A proteinuria kialakulásának örökletes okai és következményei Reusz György I. sz. Gyermekklinika

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

Kérdés

Az albumin sorsa egészségesekben Albumin szerkezete (~0.3 g/l) (A) Overview of renal handling of serum albumin in healthy subjects. (B) Basic data and configuration of serum albumin. (C) Reabsorption of filtered albumin occurs via endocytosis in the proximal tubule. Inside the cell (in lysosomes) albumin is degraded to amino acids, which are released across the basolateral membrane. Proximális tubulussejt (~0.03 g/l)

A proteinuria típusai Glomeruláris Tubuláris Túlfolyásos Postrenális Renal function and renal physiology: examples of selective glomerular proteinuria. a) Renal function, measured by glomerular filtration rate (GFR): This is the volume of fluid filtered from the renal glomerular capillaries into Bowman's capsule per unit time; originally determined by injecting inulin into the plasma. Since inulin is not reabsorbed by the kidney after glomerular filtration, its rate of excretion is directly proportional to the rate of filtration of water and solutes across the glomerular filter. b) Renal physiology, measured by urinary proteins: The glomerulus is responsible for blood filtration and retains larger proteins. Smaller, negatively charged proteins of a size that allow them to just pass through the glomerular pores (i.e., albumin, 67 kD or transferrin, 90 kD) are physiologically repelled by the negatively charged glomerular basal membrane (anionic filter). The renal tubule reabsorbs the small molecules that pass freely through the glomerulus. Postrenal contamination is characterized by serum proteins leaking into the urine, indicated by the presence of urinary α-2-macroglobulin (720 kD). The figure shows selective glomerular proteinuria, corresponding to Fig. 4b

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

A glomerulus elektronmicros-copos képe résmembrán * P A glomerulus elektronmicros-copos képe résmembrán Podocyta P basalis membrán BM endothel ET résmembrán RM ET BM RM P BM ET 4/4/2017 Reusz Gy: Glomeruláris szindrómák

Rodenwald R:, Cell Biol, 1974, Tryggvason et al 4/4/2017

A podocyta-résmembrán-egység Filtrációs barrier: Endothelium Glomerularis basalis membran Podocyta- résmembran Szelectiv filter érzékelő- jelátvívő functio lábnyúlvány A nephrosis szindróma egy olyan glomeruláris megbetegedés amely a filtrációs barrier károsodásával jár. A filtrációs barrier fesztrált endothelből, bazális membránból és a podocyta-résmembrán egységből áll. Ezen struktúrák károsodása következtében megnő a membrán permeabilitása, amely fokozott porteinuriához vezet. Ez hypoproteinaemiával és hyperlipidaemiával jár. Az ödéma csökkent kapilláris kolloid ozmotikus nyomás miatt alakul ki. A szteroid terápiára adott válasz alapján a nephrosis szindrómát két csoportra lehet osztani: A gyermekek 80%-a reagál a szteroid terápiára, tehát a betegségük szteroid szenzitív 20%-ban szteroid rezisztens nephrosis szindrómában szenvednek. 4/4/2017 Reusz Gy: Glomeruláris szindrómák

Reusz Gy: Glomeruláris szindrómák Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography Jorma Wartiovaara,et al: J. Clin. Invest. 114:1475–1483 (2004). 4/4/2017 Reusz Gy: Glomeruláris szindrómák

A podocita résmembrán síkban és térben Electron-Microscopical Imaging (Panels A and B) and Electron-Tomographic Imaging (Panel C) of the Podocyte Slit Diaphragm. In Panel A, a cross section of a human glomerular capillary shows filtration slits with slit diaphragms (arrows) between the podocyte foot processes (FPs). The glomerular basement membrane (GBM), and an endothelial cell (E) are also shown. The scale bar represents 250 nm. (Modified from Lahdenkari et al.58 with the permission of the publisher.) Panel B shows slit diaphragms (arrows) at a higher magnification. The scale bar represents 150 nm. (Modified from Lahdenkari et al.58 with the permission of the publisher.) Panel C shows a thin, three-dimensional electron tomogram of the mouse slit diaphragm (SD) en face. Cross-strands (arrows) extend from cross-cut podocyte surface membranes (M) to a central density (CD), forming lateral pores (Ps). The tomogram is a surface-rendered reconstruction. For comparison with the diameter of the pores at the same magnification, a space-filled model (yellow) of the crystal structure of a serum albumin molecule has been superimposed. The scale bar represents 10 nm. (Modified from Wartiovaara et al.59 with the permission of the publisher.)

