ELSEVIER : Ubiquitination-dependent CARM1 degradation facilitates Notch1-mediated podocyte apoptosis in diabetic nephropathy

( Dong Gil Kim ),( Seulki Lim ),( Min Jung Park ),( Joo Hee Choi ),( Jong Choon Kim ),( Ho Jae Han ),( Kyung Chul Yoon ),( Kwon Seop Kim ),( Jae Hyang Lim ),( Soo Hyun Park ) 전남대학교 약품개발연구소 2014 약품개발연구지 Vol.23 No.-

Podocyte apoptosis induced by hyperglycemia is considered a critical factor in the development of diabetic nephropathy. Recent studies have implicated Notch signaling in podocyte apoptosis; however, its regulatory mechanisms are not fully understood. In this study. we found that high-glucose treatment increased Notchl and jagged-l expression, the transcriptional activity of Hes. and podocyte apoptosis, and decreased the expression of coactivator-associated arginine methyltransferase 1 (CARMI) in rat podocytes. Transient transfection of CARMI reversed high-glucose-induced Notchl expression. the transcriptional activity of Hes, and podocyte apoptosis. Moreover. the silendng of CARMI using siRNA increased Notchl expression. the transctiptional activity of Hes, and podocyte apoptosis. However, the Glu266-mediated enzymatic activity of CARMI was not necessary for Notch signaling activation and podocyte apoptosis. Here, we demonstrate that AMP-activated protein kinase alpha (AMPKa) and cannabinoid receptor 1 (CB,R) are regulated by CARMI and that high-glucose-induced podocyte apoptosis is mediated by a CARMl-AMPKa-Notchl-CB,R signaling axis. We also show that highglucose-induced CARMI downregulation is due to ubiquitination-dependent CARMI degradation. Finally, we demonstrate that CARMI expression in podocytes was diminished in rats with streptozotocin-induced diabetes compared to vehicle-treated rats. Together, our data provide evidence that ubiquitination-dependent CARMI degradation in podocytes in diabetes promotes podocyte apoptosis via Notch 1 activation. Strategies to preserve CARMI expression or reduce the enzymatic activity of a ubiquitin ligase specific for CARMI could be used to prevent podocyte loss in diabetic nephropathy. ⓒ2014 Published by Elsevier lnc.

Effects of Angiotensin II on Podocyte Synaptopodin: Role of AMP Kinase

하태선,박혜영,최지영,남자애,안희열 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.4

AMP-activated protein kinase (AMPK), as a sensor of cellular energy status, plays an important role in the pathophysiology of diabetes and its complications. As AMPK is also expressed in podocytes, podocyte AMPK would be an important contributing factor in the development of podocyte injury. We investigated the roles of AMPK in the pathological changes of podocyte synaptopodin induced by angiotensin II (Ang II), a major injury inducer. Mouse podocytes were incubated in media containing various concentrations of Ang II and AMPK-modulating agents, and the changes of synaptopodin were analyzed by confocal imaging and Western blotting. Ang II and compound C, an AMPK inhibitor, concentrated and re-localized synaptopodin from peripheral cytoplasm to internal cytoplasm portion in podocytes. Ang II also reduced synaptopodin protein and mRNA, which were reversed by metformin and 5-aminoimidazole-4-carboxamide ribonucleoside. Losartan, an Ang II type 1 receptor antagonist, also recovered synaptopodin mRNA, which was suppressed by Ang II. We suggest that Ang II induces the relocation and suppression of podocyte synaptopodin by the suppression of AMPK and via Ang II type 1 receptor, which would be an important mechanism in Ang II-induced podocyte phenotypical changes.

