GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

김상훈 Korea University 2016 국내박사

The foot processes of podocytes, which are interdigitated to form the slit diaphragm, are crucial for the glomerular filtration barrier. Glucocorticoid-induced transcript 1 (GLCCI1) is transcriptionally regulated, but its signaling pathway in podocytes is unknown. The main objective of this study was to investigate the regulation of podocyte foot process proteins and to investigate the role of GLCCI1 in the phosphoinositide 3-kinase (PI3K) pathway, using high glucose-induced podocytes and streptozotocin-induced diabetic rats. In podocytes and rat kidneys, GLCC1, similar to other podocyte-specific proteins (nephrin, podocin, synatopodin, and podocalyxin), was found to be highly specific for the glomerulus and podocyte foot processes as determined by RT-PCR, Western blotting, immunofluorescence, and immunoelectron microscopy analyses. In addition, decreased GLCCI1 expression level under the hyperglycemic conditions restored with treatment of PI3K inhibitor (wortmannin). Moreover, immunofluorescence confirmed that GLCCI1 was co-localized with nephrin and synaptopodin, both in vivo and in vitro. Finally, immunoelectron microscopy data from streptozotocin-induced diabetic rats showed that GLCCI1 was also localized in podocyte foot processes. Hence, GLCCI1 is a component of podocyte foot processes, and its expression appears to be regulated via the PI3K pathway.

Fumarate modulates phospholipase A2 receptor autoimmunity-induced podocyte injury in membranous nephropathy

