Regulation of cytosolic NADP^(+)-dependent isocitrate dehydrogenase in diabetic nephropathy : 당뇨성 신증 (Diabetic Nephropathy)에서 세포질형 NADP^(+)-dependent Isocitrate Dehydrogenase (IDPc)의 조절 기능 연구

이소현 경북대학교 대학원 2007 국내박사

산화적 스트레스는 당뇨병의 발병과 당뇨성 신증을 포함한 당뇨병의 합병증 진행에 있어 중요한 매개체로 인식되고 있다. 앞선 보고에 따르면, 당이 높은 상태에 이르면 활성산소종의 증가와 항산화계 방어의 손상 뿐 아니라, 특히 NADPH/NADP^(+)의 비율과 GSH의 수준과 같은 세포 내 환원력에도 영향을 미친다. 여러 조직 사이에서의 IDPc 발현 양상을 살펴본 결과, 신장의 근위세뇨관 부위에서 조직 특이적으로 발현하였다. 한편, IDPc는 25 mM 농도의 당을 24 시간 처리하였을 때, 정상 농도인 5.6 mM 처리 시 보다 IDPc의 효소활성도가 40% 이상 증가하는데, 이는 당의 처리농도에 비례하여 증가하고 또한, 처리지속 시간에 비례하여 증가함을 보인다. 높은 당에 의해 증가된 IDPc의 효소활성은 활성산소종의 scarvenger로 알려져 있는 NAC나 flavonoid계열로 항산화 효능이 알려져 있는 quercetin과 같은 항산화제를 처리함으로써 감소하였다. 이는 높은 당 자체의 삼투압 현상에 의한 변화가 아님을 L-형의 포도당을 처리함으로써 확인하였고, 높은 농도의 당 처리에 의한 IDPc의 효소 활성 증가는 IDPc 단백질 수준에서의 발현 증가에 기인한 것임을 확인하였다. 높은 당에 의해 유도되는 산화적 스트레스에 대한 IDPc의 기능 연구를 위하여 IDPc 형질 전환 세포주를 제작하였다. IDPc cDNA를 retroviral vector인 LNCX-vector에 정방향으로 삽입한 뒤, 설치류의 근위세뇨관 세포주인 OK 세포에 형질 전환하여 IDPc의 발현이 증가되도록 한 세포주와 IDPc cDNA를 역방향으로 삽입하여 IDPc의 발현이 억제되도록 한 세포주를 각각 제작하였다. IDPc의 발현이 증가된 세포주에서 고농도의 당에 의한 산화적 피해가 감소하였음을 활성산소종의 생성량 측정을 비롯하여 지질 과산화정도와 단백질 산화정도 측정을 통하여 확인하였다. 또한, 실제 당뇨 환자의 신장 조직에서도 나타나는 신장 비대의 원인이 되는 ECM 분자들의 축적이 IDPc의 발현이 증가된 세포주에서 감소되었다. IDPc는 G6PD, malic enzyme과 함께 세포 내 NADPH를 생성하는데, 이 NADPH는 세포 내 가장 풍부하게 존재하는 항산화제인 GSH의 재생에 중요한 요소로 작용한다. 구축한 IDPc의 형질전환 세포주 사이에서의 NADPH 생성량과 GSH의 양을 측정한 결과, 25 mM 농도의 당 처리 이후, IDPc의 발현이 높은 세포에서 대조군보다 1.8 배의 높은 NADPH 생성량을 확인하였으며, 환원형인 GSH의 수준도 높아져 있음을 확인하였다. IDPc의 발현 양상을 당뇨성 신증 동물 모델에서도 알아보기 위하여 STZ를 처리하여 제 1형 당뇨모델을 유도하였으며, 유전적으로 변형된 db/db 동물모델을 제 2형 당뇨모델로 사용하였다. 각각 항산화제로 NAC와 quercetin을 처리하고, 당뇨치료제로 insulin과 rosiglitazone을 투여하였다. 당뇨성 신증의 충분한 유도를 확인하기 위하여, 신장의 비대를 전체 동물 몸무게에 대한 신장의 무게를 비율로 나타내었다. 그 결과, 제 1형, 2형 모두에서 당뇨성 신증이 충분히 유도되었음을 확인하였고, 조직학적인 IDPc의 발현을 살펴본 결과, 당뇨성 신증의 진행으로 IDPc의 발현량이 증가하였으며, 이는 각각의 치료제 처리에 의하여 발현량이 감소하였다. 신장 조직의 추출물에서도 같은 결과를 얻었으나, quercetin과 rosiglitazone의 IDPc에 대한 효과는 적었다. 위의 결과들을 종합해 볼 떄, IDPc는 당뇨성 신증이 유발됨으로써 효소의 활성이 떨어지는 다른 항산화 효소와 달리, 당뇨성 신증의 진행으로 효소활성이 증가하여 더 많은 산화적 스트레스로부터 신장을 방어하는데, 이는 IDPc가 신장내 근위세뇨관 부위에서 NADPH를 생성함으로써 항산화 작용을 하는 GSH에 환원력을 공급해 주는 기작으로 작용하기 떄문으로 결론 내릴 수 있다. Oxidative stress is widely recognized as a key mediator for the pathogenesis of diabetes, and its complications including diabetic nephropathy. Previous studies show that hyperglycemia not only increased production of free radicals and impaired antioxidant defenses but also affected cellular redox potential, particularly the NADPH/NADP^(+) ratio and GSH levels. Therefore, expression of IDPc in various tissues were analyzed. As a result, IDPc is expressed in specific localization of kidney tissue, especially in proximal convoluted tubular cell. By hyperglycemic condition, IDPc activity is induced in both dose-dependent