Neuronal Proteins Are Novel Components of Podocyte Major Processes and Their Expression in Glomerular Crescents Supports Their R

http://www.kidney-international.org basic research & 2012 International Society of Nephrology

Neuronal proteins are novel components of podocyte major processes and their expression in glomerular crescents supports their role in crescent formation Laleh Sistani1, Patricia Q. Rodriguez1, Kjell Hultenby2, Mathias Uhlen3, Christer Betsholtz4, Hannu Jalanko5, Karl Tryggvason1, Annika Wernerson6 and Jaakko Patrakka1

1Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; 2Division of Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; 3Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; 4Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; 5Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland and 6Division of Renal Medicine, Department of Clinical Science, Technology and Intervention, Karolinska Institutet, Stockholm, Sweden

The podocyte has a central role in the glomerular filtration The glomerulus comprises a tangled network of capillaries barrier typified by a sophisticated morphology of highly surrounded by a urinary space and Bowman’s capsule. organized primary (major) and secondary (foot) processes. The filtration of primary urine occurs through the capillary The molecular makeup of foot processes is well wall of the glomerulus. This filtration barrier is essentially characterized, but that of major processes is poorly known. composed of three layers: (1) the glomerular basement Previously, we profiled the glomerular transcriptome membrane, which serves as a foundation and support; through large-scale sequencing and microarray profiling. (2) fenestrated endothelial cells, located on the inside; and Unexpectedly, the survey found expression of three neuronal (3) visceral epithelial cells (podocytes), located on the outside proteins (Huntingtin interacting protein 1 (Hip1), neurofascin of the glomerular capillary.1 (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in The podocyte is a highly specialized epithelial cell with a the glomerulus. These proteins were expressed exclusively by remarkably sophisticated architecture.2 It has a prominent podocytes, wherein they localized to major processes as cell body with large cytoplasmic projections named major verified by RT–PCR, western blotting, immunofluorescence, processes, which further extend into smaller processes known and immunoelectron microscopy. During podocyte as foot processes. The foot processes interdigitate as they development, these proteins colocalized with vimentin, wrap around and envelope the capillaries. The adjacent foot confirming their association with major processes. Using processes interact and are connected by a specialized cell–cell immunohistochemistry, we found coexpression of Hip1 junction termed the slit diaphragm.2–4 Mutations in slit and Olfml2a along with the recognized podocyte markers diaphragm proteins cause inherited forms of proteinuria,1 synaptopodin and Pdlim2 in glomerular crescents of human which underlines the importance of these structures in the kidneys, indicating the presence of podocytes in these filtration barrier. lesions. Thus, three neuronal proteins are highly expressed in Podocytes are dynamic cells with a prominent cyto- podocyte major process. Using these new markers we found skeleton that can effectively respond to changes in their that podocytes contribute to the formation of glomerular extracellular environment.5 This cytoskeletal machinery is crescents. vital for a sustained kidney filtration, as defects in Kidney International (2012) 83, 63–71; doi:10.1038/ki.2012.321; cytoskeleton-regulating proteins of podocyte foot processes published online 22 August 2012 result in proteinuria.5 Bundles of microtubules and inter- KEYWORDS: cytoskeleton; glomerulopathy; podocyte mediate filaments support major processes, whereas the cytoskeleton of foot processes is composed mainly of actin filaments.2,5,6 This feature confirms an obvious morphologi- cal difference between these two structures, thus contributing to their molecular and functional divergence. The molecular composition of foot process is rather well characterized.4 This Correspondence: Jaakko Patrakka, Division of Pathology, Karolinska includes basal, slit diaphragm, and apical plasma membrane University Hospital Huddinge, 14186 Stockholm, Sweden. protein complexes connected by a number of linker proteins 4,5 E-mail: [email protected] and actin cytoskeleton. In contrast to this, the mole- Received 5 January 2012; revised 20 June 2012; accepted 19 July 2012; cular nature of major processes is still poorly understood. published online 22 August 2012 Besides microtubules and the intermediate filament protein

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vimentin,2,6 the components that form these cellular projec- RT–PCR WB tions are largely unknown. We have previously identified several highly glomerulus- enriched transcripts using large-scale sequencing and microarray profiling.7 In this study, we are characterizing further three of these glomerular transcripts. All three proteins in the Glomerulus Rest of kidney Glomerulus Rest of kidney kidney are expressed only by podocytes, wherein they localize to major processes. The specific expression in podocyte major Hip1 350 bp 115 kDa 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 Nfasc 350 bp 185 kDa has been a matter of controversy.

