Search

Background/aims Angiotensin II (Ang II) induces podocyte injury resulting in apoptosis in vitro and in vivo. However, the relationship between autophagy and apoptosis in Ang II-induced podocyte injury is unknown and the role of Ang II-induced autophagy in podocyte survival or death remains unclear. We investigated the sequential relationship between autophagy and apoptosis in Ang II-induced podocytes as well as the role of phosphatidylinositide 3-kinase (PI3-kinase). Methods Mouse podocytes were incubated in media containing various concentrations of Ang II and at different incubation times. The changes of podocyte autophagy and apoptosis were observed by electron microscopy, confocal imaging, western blotting, and FACS assay according to the presence of Ang II. Results Ang II enhanced the podocyte expression of the autophagic proteins, LC3A/B-II and beclin-1, and also increased the number of autophagosomes compared with control cells at early phase of 12 hours in a dose-dependent manner. This effect was inhibited by pretreatment with 3-methyladenine (3-MA), a PI3-kinase class III inhibitor. Thereafter, the Ang II-induced enhancement in autophagy decreased, whereas, podocyte apoptosis appeared later at 24 hours in concentration- and time-dependent manners in FACS and TUNEL assays. 3-MA and LY294002, a pan PI3-kinase inhibitor, further increased Ang II-induced podocyte apoptosis. Suppression of autophagy by Atg5 siRNA could induce podocyte apoptosis and further augment high-dose Ang II-induced podocyte apoptosis. Conclusion These findings suggest that Ang II promotes autophagy in podocytes before apoptosis as an early adaptive cytoprotective mechanism for podocyte survival after Ang II treatment, and the transitional imbalance between autophagy and apoptosis causes podocyte injury.

CD2-associated protein (CD2AP), an adaptor protein, plays several important roles in podocyte function, linking slit diaphragms to actin-based cytoskeleton and sending survival signals. Here, we investigated whether ginseng total saponin (GTS) had a protective role in the changes of podocyte CD2AP protein and podocyte apoptosis under in vitro diabetic conditions. Conditionally immortalized mouse podocytes cultured with normal glucose (5 mM) or high glucose (30 mM) and with or without advanced glycosylation end products were treated with GTS. We found that CD2AP co-localized with the F-actin fibers in podocyte cytoplasm using confocal imaging; however, diabetic conditions caused the podocytes to diminish and conglomerate CD2AP stainings in the peripheral cytoplasm, which were recovered by GTS. Diabetic conditions also suppressed CD2AP protein levels at 6 and 24 h in western blotting. These phenotypical changes of CD2AP protein were mitigated by GTS. Diabetic conditions also induced podocyte apoptosis at 24 h, which were attenuated by GTS. These findings provide a novel mechanism that diabetic conditions induce quantitative and qualitative changes of podocyte CD2AP protein and apoptosis, which would be restored by GTS.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P(meta)=6.71E-28, OR=1.88) and TNFSF15 (P(meta)=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Advances in podocytology and genetic techniques have expanded our understanding of the pathogenesis of hereditary steroid-resistant nephrotic syndrome (SRNS). In the past 20 years, over 45 genetic mutations have been identified in patients with hereditary SRNS. Genetic mutations on structural and functional molecules in podocytes can lead to serious injury in the podocytes themselves and in adjacent structures, causing sclerotic lesions such as focal segmental glomerulosclerosis or diffuse mesangial sclerosis. This paper provides an update on the current knowledge of podocyte genes involved in the development of hereditary nephrotic syndrome and, thereby, reviews genotype-phenotype correlations to propose an approach for appropriate mutational screening based on clinical aspects.

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.

Puromycin aminonucleoside (PAN)-induced nephrosis is a widely studied animal model of human idiopathic nephrotic syndrome because PAN injection into rats results in increased glomerular permeability with the characteristic ultrastructural changes in podocytes similar to human nephrosis. To investigate the role of zonula occludens (ZO)-1 and oxidative stress on PAN-induced podocyte phenotypical changes and hyperpermeability in vitro, we cultured rat and mouse podocytes and treated with various concentrations of PAN. PAN treatment increased oxidative stress level of podocytes significantly with the induction of Nox4. In addition, PAN changed the ultrastructure of podocytes, such as shortening and fusion of microvilli, and the separation of intercellular gaps, which were improved by anti-oxidative vitamin C and Nox4 siRNA. PAN also disrupted the intercellular linear ZO-1 staining and induced inner cytoplasmic re-localization of ZO-1 protein, resulting in increased podocyte intercellular permeability. PAN reduced ZO-1 protein amount and mRNA expression in a dose-dependent manner, which means that PAN could also modulate ZO-1 protein transcriptionally. However, the decreased ZO-1 protein of podocytes by PAN was improved by Nox4 siRNA transfection. Furthermore, vitamin C mitigated the quantitative and distributional disturbances of ZO-1 protein caused by PAN. Our results demonstrate that the phenotypical changes of intercellular ZO-1 by oxidative stress via Nox4 likely contribute to the glomerular hyperpermeability caused by PAN.

