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Keywords:

  • nephrotic syndrome;
  • podocyte;
  • proteinuria

Abstract

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

SUMMARY:  The podocyte is a highly specialized cell that plays a key role in regulating the glomerular filtration barrier. A number of advances have been made in recent years, linked to the discovery of single-gene defects in hereditary glomerular disease, which highlight the role of this cell in preventing proteinuria. This article reviews the molecular biology of the podocyte, focusing on known genetic abnormalities.

One of the major roles of the kidney is to allow the passage of water and solutes while preventing the passage of albumin and other key blood proteins. The podocyte is the final layer of the glomerular filtration barrier with cells forming primary and secondary processes that interdigitate and encircle the glomerular basement membrane (GBM) and endothelium (Fig. 1). These three components act together to regulate filtration; however, recent advances have identified the key contribution of the podocyte in preventing proteinuria. The current article reviews the findings that have identified the podocyte as an important regulator in the process of ultrafiltration and a target in the pathogenesis of renal disease.

image

Figure 1. A simplified schematic of the glomerular filtration barrier. The podocyte, glomerular basement membrane and capillary endothelium work together to filter blood and produce ‘proto-urine’ in Bowman's space.

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PODOCYTE DEVELOPMENT

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

Dysregulation of a number of transcription factors contributing to podocyte morphogenesis have been shown to be associated with renal disease (Table 1).1 Wilms tumour suppressor gene 1 (Wt1) has two alternatively spliced exons, which encode four major isoforms of a transcription factor and RNA-binding protein (WT1) required for kidney development.2 WT1 is initially expressed in the metanephric mesenchyme; however, as kidney development proceeds, WT1 becomes limited to the podocyte. Wt1 knockout mice are unable to develop a ureteric bud leading to failure of kidney development.3 Mutations of Wt1 can lead to a number of gonadal and renal abnormalities in humans. Denys–Drash syndrome, characterized by early onset mesangial sclerosis, male pseudohermaphroditism and Wilms tumour, is due to mutations in exon eight and nine of the Wt1 gene, which affect DNA-binding in zinc finger domains of the transcription factor. Conflicting data exist suggesting that this mutation might also have a potential effect on podocalyxin, a podocyte-specific cell surface protein and also a potential effect on glomerular vasculature.4 Frasier syndrome, characterized by focal glomerulosclerosis, male pseudohermaphroditism with complete sex reversal and gonadal tumour, is due to mutations at the splice donor site of the Wt1 gene, which would normally allow three amino acids, lysine-threonine-serine (KTS), to be inserted between exon 9 and 10. This alters the DNA-binding capacity of the transcription factor. Interestingly, knockout mice with one normal and one –KTS allele mimic the Frasier phenotype and display a decreased expression of nephrin, a podocyte-specific protein found in the slit diaphragm.5 The paired box gene 2 (PAX2) encodes a DNA-binding transcription factor expressed in the ear, eye, central nervous system and urogenital system. Mutations of the gene lead to the renal–coloboma syndrome, which is characterized by abnormal optic nerve and renal development. PAX2 knockout mice fail to develop kidneys, ureters or genital tracts.6 PAX2 has been shown to interact with WT1 in initiating kidney morphogenesis.7 This clearly displays the subtle and complex role of the Wt1 gene in podocyte differentiation.

Table 1.  Podocyte transcription factors associated with human glomerular disease
GeneTranscription factorSyndromeClinical features
  1. FSGS, focal segmental glomerular sclerosis.

WT1WT1Denys–Drash syndrome, Frasier syndromeNephrotic syndrome (mesangial sclerosis), Wilm's tumour, genital abnormalites Nephrotic syndrome (FSGS), gonadal tumour, ambiguous to normal female genitalia
PAX2PAX2Renal–coloboma syndromeOptic nerve colobomas, renal hypoplasia, vesico-ureteric reflux
LMX1BLMX1BNail–patella syndromeDystrophic nails, absent or hypoplastic patellae, haematuria, proteinuria, renal failure

The LMX1B gene encodes a LIM homeodomain transcription factor expressed in podocytes.8 Mutations of this gene cause nail–patella syndrome, which is characterized by dysplastic nails, absent or hypoplastic patellae, and haematuria and proteinuria. Studies in the LMX1B knockout mice show abnormal differentiation of the podocyte with an absence of foot process and slit diaphragm formation, as well as glomerular basement membrane abnormalities.9

Animal models have identified other transcription factors of potential significance in podocyte differentiation including Pod1, a basic-helix-loop-helix transcription factor expressed in the podocyte;10 Kreisler, a basic leucine domain zipper transcription factor highly expressed in developing and mature podocytes;11 and Mf2, a forkhead/winged helix transcription factor also expressed in developing and mature podocytes.12 The role of these transcription factors in human disease is as yet unclear.

THE SLIT DIAPHRAGM

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

The slit diaphragm represents the only cell–cell contact between mature podocytes, and delineates the basal and apical domains. The study of hereditary nephrotic syndromes has led to the discovery of a number of novel cell proteins, localized to the slit diaphragm, which have highlighted the role of the podocyte in ultrafiltration (Table 2).

