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.     

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture

Summary The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidneyin vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidneyin vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.     

Sieve structure of slit diaphragms of podocytes and pore cells of gastropod molluscs

Summary The ultrastructure of the slit diaphragms between the pedicels of the podocytes of the prosobranch Viviparus viviparus and between the cytoplasmic tongues of the haemocyanin producing pore cells of the pulmonate Lymnaea stagnalis was investigated. In both cell types 2 diaphragms are present in the slits. They form a 3-dimensional sieve structure with holes of respectively 90 × 110 Å (podocyte) and 200 × 220 Å (pore cell). Injection experiments showed that the size of the holes of the pore cell sieve matches that of particles which can be ingested by this cell type. The substructure of the sieves of the molluscs is compared to that of the 2-dimensional sieve of the podocytes of the mouse and the rat.The authors thank Mrs. J.E. Vlugt-van Dalen for technical assistance and Mr. G.W.H. van der Berg for drawing the diagrams. Thanks are furthermore due to Miss B.E.C. Plesch for correcting the English text     

The fine structure of the kidney of the hagfish (Myxine glutinosa L.)

Summary The fine structure of the kidney (glomerulus and archinephric duct) of the hagfish, Myxine glutinosa (L.) was studied in thin sections and by freeze-fracture technique.The glomerular filtration barrier is similar to that of mammalian kidneys. However, endothelial fenestrations are relatively scanty and the basement membranes of endothelial cells and podocytes always appear separated by a layer of collagen fibrils and microfibrils often surrounding numerous and extended mesangial cells. Between podocytes and their processes maculae occludentes and peculiar junctions of another type occur.The zonulae occludentes between epithelial cells of the archinephric duct are composed of five or more strands, occasionally of only one or two.Supported in part by Deutsche Forschungsgemeinschaft (SFB 146, STO B4) and NIH (ISOI-RR 05764).We are grateful to Dipl.-Ing. G. Wermbter for her helpful criticism and to Mr. H. Heidreich for his excellent technical assistance.     

Structure of the cumulus oophorus at the time of fertilization

Summary The paired external glomus of the fully developed pronephros has been studied in early larvae (ammocoetes) of 2 lamprey species, Lampetra fluviatilis and Petromyzon marinus, several weeks after hatching and newly hatched, by use of light-, scanning (SEM) and transmission (TEM) electron microscopy. Three weeks after hatching the glomus is a complex of capillary loops supplied by a single arteriole branching from the aorta. The glomus consists of 3 cell types: podocytes, fenestrated endothelium, and mesangial cells. A basement membrane, which has a close contact to the podocytes, is the only continuous barrier between blood and the coelomic cavity. The glomus exhibits all fine-structural elements known to be essential for function in the glomeruli of other vertebrates. We therefore assume the pronephric glomus of lampreys to be functional in ultrafiltration, with the ultrafiltrate released into the coelomic cavity. In newly hatched larvae, the structure of the glomus is not fully developed. In this earlier stage several afferent arterioles supply each glomus. The endothelial cells in the glomar capillaries still lack regular epithelial organization and resemble mesenchymal cells. However, the presence of typical podocytes stretching over a continuous basement membrane suggests that the tissue is already capable of ultrafiltration.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing     

Cell cycle re-entry sensitizes podocytes to injury induced death

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.     

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.     

Anatomy and ultrastructure of the excretory system of a heart-bearing and a heart-less sacoglossan gastropod (Opisthobranchia, Sacoglossa)

The microanatomy and ultrastructure of the excretory system of the Sacoglossa have been investigated from two species by means of semithin serial sections, reconstructions and transmission electron microscopy. Whereas Bosellia mimetica shows a functional metanephridial system consisting of a heart with ventricle and auricle in a pericardium and a single kidney, Alderia modesta lacks heart and pericardium, possessing only several haemocoelic sinuses and a very long kidney. In B. mimetica podocytes as the site of ultrafiltration could be detected in the pericardial epithelium lining the auricular wall. The flat epithelium of the kidney with extensive basal infoldings and a dense microvillous border towards the luminal surface serves to modify the ultrafiltrate. In A. modesta podocytes are absent. Solitary rhogocytes (pore cells), the fine structure of which strongly resembles podocytes (meandering slits with diaphragms covered by extracellular matrix), occur in B. mimetica and A. modesta, representing additional loci of ultrafiltration. The presence of podocytes situated in the epicardial wall of the auricle is regarded as plesiomorphic for the Mollusca and confirmed for the Sacoglossa in this study, contradicting earlier assumptions of the loss of the primary site of ultrafiltration in the ancestors of the Opisthobranchia. In contrast to the likewise heart-less Rhodopidae with a pseudoprotonephridial ultrafiltration system, A. modesta shows no further modifications of the excretory system. Accepted: 7 May 2001     

