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Commentary |
From the Department of Medicine, Renal Division and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri
The nature of the
glomerular filtration barrier is currently one of the most intensely
studied and exciting problems in nephrology research, primarily because
of the relatively recent discoveries that two novel and two previously
known genes are intimately involved in glomerular filtration. The two
novel genes, NPHS1 and NPHS2 (encoding nephrin
and podocin, respectively) were identified by positional cloning.
NPHS1 is mutated in congenital nephrotic syndrome of the
Finnish type,1
and NPHS2 is mutated in steroid
resistant nephrotic syndrome.2
The other two genes,
previously characterized but not known to be involved in filtration,
are ACTN4, encoding 
-actinin-4, and Cd2ap,
encoding CD2-associated protein (CD2AP). ACTN4 was found to
be mutated in several families exhibiting inherited focal segmental
glomerulosclerosis,3
and knockout mice lacking CD2AP
exhibit congenital nephrotic syndrome.4
Several recent
reviews have addressed the potential roles of these molecules in
glomerular filtration.5-10
All four of these genes have been shown to be expressed by podocytes, the specialized epithelial cells that lie atop the glomerular basement membrane (GBM) in the urinary space. Podocytes make up the final cell layer across which the glomerular ultrafiltrate must pass before flowing down the tubular portions of the nephron toward the ureter and bladder. Podocytes elaborate long, regularly spaced, interdigitated foot processes that enwrap the glomerular capillaries. Near the GBM, the foot processes are connected by a thin structure termed the glomerular slit diaphragm. The existence of the slit has been known at an ultrastructural level for decades,11 but until recently its molecular composition and role in glomerular filtration had been a mystery.
Several groups have shown by immunoelectron microscopy (IEM) that
nephrin is found at the slit diaphragm.12-15
Nephrin is a
transmembrane protein with a substantial extracellular domain, and both
humans and mice lacking nephrin are born without typical slit
diaphragms and exhibit massive proteinuria.1,16
Together,
these data suggest that nephrin is an actual component of the slit and
lend support to the hypothesis that the slit has a crucial role in
glomerular filtration. The localization of CD2AP and -actinin-4
proteins has also been investigated by immunohistological methods.
CD2AP is an adapter molecule17
that localizes to podocytes
and is capable of binding to the cytoplasmic tail of nephrin, perhaps
linking the slit diaphragm to the podocyte cytoskeleton and providing
stability to what might otherwise be a fragile structure.4
Our more recent studies demonstrate by IEM that CD2AP is present at the
slit and binds to nephrin via its carboxyl terminal
domain.18
Other IEM studies are consistent with these
results but suggest that CD2AP is more widely distributed in the foot
process,19
in agreement with our original
immunofluorescence data.4
-actinin-4 is a member of a
family of proteins that cross-link and anchor actin filaments, and it
was shown to be associated with the actin cytoskeleton in podocyte foot
processes.3,20
The fact that mutations in ACTN4
cause glomerulosclerosis suggests that -actinin-4 plays a crucial
role in maintaining the structure of podocyte foot processes and/or the
slit diaphragm via the actin cytoskeleton.3
But what about the localization of podocin, the fourth member of this genetically defined quartet? The article by Corinne Antignac and colleagues21 published in this issue of The American Journal of Pathology clearly confirms that podocin, like nephrin, is present in the podocyte plasma membrane in the area of the slit diaphragm. The authors made several antisera that specifically recognize podocin and showed by both high-resolution confocal microscopy and IEM that podocin is found in foot processes and concentrated at the slit diaphragms. Thus, this entire quartet of molecules localizes to podocyte foot processes, with nephrin, CD2AP, and podocin being present at slit diaphragms. The fact that mutations in any of the four genes causes proteinuria and renal failure indicates that podocytes, their foot processes, and the intervening slits are likely the most critical components of the glomerular filtration barrier.
With regard to other molecules at the slit, it has been known for a long time that ZO-1, a protein normally associated with tight junctions, is localized to the cytoplasmic face of the slit.22 This led to the conclusion that the slit diaphragm is a modified tight junction. However, it was shown by IEM that P-cadherin localizes to the extracellular portion of the slit diaphragm and co-localizes with ZO-1 on the cytoplasmic face of the slit, suggesting that the slit has features of adherens junctions, which can also contain ZO-1.23 However, the importance of P-cadherin at the slit remains to be demonstrated. The facts that 1) knockout mice lacking P-cadherin are viable and fertile with no reported kidney defects,24 and 2) humans with a mutation in CDH3, the gene encoding P-cadherin, have hair and retinal but not kidney defects25 suggests that any role P-cadherin plays in glomerular filtration is either subtle or at least partially redundant. Indeed, another member of the cadherin superfamily, FAT, has also been localized to slit diaphragms.26 However, a role for FAT in kidney function has not yet been addressed. Finally, Neph1, the gene encoding NEPH1, a protein with weak homology to nephrin, is transcribed by podocytes, and knockout mice lacking NEPH1 develop proteinuria. Most Neph1-/- mice die between 1 and 12 days after birth, but a few live up to 8 weeks of age.27 It will be very interesting to determine the subcellular localization of NEPH1 and the mechanisms leading to the onset of proteinuria. Unlike Nphs1, Neph1 is also expressed by mesangial, proximal tubular, and collecting duct cells (and in many other tissues besides kidney).27 Thus, NEPH1 may have functions in the kidney quite distinct from those attributed to nephrin.