Current concepts of the podocyte in nephrotic syndrome

( Wen Y. Ding ),( Moin A. Saleem ) 대한신장학회 2012 Kidney Research and Clinical Practice Vol.31 No.2

Nephrotic syndrome is a disorder of the glomerular filtration barrier, and central to the filtration mechanism of the glomerular filtration barrier is the podocyte. We are starting to better understand how this cell, with its unique architectural features, fulfils its exact filtration properties. The multiprotein complex between adjacent podocyte foot processes, the slit diaphragm, is essential to the control of the actin cytoskeleton and cell morphology. Many of the proteins within the slit diaphragm, including nephrin, podocin, transient receptor potential-6 channel, and a-actinin-4, have been identified via genetic studies of inherited nephrotic syndromes. Signaling from slit diaphragm proteins to the actin cytoskeleton is mediated via the Rho GTPases. These are thought to be involved in the control of podocyte motility, which has been postulated as a focus of proteinuric pathways. Nephrotic syndrome is currently treated with immunosuppressive therapy, with significant adverse effects. These therapies may work in nephrotic syndrome due to specific effects on the podocytes. This review aims to describe our currentunderstanding of the cellular pathways and molecules within the podocyte relevant to nephrotic syndrome and its treatment. With our current knowledge of the cellular biology of the podocyte, there is much hope for targeted therapies for nephrotic syndromes.

Effects of Angiotensin II on Podocyte Synaptopodin: Role of AMP Kinase

Tae-Sun Ha, Hye-Young Park, Ji-Young Choi, Ja-Ae Nam, Hee-Yul Ahn 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.4

As a sensor of cellular energy status, AMP-activated protein kinase (AMPK) plays an important role in the pathophysiology of diabetes and its complications. Because AMPK is also expressed in podocytes, podocyte AMPK would be an important factor contributing to development of podocyte injury. We investigated the roles of AMPK in the pathological changes of podocyte synaptopodin induced by angiotensin II (Ang II), a major injury inducer. Mouse podocytes were incubated in media containing various concentrations of Ang II and AMPK-modulating agents, and the changes of synaptopodin were analyzed by confocal imaging and Western blotting. Ang II and compound C, an AMPK inhibitor, concentrated and re-localized synaptopodin from peripheral cytoplasm to the internal cytoplasm portion in podocytes. Ang II also reduced synaptopodin protein and mRNA, which were reversed by metformin and 5-aminoimidazole-4-carboxamide ribonucleoside. Losartan, an Ang II type 1 receptor antagonist, also recovered synaptopodin mRNA, which was suppressed by Ang II. We suggest that Ang II induces the relocation and suppression of podocyte synaptopodin by suppression of AMPK and via Ang II type 1 receptor, which would be an important mechanism in Ang II-induced podocyte phenotypical changes.

Angiotensin II induces endoplasmic reticulum stress in podocyte, which would be further augmented by PI3-kinase inhibition

하태선,박혜영,성수빈,안희열 대한고혈압학회 2015 Clinical Hypertension Vol.21 No.12

Introduction: Angiotensin II (Ang II) contributes to the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. On renal effects, Ang II plays an important role in the development of proteinuria and glomerulosclerosis by the modification of podocyte molecules and cell survival. In the present study, we investigated the effect of Ang II on endoplasmic reticulum (ER) stress in podocytes. Methods: We cultured mouse podocytes with increasing doses of Ang II and evaluated ER stress markers by Western blotting. Results: Ang II increased Bip protein, an ER chaperone, in a dose-dependent manner at 24 h, which was ameliorated by losartan, an angiotensin II type 1 receptor antagonist. Ang II also increased ER stress markers, such as phospho-PERK, phospho-eIF2α, and ATF4 proteins of podocyte, significantly in a dose-dependent manner at 24 h. Increased phospho-PERK and ATF4 proteins were further augmented by phosphoinositide 3 (PI3)-kinase inhibitor, LY294002, which suggested that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway. Discussion: These studies suggest that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway, which would contribute to the development of podocyte injury induced by Ang II, and the augmentation of PI3-kinase would be a therapeutic target.

