- Email: [email protected]
It's not all about nephrin
co m m e nt a r y
faithful animal models of human disease that are now becoming available through the application of molecular biological techniques, we can more closely examine these different pathways and where they ultimately converge to yield a common morphologic picture, and in doing so add a new dimension to the study of glomerular and other diseases.
17.
ACKNOWLEDGMENTS I thank Laura Barisoni for her helpful comments on the manuscript.
18.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
Weiss MA, Daquioag E, Margolin G et al. Nephrotic syndrome, progressive irreversible renal failure, and glomerular “collapse”: a new clinicopathologic entity? Am J Kidney Dis 1986; 7: 20–28. Valeri A, Barisoni L, Appel GB et al. Idiopathic collapsing focal segmental glomerulosclerosis: a clinicopathologic study. Kidney Int 1996; 50: 1734–1746. Haas M, Spargo BH, Coventry S. Increasing incidence of focal-segmental glomerulosclerosis among adult nephropathies: a 20-year renal biopsy study. Am J Kidney Dis 1995; 26: 740–750. D’Agati VD, Fogo AB, Bruijn JA et al. Pathologic classification of focal segmental glomerulosclerosis: a working proposal. Am J Kidney Dis 2004; 43: 368–382. Barisoni L, Schnaper HW, Kopp JB. A proposed taxonomy for the podocytopathies: a reassessment of the primary nephrotic diseases. Clin J Am Soc Nephrol 2007; 2: 529–542. Thomas DB, Franceschini N, Hogan SL et al. Clinical and pathologic characteristics of focal segmental glomerulosclerosis pathologic variants. Kidney Int 2006; 69: 920–926. Chun MJ, Korbet SM, Schwartz MM et al. Focal segmental glomerulosclerosis in nephrotic adults: presentation, prognosis, and response to therapy of the histologic variants. J Am Soc Nephrol 2004; 15: 2169–2177. Albaqumi M, Soos TJ, Barisoni L et al. Collapsing glomerulopathy. J Am Soc Nephrol 2006; 17: 2854–2863. Kaplan JM, Kim SH, North KN et al. Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000; 24: 251–256. Henderson JM, al-Waheeb S, Weins A et al. Mice with altered α-actinin-4 expression have distinct morphologic patterns of glomerular disease. Kidney Int 2008; 73: 741–750. Barisoni L, Kriz W, Mundel P et al. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. J Am Soc Nephrol 1999; 10: 51–61. Michaud J-L, Lemieux LI, Dube M et al. Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant αactinin-4. J Am Soc Nephrol 2003; 14: 1200–1211. Dijkman H, Smeets B, van der Laak J et al. The parietal epithelial cell is crucially involved in human idiopathic focal segmental glomerulosclerosis. Kidney Int 2005; 68: 1562–1572. Dijkman HBPM, Weening JJ, Smeets B et al. Proliferating cells in HIV and pamidronate-
Kidney International (2008) 73
15. 16.
associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells. Kidney Int 2006; 70: 338–344. Bariety J, Mandet C, Hill GS et al. Parietal podocytes in normal human glomeruli. J Am Soc Nephrol 2006; 17: 2770–2780. Bariety J, Nochy D, Mandet C et al. Podocytes undergo phenotypic changes and express macrophage-associated markers in idiopathic collapsing glomerulopathy. Kidney Int 1998; 53: 918–925. Perry J, Ho M, Viero S et al. The intermediate filament nestin is highly expressed in normal human podocytes and podocytes in glomerular disease. Pediatr Dev Pathol 2007; 10: 369–382. Zhong J, Zuo Y, Ma J et al. Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.
