Letteratura scientifica selezionata sul tema "Glomerular parietal epithelial cell"

Glomerular epithelial cells (GEC) in culture are derived from intact isolated glomeruli. Although there is general agreement about distinguishing GEC from mesangial and endothelial cells, there is still uncertainty regarding the visceral versus parietal origin of cultured GEC. If these cells are to provide a useful model system, it is necessary to establish well-defined cell populations. The purpose of this study was to evaluate this important issue by comparing the characteristics of cultured GEC with glomerular epithelium from rat kidney sections. By electron microscopy, GEC were polygonal, with cilia and desmosomes between cells, similar to parietal cells in situ. Because intermediate filaments are specifically expressed in differentiated cells in the kidney, the expression of intermediate filaments in cultured GEC were compared with those of intact glomeruli. Cultured GEC are positive for cytokeratin and negative for vimentin and desmin, identical to parietal cells in situ. In contrast, podocytes are positive for vimentin and desmin and negative for cytokeratin. In vivo, podocytes express gp330 and puromycin-aminonucleoside (PAN) susceptibility, which are used as markers for cultured GEC. Immunoperoxidase staining of rat kidney sections with monoclonal anti-gp330 demonstrated gp330 localization to the cell surface and coated pits of the parietal cells, similar to its localization in podocytes. The presence of gp330 in cultured GEC was confirmed by immunoblot. PAN administration to rats induced vacuolization and detachment from the basement membrane in the parietal cells of Bowman's capsule, similar to the cytotoxicity observed in podocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex hormones help in maintaining proper immunity as well as renal homeostasis in mammals, and these multi-functional properties characterize the onset of sex-dependent diseases. To clarify the contribution of sex hormones to autoimmune disease-related renal pathogenesis, BXSB/MpJ- Yaa was investigated as a murine autoimmune glomerulonephritis model. BXSB/MpJ- Yaa and its wild-type, BXSB/MpJ- Yaa+ were castrated or sham-operated at three weeks and examined until six months of age. Both castrated strains showed significantly lower serum testosterone levels and body weights than sham-operated mice. Castration did not change the disease phenotypes in BXSB/MpJ- Yaa+. At three months, both sham-operated and castrated BXSB/MpJ- Yaa manifested splenomegaly, autoantibody production, and glomerulonephritis, and castrated BXSB/MpJ- Yaa tended to show heavier spleen weights than the sham-operated group. At six months, both the treated BXSB/MpJ- Yaa showed equivalent autoimmune disease conditions; however, castrated mice clearly showed milder glomerular sclerotic lesions than the sham-operated groups. Urinary albumin excretion in castrated BXSB/MpJ- Yaa was significantly milder than in sham-operated mice at four months, but those of both the treated BXSB/MpJ- Yaa were comparable at six months. The examined renal histopathological indices in parietal epithelial cells were remarkably altered by castration. Briefly, castration decreased the height of parietal epithelial cells and total parietal epithelial cell number in BXSB/MpJ- Yaa at six months. For immunostaining, parietal epithelial cells facing the injured glomeruli of BXSB/MpJ- Yaa expressed CD44, an activated parietal epithelial cell marker, and CD44-positive parietal epithelial cells showed nuclear localization of the androgen receptor and proliferation marker Ki67. CD44- or Ki67-positive parietal epithelial cells were significantly fewer in castrated group than in sham-operated BXSB/MpJ- Yaa at six months. Further, quantitative indices for CD44-positive parietal epithelial cell number and frequency in renal corpuscles positively correlated with glomerular sclerotic severity in BXSB/MpJ- Yaa. In conclusion, androgen seemed to have an effect on both systemic immunity and renal morpho-function; however, the effect on the latter could be more clearly observed in BXSB/MpJ- Yaa, as parietal epithelial cell activation resulted in glomerular sclerosis.

Kidney aging is accompanied by characteristic changes in the glomerulus, but little is known about the effect of aging on glomerular parietal epithelial cells (PECs), nor if the characteristic glomerular changes in humans and rats also occur in very old mice. Accordingly, a descriptive analysis was undertaken in 27-mo-old C57B6 mice, considered advanced age. PEC density was significantly lower in older mice compared with young mice (aged 3 mo), and the decrease was more pronounced in juxtamedullary glomeruli compared with outer cortical glomeruli. In addition to segmental and global glomerulosclerosis in older mice, staining for matrix proteins collagen type IV and heparan sulfate proteoglycan were markedly increased in Bowman's capsules of older mouse glomeruli, consistent with increased extracellular matrix production by PECs. De novo staining for CD44, a marker of activated and profibrotic PECs, was significantly increased in aged glomeruli. CD44 staining was more pronounced in the juxtamedullary region and colocalized with phosphorylated ERK. Additionally, a subset of aged PECs de novo expressed the epithelial-to-mesenchymal transition markers α-smooth muscle and vimentin, with no changes in epithelial-to-mesenchymal transition markers E-cadherin and β-catenin. The mural cell markers neural/glial antigen 2, PDGF receptor-β, and CD146 as well as Notch 3 were also substantially increased in aged PECs. These data show that mice can be used to better understand the aging kidney and that PECs undergo substantial changes, especially in juxtamedullary glomeruli, that may participate in the overall decline in glomerular structure and function with advancing age.

