NDT Advance Access originally published online on June 5, 2007
Nephrology Dialysis Transplantation 2007 22(8):2124-2128; doi:10.1093/ndt/gfm344
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Molecular architecture of the glomerular slit diaphragm: lessons learnt for a better understanding of disease pathogenesis
National Center of Sensor Research/BioAnalytical Sciences, Dublin City University, Ireland
Correspondence and offprint requests to: Harry Holthöfer, MD, PhD, Biomedicum Helsinki, University of Helsinki, PB 63, FI-00014, Finland. Email: harry.holthofer{at}helsinki.fi
Keywords: cell-adhesion molecules; intercellular junctions; proteinuria; podocyte
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Introduction |
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Top
Introduction
The slit diaphragm of...For more than three decades, the molecular composition of the interpodocyte slit diaphragm of the glomerular filtration barrier has remained elusive. The first electron microscopic studies described the slit diaphragm as a porous, ‘zipper-like’ structure [1], but it was not until 1998 that the first transmembrane molecule of the slit diaphragm was identified [2]: Nephrin is a cell surface receptor of the immunoglobulin superfamily participating in cell–cell adhesion and signalling functions [3]. Mutations in nephrin lead to the congenital nephrotic syndrome of the Finnish type (CNF), suggesting that nephrin is of pivotal importance for maintaining the filtration barrier. In recent years, the mapping of the genetic background of other inherited and acquired nephropathies and the generation of transgenic animal models have led to the beginning of a new era in nephrology, also promising new targeted therapies and advanced diagnostics. In the present review, the main recent findings exploring the molecular architecture of the glomerular filter itself and their role in proteinuric glomerular diseases are reviewed.
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The slit diaphragm of the glomerular capillary wall |
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Proteinuria is the hallmark of glomerular damage. While there is strong evidence that even a minute loss of albumin in urine reflects a first perturbation of the glomerular filtration barrier, there also appears to be a direct link between proteinuria and vascular damage. Thus, proteinuria is a risk factor for both progressive renal disease and cardiovascular disease [4]. Either the protein leakage through the glomerular capillary wall itself, or direct spreading of glomerular damage to the extraglomerular space appears to lead to a sequence of downstream events including interstitial inflammation and, finally, the loss of nephron function [5].
Podocytes interlinked by the slit diaphragms constitute a continuous outermost layer in the glomerular filtration barrier (Figure 1). This barrier has size- and charge-selective properties, and the loss (internalization) of the slit diaphragm associates with disruption of the filter, as reflected by proteinuria [6]. The exact mechanisms and causalities remain to be described in detail. Rodewald and Karnovsky [1] suggest that the slit diaphragm is a zipper-like structure that functions as a sieve, with a pore size smaller than albumin. Recent studies using electron tomography have suggested that, like many other biologic membranes, the slit diaphragm is composed of an organized network of winding strands, possibly constituting the base into which various molecular components are intertwined.
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Since the slit diaphragm appears as the final seal to prevent loss of circulating proteins into urine, this unique intercellular junction has been the target for increasing research interest. At the molecular level, the slit diaphragm has been referred to as a modified ‘adherens junction’ by Reiser et al. [7]. Indeed, this junctional complex has components typical for tight junctions (ZO-1) [8] as well as adherens junctions (P-cadherin [9]).
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Nephrin forms the scaffold for intertwining slit molecules |
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Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disorder, characterized by massive proteinuria and nephrosis already in utero [10]. The process that begun from the first descriptions of this rare, but severe paediatric disease led to the final cloning of the causative NPHS1 gene, in 1998 [2]. The gene product of NPHS1, nephrin, is a type-1 transmembrane protein belonging to the immunoglobulin superfamily of adhesion proteins. Nephrin was the first identified integral membrane protein the slit diaphragm. Several studies have provided support for the hypothesis that the outermost N-terminal Ig-like domains of nephrin from aadjacent podocytes interact homotypically [11,12], linking the foot processes dynamically together. This interaction most likely provides the structural but also functional scaffold for the slit diaphragm. Nephrin has shown to interact in podocytes with CD2AP [13] and podocin [14] via its intracellular domain, linking the slit-associated protein complex ultimately to podocyte cytoskeleton [15]. The interactions of this protein complex with the cytoskeleton appear to provide the basis for translating extracellular events to rapid shape changes characterizing proteinuric diseases. The mechanisms by which this is achieved are presently under active research and could provide a future site for targeted pharmacological interventions.
Expression of nephrin is regulated at different levels: usage of alternative splicing [16] and a naturally occurring antisense gene [17], supposedly needed for fine-tuning of the tissues-specific presence of nephrin. Injection of antinephrin antibodies results in massive proteinuria in rats [18]. Interestingly, a substantial amount of CNF patients treated with transplantation show a recurrence of nephrotic syndrome due to induction of autoimmunity to nephrin in the transplant and subsequently emerging circulating antinephrin antibodies [19]. Nephrin has been proposed as an early marker of podocyte damage [20], reflecting the patency of the filtration barrier even more accurately than microalbuminuria, particularly in diabetes.
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Signalling at the slit diaphragm |
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Since the first reports, it has become clear that one of the basic functions of the slit diaphragm-associated molecules include outside-in signalling. In nephrin, these functions are enabled by the nine tyrosine residues of the intracellular domain, phosphorylated at ligand binding [21]. Other ligands for extracellular nephrin remain to be discovered but obviously offer, in addition to the molecular understanding of the filtration barrier machinery, another intriguing future site of targeted therapies. Interestingly, oligomerized nephrin associates with signaling microdomains, lipid rafts, in a cholesterol-dependent manner [22]. In-vivo injection of antibodies against 9-O-acetylated GD3 ganglioside of podocytes led to rapid morphological changes at the filtration slit area, closely resembling foot process effacement. At the same time, nephrin dislocated to the apical pole of the narrowed filtration slits and was tyrosine phosphorylated [22]. It has subsequently been shown that clustering of extracellular nephrin with antinephrin antibodies in-vitro disrupts cell–cell contacts [23] and leads to phosphorylation of nephrin by Src family kinases [24].