Kidney Int 2005; 68: 1048–1060. 19. Weins A, Kenlan P, Herbert S et al. Mutational and biological analysis of α-actinin-4 in focal segmental glomerulosclerosis. J Am Soc Nephrol 2005; 16: 3694–3701. 20. Gherardi D, D’Agati V, Chu T-HT et al. Reversal of collapsing glomerulopathy in mice with the cyclin-dependent kinase inhibitor CYC202. J Am Soc Nephrol 2004; 15: 1212–1222. 21. He JC, Lu T-C, Fleet M et al. Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway. J Am Soc Nephrol 2007; 18: 93–102. 22. Tandon R, Levental I, Huang C et al. HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties. Am J Physiol Renal Physiol 2007; 292: F701–F710.
see original article on page 697
It’s not all about nephrin M Simons1 and TB Huber2 Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type. The gene product nephrin is a structural component of the glomerular slit diaphragm formed by neighboring podocytes. Nephrin has also been suggested to be involved in signaling processes that are important for podocyte survival and differentiation. A new study by Doné et al. reports that the absence of nephrin leads to the lack of slit diaphragms but does not affect podocyte apoptosis and gene expression patterns. Kidney International (2008) 73, 671–673. doi:10.1038/sj.ki.5002798
Ultrafiltration of plasma in the renal glomeruli is a major function of the kidneys. During an average human lifetime, the glomeruli produce as much as 5 million liters of primary urine. As the primary urine is virtually protein free, this means that more than 200,000 kg of albumin has to be prevented from crossing the glomerular filtration barrier. 1Mount Sinai School of Medicine, Department
of Developmental and Regenerative Biology, New York, New York, USA; and 2Renal Division, University Hospital Freiburg, Freiburg, Germany Correspondence: M Simons, Mount Sinai School of Medicine, Department of Developmental and Regenerative Biology, One Gustave L. Levy Place, New York, New York 10029, USA. E-mail: [email protected]. Or: TB Huber, Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. E-mail: [email protected]
The glomerular filter accomplishing this tremendous filtration comprises a fenestrated endothelium, the glomerular basement membrane, and podocytes, highly specialized epithelial cells of the kidney. These podocytes elaborate long, regularly spaced, interdigitated foot processes that completely surround the glomerular capillaries and form an approximately 40-nm-wide filtration slit. The filtration slit is bridged by an electron-dense membrane-like structure, the slit diaphragm (Figures 1–3). The molecular identity of the slit diaphragm remained elusive for decades until the Tryggvason group identified nephrin as a critical component of the slit membrane. 1 Nephrin was discovered through positional cloning of the NPHS1 gene, mutated in patients with congenital nephrotic syndrome of the 671
co mmentar y
Figure 1 | Schematic diagram of the glomerular slit diaphragm. Nephrin undergoes homophilic interaction on neighboring podocyte foot processes. The intercellular junction also contains the adhesion molecule NEPH-1. The slit diaphragm is firmly anchored to the underlying actin cytoskeleton and is involved in several cellular processes. (Adapted from ref. 13.)
Finnish type. 1 It is a transmembrane adhesion protein of the immunoglobulin superfamily.1 Lack of nephrin results in a loss of the slit diaphragm and massive proteinuria in utero. 2 Since then a growing number of slit diaphragm proteins have been identified, including ZO-1, podocin, CD2AP, Neph1–Neph3, P-cadherin, Densin-180, and FAT1. 3 On the basis of the domain structure and the absence of slit diaphragms in NPHS1 mutant patients and also nephrin knockout mice, it has been suggested that nephrin is a structural molecule that undergoes homophilic interaction on the neighboring podocytes4 (Figure 1). In addition, nephrin has been shown to be involved in signaling processes. Through phosphorylation of its C terminus, nephrin engages in cytoskeletal rearrangements as well as in antiapoptotic signaling. The signaling properties of nephrin seem to require the interplay with other slit diaphragm proteins such as podocin and CD2AP.3 However, it remains to be determined exactly what downstream events are triggered by nephrin signaling in vivo and whether these are necessary for the development and/or the maintenance of the filtration barrier. Doné et al.5 (this issue) now provide evidence that nephrin is dispensable for early differentiation and survival signaling.5 In nephrin-null mice, the proliferation and apoptosis rate in the glomerulus remained unaffected.5 Transcriptional 672
