NDT Advance Access originally published online on September 2, 2006
Nephrology Dialysis Transplantation 2007 22(1):37-39; doi:10.1093/ndt/gfl485
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Dynamics at the slit diaphragm—is nephrin actin'?*
RWTH Aachen, Division of Nephrology, Internal Medicine 2, Pauwelsstr. 30, 52074 Aachen, Germany
Correspondence and offprint requests to: Marcus Johannes Moeller, MD, RWTH Aachen, Division of Nephrology, Internal Medicine 2, Pauwelsstr. 30, 52074 Aachen, Germany. Email: mmoeller{at}ukaachen.de
Keywords: actin; FAT1; foot process; Nck; nephrin; podocyte
Two manuscripts published by Verma et al. in the Journal of Clinical Investigation and by Jones et al. in Nature show that nephrin interacts with the actin polymerization complex via the adaptor protein Nck at the slit diaphragm.
The glomerular filter has remained one of the most intriguing mysteries in the field of nephrology. How is it possible that the kidney filters millions of litres of fluid from the plasma in a lifetime and never clogs? How can plasma proteins be so efficiently retained within the serum by this nano-structure? And why does a glomerulus become proteinuric in diseased states?
The glomerular filter is composed of three major components: the fenestrated endothelial cells, the glomerular basement membrane and the podocytes, which form multiple interdigitating foot processes. Podocytes form an unusual intercellular junction, termed the slit diaphragm, that spans the filtration slits through which the primary filtrate passes. It is very likely that all three layers are essential for the integrity of the glomerular filtration barrier. However, the individual contribution of each component to the glomerular filter still remains a matter of debate. During the past few years, the podocyte has become a focus of interest for several reasons (Figure 1). First, it was observed that proteinuric states are associated with morphological changes of the interdigitating podocyte foot processes (termed ‘foot processes effacement’) [1], leading to the concept that the podocyte is involved in the progression of proteinuric states [2]. Podocyte foot processes are actin-based structures. Within the foot processes, a cortical actin network can be distinguished from a bundle of linear actin bundles along the dorsal aspect of the foot processes [3]. The width of the filtration slits is tightly regulated (40 nm) and the complex of proteins that make up the slit diaphragm is believed to represent a central signalling platform for the control of actin dynamics within foot processes. Indeed, the atypical cadherin FAT1, present at the slit diaphragm, was previously shown to regulate actin dynamics via its interaction with Ena/VASP proteins [4].
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Second, genetic analysis of patients affected by inherited glomerular disease or genetically modified animal models has identified a number of proteins that are essential for an intact glomerular filtration barrier. Notably, most of these proteins were found to be expressed within podocytes. Within the podocyte, most of the identified proteins turned out to be integral parts of the slit diaphragm (most notably nephrin, Neph-family proteins and FAT1 [5–7]) or to be closely associated with it (podocin, TRPC6 and others [8–10]).
Third, a number of tools and reagents have been developed that allow experimental manipulation of the podocyte. Conditionally immortalized cell lines derived from podocytes have become available from humans and rodents. More recently, it has become possible to manipulate podocytes in vivo in transgenic animals (mice and rats), overcoming some of the shortcomings of podocyte cell lines [11].
In order to understand how the glomerular filtration barrier works, much research activity has been focused on proteins that are essential for the integrity of the glomerular filtration barrier. Of these, the protein that attracted the most attention was undoubtedly nephrin, the first integral component of the slit diaphragm, identified only 8 years ago by Kestila et al. [7], from Karl Tryggvason's group. After this short period of time, the function of nephrin is still not resolved. However, two recent studies by Verma et al. [12] and Jones et al. [13] have now elegantly investigated the down-stream effects that follow after nephrin has been activated.
Nephrin belongs to the immunoglobulin (Ig) superfamily comprising a large number of proteins that may function as receptors as well as cell-adhesion molecules (and others). Similar to other Ig-superfamily receptors, nephrin can be ‘activated’ in response to a signal when it is phosphorylated by src-family kinases fyn and yes [14–17]. Both the protein kinases may be essential for the maintenance of normal podocyte morphology, because fyn and yes-deficient mice develop a glomerular phenotype with proteinuria and podocyte effacement [15]. Verma et al. [12] and Jones et al. [13] have now shown that nephrin phosphorylation induces protein–protein interaction between the small adaptor protein Nck and nephrin. Apart from nephrin, Nck also binds to several other Ig-superfamily receptors after they have been activated and tyrosine-phosphorylated. Jones et al. [13] were able to demonstrate that Nck proteins are necessary for normal podocyte function by a podocyte-specific knockout of all Nck isoforms in experimental mice [13]. These mice developed proteinuria and podocyte effacement. However, it still remains to be resolved whether this phenotype is a consequence of impaired nephrin signalling or impairment of other potential Nck-mediated signalling pathways within the podocyte.
The two groups then went ahead to show that upon phosphorylation, nephrin recruits proteins associated with the actin polymerization complex via Nck (e.g. N-WASP which recruits the actin polymerization complex Arp2/3) (Figure 1B). Because such experiments are far too complex to be performed in vivo, the nephrin-signalling complex was introduced into a fibroblast cell line. Nephrin could be artificially activated by antibody-mediated clustering and was phosphorylated as described. Strikingly, activated nephrin complexes induced ectopic actin polymerization through Nck in these cells. By analogy, it can be assumed that the nephrin complex regulates the actin polymerization machinery also within the foot process in vivo.
To investigate the significance of this interaction further, Verma et al. [12] generated an antiserum specific for ‘activated’ phosphorylated nephrin. Surprisingly, within a mature glomerulus, no activated nephrin was detected under physiological conditions. Within developing glomeruli, on the other hand, ‘activated’ nephrin was present during the period when podocytes form foot processes (i.e. the capillary loop stage) and then disappeared as the glomerulus matured. Similarly, in a disease model that induces mild reversible podocyte effacement in mice (protamine sulphate perfusion), nephrin was found to be ‘activated’ reversibly during the injury. These experiments suggest that nephrin is ‘activated’ and coupled to the actin machinery via Nck during podocyte foot process effacement and rearrangement. Assuming that ‘activated’ nephrin is a marker for the activity of the actin machinery within the foot process, these results suggest that within a resting mature glomerulus, there may be much less actin dynamics at the slit diaphragm than generally expected.
In conclusion, another important piece in the puzzle of glomerular filtration barrier has been unravelled. Our increasing understanding of the molecular machinery within the podocyte foot process carries the prospect of identifying specific proteins or protein–protein interactions that can be used as targets for the development of novel therapeutic strategies in glomerular disease.
Conflict of interest statement. None declared.
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*Comment on Verma R, Kovari I, Soofi A, Nihalani D, Patrie K, Holzman LB. Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization. J Clin Invest 2006; 116: 1346–1359 and Jones N, Blasutig IM, Eremina V et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 2006; 440: 818–823.
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Accepted in revised form: 20. 7.06
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