocyte apoptosis under diabetic conditions.ope

Apoptosis occurs differentially according to glomerular size in diabetic kidney disease

BACKGROUND: Apoptosis; which is involved in the process of mesangial cell and podocyte loss in diabetic nephropathy; is known to be regulated by protein kinase B/Akt (Akt). A number of studies have therefore investigated the activity of Akt under diabetic conditions; but the results have not been consistent. In this study; we hypothesized that apoptosis may occur differentially and that Akt may be differentially activated according to glomerular size in diabetic kidney disease. METHODS: Fifty male Sprague-Dawley rats were injected intraperitoneally with diluent (C; n = 25) or streptozotocin (DM; n = 25). After 3 months; glomeruli were isolated using sieves with pore sizes of 250; 150; 125 and 75 μm and then classified into large glomeruli (on the 125-μm sieve; LG) and small glomeruli (on the 75-μm sieve; SG) groups. Western blot analyses for phospho-Akt; apoptosis-related molecules (Bax; Bcl-2; active fragments of Caspase-3 and phospho-p53) and cyclin-dependent kinase inhibitors were performed. CONCLUSIONS: The numbers of total cells and podocytes in isolated glomeruli were determined using transmission electron microscopy. Akt phosphorylation was significantly decreased in DM-LG; while it was significantly increased in DM-SG (P < 0.05). The ratio of Bax/Bcl-2 protein expression and active fragments of Caspase-3 and phospho-p53 protein expression were significantly increased in DM-LG compared to DM-SG and C-SG (P < 0.001 and P < 0.01; respectively). In contrast; the expression of p27(Kip1) and p21(Cip1) was significantly increased in DM-SG compared to DM-LG and C-SG (P < 0.05). The numbers of total glomerular cells and podocytes were significantly decreased in DM-LG (P < 0.05). In conclusion; these data show differential expression of Akt activity and apoptosis-related molecules according to glomerular size in diabetic nephropathy; suggesting that apoptosis may be more operative in more hypertrophic glomeruli; resulting in fewer glomerular cells and podocytes in diabetic nephropathy.ope

Podocyte biology in diabetic nephropathy

Glomerular visceral epithelial cells, namely podocytes, are highly specialized cells and give rise to primary processes, secondary processes, and finally foot processes. The foot processes of neighboring podocytes interdigitate, leaving between them filtration slits. These are bridged by an extracellular substance, known as the slit diaphragm, which plays a major role in establishing size-selective barrier to protein loss. Furthermore, podocytes are known to synthesize matrix molecules to the glomerular basement membrane (GBM), including type IV collagen, laminin, entactin, and agrin. Because diabetic nephropathy is clinically characterized by proteinuria and pathologically by glomerular hypertrophy and GBM thickening with foot process effacement, podocytes have been the focus in the field of research on diabetic nephropathy. As a result, many investigations have demonstrated that the diabetic milieu per se, hemodynamic changes, and local growth factors such as transforming growth factor-beta and angiotensin II, which are considered mediators in the pathogenesis of diabetic nephropathy, induce directly and/or indirectly hypertrophy, apoptosis, and structural changes, and increase type IV collagen synthesis in podocytes. This review explores some of the structural and functional changes of podocytes under diabetic conditions and their role in the development and progression of diabetic nephropathy.ope