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Electron micrographs of podocyte foot processes. Electron microscopic specimens were made by use of either the modified cold dehydration technique ( A,B ) or isolated glomeruli with detergent treatment ( C,D ). ( A ) In the perpendicular sections of the foot processes, the cross-sections of the actin bundle (arrows) are recognized above the level of slit diaphragms (arrowheads). The actin bundle is not directly in contact with the plasma membrane. Amorphous material is seen in the area surrounding the actin bundle. En, endothelial cell; GBM, glomerular basement membrane. ( B ) Horizontal sections of the foot processes (Fp) show the actin bundles (closed arrows) running longitudinally in the foot processes. The actin bundles of neighboring foot processes are connected to each other by arcuate bundles (open arrows) in the primary process (Pp). ( C ) In cross-sections of the foot processes, the cortical actin network (asterisk) is recognized between the plasmalemma and the actin bundle (arrow). The electron-dense materials at the insertion site of the slit diaphragms (arrowheads) are in contact with the cortical actin network. GBM, glomerular basement membrane. ( D ) In the horizontal sections, the longitudinal sections of the actin bundle (closed arrows) and the arcuate bundle (open arrow) are shown. The cortical actin network (asterisk) is in contact with the electron-dense materials at the insertion site of the slit diaphragm (arrowheads). ( E ) Schematic drawing of the actin filament organization of the foot process showing actin bundle (AB) and cortical actin network (CAN). En, endothelial cell; GBM, glomerular basement membrane; SD, slit diaphragm. Bars ϭ 200 nm.
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The foot processes of podocytes possess abundant microfilaments and modulate glomerular filtration. We investigated the actin filament organization of foot processes in adult rat podocytes and the formation of the actin cytoskeletal system of immature podocytes during glomerulogenesis. Electron microscopy revealed two populations of actin cytoskele...
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Context 1
... major cytoskeleton in the foot processes of podo- cytes consisted of actin filaments, which were repre- sented either by dense actin bundles or by a cortical network, as visualized by a modified cold dehydration technique using en bloc staining with OTE as de- scribed in Materials and Methods. The dense bundles of actin filaments were found predominantly at the central portion of the upper cytoplasm of the foot pro- cesses of podocytes, above the level of the slit dia- phragm ( Figure 1A). The actin bundles ran longitudi- nally in the foot processes extending from the primary processes, as visualized in specimens sectioned parallel to the base of the cells ( Figure 1B). ...
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... dense bundles of actin filaments were found predominantly at the central portion of the upper cytoplasm of the foot pro- cesses of podocytes, above the level of the slit dia- phragm ( Figure 1A). The actin bundles ran longitudi- nally in the foot processes extending from the primary processes, as visualized in specimens sectioned parallel to the base of the cells ( Figure 1B). The cortical cyto- plasm of foot processes between the actin bundles and cell membranes was occupied by the cortical network of actin filaments, which were sparse in the apical cy- toplasm and moderately dense in the basal cytoplasm. ...
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... apical cortical cyto- plasm between the actin bundles and the apical cell membranes contained sparse filaments that were con- nected with the bundles and membranes. The basal cortical cytoplasm of foot processes contained a fine meshwork of microfilaments made up of actin (Fig- ures 1C and 1D). The filaments of the basal cortical actin network were associated with dense plaques at the base of the slit diaphragms ( Figure 1C). ...
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... basal cortical cytoplasm of foot processes contained a fine meshwork of microfilaments made up of actin (Fig- ures 1C and 1D). The filaments of the basal cortical actin network were associated with dense plaques at the base of the slit diaphragms ( Figure 1C). The fila- ments of the cortical actin network were frequently merged with those of actin bundles, and therefore clear demarcation of these two populations of actin filaments was not always possible. ...
Context 5
... visualize the pre- cise actin cytoskeletal structure, we adopted the modi- fied cold dehydration technique using OTE en bloc staining for natural tissue and detergent treatment for isolated glomeruli ( Kurihara et al. 1998). On the basis of the present results, we show that the actin cytoskel- etal structure in the foot processes of adult podocytes is divided into two populations: actin bundles and a cortical actin network ( Figure 1E). The actin bundle runs along the longitudinal axis of the foot process above the level of the slit diaphragm, while the corti- cal actin network distributes beneath the plasma membrane of the foot process. ...