and time-dependent manners. IDPc activity is also regulated by antioxidants, such as NAC, quercetin. To reveal the role of IDPc against hyperglycemia-induced oxidative stress, IDPc transfected cell lines were prepared. Mouse IDPc cDNA were transfected in sense or antisense orientation in murine proximal tubular cell line, OK cells, to performed permant cell lines. In IDPc transfectants, other antioxidative enzyme activities were not changed, except G6PD. IDPc transfectant cells were exposed to HG, SOD, CAT, GRd and G6PD enzyme activities all decreased similar level without any effects of IDPc. Different activity pattern was shown that IDPc induced by hyperglycemia both in activity and protein expression level, but, SOD, CAT, GRd, and G6PD inactivated by hyperglycemia. With these data, we can be sure to assess IDPc will defense against hyperglycemia in OK cell. Overexpression of IDPc in OK cells reduced hyperglycemia-induced oxidative damage. IDPc effectively reduced the levels of ROS generation, proteins and lipid oxidation and ECM molecule accumulation in OK cell. Especially, pLNCX-IDPc antisense cells incubated with 25 mM glucose for 48 hr, collagen synthesis rate increased 1.9-fold compared with that of 5.6 mM glucose treated cell. By the way, NADPH is essential for reducing power to recycling GSH, and IDPc, G6PD, malic enzyme has been reported to produce NADPH. To confirm that the effect of IDPc on hyperglycemia-induced oxidative damage, the level of NADPH and GSH were measured. As a results, IDPc overexpressing cells produce larger amounts of NADPH and GSH in the cytosol. These results indicated that IDPc as a major NADPH producer in cytosol plays a critical role in cellular defense against hyperglycemia-induced oxidative damage. To investigated about the regulation of IDPc in in vivo animal model of diabetic nephropathy, STZ-induced for type I diabetic model and genetically modified db/ db mice for type II diabetic model were used. Examined the full-induced diabetic nephropathy by kidney hypertrophy. Kidney hypertrophy is caused by glomerular hypertrophy or glomerular basemembrane thickening. This feature measeured by the ratio of kidney weight per total body weight. In fully induced diabetic nephropathy model, ROS generation increased with the progression of diabetic, and regulated by NAC, insulin in STZ-induced diabetic nephropathy, regulated by quercetin, rosigilitazone in db/db mice with diabetic nephropathy. Also, lipid peroxidation level was regulated by antioxidants. IDPc expression, performed by immunohistochemistry, in renal tissue was increased by the progression of diabetic nephropathy both type I and type II models, however, IDPc expression was reduced by the treatment of antioxidants or insulin, rosiglitazone.

