New aspects of the possible sites of ultrafiltration in annelids (oligochaeta)

Electron microscopic investigations of blood vessels were conducted to show sites of filtration such as podocytes or fenestrated endothelia. The endothelia of the blood vessels of Aelosoma hemprichi, Nais elinguis, Dero obtusa and Enchytraeus buchholzi consist of myoendothelial cells, chloragocytes and podocytes. The podocytes form large archs over a considerable area of the vessels. On the lumen side of the vessel there are several columnar processes which split into numerous small pedicels. The gaps between the adjacent pedicles are bridged by slit membranes. The podocytes are restricted to the front part of the ventral vessel. They are presumed to form a filtration surface. Furthermore, some parts of the ventral vessel are formed by a fenestrated endothelium, mainly in Enchytraeus buchholzi. In the vascular system of E. buchholzi two separate filtration sites were found. Additionally to the filtration site between ventral vessel and coelomic cavity a second filtration site was found in the front part of the body between blood sinus and coelomic cavity. In such areas the basement membrane is the only continuous layer between the blood vessel and the coelomic cavity. Its thickness is in the range of 40 nm. Possible filtration sites in the form of podocytes and irregular fenestrations could be localized at the border between the blood compartment and the coelomic compartment. It can be presumed that the primary urine may be formed by ultrafiltration of blood.     

Ultrastructure of the metanephridial system in Aeolosoma bengalense (Annelida)

Summary The excretory system of Aeolosoma bengalense has been examined by light and electron microscopy. The system consists of seven serially arranged paris of metanephridia and six pairs of podocytes (referring to the first zoid of an animal chain). The podocytes surround blood spaces of the alimentary canal forming dorsoventrally running loops that emerge on both sides of it. The two elements of the system have a correlative position, each podocyte extending in close proximity to the funnel of a metanephridium. Only in the region of the first metanephridia are podocytes lacking. The nephrostome of the metanephridia consists of two cells, an inner one, the terminal duct cell, and an outer one enwrapping it, called the mantle cell. Nephrostomal cilia that extend into the coelomic space arise exclusively from the rim of the mantle cell whereas those of the terminal duct cell arranged on its luminal surface protrude into the canal forming a flame. The nephridial canal is ciliated throughout and is either intra- or extracellular. Its initial loops aggregate to form a compact organ, the nephridial body. The middle part of the duct constitutes a loop that ascends at each side of the alimentary canal where it is in intimate contact with its blood spaces. Ultrastructural features of the duct cells suggest a reabsorptive function in two regions, the nephridial body and the uppermost part of the loop. The terminal part of the duct passes through the nephridial body and opens ventrolaterally. Generally, the transverse vascular loops at the gut consist of one podocyte each. In the oesophageal region, where only one pair of podocytes is present, the loops connect the dorsal with the ventral longitudinal vessel. Three pairs of podocytes are present in the dilated region of the intestine emerging from its lateral wall and joining the median ventral vessel or blood spaces near by. In the hind gut, where two pairs of podocytes occur, the loops arise from the dorsolateral part and enter directly the ventral vessel. Cytological features of podocytes resemble those of other animals. The results are discussed on the basis of current theories on the function and the phylogenetic significance of excretory systems in the Annelida.Abbreviations bl basal lamina - bs blood space - bv blood vessel - cf ciliary flame - ci cilia - co connection of the vascular loop with the intestinal blood space - cu cuticle - db dense body - dc duct cell - di dictyosome - za zonula adhearens - dv dorsal vessel - ecb epicuticular body - ev endocytotic vesicle - ic intestinal cell - ici inner cilia - iv intestinal vessel - lm longitudinal muscle - mc mantle cell - mg midgut - mi mitochondrion - mv microvilli - nu nucleus - oci outer cilia - oe oesophagus - pc podocyte - pe pedicel - pel primary elongation of the podocyte - sm slit membrane - tc terminal duct cell - ve vesicle with heterogeneous contents - vv ventral vessel     

Microscopic Anatomy and Ultrastructure of a Polian Vessel in the Sipunculan Thysanocardia nigra Ikeda, 1904 from the Sea of Japan

The microscopic anatomy and ultrastructure of a Polian vessel have been studied in the sipunculan Thysanocardia nigra Ikeda, 1904 from the Sea of Japan using the methods of histology and electron microscopy. We describe ultrastructural features of the inner and outer coelothelium, which is constructed of podocytes and multiciliary cells. Between the processes of the podocyte cells, we found double diaphragms that are considered characteristic macromolecular filters. We conclude from an analysis of the ultrastructural features of the vessel wall that coelomic fluid may be filtered from the tentacular coelom to the trunk coelom via the wall of the Polian vessel.     

Ultrastructural evidence of the excretory function in the asteroid axial organ (Asteroidea,Echinodermata)

The ultrastructure of the axial organ of Asterias amurensis has been studied The organ is a network of canals of the axial coelom separated by haemocoelic spaces. The axial coelom is lined with two types of monociliary cells: podocytes and musculo-epithelial cells. Podocytes form numerous basal processes adjacent to the basal lamina on the coelomic side. Musculo-epithelial cells form processes running along the basal lamina. Some bundles of these processes wrapped in the basal lamina pass through haemocoelic spaces between neighboring coelomic canals. It is hypothesized that the axial organ serves for filtration of fluid from haemocoelic spaces into the axial coelom cavity, from which urine is excreted through the madreporite to the exterior.     