Background Proteinuria is typically accompanied by structural and compositional changes of the foot processes and of the slit diaphragms between podocytes. CD2-associated protein (CD2AP) in podocytes serves as an adaptor protein binding to nephrin and podocin, anchoring these slit diaphragm proteins to actin filaments of podocyte cytoskeleton and sending signals inward or outward. Methods In the present study, we prepared streptozotocin-induced diabetic renal tissues and cultured podocytes in diabetic conditions to investigate podocyte phenotypical changes, including quantitative and distributional changes of CD2AP protein and search for the signalling mechanisms in diabetic conditions. We prepared cultured rat glomerular epithelial cells and mouse podocytes to study how high glucose and advanced glycosylation end products (AGE) induce phenotypical changes of cultured podocyte, under (1) normal glucose (5 mM, = control), (2) high glucose (30 mM), (3) AGE-added or (4) high glucose plus AGE-added conditions. Results According to diabetic duration, density of CD2AP in renal tissue of experimental diabetic nephropathy became conglomerulated and diminished. In cultured podocytes, CD2AP co-localized with nephrin and zonula occludens-1 by confocal imaging. High glucose and high glucose plus AGE induced the relocalization and concentration of CD2AP at internal cytoplasmic and perinuclear areas of podocytes. High glucose plus AGE-added condition also decreased CD2AP protein amount and its mRNA expression compared with normal glucose or osmotic control conditions. In addition, LY294002, a phosphoinositide 3-kinase inhibitor, prevented the quantitative and distributional changes of CD2AP induced by high glucose and AGE. Conclusions These findings suggest that diabetic conditions induce the phenotypical changes of podocyte CD2AP possibly via phosphoinositide 3-kinase/Akt signalling. Copyright © 2014 John Wiley & Sons, Ltd.

Background: The actin cytoskeleton in podocytes is essential for the maintenance of its normal structure and function. Its disruption is a feature of podocyte foot-process effacement and is associated with proteinuria. α-Actinin-4 in podocytes serves as a linker protein binding the actin filaments of the cytoskeleton. Methods: To investigate the effect of ginseng total saponin (GTS) on the pathological changes of podocyte α-actinin-4 induced by diabetic conditions, we cultured mouse podocytes under normal glucose (5mM) or high glucose (HG, 30mM) conditions, with or without the addition of advanced glycosylation end products (AGE), and treated with GTS. Results: In confocal imaging, α-actinin-4 colocalized with the ends of F-actin fibers in cytoplasm, but diabetic conditions disrupted F-actin fibers and concentrated α-actinin-4 molecules at the peripheral cytoplasm. GTS upregulated α-actinin protein in a time- and dose-dependent manner, and suppressed the receptor for AGE levels in western blotting. Diabetic conditions, including HG, AGE, and both together, decreased cellular α-actinin-4 protein levels at 24 h and 48 h. Such quantitative and qualitative changes of α-actinin-4 protein induced by diabetic conditions were mitigated by GTS. Conclusion: These findings imply that both HG and AGE have an influence on the distribution and amount of α-actinin-4 in podocytes that can be recovered by GTS.

Hypertrophy is a prominent feature of damaged podocytes in diabetic kidney disease (DKD). mTORC1 hyperactivation leads to podocyte hypertrophy, but the detailed mechanism of how mTORC1 activation occurs under pathological conditions is not completely known. Moreover, reduced nephrin tyrosine phosphorylation has been observed in podocytes under pathological conditions, but the molecular mechanism linking nephrin phosphorylation and pathology is unclear so far. In this study, we observed a significant increase in C1-Ten level in diabetic kidney and in high glucose-induced damaged podocytes. C1-Ten acts as a protein tyrosine phosphatase (PTPase) at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. Furthermore, C1-Ten causes podocyte hypertrophy and proteinuria by increasing mTORC1 activity in vitro and in vivo. These findings demonstrate the relationship between nephrin dephosphorylation and the mTORC1 pathway, mediated by C1-Ten PTPase activity. We suggest that C1-Ten contributes to the pathogenesis of DKD by inducing podocyte hypertrophy under high glucose conditions.