조형아 서울대학교 대학원 2021 국내박사

Downstream mechanisms that lead to podocyte injury mediated by phospholipase A2 receptor (PLA2R) autoimmunity remain elusive. Accumulating evidence has been shown that increased production of reactive oxygen species (ROS) can cause podocyte injury. Also, the activation of the complement system which involved the ROS generation has been identified in membranous nephropathy (MN). Among the molecules related to ROS, fumarate was recently known to be involved in the podocyte injury. Here, the role of fumarate in modulating phospholipase A2 receptor-mediated podocyte injury was investigated in in vitro model of PLA2R-associated MN. Fumarate hydratase, which hydrolyzes fumarate, colocalized with podocalyxin, and its expression was lower in glomerular sections from patients with PLA2R-associated MN than in those from healthy individuals, patients with non-PLA2R associated MN or minimal change disease. To elucidate the role of fumarate in podocyte injury in in vitro model of PLA2R-associated MN, the immunoglobulin G (IgG) was purified from the serum of patients with PLA2R-associated MN (MN-IgG) and a healthy control (Con-IgG). The purified MN-IgG reacted with the 153 kDa band when the lysates of primary cultured human podocytes were immunoblotted, but Con-IgG did not. Podocytes stimulated with MN-IgG decreased expression of fumarate hydratase and increased fumarate levels. These changes were coupled to alterations in the expression of molecules involved in the phenotypic profile of podocytes (WT1, ZO-1, Snail, and Fibronectin), an increase in albumin flux across the podocyte layer and the production of ROS in podocytes. However, overexpression of fumarate hydratase ameliorated these alterations. Furthermore, knockdown of fumarate hydratase exhibited synergistic effects with MN-IgG treatment. Thus, fumarate may promote changes in the phenotypic profiles of podocytes after the development of PLA2R autoimmunity. These findings suggest that fumarate could serve as a potential target for the treatment of PLA2R-associated MN. 막성신증에서 phospholipase A2 (PLA2) 수용체 자가면역에 따른 족세포 손상의 기전에 대해서는 밝혀져 있지 않다. 족세포 손상을 일으키는 기전 중 활성산소종이 작용할 것이라는 기존의 연구가 있으며 막성신증에서 활성산소종을 생성하는 보체계 활성화가 알려져 있다. 활성산소종과 관련된 물질 중 푸마르산은 최근 연구에서 족세포 손상에 관여하는 것으로 밝혀졌다. 본 연구에서는 PLA2 수용체 관련 막성신증 세포 모델에서 PLA2R 수용체 자가면역 유도에 따른 족세포 손상의 기전에 푸마르산이 관여한다는 가설을 세웠다. 푸마르산을 대사하는 효소인 푸마라아제 활성이 정상 신장조직의 사구체에서는 포도칼릭신과 병합되어 잘 나타났지만, PLA2 수용체 관련 막성신증 환자의 신장조직의 사구체에서는 정상 신장조직, PLA2 수용체 비관련 막성신증 환자, 미세변화신증 환자의 신장조직에서의 사구체 푸마라아제 활성과 비교하여 감소하는 것을 확인하였다. 생체 외 실험을 위해 막성신증 환자 중에서 혈청 항 PLA2 수용체 항체 역가가 양성인 환자와 정상인에게서 혈청을 얻어 각각의 혈청으로부터 면역 글로불린 G 를 정제하였다. 세포 실험을 위해서 신장절제술을 받은 환자에게서 얻은 신장조직에서 일차 배양한 사람의 족세포를 사용하였다. 각각의 면역 글로불린 G 와, 사람 족세포에서 추출한 단백질을 이용하여 웨스턴 블랏을 시행하였고 항 PLA2 수용체 항체 역가가 양성인 환자의 혈청에서 정제한 면역 글로불린 G 로 자극한 족세포에서만 항 PLA2 수용체 밴드를 153 kDa 위치에서 확인하였다. 항 PLA2 수용체 항체 역가가 양성인 환자의 혈청에서 정제한 면역 글로불린 G 로 자극한 족세포에서는 푸마라아제 활성이 감소한 것을 웨스턴 블랏으로 확인하였고 정상인 혈청에서 정제한 면역 글로불린 G 로 자극한 족세포와 비교하여 푸마르산 농도가 증가하였다. 이러한 변화는 족세포 형질전환에 관여하는 물질인 WT1, ZO-1 의 감소, Snail, 그리고 Fibronectin의 증가, 그리고 족세포 층을 가로지르는 알부민 유량의 증가, 활성산소종의 증가와 동반하였다. 이러한 족세포내의 WT1, ZO-1 의 단백질 발현의 감소는 푸마라아제 lentiviral particles를 처리하여 족세포의 푸마라아제 활성을 증가시켰을 때에는 회복되었으며 증가되었던 Snail 과 Fibronectin 단백질 발현과 활성산소종의 생성은 감소하였다. Small interfering RNA 를 사용하여 족세포에서 푸마라아제 활성을 감소시켰을 때에는 항 PLA2 수용체 항체 역가가 양성인 환자의 혈청에서 정제한 면역 글로불린 G 로 자극한 족세포에서 감소한 푸마라아제 활성이 더욱 감소하였고 더불어 WT1, ZO-1 의 약화된 발현이 더욱 약화되고, 증가한 fibronectin 과 Snail 의 단백질 발현 활성산소종의 생성이 더욱 증가하는 것을 확인하였다. 본 연구에서는 PLA2 수용체 매개 자가면역에 따라 족세포에서 감소되는 푸마라아제의 활성, 증가된 푸마르산의 결과로 족세포 표현형 변화가 유도되어 족세포 손상에 영향을 미치는 것을 확인하였다. 이러한 결과는 푸마르산이 PLA2 수용체 관련 막성신증 치료의 표적이 될 수 있는 것을 시사한다.

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.

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

김동기 연세대학교 대학원 2010 국내박사

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.