Abstract. This study examined six cases of idiopathic nephrotic syndrome with primary lesions of focal segmental glomerulosclerosis (FSGS) that relapsed after renal transplantation. The glomerular lesions comprised the cellular, the collapsing, and the scar variants of FSGS and showed shedding of large round cells into Bowman's space and within the tubular lumens. Immunohistochemistry and confocal laser microscopy carried out on kidneys with FSGS relapse disclosed several phenomena. (1) Some podocytes that expressed podocalyxin, synaptopodin, and glomerular epithelial protein-1 were detached from the tuft and were free in the urinary space. (2) In the cellular variant, most podocytes had lost podocyte-specific epitopes (podocalyxin, synaptopodin, glomerular epithelial protein-1, Wilm's tumor protein-1, complement receptor-1, and vimentin). In the scar variant, these podocyte markers were absent from cobblestone-like epithelial cells and from pseudotubules. (3) Podocytes had acquired expression of various cytokeratins (CK; identified by the AE1/AE3, C2562, CK22, and AEL-KS2 monoclonal antibodies) that were not found in the podocytes of control glomeruli. Parietal epithelial cells expressed AE1/AE3 CK that were faintly, if ever, found on the parietal epithelial cells of normal glomeruli. (4) Numerous cells located at the periphery of the tuft or free in Bowman's space and within tubular lumens expressed macrophagic epitopes (identified by PGM1 [CD68], HAM56, and 25F9 monoclonal antibodies). These macrophage-like cells expressed the activation epitopes HLA-DR and CD16. (5) A number of these cells coexpressed podocalyxin + AE1/AE3 CK, podocalyxin + CD68, and CD68 + AE1/AE3. These findings suggest that in primary FSGS relapsing on transplanted kidneys, some “dysregulated” podocytes, occasionally some parietal epithelial cells, and possibly some tubular epithelial cells undergo a process of transdifferentiation. This process of transdifferentiation was especially striking in podocytes that acquired macrophagic and CK epitopes that are absent from normal adult and fetal podocytes.

Mitotic spindle assembly checkpoint protein 2 (MAD2B), a well-known anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase-ζ, is critical for mitotic control and DNA repair. Previously, we detected a strong increase of MAD2B in the glomeruli from patients with crescentic glomerulonephritis and anti-glomerular basement membrane (anti-GBM) rats, which predominantly originated from activated parietal epithelial cells (PECs). Consistently, in vitro MAD2B was increased in TNF-α-treated PECs, along with cell activation and proliferation, as well as extracellular matrix accumulation, which could be reversed by MAD2B genetic depletion. Furthermore, we found that expression of S phase kinase-associated protein 2 (Skp2), an APC/CCDH1 substrate, was increased in the glomeruli of anti-GBM rats, and TNF-α-stimulated PECs and could be suppressed by MAD2B depletion. Additionally, genetic deletion of Skp2 inhibited TNF-α-induced PEC activation and dysfunction. Finally, TNF-α blockade or glucocorticoid therapy administered to anti-GBM rats could ameliorate MAD2B and Skp2 accumulation as well as weaken PEC activation. Collectively, our data suggest that MAD2B has a pivotal role in the pathogenesis of glomerular PEC activation and crescent formation through induction of Skp2 expression.

We previously reported that in a model of spontaneously progressive glomerular injury with early podocyte loss, abnormal migration, and proliferation of glomerular parietal epithelial progenitor cells contributed to the formation of synechiae and crescentic lesions. Here we first investigated whether a similar sequence of events could be extended to rats with adriamycin (ADR)-induced nephropathy. As a second aim, the regenerative potential of therapy with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells was evaluated. In ADR-treated rats, decrease of WT1+ podocyte number due to apoptosis was associated with reduced glomerular expression of nephrin and CD2AP. As a consequence of podocyte injury, glomerular adhesions of the capillary tuft to the Bowman's capsule were observed, followed by crescent-like lesions and glomerulosclerosis. Cellular components of synechiae were either NCAM+ parietal progenitor cells or nestin+ podocytes. In ADR rats, repeated injections of MSCs limited podocyte loss and apoptosis and partially preserved nephrin and CD2AP. MSCs attenuated the formation of glomerular podocyte-parietal epithelial cell bridges and normalized the distribution of NCAM+ progenitor cells along the Bowman's capsule, thereby reducing glomerulosclerosis. Finding that MSCs increased glomerular VEGF expression and limited microvascular rarefaction may explain the prosurvival effect by stem cell therapy. MSCs also displayed anti-inflammatory activity. Coculture of MSCs with ADR-damaged podocytes showed a functional role of stem cell-derived VEGF on prosurvival pathways. These data suggest that MSCs by virtue of their tropism for damaged kidney and ability to provide a local prosurvival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.