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Citations
... Both the SD and FA function as mechanical anchors and major signaling sites for the cytoskeleton [6], which is a highly adaptable and dynamic structure. While microtubules and intermediate filaments predominate in the cell body and primary cell processes, longitudinal bundles of microfilaments containing actin, myosin, and alpha-actinin are seen in the FPs [7][8][9][10]. ...
The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers.
... The slit diaphragm (SD) is a cell-cell adhesion structure that connects adjacent FPs and functions as the filtration barrier to urinary protein loss. [3][4][5][6] Under pathological conditions, podocytes manifest FP effacement (process loss) characterized by a broadening of the processes with disruption of the SD. The FP effacement appears to be a common finding underlying proteinuria. ...
... Inactivation of Rac1 by FilGAP may reduce the antagonistic effect of RhoA/ROCK/myosin II by Rac1. 28,32,69 Although it is debatable whether the actin stress fiber observed in culture podocytes is consistent with the structure of actin filaments in vivo, 3 these findings suggest that FilGAP inactivates Rac1 and is responsible for maintaining the actin-cytoskeletal integrity of podocytes. ...
The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin‐driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase‐activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin‐based protein complex closely associated with stress fibers, that mediates cell‐extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process‐like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP‐depleted podocyte cells was rescued by inhibition of Rac1 or P21‐activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell‐ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.
... Its structure is maintained by intracellular microtubules and actin filaments. Distribution of cytoskeletons is different significantly, such as foot processes composed of actin molecule especially the F-form actin (F-actin) [49]. There are three groups of podocyte cytoskeleton-associated proteins acting with F-actin to influence podocyte cytoskeleton. ...
Diabetic nephropathy (DN), an important complication of diabetic microvascular disease, is one of the leading causes of end-stage renal disease (ESRD), which brings heavy burdens to the whole society. Podocytes are terminally differentiated glomerular cells, which act as a pivotal component of glomerular filtration barrier. When podocytes are injured, glomerular filtration barrier is damaged, and proteinuria would occur. Dysfunction of podocytes contributes to DN. And degrees of podocyte injury influence prognosis of DN. Growing evidences have shown that epigenetics does a lot in the evolvement of podocyte injury. Epigenetics includes DNA methylation, histone modification, and non-coding RNA. Among them, histone modification plays an indelible role. Histone modification includes histone methylation, histone acetylation, and other modifications such as histone phosphorylation, histone ubiquitination, histone ADP-ribosylation, histone crotonylation, and histone β-hydroxybutyrylation. It can affect chromatin structure and regulate gene transcription to exert its function. This review is to summarize documents about pathogenesis of podocyte injury, most importantly, histone modification of podocyte injury in DN recently to provide new ideas for further molecular research, diagnosis, and treatment.
... Podocytes have a unique architecture with foot processes that extend from their cell bodies, interdigitate and form slit diaphragms facilitating size and charge-selective filtration and preventing the loss of plasma proteins 4,5 . In health, podocyte shape is maintained by a complex, highly regulated actin cytoskeleton, which supports the foot processes 6,7 , and anchors the cell to the GBM 8 . During glomerular disease, the podocyte cytoskeleton becomes disorganised often leading to podocyte loss, impaired filtration and leakage of plasma proteins, such as albumin, into the urine 7,[9][10][11] . ...
Plasma ultrafiltration in the kidney occurs across glomerular capillaries, which are surrounded by epithelial cells called podocytes. Podocytes have a unique shape maintained by a complex cytoskeleton, which becomes disrupted in glomerular disease resulting in defective filtration and albuminuria. Lack of endogenous thymosin β4 (TB4), an actin sequestering peptide, exacerbates glomerular injury and disrupts the organisation of the podocyte actin cytoskeleton, however, the potential of exogenous TB4 therapy to improve podocyte injury is unknown. Here, we have used Adriamycin (ADR), a toxin which injures podocytes and damages the glomerular filtration barrier leading to albuminuria in mice. Through interrogating single-cell RNA-sequencing data of isolated glomeruli we demonstrate that ADR injury results in reduced levels of podocyte TB4 . Administration of an adeno-associated viral vector encoding TB4 increased the circulating level of TB4 and prevented ADR-induced podocyte loss and albuminuria. ADR injury was associated with disorganisation of the podocyte actin cytoskeleton in vitro, which was ameliorated by treatment with exogenous TB4. Collectively, we propose that systemic gene therapy with TB4 prevents podocyte injury and maintains glomerular filtration via protection of the podocyte cytoskeleton thus presenting a novel treatment strategy for glomerular disease.
