scholarly journals POROUS SUBSTRUCTURE OF THE GLOMERULAR SLIT DIAPHRAGM IN THE RAT AND MOUSE

1974 ◽  
Vol 60 (2) ◽  
pp. 423-433 ◽  
Author(s):  
Richard Rodewald ◽  
Morris J. Karnovsky
Keyword(s):  
Serum Albumin ◽  
Plasma Membranes ◽  
Total Surface Area ◽  
Slit Diaphragm ◽  
Physiological Data ◽  
Filtration Barrier ◽  
Central Filament ◽  
Cross Bridges ◽  
Tracer Experiments ◽  
Glomerular Capillaries

The highly ordered, isoporous substructure of the glomerular slit diaphragm was revealed in rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde. The slit diaphragm was similar in both animal species and appeared as a continuous junctional band, 300–450 Å wide, consistently present within all slits formed by the epithelial foot processes. The diaphragm exhibited a zipper-like substructure with alternating, periodic cross bridges extending from the podocyte plasma membranes to a central filament which ran parallel to and equidistant from the cell membranes. The dimensions and spacing of the cross bridges defined a uniform population of rectangular pores approximately 40 by 140 Å in cross section and 70 Å in length. The total area of the pores was calculated to be about 2–3% of the total surface area of the glomerular capillaries. Physiological data indicate that the glomerular filter functions as if it were an isoporous membrane which excludes proteins larger than serum albumin. The similarity between the dimensions of the pores in the slit diaphragm and estimates for the size and shape of serum albumin supports the conclusion from tracer experiments that the slit diaphragm may serve as the principal filtration barrier to plasma proteins in the kidney.

scholarly journals Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney

1975 ◽  
Vol 65 (1) ◽  
pp. 233-236 ◽  
Author(s):  
MJ Karnovsky ◽  
GB Ryan
Keyword(s):  
Plasma Proteins ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Slit Diaphragm ◽  
Renal Glomerulus ◽  
Regular Array ◽  
Filtration Barrier ◽  
Normal Rat ◽  
Cross Bridges

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.

scholarly journals Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane

1978 ◽  
Vol 77 (2) ◽  
pp. 323-328 ◽  
Author(s):  
WW Franke ◽  
C Grund ◽  
E Schmid ◽  
E Mandelkow
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Membrane Surface ◽  
Hexagonal Lattice ◽  
Interparticle Distance ◽  
Macromolecular Complexes ◽  
Inner Surface ◽  
Cross Bridges ◽  
Plasma Membrane Surface

In cultured cells of the rat kangaroo PtK2 line, veils of the cell surface were observed which consisted of only plasma membrane and paracrystalline arrays of membrane-associated particles sandwiched in between. These membrane-to-membrane cross-bridging 9-to 11-nm wide particles were somewhat coumellar-shaped and were arranged on a hexagonal lattice with an interparticle distance of 16nm. At higher magnification, they revealed an unstained core, thus suggesting a ringlike substructure. Similar arrays of paracrystal-containing veils, which were rather variable in size and frequency, were also observed in other cultured cells. It is hypothesized that these paracrystals represent protein macromolecular complexes associated with the inner plasma membrane surface which crystallize when plasma membranes come into close intracellular contact and other components of the subsurface network are removed.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

2021 ◽  
pp. ASN.2020040501
Author(s):  
Annika Möller-Kerutt ◽  
Juan E. Rodriguez-Gatica ◽  
Karin Wacker ◽  
Rohan Bhatia ◽  
Jan-Peter Siebrasse ◽  
...  
Keyword(s):  
Er Stress ◽  
Fluorescent Protein ◽  
Glycosylation Site ◽  
Slit Diaphragm ◽  
Glycosylation Pattern ◽  
Steroid Resistant ◽  
Steroid Resistant Nephrotic Syndrome ◽  
Er Stress Response ◽  
Filtration Barrier ◽  
Protein Variants

BackgroundCrumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identifiedCRB2mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).MethodsTo study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze theseCrb2podKOknockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein–tagged CRB2 wild type and mutants.ResultsCrb2podKOmice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conservedO-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.ConclusionsCrb2 is an essential component of the podocyte’s slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

scholarly journals Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique.