ClC-5 with megalin (LRP2), cubilin, Disabled 2 (Dab2), and Amnionless (AMN), is part of the molecular complex involved at proximal tubular level in the endocytic re-uptake of low-molecular-weight proteins and albumin. ClC-5, megalin and cubilin expression in podocytes of human renal biopsies was already reported. Moreover, it was demonstrated that podocytes are able to internalize albumin through an endocytic process. It is reasonable to assume that there should be an involvement of this system in protein uptake mediated by podocytes. A disruption of this system can lead to proteinuria which is one of the first manifestation of kidney disease in Systemic Lupus Erythematous (SLE). Aims of this study were to explore the presence of the tubular endocytic machinery components also in human podocytes in vitro and to evaluate whether albumin modulates this system. Moreover, we wanted to verify and quantify the expression of ClC-5, megalin and cubilin in both glomerular and tubular compartments in renal biopsies of patients presenting Lupus nephritis and observing the presence of a relationship with clinical data. To verify the presence of an uptake mechanism in human podocytes in vitro, we performed time lapse experiments with a low dose of FITC albumin (10 µg/ml). We observed albumin internalization starting from 2 to 15 hours. To evaluate the uptake kinetic, we stimulated podocytes at different time (30 min and 2 hours) and doses (10 µg/ml, 100 µg/ml and 1 mg/ml) at 37°C and 4°C. We observed a significant dose dependent increase in fluorescence vs controls after 2 hours’ stimulation with a typical receptor-mediated kinetic since it is inhibited at 4 °C. Moreover, we disclosed the presence of ClC-5, Dab2 and AMN beyond megalin and cubilin, in this in vitro system using immunohistochemistry (IHC) and immunofluorescence (IF) techniques, highlighting a co-localization between albumin and both receptors. To analyze whether the proteinuric environment modulates CLCN5, LRP2, CUBN, DAB2 and AMN expression, we stimulated human podocytes with increasing concentrations of BSA (range 10 µg/ml - 30 mg/ml) and we evaluated the mRNA expression at different time points (2, 4, 8, 24, 48 and 72 hours). Using Real Time PCR we observed a significant time and dose dependent increase in CLCN5, CUBN and AMN expression and an up-regulation of DAB2 only at 24 hours. We collected 23 SLE renal biopsies, 6 control biopsies and 1 case of Minimal Change Disease. As clinical parameters we considered proteinuria and pharmacological therapy. IHC and IF were used to analyze ClC-5, megalin and cubilin protein expression in serial sections. Morphometric quantification revealed a direct correlation between tubular and glomerular expression of all molecules in SLE patients, highlighting a relationship between glomerular and tubular compartments independently from proteinuria levels. Furthermore, preliminary data on patients without ACEi/ARB and immunosuppressive drugs disclosed a positive trend among these molecules at glomerular level. Interestingly, we revealed megalin and cubilin expression in hypertrophic PECs of some SLE patients and characterization experiments identified a subpopulation with an intermediate phenotype between mature and progenitor cells. In conclusion, for the first time we demonstrated that human podocytes are naturally committed to perform albumin endocytosis via a receptor-mediated mechanism. Moreover, protein overload upregulates CUBN, AMN and CLCN5 in these cells. Functional studies regarding the role of cubilin in albumin uptake underlined its participation in this mechanism even if this is not the only pathway involved. Further studies will be necessary to analyze which may be the partner(s) of the CUBAM complex in this mechanism. For the first time we demonstrated the presence of ClC-5, megalin and cubilin in glomeruli of patients with SLE and MCD in addition to controls confirming in vitro data. Furthermore, in SLE biopsies we highlighted a strong correlation between the two renal compartments in the expression of the protein uptake system, supporting the idea of a partnership between tubular and glomerular cells in albumin uptake via the same mechanism of internalization. In addition, preliminary data on patients without ACEi/ARB and immunosuppressive drugs lead us to suppose that the pharmacological therapy could affect the expression of this system, in particular at glomerular level. The differences observed from in vivo and in vitro data, especially on megalin expression, underline the involvement of other glomerular cell types in addition to podocytes in protein uptake. Finally, megalin and cubilin expression in PECs of SLE patients is a real interesting but complex data, since characterization experiments identified a subpopulation with an intermediate phenotype between mature and progenitor cells. Further studies will be performed to better characterize the role of these double-positive cells and their correlation with clinical data and/or disease progression.
ClC-5, megalina (LRP2), cubilina, Disabled 2 (Dab2) ed Amnionless (AMN) fanno parte del complesso molecolare coinvolto a livello del tubulo prossimale nel recupero delle proteine a basso peso molecolare e dell’albumina mediante endocitosi. È già stata riportata la presenza di ClC-5, megalina e cubilina a livello dei podociti in biopsie renali umane. Inoltre è stato dimostrato che i podociti sono in grado di internalizzare l’albumina attraverso un meccanismo di endocitosi. Il mancato funzionamento di questo sistema può portare a proteinuria, che è una delle prime manifestazioni del coinvolgimento renale nel Lupus Eritematoso Sistemico (LES). È quindi ragionevole supporre che vi possa essere un coinvolgimento di questo sistema nel meccanismo di uptake delle proteine da parte dei podociti. Gli scopi di questo studio sono stati di esplorare la presenza dei componenti del sistema tubulare di endocitosi delle proteine in podociti umani in coltura e di valutare se e come l’albumina ne modulasse l’espressione. Inoltre, si è voluto indagare l’espressione di ClC-5, megalina e cubilina sia a livello glomerulare che tubulare in biopsie renali di pazienti con nefrite lupica, valutando una possibile relazione con i dati clinici. Abbiamo verificato la presenza di un meccanismo di uptake in podociti umani in coltura attraverso esperimenti di time-lapse con basse dosi di FITC-BSA (10 µg/ml) ed abbiamo osservato l’inizio del processo di internalizzazione in un periodo di tempo variabile dalle 2 alle 15 ore. Per caratterizzare il tipo di cinetica di uptake della FITC-BSA, i podociti sono stati stimolati a differenti tempi (30 min e 2 ore) e dosi (10 µg/ml, 100 µg/ml and 1 mg/ml) mantenendo la coltura a 37°C o 4°C. Si è osservato un aumento significativo della fluorescenza dose-dipendente rispetto al controllo dopo 2 ore dalla stimolazione con una tipica cinetica di internalizzazione recettore-mediata poiché veniva inibita a 4°C. Abbiamo osservato la presenza di ClC-5, Dab2 e AMN oltre a quella di megalina e cubilina in podociti umani in coltura in condizioni basali mediante tecniche di immunoistochimica (IHC) ed immunofluorescenza (IF) ed abbiamo dimostrato la co-localizzazione dei due recettori con l’albumina fluorescente. Per valutare se l’ambiente proteinurico fosse in grado di modulare l’espressione di CLCN5, LRP2, CUBN, DAB2 ed AMN, i podociti umani sono stati stimolati con concentrazioni crescenti di BSA (range 10 µg/ml - 30 mg/ml) e l’espressione dell’RNA messaggero è stata valutata a tempi diversi (2, 4, 8, 24, 48 and 72 hours). Mediante analisi in Real Time PCR, abbiamo osservato un aumento significativo tempo e dose-dipendente di CLCN5, CUBN ed AMN ed un aumento di DAB2 solamente alle 24 ore. Abbiamo raccolto 23 biopsie renali di pazienti con LES, 6 biopsie di controllo ed un caso di Minimal Change Disease. Come parametri clinici abbiamo considerato la proteinuria e la terapia farmacologica. Mediante IHC ed IF abbiamo analizzato l’espressione proteica di ClC-5, megalina e cubilina in sezioni seriali. La quantificazione eseguita mediante analisi morfometrica ha rivelato una correlazione diretta dell’espressione di tutte le molecole in analisi tra il compartimento tubulare e glomerulare, evidenziando una stretta relazione tra i due compartimenti indipendentemente dai livelli di proteinuria. Inoltre, dati preliminari su pazienti privi di terapia farmacologica (ACEi/ARB o immunosoppressivi) hanno mostrato un trend positivo tra l’espressione di queste molecole a livello glomerulare. Curiosamente, abbiamo evidenziato l’espressione di megalina e cubilina in cellule parietali della capsula (PECs) con morfologia ipertrofica in alcuni pazienti LES che, mediante esperimenti di caratterizzazione, abbiamo identificato come una nuova sottopopolazione con un fenotipo intermedio tra cellule mature e progenitrici. Concludendo, per la prima volta abbiamo dimostrato che i podociti umani sono naturalmente predisposti ad effettuare l’endocitosi dell’albumina attraverso un meccanismo recettore-mediato. Inoltre, l’overload proteico è in grado di aumentare l’espressione di CLCN5, CUBN ed AMN in queste cellule. Studi funzionali per dimostrare il ruolo di cubilina nel processo di uptake dell’albumina hanno sottolineato la sua partecipazione in questo meccanismo anche se, verosimilmente, non è l’unico pathway coinvolto. Ulteriori studi saranno necessari per analizzare quali altre molecole possano essere chiamate in causa in questo meccanismo. Per la prima volta abbiamo dimostrato la presenza di ClC-5, megalina e cubilina in glomeruli di pazienti con LES, MCD e controlli, confermando i dati in vitro. Inoltre, nelle biopsie dei pazienti LES abbiamo evidenziato una stretta relazione tra i due compartimenti renali nell’espressione dei componenti di questo sistema, supportando l’idea di una partnership tra cellule tubulari e glomerulari nell’uptake dell’albumina attraverso lo stesso meccanismo di internalizzazione. In aggiunta, dati preliminari ottenuti da pazienti privi di terapia con ACEi/ARB o immunosoppressivi ci ha fatto supporre che il trattamento farmacologico possa influire sull’espressione di questo sistema a livello glomerulare. Le differenze osservate tra lo studio in vivo e quello in vitro, in particolare riguardo l’espressione di megalina, suggeriscono il coinvolgimento di altre cellule del glomerulo oltre ai podociti. Infine, l’espressione di megalina e cubilina nelle PECs dei pazienti LES è un dato molto interessante ma complesso, poiché gli esperimenti di caratterizzazione hanno identificato una sottopopolazione con un fenotipo intermedio tra cellule mature e progenitrici. Ulteriori studi dovranno essere condotti per meglio caratterizzare il ruolo di queste cellule con doppia positività e la loro correlazione con i dati clinici o di progressione della malattia.

