Academic literature on the topic 'Glomerular parietal epithelial cells'

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)

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.

A comparative study of the localization of 125I-labeled atrial natriuretic factor (ANF) and 125I-labeled angiotensin II (ANG II) binding sites in the glomerulus of the rat, after an intravascular injection, has been done by ultrastructural radioautography. 125I-ANF binding sites are localized predominantly on the podocytes of the visceral epithelium (63%) followed by the endothelium of capillaries (14%), the parietal epithelium (13%), and finally mesangial cells (10%). In a comparative study, it was confirmed that 125I-ANG II uptake is localized predominantly on mesangial cells (60%) followed by epithelial visceral cells (23%) and the endothelium of capillaries (16%). Using isolated rat glomeruli, it was confirmed that ANG II decreases glomerular size (maximum effect of 15%) with an apparent half maximum effective concentration (EC50) between 10(-9) and 10(-8) M. Although ANF alone has no apparent effect on glomerular size, it inhibits the contractile effect of ANG II with a half maximum inhibitory concentration (IC50) between 10(-11) and 10(-10) M. These results suggest that an intraglomerular mechanism other than glomerular arteriolar resistance may be involved in the modulation of glomerular filtration rate by ANF. The presence of 125I-ANF uptake mainly in foot processes of visceral epithelial cells of glomeruli in vivo and the inhibition of ANG II decrease in glomerular size by ANF in vitro raise the possibility that ANF may regulate the ultrafiltration coefficient by two mechanisms: modulation of glomerular permeability, and surface area.

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.

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.

The precise localization of the calcitriol (1 alpha,25-dihydroxyvitamin D3) receptor (VDR) and the 25-hydroxyvitamin D3 [25(OH)D3] 24-hydroxylase cytochrome P-450 in the human kidney is unknown. Using newly developed polyclonal antibodies against the human VDR, we demonstrate that the receptor is present in cells of the distal tubule, the collecting duct, the proximal tubule, and in the parietal epithelial cells of the glomerulus. In the distal tubule and collecting duct not all cells contain epitopes for the receptor. The protein is not detected in glomerular capillaries, in the glomerular mesangium, in the interstitium, or in blood vessels. Specific polyclonal antibodies directed against the 25(OH)D3 24-hydroxylase cytochrome P-450 demonstrate epitopes for the cytochrome in cells of the proximal tubule, the distal tubule, glomerular parietal epithelial cells, and mesangial cells. The protein is absent from interstitial cells. Calbindin D28k is present exclusively in principal cells of the distal tubule and collecting duct. In the human kidney, the VDR is present in cells where vitamin D-inducible proteins are found; conversely it is absent from cells where vitamin D-dependent proteins are not present.

