Skip Navigation


NDT Advance Access originally published online on September 24, 2007
Nephrology Dialysis Transplantation 2007 22(12):3408-3410; doi:10.1093/ndt/gfm403
This Article
Right arrow Extract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
22/12/3408    most recent
gfm403v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Aaltonen, P.
Right arrow Articles by Holthöfer, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aaltonen, P.
Right arrow Articles by Holthöfer, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


The nephrin-based slit diaphragm: new insight into the signalling platform identifies targets for therapy

Petri Aaltonen1 and Harry Holthöfer1,2

1The Haartman Institute, University of Helsinki, Finland and 2Center for BioAnalytical Sciences, Dublin City University, Ireland

Correspondence and offprint requests to: Harry Holthöfer, MD, PhD, Professor, Center for BioAnalytical Sciences, Dublin City University, Ireland. Email: harry.holthofer{at}dcu.ie

Keywords: glomerulus; filtration barrier; slit diaphragm; nephrin; signal transduction; proteinuria; molecular biology; protein complexes



   Introduction
Top
 Introduction
Focus on the podocyte
 The nephrin-link, a target...
 Conclusion
 References
 
The global epidemic of chronic kidney disease (CKD) is progressing at an alarming rate. Ranging from mild to a most severe state, already up to 11% of the general population is affected in the US, Australia, Japan and Europe [1,2]. With a simultaneous steady increase of type II diabetes and its associated kidney complications, particularly in India, China and South-East Asia, kidney-related diseases globally are rapidly escaping any present treatment options and resources.

In most cases of CKD, the first clinical sign is proteinuria. Thus, a better understanding of mechanisms controlling the kidney permeability barrier in health and disease is essential to design better early diagnostics and therapies, to act while permanent damage may still be preventable. The key to success should be in bringing the latest genetic and molecular findings efficiently into translational platforms and finally into clinical verification. The meticulously collected large patient databases should be an essential part of this process.



   Focus on the podocyte
Top
 Introduction
 Focus on the podocyte
The nephrin-link, a target...
 Conclusion
 References
 
While some key molecules of the filtration barrier have been identified and firmly documented and their number is rapidly increasing [3–5], crucial questions on their interactions and functionality in disease conditions remain unanswered.

The morphologically distinct glomerular visceral epithelial cell, podocyte, has proven to be an irreplaceable cell type maintaining permeability properties of the filtration barrier. Obviously, the other filter components, including the glomerular basement membrane and the endothelial cells of glomerular capillaries, are also major contributors to the permeability properties, Podocyte is a cell with distinct domains, including the interdigitating foot processes, their strict division to apical and basal aspects and the interconnecting filtration slits, together with dynamic connections into the cytoskeletal machinery. Interestingly, this complex cell has provided some recent important clues on how some of the thus far identified core molecules play together, at least in vitro.

The inducer of the ongoing rapid progress in revealing molecular mechanisms of proteinuria was, after years of convergent findings, the identification of NPHS1 gene-encoding nephrin [6], the key protein mutated in the congenital nephrotic syndrome of the Finnish type, the prototype disease of pure proteinuria. The full role of nephrin in various more common clinical entities is still evolving, but already, results suggest its functional involvement e.g. in diabetes [7,8] and hypertension [9]. What also makes nephrin so interesting is its apparent role as a molecular scaffold at the slit diaphragm, serving to link molecules like podocin, CD2AP and others [10,11]. Thus, an ever deeper understanding of detailed nephrin functions may also be the key to understanding the pathophysiological role of the whole molecular complex maintaining the patent filtration properties.

What, then, makes nephrin and this molecular complex in the slit diaphragm so special? First, nephrin structure appears to be preserved throughout evolution [12]. From studies in lower vertebrates and invertebrates, hints of its linkage to cell-cell interactions are emerging [13,14]. Also, nephrin is shown to have distinct outside-in and inside-out signalling functions [11,15]. However, the in vivo ligands and effects need to be characterized in detail. As is true for many key functional molecules, nephrin has also shown to present with unique patterns of regulation, including alternative usage of exons to yield tissue specificity [16]. Nephrin mRNA also shows a typical splicing pattern apparently regulated in different disease entities [17], and a very unique regulation by natural antisense mRNA [18] and bidirectional regulation with a closely related molecule, filtrin [19]. It has also become evident that nephrin may leak out from podocytes in distinct ailing circumstances and, subsequently, be found in urine in typical fragmented patterns [7,20]. Whether these truly are shed or actively shed by-products of an active vesicular transport process from podocyte foot processes as suggested earlier [21], secreted from perturbing podocytes in specific exosomes, or are released during apoptotic process after podocyte detachment and leakage into urine [22], is currently uncertain.