La glomérulonéphrite extra-capillaire (GEC) est une maladie glomérulaire sévère associée à un risque majeur d’insuffisance rénale chronique terminale. Le traitement principal (immunosuppresseur) est associé à des effets secondaires importants, et aucun traitement ne cible à ce jour les mécanismes locaux impliqués dans la formation du croissant extracapillaire.Des études récentes ont identifié les cellules épithéliales pariétales (PEC) glomérulaires comme un composant majeur du croissant. De plus, par sa capacité à s’activer, se dédifférencier, proliférer et migrer, la PEC joue un rôle à part entière dans les mécanismes de formation de cette lésion. La protéine de choc thermique 27 (HSP27/HSPB1) est une protéine ubiquitaire, dont l’expression est induite par divers types de stress. Elle est impliquée dans les fonctions de prolifération, de dédifférenciation et de migration au cours de pathologies cancéreuses notamment. Son importance a mené au développement en oncologie d’un oligonucléotide antisens (OGX-427) dirigé contre le transcrit d’HSPB1. De plus, récemment, l'Ivermectine, un antiparasitaire, a été identifiée comme inhibant la phosphorylation et la fonction d’HSP27. Dans ce travail, nous montrons la surexpression d’HSP27 au sein des croissants au cours des GEC chez l’homme et l’animal. Cette surexpression est identifiée principalement au sein de PEC activées. De plus, le taux sérique d’HSP27 est associé à l’activité des GEC chez l’homme. De façon intéressante, HSP27 n’apparaît pas seulement comme un biomarqueur d’activité mais également comme un acteur du développement des croissants : l’inhibition d’HSP27 par OGX 427 limite les lésions extracapillaires dans un modèle de GEC.En parallèle, nous identifions les mécanismes expliquant le rôle pathogène d’HSP27 au sein des PECs. En effet, l'inhibition d’HSP27 par OGX-427 in vitro diminue l’activation des PECs et leur migration. Par ailleurs, l’utilisation de l’ivermectine reproduit les résultats observés sous OGX-427 avec une baisse de l’activation et de la migration des PECs, et une diminution des lésions extracapillaires chez l’animal. Cependant, le traitement par OGX-427 majore les lésions de microangiopathies thrombotiques chez l’animal. De plus, HSP27 module in vitro la migration des cellules endothéliales médiée par la signalisation du VEGFR2. En conclusion, ce travail démontre l’importance pathogénique d’HSP27 au cours de la GEC. Il identifie ainsi une nouvelle voie physiopathologique associée à l’activation des PEC et au développement des croissants. De plus, cette étude met en évidence l’importance d’HSP27 dans l’homéostasie endothéliale. Outre le rôle potentiel d’HSP27 comme biomarqueur d’activité des GEC, cette étude identifie de nouvelles cibles thérapeutiques des GEC chez l’homme
Crescentic glomerulonephritis (CG) is an aggressive glomerular disease associated with severe kidney outcome. While the main treatment consisting in immunosuppressive therapy is associated with major side-effects in frail patients, to date no drugs targeting the mechanisms directly involved in epithelial crescent formation are available. Indeed, recent studies identified parietal epithelial cells (PEC) as a major component of crescent formation.In this study, we showed that Heat shock protein 27 (HSP27/HSPB1), a stress-inducible protein involved in cancer cell proliferation and migration, is overexpressed in activated PEC of crescentic lesions both in in nephrotoxic nephritis mouse model and humans. Moreover, circulating HSP27 is associated with disease activity in humans with biopsy-proven CG. HSP27 inhibition through OGX-427, an antisens oligonucloeotide targeting HSPB1 mRNA, and Ivermectin (which inhibits HSP27 dimerization) reduce crescent formation in experimental CG model and PEC activation and migration in vitro. However, in NTN murine model, HSP27 inhibition with OGX-427 but no with Ivermectin was associated with a worsening of endothelial injury.Overall, these results identify HSP27 as a new pathogenic protein during crescent formation and a biomarker of disease activity in human CG. Optimal therapeutic option inhibiting HSP27 with the aim to reduce molecular processes leading to proliferative glomerulonephritis remains to be determined