profiling revealed only minor changes in gene expression, including that of genes that are critical for podocyte function.5 Except for broadening of foot processes, the overall organization of podocyte architecture appeared to be intact in the absence of nephrin. On the junctional level, however, the podocytes compensated for the lack of nephrin with upregulation of tight junction proteins such as claudin 3.5 In light of recent findings on the role of nephrin in diverse signaling processes, nephrin’s overall low impact on podocyte differentiation is rather surprising. During glomerular development, the podocytes arise from a columnar visceral epithelium characteristic of the S-shaped body stage. In the capillary loop stage, the future podocytes begin to elaborate their foot
process morphology (Figure 2). This process is accompanied by the conversion of apical tight junctions into more basally located slit diaphragms — in fact, the retrograde conversion is often seen under pathological conditions. During normal development, an increased phosphotyrosine signal can be detected in the early slit diaphragms.6 Phosphorylated tyrosine motifs on the cytoplasmic tail of nephrin have been shown to serve as docking sites for SH2containing proteins of the Nck family.7,8 It has therefore been suggested that the slit diaphragm maturation, including its firm anchorage to the actin cytoskeleton, is reinforced through outside-in signaling occurring at the junctions or even from the neighboring podocytes. According to the findings by Doné et al., 5 this signaling might not involve transcriptional regulation. At least during development, the podocytes still maintain a normal gene expression profile in the absence of slit diaphragms. This suggests that the signaling occurring at the slit diaphragm has only local consequences, for example on the actin cytoskeleton. However, it is still an open question whether this is true for situations occurring outside the developmental context, in particular in stress situations such as hyperfiltration or during repair processes. It is interesting to see that podocytes still can form junctions and foot processes in the absence of nephrin. The nature of nephrin-deficient junctions still needs to be studied in more detail. It seems, however, that podocytes retain
Figure 2 | Podocyte development. During development, podocytes (green) arise from simple columnar epithelial cells (blue). Their tight junctions convert into more basally located slit diaphragms during this process. (Adapted from ref. 14.) Kidney International (2008) 73
co m m e nt a r y
Figure 3 | Scanning electron micrograph of adult podocytes demonstrating the elaborate foot process network that surrounds the glomerular capillaries.
their tight junctions from earlier stages of development, which is reflected by the upregulation of claudin 3. Nephrin probably makes the difference between the observed cell junction and the slit diaphragm; however, nephrin is not necessary for the assembly of the cell junction per se. At least on the lightmicroscopy level, the junctions also contain other components of the later slit diaphragm, such as podocin and CD2AP. 5 But without nephrin these proteins fail to organize the podocyte’s filtration capacity, which is highlighted by the massive proteinuria seen in NPHS1-mutated patients and mice. NEPH-1, another immunoglobulinsuperfamily protein of the slit diaphragm with homology to nephrin, obviously also fails to compensate for the loss of nephrin. 9 Likewise, in NEPH-1-deficient mice, nephrin alone cannot prevent slit diaphragm loss and proteinuria. With respect to the foot processes there seems to be a slight difference in both mouse models. Whereas it appears that nephrin-deficient mice still develop broadened foot processes, NEPH-1 deficiency leads to a complete foot process retraction or ‘foot process effacement’ in newborn mice.9 In Drosophila, the NEPH-1 homologue Irre-C functions as an axon guidance molecule that organizes correct projections of visual fibers in the optic chiasms of Drosophila Kidney International (2008) 73