Role of LPAR1 Signaling in the Development of Diabetic nephropathy in db/db Mice

LI HUI YING 가천대학교 대학원 2016 국내박사

PARTⅠ Lysophosphatidic acid (LPA) is known to regulate various biological responses by binding to LPA receptors (LPARs). Previously, it was reported that serum level of LPA is elevated in the diabetic conditions, but the involvement of LPA in development of diabetes and its complications remains unknown. We investigated the role of LPA signaling in diabetic nephropathy and the molecular mechanisms involved. The mRNA level of LPAR1 was significantly increased in both high-glucose maintained mesangial cells (SV40 MES13) and the kidney cortex of diabetic db/db mice. Increased albuminuria, serum creatinine, glomerular tuft area and glomerular volume were observed in db/db mice; this was reduced by LPAR1 antagonist (ki16425) treatment. TGFβ expression was upregulated in SV40 MES13 cells by LPA stimulation or in the kidney cortex of db/db mice, but which were blocked by ki16425 treatment. LPA treatment of SV40 MES13 cells increased phosphorylated glycogen synthase kinase (GSK)3β at ser9 and induced translocation of sterol regulatory element-binding protein (SREBP)1 into the nucleus. Blocking phosphorylation of GSK3β inhibited SREBP1 activation and consequently blocked LPA-induced TGFβ expression in SV40 MES13 cells. Phosphorylated GSK3β and nuclear SREBP1 accumulation were increased in kidney cortex of db/db mice, and ki16425 treatment reduced these pathways. These results suggest that LPAR1 signaling increased TGFβ expression via GSK3β phosphorylation and SREBP1 activation, contributing to the development of diabetic nephropathy. PARTⅡ Renal mesangial cell hyperproliferation is a basic pathological change in DN in the early stage, followed by the accumulation of extracellular matrix and thickness of glomerular basement membrane and lead to the glomerulosclerosis. Lysophosphatidic acid (LPA), a major member of the bioactivities including renal mesangial cell proliferation, but the mechanism remains unknown. We investigated the effect of LPA on SV40 MES13 cell proliferation by CCK-8 assay and showed that LPA treatment significantly increases cell proliferation both of does and time dependently. Changes of cell cycle-related molecules by LPA were examined by q-RT PCR and western blot. The results showed that the expression of cyclinD1 and CDK4 are upregulated by LPA treatment, whereas, the expression of p27 is downregulated. Moreover, LPA significantly increases the expression of keuppel-like factor (KLF) 5 at both of the transcription (mRNA) and translation (protein) levels as evaluated by q-RT PCR and western blot. However, RNAi silence of KLF5 abolished LPA induced p27 reduction as well as SV40 MES13 cell proliferation, examined by each of western blot and CCK- assay. In addition, LPA also increased the expression of early growth response factor (Egr) 1 which known as the transcription factor of KLF5, in both of the transcription (mRNA) and translation (protein) levels as evaluated by q-RT PCR and western blot. Finally, the activation of MAPKs (phospho-Erk, phospho-JNK, phospho-p38), which known as upstream pathway of Egr1 was also increased by LPA treatment analyzed by western blot. Altogether, these data demonstrated that LPA-induced renal mesangial cell hyperproliferation is through Egr1-mediated KLF5 expression as well as its upstream pathway mitogen-activated protein kinase (MAPK) such as phospho Erk, phospho p38 and phospho JNK.

Taurine ameliorates the progression of diabetic nephropathy in type 2 diabetic rat model

고장현 Graduate School, Yonsei University 2012 국내박사

Diabetic nephropathy is the most common diabetic microvascular complication and the most common cause of end-stage renal disease. It is believed to be caused by diverse factors, and a variety of mechanisms have been suggested. The overexpression of vascular endothelial growth factor (VEGF) is known to participate in the pathogenesis of diabetic nephropathy. Taurine has some beneficial effects on diabetic nephropathy, but its mechanisms are still unknown. In the present study, the preventive effect and mechanism of taurine on diabetic nephropathy were investigated. Taurine was administered to Otsuka-Long-Evans-Tokushima fatty (OLETF) rats during 20 weeks. Blood glucose and urine albumin creatinine ratio (ACR) were measured. Glomerular basement membrane (GBM) thickness and slit pore numbers of podocytes were measured by electron microscopy. Renal VEGF mRNA and protein synthesis were evaluated by quantitative real time-polymerase chain reaction (qRT-PCR) and western blot immunostaining. Nephrin and VEGF expression were determined in podocytes cultured with high glucose. Fasting blood glucose, twenty four hours urinary albumin and ACR were reduced, and insulin and homeostasis model assessment of beta-cell function (HOMA-β) were increased in taurine treated diabetic group compared with those in diabetic control group. In taurine treated diabetic group, GBM thickness was decreased and open slit pore numbers were increased. Taurine effectively lowered the elevated VEGF mRNA levels in diabetic group. In podocytes cultured with taurine and high glucose, reactive oxygen species (ROS) formation and VEGF mRNA expression were reversed to the control levels. Nephrin mRNA expressions were increased in podocytes cultured with taurine and high glucose. These data support that taurine prevents the progression of diabetic nephropathy possibly through the recovery of nephrin and insulin secretion, and the downregulation of renal VEGF expression.