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

Introduction : La glomérulonéphrite à croissant (cGN) est une forme avancée de glomérulopathie commune à plusieurs maladies rénales d'origine immunitaire. La cGN se caractérise par une prolifération des cellules épithéliales pariétales glomérulaires (PEC), formant des croissants dans la chambre urinaire et conduisant en l'absence de traitement à l'insuffisance rénale terminale. Dans le cadre de ma thèse, j'ai particulièrement étudié la claudine-1 (CLDN1), une protéine transmembranaire impliquée dans les jonctions serrées, qui peut être exposée en dehors de ces jonctions serrées et activer des voies profibrotiques. Nous savons que la CLDN1 est exprimée spécifiquement par les PEC dans le glomérule, étant donné l'importance de l'activation des PEC dans les lésions glomérulaires, un anticorps monoclonal ciblant la CLDN1 pourrait être une cible thérapeutique pertinente et innovante. Par ailleurs, des biomarqueurs spécifiques des PEC activées, tels que CD44 et CD9, ont été mis en évidence dans les cGN. Les souris déficientes en CD44 n'étant pas capables d'induire de cGN et la délétion spécifique de CD9 dans les PEC prévenant les lésions tissulaires de la cGN. Malgré leurs rôles primordiaux dans la physiopathologie des cGN, leur expression dans la pathologie glomérulaire humaine n'est pas connue, tout comme leur corrélation avec le pronostic rénal. Le premier objectif de cette thèse est donc d'étudier l'expression des biomarqueurs des PEC (CLDN1, CD9 et CD44) en fonction des lésions histologiques glomérulaires les cGN humaines (néphropathie à IgA (IgAN) et vascularite à ANCA (vANCA)), leur rapport avec la perte et la dédifférenciation podocytaire et avec le pronostic néphrologique des patients. Le deuxième objectif de cette thèse est d'étudier le potentiel bénéfice de cibler la CLDN1 avec l'anticorps anti-CLDN1 dans un modèle murin de cGN. Méthode : L'expression de CLDN1, CD9 et CD44 dans les tissus rénaux de patients atteints de IgAN et vANCA a été analysée grâce à une technique d'immunofluorescence multiplex (méthode OPAL). La corrélation entre l'expression de CLDN1, CD9 et CD44 et les critères cliniques (DFGe, protéinurie), les lésions histologiques, les biomarqueurs de dédifférenciation podocytaire (p57 et WT1) et le pronostic néphrologique a été étudiée. Une analyse en transcriptomique spatiale des croissants glomérulaires exprimant CLDN1 et CD44 dans les reins de patients atteints de cGN a été réalisée. Des études de preuve de concept utilisant des anticorps anti-CLDN1 ont été effectuées dans un modèle préclinique de cGN. Résultats : Dans les tissus (n=131) de patients atteints de vANCA et IgAN, l'immunofluorescence en multiplex a révélé une surexpression de CLDN1 dans les croissants glomérulaires ainsi que dans les lésions fibreuses. Au moment de la biopsie rénale diagnostique, l'expression de CLDN1 dans les glomérules était corrélée à la perte de podocytes (mesurée par l'expression de P57). L'augmentation de l'expression des cellules doubles positives (CLDN1+ et CD44+) était statistiquement significativement associée à un mauvais pronostic rénal chez les patients atteints de vANCA et d'IgAN avec un suivi médian de 2,5 et 6.9 ans respectivement. L'analyse en transcriptomique spatiale a mis en évidence l'association entre la présence de PEC « activées » (CLDN1+/CD44+) et l'augmentation de l'expression des gènes de la matrice extracellulaire. Dans le modèle murin de cGN, nous avons mis en évidence qu'un anticorps anti-CLDN1 avait la capacité de se lier aux PEC activés, sa cible thérapeutique, et de réduire significativement de l'albuminurie chez les souris traitées. Conclusion : Nos résultats suggèrent un rôle fonctionnel de CLDN1 dans la pathogenèse de la cGN, fournissant une preuve de concept préclinique pour les anticorps anti-CLDN1. Ces données suscitent des questions mécanistiques et suggèrent que des anticorps anti CLDN1 pourrait représente une approche thérapeutique novatrice chez les patients atteints de cGN
Introduction: Crescentic glomerulonephritis (cGN) is the final mode of kidney injury common to several immune-mediated kidney diseases. cGN is characterized by extensive glomerular parietal epithelial cells (PEC) proliferation, forming crescents in urinary space and leading, if untreated, to end-stage kidney disease (ESKD). In a pathological context, claudin-1 (CLDN1), a transmembrane protein involved in epithelial tight junctions, can be exposed outside the tight junctions and mediate pro-fibrotic pathways and extracellular matrix (ECM) remodeling as described in other cell types. CLDN1 is expressed explicitly by PEC in the glomerulus and given the importance of PEC activation in glomerular injury, we evaluated whether a monoclonal antibody targeting CLDN1 could represent a relevant and innovative therapeutic tool. Additionally, specific biomarkers of PEC activation, such as CD44 and CD9, have been studied in cGN. CD44-deficient mice display attenuated experimental cGN, and specific deletion of CD9 in PEC prevents tissue damage in experimental cGN. However, CD9 and CD44 expression in human glomerular cGN (IgA nephropathy (IgAN) and ANCA vasculitis (AAV)) is unknown, as is their correlation with podocyte loss, histological lesions, and renal outcome. The first objective of this study was to evaluate PEC biomarkers (CLDN1, CD9, and CD44) expression in human cGN, their association with glomerular histological lesions, podocyte loss and dedifferentiation, and renal outcome. The second objective is to evaluate the potential benefit of targeting CLDN1 with an anti-CLDN1 Ab in a cGN mouse model. Method: CLDN1, CD9, and CD44 expression in renal tissues of cGN patients was analyzed using kidney multichannel immunofluorescence staining and spatial transcriptomics. Correlation between CLDN1, CD9, and CD44 expression and clinical endpoints (eGFR, proteinuria), histological lesions, biomarkers of podocyte dedifferentiation (p57 and WT1), and renal outcome were studied. A spatially resolved molecular roadmap from CLDN1+/CD44+ crescentic glomeruli was conducted. Proof-of-concept studies using anti-CLDN1 monoclonal antibody were performed in the Matsugi model of cGN. Results: In tissues of 131 patients with IgAN and AAV, multichannel immunofluorescence revealed up-regulated CLDN1 expression by crescents and fibrous lesions. At the time of diagnostic kidney biopsy, glomerular CLDN1 expression was correlated with podocyte loss (measured by p57 expression) and glomerular fibronectin area. The increase of proportion of double-positive (CLDN1+ and CD44+) cells was statistically significantly associated with poor renal outcome (50% decline in estimated glomerular filtration rate (eGFR) or ESKD) in patients with ANCA vasculitis and IgA nephropathy, with a median follow-up of 2.5 and 6.9 years, respectively. Spatial transcriptomics analysis highlighted the association between CLDN1+/CD44+ crescentic glomeruli and extracellular matrix genes. In the cGN murine model, we demonstrated that anti-CLDN1 antibody could bind to activated PEC, its therapeutic target and significantly reduced albuminuria in treated mice. Conclusion: Our results suggest a functional role of CLDN1 in the pathogenesis of cGN providing a preclinical proof-of-concept for the use of anti-CLDN1 antibodies as a novel therapeutic approach in patients with cGN