optic ganglia.10 The analogy of axonal projections to podocyte processes is tempting and leads to the suggestion that NEPH-1 might contribute (more than nephrin) to foot process migration and interdigitation. Podocyte depletion through apoptosis seems to be a common theme in the development of glomerulosclerosis. Recent studies identified antiapoptotic AKT-mediated signaling pathways initiated by the slit diaphragm molecules nephrin and CD2AP as well as by integrins and integrin-linked kinase (ILK) at the podocyte sole, suggesting that these molecules synergistically contribute to a ‘survival tone’ in podocytes.3 In agreement with these findings, loss of CD2AP results in increased apoptosis rates of podocytes in 3- to 4-week-old mice.11,12 Doné et al.5 now demonstrate that loss of nephrin does not result in an impairment of podocyte viability. This suggests that nephrin-mediated survival signaling, at least during glomerular development, can be compensated for by other molecules such as ILK and CD2AP. 5 Future studies will have to determine the impact of nephrin-mediated AKT signaling in adulthood and/ or under glomerular disease conditions in vivo. This new study by the Tryggvason group 5 emphasizes the importance of in vivo models for gaining knowledge in glomerular biology. The study of dynamic signaling events in mammals has been hampered by the low accessibility of the podocytes and their tiny foot processes. A very promising approach might include the use of lower model organisms that are genetically tractable and offer high-resolution in vivo analysis. Nephrin and NEPH protein complexes have been found to be critical regulators of cell adhesion and cell signaling not only at the kidney filtration barrier in mammals, but also in the zebrafish pronephros, the pupal eye, optic nerves, and somatic muscles of Drosophila, or during synaptogenesis in Caenorhabditis elegans.3 However, in order to obtain a meaningful understanding of podocyte biology and pathobiology, all findings will eventually have to be correlated with good mouse
models. The nephrin knockout mouse accurately models congenital nephrotic syndrome of the Finnish type and is therefore an important part of the podocyte toolbox. Genetic interaction studies with other important podocyte genes combined with conditional knockout strategies represent inevitable future steps in grasping the entire genetic network of the podocyte. This network may not be all about nephrin, but it certainly still cannot function without it. REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
Kestilä 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. Putaala H, Soininen R, Kilpelainen P et al. The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 2001; 10: 1–8. Huber TB, Benzing T. The slit diaphragm: a signaling platform to regulate podocyte function. Curr Opin Nephrol Hypertens 2005; 14: 211–216. Wartiovaara J, Ofverstedt LG, Khoshnoodi J et al. Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 2004; 114: 1475–1483. Doné SC, Takemoto M, He L et al. Nephrin is involved in podocyte maturation but not survival during glomerular development. Kidney Int 2008; 73: 697–704. Kurihara H, Anderson JM, Farquhar MG. Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes. Am J Physiol 1995; 268: F514–F524. 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. Verma R, Kovari I, Soofi A et al. Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization. J Clin Invest 2006; 116: 1346–1359. 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. Schneider T, Reiter C, Eule E et al. Restricted expression of the irreC-rst protein is required for normal axonal projections of columnar visual neurons. Neuron 1995; 15: 259–271. Huber TB, Hartleben B, Kim J et al. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol 2003; 23: 4917–4928. Schiffer M, Mundel P, Shaw AS et al. A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-betainduced apoptosis. J Biol Chem 2004; 279: 37004–37012. Tryggvason et al. N Engl J Med 2006; 354: 1387– 1401. Dressler, GR. The cellular basis of kidney development. Annu Rev Cell Dev Biol 2006; 22: 509–529.
673
faithful animal models of human disease that are now becoming available through the application of molecular biological techniques, we can more closely examine these different pathways and where they ultimately converge to yield a common morphologic picture, and in doing so add a new dimension to the study of glomerular and other diseases.
17.
ACKNOWLEDGMENTS I thank Laura Barisoni for her helpful comments on the manuscript.
