S U M M A R YThe 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 ␣ -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 ␣ -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.

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.

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.

Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging, a powerful tool for analyzing the 3D cellular ultrastructure, to precisely reveal the morphological process of podocyte development, such as the formation of foot processes. Development of foot processes gives rise to three morphological states: the primitive, immature and mature foot processes. Immature podocytes were columnar in shape and connected to each other by the junctional complex, which migrated toward the basal side of the cell. When the junctional complex was close to the basement membrane, immature podocytes started to interdigitate with primitive foot processes under the level of junctional complex. As primitive foot processes lengthened, the junctional complex moved between primitive foot processes to form immature foot processes. Finally, the junctional complex was gradually replaced by the slit diaphragm, resulting in the maturation of immature foot processes into mature foot processes. In conclusion, the developmental process of podocytes is now clearly visualized by block-face SEM imaging.

Studies have revealed many analogies between podocytes and neurons, and these analogies may be key to elucidating the pathogenesis of podocyte injury. Cathepsin D (CD) is a representative aspartic proteinase in lysosomes. Central nervous system neurons in CD-deficient mice exhibit a form of lysosomal storage disease with a phenotype resembling neuronal ceroid lipofuscinoses. In the kidney, the role of CD in podocytes has not been fully explored. Herein, we generated podocyte-specific CD-knockout mice that developed proteinuria at 5 months of age and ESRD by 20-22 months of age. Immunohistochemical analysis of these mice showed apoptotic podocyte death followed by proteinuria and glomerulosclerosis with aging. Using electron microscopy, we identified, in podocytes, granular osmiophilic deposits (GRODs), autophagosome/autolysosome-like bodies, and fingerprint profiles, typical hallmarks of CDdeficient neurons. CD deficiency in podocytes also led to the cessation of autolysosomal degradation and accumulation of proteins indicative of autophagy impairment and the mitochondrial ATP synthase subunit c accumulation in the GRODs, again similar to changes reported in CD-deficient neurons. Furthermore, both podocin and nephrin, two essential components of the slit diaphragm, translocated to Rab7-and lysosome-associated membrane glycoprotein 1-positive amphisomes/autolysosomes that accumulated in podocyte cell bodies in podocyte-specific CD-knockout mice. We hypothesize that defective lysosomal activity resulting in foot process effacement caused this accumulation of podocin and nephrin. Overall, our results suggest that loss of CD in podocytes causes autophagy impairment, triggering the accumulation of toxic subunit c-positive lipofuscins as well as slit diaphragm proteins followed by apoptotic cell death.

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.

We investigated the actin filament organization and immunolocalization of actin-binding proteins (alpha-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 alpha-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.

Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3), capillary loop (95 ± 4%, n =  4), and maturing glomerulus (76 ± 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 ± 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-010-0983-7) contains supplementary material, which is available to authorized users.