Diabetic conditions modulate the adenosine monophosphate-activated protein kinase of podocytes

( Tae Sun Ha ),( Hye Young Park ),( Ja Ae Nam ),( Gi Dong Han ) 대한신장학회 2014 Kidney Research and Clinical Practice Vol.33 No.1

Background: Adenosine monophosphate-activated protein kinases (AMPKs), as a sensor of cellular energy status, have been known to play an important role in the pathophysiology of diabetes and its complications. Because AMPKs are known to be expressed in podocytes, it is possible that podocyte AMPKs could be an important contributing factor in the development of diabetic proteinuria. We investigated the roles of AMPKs in the pathological changes in podocytes induced by high-glucose (HG) and advanced glycosylation end products (AGEs) in diabetic proteinuria. Methods: We prepared streptozotocin-induced diabetic renal tissues and cultured rat and mouse podocytes under diabetic conditions with AMPK-modulating agents. The changes in AMPKα were analyzed with confocal imaging and Western blotting under the following conditions: (1) normal glucose (5mM, =control); (2) HG (30mM); (3) AGE-added; or (4) HG plus AGE-added. Results: The density of glomerularphospho-AMPKα in experimental diabetic nephropathy decreased as a function of the diabetic duration. Diabetic conditions including HG and AGE changed the localization of phospho-AMPKα from peripheral cytoplasm to internal cytoplasm and peri- and intranuclear areas in podocytes. HG reduced the AMPKα (Thr172) phosphorylation of rat podocytes, and similarly, AGEs reduced the AMPKα (Thr172) phosphorylation of mouse podocytes. The distributional and quantitative changes in phospho-AMPKα caused by diabetic conditions were preventable using AMPK activators, metformin, and 5-aminoimidazole-4-carboxamide-1β-riboside. Conclusion: We suggest that diabetic conditions induce the relocation and suppression of podocyte AMPKα, which would be a suggestive mechanism in diabetic podocyte injury.

일측요관폐쇄 생쥐에서 발생된 족세포의 세포자멸사와 TGF β1-LAP의 역할

이동원 ( Dong Won Lee ),강미선 ( Mi Seon Kang ),강선우 ( Sun Woo Kang ),김양욱 ( Yang Wook Kim ),김영훈 ( Yeong Hoon Kim ) 대한신장학회 2008 Kidney Research and Clinical Practice Vol.27 No.3

목적: TGF-β1이 신장에서 간질 섬유화와 족세포의 손상을 일으키는 데 중요한 역할을 함이 잘 알려져 있다. 본 저자들은 일측요관폐쇄로 신섬유화를 일으킨 실험동물에서 복강내 TGF-β1 LAP의 투여로 신섬유화와 족세포의 세포 자멸사를 막을 수 있는지를 알아보았다. 방법: 24 마리의 생쥐에게 좌측 요관 결찰술을 시행하였다. 그 중 12마리의 생쥐와 수술 받지 않은 8마리 중 4마리의 생쥐에게 TGF-β1 LAP를 복강내로 주사하였다. 수술 후 3, 7, 14, 21일에 생쥐들을 수술군과 비수술군 각각 3마리, 1마리씩 희생시켜 좌측 신장의 조직을 얻었다. 조직학적 검사를 하여 족세포의 세포자멸사를 관찰하였다. 결과: 신우의 확장과 간질 섬유화는 시간이 경과함에 따라 비례하여 증가됨이 관찰되었다. LAP를 투여한 실험군에서는 대조군에 비해 족세포의 세포 자멸사가 적었고, WT-1의 발현도 유지되었다. 또한 실험군에서 대조군에 비해 단백뇨도 적었다. 결론: 유전자 재조합된 LAP의 복강 내 투여는 요관 결찰술을 받은 생쥐의 14일까지의 TGF-β1의 발현을 일부 억제하고 신장의 손상을 지연시키며 족세포의 소실을 막을 수 있었다. Purpose: It was well known that transforming growth factor (TGF)-β1 plays a pivotal role in interstitial fibrosis and loss of podocyte. We explored the effects of exogenous administration of TGF-β1 latency-associated peptide (LAP) in a model of renal fibrosis induced by unilateral ureteral obstruction (UUO) and examined whether TGF-β1 LAP can inhibit apoptosis of podocyte. Methods: Twenty four male BALB/c mice were unilaterally obstructed of proximal ureters by ligation. Half of the mice with operation and half of 8 control were administered recombinant human LAP intraperitoneally. One to three mice per group were euthanized on days 3, 7, 14, and 21 after operation for observation of renal fibrosis and apoptosis of podocyte. Results: Interstitial fibrosis was less severe in LAP-treated group. Obstructed kidneys from LAP-untreated mice had more glomerular apoptotic podocytes (TUNEL assay) compared to LAP-treated mice at day 7, 14, and 21 after operation. Conclusion: Intraperitoneal administration of TGF-β1 LAP prevents the loss of podocyte & renal damage partially up to day 14 after operation.