18.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
Weiss MA, Daquioag E, Margolin G et al. Nephrotic syndrome, progressive irreversible renal failure, and glomerular “collapse”: a new clinicopathologic entity? Am J Kidney Dis 1986; 7: 20–28. Valeri A, Barisoni L, Appel GB et al. Idiopathic collapsing focal segmental glomerulosclerosis: a clinicopathologic study. Kidney Int 1996; 50: 1734–1746. Haas M, Spargo BH, Coventry S. Increasing incidence of focal-segmental glomerulosclerosis among adult nephropathies: a 20-year renal biopsy study. Am J Kidney Dis 1995; 26: 740–750. D’Agati VD, Fogo AB, Bruijn JA et al. Pathologic classification of focal segmental glomerulosclerosis: a working proposal. Am J Kidney Dis 2004; 43: 368–382. Barisoni L, Schnaper HW, Kopp JB. A proposed taxonomy for the podocytopathies: a reassessment of the primary nephrotic diseases. Clin J Am Soc Nephrol 2007; 2: 529–542. Thomas DB, Franceschini N, Hogan SL et al. Clinical and pathologic characteristics of focal segmental glomerulosclerosis pathologic variants. Kidney Int 2006; 69: 920–926. Chun MJ, Korbet SM, Schwartz MM et al. Focal segmental glomerulosclerosis in nephrotic adults: presentation, prognosis, and response to therapy of the histologic variants. J Am Soc Nephrol 2004; 15: 2169–2177. Albaqumi M, Soos TJ, Barisoni L et al. Collapsing glomerulopathy. J Am Soc Nephrol 2006; 17: 2854–2863. Kaplan JM, Kim SH, North KN et al. Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 2000; 24: 251–256. Henderson JM, al-Waheeb S, Weins A et al. Mice with altered α-actinin-4 expression have distinct morphologic patterns of glomerular disease. Kidney Int 2008; 73: 741–750. Barisoni L, Kriz W, Mundel P et al. The dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and HIV-associated nephropathy. J Am Soc Nephrol 1999; 10: 51–61. Michaud J-L, Lemieux LI, Dube M et al. Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant αactinin-4. J Am Soc Nephrol 2003; 14: 1200–1211. Dijkman H, Smeets B, van der Laak J et al. The parietal epithelial cell is crucially involved in human idiopathic focal segmental glomerulosclerosis. Kidney Int 2005; 68: 1562–1572. Dijkman HBPM, Weening JJ, Smeets B et al. Proliferating cells in HIV and pamidronate-
Kidney International (2008) 73
15. 16.
associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells. Kidney Int 2006; 70: 338–344. Bariety J, Mandet C, Hill GS et al. Parietal podocytes in normal human glomeruli. J Am Soc Nephrol 2006; 17: 2770–2780. Bariety J, Nochy D, Mandet C et al. Podocytes undergo phenotypic changes and express macrophage-associated markers in idiopathic collapsing glomerulopathy. Kidney Int 1998; 53: 918–925. Perry J, Ho M, Viero S et al. The intermediate filament nestin is highly expressed in normal human podocytes and podocytes in glomerular disease. Pediatr Dev Pathol 2007; 10: 369–382. Zhong J, Zuo Y, Ma J et al. Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.
Kidney Int 2005; 68: 1048–1060. 19. Weins A, Kenlan P, Herbert S et al. Mutational and biological analysis of α-actinin-4 in focal segmental glomerulosclerosis. J Am Soc Nephrol 2005; 16: 3694–3701. 20. Gherardi D, D’Agati V, Chu T-HT et al. Reversal of collapsing glomerulopathy in mice with the cyclin-dependent kinase inhibitor CYC202. J Am Soc Nephrol 2004; 15: 1212–1222. 21. He JC, Lu T-C, Fleet M et al. Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway. J Am Soc Nephrol 2007; 18: 93–102. 22. Tandon R, Levental I, Huang C et al. HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties. Am J Physiol Renal Physiol 2007; 292: F701–F710.