Table 2.  Podocyte proteins associated with human glomerular disease
GeneProteinSyndrome
  1. FSGS, focal segmental glomerular sclerosis.

NPHS1NephrinCongenital nephrotic syndrome of the Finnish type
NPHS2PodocinFSGS, microalbuminuria
CD2APCD2APFSGS
ACTN4α-actinin 4FSGS

Congenital nephrotic syndrome of the Finnish type (CNF) is characterized by massive proteinuria in utero and nephrotic syndrome at birth. It is an autosomal-recessive condition, affecting 1 in 10 000 births in Finland, which has been described in various other ethnic groups worldwide. Two discrete mutations of the NPHS1 gene have been found to account for the majority of patients with CNF: Fin major (nt121delCT) and Fin minor (R1109X).13 The NPHS1 gene codes for nephrin, a transmembrane protein that localizes to the slit diaphragm. It consists of eight immunoglobulin-like domains and one fibronectin-like motif that are extracellular, a transmembrane region, and have a short cytoplasmic tail.14 The Fin major mutation results in a truncated protein that lacks membranous and extramembranous components of nephrin, while the Fin minor mutation results in a nephrin that lacks a cytoplasmic component. Injection of mAb 5-1-6, an anti-nephrin antibody, in rats was found to cause massive proteinuria.15 Inactivation of the NPHS1 gene in mice has been shown to lead to massive proteinuria and death.16 In addition to playing a role in maintaining the structure of the slit diaphragm, cell culture models have shown that nephrin also appears to play a role as a signalling molecule activating a mitogen-associated protein kinase cascade.17 Although results are variable and subject numbers are small, clinical studies in humans have shown a reduction of both mRNA and protein expression of nephrin in acquired proteinuric renal disease.18–21

The NPHS2 gene codes for podocin, a hairpin-like membrane protein with intracellular termini. A variety of frameshift, stop codon or missense mutations have been described. Podocin was initially described as mutated in a form of autosomal-recessive, steroid-resistant nephrotic syndrome characterized by childhood-onset proteinuria with focal segmental glomerular sclerosis (FSGS), which progresses to end-stage renal failure.22 More recently, mutations have been described with adult onset FSGS23 and microalbuminuria in the general population.24 Podocin localizes to the slit diaphragm with nephrin, in lipid rafts, where it acts as a scaffolding protein and enhances the signalling activity of nephrin.17

CD2-associated protein (CD2AP) was originally described as an adaptor protein responsible for recruiting CD2 to the junction of T-cells and antigen-presenting cells during the immune response.25 Interestingly, CD2AP knockout mice were found to develop massive proteinuria. CD2AP localizes to the slit diaphragm with nephrin and podocin,26 and plays a role in intracellular signalling.27 Intriguingly, it has been shown in knockout mice that CD2AP might play a role in transforming growth factor-β (TGF-β)-induced podocyte apoptosis.28 Two human patients with splice variation mutations in the CD2AP gene have been found with idiopathic FSGS,29 suggesting CD2AP might have a role in modulating human glomerular disease.

A novel family of proteins, NEPH1, 2 and 3 have been identified in human podocytes.30 These proteins are structurally similar to nephrin: they have five extracellular immunoglobulin-like domains, a transmembrane domain and a short cytoplasmic tail, and have been found to interact with podocin. Their significance in human disease in unclear; however, NEPH1 has been shown to localize to the slit diaphragm and NEPH1 knockout mice develop massive proteinuria. Intriguingly, the NEPH3 gene is located adjacent to the NPHS1 gene; it may be that patients with congenital nephrotic syndrome with linkage to the NPHS1 gene might have mutations of the NEPH3 gene.

A number of other molecules have been found to localize to the slit diaphragm, including zonula occludens 1 (ZO-1), P-cadherin, α-, β- and γ-catenins and FAT. Their role in human glomerular disease is unclear.

THE ACTIN CYTOSKELETON

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

The structure of the podocyte is maintained by a complex cytoskeleton consisting of actin, myosin, α-actinin-4, talin and vinculin. The ACTN4 gene encodes α-actinin-4, a 100 kDa protein that forms head-to-tail homodimers that crosslinks and bundles filamentous actin. Point mutations in the ACTN4 gene in humans have been shown to be associated with an autosomal-dominant form of FSGS.31 Patients have a variable phenotype, with some patients developing progressive renal failure, others developing moderate proteinuria and a few displaying normal renal function. Mouse models suggest that mutant α-actinin-4 displays protein misfolding and accelerated degradation leading to abnormal aggregation and function.32

Synaptopodin is a proline-rich protein unique to the podocyte and brain that interacts with filamentous actin.33 Expression of this protein has been shown to be lost early in proteinuric renal diseases, such as FSGS and minimal change disease.34

THE APICAL DOMAIN

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

The apical membrane of the podocyte, found above the slit diaphragm, forms another domain. It is highly negatively charged related to the presence of podocalyxin. Podocalyxin links to the actin cytoskeleton via a sodium-hydrogen exchanger regulatory factor (NHERF-2) and ezrin. Animal models have shown the importance of these proteins; however, their exact role in contributing to human disease is as yet unclear.

