Filtration Slit Diaphragm Research Articles

Urinary Claudin-1 Level as a Marker of Podocyte Injury in Patients with Proteinuria

Abstract Background The biology of claudins is a rapidly evolving field, and many intriguing questions remain unanswered. Although it had been assumed that the reason there are ≥24 isoforms of claudin is that each one has distinct permeability properties. The nephron displays a wide spectrum of claudins, whose distribution varies in each tubular segment. In diabetic nephropathy and glomerulonephritis the gene expression of claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit diaphragm (cell-cell junction made by the glomerular podocytes) and the appearance of TJ-like structures between the foot processes causing further podocytes injury and proteinuria. Aim of the work to assess urinary claudin -1 level as a marker of podocyte injury in patients with proteinuria. Patients and Methods it is a case control study which was conducted upon 90 subjects who were divided into three groups: group I included 30 patients with type II DM, group II included 30 patients with glomerulonephritis and group III had 30 healthy subjects as controls. Urinary claudin-1 level was measured by Enzyme linked Immunosorbent Assay (ELISA) Results In this study, we found that urinary claudin-1 level was significantly higher in diabetics group and GN group than in control group. In comparison between GN group and diabetic group, we found that urinary claudin-1 level was higher in GN group than in diabetics group but with no statistically significant difference between the two groups. Conclusion urinary claudin-1 level was significantly higher in diabetics and GN group and has positive correlation with uACR. So it can be used as marker of podocytes injury and proteinuria

Sodium hydrogen sulfide (NaHS) ameliorates alterations caused by cisplatin in filtration slit diaphragm and podocyte cytoskeletal in rat kidney.

Background: Hydrogen sulfide (H2S) has been shown to have a protective role in various kidney disorders. Objectives: This study investigated the molecular mechanism of NaHS (a H2S donor) in treating on the renal damage induced by cisplatin (CP).Materials and Methods: Thirty-two male rats were randomly divided into 4 groups: Normal control group (group A)‚ NaHS group (group B) which received 200 µg/kg/d (intraperitoneal injection; i.p.) for 15 days‚ CP group (group C) which rats were injected with CP (5 mg/kg, single dose, i.p.), and CP + NaHS group (group D) (5 mg/kg and 200 µg/kg, respectively, i.p.). Samples of urine and serum, tissue of kidney were collected for analysis after treatments for 15 days. Morphological changes were elevated under light microscope‚ protein expression of desmin and nephrin were determined by immunohistochemistry and western blotting and also malondialdehyde (MDA) level was determined by spectrophotometer. Results: Compared to the CP group, NaHS treatment mitigated histological damages, decreased 24-hour urine protein excretion, serum urea and creatinine as well as MDA level. NaHS treatment increased protein levels of nephrin. Moreover, NaHS treatment decreased protein levels of desmin. Conclusions: NaHS can ameliorate CP -induced renal damage in rats which is associated with the increase in nephrin protein expression, and the decrease in MDA level and desmin protein expression.

Effect of Pravastatin on Levels of Filtration Slit Diaphragm Protein and Oxidative Stress in Doxorubicin- Induced Nephrotoxicity

Abstract: Introduction: Nephrotoxicity is one of the complications the use of doxorubicin (DXR) which may be caused by the formation of free radicals. The aim of this study was to investigate the effects of pravastatin (PS) on doxorubicin-induced nephrotoxocity. Methods: The rats were divided into control, doxorubicin (15 mg/kg, i.p.), PS (20 mg/kg orally, 5 days prior to DXR injection) + DXR and DXR + PS (5 days after DXR injection) groups. At the end of the 20th day, kidneys were removed for evaluation. Podocin mRNA and protein were determined by real–time PCR and Western blotting. Also, the level of tissue’s malondialdehyde (MDA), catalase (CAT), Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were determined. Results: Adminstration of pravastatin increased (P < 0.005) the mRNA and protein expression of podocin and the activities of CAT, SOD and GPx which were decreased in doxorubicin group. Pravastatin also, reduced (P < 0.005) the elevated MDA level in doxorubicin group. Conclusion: The present study showed that that pravastatin can improve doxorubicin-induced nephrotoxicity in rats which is associated with the increase in expression of podocin and antioxidant enzymes activities, and decrease in level of MDA. Key words: Pravastatin, Doxorubicin, Nephrotoxicity, Oxidative stress, Podocin.