see original article on page 697
It’s not all about nephrin M Simons1 and TB Huber2 Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type. The gene product nephrin is a structural component of the glomerular slit diaphragm formed by neighboring podocytes. Nephrin has also been suggested to be involved in signaling processes that are important for podocyte survival and differentiation. A new study by Doné et al. reports that the absence of nephrin leads to the lack of slit diaphragms but does not affect podocyte apoptosis and gene expression patterns. Kidney International (2008) 73, 671–673. doi:10.1038/sj.ki.5002798
Ultrafiltration of plasma in the renal glomeruli is a major function of the kidneys. During an average human lifetime, the glomeruli produce as much as 5 million liters of primary urine. As the primary urine is virtually protein free, this means that more than 200,000 kg of albumin has to be prevented from crossing the glomerular filtration barrier. 1Mount Sinai School of Medicine, Department
of Developmental and Regenerative Biology, New York, New York, USA; and 2Renal Division, University Hospital Freiburg, Freiburg, Germany Correspondence: M Simons, Mount Sinai School of Medicine, Department of Developmental and Regenerative Biology, One Gustave L. Levy Place, New York, New York 10029, USA. E-mail: [email protected]. Or: TB Huber, Renal Division, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. E-mail: [email protected]
The glomerular filter accomplishing this tremendous filtration comprises a fenestrated endothelium, the glomerular basement membrane, and podocytes, highly specialized epithelial cells of the kidney. These podocytes elaborate long, regularly spaced, interdigitated foot processes that completely surround the glomerular capillaries and form an approximately 40-nm-wide filtration slit. The filtration slit is bridged by an electron-dense membrane-like structure, the slit diaphragm (Figures 1–3). The molecular identity of the slit diaphragm remained elusive for decades until the Tryggvason group identified nephrin as a critical component of the slit membrane. 1 Nephrin was discovered through positional cloning of the NPHS1 gene, mutated in patients with congenital nephrotic syndrome of the 671
co mmentar y
Figure 1 | Schematic diagram of the glomerular slit diaphragm. Nephrin undergoes homophilic interaction on neighboring podocyte foot processes. The intercellular junction also contains the adhesion molecule NEPH-1. The slit diaphragm is firmly anchored to the underlying actin cytoskeleton and is involved in several cellular processes. (Adapted from ref. 13.)
Finnish type. 1 It is a transmembrane adhesion protein of the immunoglobulin superfamily.1 Lack of nephrin results in a loss of the slit diaphragm and massive proteinuria in utero. 2 Since then a growing number of slit diaphragm proteins have been identified, including ZO-1, podocin, CD2AP, Neph1–Neph3, P-cadherin, Densin-180, and FAT1. 3 On the basis of the domain structure and the absence of slit diaphragms in NPHS1 mutant patients and also nephrin knockout mice, it has been suggested that nephrin is a structural molecule that undergoes homophilic interaction on the neighboring podocytes4 (Figure 1). In addition, nephrin has been shown to be involved in signaling processes. Through phosphorylation of its C terminus, nephrin engages in cytoskeletal rearrangements as well as in antiapoptotic signaling. The signaling properties of nephrin seem to require the interplay with other slit diaphragm proteins such as podocin and CD2AP.3 However, it remains to be determined exactly what downstream events are triggered by nephrin signaling in vivo and whether these are necessary for the development and/or the maintenance of the filtration barrier. Doné et al.5 (this issue) now provide evidence that nephrin is dispensable for early differentiation and survival signaling.5 In nephrin-null mice, the proliferation and apoptosis rate in the glomerulus remained unaffected.5 Transcriptional 672
profiling revealed only minor changes in gene expression, including that of genes that are critical for podocyte function.5 Except for broadening of foot processes, the overall organization of podocyte architecture appeared to be intact in the absence of nephrin. On the junctional level, however, the podocytes compensated for the lack of nephrin with upregulation of tight junction proteins such as claudin 3.5 In light of recent findings on the role of nephrin in diverse signaling processes, nephrin’s overall low impact on podocyte differentiation is rather surprising. During glomerular development, the podocytes arise from a columnar visceral epithelium characteristic of the S-shaped body stage. In the capillary loop stage, the future podocytes begin to elaborate their foot
process morphology (Figure 2). This process is accompanied by the conversion of apical tight junctions into more basally located slit diaphragms — in fact, the retrograde conversion is often seen under pathological conditions. During normal development, an increased phosphotyrosine signal can be detected in the early slit diaphragms.6 Phosphorylated tyrosine motifs on the cytoplasmic tail of nephrin have been shown to serve as docking sites for SH2containing proteins of the Nck family.7,8 It has therefore been suggested that the slit diaphragm maturation, including its firm anchorage to the actin cytoskeleton, is reinforced through outside-in signaling occurring at the junctions or even from the neighboring podocytes. According to the findings by Doné et al., 5 this signaling might not involve transcriptional regulation. At least during development, the podocytes still maintain a normal gene expression profile in the absence of slit diaphragms. This suggests that the signaling occurring at the slit diaphragm has only local consequences, for example on the actin cytoskeleton. However, it is still an open question whether this is true for situations occurring outside the developmental context, in particular in stress situations such as hyperfiltration or during repair processes. It is interesting to see that podocytes still can form junctions and foot processes in the absence of nephrin. The nature of nephrin-deficient junctions still needs to be studied in more detail. It seems, however, that podocytes retain
Figure 2 | Podocyte development. During development, podocytes (green) arise from simple columnar epithelial cells (blue). Their tight junctions convert into more basally located slit diaphragms during this process. (Adapted from ref. 14.) Kidney International (2008) 73
co m m e nt a r y
Figure 3 | Scanning electron micrograph of adult podocytes demonstrating the elaborate foot process network that surrounds the glomerular capillaries.