Ginseng total saponin regulates podocyte P-cadherin/β-catenin unit in diabetic condition

Tae-Sun Ha, Jin-Seok Lee, Hye-Young Park, Ji-Young Choi, Ja-Ae Nam 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.3

Proteinuric conditions demonstrate structural and compositional changes of the foot processes and slit diaphragms between podocytes. β-Catenin in podocytes serves as an adapter protein anchoring P-cadherin of slit diaphragms to actin filaments of the podocyte cytoskeleton. To investigate the effect of ginseng total saponin (GTS) on pathologic changes of podocyte P-cadherin/β-catenin unit induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) advanced glycosylation end products (AGE)-added; or 4) HG plus AGE-added conditions and treated with GTS. In confocal imaging, β-catenin colocalized with P-cadherin predominantly at intercellular junction area. However, diabetic conditions relocalized and concentrated both molecules at perinuclear cytoplasmic area. In Western blotting, diabetic conditions, especially HG plus AGE-added condition, also decreased cellular β-catenin protein levels at 6, 24, and 48 hours. GTS improved such quantitative and qualitative changes of β-catenin. These findings imply that HG plus AGE have an influence on the redistribution and amount of P-cadherin/ β-catenin unit of podocytes, which can be reversed by GTS.

Nonimmunologic targets of immunosuppressive agents in podocytes

( Tae Hyun Yoo ),( Alessia Fornoni ) 대한신장학회 2015 Kidney Research and Clinical Practice Vol.34 No.2

Proteinuria is a characteristic finding in glomerular diseases and is closely associated with renal outcomes. In addition, therapeutic interventions that reduce proteinuria improve renal prognosis. Accumulating evidence has demonstrated that podocytes act as key modulators of glomerular injury and proteinuria. The podocyte, or glomerular visceral epithelial cell, is a highly specialized and differentiated cell that forms interdigitated foot processes with neighboring podocytes, which are bridged together by an extracellular structure known as the “slit diaphragm” (SD). The SD acts as a size- and charge-selective barrier to plasma protein. Derangement of SD structure or loss of SD-associated protein results in podocyte injury and proteinuria. During the past decades, several immunemodulating agents have been used for the treatment of glomerular diseases and for the reduction of proteinuria. Interestingly, recent studies have demonstrated that immunosuppressive agents can have a direct effect on the SD-associated proteins and stabilize actin cytoskeleton in podocyte and have therefore introduced the concept of nonimmunologic mechanism of renoprotection by immunomodulators. This review focuses on the evidence that immuno-modulating agents directly target podocytes.

Ginseng total saponin regulates podocyte P-cadherin/β-catenin unit in diabetic condition

Jin-Seok Lee,Hye-Young Park,Ji-Young Choi,Ja-Ae Nam,하태선 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.3

Proteinuric conditions demonstrate structural and compositional changes of the foot processes and slit diaphragms between podocytes. β-Catenin in podocytes serves as an adapter protein anchoring P-cadherin of slit diaphragms to actin filaments of podocyte cytoskeleton. To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte P-cadherin/β-catenin unit induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) advanced glycosylation end products (AGE)-added; or 4) HG plus AGE-added conditions and treated with GTS. In confocal imaging, β-catenin colocalized with P-cadherin at intercellular junction area, predominantly. However, diabetic conditions relocalized and concentrated both molecules at perinuclear cytoplasmic area. In Western blotting, diabetic conditions, especially HG plus AGE-added condition, also decreased cellular β-catenin protein levels at 6, 24, and 48 hours. GTS improved such quantitative and qualitative changes of β-catenin. These findings imply that HG plus AGE have an influence on the redistribution and amount of P-cadherin/β-catenin unit of podocytes, which can be reversed by GTS.