1987 ◽  
Vol 105 (4) ◽  
pp. 1771-1780 ◽  
Author(s):  
T Nakata ◽  
N Hirokawa
Keyword(s):  
Actin Filaments ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Electron Microscopic Level ◽  
Immunocytochemical Study ◽  
Cytoskeletal Reorganization ◽  
Electron Microscopic ◽  
Activated Platelets ◽  
Mixed Polarity ◽  
Cross Bridges

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.

Pathogenesis of proteinuria in minimal change disease and focal segmental glomerulosclerosis

2018 ◽  
Author(s):  
Patrick Niaudet ◽  
Alain Meyrier
Keyword(s):  
Serum Albumin ◽  
Focal Segmental Glomerulosclerosis ◽  
Minimal Change Disease ◽  
Minimal Change ◽  
Slit Diaphragm ◽  
Segmental Glomerulosclerosis ◽  
Podocyte Loss ◽  
Glomerular Filtrate

It is now well established that the podocyte, and in particular the slit diaphragm structure, are critical to the barrier to serum albumin entering glomerular filtrate in large quantities. In minimal change disease there is proteinuria without podocyte death, whereas in focal segmental glomerulosclerosis there is not only podocyte dysfunction but also podocyte loss.

scholarly journals Early Glomerular Filtration Defect and Severe Renal Disease in Podocin-Deficient Mice

2004 ◽  
Vol 24 (2) ◽  
pp. 550-560 ◽  
Author(s):  
Séverine Roselli ◽  
Laurence Heidet ◽  
Mireille Sich ◽  
Anna Henger ◽  
Matthias Kretzler ◽  
...  
Keyword(s):  
Renal Disease ◽  
Glomerular Filtration ◽  
Molecular Mechanisms ◽  
Slit Diaphragm ◽  
Vascular Lesions ◽  
Suitable Model ◽  
Glomerular Diseases ◽  
Filtration Barrier ◽  
Severe Renal Disease

ABSTRACT Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2 −/−) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2 −/− mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2 −/− kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.

scholarly journals Myosin 1e is a component of the glomerular slit diaphragm complex that regulates actin reorganization during cell-cell contact formation in podocytes

2013 ◽  
Vol 305 (4) ◽  
pp. F532-F544 ◽  
Author(s):  
J. Bi ◽  
S. E. Chase ◽  
C. D. Pellenz ◽  
H. Kurihara ◽  
A. S. Fanning ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Molecular Motor ◽  
Subcellular Fractionation ◽  
Slit Diaphragm ◽  
Cell Junctions ◽  
Cell Contact ◽  
Filtration Barrier ◽  
Renal Glomeruli ◽  
Actin Cables ◽  
Cell Cell

Glomerular visceral epithelial cells, also known as podocytes, are critical to both normal kidney function and the development of kidney disease. Podocyte actin cytoskeleton and their highly specialized cell-cell junctions (also called slit diaphragm complexes) play key roles in controlling glomerular filtration. Myosin 1e (myo1e) is an actin-based molecular motor that is expressed in renal glomeruli. Disruption of the Myo1e gene in mice and humans promotes podocyte injury and results in the loss of the integrity of the glomerular filtration barrier. Here, we have used biochemical and microscopic approaches to determine whether myo1e is associated with the slit diaphragm complexes in glomerular podocytes. Myo1e was consistently enriched in the slit diaphragm fraction during subcellular fractionation of renal glomeruli and colocalized with the slit diaphragm markers in mouse kidney. Live cell imaging studies showed that myo1e was recruited to the newly formed cell-cell junctions in cultured podocytes, where it colocalized with the actin filament cables aligned with the nascent contacts. Myo1e-null podocytes expressing FSGS-associated myo1e mutant (A159P) did not efficiently assemble actin cables along new cell-cell junctions. We have mapped domains in myo1e that were critical for its localization to cell-cell junctions and determined that the SH3 domain of myo1e tail interacts with ZO-1, a component of the slit diaphragm complex and tight junctions. These findings suggest that myo1e represents a component of the slit diaphragm complex and may contribute to regulating junctional integrity in kidney podocytes.