their tight junctions from earlier stages of development, which is reflected by the upregulation of claudin 3. Nephrin probably makes the difference between the observed cell junction and the slit diaphragm; however, nephrin is not necessary for the assembly of the cell junction per se. At least on the lightmicroscopy level, the junctions also contain other components of the later slit diaphragm, such as podocin and CD2AP. 5 But without nephrin these proteins fail to organize the podocyte’s filtration capacity, which is highlighted by the massive proteinuria seen in NPHS1-mutated patients and mice. NEPH-1, another immunoglobulinsuperfamily protein of the slit diaphragm with homology to nephrin, obviously also fails to compensate for the loss of nephrin. 9 Likewise, in NEPH-1-deficient mice, nephrin alone cannot prevent slit diaphragm loss and proteinuria. With respect to the foot processes there seems to be a slight difference in both mouse models. Whereas it appears that nephrin-deficient mice still develop broadened foot processes, NEPH-1 deficiency leads to a complete foot process retraction or ‘foot process effacement’ in newborn mice.9 In Drosophila, the NEPH-1 homologue Irre-C functions as an axon guidance molecule that organizes correct projections of visual fibers in the optic chiasms of Drosophila Kidney International (2008) 73
optic ganglia.10 The analogy of axonal projections to podocyte processes is tempting and leads to the suggestion that NEPH-1 might contribute (more than nephrin) to foot process migration and interdigitation. Podocyte depletion through apoptosis seems to be a common theme in the development of glomerulosclerosis. Recent studies identified antiapoptotic AKT-mediated signaling pathways initiated by the slit diaphragm molecules nephrin and CD2AP as well as by integrins and integrin-linked kinase (ILK) at the podocyte sole, suggesting that these molecules synergistically contribute to a ‘survival tone’ in podocytes.3 In agreement with these findings, loss of CD2AP results in increased apoptosis rates of podocytes in 3- to 4-week-old mice.11,12 Doné et al.5 now demonstrate that loss of nephrin does not result in an impairment of podocyte viability. This suggests that nephrin-mediated survival signaling, at least during glomerular development, can be compensated for by other molecules such as ILK and CD2AP. 5 Future studies will have to determine the impact of nephrin-mediated AKT signaling in adulthood and/ or under glomerular disease conditions in vivo. This new study by the Tryggvason group 5 emphasizes the importance of in vivo models for gaining knowledge in glomerular biology. The study of dynamic signaling events in mammals has been hampered by the low accessibility of the podocytes and their tiny foot processes. A very promising approach might include the use of lower model organisms that are genetically tractable and offer high-resolution in vivo analysis. Nephrin and NEPH protein complexes have been found to be critical regulators of cell adhesion and cell signaling not only at the kidney filtration barrier in mammals, but also in the zebrafish pronephros, the pupal eye, optic nerves, and somatic muscles of Drosophila, or during synaptogenesis in Caenorhabditis elegans.3 However, in order to obtain a meaningful understanding of podocyte biology and pathobiology, all findings will eventually have to be correlated with good mouse
models. The nephrin knockout mouse accurately models congenital nephrotic syndrome of the Finnish type and is therefore an important part of the podocyte toolbox. Genetic interaction studies with other important podocyte genes combined with conditional knockout strategies represent inevitable future steps in grasping the entire genetic network of the podocyte. This network may not be all about nephrin, but it certainly still cannot function without it. REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
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