Download Molecular architecture of the glomerular slit

Nephrol Dial Transplant (2007) 22: 2124–2128
doi:10.1093/ndt/gfm344
Advance Access publication 5 June 2007
Editorial Review
Molecular architecture of the glomerular slit diaphragm: lessons learnt
for a better understanding of disease pathogenesis
Harry Holthöfer
National Center of Sensor Research/BioAnalytical Sciences, Dublin City University, Ireland
Keywords: cell-adhesion molecules; intercellular
junctions; proteinuria; podocyte
Introduction
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.
The slit diaphragm of the glomerular
capillary wall
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
Correspondence and offprint requests to: Harry Holthöfer, MD,
PhD, Biomedicum Helsinki, University of Helsinki, PB 63,
FI-00014, Finland. Email: [email protected]
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.
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]).
Nephrin forms the scaffold for intertwining slit
molecules
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
ß The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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Molecular architecture of the glomerular slit diaphragm
2125
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.
A
Urinary space
Slit diaphragm
Podocyte foot process
Glomerular basement membrane
Endothelium
Signalling at the slit diaphragm
B
Urinary space
ZO-1
Filtrin
Podocyte foot process
NEPH1/2
Slit diaphragm
Nephrin
Podocin
Actin
Fyn
CD2AP
FAT
Beta-catenin
P-cadherin
Densin
Alpha-actinin-4
Synaptopodin
Fig. 1. Schematic picture of podocyte architechture (A).
In (B), key slit area molecules and their interlinkages are shown.
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 slitassociated 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.
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].
Nephrin-like proteins are essential for cell–cell
adhesion at the slit diaphragm
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, NEPH1deficient 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
2126
[25] suggesting that it is a shared linker for many of the
essential slit diaphragm molecules.
P-cadherin and FAT1 localize to the slit
diaphragm
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.
Intracellular scaffold and adaptor proteins of
podocytes
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.
H. Holthöfer
a-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.
Conclusion
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?
Acknowledgements. Figure 1 is courtesy of Johanna RintaValkama (University of Helsinki) which is gratefully acknowledged.
Conflict of interest statement. None declared.
References
1. Rodewald R, Karnovsky MJ. Porous substructure of the
glomerular slit diaphragm in the rat and mouse. J Cell Biol
1974; 60: 423–433
2. 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
3. Lenkkeri U, Mannikko M, McCready P et al. Structure of the
gene for congenital nephrotic syndrome of the finnish type
(NPHS1) and characterization of mutations. Am J Hum Genet
1999; 1: 51–61
Molecular architecture of the glomerular slit diaphragm
4. Remuzzi G, Benigni A, Remuzzi A. Mechanisms of progression
and regression of renal lesions of chronic nephropathies and
diabetes. J Clin Invest 2006; 2: 288–296
5. Kriz W, LeHir. Pathways to nephron loss starting from
glomerular diseases-insights from animal models. Kidney Int
2005; 67: 404–419
6. Tryggvason K. Unraveling the mechanisms of glomerular
ultrafiltration: nephrin, a key component of the slit diaphragm.
J Am Soc Nephrol 1999; 10: 2440–2445
7. Reiser J, Kriz W, Kretzler M, Mundel P. The glomerular slit
diaphragm is a modified adherens junction. J Am Soc Nephrol
2000; 11: 1–8
8. Schnabel E, Anderson JM, Farquhar MG. The tight junction
protein ZO-1 is concentrated along slit diaphragms of the
glomerular epithelium. J Cell Biol 1990; 111: 1255–1263
9. Balda MS, Matter K. Transmembrane proteins of tight
junctions. Semin Cell Dev Biol 2000; 11: 281–299
10. Hallman N, Hjelt L, Ahvenainen EK. Nephrotic syndrome in
newborn and young infants. Annales Paediatriae Fenniae 1956;
2: 227–241
11. Gerke P, Huber TB, Sellin L, Benzing T, Walz G.
Homodimerization and heterodimerization of the glomerular
podocyte proteins nephrin and NEPH1. J Am Soc Nephrol 2003;
14: 918–926
12. Barletta GM, Kovari IA, Verma RK, Kerjaschki D,
Holzman LB. Nephrin and Neph1 Co-localize at the Podocyte
Foot Process Intercellular Junction and Form cis Heterooligomers. J Biol Chem 2003; 278: 19266–19271
13. Shih NY, Li J, Cotran R, Mundel P, Miner JH, Shaw AS.
CD2AP localizes to the slit diaphragm and binds to nephrin
via a novel C-terminal domain. Am J Pathol 2001; 159:
2303–2308
14. Boute N, Gribouval O, Roselli S et al. NPHS2, encoding the
glomerular protein podocin, is mutated in autosomal recessive
steroid-resistant nephrotic syndrome. Nat Genet 2000; 24:
349–354
15. Yuan H, Takeuchi E, Taylor GA, McLaughlin M, Brown D,
Salant DJ. Nephrin dissociates from actin, and its expression is
reduced in early experimental membranous nephropathy. J Am
Soc Nephrol 2002; 13: 946–956
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
17. 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
18. Orikasa M, Matsui K, Oite T, Shimizu F. Massive proteinuria
induced in rats by a single intravenous injection of a monoclonal
antibody. J Immunol 1988; 141: 807–814
19. Wang SX, Ahola H, Palmen T, Solin ML, Luimula P,
Holthofer H. Recurrence of nephrotic syndrome after transplantation in CNF is due to autoantibodies to nephrin. Exp
Nephrol 2001; 9: 327–331
20. Pätäri A, Forsblom C, Havana M et al. Nephrinuria in Diabetic
Nephropathy of Type 1 Diabetes. Diabetes 2003; 52: 2969–2674
21. Verma R, Wharram B, Kovari I et al. Fyn binds to and
phosphorylates the kidney slit diaphragm component nephrin. J
Biol Chem 2003; 278: 20716–20723
22. 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
23. Khoshnoodi J, Sigmundsson K, Ofverstedt LG et al. Nephrin
Promotes Cell-Cell Adhesion through Homophilic Interactions.