Fin–major nephrin mutáció hereditaer nephrosisban mutáns ép ép mutáns Glomerular Phenotype in a Control Subject and in a Patient with the Fin-Major Nephrin Mutation and the Congenital Nephrotic Syndrome of the Finnish Type (CNF). Panel A shows scanning electron micrographs of podocytes (Ps) on glomerular capillaries of the normal human kidney, with long processes that branch into well-organized, interdigitating foot processes (FP) (inset). The scale bars represent 5 μm in Panel A and 1 μm in the inset. As shown in Panel B, the podocytes in a patient with CNF are flattened, with only a few, wide foot processes. The scale bar represents 1 μm. Panel C shows a transmission electron micrograph of a cross section of a normal glomerular capillary. The foot processes are approximately 250 nm wide and are separated by filtration slits (arrows) containing a slit diaphragm. The scale bar represents 200 nm. As shown in Panel D, the flattened and fused (effaced) foot processes of a patient with CNF line the glomerular basement membrane, and the filtration slits (arrow) are far apart. The scale bar represents 200 nm. Panel E shows the boxed portion of Panel C at a higher magnification of Panel C: regular filtration slits (arrows) approximately 40 nm wide are bridged by a thin slit diaphragm. The scale bar represents 100 nm. Panel F shows the boxed portion of Panel D at a higher magnification of Panel D; no slit-diaphragm line is visible, and only faint fuzzy material can be seen in a narrow and elongated filtration slit (arrow). The scale bar represents 100 nm. Panel G shows a tomogram of a typical filtration slit in a glomerulus of a patient with CNF. The slit, which is normally about 40 nm wide, is only about 10 nm wide; it has no organized slit-diaphragm structure, but only some short, unorganized strands. M denotes the podocyte surface membrane. The scale bar represents 5 nm. (Panels A through F are modified from Lahdenkari et al.58 with the permission of the publisher, and Panel G is modified from Wartiovaara et al.59 with the permission of the publisher.) mutáns ép mutáns

CyA

A podociták válasza mechanikus stresszre Endlich N és mtsai J Am- Soc Nephrol, 2001, 12: 413-422 4/4/2017 Reusz Gy: Glomeruláris szindrómák

A glomerulus capilláris és a podocyta remisszióban és nephrosisban Podocyta sejttest = P Lábnyúlványok = L Pseudovillusok = V V P L L P 4/4/2017 V Reusz Gy: Glomeruláris szindrómák

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

Megalin-cubilin és albumin ko-lokalizációja Meg-alb Meg-Cub Co-localization of megalin, cubilin, and albumin in the endocytic compartment of the mouse renal proximal tubule. A Double immunofluorescence labeling for megalin (green) and cubilin (red). B Double immunofluorescence labeling for megalin (green) and albumin (red). The yellow color illustrates the co-localization of megalin, cubilin, and albumin in the apical part of the cell. C Immunogold labeling for megalin (5-nm gold particles) and cubilin (10-nm gold particles). D Immunogold labeling for megalin (5-nm gold particles) and albumin (10-nm gold particles). Labeling shows co-localization of megalin, cubilin, and albumin in apical coated pits (CP), endosomes (E), microvilli (MV), and dense apical tubules (DAT). A and B, bars=10 mm, C and D, bars=0.2 mm

Az albumin transztubuláris transzportja = Dent = Lowe-Dent2 Figure 2 (A) The endocytic pathway of albumin. The two photomicrographs show the endosomal localization of FITC-albumin after 15 min incubation and the diffuse distribution of the degradation products of FITC-albumin after 120 min [experiments were performed in proximal tubule-derived opposum kidney (OK) cells]. EEV, early endocytic vesicle; SE, sorting endosome; LE, late endosome; RE, recycling endosome; LY, lysosome. (B) Scheme of the megalin-cubilin complex at the apical membrane. (C) Model showing the components that contribute to vesicular pH homeostasis. βH, buffer capacity for protons;X+,Y−, unknown ions that may serve as counterions.

Megalin és cubilin A proximális tubulus endocitotikus receptorai Albumin IgG könnyű lánc Hgb, Myoglobin D-vit kötő feh Retinol kf Transcobalamin-B12 Beta-2 mg Alfa-1 mg EGF Apo-H Prolaktin Inzulin PTH Lizozim Citokróm-C amiláz Megalin és cubilin A proximális tubulus endocitotikus receptorai Apo A-1 Transzferrin cubilin megalin Schematic presentation of megalin and cubilin—the endocytic receptors responsible for protein reabsorption in the proximal tubule. Specific as well as shared ligands for the two receptors are depicted. Clathrin-dependent internalization of megalin occurs via its cytoplasmic domain. The binding between megalin and cubilin probably enables cubilin ligand internalization internalizáció