Nephrin missense mutations: induction of endoplasmic reticulum stress and cell surface rescue by reduction in chaperone interactions

Nephrin, an important component of the podocyte filtration slit diaphragm, plays a key role in the maintenance of glomerular permselectivity. Mutations in nephrin lead to proteinuria and congenital nephrotic syndrome. Nephrin undergoes posttranslational modifications in the endoplasmic reticulum (ER) prior to export to the plasma membrane. We examined the effects of human nephrin disease-associated missense mutations on nephrin folding in the ER and on cellular trafficking in cultured cells. Compared with wild-type (WT) nephrin, the mutants showed impaired glycosylation and enhanced association with the ER chaperone, calnexin, as well as accumulation in the ER. Nephrin mutants demonstrated enhanced ubiquitination, and they underwent ER-associated degradation. Certain nephrin mutants did not traffic to the plasma membrane. Expression of nephrin mutants resulted in the stimulation of the activating transcription factor-6 pathway of the unfolded protein response, and an increase in the ER chaperone, Grp94. We treated cells with castanospermine (an inhibitor of glucosidase I) in order to decrease the association of nephrin mutants with calnexin. Castanospermine increased plasma membrane expression of nephrin mutants; however, full glycosylation and signaling activity of the mutants were not restored. Modulation of ER quality control mechanisms represents a potential new approach to development of therapies for proteinuric kidney disease, including congenital nephrotic syndrome.

High resolution helium ion scanning microscopy of the rat kidney.

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells.

CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney

Background. CRIM1 is a plasma membrane bound protein containing six cysteine-rich repeats (CRR). Through these, CRIM1 has been shown to interact with a subgroup of the TGF-β superfamily, the bone morphogenic proteins (BMP) isoforms 2, 4 and 7. The probable action is to modulate the signalling properties of these factors. CRIM1 has also been shown to regulate the release of VEGFA by podocytes during renal organogenesis. Knock-out studies in mice have shown that CRIM1 is critically involved in the development of the central nervous system, eye and kidney. Replacement of CRIM1 with a defective version leads to renal dysgenesis and perinatal death. We have analysed the distribution of CRIM1 in adult human renal tissue.Methods. To this end, we have used immunofluorescence, immunohistochemistry and immunoelectron microscopy. We performed western blotting for the CRIM1 protein, using lysates from isolated glomerular podocytes and human renal tissue homogenate. By using quantitative PCR, we compared the CRIM1 mRNA levels in podocytes, human renal tissue homogenate, primary human renal proximal tubular epithelial cells and primary human pulmonary artery smooth muscle cells.Results. The results show that in the human adult kidney, CRIM1 is mainly expressed in the glomerular podocytes and is associated with the insertional region of the filtration slit diaphragm (SD) of the podocyte pedicles.Conclusions. CRIM1 is a protein that should be added to the list of proteins associated with the podocyte filtration SD and with the probable action of modulating BMP and VEGFA signalling.

The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation1 ‘nephron-like’ features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors2. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity with the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialised filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly orthologues of the major constituents of the slit diaphragm, including nephrin, neph1, CD2AP, ZO-1 and podocin are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find the nephrocyte diaphragm is completely lost in flies mutant for nephrin or neph1 orthologues, a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in the human kidney disease NPHS1) or neph1. These changes drastically impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting the two cell types are evolutionarily related and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

Targeted Downregulation of Extracellular Nephrin in Human IgA Nephropathy

Background: The identification of nephrin and CD2AP, podocyte proteins which modulate the properties of glomerular barrier selectivity, has made it possible to unravel the mechanisms undergoing foot process effacement and proteinuria. Here we explored the role of nephrin and CD2AP together with the integrity of the slit diaphragm in the pathogenesis of proteinuria in patients with acquired glomerular diseases. Methods: Nephrin mRNA and protein expression were systematically evaluated in 28 renal biopsy samples from adult patients with primary glomerular disease and proteinuria by in situ hybridization and immunohistochemistry using antibodies directed against extra- and intracellular nephrin and compared with biopsy samples from normal controls. CD2AP protein expression by immunohistochemistry was also assessed. Morphometrical analysis of the filtration slit was performed by transmission electron microscopy. Results: Nephrin mRNA and expression of the extracellular nephrin were markedly reduced in IgA nephropathy. No changes were found in patients with minimal change nephrosis and focal segmental glomerulosclerosis. The staining of intracellular nephrin and CD2AP did not change among patients. A comparable frequency of the filtration slits was observed in all patient groups, except in minimal change disease patients, due to extensive foot process effacement. The percentage of slit diaphragms with a filamentous image was markedly reduced in IgA nephropathy, but was comparable to controls in minimal change disease. Slit pore width showed a tendency to decrease both in patients with minimal change disease and IgA nephropathy. Conclusions: These results indicate that the ultrastructure of the filtration slit diaphragm is altered in patients with IgA nephropathy as a consequence of targeted downregulation of extracellular nephrin. Further studies are needed to evaluate the pathophysiological meaning of nephrin abnormality in IgA nephropathy and how these changes can be modulated by antiproteinuric therapy.

Ultrastructural model for size selectivity in glomerular filtration.

A theoretical model was developed to relate the size selectivity of the glomerular barrier to the structural characteristics of the individual layers of the capillary wall. Thicknesses and other linear dimensions were evaluated, where possible, from previous electron microscopic studies. The glomerular basement membrane (GBM) was represented as a homogeneous material characterized by a Darcy permeability and by size-dependent hindrance coefficients for diffusion and convection, respectively; those coefficients were estimated from recent data obtained with isolated rat GBM. The filtration slit diaphragm was modeled as a single row of cylindrical fibers of equal radius but nonuniform spacing. The resistances of the remainder of the slit channel, and of the endothelial fenestrae, to macromolecule movement were calculated to be negligible. The slit diaphragm was found to be the most restrictive part of the barrier. Because of that, macromolecule concentrations in the GBM increased, rather than decreased, in the direction of flow. Thus the overall sieving coefficient (ratio of Bowman's space concentration to that in plasma) was predicted to be larger for the intact capillary wall than for a hypothetical structure with no GBM. In other words, because the slit diaphragm and GBM do not act independently, the overall sieving coefficient is not simply the product of those for GBM alone and the slit diaphragm alone. Whereas the calculated sieving coefficients were sensitive to the structural features of the slit diaphragm and to the GBM hindrance coefficients, variations in GBM thickness or filtration slit frequency were predicted to have little effect. The ability of the ultrastructural model to represent fractional clearance data in vivo was at least equal to that of conventional pore models with the same number of adjustable parameters. The main strength of the present approach, however, is that it provides a framework for relating structural findings to the size selectivity of the glomerular barrier.

Ultrastructure of the coxal gland of the horseshoe crab Limulus polyphemus: Evidence for ultrafiltration and osmoregulation

Electron microscopic examination of the paired coxal glands of the horseshoe crab Limulus polyphemus, focusing on urinary and vascular channels, shows six morphologically distinct regions. Each of four nephridial lobes consists of two cortical layers surrounding a medulla. The outer and inner cortexes contain blood vessels separated by a basement membrane from the urinary space lined by podocytes. Podocyte foot processes are applied to the basement membrane, interdigitate with those from other podocytes, and have a filtration slit diaphragm between them. Cortical morphology demonstrates ultrafiltration of blood, a previously undescribed function of the gland, as well as possible endocytic reabsorption of materials by the podocytes. The medulla drains into the stolon connecting the four lobes. These two areas have urinary tubules of cuboidal epithelium featuring microvillous-like apical projections, cytoplasmic vesicles and vacuoles, elaborate lateral interdigitations with septate junctions, and basal invaginations containing numerous mitochondria. These tubules are closely surrounded by blood channels, lined by a basement membrane containing embedded support cells. The medulla and stolon morphology are suggestive of both ion transport and water movement, in keeping with the gland's role in osmoregulation. The stolon empties into the end sac in the base of the most posterior lobe. It is lined by tall epithelium exhibiting apical overlap, blunt projections into the lumen, apparent endocytic vesicles, and basal plasma membrane infoldings with mitochondria. The end sac drains into the conducting nephric duct, the proximal end of which is lined by a cuticle. J. Morphol. 234:233–252, 1997. © 1997 Wiley-Liss, Inc.

Ultrastructure of the coxal gland of the horseshoe crab Limulus polyphemus: Evidence for ultrafiltration and osmoregulation.

Electron microscopic examination of the paired coxal glands of the horseshoe crab Limulus polyphemus, focusing on urinary and vascular channels, shows six morphologically distinct regions. Each of four nephridial lobes consists of two cortical layers surrounding a medulla. The outer and inner cortexes contain blood vessels separated by a basement membrane from the urinary space lined by podocytes. Podocyte foot processes are applied to the basement membrane, interdigitate with those from other podocytes, and have a filtration slit diaphragm between them. Cortical morphology demonstrates ultrafiltration of blood, a previously undescribed function of the gland, as well as possible endocytic reabsorption of materials by the podocytes. The medulla drains into the stolon connecting the four lobes. These two areas have urinary tubules of cuboidal epithelium featuring microvillous-like apical projections, cytoplasmic vesicles and vacuoles, elaborate lateral interdigitations with septate junctions, and basal invaginations containing numerous mitochondria. These tubules are closely surrounded by blood channels, lined by a basement membrane containing embedded support cells. The medulla and stolon morphology are suggestive of both ion transport and water movement, in keeping with the gland's role in osmoregulation. The stolon empties into the end sac in the base of the most posterior lobe. It is lined by tall epithelium exhibiting apical overlap, blunt projections into the lumen, apparent endocytic vesicles, and basal plasma membrane infoldings with mitochondria. The end sac drains into the conducting nephric duct, the proximal end of which is lined by a cuticle. J. Morphol. 234:233-252, 1997. © 1997 Wiley-Liss, Inc.