Neuronal proteins are novel components of podocyte major processes and their expression in glomerular crescents supports their role in crescent formation
2012; Elsevier BV; Volume: 83; Issue: 1 Linguagem: Inglês
10.1038/ki.2012.321
ISSN1523-1755
AutoresLaleh Sistani, Patricia Q. Rodriguez, Kjell Hultenby, Mathias Uhlén, Christer Betsholtz, Hannu Jalanko, Karl Tryggvason, Annika Wernerson, Jaakko Patrakka,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoThe podocyte has a central role in the glomerular filtration barrier typified by a sophisticated morphology of highly organized primary (major) and secondary (foot) processes. The molecular makeup of foot processes is well characterized, but that of major processes is poorly known. Previously, we profiled the glomerular transcriptome through large-scale sequencing and microarray profiling. Unexpectedly, the survey found expression of three neuronal proteins (Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in the glomerulus. These proteins were expressed exclusively by podocytes, wherein they localized to major processes as verified by RT–PCR, western blotting, immunofluorescence, and immunoelectron microscopy. During podocyte development, these proteins colocalized with vimentin, confirming their association with major processes. Using immunohistochemistry, we found coexpression of Hip1 and Olfml2a along with the recognized podocyte markers synaptopodin and Pdlim2 in glomerular crescents of human kidneys, indicating the presence of podocytes in these lesions. Thus, three neuronal proteins are highly expressed in podocyte major process. Using these new markers we found that podocytes contribute to the formation of glomerular crescents. The podocyte has a central role in the glomerular filtration barrier typified by a sophisticated morphology of highly organized primary (major) and secondary (foot) processes. The molecular makeup of foot processes is well characterized, but that of major processes is poorly known. Previously, we profiled the glomerular transcriptome through large-scale sequencing and microarray profiling. Unexpectedly, the survey found expression of three neuronal proteins (Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in the glomerulus. These proteins were expressed exclusively by podocytes, wherein they localized to major processes as verified by RT–PCR, western blotting, immunofluorescence, and immunoelectron microscopy. During podocyte development, these proteins colocalized with vimentin, confirming their association with major processes. Using immunohistochemistry, we found coexpression of Hip1 and Olfml2a along with the recognized podocyte markers synaptopodin and Pdlim2 in glomerular crescents of human kidneys, indicating the presence of podocytes in these lesions. Thus, three neuronal proteins are highly expressed in podocyte major process. Using these new markers we found that podocytes contribute to the formation of glomerular crescents. The glomerulus comprises a tangled network of capillaries surrounded by a urinary space and Bowman's capsule. The filtration of primary urine occurs through the capillary wall of the glomerulus. This filtration barrier is essentially composed of three layers: (1) the glomerular basement membrane, which serves as a foundation and support; (2) fenestrated endothelial cells, located on the inside; and (3) visceral epithelial cells (podocytes), located on the outside of the glomerular capillary.1.Tryggvason K. Patrakka J. Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria.N Engl J Med. 2006; 354: 1387-1401Crossref PubMed Scopus (444) Google Scholar The podocyte is a highly specialized epithelial cell with a remarkably sophisticated architecture.2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1202) Google Scholar It has a prominent cell body with large cytoplasmic projections named major processes, which further extend into smaller processes known as foot processes. The foot processes interdigitate as they wrap around and envelope the capillaries. The adjacent foot processes interact and are connected by a specialized cell–cell junction termed the slit diaphragm.2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1202) Google Scholar, 3.Welsh G.I. Saleem M.A. Nephrin-signature molecule of the glomerular podocyte?.J Pathol. 2010; 220: 328-337PubMed Google Scholar, 4.Patrakka J. Tryggvason K. New insights into the role of podocytes in proteinuria.Nat Rev Nephrol. 2009; 5: 463-468Crossref PubMed Scopus (139) Google Scholar Mutations in slit diaphragm proteins cause inherited forms of proteinuria,1.Tryggvason K. Patrakka J. Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria.N Engl J Med. 2006; 354: 1387-1401Crossref PubMed Scopus (444) Google Scholar which underlines the importance of these structures in the filtration barrier. Podocytes are dynamic cells with a prominent cytoskeleton that can effectively respond to changes in their extracellular environment.5.Oh J. Reiser J. Mundel P. Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome.Pediatr Nephrol. 2004; 19: 130-137Crossref PubMed Scopus (68) Google Scholar This cytoskeletal machinery is vital for a sustained kidney filtration, as defects in cytoskeleton-regulating proteins of podocyte foot processes result in proteinuria.5.Oh J. Reiser J. Mundel P. Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome.Pediatr Nephrol. 2004; 19: 130-137Crossref PubMed Scopus (68) Google Scholar Bundles of microtubules and intermediate filaments support major processes, whereas the cytoskeleton of foot processes is composed mainly of actin filaments.2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1202) Google Scholar, 5.Oh J. Reiser J. Mundel P. Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome.Pediatr Nephrol. 2004; 19: 130-137Crossref PubMed Scopus (68) Google Scholar, 6.Drenckhahn D. Franke R.P. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.Lab Invest. 1988; 59: 673-682PubMed Google Scholar This feature confirms an obvious morphological difference between these two structures, thus contributing to their molecular and functional divergence. The molecular composition of foot process is rather well characterized.4.Patrakka J. Tryggvason K. New insights into the role of podocytes in proteinuria.Nat Rev Nephrol. 2009; 5: 463-468Crossref PubMed Scopus (139) Google Scholar This includes basal, slit diaphragm, and apical plasma membrane protein complexes connected by a number of linker proteins and actin cytoskeleton.4.Patrakka J. Tryggvason K. New insights into the role of podocytes in proteinuria.Nat Rev Nephrol. 2009; 5: 463-468Crossref PubMed Scopus (139) Google Scholar,5.Oh J. Reiser J. Mundel P. Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome.Pediatr Nephrol. 2004; 19: 130-137Crossref PubMed Scopus (68) Google Scholar In contrast to this, the molecular nature of major processes is still poorly understood. Besides microtubules and the intermediate filament protein vimentin,2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1202) Google Scholar,6.Drenckhahn D. Franke R.P. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.Lab Invest. 1988; 59: 673-682PubMed Google Scholar the components that form these cellular projections are largely unknown. We have previously identified several highly glomerulus-enriched transcripts using large-scale sequencing and microarray profiling.7.Takemoto M. He L. Norlin J. et al.Large-scale identification of genes implicated in kidney glomerulus development and function.EMBO J. 2006; 25: 1160-1174Crossref PubMed Scopus (181) Google Scholar In this study, we are characterizing further three of these glomerular transcripts. All three proteins in the kidney are expressed only by podocytes, wherein they localize to major processes. The specific expression in podocyte major processes suggests that they have a dedicated role in the formation of these peculiar cellular projections. Furthermore, we use these novel podocyte markers to demonstrate that podocytes are present in glomerular crescents, an issue that has been a matter of controversy. Previously, we have profiled the glomerular transcriptome through large-scale sequencing and microarray profiling.7.Takemoto M. He L. Norlin J. et al.Large-scale identification of genes implicated in kidney glomerulus development and function.EMBO J. 2006; 25: 1160-1174Crossref PubMed Scopus (181) Google Scholar In this approach, we chose three transcripts, Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a), which were enriched in the glomerulus, for a more detailed analysis. These genes were chosen for further studies because their biology was poorly understood, and their role and expression in the kidney were completely uncharacterized. First, we analyzed the expression of Hip1 in the kidney through reverse transcriptase–PCR (RT–PCR). The PCR product for Hip1 was amplified in glomeruli, whereas no expression was detected in the rest of the kidney lacking glomeruli (Figure 1). The control experiment with glyceraldehyde 3–phosphate dehydrogenase showed strong signal in both lanes, whereas podocin, a known glomerulus-specific