당뇨병성 신증의 발병기전으로 podocyte에서 형질전환인자의 역할

강영선 고려대학교 대학원 2002 국내박사

Background: According to the development of methods in podocyte cell culture several studies for the role of podocyte in the progression of glomerulosclerosis have been recently reported. But there is few report for the regulation of TGFβ1 synthesis and type Ⅳ collagen production in podocyte in diabetic nephropathy. Object: We investigated the effects of high glucose and TGFβ1 in culture medium on TGFβ and type Ⅳ collagen production and whether their production is dependent on protein kinase C(PKC) pathway in cultured mouse podocyte cell line. Methods: Conditionally immortalized mouse podocytes with a temperature-sensitive variant of SV40 large T antigen were cultivated. To propagate podocytes, cells were cultivated at 33℃ and treated with γ-interferon(permissive condition). And to induce differentiation, podocytes were changed at 37℃ and deprived of γ-interferon(non-permissive condition). The effects of high glucose and TGFβ1 in culture media on procollagen α1(PCα1(Ⅳ)) and their relationships to PKC pathway were examined. The mRNA expression and protein production of TGFβ1 and PCα1(Ⅳ) were assayed by reverse transcription - polymerase chain reaction(RT-PCR) and western analysis. Results: Compared with normal glucose(NG, 5.5mM), high glucose exposure(HG, 15, 30mM) increased the mRNA expression and protein production of TGFβ1 and PCα1(Ⅳ), but without a statistic significance: TGFβ1(after 6hours: 69.62±9.00 vs 83.48±7.82 vs 74.49±24.73, after 24hours: 65.06±20.55 vs 68.01±24.35 vs 94.23±13.14), PCα1(Ⅳ)(after 6hours: 109.94±10.43 vs 102.00±6.68 vs 138.65±39.83, after 24hours: 105.88±9.53 vs 83.95±1.12 vs 109.14±3.29, after 72hours: 99.18±5.30 vs 92.93±6.33 vs 109.25±4.11). TGFβ1 significantly decreased the expression of PCα1(Ⅳ). Calphostin C treatment further stimulated the increase of PCα1(Ⅳ) production induced by HG and inhibited the decreased mRNA expression of PCα1(Ⅳ) induced by TGFβ1 administration. Conclusion: We suggest that TGFβ1 have an important role in podocyte in the pathogenesis of diabetic nephropathy. The HG-induced increases of procollagen α1 type Ⅳ collagen seems to be negatively regulated by TGF β1 and PKC pathway and possibly another pathway will positively regulate the production of PCα1(Ⅳ), and these pathways may have a different effect on collagen synthesis dependent on the renal cell type.

(The) reno-protective effect by PI3K inhibitor (wortmannin) on STZ-induced proteinuric kidney disease rats

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

Diabetic nephropathy (DN) is the most common cause of renal failure. Although many studies are now in progress, the mechanism of injury in DN is poorly understood. Podocytes are cells in the kidneys that wrap around the capillaries of the glomerulus. Recent studies have shown that podocytes are functionally and structurally injured in early DN. PI3K inhibitor (LY294002) simultaneously regulate Rac1 and Cdc42 that associated with actin cytoskeleton of kidney podocytes. The main objective of this study is administered wortmannin, the other PI3K inhibitor to streptozotocin (STZ, 70 mg/kg)-induced DN Sprague-Dawley (SD) rats in vivo and confirm that whether there’ll be improvement effect of DN. After STZ administration, to identify of DN, body weight and blood glucose level were measured every week. Wortmannin (1 mg/kg) was administered to STZ-induced DN rats every day during 8 weeks. To demonstrate reno-protective role of PI3K inhibitor on STZ-induced DN rats, albuminuria and albumin to creatinine ratio (ACR) level were measured using ELISA kit and pathological changes by Periodic acid-Schiff (PAS) staining were examined. Albuminuria value of diabetes group was 3.5 mg/24h, whereas wortmannin group was 1.75 mg/24h. Also, ACR value of diabetes group was 42 albumin mg/creatinine g, whereas wortmannin group was 20 albumin mg/creatinine g. These value was significantly decreased in wortmannin group. Expression level changes of nephrin, podocin and Rac1/Cdc42 by wortmannin administration were confirmed with Western blotting. Nephrin, podocin and Rac1/Cdc42 band intensity value of diabetes group were 56, 55 and 54% of control respectively, whereas wortmannin group value were 80, 86 and 84% of control respectively. As a results, expression level of nephrin, podocin and Rac1/Cdc42 of wortmannin group were increased when compared with diabetes group. Also, expression and localization of podocyte marker proteins, nephrin, podocin and desmin were confirmed by immunofluorescence. Therefore, the wortmannin have an effective on treatment of DN through reno-protection.

Heme oxygenase-1 expression is increased to protect podocyte apoptosis under diabetic conditions