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

Visceral glomerular epithelial cells (GEC), also known as podocytes, are essential components of the kidney glomerulus, and play a pivotal role in maintaining normal glomerular permselectivity. The regulation of the actin cytoskeleton is central to podocyte morphology and function. In the rat model of membranous nephropathy, passive Heymann nephritis (PHN), heterologous antibody binds to GEC antigens. Following immune complex formation the complement system is activated with the assembly of the C5b-9 membrane attack complex. In GEC, C5b-9 induces sublytic injury associated with morphological changes and proteinuria. We have previously reported that complement activates RhoA, member of the Rho family of small GTPases, in vitro and in vivo. The current study addresses the role of GEF-H1, a Rho GEF protein, in complement-mediated RhoA activation in GEC injury. In glomeruli from rats with PHN and cultured GEC, complement stimulation increased GEF-H1 activity, in a parallel fashion with RhoA activation. Activation of GEF-H1 by complement was at least partially dependent on the extracellular signal-regulated kinases (ERK) pathway, but not on the epidermal growth factor (EGF) receptor, or Src-family kinases, or microtubules. To address the functional effects of GEF-H1, GEC were transduced with lentivirus-mediated shRNA of GEF-H1, to knockdown the expression. GEC transduced with shRNA of GEF-H1 significantly blocked RhoA activation and augmented cytotoxicity in response to complement stimulation, implicating a cytoprotective effect of GEF-H1 on GEC in complement-mediated injury.
Les cellules glomérulaires épithéliales viscérales (CGE), ou podocytes, sont des composantes essentielles du glomérule du rein, et jouent un rôle important dans le maintien de la permsélectivité glomérulaire. La régulation du cytosquelette d'actine est à la base du fonctionnement normal du podocyte, ainsi qu'à sa morphologie. Dans le modèle de rat de la néphropathie membraneuse, passive Heymann nephritis (PHN), les anticorps hétérologues se lient aux antigènes des podocytes. Après formation de complexes immuns, le système du complément est activé avec l'assemblage du complexe d'attaque membranaire C5b-9. Dans les CGE, C5b-9 induit des effets sublytiques associés à des modifications morphologiques et à la protéinurie. Nous avons déjà signalé que le complément active RhoA, membre de la famille Rho des petites GTPases, in vitro et in vivo. La présente étude porte sur le rôle de GEF-H1, une protéine de la famille des Rho GEF, dans l'activation de RhoA dans les CGE méditée par le complément. Dans les glomérules de rats avec PHN et dans les CGE cultivés in vitro, la stimulation du complément a augmenté l'activité de GEF-H1, d'une façon parallèle avec l'activation de RhoA. L'activation du GEF-H1 par le complément a été au moins partiellement dépendante de l'extracellular signal-regulated kinase (ERK), mais pas du facteur de croissance épidermique (EGF), de la famille des kinases Src, ou des microtubules. Pour faire face aux effets fonctionnels de GEF-H1, CGE ont été infectées par des lentivirus contenants un petit ARN en épingle à cheveu, pour réduire l'expression de GEF-H1. Les CGE infectées par les lentivirus ont bloqué de manière significative l'activation de RhoA et ont augmenté la toxicité en réponse à la stimulation du complément, impliquant un effet protecteur de GEF-H1 sur les CGE.