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Nephrin-like proteins are essential for cell–cell adhesion at the slit diaphragm |
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Nephrin-like proteins include three closely related transmembrane proteins of substantial similarity to nephrin which contribute to the slit diaphragm structure. Like nephrin, they are preferentially expressed in podocytes [25,26], and NEPH1 and NEPH2 have been determined to localize closely to the slit diaphragm [12,27]. In addition, NEPH1-deficient mice suffer from perinatal lethality and proteinuria together with foot process effacement [28], thus resembling the findings in nephrin-deficient mice [29]. The extracellular nephrin core interacts both with NEPH1 and NEPH2, whereas they do not appear to interact homotypically with each other like nephrin [12,30]. Nephrin-like proteins also interact with ZO-1, the first identified molecule of the slit diaphragm area. Interestingly, NEPH2 is cleaved from podocytes by locally activated metalloproteinases and is released into the urine [27]. Podocin also interacts with the intracellular domains of NEPH1, NEPH2 and filtrin [25] suggesting that it is a shared linker for many of the essential slit diaphragm molecules.
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P-cadherin and FAT1 localize to the slit diaphragm |
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P-cadherin may contribute to the molecular complex with defined functions for the maintenance of the filtration slit barrier [31]. However, since P-cadherin deficient mice and humans with mutation in the P-cadherin gene show no kidney phenotype, it obviously does not have a direct role in glomerular filtration.
FAT1 is a distinct member of the cadherin superfamily of adhesion molecules and has been localized to the slit area, co-localizing with nephrin as well as with ZO-1 [32]. Mice lacking FAT1 are born with effaced foot processes without slit diaphragms and die within 48 h post-natally [33]. FAT1 is possibly needed for the intercellular adhesion framework between foot processes, while at the same time severing the intercellular gap distance; as supported by findings in the FAT1 knock-out model [33], this molecule is apparently centrally involved in the organizaation of actin dynamics.
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Intracellular scaffold and adaptor proteins of podocytes |
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CD2-associated protein (CD2AP) was initially described in the T-lymphocyte CD2 protein complex [13,34], but it is also present in the slit diaphragm area. It links intracellular nephrin by the C-terminal domain. Importantly for the signalling function of the complex, the N-terminal part of CD2AP potentiates nephrin-induced AKT-mediated signalling [34]. The CD2AP-knockout mice present with a severe kidney phenotype and defects in foot process formation [34]. CD2AP and nephrin may mediate their functions via connections to the actin cytoskeleton, as evidenced by the shape changes typical for the podocyte in proteinuria.
Podocin was initially discovered in autosomal recessive and in sporadic familiar focal segmental glomerulosclerosis patients [14]. Podocin-knockout mice also show antenatal proteinuria, foot process effacement and reduced nephrin expression [35]. Podocin is an integral membrane protein and, unlike nephrin, appears strictly localized in podocytes. Podocin binds by its C-terminal domain with CD2AP and nephrin to form a scaffolding complex of the slit diaphragm, but also enhances the mitogen-activated protein kinase cascades by recruiting nephrin into lipid rafts [36,37] and the subsequent signalling. Thus, these two molecules appear to be functionally strongly interacting. Podocin also interacts with the intracellular domains of NEPH1, NEPH2 and filtrin [25], suggesting that it is the shared linker for many of the essential slit diaphragm molecules.
-Actinin-4 is a cytosolic actin-filament cross-linking protein. In autosomal dominant familial focal segmental glomerulosclerosis, a-actinin-4 is typically mutated [38], suggesting that a-actinin-4 is a robust linker molecule, mediating and modulating interactions of the slit diaphragm domain molecules with the actin cytoskeleton of podocytes.
Densin is a heavily sialylated post-synaptic protein of the brain. Interestingly, it is among the growing group of molecules only shared with the podocyte and the neurons [39]. Densin interacts either directly, or possibly via some as yet unrecognized intermediates, with nephrin and localizes strictly to the slit diaphragm area. Interestingly, densin mRNA and protein expression is increased in CNF kidneys [39], suggesting that its expression may account for compensatory mechanisms in kidney damage.
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Conclusion |
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While being a key element in the glomerular filtration barrier and a direct target for damages in hypertension, diabetes and other proteinuric diseases, the podocyte, and particularly the slit diaphragm, have remained poorly understood cells. They share remarkable morphological and molecular similarities with neurons and cells of the immunological systems. They are terminally differentiated, with extremely tight growth control, being virtually the only cell type in the body without known malignant transformation. Few pharmaceutically effective molecules have been available for treating diseases deriving from podocyte dysfunction and its consequences. With the milestone discovery of nephrin, its cell biology and the avalanche of subsequent molecular components maintaining the glomerular filtration barrier, we are rapidly entering a new era identifying a set of pharmacologic targets. Will the new predictive diagnostics and therapies be developed fast enough to combat the avalanche of chronic kidney disease so often starting from podocyte dysfunction?
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Acknowledgements |
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Figure 1 is courtesy of Johanna Rinta-Valkama (University of Helsinki) which is gratefully acknowledged.
Conflict of interest statement. None declared.
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Accepted in revised form: 4. 5.07
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