transcript, gave significantly stronger signal in the glomerular fraction. Next, we generated a polyclonal antibody against the human Hip1 protein. By using western blotting of human kidney lysates, our anti-Hip1 antibody detected a band of expected size (∼115–kDa) in the glomerulus as opposed to the rest of the kidney, which appeared completely negative (Figure 1). A similar expression pattern was observed for nephrin, a known podocyte-specific protein. The control experiment with β-actin antibody showed clear signal in both lysates. In immunofluorescence, Hip1 antibody showed intense staining in the glomeruli, and no signal was detected in the extraglomerular areas (Figures 2a and 3a). Double staining with foot process marker nephrin showed that Hip1 was located at the side of the urinary space of nephrin (Figure 3b and c). This indicated expression in podocytes. Occasionally, some overlapping reactivity with nephrin was observed (arrow, Figure 3c). Double labeling with vimentin showed that Hip1 colocalized with this major process marker (Figure 3d). Immunoelectron microscopy was used to confirm the subcellular localization of Hip1. Gold label for Hip1 was observed mostly in major processes (Figure 4a). Quantitatively, we detected a total of 176 gold labels in glomeruli, of which 96 (55%) were located in major processes. In addition, few labels were occasionally detected in foot processes (Figure 4a). No significant signal for Hip1 was found outside podocytes.Figure 3Expression of novel podocyte proteins in the glomerulus as shown with double immunofluorescence labeling. Double immunolabeling was performed with foot process marker nephrin (red) and major process marker vimentin (red). DAPI (4,6-diamidino-2-phenylindole; a nuclear marker) staining is shown in blue. (a) Staining for Huntingtin interacting protein 1 (Hip1) in the kidney shows a positive reactivity exclusively in the glomerulus. (b, c) Double labeling with nephrin shows only rare (arrow) overlapping. Hip1 is observed at the urinary side of nephrin. (d) Double labeling with vimentin shows overlapping immunoreactivity (yellow) in major processes. (e) Staining for neurofascin (Nfasc) gives an intense positive signal exclusively in the glomerulus. (f, g) Double labeling with nephrin does not show any overlapping immunoreactivity, because Nfasc staining is found at the urinary side of nephrin. (h) Double labeling with vimentin shows overlapping reactivity (yellow) in major processes. (i) Staining for olfactomedin-like 2a (Olfml2a) in the kidney shows specific reactivity in the glomerulus. (j, k) Double labeling with nephrin does not show any overlapping immunoreactivity. Staining for Olfml2a is located at the urinary side of nephrin. (l) Double labeling with vimentin shows overlapping reactivity (yellow) in major processes. CS, capillary space; US, urinary space. Original magnifications: (a, b, e, f, i, j) × 400; (c, d, g, h, k, l) × 2000.View Large Image Figure ViewerDownload (PPT)Figure 4The subcellular distribution of novel podocyte proteins in the glomerulus by immunoelectron microscopy. (a) Immunogold particles (arrowheads) for Huntingtin interacting protein 1 (Hip1) are found mostly in the major processes of podocytes. Occasionally, label is found in foot processes. (b) Immunogold particles (arrowheads) for olfactomedin-like 2a (Olfm12a) were almost exclusively found in major processes. Bars=500nm. FP, foot processes; GBM, glomerular basement membrane; MP, major processes.View Large Image Figure ViewerDownload (PPT) To confirm the association of Hip1 with major processes, we investigated the expression of Hip1 during podocyte development. The two earliest stages of development, vesicle and S-shaped stages, showed no Hip1 expression (data not shown). In the capillary stage glomerulus, a stage wherein the formation of major processes begins, immunoreactivity for Hip1 was first detected (Figure 5a and b). At this stage, Hip1 colocalized with vimentin on the basal aspect of immature podocytes. In maturing-stage glomerulus, staining for Hip1 colocalized with vimentin both on the basal aspects and at the apical membrane of developing podocytes (Figure 5c). Taken together, Hip1 colocalized with major process protein vimentin through glomerulogenesis, which supports the idea that Hip1 associates with these cellular projections. In RT–PCR, Nfasc was detected in the glomerulus, whereas no fragment was observed in the rest of the kidney lacking glomeruli (Figure 1). In western blot analysis, a band of the expected size (∼185kDa, see ref. 8.Dijkman H.B. Weening J.J. Smeets B. et al.Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells.Kidney Int. 2006; 70: 338-344Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) in the glomerular fraction was detected (Figure 1). Consistent with RT–PCR results, no band was visible in the sample obtained from the rest of the kidney. Immunofluorescence was performed using two different Nfasc antibodies, and both showed similar results (data not shown). In adult human kidney, strong staining for Nfasc was seen in the glomerulus, whereas no immunoreactivity was observed outside of glomeruli (Figures 2b and 3e). In the glomerulus, Nfasc was detected at the urinary side of nephrin staining, and no overlapping reactivity was observed for these proteins (Figure 3f and g). Instead, Nfasc appeared to localize to the major processes, as it showed overlapping reactivity with vimentin (Figure 3h). In immunoelectron microscopy experiments, no reliable signal was observed for Nfasc. During glomerulogenesis, vesicle and S-shaped-stage glomeruli (the earliest developmental stages of the glomerulus) showed no Nfasc immunoreactivity (data not shown). The signal for Nfasc was first observed at the capillary-stage glomerulus, where the staining colocalized with the major process marker vimentin (Figure 5d and e). In maturing glomeruli, Nfasc expression was detected both on the basal aspects and at the apical membrane of developing podocytes, again, colocalizing with vimentin (Figure 5f). The similar distribution of Nfasc with vimentin throughout podocyte maturation supports the idea that Nfasc associates with major processes. RT–PCR analysis showed the expression of Olfml2a only in the glomerular fraction in comparison with the rest of the kidney lacking glomeruli (Figure 1). In the western blot analysis, a band of ∼110kDa was detected in the glomerular fraction. No band was observed in the rest of the kidney fraction (Figure 1). Immunofluorescence on adult kidney samples showed intense staining in the glomerulus, whereas no immunoreactivity was seen outside of the glomeruli (Figures 2c and 3i). Double labeling with nephrin did not demonstrate colocalization (Figure 3j and k), as the Olfml2a signal was found on the side of the urinary space from nephrin. Double staining with vimentin revealed overlapping reactivity (Figure 3l), indicating the presence of Olfml2a in major processes. To confirm the subcellular location, immunoelectron microscopy experiments were performed. This demonstrated that the label for Olfml2a was mostly observed in major processes. The labeling frequency was generally rather weak, but from a total of 27 gold labels observed in glomeruli 23 (85%) were found in major processes (Figure 4b). Thus, Olfml2a was exclusively expressed by podocytes in the kidney, wherein it localized to major processes. During glomerulogenesis, the vesicle-stage glomerulus did not show any immunoreactivity for Olfml2a (data not shown). Surprisingly, at the S-shaped stage, Olfml2a presented positive signal in the invading endothelial cells located in the glomerular cleft (data not shown). In later stages, more mature endothelial cells did not express Olfml2a (Figure 5g–i). Instead, at the capillary stage, a clear signal for Olfml2a was observed at the basal aspects of developing podocytes (Figure 5g and h), wherein Olfml2a colocalized with vimentin. Similarly, at the maturing-stage glomerulus, Olfml2a colocalized with vimentin on the basal aspects and at the apical surface of developing podocytes (Figure 5h). Thus, Olfml2a colocalized with the major process protein vimentin throughout podocyte development, which strongly supports the idea that this protein associates with major processes. The presence or absence of podocytes in glomerular crescents has been a controversial issue.9.Nitta K. Horita S. Honda K. et al.Glomerular expression of cell-cycle-regulatory proteins in human crescentic glomerulonephritis.Virchows Arch. 1999; 435: 422-427Crossref PubMed Scopus (46) Google Scholar,10.Singh S.K. Jeansson M. Quaggin S.E. New insights into the pathogenesis of cellular crescents.Curr Opin Nephrol Hypertens. 