Koichiro Ichimura

Actin filament organization of foot processes in rat podocytes.

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 cytoskeletons in foot processes of adult podocytes. One is the actin bundle running above the level of slit diaphragms and the other is the cortical actin network located beneath the plasmalemma. Immunogold labeling for actin-binding proteins demonstrated that oí-actinin and synaptopodin were localized in the actin bundle, whereas cortactin was in the cortical actin network. Immunofluorescence labeling for actin-binding proteins in immature podocyte showed that α-actinin was localized at the level of the junctional complex, whereas cortactin was distributed beneath the entire plasmalemma. Synaptopodin was first observed along the basal plasmalemma from the advanced S-shaped body to the capillary loop stage. We conclude that foot processes have specialized actin filamentous organization and that its establishment is associated with the expression and redistribution of actin-binding proteins during development.

Glomerular Endothelial Cells Form Diaphragms during Development and Pathologic Conditions

Unlike most fenestrated capillary endothelial cells, adult glomerular endothelial cells (GEnC) are generally thought to lack diaphragms at their fenestrae, but this remains controversial. In this study, morphologic and immunocytochemical analyses demonstrated that, except for a small fraction, GEnC of adult rats lacked diaphragmed fenestrae, which contain the transmembrane glycoprotein PV-1. In contrast, the GEnC in embryonic rats exhibited diaphragmed fenestrae and expressed PV-1 protein. The luminal surface of the fenestral diaphragm possesses a high density of anionic sites, thereby compensating for the functional immaturity of the embryonic glomerular filtration barrier. In addition, GEnC with diaphragmed fenestrae and PV-1 expression were significantly increased in adult rats with Thy-1.1 nephritis, presumably reflecting a process of restorative remodeling of the glomerular capillary tuft after injury; therefore, the reappearance of PV-1 expression and diaphragmed fenestrae may serve as a marker of glomerular capillary remodeling.

Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

Actin filament organization of foot processes in vertebrate glomerular podocytes

We investigated the actin filament organization and immunolocalization of actin-binding proteins (α-actinin and cortactin) in the podocyte foot processes of eight vertebrate species (lamprey, carp, newt, frog, gecko, turtle, quail, and rat). Three types of actin cytoskeleton were found in these foot processes. (1) A cortical actin network with cortactin filling the space between the plasma membrane and the other actin cytoskeletons described below was found in all of the species examined here. The data indicated that the cortical actin network was the minimal essential actin cytoskeleton for the formation and maintenance of the foot processes in vertebrate podocytes. (2) An actin bundle with α-actinin existing along the longitudinal axis of foot process above the level of slit diaphragms was only observed in quail and rat. (3) An actin fascicle consisting of much fewer numbers of actin filaments than that of the actin bundle was observed in the species other than quail and rat, but at various frequencies. These findings suggest that the actin bundle is an additional actin cytoskeleton reflecting a functional state peculiar to quail and rat glomeruli. Considering the higher intraglomerular pressure and the extremely thin filtration barrier in birds and mammals, the foot processes probably mainly protect the thinner filtration barrier from the higher internal pressure occurring in quail and rat glomeruli. Therefore, we consider that the actin bundle plays a crucial role in the mechanical protection of the filtration barrier. Moreover, the actin fascicle may be a potential precursor of the actin bundle.

Wtip and Vangl2 are required for mitotic spindle orientation and cloaca morphogenesis

Summary Defects in cilia and basal bodies function are linked to ciliopathies, which result in kidney cyst formation. Recently, cell division defects have been observed in cystic kidneys, but the underlying mechanisms of such defects remain unclear. Wtip is an LIM domain protein of the Ajuba/Zyxin family, but its role in ciliogenesis during embryonic development has not been previously described. We report Wtip is enriched in the basal body and knockdown of wtip leads to pronephric cyst formation, cloaca malformation, hydrocephalus, body curvature, and pericardial edema. We additionally show that wtip knockdown embryos display segment-specific defects in the pronephros: mitotic spindle orientation defects are observed only in the anterior and middle pronephros; cloaca malformation is accompanied by a reduced number of ciliated cells; and ciliated cells lack the striated rootlet that originates from basal bodies, which results in a lack of cilia motility. Our data suggest that loss of Wtip function phenocopies Vangl2 loss of function, a core planar cell polarity (PCP) protein located in the basal body protein. Furthermore, we demonstrate that wtip and vangl2 interact genetically. Taken together, our results indicate that in zebrafish, Wtip is required for mitotic spindle orientation in the anterior and middle of the pronephros, cloaca morphogenesis, and PCP, which may underlie the molecular etiology of ciliopathies.