Low molecular weight heparin prevents loss of nephrin and podocin on rats with early diabetic nephropathy

장영우 School of Life Sciences and Biotechnology, Korea U 2011 국내석사

In this study, a heparin derivative, low-molecular-weight heparin (LMWH), was tested for its ability to afford reno-protection in an established rat model of diabetic nephropathy (DN). Two groups (DN, diabetic nephropathy; DNH, diabetic nephropathy with heparin) of Sprague-Dawley (SD) rats (190±10 g) received a single intraperitoneal injection of streptozotocin (70 mg/kg) to induce DN, and one of them received low-molecular-weight heparin (500 ug/day/rat) treatment for 8 weeks. After inducing DN, there are no effects of body weight and plasma glucose level. To determine 24-hour microalbuminuria, we measured microalbuminuria by ELISA. Excretion of urinary albumin was markedly increased on DN group (3.5 mg/24h), whereas administration of heparin reduced the urinary albumin (0.8 mg/24h). Also, ACR was markedly increased on DN group (40 albumin mg/creatinine g), whereas administration of heparin reduced the ACR (10 albumin mg/creatinine g). The kidney tissue was subjected to histopathologic examination by Periodic Acid-Schiff (PAS) staining. The pathological change was observed in glomerulus of DNH group compare to DN group. Protein expression level changes of nephrin, podocin and GRK2 by LMWH administration were confirmed with Western blotting. LMWH supplemented loss of nephrin and podocin by 18% and reduced the GRK activation by 10%. These results will provide better understanding of mechanism for LMWH in DN therapy.

Association between heme oxygenase-1 promoter polymorphisms and the development of diabetic nephropathy in type 2 diabetes

이은영 Graduate School, Yonsei University 2014 국내박사

Kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions

곽승재 Graduate School, Yonsei University 2010 국내박사

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.

Effects of all-trans-retinoic acid on the treatment of type 2 diabetic nephropathy