Megalin és cubilin A proximális tubulus endocitotikus receptorai The megalin- and cubilin-mediated uptake of three vitamin carrier protein complexes: vitamin D binding protein (DBP)-vitamin D3, transcobalamin (TC)-vitamin B12, and retinol binding protein (RBP)-retinol in renal proximal tubule. Following receptor-mediated endocytosis via apical coated pits, the complexes accumulate in lysosomes for degradation of the proteins, while the receptors recycle to the apical plasma membrane via dense apical tubules. As illustrated here and detailed in the text, megalin mediates the uptake of cubilin and its ligands. Whether the two receptors are constitutively associated in the plasma membrane and remain associated during recycling in dense apical tubules is not known. Whereas TC and RBP apparently bind exclusively to megalin, DBP binds with similar affinity to bothmegalin and cubilin. The intracellular processing of the vitamins may include modifications such as hydroxylation of 25(OH)D3 to 1,25-(OH)2D3, and metabolism of B12. The mechanisms for the cellular release of the vitamins remain to be clarified.

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

Glomeruláris kórképek

Podocyta-diaphragma molekuláris felépítése Cat=Catenin; CD=CD2 asszociált protein; Ez= ezrin; FAK=fokális adhésiós kináz; ILK=integrinhez kötött kináz; M=myosin; PC=podocalycin; S=synaptopodin; TPV= talin, paxillin, vinculin; U=utrophin; Z=Z-1; FSGS= focalis segmantalis glomerulosclerosis; TRCP6=Transient receptor potential cation channel, subfamily C, member 6; CNF=finn típusú congenitális nephrosis AR-FSGS TRPC6 AD-FSGS CNF AR-AD-Alport 4/4/2017 Reusz Gy: Glomeruláris szindrómák

Néhány ismert mutáció familiáris nephrosis syndromában Finn típusú nephrosis: NPHS1 - nephrin 65% Fin-maj: 2 bp deléció a 2-es exonban 8% Fin-min: nonsens mutáció a 26-os exonban 16% heterozigóta: a fenti mutációk egyike + további, még nem identifikált mutációt nem finn típusú nephrosis nephrin további mutációi Familiáris FSGS: AR: NPHS2 - podocin: mechanotransductio AD: ACTN4 - alfa-actinin 4: aktin filamentumok keresztkötései AD: TRCP6 – ion chatorna: mechanotransductióban szerep 4/4/2017 Reusz Gy: Glomeruláris szindrómák

Proteinuria szindrómákban Köröm-patella szindróma AD, basalis mb megvastagszig, felrostozódik, fibrilláris depositumok; proteinuria, patella hiány, köröm abrormalitások LMX1B mutáció : szabályozó fehérje, a nephrin, podocin, CD2AP és a kollagén alfa-3(IV) és alfa-4(IV) láncok expresszióját befolyásolja Pierson szindróma AR, Congenitális nephrosis (DMS) és microcoria, veselégtelenség 2 hónapos kor előtt Laminin béta-2 lánc mutációja Denys-Drash és Frasier szindróma AD, + Drash syndroma (korai DMS, CRF: 3 éves korig+Wilms tumor+genitális anomáliák) + Frasier syndroma (későbbi FSGS, CRF: 2-3 évtized+Gonadoblastoma+genitális anomáliák) 11p13: WT1 gén: tumor suppresszor, ill. embryonalis regulátor fehérje

Tubuláris kórképek

Proximális tubulus transzport defektusai Albumin + LMW protein Proximális tubulus transzport defektusai Dent Dent 1: CLCN5 X rec. Nephrolith X rec. Hypophosphatemia X rec. Tub. proteinuria Dent 2 OCRL (inositol 5′ phosphatase) Lowe OCRL Fanconi Lumen Vér Mutations of the inositol 5′ phosphatase oculocerebrorenal syndrome of Lowe (OCRL) give rise to the congenital X-linked disorders oculocerebrorenal syndrome of Lowe and Dent disease, two conditions giving rise to abnormal kidney proximal tubule reabsorption, and additional nervous system and ocular defects in the case of Lowe syndrome. Here, we identify two closely related endocytic proteins, Ses1 and Ses2, which interact with the ASH-RhoGAP–like (ASPM-SPD-2-Hydin homology and Rho-GTPase Activating Domain-like) domain of OCRL. The interaction is mediated by a short amino acidmotif similar to that used by the rab-5 effector APPL1 (Adaptor Protein containing pleckstrin homology [PH] domain, PTB domain and Leucine zipper motif 1) APPL1 for OCRL binding. Ses binding is mutually exclusive with APPL1 binding, and is disrupted by the samemissensemutations in the ASHRhoGAP– likedomain that also disruptAPPL1binding. Like APPL1, Ses1 and -2 are localized on endosomes but reside on different endosomal subpopulations. These findings define a consensus motif (which we have called a phenylalanine and histidine [F&H]motif) for OCRL binding and are consistent with a scenario in which Lowe syndrome and Dent disease result from perturbations at multiple sites within the endocytic pathway Lowe

vázlat A fehérje sorsa a nephronban A proteinuria típusai A filtrációs barrier felépítése A tubuláris reabszoprtió mechanizmusa Örökletes kórképek Klinikai következmények