2011; 20: 258-262Crossref PubMed Scopus (30) Google Scholar We used our novel podocyte markers to clarify this interesting question. Immunoreactivity for Hip1 was observed in 14 of the 26 crescents identified in our biopsy material (Figure 6a and b). In line with this, Olfml2a staining was clearly detected in 21 of the 31 glomerular crescents identified (Figure 6c and d). All glomerular crescents positive for Hip1 and Olfml2a expression were cellular crescents, and less than half of the cells in these lesions showed positive immunoreactivity (Figure 6a–d, data not shown). Anti-Nfasc antibodies did not give a reliable signal in the paraffin-embedded material (data not shown). To validate our results, we analyzed the expression of synaptopodin and Pdlim2 in glomerular crescents. These proteins are molecular components of podocyte foot processes, and, importantly, in the kidney, they are expressed only by podocytes.2.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1202) Google Scholar,11.Sistani L. Duner F. Udumala S. et al.Pdlim2 is a novel actin-regulating protein of podocyte foot processes.Kidney Int. 2011; 80: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Along with an established podocyte marker synaptopodin, we chose Pdlim2 as a marker, because during podocyte development it is expressed earlier than other foot process markers, such as nephrin and synaptopodin (data not shown). Here, immunostaining with anti-synaptopodin antibody demonstrated a positive signal in 25 out of 40 glomerular crescents identified (Figure 6e and f). Similarly, anti-Pdlim2 antibody demonstrated a positive staining in 30 of the 43 glomerular crescents identified (Figure 6g and h). All these crescents were classified as cellular crescents. Taken together, cells in glomerular crescents showed positivity for four podocyte markers. Finally, to exclude that the cells showing positivity for podocyte markers would originate from parietal epithelial cells, we stained consecutive sections for cytokeratin 8 (CK8), a marker for activated parietal epithelial cells.8.Dijkman H.B. Weening J.J. Smeets B. et al.Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells.Kidney Int. 2006; 70: 338-344Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Staining for CK8 was detected in glomerular crescents, but these cells remained negative for synaptopodin (Supplementary Figure S1 online). In contrast, synaptopodin-positive cells in glomerular crescents did not show CK8 staining. Download .jpg (.27 MB) Help with files Supplementary Figure 1 Mutations in podocyte-enriched genes are the cause of inherited forms of proteinuria.1.Tryggvason K. Patrakka J. Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria.N Engl J Med. 2006; 354: 1387-1401Crossref PubMed Scopus (444) Google Scholar These genes show restricted expression profiles outside of the podocyte, which reflects the unique structure and function of podocytes in the filtration barrier. In this study, we have identified three novel highly podocyte-associated proteins: Hip1, Nfasc, and Olfml2a. In the kidney, these proteins are expressed exclusively in podocytes, wherein they localize to major processes. This is intriguing, as the molecular composition of these peculiar cytoplasmic extensions is poorly known. The restricted expression in major processes suggests that these proteins have a specialized functional role in these cellular processes. Two of the proteins, Hip1 and Nfasc, have been previously identified in neurons. Thus, these proteins can be added to a long list of proteins shared by podocytes and neuronal cells. Recently, Brunskill et al.12.Tait S. Gunn-Moore F. Collinson J.M. et al.An oligodendrocyte cell adhesion molecule at the site of assembly of the paranodal axo-glial junction.J Cell Biol. 2000; 150: 657-666Crossref PubMed Scopus (266) Google Scholar characterized podocyte transcriptome through microarray profiling and could similarly confirm the neuronal-like molecular signature of podocytes. This is not surprising as podocytes and neurons share many structural features. Hip1 has previously been studied mostly in the brain. It was first described via its interaction with the Huntington's disease–causing protein, Huntingtin.13.Brunskill E.W. Georgas K. Rumballe B. et al.Defining the molecular character of the developing and adult kidney podocyte.PloS One. 2011; 6: 1-12Crossref Scopus (103) Google Scholar Hip1 is known to be an endocytic adaptor protein and has a role in clathrin-mediated vesicle trafficking.14.Kalchman M.A. Koide H.B. McCutcheon K. et al.HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain.Nat Genet. 1997; 16: 44-53Crossref PubMed Scopus (320) Google Scholar,15.Legendre-Guillemin V. Wasiak S. Hussain N.K. et al.ENTH/ANTH proteins and clathrin-mediated membrane budding.J Cell Sci. 2004; 117: 9-18Crossref PubMed Scopus (167) Google Scholar It is present in a complex with glutamate receptors, and studies in knockout animals have shown that Hip1 facilitates glutamate signaling in neurons.16.Metzler M. Legendre-Guillemin V. Gan L. et al.HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2.J Biol Chem. 2001; 276: 39271-39276Crossref PubMed Scopus (153) Google Scholar In this study, we identified Hip1 expression in podocyte major processes. As glutamate signaling has been reported to have a crucial role in podocytes,17.Metzler M. Li B. Gan L. et al.Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking.EMBO J. 2003; 22: 3254-3266Crossref PubMed Scopus (100) Google Scholar it is possible that Hip1 promotes glutamate signaling in podocytes. In this respect, the localization of Hip1 to major processes is somewhat surprising, because in podocytes the slit diaphragm is thought to be involved in glutamate signaling. During podocyte development, we observed Hip1 expression as early as the capillary-stage glomerulus. This is in contrast to the expression of other glutamate signaling proteins, which are not expressed in immature podocytes.17.Metzler M. Li B. Gan L. et al.Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking.EMBO J. 2003; 22: 3254-3266Crossref PubMed Scopus (100) Google Scholar This, together with the subcellular localization, suggests that Hip1 may have another role than promoting glutamate signaling in podocytes. Nfasc is a member of the L1 cell adhesion transmembrane molecules.18.Giardino L. Armelloni S. Corbelli A. et al.Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.J Am Soc Nephrol. 2009; 20: 1929-1940Crossref PubMed Scopus (69) Google Scholar The characteristic of L1 cell adhesion transmembrane molecules is an extracellular domain composed of repetitive immunoglobulin-like and fibronectin-like domains. The cytoplasmic domain binds ankyrin and a member of ezrin–radixin–moesin proteins, and through these, L1 cell adhesion transmembrane molecules are linked to the actin cytoskeleton.18.Giardino L. Armelloni S. Corbelli A. et al.Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.J Am Soc Nephrol. 2009; 20: 1929-1940Crossref PubMed Scopus (69) Google Scholar In addition, the cytoplasmic tail of Nfasc binds doublecortin, a microtubule-binding/stabilizing protein.19.Herron L.R. Hill M. Davey F. et al.The intracellular interactions of the L1 family of cell adhesion molecules.Biochem J. 2009; 419: 519-531Crossref PubMed Scopus (43) Google Scholar This interaction may have an important role in microtubular dynamics, because a mutation in the drosophila ortholog of Nfasc impairs the assembly of microtubules in neurons.20.Kizhatil K. Wu Y.X. Sen A. et al.A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin.J Neurosci. 2002; 22: 7948-7958Crossref PubMed Google Scholar In line with this, Nfasc knockout mice die soon after birth and display severe defects in the formation of axons.21.Godenschwege T.A. Kristiansen L.V. Uthaman S.B. et al.A conserved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.Curr Biol. 2006; 16: 12-23Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Importantly, Nfasc has previously been thought to be specific for the neuronal tissue, where three major isoforms, NF155, NF166, and NF186 (named after the sizes detected in sodium dodecyl sulfate–page gel), have been identified.21.Godenschwege T.A. Kristiansen L.V. Uthaman S.B. et al.A conserved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.Curr Biol. 2006; 16: 12-23Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar In this report, we have identified Nfasc as a novel component of major processes in podocytes. In western blotting, the size of glomerular Nfasc is ∼185kD, which probably represents the NF186 isoform. The restricted expression of Nfasc in podocyte major processes is likely linked to the presence of an extensive microtubular system in these cellular processes. It is possible that Nfasc has a role in the microtubular dynamics of major processes, similar to that seen in drosophila.20.Kizhatil K. Wu Y.X. Sen A. et al.A new activity of doublecortin in recognition of the phospho-FIGQY tyrosine in the cytoplasmic domain of neurofascin.J Neurosci. 2002; 22: 7948-7958Crossref PubMed Google Scholar In fact, we have identified Nfasc-binding protein doublecortin in major processes (L Sistani et al., unpublished data), which could link Nfasc to microtubules. At present, we are generating a podocyte-specific knockout mouse line for Nfasc to elucidate the functional role of this protein in the kidney. Olfml2a is a member of olfactomedin domain–containing proteins. On the basis of its amino-acid sequence, Olfml2a is predicted to be a secreted protein associating with the plasma membrane. The function and expression pattern of Olfml2a is unknown. It is noteworthy that the olfactomedin domain of gliomedin, a protein that is found exclusively in the nodes of Ranvier, binds to neurofascin, and this interaction stabilizes these structures.22.Sherman D.L. Tait S. Melrose S. et al.Neurofascins are required to establish axonal domains for saltatory conduction.Neuron. 