RESULTS 350 bp Previously, we have profiled the glomerular transcriptome Olfml2a 110 kDa through large-scale sequencing and microarray profiling.7 In this approach, we chose three transcripts, Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a), which were enriched in the Podocin/Nephrin 600 bp 185 kDa 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. β GAPDH/ -actin 600 bp 40 kDa Expression of Hip1 in mature and developing kidney First, we analyzed the expression of Hip1 in the kidney Figure 1 | Expression of Huntingtin interacting protein 1 through reverse transcriptase–PCR (RT–PCR). The PCR (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a product for Hip1 was amplified in glomeruli, whereas no (Olfml2a) in the kidney as analyzed with reverse expression was detected in the rest of the kidney lacking transcriptase–PCR (RT–PCR) and western blotting (WB). We compared the expression in the glomerular fraction with the glomeruli (Figure 1). The control experiment with glycer- rest of the kidney depleted of glomeruli. The transcripts are all aldehyde 3–phosphate dehydrogenase showed strong signal in amplified in the glomeruli, whereas no (or very weak) expression both lanes, whereas podocin, a known glomerulus-specific is detected in the rest of the kidney. The control reaction with transcript, gave significantly stronger signal in the glomerular podocin, a known glomerulus-specific transcript, is detected in the glomerular fraction, whereas glyceraldehyde 3–phosphate fraction. Next, we generated a polyclonal antibody against the dehydrogenase (GAPDH) is detected in both the glomerular human Hip1 protein. By using western blotting of human fractions and the rest of the kidney fractions. In WB, the kidney lysates, our anti-Hip1 antibody detected a band of antibodies directed against Hip1, Nfasc, and Olfml2a show B immunoreactivity in the glomerular fraction, whereas no expected size ( 115–kDa) in the glomerulus as opposed to (or very weak) band is visible in the rest of the kidney lysate. the rest of the kidney, which appeared completely negative The sizes of proteins detected are as follows: Hip1 B115 kDa, (Figure 1). A similar expression pattern was observed for Nfasc B185 kDa, and Olfml2a B110 kDa. The control reaction nephrin, a known podocyte-specific protein. The control with nephrin antibody recognizes the protein with the expected size (B185 kDa) in the glomerular fraction. b-Actin antibody, used experiment with b-actin antibody showed clear signal in both as a loading control, recognizes a protein B40 kDa in both lanes. 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 glomeruli, of which 96 (55%) were located in major with foot process marker nephrin showed that Hip1 was processes. In addition, few labels were occasionally detected located at the side of the urinary space of nephrin (Figure 3b in foot processes (Figure 4a). No significant signal for Hip1 and c). This indicated expression in podocytes. Occasionally, was found outside podocytes. some overlapping reactivity with nephrin was observed To confirm the association of Hip1 with major processes, (arrow, Figure 3c). Double labeling with vimentin showed we investigated the expression of Hip1 during podocyte that Hip1 colocalized with this major process marker development. The two earliest stages of development, vesicle (Figure 3d). Immunoelectron microscopy was used to and S-shaped stages, showed no Hip1 expression (data not confirm the subcellular localization of Hip1. Gold label for shown). In the capillary stage glomerulus, a stage wherein Hip1 was observed mostly in major processes (Figure 4a). the formation of major processes begins, immunoreactivity Quantitatively, we detected a total of 176 gold labels in for Hip1 was first detected (Figure 5a and b). At this stage,

64 Kidney International (2012) 83, 63–71 L Sistani et al.: Hip1, Nfasc, and Olfml2a in podocytes and glomerular crescents basic research

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.