Glomerular epithelial cells (GECs) are intrinsic components of the kidney glomerulus and are in contact with extracellular matrix (ECM). Under normal conditions, there is little turnover of GECs, but in glomerular injury, apoptosis and proliferation of GECs, and expansion of ECM may lead to sclerosis and impaired glomerular function and/or permselectivity. In order to better understand how adhesion to ECM modulates GEC survival and proliferation, we monitored the activation of the intracellular signaling pathways activated by adhesion to ECM (collagen), along with their coordinate modulation of the growth factor signaling pathways. Adhesion to collagen resulted in the activation of a focal adhesion kinase (FAK)/Src/Grb2 complex, thus activating extracellular signal-regulated kinase (ERK), possibly within focal adhesions. The ERK pathway was necessary to promote survival and growth factor-dependent proliferation. ECM-induced ERK activation was also necessary to achieve and maintain a cortical F-actin structure. There appeared to be a reciprocal relationship between collagen-induced cortical F-actin assembly and collagen-dependent survival signaling, including ERK activation. Moreover, maintenance of the cortical F-actin architecture was associated with increased turnover of inositol phospholipids. Analogous signals for GEC survival are activated in experimental focal segmental glomerulosclerosis (FSGS) in vivo. These studies will provide insights into novel therapeutic approaches to preserving intact glomerular permselectivity.