2005; 48: 737-742Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar As Olfml2a contains one olfactomedin domain, it is possible that a similar interaction exists in podocytes. More so, this view draws a link to the fact that both Olfml2a and neurofascin are found only in major processes. Thus, Olfm2a, neurofascin, and doublecortin may serve as an important protein complex in major processes, stabilizing microtubular structures. Unfortunately, similar to neurofascin, Olfml2a is not expressed by immortalized podocyte cells, which makes it difficult to study the function of these proteins in cell culture. Crescentic lesions are often observed in inflammatory diseases affecting the glomerulus, and generally they are considered as a histologic sign of severe injury.10.Singh S.K. Jeansson M. Quaggin S.E. New insights into the pathogenesis of cellular crescents.Curr Opin Nephrol Hypertens. 2011; 20: 258-262Crossref PubMed Scopus (30) Google Scholar The glomerular crescents are recognized as a heterogeneous composition of cells and matrix, including leukocytes, renal cells, and fibrin—all found between the capillary tuft and Bowman's capsule. However, the cellular composition of crescents is a controversial subject, mainly because the presence and the role of podocytes in the formation of these lesions has remained unclear.10.Singh S.K. Jeansson M. Quaggin S.E. New insights into the pathogenesis of cellular crescents.Curr Opin Nephrol Hypertens. 2011; 20: 258-262Crossref PubMed Scopus (30) Google Scholar In the past, podocytes have not been regarded to take part in the formation of the glomerular crescent. This idea has been supported by studies that do not show any expression of podocyte markers in glomerular crescents.9.Nitta K. Horita S. Honda K. et al.Glomerular expression of cell-cycle-regulatory proteins in human crescentic glomerulonephritis.Virchows Arch. 1999; 435: 422-427Crossref PubMed Scopus (46) Google Scholar, 23.Eshed Y. Feinberg K. Poliak S. et al.Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.Neuron. 2005; 47: 215-229Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 24.Usui J. Kanemoto K. Tomari S. et al.Glomerular crescents predominantly express cadherin-catenin complex in pauci-immune-type crescentic glomerulonephritis.Histopathology. 2003; 43: 173-179Crossref PubMed Scopus (14) Google Scholar However, data reporting the presence of podocytes in these structures have emerged.25.Patrakka J. Ruotsalainen V. Ketola I. et al.Expression of nephrin in pediatric kidney diseases.J Am Soc Nephrol. 2001; 12: 289-296PubMed Google Scholar,26.Thorner P.S. Ho M. Eremina V. et al.Podocytes contribute to the formation of glomerular crescents.Journal Am Soc Nephrol. 2008; 19: 495-502Crossref PubMed Scopus (107) Google Scholar In particular, the data gained from a mouse model in which podocytes have been genetically tagged suggest that podocytes are present in glomerular crescents.26.Thorner P.S. Ho M. Eremina V. et al.Podocytes contribute to the formation of glomerular crescents.Journal Am Soc Nephrol. 2008; 19: 495-502Crossref PubMed Scopus (107) Google Scholar In this study, our data highlight the presence of podocytes in the glomerular crescents of human patients. In the renal biopsies examined, Hip1 and Olfml2a (identified as major processes markers), as well as pdlim2 and synaptopodin (foot processes markers), were clearly detected in the cellular components of glomerular crescents. The presence of these markers in these lesions suggests the participation of podocytes in the pathological cascade of the glomerular crescent formation. It is noteworthy that we also detected another subpopulation of cells in crescents that were positive for CK8, a marker for activated parietal epithelial cells, supporting the idea that these cells also contribute to the formation of crescents. The conflicting data reported on the presence of podocytes and the lack of expression of various podocyte markers in the glomerular crescents is an interesting question. One reason for the contradictory results is that podocytes dedifferentiate or transdifferentiate and lose their podocyte-specific markers in parallel with the progression