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.

The glomerular basement membrane (GBM) is a condensed network of extracellular matrix molecules which provides a scaffold and niche to support the function of the overlying glomerular cells. Within the glomerulus, the GBM separates the fenestrated endothelial cells, which line capillary walls from the epithelial cells or podocytes, which cover the outer aspect of the capillaries. In common with basement membranes throughout the body, the GBM contains core components including collagen IV, laminins, nidogens, and heparan sulphate proteoglycans. However, specific isoforms of these proteins are required to maintain the integrity of the glomerular filtration barrier.Across the spectrum of glomerular disease there is alteration in glomerular extracellular matrix (ECM) and a number of histological patterns are recognized. The GBM can be thickened, expanded, split, and irregular; the mesangial matrix may be expanded and glomerulosclerosis represents a widespread accumulation of ECM proteins associated with loss of glomerular function. Whilst histological patterns may follow a sequence or provide diagnostic clues, there remains limited understanding about the mechanisms of ECM regulation and how this tight control is lost in glomerular disease. Monogenic disorders of the GBM including Alport and Pierson syndromes have highlighted the importance of both collagen IV and laminin isoforms and these observations provide important insights into mechanisms of glomerular disease.

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.

Abstract Polycystic kidney disease (PKD) is a genetic or acquired disorder with progressive dilatation of multiple tubular segments and/or enlargement of glomerular capsules, accompanied by fluid accumulation, growth of non-neoplastic epithelial cells, and deranged remodelling of the extracellular matrices. This results ultimately in a certain degree of renal functional impairment, which potentially can regress to normal after removal of the inductive agent(s) in experimental animal models (Carone et al. 1994; Kanwar and Carone 1984). These localized events with seg mental dysmorphogenetic lesions are conceivably related to an aberration of one or more factors which maintain normal tubular and glomerular morphological features.

62-year-old man with shortness of breath and an abnormal chest CT Axial 3D SPGR postgadolinium images (Figure 13.8.1) demonstrate diffuse thickening and enhancement of the left pleura, with a few minimally enhancing, focal right-sided pleural plaques. Malignant pleural mesothelioma Malignant pleural mesothelioma is a rare neoplasm that originates from the mesothelial cells lining the visceral and parietal pleura. The incidence of malignant pleural mesothelioma in the United States is 15 cases per million; there is a strong correlation with asbestos exposure. Malignant pleural mesothelioma is divided into 3 histologic subtypes: epithelial (55%-65%), sarcomatoid (10%-15%), and mixed (20%-35%). Patients with epithelial malignant pleural mesothelioma have the best prognosis, and among those with limited disease who undergo extrapleural pneumonectomy (removal of the pleura, lung, hemidiaphragm, and part of the pericardium), survival is longer (5-year survival, 39%) than among all patients (median survival, 8-18 months after diagnosis)....