The notion that the glomerular slit diaphragm merely serves as a filter to keep albumin and other plasma proteins from entering the urinary space is being challenged by evidence that slit-associated molecules are involved in signaling events. For example, Huber and colleagues have shown that transfection of nephrin into 293T cells initiates activation of AP-1 transcriptional activity and of stress-activated p38 and c-Jun N-terminal protein kinases.28 Interestingly, co-transfection of podocin synergistically increased nephrin-initiated AP-1 activation,28 suggesting that nephrin and podocin may cooperate at the slit to mediate signaling. Furthermore, two groups have shown that nephrin is associated with lipid rafts, specialized cholesterol-rich membrane domains associated with signaling molecules and signaling events.29,30 Podocin is also associated with lipid rafts and interacts directly with nephrin and CD2AP at the slit diaphragm.31
What of the forgotten role of the GBM in glomerular filtration? With
all of the focus on podocytes and glomerular slit diaphragms, less
attention is being paid to the GBM. Although the GBM may not be the
major size-selective filter, its negative charge—imparted by the
presence of heparan sulfate proteoglycans such as agrin—is likely
important in establishing a charge-selective barrier to negatively
charged plasma proteins. In addition, the unique molecular structure of
the GBM seems to be crucial for maintaining podocyte homeostasis.
Mutations that alter the composition of the GBM and that result in
filtration defects may affect podocytes directly and filtration
indirectly. The GBM is normally composed of laminin-11 (5ß2
1),
the collagen 3, 4, and 5(IV) chains, agrin, and nidogen, as
well as other matrix molecules.32
Knockout mice lacking
the laminin ß2 chain have no laminin-11 in their GBMs and develop
massive proteinuria at 7 days of age.33
Laminin-11 may
therefore be necessary for proper podocyte adhesion and for maintenance
of foot process architecture. Similarly, mice, humans, and dogs with
collagen IV gene mutations exhibit Alport syndrome, which is
characterized by later onset filtration defects.34,35
The
GBM becomes thickened and split, and novel laminin chains, such as 2
and ß1, accumulate within it.36,37
These ectopic
laminins may provide atypical signals to the overlying podocytes that
disrupt their behavior, leading to foot process effacement and
proteinuria. Indeed, reduced levels of laminin 2 in Alport GBM was
associated with a reduction in podocyte damage.36
In conclusion, it is clear that the glomerular slit diaphragm is a major, if not the most important, component of the kidney’s ultrafiltration barrier. The finding that podocin is concentrated there reinforces this notion, because mutations in NPHS2, the gene encoding podocin, cause nephrotic syndrome and glomerulosclerosis. Determining the function of podocin will be challenging, given that podocin is a member of the stomatin family of proteins, about which little is known. Nevertheless, the generation of knockout mice lacking podocin, which is presumably underway, will allow for those lines of experimentation that are not possible with human patients, but that will hopefully lead to a better understanding of podocin and the structure and function of the glomerular slit diaphragm.
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Address reprint requests to Dr. Jeffrey H. Miner, Renal Division, Box 8126, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: minerj{at}pcg.wustl.edu
Accepted for publication October 31, 2001.
References
This article has been cited by other articles:
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  A. V. Cybulsky, R. J. Quigg, and D. J. Salant Experimental membranous nephropathy redux Am J Physiol Renal Physiol, October 1, 2005; 289(4): F660 - F671. [Abstract] [Full Text] [PDF]
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 T. B. Huber, M. Simons, B. Hartleben, L. Sernetz, M. Schmidts, E. Gundlach, M. A. Saleem, G. Walz, and T. Benzing Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains Hum. Mol. Genet., December 15, 2003; 12(24): 3397 - 3405. [Abstract] [Full Text] [PDF]
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 Y. Kikkawa, I. Virtanen, and J. H. Miner Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin {alpha}5 in the glomerular basement membrane J. Cell Biol., April 14, 2003; 161(1): 187 - 196. [Abstract] [Full Text] [PDF]
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 J. A. Kreidberg Podocyte Differentiation and Glomerulogenesis J. Am. Soc. Nephrol., March 1, 2003; 14(3): 806 - 814. [Full Text] [PDF]
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P. Mundel and S. J. Shankland Podocyte Biology and Response to Injury J. Am. Soc. Nephrol., December 1, 2002; 13(12): 3005 - 3015. [Full Text] [PDF]
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 E. Yaoita, J. Yao, Y. Yoshida, T. Morioka, M. Nameta, T. Takata, J.-i. Kamiie, H. Fujinaka, T. Oite, and T. Yamamoto Up-Regulation of Connexin43 in Glomerular Podocytes in Response to Injury Am. J. Pathol., November 1, 2002; 161(5): 1597 - 1606. [Abstract] [Full Text] [PDF]
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