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to breakdown of glomerular peimselectivity and proteinuria. This study addresses mechanisms that limit complement-mediated injury, focusing on Ras. Complement-mediated injury was attenuated in cultured GEC expressing a constitutively active form of Ras (V12Ras), compared with Neo (control) GEC. V12Ras GEC showed constitutive activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways, but inhibition of these pathways did not reverse the protective effect of Ras. V12Ras GEC showed smaller and rounder morphology, decreased F- to G-actin ratio, decreased activity of the Rho GTPase, Rac, and decreased Src activity. In V12Ras GEC, disruption or stabilization of the F-actin cytoskeleton reversed the protective effect of V12Ras on complement-mediated injury. Thus, the protective effect of V12Ras may be dependent on remodeling of the actin cytoskeleton. Furthermore, the reduction of Src activity due to Ras activation may alter the equilibrium in activities of Rho GTPases, a family of proteins known regulate the actin cytoskeleton. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.

In glomerular epithelial cells (GEC), the actin cytoskeleton is a key determinant of cell morphology and functions, including permselectivity. Complement C5b-9 induces sublytic GEC injury associated with GEC morphological changes and proteinuria. This study addressed the role of Rho GTPases in complement-mediated GEC injury. We demonstrated that the amount of active RhoA increased; while the amount of active Rac1 and Cdc42 were decreased in C5b-9 mediated sublytic GEC injury both in vitro and in glomeruli from rats with PHN in vivo. Complement mediated inactivation of p190RhoGAP may contribute to complement-induced RhoA activation. Overexpression of constitutively active or dominant negative mutants of RhoA, Rac1 and Cdc42 distinctly altered GEC morphology and F-actin pattern. Complement caused changes in GEC actin cytoskeleton, at least in part mediated by a downstream kinase of RhoA--Rho kinase (ROCK). Activation of RhoA exacerbated complement-mediated cytotoxicity in GEC, while inhibition of ROCK attenuated it.

Eicosanoids play important roles in normal renal physiology and inflammation. Release of arachidonic acid from phospholipids by PLA2 is the rate-limiting step in eicosanoid synthesis. Activation of cPLA2 involves phosphorylation and calcium-dependent translocation from the cytosol to the membrane. cPLA 2 is reported to translocate to nuclear membranes after cell activation, however, other potentially relevant subcellular membranes have not been clearly defined. In the present study, we examined the association of cPLA2 with membranes of organelles, using a glomerular epithelial cell line that overexpresses cPLA2. Unstimulated cells, or cells stimulated with the Ca2+ ionophore, ionomycin, plus phorbol myristate acetate (PMA; 30min) were disrupted by homogenization in Ca2+-free buffer, and were subjected to low-speed centrifugation to pellet nuclei and unbroken cells. The post-nuclear supernatants were then subjected to sucrose or iodixanol gradient centrifugation. After collecting fractions from gradients, the distribution of cPLA2 and organelles was analyzed by immunoblotting and quantification by densitometry, using antibodies to cPLA2, calnexin (marker of endoplasmic reticulum), Hsp60 (mitochondria), and neu (plasma membrane), or by beta 1,4-galactosyltransferase enzyme assay (golgi). In cells stimulated with ionomycin + PMA, analytical fractionation showed that cPLA2 colocalized with plasma membrane and endoplasmic reticulum. There was no association with mitochondria or golgi. Significantly more cPLA 2 was associated with plasma membrane and endoplasmic reticulum in stimulated cells, as compared with unstimulated, however, a readily detectable amount of cPLA2 was also membrane-associated under resting conditions. The pattern of cPLA2-membrane-association in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin + PMA. Thus, release of arachidonic acid by cPLA2 may be due to hydrolysis of phospholipids at multiple sub