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.

OBJECTIVE AND DESIGN: Interleukin-13 (IL-13) has recently been reported to be a potential cytokine in the pathogenesis of minimal-change nephrotic syndrome (MCNS). However, the mechanistic insights associated with podocyte dysfunction mediated by IL-13-induced changes in various slit diaphragm (SD) and cytoskeletal molecules have not yet been shown in cultured human podocytes in vitro.MATERIALS: Human conditionally immortalized podocytes were used.TREATMENT: Podocytes were incubated with various concentrations of IL-13 during the indicated time periods (6, 12, and 24 h) and montelukast was administered with the dose of 0.1 μg.RESULTS: Treatment of IL-13 resulted in a progressive decrease in distinct processes or projections of the human podocytes and high dose of IL-13 increased podocyte permeability in vitro at 6 h. IL-13 had a substantial impact on the redistribution and rearrangement of zonula occludens (ZO)-1, synaptopodin, α-actinin, CD2-associated protein (CD2AP) in podocytes and disrupted the cytoskeletal connections in a concentration-dependent manner on confocal microscopy. IL-13 also down-modulated ZO-1, synaptopodin, α-actinin, CD2AP, and p130Cas at protein levels and upregulated β-catenin and B7-1 in podocytes. Furthermore, we demonstrated that down-modulated changes in various SD and cytoskeletal structures of human podocytes induced by IL-13 was significantly restored after treatment with montelukast with upregulation of B7-1.CONCLUSION: Our results suggest that targeting IL-13 may be one of the important cytokines in the pathogenesis of MCNS and targeting IL-13 could be one of the potential therapeutic strategies in MCNS.

Proteinuric conditions demonstrate structural and compositional changes of the foot processes and slit diaphragms between podocytes. p130Cas in podocytes serves as an adapter protein anchoring glomerular basement membrane to actin filaments of podocyte cytoskeleton. To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte p130Cas induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, control); 2) high glucose (HG, 30 mM); 3) advanced glycosylation endproducts (AGE)-added; or 4) HG plus AGE-added conditions and treated with GTS. In confocal imaging, p130Cas colocalized with zonula occludens-1 and synaptopodin connecting to F-actin. However, diabetic conditions relocalized p130Cas molecules at perinuclear cytoplasmic area and reduced the intensity of p130Cas. In Western blotting, diabetic conditions, especially HG plus AGE-added condition, decreased cellular p130Cas protein levels at 24 and 48 h. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of p130Cas of podocytes, which can be reversed by GTS.

Purpose Puromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro. Methods We observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction. Results In the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants. Conclusion These findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.

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.

Early diabetic nephropathy is characterized by glomerular hyperpermeability as a result of impaired glomerular filtration structure caused by hyperglycemia, glycated proteins or irreversible advanced glycosylation endproducts (AGE). To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte ZO (zonula occludens)-1 protein and podocyte permeability induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) AGE-added; or 4) HG plus AGE-added conditions and treated with GTS. HG and AGE increased the dextran filtration of monolayered podocytes at early stage (2-8 hr) in permeability assay. In confocal imaging, ZO-1 colocalized with actin filaments and β-catenin at cell contact areas, forming intercellular filtration gaps. However, these diabetic conditions suppressed ZO-1 immunostainings and disrupted the linearity of ZO-1. In Western blotting, diabetic conditions also decreased cellular ZO-1 protein levels at 6 hr and 24 hr. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of ZO-1 protein of podocytes thereby causing hyperpermeability at early stage, which can be reversed by GTS.

Regardless of the underlying disease, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. To investigate how high-glucose (HG) and advanced glycosylation end products (AGE) induce podocyte phenotypical changes, including quantitative and distributional changes of zonula occludens (ZO)-1 protein and search for the signaling mechanisms, we cultured rat glomerular epithelial cells (GEpC) and mouse podocytes under: (1) normal glucose (5 mM, control); (2) HG (30 mM); (3) AGE-added; or (4) HG plus AGE-added conditions. HG plus AGE increased the permeability of monolayered GEpCs and induced ultrastructural separation between confluent GEpCs. ZO-1 moved to inner actin filament complexes in both AGE- and/or HG by confocal imaging. HG plus AGE-added condition also decreased ZO-1 protein amount and mRNA expression compared to normal glucose or osmotic control conditions. We could also confirm the induction of RAGE (receptor for AGE) and PI3-K/Akt signaling pathway by AGE and HG. In addition, LY294002, a PI3-K inhibitor, could prevent the quantitative and distributional changes of ZO-1 and RAGE and the increased permeability induced by HG and AGE. These findings suggest that diabetic conditions induce the podocyte ZO-1 changes via RAGE and PI3-K/Akt signaling, leading to increased permeability.