Expression of Nfasc in mature and developing kidney 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 (B185 kDa, see ref. 8) in the glomerular fraction was detected (Figure 1). Consistent with RT–PCR Hip1 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 Nfasc 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.

Expression of Olfml2a in mature and developing kidney 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 B110 kDa was detected in the glomerular fraction. No band was observed in the rest of the kidney fraction (Figure 1). Olfml2a Immunoﬂuorescence on adult kidney samples showed intense staining in the glomerulus, whereas no immuno- Figure 2 | Expression of novel podocyte proteins, Huntingtin reactivity was seen outside of the glomeruli (Figures 2c interacting protein 1 (Hip1), neurofascin (Nfasc), and and 3i). Double labeling with nephrin did not demonstrate olfactomedin-like 2a (Olfml2a), in the kidney as seen with colocalization (Figure 3j and k), as the Olfml2a signal was immunofluorescence staining. (a–c) All antibodies show intense signal in the glomerular tufts of mature human kidney, whereas found on the side of the urinary space from nephrin. no significant signal is detected outside of the glomeruli. Double staining with vimentin revealed overlapping reacti- (a–c) Original magnification 80. vity (Figure 3l), indicating the presence of Olfml2a in major

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Hip1 Nfasc Olfml2a Nephrin

US US

US CS CS CS Nephrin Vimentin

Figure 3 | Expression 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. processes. To conﬁrm the subcellular location, immuno- (Figure 4b). Thus, Olfml2a was exclusively expressed by electron microscopy experiments were performed. This podocytes in the kidney, wherein it localized to major demonstrated that the label for Olfml2a was mostly observed processes. in major processes. The labeling frequency was generally During glomerulogenesis, the vesicle-stage glomerulus rather weak, but from a total of 27 gold labels observed did not show any immunoreactivity for Olfml2a (data in glomeruli 23 (85%) were found in major processes not shown). Surprisingly, at the S-shaped stage, Olfml2a

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synaptopodin antibody demonstrated a positive signal in MP 25 out of 40 glomerular crescents identified (Figure 6e and f). MP 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 FP FP crescents showed positivity for four podocyte markers.