이상철 Graduate School, Yonsei University 2008 국내박사

Background: Diabetic nephropathy, the leading cause of end-stage renal disease in many countries, is clinically characterized by proteinuria. 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 reactive oxygen species-mediated apoptosis is known to be involved in the process of podocyte loss under diabetic conditions. Heme oxygenase-1 (HO-1), a microsomal enzyme, has been regarded as an anti-oxidant and to be upregulated to alleviate the deleterious consequences of oxidant injury. Previous studies have demonstrated that HO-1 expression is increased in experimental diabetic glomeruli, but the functional significance of this increase has not yet been explored. In this study, to elucidate the role of HO-1 in podocyte apoptosis under diabetic conditions, I investigated not only the expression of HO-1 but also podocyte apoptosis in experimental diabetic glomeruli and in high glucose-stimulated cell before and after inhibition of HO activity.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 IP Zinc protoporphyrin (ZnPP, 50 mol/kg/day), an HO-1 inhibitor, for 6 weeks. In vitro, immortalized mouse podocytes were cultured in media containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without HO-1 siRNA. Real-time PCR for HO-1 mRNA expression and Western blotting for HO-1, Bax, Bcl-2, and active fragments of caspase-3 protein expression were performed with sieved glomeruli and cell lysates. In addition, TUNEL assay and immunofluorescence (IF) staining for active fragments of caspase-3 were conducted with renal tissue and Hoechst 33342 staining with cultured podocytes.Results: Urinary albumin excretion was significantly higher in DM (1.18±0.11 mg/day) compared to C rats (0.32±0.04 mg/day) (p<0.05), and was further increased in DM rats by ZnPP (1.59±0.19 mg/day) (p<0.05). HO-1 mRNA and protein expression were increased in diabetic glomeruli and in HG-stimulated podocytes, and these increases were inhibited by ZnPP and HO-1 siRNA, respectively. The ratios of Bax/Bcl-2 protein expression were significantly higher in diabetic glomeruli and in podocytes exposed to HG, which were further increased by ZnPP and HO-1 siRNA, respectively. Western blotting with active fragments of caspase-3 showed a similar pattern to the ratios of Bax/Bcl-2 protein expression. TUNEL positive-stained nuclei were also significantly increased in DM (1.30.2) compared to C glomeruli (0.40.1) (p<0.05), and were further increased in DM glomeruli by ZnPP (2.20.3) (p<0.05). In addition, double IF staining with active fragments of caspase-3 and synaptopodin revealed that active fragments of caspase-3 expression was most prominent in podocytes of DM+ZnPP rats. Apoptotic cells assessed by Hoechst 33342 staining were also significantly increased in HG-stimulated podocytes compared to NG cells, which were further increased in HG+siRNA group.Conclusion: This study suggests that HO-1 expression is increased in experimental diabetic glomeruli and in HG-stimulated podocytes, and the increase in HO-1 expression plays a protective role against podocyte apoptosis under diabetic conditions. 