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.

Background Proteinuria results from increased glomerular permeability and is associated with retraction and effacement of the highly specialized interdigitating podocyte foot processes. In podocytes (glomerular epithelial cells [GECs]), p130Cas localizes diffusely to the cytoplasm and connects focal adhesion proteins and kinases to the glomerular basement membrane and adapter proteins of slit diaphragm insertion sites. Methods To investigate whether high-glucose (HG) and advanced glycosylation end products (AGEs) induce quantitative and distributional changes of podocyte p130Cas protein, a docking protein connecting F-actin fibers to the glomerular basement membrane and adapter proteins, we cultured rat GECs (r-GECs) and mouse GECs (m-GECs) under (i) normal glucose (5 mM = control), (ii) HG (30 mM), (iii) AGE-added or (iv) HG plus AGE-added conditions. We also prepared streptozotocin-induced diabetic renal tissues. Results We found that p130Cas stainings were located in peripheral cytoplasm of r-GECs and m-GECs as dots in linear alignment, partially colocalized with actin-binding proteins such as synaptopodin and alpha-actinin, and locally connected to the ends of actin filaments. In diabetic conditions, the intensities of p130Cas stainings were diminished in more pathological HG plus AGE-added condition of r-GECs and m-GECs and in chronic diabetic renal tissues. p130Cas protein was decreased significantly by HG, AGE and HG plus AGE in both cells compared with normal glucose or osmotic control conditions. p130Cas mRNA expression levels were also suppressed similarly in diabetic conditions. Conclusion We suggest that diabetic conditions modulate the quantitative and distributional changes of podocyte p130Cas and therefore affect the filtration function of podocytes.

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.

Podocytes covering the glomerular basement membrane over the glomerular capillary consist of three morphologically and functionally different segments, the cell body, major processes and extending finger-like foot processes (FPs). The FPs of neighboring podocytes are connected by a continuous adherent junction structure named the slit diaphragm (SD). The extracellular SD is linked to the intracellular, a highly dynamic, cytoskeleton through adaptor proteins. These adaptor proteins, such as CD2-associated protein, zonula occludens 1, β-catenin, Nck and p130Cas, located at the intracellular SD insertion area near lipid rafts, have important structural and functional roles. Adaptor proteins in podocytes play important roles as a structural component of the podocyte structure, linking the SD to the cytoskeletal structure and as a signaling platform sending signals from the SD to the actin cytoskeleton. This review discusses the roles of adaptor proteins in the podocyte cytoskeletal structure and signaling from the SD to the actin cytoskeleton.

High glucose levels can change podocyte gene expression and subsequently induce podocyte damage through altered glucose metabolism. l-Carnitine is known to play a beneficial role in diabetes; however, there are no studies on the effects of l-carnitine on podocyte alteration under high glucose conditions. This study investigated whether l-carnitine can attenuate diabetic podocyte injury through the prevention of loss of slit diaphragm proteins. The l-carnitine treatment group showed increased glucose uptakes compared to the control group, suggesting that glucose utilization in the podocytes was increased by l-carnitine. l-Carnitine treatment also prevented decreased mRNA expressions of nephrin and podocin in the high glucose-stimulated podocytes. However, mRNA expressions of CD2AP and α-actinin-4 were not significantly changed by the high glucose conditions. When these data are taken together, l-carnitine can increase glucose uptake in podocytes under high glucose conditions, and its mechanism may be at least partly related to the up-regulation of nephrin and podocin. Our results help clarify the beneficial effects of l-carnitine in diabetic nephropathy.