Les mécanismes à l’origine de la genèse des lésions extracapillaires au cours de la hyalinose segmentaire et focale (HSF) et de la glomérulonéphrite rapidement progressive (GNRP) restent imparfaitement compris.L’invasion du flocculus par les cellules épithéliales pariétales glomérulaires (PEC) est un évènement-clé, imparfaitement élucidé, à l’origine de la destruction glomérulaire et de la survenue de l’insuffisance rénale.Nous montrons, ici, que l’expression de la tétraspanine CD9 augmente de manière importante au sein des PEC, au cours de modèles murins de GNRP et de HSF ainsi qu’au sein des reins de patients atteints de ces pathologies. La délétion génique de Cd9 au sein des PEC est un facteur protecteur au cours de modèles murins de GNRP et de HSF.D’um point de vue mécanistique, l’absence de CD9 empêche la migration orientée des PEC en direction du capillaire glomérulaire ainsi que l’expression de CD44 et de l’intégrine β1.Ce travail met en évidence le rôle essentiel de l’expression de novo de CD9 au sein des PEC comme signal commun d’un changement phénotypique des PEC au cours de la GNRP et de la HSF et pourrait offrir un nouvel arsenal thérapeutique potentiel dans ces maladies
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PEC) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PEC in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PEC prevents glomerular damage in CGN and FSGS mouse models.Mechanistically, CD9 deficiency prevents the oriented migration of PEC into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions

碩士
國立宜蘭大學
生物技術研究所碩士班
97
Glomerulonephritis (GN) is the major cause of end-stage renal diseases worldwide. They are considered to be consequences of injury primarily to the glomerulus. Because of noninvasiveness and simplicity of specimen collection, human urine is one of the most useful biofluids for proteomics study. Identification of urinary biomarkers may lead to the development of a simple diagnostic test for earlier disease detection, or improvement in prognosis. In the current study, we seek to discover urine biomarkers for glomerular nephritis, including poststreptococcal glomerulonephritis (PSGN), Henoch-Schoenlein purpura nephritis (HSPN) and immunoglobulin A nephropathy (IgAN) using gel-based (2-DE) and gel-free proteomics approaches. Four significantly altered proteins were identified by LC-ESI-Q-TOF-MS/MS as Tamm-Horsfall protein, precursor of Alpha1-acid glycoprotein 1 also called orosomucoid-1, thioredoxin peroxidase B, and chain B of haemoglobin. Western blot and 2-D western analyses were performed to evaluate the sensitivity and specificity of these proteins. Among the four proteins, Alpha1-acid glycoprotein 1 and thioredoxin peroxidase B were considered potential biomarkers for glomerulonephritis. Furthermore, in order to evaluate the possible mechanism of renal tubular cell injury caused by Chinese herb nephropathy (CHN), aristolochic acid (AA) was applied to treat human renal proximal tubular epithelial cell line, HK-2. AA is a natural nephrotoxant from Aristolochiaceae plants which is widely used in traditional Chinese medicine (TCM). Previous studies have shown that AA is responsible for CHN (also called aristolochic acid nephropathy, AAN), a rapidly progressive interstitial nephropathy. In Asian countries, where TCMs are very popular, the complexity of the pharmacopoeia represents a high risk for AAN because of the frequent substitution of the botanical products by AA-containing herbs. In the present study, we applied gel-based proteomics (2-DE) to reveal the mechanism of AA-induced cytotoxicity for HK-2 cell line. Seven significantly altered proteins were identified by LC-ESI-Q-TOF-MS/MS as myosin, chaperonin, heat shock 70 kDa protein 8, tropomyosin 3, vimentin, beta actin, and calreticulin precursor. These protein are involved in cytoskeleton structure and transdifferentiation, indicating that AA may trigger cytoskeleton reorganization and cell transdifferentiation of HK-2 cell.

The glomerulus performs its functions with three major cell types. Endothelial cells and visceral epithelial cells (podocytes) lie on the inside and outside of the glomerular basement membrane, and together these three structures form the glomerular filtration barrier. Mesangial cells sit in the axial region. Pathologies of all these regions and cell types can be identified. Parietal epithelial cells lining Bowman’s capsule participate in crescent formation, and at the tubular pole some of these cells seem to represent a stem cell population for tubular cells and podocytes. The extraglomerular mesangium and juxtaglomerular apparatus complete the description of the glomerular corpuscle. The structure of these elements, and how they relate to function, are illustrated in detail.

Crescent formation refers to the appearance of proliferating cells in Bowman’s space in response to severe glomerular inflammation. Any aggressive ‘nephritic’ diseases that cause basement membrane breaks may provoke this. Specific serum proteins appear to be responsible for provoking crescent formation as it is largely abolished by defibrination in animal models. The cells in the crescent are initially mostly hypertrophying and proliferating parietal epithelial cells that normally line Bowman’s capsule. Foci of proliferation of these cells (extracapillary proliferation) are the first steps of crescent formation. Monocytes are frequently seen in established crescents. At this stage recovery of glomerular structure and function is possible in many circumstances. However, if Bowman’s capsule is ruptured, fibroblast ingress followed by fibrosis and glomerulosclerosis are likely. Crescentic nephritis is a histological description, but it fits closely with the clinical picture of rapidly progressive glomerulonephritis (RPGN), in which renal function is lost over days to weeks. The diseases most likely to cause this clinical picture are small vessel vasculitis, anti-GBM disease, lupus nephritis, and post-infectious glomerulonephritis. Any ‘nephritic’ disease may provoke crescent formation, but it is frequently encountered in immunoglobulin A nephropathy/Henoch–Schönlein purpura, and in post-infective glomerulonephritis. Recognizing the clinical picture is important as aggressive immunosuppression can be effective in saving glomerular function in some of the conditions causing it.