김철식 Graduate School, Yonsei University 2007 국내박사

당뇨병성 신증은 만성 신부전의 가장 흔한 원인 질환으로 초기에 기저막 비후와 사구체 간질세포의 증식을 보이다 사구체 경화와 세뇨관 간질의 섬유화를 동반하는데 고혈당, advanced glycation end-product (AGE), polyol 대사 이상, 산화성 스트레스, transforming growth factor-β1 (TGF-β1)의 활성화 등 다양한 인자들이 서로 복합적으로 이 질환의 생성에 관여한다고 알려져 있다.Retinoic acid는 비타민 A의 유도체로, 식이 섬유 중에 포함되어 있는 지용성 필수 인체 구성물질인 비타민 A가 retinol 형태로 간에 저장된 후 retinoic acid로 전환되어 조직으로 이동하여 정상적인 성장이나 조직의 유지, 면역기능에 관여한다. 비타민 A는 인체 내에서 trans와 cis 두 가지 활성형으로 전환되어 세포질내의 retinoic acid A receptor (RAR)와 retinoic X receptor (RXR)와 반응하여 복합체를 형성한 후 핵 내로 들어가 retinoic acid response element를 자극하는 전사인자의 역할을 통해 항증식, 항염증 작용을 한다고 알려져 있다. 최근 all trans retinoic acid (ATRA)가 당뇨병쥐에서 사구체 간질세포의 증식을 억제하며, 사구체의 염증 반응을 감소시켜 신장의 손상을 방지하는 것으로 보고되었다. 또한 ATRA는 지질의 산화를 억제하는 증 제2형 당뇨병의 진행을 예방해주는 효과가 있는 것으로 보고되고 있다.본 연구에서는 제 2형 당뇨병 동물모델인 Otsuka Long Evans Tokushima Fatty (OLETF) 쥐에서 ATRA를 투여하여 요중 알부민 배설량을 측정하여 당뇨병성 신증의 진행이 호전되는지를 알아보고, ATRA의 작용 기전을 이해하고자 배양된 백서의 사구체 간질세포(rat mesangial cells, RMCs)에서 고혈당 자극을 주었을 때 TGF-β1이 증가하는지, 그리고 증가된 TGF-β1이 ATRA 투여로 억제되는지를 알아보았다. 또한 ATRA 투여 후 TGF-β1의 세포 내 신호전달경로의 상부에 있는 protein kinase-C (PKC), 산화성 스트레스(reactive oxygen species, ROS)의 변화를 관찰하였다.생후 6주된 OLETF 쥐를 온도가 25℃, 실내습도가 40~60%로 잘 유지되는 사육실에서 충분한 사료 및 식수를 제공하면서 사육하고 28주령이 되는 시기에 경구 당부하검사를 시행하여 자연 발생적으로 당뇨병이 발생하였음을 확인하였다. 제2형 당뇨병이 발생한 28주령의 OLETF 쥐 20마리에 16주 동안 kg 당 10 mg의 ATRA를 DMSO 및 셀룰로오스에 녹여 Sonde를 이용하여 투여하였고 같은 기간 동안 OLETF 쥐 20마리와 정상 대조군인 LETO 쥐군에 vehicle을 투여하였다.28주령의 공복 및 식후 혈당은 OLETF 쥐군에서 LETO 쥐군에 비해 높았다. 한편 44주령 때의 체중은 LETO 쥐(561.0 ± 40.4 gram)보다 OLETF 쥐의 체중이 높았으며 ATRA를 투여한 군의 체중(648.0 ± 67.7 gram)이 투여하지 않은 군의 체중(697.5 ± 88.4 gram)에 비해 낮았으나 통계적인 의미는 없었다. ATRA 투여 16주 후의 혈당은 LETO 쥐의 혈당(105.4 ± 14.4 mg/dL)보다 OLETF 쥐의 혈당이 높았고 ATRA를 투여한 OLETF 쥐의 혈당(151.0 ± 25.1 mg/dL)이 투여하지 않은 쥐(178.5 ± 38.3 mg/dL)에 비해 의미있게 낮았다 (P < 0.05). 또한 LETO 쥐에 비해 OLETF 쥐의 총콜레스테롤, 중성지방이 높았으나 OLETF 쥐에서 ATRA를 투여한 군과 투여하지 않은 군의 차이는 없었다. ATRA를 투여한 지 16주가 되는 44주령의 시기에 하루 요중 알부민 배설량을 측정한 결과 LETO 쥐의 요중 알부민 배설량(0.01 ± 0.01 mg/mgCr)이 OLETF 쥐보다 적었으며 ATRA를 투여한 OLETF 쥐의 요중 알부민 배설량(0.07 ± 0.03 mg/mgCr)이 투여하지 않은 OLETF 쥐(0.17 ± 0.15 mg/mgCr)보다 적었다 (P < 0.01). 한편 약제의 부작용을 알아보기 위해 시행한 혈액 및 생화학 검사 결과 ATRA가 신장, 간, 혈액에 미치는 부작용은 관찰되지 않았다.배양된 백서의 사구체 간질세포에서 고농도의 포도당은 저농도의 포도당에 비해 TGF-β1을 증가시켰다. ATRA를 10-8, 10-7, 10-6, 10-5 M의 농도로 각각 처리를 하고 또한 0시간, 6시간, 24시간, 48시간 동안 ATRA (10-5 M)를 처리한 결과 TGF-β1의 농도를 시간 및 농도 의존적으로 감소를 시켰다. 또한 같은 세포에서 ATRA는 PKC-α, β, δ의 발현을 용량 의존적으로 감소시켰으며 세포 내 ROS 또한 용량, 시간 의존적으로 감소시켰다.이 연구결과 제2형 당뇨병 모델 동물에서 16주 동안 ATRA를 투여한 결과 투여하지 않은 대조군에 비해 혈당 및 인슐린저항성이 개선됨이 관찰되었으며 요중 알부민 배설량이 낮게 나타났다. 또한 배양된 백서의 사구체 간질세포에서 ATRA는 고농도 포도당에 의해 증가된 TGF-β1 생산을 감소시켰다. 또한 이러한 ATRA의 TGF-β1 감소 기전에는 PKC, ROS의 감소와 관련이 되어있음을 알았다. 우리의 연구 결과는 ATRA가 당뇨병성 신증의 치료제로서의 가능한 역할이 있음을 시사한다. Diabetic nephropathy is the leading cause of kidney disease in patients starting renal replacement therapy and affects approximately 40% of patients with diabetes mellitus. It increases the risk of death, mainly from cardiovascular causes, and is defined by increased urinary albumin excretion (UAE) in the absence of other renal diseases. About fifty percent of diabetic subjects develop microalbuminuria, which progresses towards established diabetic nephropathy in one third of the patients. Hyperglycemia, advanced glycation end-product (AGE), increased polyol pathway, oxidative stress, and the activation of transforming growth factor-β1 (TGF-β1) are interrelated in the pathogenesis of