AIM: To investigate the molecular basis for the phenotypic alterations of glomerular epithelial cells (GEpC, podocytes), involving cytoskeletal changes especially on alpha-actinin-4 as a candidate regulating the barrier to protein filtration and the podocyte actin cytoskeleton. METHODS: To examine the effects of glucose and advanced glycosylated end-products (AGE) on alpha-actinin-4, the author cultured rat GEpC on AGE- or BSA-coated plates under normal (5 mmol) and high glucose (30 mmol) conditions and examined the distribution of alpha-actinin by confocal microscope and measured the change in alpha-actinin-4 production by western blotting and reverse transcription-polymerase chain reaction. RESULTS: Confocal microscopy indicated that alpha-actinin-4 moved from the peripheral cytoplasm to inner actin filament complexes in the presence of AGE and high glucose. These changes might be related to the fusion of microvilli of cell surface examined by electron microscopy. In western blot analysis, AGE significantly decreased the amount of alpha-actinin by 28.1%. Furthermore, the combination of high glucose and AGE decreased the amount of alpha-actinin more significantly by 53.6% compared with that of the control. The mRNA expression for alpha-actinin-4 was not changed with high glucose or AGE-coated surfaces; however, when added, the combination of high glucose and AGE significantly decreased the expression of alpha-actinin-4 mRNA by 15.7% compared with that of the control. CONCLUSION: The author suggests that both high glucose and AGE (either individually or in combination) induce the cytoplasmic translocation and the combination suppresses the production of alpha-actinin-4 at the transcriptional level with post-translational modification and these in vitro changes may explain the cytoskeletal changes of GEpC in diabetic conditions.

목적: 단백뇨 질환의 실험모델인 puromycin aminonucleoside (PAN)에서 관찰할 수 있는 족세포의 병리학적 이상에 있어서 P-cadherin의 변화를 생체 외 족세포 배양실험을 통하여 알아보고자 하였다. 방법: PAN에 의한 족세포의 P-cadherin의 변화를 생체 외 배양실험을 통해 알아보고자 백서 사구체 상피세포(GEpC)를 배양하여 다양한 농도의 PAN을 투여하여 confocal 현미경을 통하여 P-cadherin의 분포를 관찰하였고, Western blotting 과 RT-PCR을 사용하여 P-cadherin 발현의 변화를 관찰하였다. 결과: 외곽세포질에 분포하는 P-cadherin은 단일세포 혹은 응집환경에서 PAN의 농도가 올라갈수록 내부로 응집되는 현상를 볼 수 있었다. Western 분석에서, P-cadherin 단백양은 PAN에 농도-의존적으로, 특히, 고농도인 50 mg/mL에서 24시간과 48시간이 노출 조건에서 각각 21.9% (P 【Purpose: To test whether the expression of P-cadherin, a component of slit diaphragms between podocyte foot processes, would be altered by puromycin aminonucleoside (PAN) in a cultured podocyte in vitro. Methods: Rat glomerular epithelial cells (GEpC) were cultured with various concentrations of PAN. The distribution of P-cadherin was examined with a confocal microscope. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to measure the change in P-cadherin expression. Results: This study found that P-cadherin was concentrated in the inner and peripheral cytoplasm with high concentrations of PAN under immunofluorescence views. Western blotting of GEpC revealed that PAN induced a decrease of P-cadherin in dose- and time-dependent manners. A high dose ( $50{\mu}g/mL$ ) of PAN decreased P-cadherin expression by 21.9% at 24 h (P $50{\mu}g/mL$ ) of PAN also decreased P-cadherin mRNA expression, similar to protein suppression, by 23.5% at 48 h (P】

Purpose : This study was designed to clarify the mechanism of proteinuria in nephrotic syndrome patients by using puromycin aminonucleoside (PAN) nephrosis model. Methods : Following administration of various concentrations of PAN and antioxidants we observed the changes of podocyte cytoskeletons in cultured rat glomerular epithelial cells (GEpC) by method of scanning electron microscope, reactive oxyten species (ROS) analysis, permeability assay, confocal microscope, and Western blot assay. Results : PAN not only induced the ultrastructural changes of GEpC, such as shortening and fusion of microvilli, but also separated the intercellular gaps and linear ZO-1. PAN induced oxidative stresses in time and dose dependent manners and increases of intercellular permeability in anti-oxidants inhibitable manners. High concentration of PAN induced not only actin polymerization and disorganization, but also the conglomerulation and internal dislocation of α-actinin protein. The intensities of fluorescences of ZO-1 protein were diminished and internalized by PAN in a dose-dependent manner, which were inhibited by anti anti-oxidants. Conclusion : PAN induced the changes of podocytes cytoskeleton and junctional barriers by way of increasing ROS in GEpC that resulted in increasing their permeability in a antioxidatn-inhibitable manner. Glomerular hyperpermeability induced by PAN mediateing through oxidative stresses is thought to take part in the mechanism of proteinuria in nephrotic syndrome.