Trb2, a mouse homolog of tribbles, is dispensable for kidney and mouse development

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.     

Expression of intermediate filaments of podocytes within nephrotic syndrome glomerulopathies in children

The study attempted to define characteristics of renal podocytes in nephrotic syndrome glomerulopathies in children with and without glomerular immaturity based on the histochemical expression of cytokeratin 18 (CK 18) and vimentin. Material consisted of 29 renal biopsies performed in the Department of Pediatric Nephrology, Poznan University of Medical Sciences, between 1991 and 2000. The study group included 16 children with mesangial glomerulonephritis (MesGN) and signs of glomerular immaturity and 13 children with MesGN without signs of glomerular immaturity. The control tissue was derived from macroscopically normal renal cortex taken from kidneys resected for localised neoplasms (n=3). In the control samples, the immunocytochemical expression of CK 18 was found only in epithelial cells of proximal and distal tubules. Vimentin was present in all podocytes, some mesangial cells and endothelium. In all cases of children with MesGN with signs of immaturity, both CK 18-positive and vimentin-positive podocytes were found. In all cases of MesGN without immaturity we revealed CK 18-negative podocytes but with distinct vimentin-positive expression. Reorganisation of cytoskeletal proteins within immature podocytes may be associated with the unfavourable clinical course of nephrotic syndrome in children. The application of antibodies against intermediate filaments may help to differentiate between mature and immature forms of MesGN.     

Role of nephrin in podocyte injury induced by angiotension II

Objective: To investigate the function of nephrin in podocytes and its relation to proteinuria in kidney diseases, and to study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury. Methods: Experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8?h, 24?h and 48?h. Detecting nephrin mRNA and protein expression changes by real time PCR (RT-PCR) and western blotting at different time points. Results: Podocyte cell bodies were significantly reduced after Ang II injury (p?<?0.01), losartan directly reduces the rate of apoptotic podocytes induced by Ang. Apoptotic podocytes may related to the decrease of nephrin mRNA and protein expressions, losartan reduced the apoptosis and proteinuria by declining nephrin mRNA and protein expressions. Conclusion: Ang II induced podocyte injury caused abnormal expression and distribution of nephrin in podocytes, losartan maybe maintain the stability of nephrin expression and the integrity of hole diaphragm (SD) structure and function by blocking the signal path, playing a important role in protection mechanisms of anti-proteinuria. Our findings provide some possible clues for further exploring the pharmacological targets to the proteinuria. These novel findings provide new insights into the beneficial effects of losartan on podocytes directly.     

MDM2 is implicated in high‐glucose‐induced podocyte mitotic catastrophe via Notch1 signalling

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in ‘post‐mitosis’ state and unable to regenerate. Re‐entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi‐nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up‐regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p‐H3. Genetic deletion of MDM2 partly reversed HG‐induced mitotic phase re‐entering of podocytes. Moreover, HG‐induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin‐3a, an inhibitor of MDM2‐p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG‐mediated podocyte MC. In conclusion, high glucose up‐regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG‐induced MC in podocytes.     

Free-swimming Cellular Complexes in the Coelom of the Sipunculid Thysanocardia nigra Ikeda, 1904 (Sipuncula)

The ultrastructural characteristics of coelomic cell complexes in the coelomic fluid were investigated with the use of transmission electron microscopy on the example of Japanese sipunculid. In the sipunculid coelom, complexes consisting of several cells were found for the first time: the central glandular cell and the outer layer of podocytes. Peculiar cell complexes (urns), comprising by ciliary and granular cells, were described in Thysanocardia for the first time. It had been proposed that both types of coelomic cell complexes dissociated from extensive chloragogenic tissue clusters on the intestine surface of Th. nigra. The variety of cell complexes in the coelom of other sipunculid is discussed.     