... Ion channels, adhesion proteins, and proteins associated with the cytoskeleton are all critical elements to transduce the mechanical forces into biochemical signals in podocytes (4). The well-organized actin cytoskeletons of foot processes coordinating with the delicate slit diaphragms (SDs) play important roles in maintaining normal structure and function of podocytes (4)(5)(6). Linker proteins such as CD2-associated protein and the noncatalytic region of tyrosine kinase proteins are important to link the fine-tuned structure of SDs to the actin cytoskeleton podocyte (7,8). Mutations of many genes encoding the intrinsic SD components or their interaction partners have been reported to mediate the development of proteinuria and progressive glomerular injury (9). ...
Podocytes are an important component of the glomerular filtration barrier, and maintenance of the integrity of its highly specified structure and function is critical for normal kidney function. YAP/TAZ are two crucial effectors of the Hippo signaling pathway, and recent studies have shown that podocyte-specific YAP deletion causes podocyte apoptosis and development of FSGS followed by progressive renal failure. In the current study, we investigated a potential role of the YAP paralog, TAZ in podocytes. TAZ was found to be constitutively active in podocytes, and the mice with podocyte specific deletion of TAZ (Taz podKO ) developed proteinuria starting at 4 weeks of age, and had increased podocyte apoptosis. Utilizing primary cultured podocytes or immortalized mouse podocytes from Taz flox/flox mice, we found that TAZ is a transcriptional activator for TEAD-dependent expression of synaptopodin, ZO-1 and ZO-2. This is the first study to determine that TAZ plays an important role in maintenance of the structure and function of podocytes .
... In the past, it was shown that the morphology of the podocyte foot processes is mainly determined by the actin cytoskeleton. 4,[52][53][54] We therefore wanted to analyze whether the expression of filamin A is essential for proper foot process morphology as well as for the attachment of podocytes to the GBM. In order to do so, we studied the F-actin cytoskeleton of cultured podocytes after the specific knockdown (Flna KD) or knockout (Flna KO) of filamin A. The experiments revealed that Flna KD/KO induces a significant reorganization as well as a disassembly of actin stress fibers. ...
Glomerular hypertension induces mechanical load to podocytes, often resulting in podocyte detachment and the development of glomerulosclerosis. Although it is well known that podocytes are mechanosensitive, the mechanosensors and mechanotransducers are still unknown. Since filamin A, an actin‐binding protein, is already described to be a mechanosensor and mechanotransducer, we hypothesized that filamins could be important for the outside‐in signaling as well as the actin cytoskeleton of podocytes under mechanical stress. In this study, we demonstrate that filamin A is the main isoform of the filamin family that is expressed in cultured podocytes. Together with filamin B, filamin A was significantly up‐regulated during mechanical stretch (3 days, 0.5 Hz, and 5% extension). To study the role of filamin A in cultured podocytes under mechanical stress, filamin A was knocked down (Flna KD) by specific siRNA. Additionally, we established a filamin A knockout podocyte cell line (Flna KO) by CRISPR/Cas9. Knockdown and knockout of filamin A influenced the expression of synaptopodin, a podocyte‐specific protein, focal adhesions as well as the morphology of the actin cytoskeleton. Moreover, the cell motility of Flna KO podocytes was significantly increased. Since the knockout of filamin A has had no effect on cell adhesion of podocytes during mechanical stress, we simultaneously knocked down the expression of filamin A and B. Thereby, we observed a significant loss of podocytes during mechanical stress indicating a compensatory mechanism. Analyzing hypertensive mice kidneys as well as biopsies of patients suffering from diabetic nephropathy, we found an up‐regulation of filamin A in podocytes in contrast to the control. In summary, filamin A and B mediate matrix‐actin cytoskeleton interactions which are essential for the adaptation of cultured podocyte to mechanical stress.
... The development of the glomerular capillary system starts from the S-shaped body stage of glomerulogenesis (Reeves et al., 1978;Ichimura et al., 2003). The growth process of the glomerular capillary system is considered to be mediated by two types of angiogenesis processes, namely, sprouting angiogenesis and intussusceptive angiogenesis. ...
Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a “seamless” tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.
... Effacement is thought to be due to a 13 breakdown in the actin cytoskeleton of the foot processes 24 . Nephrin is dephosphorylated when the slit membrane is constructed, and it binds to actin through CD2AP and podocin 25,26 . When a slit diaphragm is injured, nephrin molecules become clustered, inducing nephrin phosphorylation, actin polymerization, and the effacement of foot processes 26 . ...