Am J Pathol 2003; 163: 2337–2346
24. Lahdenperä J, Kilpeläinen P, Liu XL et al. Clustering-induced
tyrosine phosphorylation of nephrin by Src family kinases.
Kidney Int 2003; 64: 404–413
25. Sellin L, Huber TB, Gerke P, Quack I, Pavenstadt H, Walz G.
NEPH1 defines a novel family of podocin interacting proteins.
FASEB J 2003; 17: 115–117
2127
26. 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
27. Gerke P, Sellin L, Kretz O et al. NEPH2 is located at the
glomerular slit diaphragm, interacts with nephrin and is cleaved
from podocytes by metalloproteinases. J Am Soc Nephrol 2005;
16: 1693–1702
28. Donoviel DB, Freed DD, Vogel H et al. Proteinuria and
perinatal lethality in mice lacking neph1, a novel protein
with homology to nephrin. Mol Cell Biol 2001; 21:
4829–4836
29. Rantanen M, Palmen T, Patari A et al. Nephrin TRAP
Mice Lack Slit Diaphragms and Show Fibrotic Glomeruli
and Cystic Tubular Lesions. J Am Soc Nephrol 2002; 13:
1586–1594
30. Liu G, Kaw B, Kurfis J, Rahmanuddin S, Kanwar YS,
Chugh SS. Neph1 and nephrin interaction in the slit diaphragm
is an important determinant of glomerular permeability.
J Clin Invest 2003; 112: 209–221
31. Reiser J, Kriz W, Kretzler M, Mundel P. The glomerular slit
diaphragm is a modified adherens junction. J Am Soc Nephrol
2000; 11: 1–8
32. Inoue T, Yaoita E, Kurihara H et al. FAT is a component of
glomerular slit diaphragms. Kidney Int 2001; 59: 1003–1012
33. Ciani L, Patel A, Allen ND, ffrench-Constant C. Mice lacking
the giant protocadherin mFAT1 exhibit renal slit junction
abnormalities and a partially penetrant cyclopia and anophthalmia phenotype. Mol Cell Biol 2003; 23: 3575–3582
34. Shih NY, Li J, Karpitskii V et al. Congenital nephrotic
syndrome in mice lacking CD2-associated protein. Science
1999; 286: 312–315
35. Roselli S, Heidet L, Sich M et al. Early glomerular filtration
defect and severe renal disease in podocin-deficient mice. Mol
Cell Biol 2004; 24: 550–560
36. Huber TB, Kottgen M, Schilling B, Walz G, Benzing T.
Interaction with podocin facilitates nephrin signaling.
J Biol Chem 2001; 276: 41543–41546
37. Huber TB, Simons M, Hartleben B et al. Molecular basis of the
functional podocin-nephrin complex: mutations in the NPHS2
gene disrupt nephrin targeting to lipid raft microdomains.
Human Mol Genet 2003; 12: 3397–3405
38. Kaplan JM, Kim SH, North KN et al. Mutations in ACTN4,
encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000; 24: 251–256
39. Ahola H, Heikkilä E, Åström E et al. A novel protein, densin,
expressed by glomerular podocytes. J Am Soc Nephrol 2003; 14:
1731–1737
40. Winn MP, Conlon PJ, Lynn KL et al. A mutation in the TRPC6
cation channel causes familial focal segmental glomerulosclerosis. Science 2005; 308: 1801–1804
41. Takeda T. Podocyte cytoskeleton is connected to the
integral membrane protein podocalyxin through Naþ/Hþ
exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol
2003; 7: 260–269
42. Thomas PE, Wharram BL, Goyal M et al. GLEPP1, a renal
glomerular epithelial cell (podocyte) membrane protein-tyrosine
phosphatase. J Biol Chem 1994; 269: 19953–19962
43. Pavenstadt H, Kriz W, Kretzler M. Cell biology of the
glomerular podocyte. Physiol Rev 2003; 83: 253–307
44. Raats CJ, van den Born J, Bakker MA et al. Expression of agrin,
dystroglycan and utrophin in normal renal tissue and in
experimental glomerulopathies. Am J Pathol 2000; 156:
1749–1765
45. Regoli M, Bendayan M. Alterations in the expression of the
alpha3 beta 1 integrin in certain membrane domains of the
glomerular epithelial cells (podocytes) in diabetes mellitus.
Diabetologia 1997; 40: 15–22
46. Harita Y, Miyauchi N, Karasawa T et al. Altered expression of
junctional adhesion molecule 4 (JAM4) in injured podocytes.
Am J Physiol Renal Physiol 2005
2128
47. Kerjaschki D, Farquhar MG. Immunocytochemical localozation
of Heymann nephritis antigen (gp330) in glomerular epithelial
cells of normal Lewis rats. J Exp med 1983; 157: 667–686
48. Patrie KM, Drescher AJ, Goyal M, Wiggins RC, Margolis B.
The membrane associated guanylate kinase protein MAGI-1
H. Holthöfer
binds megalin and is present in glomerular podocytes. J Am Soc
Nephrol 2001; 12: 667–677
49. Rastaldi MP, Armelloni S, Berra S et al. Glomerular podocytes
contain neuron-like functional synaptic vesicles. FASEB J 2006;
20: 976–978
Received for publication: 25.10.06
Accepted in revised form: 4.5.07