Renal involvement in primary Sjögren’s syndrome (pSS) was first described in the 1960s, with reports of acquired defects in tubular function, in the setting of lymphocytic tubulointerstitial inflammation. Renal involvement in pSS is common, but is frequently undiagnosed due to its covert nature. It may manifest either as epithelial disease causing tubulointerstitial nephritis, or as an immune-complex glomerulopathy. Tubulointerstitial inflammation leads to acquired defects in tubular function, which may occur at multiple nephron segments. Treatment protocols lack evidence from randomized trials, and involve immunosuppression targeting B- and T-cells, which mediate both interstitial and glomerular disease to varying extents. In this chapter, we review the prevalence of renal involvement in pSS and its clinical manifestations, likely pathogenesis, and 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.

The basement membrane is a specialized part of the extra-cellular matrix. It is usually characterized as a scaffold for epithelial cells but in some tissues it serves other, mechanical, roles [1]. The mechanical properties of the basement membrane are mainly determined by one of its main constituents — type IV collagen. Unlike the well-known fibrous type I collagen, collagen IV assembles into planar networks (Fig. 1) [2]. The α 1(IV) and α 2(IV) collagen IV chains assemble into the so-called major chain network, present in all basement membranes. The α 3(IV), α 4(IV), α 5(IV) collagen IV chains form the minor chain network which is found only in the adult basement membranes of the kidney glomerular capillaries (GBM), ocular lens (LBM), cochlea, and the testes [3]. The minor chains have a higher number of cysteine residues, allowing them to form a higher number of lateral interactions. In the minor chain network, the greater potential to interact laterally manifests in the formation of super-coils, which are rarely observed in the major chain network [4]. Increasing the number of cross-links in a polymeric material is known to increase material stiffness; therefore, it is believed that the minor chain network confers basement membranes with additional strength and stability [5]. In the hereditary disease Alport syndrome, a mutation causes the absence of the minor chain network. The GBM and LBM of Alport patients appear weakened and unable to meet their mechanical demands, further supporting this theory [6]. The objective of this study was to evaluate the importance of cross-linking in the minor chains for the mechanical properties of type IV collagen networks, specifically in the GBM and LBM where the absence of the minor chains has an observed mechanical effect.

Female F1 hybrid of New Zealand black and white mice(NZB/W) spontaneouslydevelop an autoimmune disease characterize by afatal immune complex glomerulonephritis.Theyare considered to be a relevant model of human systemic lupus erythematosus. We observeda doubling of the concentration of TXB2 in urine at the same time when onset of proteinuria was noticed. This suggests that TXA2 synthetized by mesangial and epithelial cells of the glomeruli as well as by some inflammatory cells and platelets might be an important mediator in the pathogenesis of thi auto immune-mediated glomerular disease. Weused BM 13.505 as an long-acting TX receptor blocker for evaluating the importance of TXA2 on the development of proteinuria and compared its effects with that of the immunsuppressive agent cyclophosphamide.NZB/W mice were distributed to vehicle-treated (V-)group 20 mg/kg BM 13.505 (BM-) group and 20 mg/kgcyclophosphamide (C-) group( = 13 -14).Daily dosing by gavage was startedat the age of12 weeks. Every fourth week theurinary concentrations of proteins were measured by th biuret method and TXB2by a RIA. An increasein TXB2 was seen in the V- and BM-group, while in the C-group TXB2 was lowered. At 36 weeks of age 8of 14 animals of the V-group were proteinuria positive (>100 mg/100 ml). The study was finished at 44 weeks because more than 2/3 of the animals of the V-group haddeveloped a proteinuria. Previously four animals died and in most of other the disease was faradvanced. In the BM-group no animal had diedor showed signs of illness. However seven ofthe animals had slightly elevated protein concentrations in urine and two moderate elevated values. In the C-group no proteinuria was detected. Histological examinations of thekidneys showed a correlation in individualanimals of the V-group between the duration and extent of proteinuria and changes in the morphology of the glomeruli. In the BM-treated animals slight to moderate protein deposits were detectable, while in cyclophosphamide-treated animals glomeruli were of normal structure. This study presents someevidence that TXA2 may be an important mediator in the pathogenesis of this immune-mediated renal disease. Manifestation of this disease is delayed by the administration of thespecific TX receptor blocker BM 13.505.