What appears interesting, however, is the fact that in addition to the embryonic lethality of nephrin knock-out mouse, reintroduction of functional nephrin specifically into podocytes of this knockout mouse line appears to rescue from lethality [23]. This result suggests that it is indeed the nephrin scaffold in the kidney which is crucial to functions, while the nephrin in its other sites of expression, like the pancreatic beta cells [24] may still have some as yet identified major functions.

Together, these findings suggest that there are many good reasons to focus major research activity on the nephrin protein complex and its key role in maintenance of the functional filtration barrier.



   The nephrin-link, a target for therapy
Top
 Introduction
 Focus on the podocyte
 The nephrin-link, a target...
Conclusion
 References
 
Nephrin has been shown to mediate outside-in signalling of podocytes to maintain the connection to the intracellular actin-paved cytoskeleton responsible for the complex podocyte structure, and to participate in its rapid change in proteinuria. Enabling this signalling function, the intracellular part of nephrin has the tyrosine residues required for the action of the specific kinases of the Src family [25], including Fyn. The phosphorylation of Fyn modulates podocyte gene expression and interaction of nephrin with podocin [26].

The phosphorylation of nephrin is known to activate at least two pathways (Figure 1). First, the phosphorylation of nephrin by the Fyn kinase leads to the activation of the phosphoinositide 3-OH kinase (PI3K), as the regulatory subunit p85 of PI3K recognizes the phosphorylated nephrin-CD2AP complex and allows the catalytic subunit p110 to act on the phospholipids of the inner leaflet of the cell membrane [27]. This leads to the downstream phosphorylation and inactivation of the apoptotic factor Bad via the serine-threonine kinase AKT.


Figure 1
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. The intracellular interaction pathways of nephrin. CD2AP = CD2-associated protein, Fyn = a protein kinase of the Src-protein family, Nck = an adaptor protein important to actin polymerization, PI3K = phosphoinositide 3-OH kinase, AKT = a member of the serine/threonine protein kinase family. Bad = a proapoptotic factor. See text for further details.

 
The second pathway involves a key mediator Nck, as recently demonstrated [28,29]. It is shown that the Nck adaptor protein is involved in the regulation of actin-cytoskeletal link to nephrin. Nck proteins can bind to the nephrin phosphotyrosines via their SH2 domain, while the SH3 domains can mediate actin polymerization. The specific binding sites of Nck1 and Nck2 isotypes on nephrin have been mapped, and mutations in these sites prevent the interaction with nephrin and, subsequently, the mediation of nephrin signalling to the structurally and functionally important podocyte cytoskeleton [29].

With this information now available, completely new approaches can be designed to modulate the nephrin-cytoskeleton interaction in situations of proteinuria.



   Conclusion
Top
 Introduction
 Focus on the podocyte
 The nephrin-link, a target...
 Conclusion
References
 
Taken together, nephrin appears to be an important scaffold, into which other molecules of the slit diaphragm are associated into structural-functional complexes, which appears an important target structure for pharmacologic molecules but also for diagnostic platforms [5]. Clearly, all the previously generated transgenic and nephrin knockout mouse lines and particularly the podocyte-specific inducible nephrin knockout mouse line avoiding the embryonic lethality will be critically important in further studies. We are now beginning to understand how extracellular stimuli are transduced intracellularly in the podocyte, to maintain structural integrity of the filtration slit in response to blood pressure, proteinuria, and engagement by antibodies or other factors. Future steps to design molecules to modulate these interactions are yet to be taken.

Conflict of interest statement. None declared.



   References
Top
 Introduction
 Focus on the podocyte
 The nephrin-link, a target...
 Conclusion
 References
 