diabetic nephropathy.All-trans-retinoic acid (ATRA) has been reported to suppress interstitial proliferation as well as glomerular inflammation, and to prevent renal damage in diabetic rats. Retinoic acid has also been reported to block lipid peroxidation in streptozocin-induced diabetic rats, and there are studies that also support the protective effect of retinoic acid on the progression of type 2 diabetic nephropathy.In this study, we examined the effect of ATRA on improving diabetic nephropathy by measuring the amount of UAE after administrating ATRA to Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In order to understand the mechanism of action of ATRA, we administrated ATRA to see its inhibitory action on the production of TGF-β1 after confirming the increased production of TGF-β1 in response to high or control glucose media in cultured rat mesangial cells (RMCs). Moreover, we examined changes in protein kinase C (PKC) and reactive oxidative stress (ROS), which are located in the upstream of intracellular signaling pathway of TGF-β1 after the administration of ATRA.From 28 weeks of age, the OLETF rats weighed more than the LETO rats of the same age, while there was no difference in weights between the non-treated OLETF rat group and the ATRA-treated OLETF rat group. At 44 weeks of age, 16 weeks after the administration of ATRA, fasting glucose levels were significantly lower in the LETO rats (105.4 ± 14.4 mg/dL) than in the non-treated OLETF rats (178.5 ± 38.3 mg/dL, P < 0.01) or in the ATRA-treated OLETF rats (151.0 ± 25.1 mg/dL, P < 0.01). A decrease in serum glucose was observed in the ATRA-treated OLETF rats when compared with the non-treated OLETF rats. Compared with the LETO rats, the OLETF rats showed increased levels of total cholesterol and triglyceride. However, no significant differences in total cholesterol and triglyceride levels were found between the ATRA-treated OLETF rats and the non-treated OLETF rats. The non-treated OLETF rats might have been in the greater insulin resistance status, as demonstrated by increased insulin, C-peptide and HOMA-IR levels than the other two groups. Also, there were no remarkable differences in AST, ALT, creatinine, hemoglobin levels, white blood cell count, or platelet count between the three groups. The OLETF rats showed a higher daily UAE than the LETO rats at 44 weeks of age. In the ATRA-treated OLETF rats, daily UAE was lower than that of non-treated the OLETF rats (0.07 ± 0.03 vs. 0.17 ± 0.15 mg/mgCr, P < 0.01).After incubation of quiescent mesangial cells in media containing 30 or 5 mM of glucose, treatment with ATRA showed time- and dose-dependent decreases in TGF-β1 levels. Moreover, ATRA treatment under both low and high glucose conditions showed a dose-dependent decrease in PKC activity. Lastly, treatment with ATRA showed dose- and time-dependent decreases in DCF-sensitive cellular ROS in the RMCs.In this study, we demonstrated that the administration of ATRA resulted in a reduction of UAE in OLETF rats, cultured RMCs increased TGF-β1 synthesis in response to high glucose stimuli, and that ATRA treatment suppressed TGF-β1 synthesis induced by high glucose stimulation. It is of interest that ATRA treatment suppressed UAE and TGF-β1 synthesis, which was mediated by a significant reduction of PKC activity and ROS production. Our results suggest that ATRA has a potential therapeutic role for diabetic nephropathy.