In this study, we focused on nephrin, one of the key molecules within the slit diaphragm of podocytes, as although there have been reports on its expression in humans and rats, their presence in common marmosets has not been reported. We investigated nephrin expression and changes in glomeruli, depending on the development of spontaneous progressive glomerulonephropathy in common marmosets. Nineteen common marmosets at two to ten years of age were evaluated. The kidney was examined by microscopy with hematoxylin and eosin and immunohistochemical staining for nephrin. The lesions were classified into three grades according to a renal lesion grading system reported previously. The nephrin-positive area was measured by morphometric analysis, and the nephrin-positive ratio was calculated. Nephrin expression was observed along the glomerular capillary loop in a continuous linear pattern in renal lesion grades 0 to 2 and either discontinuous linear or coarse granular pattern in grade 3. Nephrin expression tended to decrease significantly depending on the grade of renal lesions. Alteration in nephrin expression has been suggested to play an important role in the progression of renal lesions.
... The foot processes contain a contractile apparatus including actin, myosin, actinin, talin, vinculin, and vimentin, which opposes the hemodynamic forces of the glomerular capillaries. [7,8] Podocytes' main task is to participate in glomerular filtration. The glomerular filtrate flows through endothelial fenestrae, the basement membrane, and the slit diaphragms in the spaces between the foot processes. ...
... CD2AP is found in all human tissues, although experiments on murine models have shown that normal function only requires its presence in the kidneys [12,13]. Proper interaction of the nephrin-podocin-CD2AP complex is believed to be essential for the flow of fluids, electrolytes, and proteins through the filtration slit to occur [8]. Neph1 also interacts with the protein zonula occludens-1 (ZO-1) [11]. ...
Preeclampsia (PE) is a disorder that affects 3–5% of normal pregnancies. It was believed for a long time that the kidney, similarly to all vessels in the whole system, only sustained endothelial damage. The current knowledge gives rise to a presumption that the main role in the development of proteinuria is played by damage to the podocytes and their slit diaphragm. The podocyte damage mechanism in preeclampsia is connected to free VEGF and nitric oxide (NO) deficiency, and an increased concentration of endothelin-1 and oxidative stress. From national cohort studies, we know that women who had preeclampsia in at least one pregnancy carried five times the risk of developing end-stage renal disease (ESRD) when compared to women with physiological pregnancies. The focal segmental glomerulosclerosis (FSGS) is the dominant histopathological lesion in women with a history of PE. The kidney’s podocytes are not subject to replacement or proliferation. Podocyte depletion exceeding 20% resulted in FSGS, which is a reason for the later development of ESRD. In this review, we present the mechanism of kidney (especially podocytes) injury in preeclampsia. We try to explain how this damage affects further changes in the morphology and function of the kidneys after pregnancy.
... The microscopic study of the renal glomerulus was significantly expanded by Koichiro Ichimura (Associate Professor), who studied the actin cytoskeletons in the podocytes 13) , the diaphragms of glomerular endothelial cells 14) , and with the focused ion beam scanning electron microscope (FIB-SEM) the three-dimensional configuration of the podocytes 15) . ...
Forty-one years have passed, since I began my career as anatomist at the Department of Anatomy in alma mater after graduation from the Faculty of Medicine, University of Tokyo in 1978. Twenty-nine years have passed, since I became the Professor of Anatomy at the Juntendo University. I conducted academic activities in four fields. 1) In the microscopic research on the kidney, I discovered the mechanical function of mesangial cells to counterbalance the expanding force of blood pressure in 1987. Since then I studied the microscopic morphology of renal glomerulus, and of the interstitium and blood vessels in various tissues, 2) In the macroscopic research with my colleagues, I studied the morphology of the peripheral nerves and arteries. In recent years, the isolated muscle specimens became an excellent tool to analyze the functional architecture of skeletal muscles. 3) In the history of medicine, I studied especially the history of anatomy, of medical education and of Western medicine in general. 4) In the academic and social education of the anatomy and body donation, I have published many academic textbooks, including 17 titles and 35 editions of the translated and/or supervised textbooks of anatomy, 17 titles and 26 editions authored and/or edited textbooks of anatomy and biology. I have long been engaged in the activities of Tokushi Kaibo Zenkoku Rengokai [National Confederation of Anatomy Body Donation], and served the executive director from 2002 to 2006 and the chairman from 2006 to 2010. I experienced that the body donation became increasingly popular so that the proportion of donated cadavers to the total dissected ones increased from 42% in the 1983 to more than 99% in the 2018.