  1. Meguid El Nahas A, Bello AK. Chronic kidney disease: the global challenge. Lancet (2005) 365:331–340.[Web of Science][Medline]
  2. Remuzzi G, Benigni A, Remuzzi A. Mechanisms of progression and regression of renal lesions of chronic nephropathies and diabetes. J Clin Invest (2006) 116:288–296.[CrossRef][Web of Science][Medline]
  3. Patari-Sampo A, Ihalmo P, Holthofer H. Molecular basis of the glomerular filtration: nephrin and the emerging protein complex at the podocyte slit diaphragm. Ann Med (2006) 38:483–492.[CrossRef][Web of Science][Medline]
  4. Kawachi H, Miyauchi N, Suzuki K, Han GD, Orikasa M, Shimizu F. Role of podocyte slit diaphragm as a filtration barrier. Nephrology (Carlton) (2006) 11:274–281.[CrossRef][Medline]
  5. Johnstone DB, Holzman LB. Clinical impact of research on the podocyte slit diaphragm. Nat Clin Pract Nephrol (2006) 2:271–282.[CrossRef][Web of Science][Medline]
  6. Kestila M, Lenkkeri U, Mannikko M, et al. Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell (1998) 1:575–582.[CrossRef][Web of Science][Medline]
  7. Aaltonen P, Luimula P, Astrom E, et al. Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy. Lab Invest (2001) 81:1185–1190.[Web of Science][Medline]
  8. Toyoda M, Suzuki D, Umezono T, et al. Expression of human nephrin mRNA in diabetic nephropathy. Nephrol Dial Transplant (2004) 19:380–385.[Abstract/Free Full Text]
  9. Bonnet F, Tikellis C, Kawachi H, et al. Nephrin expression in the post-natal developing kidney in normotensive and hypertensive rats. Clin Exp Hypertens (2002) 24:371–381.[CrossRef][Web of Science][Medline]
  10. Schwarz K, Simons M, Reiser J, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest (2001) 108:1621–1629.[CrossRef][Web of Science][Medline]
  11. Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol (2004) 15:1382–1391.[Free Full Text]
  12. Dworak HA, Charles MA, Pellerano LB, Sink H. Characterization of Drosophila hibris, a gene related to human nephrin. Development (2001) 128:4265–4276.[Abstract/Free Full Text]
  13. Ahola H, Wang SX, Luimula P, Solin ML, Holzman LB, Holthofer H. Cloning and expression of the rat nephrin homolog. Am J Pathol (1999) 155:907–913.[Abstract/Free Full Text]
  14. Artero RD, Castanon I, Baylies MK. The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development (2001) 128:4251–4264.[Abstract/Free Full Text]
  15. Simons M, Schwarz K, Kriz W, et al. Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm. Am J Pathol (2001) 159:1069–1077.[Abstract/Free Full Text]
  16. Holthofer H, Ahola H, Solin ML, et al. Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney. Am J Pathol (1999) 155:1681–1687.[Abstract/Free Full Text]
  17. Luimula P, Aaltonen P, Ahola H, Palmen T, Holthofer H. Alternatively spliced nephrin in experimental glomerular disease of the rat. Pediatr Res (2000) 48:759–762.[Web of Science][Medline]
  18. Ihalmo P, Rinta-Valkama J, Mai P, et al. Molecular cloning and characterization of an endogenous antisense transcript of Nphs1. Genomics (2004) 83:1134–1140.[CrossRef][Web of Science][Medline]
  19. Ihalmo P, Palmen T, Ahola H, Valtonen E, Holthofer H. Filtrin is a novel member of nephrin-like proteins. Biochem Biophys Res Commun (2003) 300:364–370.[CrossRef][Web of Science][Medline]
  20. Patari A, Forsblom C, Havana M, Taipale H, Groop PH, Holthofer H. Nephrinuria in Diabetic Nephropathy of Type 1 Diabetes. Diabetes (2003) 52:2969–2974.[Abstract/Free Full Text]
  21. Simons M, Saffrich R, Reiser J, Mundel P. Directed membrane transport is involved in process formation in cultured podocytes. J Am Soc Nephrol (1999) 10:1633–1639.[Abstract/Free Full Text]
  22. Petermann AT, Pippin J, Krofft R, et al. Viable podocytes detach in experimental diabetic nephropathy: potential mechanism underlying glomerulosclerosis. Nephron Exp Nephrol (2004) 98:e114–e123.[CrossRef][Medline]
  23. Juhila J, Lassila M, Roozendaal R, Lehtonen E, Verbeek S, Holthofer H. Inducible rat nephrin in podocytes rescues lethality of nephrin deficiency in mice. In: American Society of Nephrology Renal Week. (2006) November 17. PA, USA: Philadelphia. (Abstract F-PO859).
  24. Palmen T, Ahola H, Palgi J, et al. Nephrin is expressed in the pancreatic beta cells. Diabetologia (2001) 44:1274–1280.[CrossRef][Web of Science][Medline]
  25. Lahdenpera J, Kilpelainen P, Liu XL, et al. Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases. Kidney Int (2003) 64:404–413.[CrossRef][Web of Science][Medline]
  26. Li H, Lemay S, Aoudjit L, Kawachi H, Takano T. SRC-family kinase Fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin. J Am Soc Nephrol (2004) 15:3006–3015.[Abstract/Free Full Text]
  27. Huber TB, Hartleben B, Kim J, et al. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signalling. Mol Cell Biol (2003) 23:4917–4928.[Abstract/Free Full Text]
  28. 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.[CrossRef][Web of Science][Medline]
  29. 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.[CrossRef][Medline]
Received for publication: 21.12.06
Accepted in revised form: 29. 5.07


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Extract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
22/12/3408    most recent
gfm403v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Aaltonen, P.
Right arrow Articles by Holthöfer, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aaltonen, P.
Right arrow Articles by Holthöfer, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?