Involvement of Mesangial Cells Expressing α-Smooth Muscle Actin During Restorative Glomerular Remodeling in Thy-1.1 Nephritis

The function of actin cytoskeleton in mesangial cells (MCs) during the recovering process of injured glomeruli is not fully understood. MCs in injured glomeruli express α-smooth muscle actin (α-SMA), which is not detected in normal glomeruli. We focused on the localization of α-SMA in MCs of Thy-1. 1 nephritic rat. Expression of α-SMA in the injured glomeruli peaked at day 5 after antibody injection and then declined gradually. At day 5, MCs, where α-SMA was localized at their cytoplasmic processes situated in various positions, occupied the expanded mesangium. MCs expressing α-SMA tended to be located at the peripheral region close to the glomerular basement membrane (GBM) or endothelial cells at day 8. Localization of α-SMA within the peripheral MCs was restricted to the cytoplasmic processes radiating toward the GBM and touching it with their tips at day 8. These α-SMA-containing processes are suitable to transmit the contractile force to GBM and may contribute to normalize the expanded glomerular volume. In addition, an actin-binding protein, drebrin, was localized in all MC processes extending toward various directions throughout the course of nephritis, suggesting that drebrin is involved in the formation of MC processes.

SIRP-α-CD47 system functions as an intercellular signal in the renal glomerulus

The renal glomerulus consists of endothelial cells, podocytes, and mesangial cells. These cells cooperate with each other for glomerular filtration; however, the intercellular signaling molecules between glomerular cells are not fully determined. Tyrosine phosphorylation of slit diaphragm molecules is a key to the detection of the signal to podocytes from other cells. Although src kinase is involved in this event, the molecules working for dephosphorylation remain unclear. We demonstrate that signal-inhibitory regulatory protein (SIRP)-alpha, which recruits a broadly distributed tyrosine dephosphorylase SHP-2 to the plasma membrane, is located in podocytes. SIRP-alpha is a type I transmembrane glycoprotein, which has three immunoglobulin-like domains in the extracellular region and two SH2 binding motifs in the cytoplasm. This molecule functions as a scaffold for many proteins, especially the SHP-2 molecule. SIRP-alpha is concentrated in the slit diaphragm region of normal podocytes. CD47, a ligand for SIRP-alpha, is also expressed in the glomerulus. CD47 is located along the plasma membrane of mesangial cells, but not on podocytes. CD47 is markedly decreased during mesangiolysis, but increased in mesangial cells in the restoration stage. SIRP-alpha is heavily tyrosine phosphorylated under normal conditions; however, tyrosine phosphorylation of SIRP-alpha was markedly decreased during mesangiolysis induced by Thy1.1 monoclonal antibody injection. It is known that the cytoplasmic domain of SIPR-alpha is dephosphorylated when CD47 binds to the extracellular domain of SIRP-alpha. The data suggest that the CD47-SIRP-alpha interaction may be functionally important in cell-cell communication in the diseased glomerulus.

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants.

Medaka fish, Oryzias latipes, as a model for human obesity-related glomerulopathy.

Obesity, an ongoing significant public health problem, is a part of complex disease characterized as metabolic syndrome. Medaka and zebrafish are useful aquatic experimental animals widely used in the field of toxicology and environmental health sciences and as a human disease models. In medaka, simple feeding of a high fat diet (HFD) can induce body weight gain, excessive accumulation of visceral adipose tissue, hyperglycemia, hyperlipidemia, and steatohepatitis, which mimics human metabolic syndrome. In the present study, to explore the possibility that the adult medaka fed with HFD (HFD-medaka) can be used as an animal model for human metabolic syndrome-associated glomerular disease, including obesity-related glomerulopathy (ORG), we analyzed structural alterations and protein expression in the mesonephric kidney of HFD-medaka. We found that the histopathology was consistent with glomerulomegaly accompanied by the dilation of glomerular capillaries and proliferative expansion of the mesangium, a condition partially comparable to human ORG. Moreover, expressions of several kinds of kidney disease-related proteins (such as MYH9, SM22α) were significantly elevated. Thus, the HFD-medaka has a high potential as an animal model useful for exploring the mechanism underling human ORG.