The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.     

Possible sites of ultrafiltration in Tubifex tubifex Müller (Annelida,Oligochaeta)

Summary The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4–1 m. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50 nm. It is the only permeability barrier between blood and coelomic fluid.     

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.     

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin cytoskeleton, disruption of the filtration barrier and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context.     

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

Hemoglobins from deep-sea hydrothermal vent scaleworms of the genus Branchipolynoe: a new type of quaternary structure

Branchipolynoe symmytilida and B. seepensis are two scaleworms (Polychaeta; Polynoidae) living commensally in the mantle cavity of deep-sea hydrothermal vent and cold-seep mussels. In contrast with littoral members of this family, the two species exhibit a large amount of extracellular hemoglobin (Hb) in their coelomic fluid. Gel filtration revealed the existence of four different Hbs: one minor, high molecular mass (3x10(6) Da) Hb, V1-Hb, reminiscent of a vascular hexagonal bilayer annelid Hb; two major coelomic Hbs, C1-Hb, and C2-Hb, with unusual masses for extracellular annelid Hbs of 153 and 124 kDa respectively; and a minor probably coelomic Hb of 23 kDa (C3-Hb). Using electrospray ionization mass spectrometry, SDS-PAGE after subtilisin treatment, and tandem mass spectrometry, we showed that C1-Hb is a trimer of a 57,996 Da chain and C2-Hb is a dimer of a 57,648 Da chain, each chain being a four-domain/four-heme polypeptide. This multimeric, multidomain arrangement is unique among annelid Hbs and appears different from that of other known multidomain Hbs.     

The Structure of Anuran Podocyte—Determined by Ecology?

Abstract The podocytes of ten frog species with different habitat preference were investigated by scanning electron microscopy. The visceral epithelium within these species shows considerable variation in the branching mode of the cellular processes, in the number of pedicels and in the form of cell bodies. The presence of various podocyte cell forms within anurans of one family (e.g. within Ranidae and Discoglossidae) indicates that podocytic structure is not manifested phylogenetically. The complexity of processes and pedicel numbers are high in glomeruli of terrestrial and semiterrestrial frogs but low in aquatic and semiaquatic animals. Consequently, podocyte structure is (a) correlated with environmental conditions and (b) plays an important role in osmoregulation. Furthermore, since podocytes are suggested to serve as stabilizers of glomerular vessels, the cells of the visceral epithelium provide the structural basis for regulation of glomerular filtration rate, e.g. for glomerular intermittency.     

Stretch, tension and adhesion - adaptive mechanisms of the actin cytoskeleton in podocytes

Podocytes form an epithelial layer on the outer aspect of the basement membrane of glomerular capillaries. The interdigitating pattern of podocyte foot processes (PFPs) generates a unique and extremely long cell-cell contact area - the filtration slit. Thus, the interdigitating PFPs are the morphological basis for the high hydraulic conductivity of the glomerular capillaries. Any disturbance in this interdigitating pattern results in a drop of glomerular filtration rate impairing renal function. PFPs are based on the actin cytoskeleton, consisting of a subplasmalemmal network and a central core of filament bundles. Besides giving PFPs their morphology, the actin cytoskeleton anchors cell-cell contact and cell-matrix proteins in podocytes. Several human genetic diseases as well as transgenic mouse models provide evidence for the crucial role of the actin cytoskeleton in podocytes. Varying flow rates of the filtrate, increased glomerular capillary pressure in glomerular hypertension, and varying activation states of contractile proteins in PFPs impose a mechanical load on the actin cytoskeleton, challenging the intricate arrangement of PFPs and podocyte adhesion. Here we review data about the actin cytoskeleton of podocytes and the response of podocytes to mechanical load. From these data possible mechanisms are emerging how the actin cytoskeleton may allow podocytes to adapt to states of increased mechanical load.     

Actin-associated Proteins in the Pathogenesis of Podocyte Injury

Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies.     

Advanced glycation end products inhibit adhesion ability of differentiated podocytes in a neuropilin-1-dependent manner