PIK3C3/VPS34, the class III PtdIns 3-kinase,plays indispensable roles in the podocyte

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3^pdKO/mVps34^pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.     

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     

Immunohistochemical evidence for ecdysteroid-like material in the putative molting glands of Lithobius forficatus (Chilopoda)

Summary Ecdysteroid-like material was demonstrated by means of immunhistochemistry in the anterior body region of Lithobius forficatus with the use of an antiserum against an ecdysone-methoxim-BSA-conjugate in conjunction with a modified PAP-method (Sternberger and Joseph 1979). This material is restricted to a tissue formed by podocytes loosely surrounding the salivary glands. Earlier ultrastructural, experimental and biochemical in vitro investigations indicated that this tissue represents the ecdysial glands; this interpretation is now strengthened by immunohistochemical evidence. Reactivity within the cells occurs predominantly in cytosomes.The authors dedicate this paper to Professor Günther Cleffmann, Institut für Tierphysiologie, Justus-Liebig-Universität Giessen, on the occasion of his 60^th birthday, January 27, 1988     

Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish

Summary Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinaced), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphryna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.     

Ultrastructure of possible sites of ultrafiltration in some gastropoda,with particular reference to the auricle of the freshwater prosobranch Viviparus viviparus L.

Summary In the Gastropoda pressure-ultrafiltration of the blood is assumed to be the first step in urine formation. The most probable site of ultrafiltration is the wall of the heart. Since in other animal groups ultrafilters are characterized by a special cell type, the podocyte, the hearts of two pulmonates (Lymnaea stagnalis, Biomphalaria glabrata) and of four prosobranchs (Viviparus viviparus, Bithynia tentaculata, Ampullaria gigas, Littorina littorea) were ultrastructurally investigated, in order to establish whether or not podocytes occur in these structures. It appeared that only in the wall of the auricle of Viviparus podocytes are present. They form a layer underneath the epicardium, the epithelium covering the auricle. It is assumed that in Viviparus ultrafiltration proceeds in the auricle. The possible route of the pro-urine is discussed. The location of the ultrafilters in the other species studied remains still unknown.     

Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy

Thioredoxin-interacting protein (TXNIP) is induced by high glucose (HG), whereupon it acts to inhibit thioredoxin, thereby promoting oxidative stress. We have found that TXNIP knockdown in human renal tubular cells helped prevent the epithelial-to-mesenchymal transition (EMT). Here, we studied the potential effect of TXNIP on podocyte phenotypic alterations in diabetic nephropathy (DN) in vivo and in vitro. In conditionally immortalized mouse podocytes under HG conditions, knocking down TXNIP disrupted EMT, reactive oxygen species (ROS) production, and mammalian target of rapamycin (mTOR) pathway activation. Further, Raptor short hairpin RNA (shRNA), Rictor shRNA, and mTOR specific inhibitor KU-0063794 were used to assess if the mTOR signal pathway is involved in HG-induced EMT in podocytes. We found that Raptor shRNA, Rictor shRNA, and KU-0063794 could all restrain HG-induced EMT and ROS production in podocytes. In addition, antioxidant Tempol or N-acetylcysteine presented a prohibitive effect on HG-induced EMT in podocytes. Streptozotocin was utilized to render equally diabetic in wild-type (WT) control and TXNIP ^−/− (TKO) mice. Diabetes did not increase levels of 24-hr urinary protein, serum creatinine, blood urea nitrogen, and triglyceride in TXNIP ^−/− mice. Podocyte phenotypic alterations and podocyte loss were detected in WT but not in TKO diabetic mice. Oxidative stress was also suppressed in diabetic TKO mice relative to WT controls. Also, TXNIP deficiency suppresses the activation of mTOR in glomeruli of streptozotocin-induced diabetic mice. Moreover, TXNIP expression, mTOR activation, Nox1, and Nox4 could be detected in renal biopsy tissues of patients with DN. This suggests that decreased TXNIP could ameliorate phenotypic alterations of podocytes via inhibition of mTOR in DN, highlighting TXNIP as a promising therapeutic target.