실험적 당뇨 백서에서 사구체의 크기에 따른 유전자 발현의 차이

곽승재 연세대학교 대학원 2006 국내석사

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 m인 stainless sieve를 차례로 통과시켜 분리하였으며, 당뇨 사구체를 크기에 따라 125 m 체공의 sieve에 걸린 사구체를 큰 사구체 (large DM glomeruli, LG), 75 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. 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 m, 150 m, 125 m, and 75 m. Diabetic glomeruli from 125 m and 75 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.

알도스테론이 혈관 평활근 세포와 당뇨 신장에서 connective tissue growth factor (CTGF) 발현에 미치는 영향

한금현 高麗大學校 2005 국내박사

It is well known that aldosterone induces renal fibrosis. But, the mechanisms by which aldosterone induces fibrosis are not clear. Connective tissue growth factor (CTGF) is a candidate factor mediating fibrogenic properties. We investigated whether CTGF is related with profibrotic effects of aldosterone in diabetic nephropathy as well as in vascular smooth muscle cells on a high glucose condition. CTGF mRNA and protein expression in vascular smooth muscle cells in the presence of high glucose and aldosterone were investigated. CTGF mRNA and protein expression in the kidneys of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which were treated with aldosterone antagonist, spironolactone, also were investigated by RT-PCR, Western blot and immunohistochemistry. CTGF mRNA and protein expression in vascular smooth muscle cells was enhanced by the high glucose. Aldosterone enhanced CTGF mRNA and protein expression in vascular smooth muscle cells independent of glucose concentration, and spironolactone attenuated its expression. Diabetic nephropathy was developed in OLETF rats, and CTGF, type 1 collagen expressions and glomerulosclerosis were enhanced in the kidneys of OLETF rats, which were partially attenuated by spironolactone independent of blood pressure. CTGF mediates aldosterone-induced fibrosis in diabetic nephropathy. Attenuation of renal fibrosis by spironolactone supports the protective effects of aldosterone blockade in diabetic nephropathy.

Glucose independent pleiotropic effects of a dipeptidyl peptidase-4 inhibitor on diabetic complications

정은수 서울대학교 대학원 2016 국내박사

Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as anti-diabetic agents in clinical practice. Gemigliptin, a new and selective DPP-4 inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic complications have not yet been reported. This study evaluated the inhibitory effects of gemigliptin, a highly selective dipeptidyl peptidase-4 inhibitor, on the formation of advanced glycation end products (AGEs) and AGE cross-links with proteins in in vitro as well as in type 2 diabetic db/db mice. In in vitro assay, gemigliptin dose-dependently inhibited methylglyoxal-modified AGE-bovine serum albumin (BSA) formation (IC50 = 11.69 mM). AGE-collagen cross-linking assays showed that gemigliptin had a potent inhibitory effect (IC50 = 1.39 mM) on AGE-BSA cross-links to rat tail tendon collagen, and its activity was stronger than aminoguanidine (IC50 = 26.4 mM). In addition, gemigliptin directly trapped methylglyoxal in a concentration-dependent manner in vitro. To determine whether gemigliptin inhibits the in vivo glycation processes, gemigliptin (100 mg/kg/day) was orally administered into type 2 diabetic db/db mice for 12 weeks. Elevated serum levels of AGEs in db/db mice were suppressed by the administration of gemigliptin. These inhibitory effects of gemigliptin on the glycation process in both in vitro and in vivo suggest its therapeutic potential for ameliorating AGE-related diabetic complications. Podocytes participate in the formation and regulation of the glomerular filtration barrier. Loss of podocytes occurs during the early stages of diabetic nephropathy and impairs glomerular filtration. Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as anti-diabetic agents in clinical practice. In this study, we showed that gemigliptin, a novel DPP-4 inhibitor, reduced podocyte apoptosis in type 2 diabetic db/db mice without reducing hyperglycemia. Gemigliptin (100 mg/kg/day) was administered orally for 12 weeks in db/db mice. Blood glucose levels and albuminuria were measured. The renal cortex was collected for histological examination, and molecular assays were used to detect 8-hydroxydeoxyguanosine, advanced oxidation protein products (AOPP), the receptor for advanced glycation end products (RAGE), and integrin-linked kinase (ILK). Type 2 diabetic db/db mice exhibited albuminuria, renal histopathological changes, and podocyte loss. Administration of gemigliptin to db/db mice suppressed albuminuria, enzyme activity and expression of DPP-4, and podocyte apoptosis. The effect of gemigliptin on diabetes-induced podocyte loss was associated with the suppression of oxidative damage, AOPP accumulation, RAGE expression, and ILK expression. These results indicate the possible benefits of using gemigliptin in diabetes patients to treat renal impairment without affecting glycemic control. Retinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy. Gemigliptin (100mg/kg/day) was orally administered to the db/db mice for 12 weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5 days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50 mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4 months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin.