Nephrin--a unique structural and signaling protein of the kidney filter

Since the discovery of nephrin, the first integral component of the slit diaphragm to be identified, the podocyte slit pore has become a major focus in research concerning the glomerular filtration barrier. Nephrin is a central component of the glomerular ultrafilter, with both structural and signaling functions. The extracellular domain of nephrin and other components of the slit diaphragm seem to form a porous molecular sieve. The intracellular domain of nephrin is associated with linker proteins, such as CD2-associated protein and Nck proteins that can connect nephrin to the actin cytoskeleton. Alterations in nephrin interactions with other proteins during development or injury can lead to complex signaling reactions aimed at establishing or restoring the filter function.     

Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

Nephrin expression is increased in anti-Thy1.1-induced glomerulonephritis in rats

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.     

Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.     

Advanced oxidation protein products decrease expression of nephrin and podocin in podocytes via ROS-dependent activation of p38 MAPK

Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin.The expression of glomerular podocyte slit diaphragm(PSD)-associated proteins,nephrin and podocin,was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs.Chronic inhibi-tion of NADPH oxidase by apocynin p...     

An update: the role of Nephrin inside and outside the kidney

Nephrin is a key molecule in podocytes to maintain normal slit diaphragm structure. Nephin interacts with many other podocyte and slit diaphragm protein and also mediates important cell signaling pathways in podocytes. Loss of nephrin during the development leads to the congenital nephrotic syndrome in children. Reduction of nephrin expression is often observed in adult kidney diseases including diabetic nephropathy and HIV-associated nephropathy. The critical role of nephrin has been confirmed by different animal models with nephrin knockout and knockdown. Recent studies demonstrate that knockdown of nephrin expression in adult mice aggravates the progression of unilateral nephrectomy and Adriamycin-induced kidney disease. In addition to its critical role in maintaining normal glomerular filtration unit in the kidney, nephrin is also expressed in other organs. However, the exact role of nephrin in kidney and extra-renal organs has not been well characterized. Future studies are required to determine whether nephrin could be developed as a drug target to treat patients with kidney disease.     

Organ specific underexpression renal of Na+-dependent B0AT1 in the SHR correlates positively with overexpression of NHE3 and salt intake

Densin is a member of LAP (leucine-rich repeat and PDZ domain) protein family that localizes in kidney to slit diaphragms, which are essential components of the glomerular filtration barrier. We have previously shown that densin interacts with a crucial slit diaphragm protein, nephrin. Here, we searched for novel binding partners of densin by yeast-two hybrid assay and identified beta-catenin. The interaction was confirmed by reciprocal co-immunoprecipitation assay and the binding site in densin was determined by GST-pull down assays. The GST-tagged densin was also able to pull down P-cadherin together with beta-catenin from human kidney glomerular lysates. Furthermore, densin co-localized with beta-catenin and F-actin in cell–cell contacts in cultured mouse podocytes. During cell–cell contact disruption and reformation densin and beta-catenin were dislocated from and relocated back to plasma membrane in a similar fashion. These and our previous findings suggest that densin may associate with the cadherin-catenin and nephrin complex(es), and may be involved in the formation of the cell–cell contacts including the slit diaphragm.     

Characterization of the interactions of the nephrin intracellular domain

Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.     

CD2AP regulates SUMOylation of CIN85 in podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.     

Filtrin is a novel member of nephrin-like proteins

NPHS1 encodes nephrin, the core protein of the interpodocyte slit diaphragm of the kidney glomerulus. NPHS1 is the causative gene for congenital nephrotic syndrome of the Finnish type (CNF) with massive, treatment resistant proteinuria. We report here the establishment of a novel nephrin-like gene, NLG1 encoding filtrin, a protein with substantial homology to human nephrin. Filtrin is a type I transmembrane protein consisting of 708 amino acids. Together with the recently cloned NEPH1, NLG1 establishes a new nephrin-like subgroup of genes belonging to the immunoglobulin superfamily of cell adhesion molecules. The RNA dot blot experiment revealed that the NLG1 mRNA expression is widely distributed but most prominently observed in the pancreas and lymph nodes. The expression of NLG1 mRNA in kidney glomeruli was verified with RT-PCR. Further immunoblotting studies with antifiltrin antibody showed a specific band at 107kDa in the human and rat glomeruli. In immunofluorescence microscopy specific staining of glomeruli but also proximal and distal parts of the nephron was seen in human kidney cortex. Due to its structural similarity and sequence homology as well as partially consistent expression pattern with nephrin we propose that filtrin belongs to a functionally important complex of proteins of the glomerular filtration barrier.     

Trans-interaction of nephrin and Neph1/Neph3 induces cell adhesion that associates with decreased tyrosine phosphorylation of nephrin

Slit diaphragms are specialized junctions between glomerular epithelial cells (podocytes) that are crucial for glomerular ultrafiltration. The Ig superfamily members nephrin and Neph1 are essential components of the slit diaphragm, whereas the role of Neph1 homologue Neph3 in the slit diaphragm is unknown. In the present paper we show that Neph3 homodimerizes and heterodimerizes with nephrin and Neph1. We further investigated whether these interactions play a role in cell adhesion by using mouse L fibroblasts that lack endogenous cell-adhesion activity and found that Neph1 and Neph3 are able to induce cell adhesion alone, whereas nephrin needs to trans-interact with Neph1 or Neph3 in order to promote formation of cell-cell contacts. Tyrosine phosphorylation of nephrin was down-regulated after nephrin trans-interacted with either Neph1 or Neph3 leading to formation of cell-cell contacts. We further found that the expression of Neph3 was increased in nephrin-deficient mouse podocytes. The findings of the present paper show that nephrin and Neph1 or Neph3 trans-interactions promote cell-contact formation, suggesting that they may also function together in slit diaphragm assembly.     

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.     

Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm.     

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in?vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury. Structured digital abstract ? Sirp-alpha?physically interacts?with?Nephrin?by?anti bait coimmunoprecipitation?(View interaction) ? Sirp-alpha?physically interacts?with?Nephrin?by?anti tag coimmunoprecipitation?(View interaction).