THE PODOCYTE AND GBM JUNCTION

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

The basal domain of the podocyte attaches to the GBM via α3β1-integrin heterodimers and αβ-dystroglycan complexes. Although proteinuria is characterized by detachment of the foot processes from the GBM, no consistent alterations in integrin or dystroglycan expression have been shown in humans. However, an integrin-linked kinase has been shown to be induced in congenital nephrotic syndrome.35 This might mediate integrin-mediated cell adhesion, signalling and changes in the podocyte cytoskeleton. Figure 2 shows our current understanding of the different domains of the podocyte and how they link to the cytoskeleton.

image

Figure 2. Molecular anatomy of the podocyte foot process. A simplified schematic showing the apical domain, slit diaphragm, basal domain, cytoskeleton and some of the molecules found in the foot process of a podocyte.

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THE PODOCYTE AND ENDOTHELIUM

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

The podocyte has been shown to express vascular endothelial growth factor-A (VEGF-A), a critical factor in angiogenesis, both during glomerular development and once the podocyte is mature. VEGF receptor, fetal liver kinase 1 (Flk1), is present in glomerular endothelial cells. Pre-eclamptic women have been shown to have a reduction in VEGF-A due to the presence of a soluble VEGF receptor, fms-like tyrosine kinase 1 (Flt1).36 Injection of this receptor into rats leads to proteinuria and endotheliosis.37 Animal models, using podocyte-specific heterozygous deletion of VEGF-A, resulted in proteinuria and endotheliosis, the typical renal lesion seen in pre-eclampsia. Podocyte-specific overexpression of VEGF-164 resulted in a collapsing glomerulopathy phenotype.38

The angiopoietins (Ang) are endothelial growth factors that interact with VEGF to regulate vascular permeability. Recent evidence has shown that in both a cell culture model and mature human glomeruli, podocytes express Ang1. Protein receptor tyrosine kinase 2 (Tie2), an angiopoietin receptor, was also shown to occur on the abluminal surface of human glomerular endothelium cells.39 Thus the podocyte might also regulate the glomerular filtration barrier via the growth factors VEGF and Ang1.

CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

Except in HIV nephropathy and collapsing FSGS the differentiated podocyte is unable to proliferate. In Pima Indians with type 2 diabetes mellitus, a reduction in podocyte number has been shown to be associated with microalbuminuria and severity of the nephropathy.40 Patients with type 1 diabetes mellitus have also been shown to have a decrease in podocyte numbers.41 Once podocytes are terminally differentiated they are unable to undergo cell division; however, they may enter the cell cycle. Mature podocytes have an increased expression of cyclin-dependent kinase inhibitors, which prevent proliferation.42

It has been suggested that loss of the podocyte and its lack of ability to proliferate might contribute to progressive glomerular scarring. The hypothesis is that as well as acting as a filtration barrier it is thought the podocyte provides tensile support to the glomerular capillary loop. Podocyte loss then leads to inadequate numbers of podocytes able to cover the GBM. Denuded GBM lacking the tensile support of the podocyte might then bulge into and abut Bowman's capsule leading to tuft adhesions and segmental sclerosis. The tuft then merges with the interstitium leading to progressive sclerosis and fibrosis.43

RESEARCH TOOLS

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

Using a temperature sensitive SV40 T-antigen, immortalized murine and human podocyte cell lines have been established.44–46 These cells proliferate at 33°C and differentiate at 37°C, and display morphological and molecular features of mature podocytes. They are therefore an excellent tool for the in vitro study of podocyte biology.

An efficient, large-scale system of isolating intact glomeruli from maturing and adult mice using magnetic beads has been described.47 This should prove useful in analysing gene and protein expression in in vivo models of renal disease.

The discovery of podocyte-specific proteins such as podocin and nephrin has also allowed the development of an in vivo system for non-inducible48 and inducible49 disruption of genes in podocytes. This should allow the assessment of individual genes in podocyte function.

SUMMARY

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES

Podocytes have an elegant and complex structure, forming the final layer of the glomerular filtration barrier. Recent advances identifying genetic disorders have identified the podocyte's key role in regulating proteinuria, both by maintaining the structure of the filtration barrier and via cell signalling. A more comprehensive review of this topic has recently been published by Pavenstädt et al.;50 however, recent advances in cell culture and podocyte-specific knockout mice models are likely to lead to further understanding of the podocyte and might lead to the identification of therapeutic targets for the prevention of progressive renal disease.

REFERENCES

  1. Top of page
  2. Abstract
  3. PODOCYTE DEVELOPMENT
  4. THE SLIT DIAPHRAGM
  5. THE ACTIN CYTOSKELETON
  6. THE APICAL DOMAIN
  7. THE PODOCYTE AND GBM JUNCTION
  8. THE PODOCYTE AND ENDOTHELIUM
  9. CELL CYCLE OF THE PODOCYTE AND PROGRESSIVE RENAL INJURY
  10. RESEARCH TOOLS
  11. SUMMARY
  12. ACKNOWLEDGEMENTS
  13. REFERENCES
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