서론: 당뇨병성 신병증은 국내외적으로 말기 신부전증의 가장 흔한 원인 질환으로, 초기에는 단백뇨가 대표적인 임상적 특징이다. 최근의 보고들에 의하면 단백뇨의 발생에 사구체 여과 장벽을 구성하는 세포의 하나인 족세포가 중요한 역할을 하는 것으로 알려져 있다. 당뇨병성 신병증에서 족세포의 수 및 밀도가 감소되어 있으며 이는 사구체 여과장벽의 손상을 일으켜 결국 사구체 경화증을 초래하는 요인이 되는데, 이 과정에 당뇨 조건 하에서 생성이 증가된 ROS가 관여하는 것으로 알려져 있다. Heme oxygenase-1 (HO-1)은 미소체 효소로서 산화성 스트레스에 의한 각종 조직 손상에서 발현이 증가되며, 항산화 효과, 항염증 작용, 그리고 항세포사멸 효과가 있는 것으로 보고되고 있다. 당뇨병성 신병증에서도 HO-1의 발현 증가가 보고된 바는 있으나, 그 기능적 역할에 대한 연구는 전무한 실정이다. 이에 본 연구자는 당뇨병성 신병증에서 HO-1의 발현 증가 여부를 확인하고, 당뇨병성 신병증에서 발현이 증가되는 HO-1의 기능적 의미를 알아보기 위하여 생체 내외의 실험을 통하여 HO-1 발현 억제 전후에 족세포 사멸의 변화를 알아보고자 하였다.방법: 32마리의 Sprague-Dawley 백서를 대상으로 대조군 (16마리)과 복강 내로 streptozotocin을 투여한 당뇨군 (16마리)으로 나누었으며 각 군에서 8마리에는 HO-1의 억제제인 Zinc protoporphyrin (ZnPP, 50 μmol/kg/day)를 6주간 복강 내로 투여하였다. 또한, 불멸 생쥐 족세포 (immortalized mouse podocytes)를 정상 포도당군 (5.6 mM), 정상 포도당+만니톨군 (24.4 mM 만니톨), 정상 포도당+HO-1 siRNA 처리군, 고포도당군 (30 mM), 그리고 고포도당+HO-1 siRNA 처리군으로 나누어 배양하였다. 실험 동물로부터 분리한 사구체와 배양 족세포에서 HO-1 mRNA와 단백 발현은 각각 real-time PCR과 Western blot을 이용하여 분석하였으며, 신장 조직 내 HO-1의 단백 발현은 면역조직화학 염색을 시행하여 확인하였다. 세포사멸은 Bax, Bcl-2, 그리고 active fragments of caspase-3에 대한 Western blot을 이용하여 분석하였으며, 신장 조직을 이용한 TUNEL 염색과 배양 세포를 이용한 Hoechst 33342 염색으로도 확인하였다.결과: 24시간 뇨알부민 배설량은 대조군 (0.32±0.04 mg/day)에 비하여 당뇨군 (1.18±0.11 mg/day)에서 의미있게 많았으며 (p<0.05), ZnPP 투여 당뇨군에서의 뇨알부민 배설량이 유의하게 더 증가하였다 (1.59±0.19 mg/day) (p<0.05). 사구체 내 HO-1의 mRNA 발현은 대조군에 비하여 당뇨군에서 5.1배 증가되었으며 (p<0.01), ZnPP 투여로 당뇨군에서 발현 증가가 46.6% 억제되었다 (p<0.05). 사구체 내 HO-1의 단백 발현도 mRNA 발현 양상과 유사하였다. 세포 실험상 고포도당으로 자극한 족세포에서 HO-1의 단백 발현은 정상 포도당군에 비하여 2.3배 증가되었으며 (p<0.05), HO-1 siRNA는 HO-1의 단백 발현을 농도 의존적으로 억제시켰다. 세포사멸의 지표인 Bax/Bcl-2 단백 발현의 비와 active fragments of caspase-3의 단백 발현은 당뇨 사구체와 고포도당으로 자극한 족세포에서 의미있게 높았으며 (p<0.05), ZnPP 투여 및 HO-1 siRNA 처치로 의의있게 더 증가되었다 (p<0.01). 사구체 내 TUNEL-양성 세포 수는 대조군 (0.4±0.1)에 비하여 당뇨군 (1.3±0.2)에서 유의하게 많았으며 (p<0.05), ZnPP 투여 당뇨군 (2.2±0.3)에서 의미있게 더 증가되었다 (p<0.05). WT-1에 대한 면역조직화학 염색을 이용하여 측정한 족세포 수는 대조군 (170.0±4.2)에 비하여 당뇨군 (159.1±3.7)에서 적었으며, ZnPP를 투여한 당뇨군 (142.9±3.2)에서 의의있게 더 감소되었다 (p<0.05). Active fragments of caspase-3와 synaptopodin에 대한 이중 면역형광염색을 시행한 결과, 세포사멸이 동반된 세포가 주로 족세포임을 알 수 있었다. 배양 족세포에서 Hoechst 33342 염색을 이용하여 측정한 세포사멸이 동반된 족세포 수도 정상 포도당군 (3.7±0.1%)에 비하여 고포도당군 (11.1±0.2%)에서 유의하게 많았으며 (p<0.05), HO-1 siRNA 처리군 (17.2±0.2%)에서 의미있게 더 증가되었다 (p<0.05).결론: 당뇨병성 신병증의 동물 모델과 고포도당으로 자극한 족세포에서 HO-1의 발현은 의의있게 증가되었으며, HO-1의 발현 증가를 억제시킬 경우 족세포의 세포사멸이 유의하게 더 증가되었다. 이상의 결과들을 종합해 볼 때, 당뇨 조건 하에서 족세포 내 HO-1의 발현 증가는 세포사멸에 대한 보호 역할을 할 것으로 생각된다.