Podocyta sérülés és regeneráció

Glomeruláris epitheliális őssejtek A jó: a podocyták physiológiás pótlása The good: Glomerular epithelial stem cells regenerate podocytes. Glomerular epithelial stem cells (red) are localized at the urinary pole. A transitional cell population (podocyte progenitors, red/light blue) displays features of either glomerular epithelial stem cells or podocytes (light blue) and localize between the urinary pole and the vascular stalk. Cells that express only podocyte markers and the phenotypic features of differentiated podocytes localize at the vascular stalk of the glomerulus or within the glomerular tuft (light blue). Proposed mechanisms of podocyte regeneration are depicted in more detail in (A), (B), and (C). (A) Glomerular epithelial stem cells can self-renew and also generate novel podocytes by progressively proliferating and differentiating toward the vascular stalk. This occurs as the kidney grows, during childhood and adolescence, and might also occur after an injury, which allows for a slow generation of novel podocytes, such as after uninephrectomy. (B and C) In glomerular disorders characterized by severe podocyte death or detachment, glomerular epithelial stem cells generate cell bridges between the Bowman’s capsule and the glomerular tuft, which may allow a quick replacement of lost podocytes. (B) Cell bridges may provide a slide for the migration, proliferation, and differentiation of an adjacent progenitor and a quick replacement of lost podocytes. (C) Bridging parietal epithelial cells might also acquire podocyte markers after injury and directly replace the lost podocytes. The directions of migration, proliferation, and differentiation of glomerular epithelial stem cells to regenerate lost podocytes are indicated by the arrows.

A rossz: elégtelen regeneráció Ha a regeneráció az őssejtekből elégtelen, a podocyta veszteségre az őssejtek extracelluláris mátrixot termelnek (TGF-béta hatás) őssejt EC mátrix Leváló podo- cyta The bad: Limits and defaults of the regenerative potential of glomerular epithelial stem cells. Age, genetic alterations, and environmental factors limit the regenerative response of glomerular epithelial stem cells (red), thus impairing podocyte (light blue) replacement when the amount of injured cells is extensive. If regeneration is impaired, podocyte loss typically results in the deposition of extracellular matrix (pink), which can be produced by glomerular epithelial stem cells in response to TGF- that is secreted by injured podocytes Podo- cyta pro-genitor Leváló podo- cyta

A csúf: irányítatlan proliferáció Crescent képződés „TIP” lézió Vizelet pólus őssejt hidak Leváló podociták Makro- fág The ugly: Dysregulated glomerular epithelial stem cells create their own lesions. Aberrant proliferation of glomerular epithelial stem cells can generate hyperplastic lesions. (A) After massive podocyte injury, glomerular epithelial stem cells (red) generate cell bridges with the glomerular tuft in several areas of the glomerulus to quickly replace lost podocytes (light blue). However, numerous areas of podocyte injury distort glomerular structural integrity, thus altering the polarity of glomerular epithelial stem cell division and initiating their abnormal proliferation and the development of extracapillary hyperplastic lesions as well as crescents. Macrophages (green) can also be included within the lesions. Similar processes might occur in crescentic glomerulonephritis and collapsing glomerulopathy. (B) Replacement of podocytes under physiologic conditions follows a gradient, with neo-podocytes progressively added at the vascular stalk. Thus, the tip podocytes represent the “oldest” podocytes of the glomerular tuft, which suggests they might be more susceptible to injury related to heavy proteinuria. Glomerular epithelial stem cells may also proliferate and migrate from the urinary pole of the Bowman’s capsule toward the tuft in an attempt to replace the podocytes lost in response to heavy proteinuria, and generate the tip lesion. Podocyta- progenitor Érpólus

A pathogenesist befolyásoló tényezők proteinuria esetén Betegségre hajlamosító gének HLA Etnikum Apolipoproteinek Paraoxonáz Cytokinek és receptorok egyéb Keringő, permeabilitást fokozó faktor Progressziót befolyásoló gének ACE Etnikum ? Immunosuppresióra adott válasz ? Sclerosis hajlam Keringő, permeabilitást csökkentő faktor Proteinuria hajlam (ld. nephrin, podocyn polymorfizmus, heterozygotia) PROTEINURIA 4/4/2017 Reusz Gy: Glomeruláris szindrómák

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