of disease. Consequently, the antigens are not detected by the traditional immunohistochemistry approach in human material. This explains the fact that only some crescents were positive for podocyte markers in our study. These positive crescents were all fresh crescents, which are likely to contain podocyte cells that have maintained their differentiated phenotype. In contrast to this, the cells in more chronic lesions were negative for podocyte markers, which probably reflects the progressive dedifferentiation of podocytes. In summary, we have identified three novel podocyte proteins that are, in the kidney, highly specific to podocyte major processes. Given their specificity, these proteins are likely to have a dedicated role in the biology of major processes. Furthermore, by using new podocyte markers, we show that podocytes are present in glomerular crescents. Our findings provide novel insights into the molecular composition of major processes and show that podocytes contribute to the formation of glomerular crescents. The expression of transcripts was analyzed in the mouse glomerulus and the kidney fraction excluding the glomerulus with RT–PCR. Glomeruli were isolated using a standard perfusion method. Primer sequences were as follows: Hip1, left 5′-GCTTCATGGAGCAGTTCACA-3′, right 5′-GTCCTTGTTCACGCCATTTT-3′; Nfasc, left 5′-GAGGCCACTTTTCACAGAGC-3′, right 5′-ATAGGCAAACCAGGACAACG-3′; Olfml2a, left 5′-CTGCTGCCTGATGTGGTCTA-3′, right 5′-GTGAGCATCAGTGCCAGTGT-3′. Podocin and glyceraldehyde 3–phosphate dehydrogenase primers were used in control reactions. Polyclonal antibodies were generated by immunizing rabbits with human recombinant proteins (residues: Hip1 663–772, Olfml2a 74–164). The protein fragments were expressed and purified as described.27.Moeller M.J. Soofi A. Hartmann I. et al.Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol. 2004; 15: 61-67Crossref PubMed Scopus (157) Google Scholar Nfasc antibodies were purchased from Sigma (St Louis, MO) and Abcam (Cambridge, UK). Lysates of glomeruli and the kidneys lacking glomeruli were obtained from cadaver kidneys unsuitable for transplantation because of vascular abnormalities (from the fourth Department of Surgery of Helsinki, Finland). Glomeruli were isolated using a standard sieving method. Western blotting experiments were performed according to standard procedures. Anti-β-actin antibody (Abcam) served as a loading control. Adult human kidney samples were collected from cadaver kidneys (see above). Samples from a developing kidney were obtained from an embryo aborted at week 20 of pregnancy owing to neural tube defects and hydrocephalus. The use of human material was accepted by the local ethics committee. Immunofluorescence stainings were performed as described.28.Patrakka J. Xiao Z. Nukui M. et al.Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik.J Am Soc Nephrol. 2007; 18: 689-697Crossref PubMed Scopus (65) Google Scholar,29.Wernerson A. Duner F. Pettersson E. et al.Altered ultrastructural distribution of nephrin in minimal change nephrotic syndrome.Nephrol Dial Transplant. 2003; 18: 70-76Crossref PubMed Scopus (89) Google Scholar Double-labeling experiments were performed using vimentin (Zymed Laboratories, San Francisco, CA), synaptopodin (Progen, Heidelberg, Germany), and nephrin 50A9 (ref. 3.Welsh G.I. Saleem M.A. Nephrin-signature molecule of the glomerular podocyte?.J Pathol. 2010; 220: 328-337PubMed Google Scholar) antibodies. Immunoelectron microscopy experiments were performed as described.28.Patrakka J. Xiao Z. Nukui M. et al.Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik.J Am Soc Nephrol. 2007; 18: 689-697Crossref PubMed Scopus (65) Google Scholar Immunoperoxidase staining was performed using standard procedures on renal biopsies from patients with crescentic glomerulonephritis. The underlying diagnosis in all cases was ANCA-positive vasculitis. Additional antibodies used in immunoperoxidase stainings were as follows: pdlim2 (described previously in ref. 11.Sistani L. Duner F. Udumala S. et al.Pdlim2 is a novel actin-regulating protein of podocyte foot processes.Kidney Int. 2011; 80: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), synaptopodin (Progen), and CK8 antibody (Sigma). We are thankful to Ms Anneli Hansson for skillful technical assistance. Figure S1. Expression of cytokeratin 8 (CK8) and synaptopodin in crescentic glomerulonephritis. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .doc (.03 MB) Help with doc files Supplementary Figure Legend
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