Deciphering the pathogenic mechanism of CoQ10-deficient glomerulopathy and optimizing therapeutic effect of drugs

Yu, Seyoung 연세대학교 일반대학원 2025 국내박사

Steroid-resistant nephrotic syndrome (SRNS) is characterized by proteinuria and is commonly associated with focal segmental glomerulosclerosis (FSGS), which can result from mutations in genes involved in coenzyme Q10 (CoQ10) biosynthesis, such as PDSS2, COQ2, COQ6, and ADCK4. CoQ10 is an essential component of the mitochondrial inner membrane, playing a crucial role in electron transport in oxidative phosphorylation (OXPHOS) and protection against oxidative stress. While CoQ10 supplementation provides partial therapeutic benefits, its efficacy is inconsistent and has limitations. Therefore, it is crucial to elucidate the pathogenesis of CoQ10-deficient glomerulopathy and establish optimal treatment conditions. This study utilized CoQ10-deficient cell and animal models (podocyte-specific Adck4 knockout mice, Adck4△Podocyte) and kidney organoids to investigate the effects of CoQ10 deficiency. CoQ10 deficiency due to ADCK4 loss led to mitochondrial dysfunction, including a reduction in membrane potential (ΔΨm), which was not observed in HK-2 cells, indicating a greater susceptibility of podocytes to CoQ10 deficiency. In addition, CoQ10-deficient podocytes exhibited increased oxidative stress, further highlighting their vulnerability to mitochondrial impairment. ADCK4 directly interacts with COQ5 and plays a crucial role in stabilizing the COQ complex. Loss of ADCK4 resulted in a decrease in COQ complex protein levels and a mild reduction in OXPHOS complex III expression. Consistent with this, CoQ10-deficient kidney organoids exhibited normal overall morphology but displayed mitochondrial abnormalities in podocytes, reinforcing the importance of CoQ10 in maintaining mitochondrial homeostasis during kidney development. Single-cell RNA sequencing of kidney organoids derived from CoQ10-deficient iPSC lines (PDSS2-, COQ2-, ADCK4-Knockout) revealed preserved cell-type composition but distinct transcriptomic alterations in podocytes. Gene set enrichment analysis identified downregulation of oxidative phosphorylation and upregulation of TGF-β signaling, epithelial-to-mesenchymal transition, and apoptosis pathways, indicating mitochondrial dysfunction and early injury responses in CoQ10-deficient podocytes. Additionally, the therapeutic potential of 2,4-dihydroxybenzoic acid (2,4-diHB), 2-probucol and Aspirin were evaluated using mice