Podocyte injury can occur by a number of stimuli. Maintaining of an intact podocyte structure is essential for glomerular filtration; therefore, podocyte damage severely impairs renal function. Recently, we have reported that addition of glycated BSA [advanced glycation end products (AGE)-BSA] to differentiated murine podocytes inhibited neuropilin-1 (NRP1) expression and dramatically influenced podocyte migration ability (Bondeva T, Ruster C, Franke S, Hammerschmid E, Klagsbrun M, Cohen CD, Wolf G. Kidney Int 75: 605-616, 2009; Bondeva T, Wolf G. Am J Nephrol 30: 336-345, 2009). The present study analyzes the influence of AGEs and NRP1 on podocyte adhesion and cytoskeleton reorganization. We show that treatment with AGE-BSA significantly reduced podocyte adhesion to collagen IV, laminin, and fibronectin compared with Co-BSA (nonglycated BSA)-incubated cells, which was further augmented by transient inhibition of NRP1 expression using NRP1 short interference (si) RNA. On the other hand, forced overexpression of NRP1 markedly increased the adhesion ability of podocytes to the ECMs despite the AGE-BSA treatment. No changes were observed when podocyte adhesion to collagen I was assayed. These findings were also manifested with disorganization of podocyte actin stress fibers and decreased lamellipodia formation processes due to AGE-BSA treatment or NRP1 suppression. In addition, AGE-BSA or suppression of NRP1 both reduced the phosphorylation of focal adhesion kinase (FAK) and Erk1/2 in PMA-stimulated differentiated podocytes. Analysis of RhoA family GTPase activity demonstrated that treatment with AGE-BSA or NRP1 depletion inhibited as well the activation of the Rac-1 and Cdc42 but did not affect RhoA activity. All these effects were reversed by forced overexpression of full-length NRP1 cloned into the pcDNA3 vector in differentiated podocytes. Our study demonstrates that AGEs, in part via suppression of NRP1 expression, decreased podocyte adhesion and contribute to reduction of Rac-1 and Cdc42 GTPase activity. These effects may be further responsible for the podocytes damage and loss in diabetic nephropathy. Our findings suggest a role for NRP1 in regulating the podocyte actin cytoskeleton, and therefore reduction of NRP1 expression could be critical for podocyte function.     

Reduction of Proteinuria through Podocyte Alkalinization

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pH_i). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pH_i.     

A proteomic investigation of glomerular podocytes from a Denys-Drash syndrome patient with a mutation in the Wilms tumour suppressor gene WT1

Glomerular podocytes are essential for blood filtration in the kidney underpinned by their unique cytoskeletal morphology. An increasing number of kidney diseases are being associated with key podocyte abnormalities. The Wilms tumour suppressor gene (WT1) encodes a zinc finger protein with a crucial role in normal kidney development; and in the adult, WT1 is required for normal podocyte function. Denys-Drash Syndrome (DDS) results from mutations affecting the zinc finger domain of WT1. The aim of this study was to undertake, for the first time, a proteomic analysis of cultured human podocytes; and to analyse the molecular changes in DDS podocytes. The morphology of DDS podocytes was highly irregular, reminiscent of a fibroblastic appearance. A reference 2-D gel was generated, and 75 proteins were identified of which 43% involved in cytoskeletal architecture. The DDS and wild-type proteomes were compared by 2-D DIGE. The level of 95.6% of proteins was unaltered; but 4.4% were altered more than two-fold. A sample of proteins involved in cytoskeletal architecture appeared to be misexpressed in DDS podocytes. Consistent with this finding, overall levels of filamentous actin also appeared reduced in DDS podocytes. We conclude that one of WT1 functions in podocytes is to regulate the expression of key components and regulators of the cytoskeleton.     

足细胞发育异常及相关肾脏疾病研究进展

足细胞是肾小球滤过屏障的重要组成部分,其数量减少或功能障碍将导致肾小球滤过功能损伤和相关肾脏疾病的发生。足细胞为不可再生性细胞,其数量和功能在一定程度上取决于其正常发育。已发表的文献和本实验室的研究工作表明,遗传或不良宫内环境等原因所致的足细胞发育不良,可能导致成年后肾小球滤过功能障碍,并成为某些胎源性肾脏疾病发生或易感的病因之一,而表观遗传学机制可能参与介导足细胞发育过程中某些关键基因的表达异常。本文对足细胞结构功能和正常发育、足细胞发育异常的病因和机制、以及足细胞发育异常所致的肾脏疾病等几方面进行综述,以期对发育源性足细胞相关肾脏疾病的诊断与治疗提供借鉴与参考。     

The cytoprotective role of autophagy in puromycin aminonucleoside treated human podocytes

Autophagy is a ubiquitous catabolic process involving degradation of damaged organelles and protein aggregates. It shows cytoprotective effects in many cell types and helps to maintain cell homeostasis. In many glomerular diseases, podocyte damage leads to the disruption of the renal filtration barrier and subsequent proteinuria. Puromycin aminonucleoside (PAN) which induces podocyte apoptosis in vitro and in vivo is widely used for studying the pathophysiology of glomerular diseases. It has been shown that PAN induces autophagy in podocytes. However, the relationship between autophagy and apoptosis in PAN treated human podocytes is not known and the role of PAN-induced autophagy in podocyte survival remains unclear. Here we demonstrate that PAN induced autophagy in human podocytes prior to apoptosis which was featured with the activation of mTOR complex 1 (mTORC1). When the PAN-induced autophagy was inhibited by 3-methyladenine (3-MA) or chloroquine (CQ), podocyte apoptosis increased significantly along with the elevation of active caspase-3. Under such circumstance, the podocyte cytoskeleton was also disrupted. Collectively, our results suggested that the induced autophagy may be an early adaptive cytoprotective mechanism for podocyte survival after PAN treatment.     

Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.