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment

2005 ◽  
Vol 289 (2) ◽  
pp. F431-F441 ◽  
Author(s):  
Maribel Rico ◽  
Amitava Mukherjee ◽  
Martha Konieczkowski ◽  
Leslie A. Bruggeman ◽  
R. Tyler Miller ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Slit Diaphragm ◽  
Cell Junctions ◽  
Puromycin Aminonucleoside ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Interacting Protein ◽  
Filtration Barrier ◽  
Cell Cell

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 μg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

Stokes Flow Through a Row of Cylinders Between Parallel Walls: Model for the Glomerular Slit Diaphragm

1994 ◽  
Vol 116 (2) ◽  
pp. 184-189 ◽  
Author(s):  
M. Claudia Drumond ◽  
William M. Deen
Keyword(s):  
Stokes Flow ◽  
Stokes Equations ◽  
Slit Diaphragm ◽  
Hydraulic Permeability ◽  
Shear Stresses ◽  
Two Factors ◽  
Additional Resistance ◽  
Flow Through ◽  
Experimental Values ◽  
Glomerular Capillaries

As a model for flow through the slit diaphragms which connect the epithelial foot processes of renal glomerular capillaries, finite element solutions of Stokes equations were obtained for flow perpendicular to a row of cylinders confined between parallel walls. A dimensionless “additional resistance” (f), defined as the increment in resistance above the Poiseuille flow value, was computed for L/W≤4 and 0.1≤ R/L≤0.9, where L is half the distance between cylinder centers, W is half the distance between walls and R is the cylinder radius. Two factors contributed to f: the drag on the cylinders, and the incremental shear stresses on the walls of the channel. Of these two factors, the drag on the cylinders tended to be dominant. A more complex representation of the slit diaphragm, suggested in the literature, was also considered. The predicted hydraulic permeability of the slit diaphragm was compared with experimental values of the overall hydraulic permeability of the glomerular capillary wall.

scholarly journals Experimental membranous nephropathy redux

2005 ◽  
Vol 289 (4) ◽  
pp. F660-F671 ◽  
Author(s):  
Andrey V. Cybulsky ◽  
Richard J. Quigg ◽  
David J. Salant
Keyword(s):  
Signaling Pathways ◽  
Complement Activation ◽  
Membranous Nephropathy ◽  
Plasma Membranes ◽  
Stress Proteins ◽  
Experimental Models ◽  
Slit Diaphragm ◽  
Glomerular Epithelial Cell ◽  
Substantial Progress ◽  
Key Steps

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Active and passive Heymann nephritis (HN) in rats are valuable experimental models because their features so closely resemble human MN. In HN, subepithelial immune deposits form in situ as a result of circulating antibodies. Complement activation leads to assembly of C5b-9 on glomerular epithelial cell (GEC) plasma membranes and is essential for sublethal GEC injury and the onset of proteinuria. This review revisits HN and focuses on areas of substantial progress in recent years. The response of the GEC to sublethal C5b-9 attack is not simply due to disruption of the plasma membrane but is due to the activation of specific signaling pathways. These include activation of protein kinases, phospholipases, cyclooxygenases, transcription factors, growth factors, NADPH oxidase, stress proteins, proteinases, and others. Ultimately, these signals impact on cell metabolic pathways and the structure/function of lipids and key proteins in the cytoskeleton and slit-diaphragm. Some signals affect GEC adversely. Thus C5b-9 induces partial dissolution of the actin cytoskeleton. There is a decline in nephrin expression, reduction in F-actin-bound nephrin, and loss of slit-diaphragm integrity. Other signals, such as endoplasmic reticulum stress, may limit complement-induced injury, or promote recovery. The extent of complement activation and GEC injury is dependent, in part, on complement-regulatory proteins, which act at early or late steps within the complement cascade. Identification of key steps in complement activation, the cellular signaling pathways, and the targets will facilitate therapeutic intervention in reversing GEC injury in human MN.

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