model, with a focus on the timing of treatment. Consistent with a previous study, early treatment (at 3 months) with 2,4-diHB successfully preserved kidney function in Adck4△Podocyte mice, whereas delayed treatment (at 5 months) was ineffective. Furthermore, Probucol and Aspirin did not show significant therapeutic effects, suggesting that disease progression limits treatment efficacy and that early intervention is essential. In conclusion, this study emphasizes the role of ADCK4 in maintaining CoQ10 homeostasis and podocyte integrity, demonstrating that CoQ10 deficiency leads to mitochondrial dysfunction and progressive glomerular damage. The establishment of a CoQ10-deficient kidney organoid model offers a robust platform for investigating disease mechanisms and evaluating targeted therapeutic strategies. Notably, early intervention with 2,4-diHB effectively preserved renal function, whereas delayed treatment failed to prevent disease progression, highlighting the urgency of timely therapeutic intervention in CoQ10-associated nephropathy. 스테로이드 저항성 신증후군(SRNS)은 단백뇨를 특징으로 하며, 흔히 국소분절성 사구체경화증(FSGS)과 관련되어 나타난다. 이 질환은 PDSS2, COQ2, COQ6, ADCK4 등 코엔자임 Q10(CoQ10) 생합성과 관련된 유전자의 돌연변이로 인해 발생할 수 있다. CoQ10은 미토콘드리아 내막의 필수 성분으로, 산화적 인산화(OXPHOS) 과정에서 전자 전달과 산화 스트레스 방어에 중요한 역할을 한다. CoQ10 보충 요법은 부분적인 치료 효과를 보이기도 하지만, 효과가 일관되지 않고 한계가 있다. 따라서 CoQ10 결핍성 사구체병증의 병태생리를 규명하고 최적의 치료 조건을 확립하는 것이 필요하다. 이 연구에서는 CoQ10이 결핍된 세포 및 동물 모델(Adck4△Podocyte 마우스)과 신장 오가노이드를 이용해 CoQ10 결핍이 미치는 영향을 분석하였다. ADCK4 결실로 인한 CoQ10 결핍은 미토콘드리아 기능 이상을 초래하였고, 특히 막전위(ΔΨm)의 감소가 확인되었다. 이러한 변화는 HK-2 세포에서는 관찰되지 않아, 족세포가 CoQ10 결핍에 더 취약함을 시사한다. 또한, CoQ10 결핍 족세포에서는 산화 스트레스가 증가하였다. ADCK4는 COQ5와 직접 상호작용하며 COQ 복합체의 안정성 유지에 관여하고, ADCK4 결손 시 COQ 단백질 수준과 OXPHOS 복합체 III의 발현이 감소하는 양상이 확인되었다. CoQ10 결핍 신장 오가노이드에서는 전체적인 구조는 유지되었으나, 족세포 내 미토콘드리아 이상이 관찰되어 CoQ10이 신장 발달과 족세포 건강에 중요한 역할을 한다는 점을 보여준다. CoQ10 결핍 iPSC(PDSS2, COQ2, ADCK4-KO 유래)에서 유래한 신장 오가노이드를 단일세포 전사체 분석한 결과, 세포 구성에는 큰 변화가 없었으나, 족세포에서 특징적인 전사체 변화가 확인되었다. 산화적 인산화 경로의 하향 조절과 TGF-β 신호전달, 상피-간엽 전이, 세포사멸 경로의 상향 조절이 나타나, 미토콘드리아 기능 저하와 초기 손상 반응을 반영한다. 또한 마우스 모델을 이용하여 CoQ10 전구체 유사체인 2,4-dihyroxybenzoic acid(2,4-diHB)와 치료 후보 약물인 Probucol와 Aspirin의 치료 효과를 시점에 따라 비교하였고 그 결과, 3개월 시점에서의 조기 투여는 신장 기능을 보존했지만, 5개월 이후의 지연 투여는 효과가 없었음을 밝혔다. 이는 병이 진행된 이후에는 치료 효과가 제한됨을 의미한다. 이 연구는 ADCK4가 CoQ10 항상성과 족세포 유지에 필수적이라는 점을 강조하며, CoQ10 결핍이 미토콘드리아 기능 이상과 진행성 사구체 손상을 유도한다는 것을 입증하였다. CoQ10 결핍 신장 오가노이드는 질병 기전을 연구하고 타겟 치료제를 평가할 수 있는 유용한 플랫폼이 될 수 있다. 특히, 조기 투여의 중요성이 부각되며, CoQ10 결핍성 신증의 임상 개입 시기를 설정하는 데 중요한 근거를 제공한다.