Idiopathic nephrotic syndrome is defined by the combination of massive proteinuria, hypoalbuminaemia, hyperlipidaemia, and oedema, and of non-specific histological abnormalities of the glomeruli. Light microscopy may disclose minimal change disease, diffuse mesangial proliferation, or focal segmental glomerular sclerosis (FSGS). The two main causes of idiopathic nephrotic syndrome are characterized histologically. On electron microscopy the glomerular capillaries show a fusion of visceral epithelial cell (podocyte) foot processes and with the exception of some variants no significant deposits of immunoglobulins or complement by immunofluorescence. In a majority of children only minimal changes are seen on light microscopy. These children are referred to as having ‘minimal change disease’. In adults with idiopathic nephrotic syndrome, lesions of FSGS are more frequent.

In the intricate maze of proposed mechanisms, modifiers, modulators, and sensitizers for acute kidney injury (AKI) and diverse causes inducing it, this chapter focuses on several common and undisputable strands which do exist.Structurally, the loss of the brush border, desquamation of tubular epithelial cells, and obstruction of the tubular lumen are commonly observed, albeit to various degrees. These morphologic hallmarks of AKI are accompanied by functional defects, most consistently reflected in the decreased glomerular filtration rate and variable degree of reduction in renal blood flow, accompanied by changes in the microcirculation. Although all renal resident cells participate in AKI, the brunt falls on the epithelial and endothelial cells, the fact that underlies the development of tubular epithelial and vascular compromise.This chapter further summarizes the involvement of several cell organelles in AKI: mitochondrial involvement in perturbed energy metabolism, lysosomal involvement in degradation of misfolded proteins and damaged organelles, and peroxisomal involvement in the regulation of oxidative stress and metabolism, all of which become defective. Common molecular pathways are engaged in cellular stress response and their roles in cell death or survival. The diverse families of nephrotoxic medications and the respective mechanisms they induce AKI are discussed. The mechanisms of action of some nephrotoxins are analysed, and also of the preventive therapies of ischaemic or pharmacologic pre-conditioning.An emerging concept of the systemic inflammatory response triggered by AKI, which can potentially aggravate the local injury or tend to facilitate the repair of the kidney, is presented. Rational therapeutic strategies should be based on these well-established pathophysiological hallmarks of AKI.

Minimal-change nephrotic syndrome (MCNS) is an immune-mediated condition, usually of unknown cause, but which can sometimes be associated with Hodgkin’s disease or the use of nonsteroidal anti-inflammatory drugs. On light microscopy the glomeruli appear normal or small, and on electron microscopy there is effacement of epithelial-cell foot processes over the outer surface of the glomerular basement membrane. MCNS is the cause of about 80% of cases of nephrotic syndrome in children and 20% in adults....

Minimal-change nephrotic syndrome (MCNS) is an immune-mediated condition, usually of unknown cause. On light microscopy the glomeruli appear normal, and on electron microscopy there is effacement of epithelial cell foot processes over the outer surface of the glomerular basement membrane. MCNS is the cause of about 75% of cases of nephrotic syndrome in children and 17% in adults. Management and prognosis—treatment in adults is with prednisolone at an initial dose of 80 mg/day, then tapering. This leads to complete remission in 90 to 95% of patients, but 50 to 75% of glucocorticoid-responsive adults will have a relapse. Progression to renal failure is not expected and would call the diagnosis of MCNS into question. Focal segmental glomerulosclerosis (FSGS) is not a specific disease entity but a histological lesion, often of unknown aetiology, which is characterized by segmental areas of glomerular sclerosis. It may be (1) primary—either due to genetic mutation, or associated with an unknown circulating protein factor that causes an increase in glomerular permeability; or (2) secondary—the end product of a variety of pathological processes including glomerular hyperfiltration, healed glomerulonephritis, viral (including HIV) infection, or parasitic infection. Management and prognosis—corticosteroid and immunosuppressive therapy should be considered only in patients with primary FSGS and nephrotic syndrome. The steroid regimen is as used for MCNS, but with lesser success. Steroid-resistant cases are treated with ciclosporin (for which there is most published evidence), mycophenolate mofetil, or cyclophosphamide. Prognosis depends on histology and response to treatment.

The shape of a cell is critical for proper signaling and resultant biological function [1]. Podocytes, kidney visceral epithelial cells, have a distinctive morphology with interdigitating foot processes that wrap around the capillaries of the glomeruli and, together with endothelial cells and the basement membrane, form the glomerular filtration barrier. In addition to forming the filtration barrier, slit diaphragms that connect the alternating foot processes from two podocytes are thought to be signaling hubs that regulate cell morphology and function.