족세포에서 고포도당과 싸이클로스포린이 세포사멸에 미치는 영향

정동섭 연세대학교 대학원 2006 국내석사

싸이클로스포린 (CsA)은 신이식 후 사용하는 강력한 면역억제제로 이식신 생존율을 유의하게 호전시켰다. 그러나 CsA을 장기간 사용할 경우 구심성 신소동맥 병변, 세뇨관 위축, 세뇨관-간질성 섬유화, 그리고 사구체경화증 등이 동반되는 만성 CsA 신독성이 발생하게 되는데, 이러한 만성 CsA 신독성의 병인으로는 구심성 세동맥의 수축으로 인한 허혈과 세포사멸 등이 있다. 또한, 세포사멸은 당뇨병성 신병증의 특징 중 하나인 족세포 수의 감소와도 밀접하게 관련되어 있는 것으로 알려져 있다. 당뇨병성 신병증은 전 세계적으로 말기 신부전증의 가장 흔한 원인 질환으로, 당뇨병성 신병증에 의한 말기 신부전증 환자의 증가와 함께 신이식을 받는 당뇨병 환자도 더욱 증가할 것으로 예상된다. 그러나 당뇨병을 동반한 말기 신부전증 환자에서의 이식 후 신생존율은 감염이나 급성 거부반응으로 인하여 비당뇨병 말기 신부전증 환자에 비하여 낮은 것으로 보고되고 있다.이에 본 연구자는 당뇨병 신이식 환자에서의 낮은 신생존율과 족세포 사멸 사이의 연관성을 규명하기 위하여 고포도당과 CsA으로 자극한 족세포에서의 세포사멸을 알아보고자 하였다. 족세포를 33?에서 감마인터페론이 포함된 배양액으로 계대 배양하였으며, 이후 37?에서 감마인터페론이 없는 배양액으로 배양하면서 분화를 시켰다. 족세포의 분화를 확인한 후 우태아혈청이 없는 배양액으로 24시간 동안 배양한 다음, 5.6 mM 포도당 (NG), NG + 24.4 mM 만니톨 (NG + M), NG + 10-6 M CsA (NG + CsA), 30 mM 포도당 (HG), 그리고 HG + 10-6 M CsA (HG + CsA) 배양액으로 72시간 동안 배양하였다. mRNA 발현은 RT-PCR로, 단백 발현은 Western blot로, 그리고 세포사멸은 Hoechst 33258 염색과 FACS를 이용하여 분석하여 다음과 같은 결과를 얻었다.1. 족세포에서 Bax/Bcl-2 mRNA 발현비는 NG + CsA군과 HG + CsA군에서 NG군에 비하여 각각 3배, 2.8배 증가되었으며 (p<0.05), Bax/Bcl-2의 단백 발현비도 NG군에 비하여 NG + CsA군과 HG + CsA군에서 2.2배, 1.7배 증가되었다 (p<0.01). 그러나 고포도당이나 만니톨은 족세포의 Bax와 Bcl-2 mRNA 및 단백 발현에 영향을 미치지 않았다.2. TGF-?] mRNA 및 단백 발현은 NG군과 HG군에 비하여 NG + CsA군과 HG + CsA군에서 의의있게 증가되었다 (p<0.05). 그러나 고포도당이나 만니톨은 족세포의 TGF-?] 발현에 영향을 미치지 않았다.3. Hoechst 33258 염색에 의한 apoptotic cell의 수는 CsA 처치군에서 의의있게 증가되었으며, FACS 분석상으로도 CsA 처치군에서 apoptotic cell의 비율이 유의하게 높게 나타났다 (p<0.01).이상의 결과로 족세포에서 싸이클로스포린이 세포사멸을 증가시켰으며, 싸이클로스포린에 의한 세포사멸이 고포도당 조건 하에서 더 심화되지는 않을 것으로 생각된다 Cyclosporine A (CsA) is a potent immunosuppressant for transplantation and various autoimmune diseases, and has markedly improved the success of organ transplantation. However, the major limiting problem in the long-term use of CsA is chronic nephrotoxicity, a histologic lesion characterized by affterent arteriolopathy, tubular atrophy, tubulointerstitial fibrosis, and finally glomerulosclerosis. The pathogenesis of chronic CsA nephrotoxicity is not fully understood, but several previous studies have suggested that it is attributed to sustained ischemia and apoptosis of tubular cells, mesangial cells, and podocytes. On the other hand, apoptosis of podocytes is also considered one of the pathophysiologic events in diabetic nephropathy, resulting in decrease in podocytes number. Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) in many countries, so more diabetic ESRD patients are supposed to receive a kidney transplant. However, the renal survival rates in diabetic ESRD patients are known to be worse than non-diabetic ESRD patients due to more infection and acute rejection, in which apoptosis is known to be implicated.In this study, we evaluated the combined effect of high glucose and CsA on apoptosis in cultured podocytes to determine whether high glucose and CsA exert synergistic deleterious effects on podocytes, leading to poor renal survival in diabetic transplant patients in clinical field. Conditionally immortalized mouse podocytes were cultured under permissive conditions at 33? with ?-interferon and were subcultured, and then allowed to differentiate at 37? without ?-interferon. After confirming differentiation of podocytes and serum restriction for 24 hours, podocytes were exposed to medium containing 5.6 mM glucose (NG), NG + 24.4 mM mannitol (NG + M), NG + 10-6 M CsA (NG + CsA), 30 mM glucose (HG), or HG + 10-6 M CsA (HG + CsA) for 72 hours. mRNA expression was determined by RT-PCR, protein expression by Western blot, and apoptosis by Hoechst 33258 staining and FACS analysis. The results were as follows;1. Bax/Bcl-2 mRNA expression ratio was significantly increased in NG + CsA and HG + CsA cells by 3.0- and 2.8-fold, respectively, compared to podocytes exposed to NG medium (p<0.05). Bax/Bcl-2 protein ratio was also significantly increased in NG + CsA and HG + CsA cells by 2.2- and 1.7-fold, respectively, compared to NG podocytes (p<0.01). However high glucose and mannitol had no effect on Bax and Bcl-2 mRNA and protein expression.2. TGF-?] mRNA and protein expression were significantly higher in NG + CsA and HG + CsA podocytes than NG cells (p<0.05). However high glucose and mannitol had no effect on TGF-?] expression.3. The numbers of apoptotic cell determined by Hoechst 33258 staining were significantly increased by CsA treatment, and FACS analysis also revealed significantly higher proportion of apoptotic cells in CsA-treated groups.In conclusion, these results suggest that CsA increases apoptosis in podocytes and diabetes status may not affect CsA-induced apoptosis of podocytes.

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.