GBM GBM Finally, to exclude that the cells showing positivity for Hip1 Olfml2a podocyte markers would originate from parietal epithelial cells, we stained consecutive sections for cytokeratin 8 (CK8), Figure 4 | The subcellular distribution of novel podocyte a marker for activated parietal epithelial cells.8 Staining for CK8 proteins in the glomerulus by immunoelectron microscopy. was detected in glomerular crescents, but these cells remained (a) Immunogold particles (arrowheads) for Huntingtin interacting protein 1 (Hip1) are found mostly in the major processes of negative for synaptopodin (Supplementary Figure S1 online). podocytes. Occasionally, label is found in foot processes. In contrast, synaptopodin-positive cells in glomerular cres- (b) Immunogold particles (arrowheads) for olfactomedin-like 2a cents did not show CK8 staining. (Olfm12a) were almost exclusively found in major processes. Bars ¼ 500 nm. FP, foot processes; GBM, glomerular basement membrane; MP, major processes. DISCUSSION Mutations in podocyte-enriched genes are the cause of inherited forms of proteinuria.1 These genes show restricted presented positive signal in the invading endothelial cells expression profiles outside of the podocyte, which reflects the located in the glomerular cleft (data not shown). In later unique structure and function of podocytes in the filtration stages, more mature endothelial cells did not express Olfml2a barrier. In this study, we have identified three novel highly (Figure 5g–i). Instead, at the capillary stage, a clear signal for podocyte-associated proteins: Hip1, Nfasc, and Olfml2a. In Olfml2a was observed at the basal aspects of developing the kidney, these proteins are expressed exclusively in podocytes (Figure 5g and h), wherein Olfml2a colocalized podocytes, wherein they localize to major processes. This is with vimentin. Similarly, at the maturing-stage glomerulus, intriguing, as the molecular composition of these peculiar Olfml2a colocalized with vimentin on the basal aspects and cytoplasmic extensions is poorly known. The restricted at the apical surface of developing podocytes (Figure 5h). expression in major processes suggests that these proteins Thus, Olfml2a colocalized with the major process protein have a specialized functional role in these cellular processes. vimentin throughout podocyte development, which strongly Two of the proteins, Hip1 and Nfasc, have been previously supports the idea that this protein associates with major identified in neurons. Thus, these proteins can be added to a processes. long list of proteins shared by podocytes and neuronal cells. Recently, Brunskill et al.12 characterized podocyte transcrip- Expression of novel podocyte markers in glomerular tome through microarray profiling and could similarly crescents confirm the neuronal-like molecular signature of podocytes. The presence or absence of podocytes in glomerular crescents This is not surprising as podocytes and neurons share many has been a controversial issue.9,10 We used our novel podo- structural features. cyte markers to clarify this interesting question. Immuno- Hip1 has previously been studied mostly in the brain. It reactivity for Hip1 was observed in 14 of the 26 crescents was first described via its interaction with the Huntington’s identified in our biopsy material (Figure 6a and b). In line disease–causing protein, Huntingtin.13 Hip1 is known to be with this, Olfml2a staining was clearly detected in 21 of an endocytic adaptor protein and has a role in clathrin- the 31 glomerular crescents identified (Figure 6c and d). All mediated vesicle trafficking.14,15 It is present in a complex glomerular crescents positive for Hip1 and Olfml2a expres- with glutamate receptors, and studies in knockout animals sion were cellular crescents, and less than half of the cells in have shown that Hip1 facilitates glutamate signaling in these lesions showed positive immunoreactivity (Figure 6a–d, neurons.16 In this study, we identified Hip1 expression in data not shown). Anti-Nfasc antibodies did not give a reliable podocyte major processes. As glutamate signaling has been signal in the paraffin-embedded material (data not shown). reported to have a crucial role in podocytes,17 it is possible To validate our results, we analyzed the expression that Hip1 promotes glutamate signaling in podocytes. In this of synaptopodin and Pdlim2 in glomerular crescents. respect, the localization of Hip1 to major processes is These proteins are molecular components of podocyte foot somewhat surprising, because in podocytes the slit dia- processes, and, importantly, in the kidney, they are expressed phragm is thought to be involved in glutamate signaling. only by podocytes.2,11 Along with an established podocyte During podocyte development, we observed Hip1 expression marker synaptopodin, we chose Pdlim2 as a marker, because as early as the capillary-stage glomerulus. This is in contrast during podocyte development it is expressed earlier than to the expression of other glutamate signaling proteins, other foot process markers, such as nephrin and synapto- which are not expressed in immature podocytes.17 This, podin (data not shown). Here, immunostaining with anti- together with the subcellular localization, suggests that Hip1

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Capillary Capillary Maturing Vimentin Vimentin

bs Hip1

bs Nfasc

bs Olfml2a

Figure 5 | Expression of novel podocyte proteins in human glomerulogenesis as shown with immunofluorescence staining. The sections were stained for Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a; green) for major process marker vimentin (red) and nuclear marker 4,6-diamidino-2-phenylindole (DAPI; blue). (a, b) Hip1 expression is first detected during the capillary stage. At this stage, Hip1 is localized at the basal aspects of developing podocytes, wherein it colocalizes (yellow) with vimentin. (c) In maturing-stage glomeruli, Hip1 colocalizes with vimentin at the basal aspects and the apical membrane of developing podocytes. (d, e) Nfasc expression is first detected at the capillary stage, wherein Nfasc is seen at the basal aspects of immature podocytes. Vimentin colocalizes with Nfasc at this stage. (f) In maturing-stage glomeruli, staining for Nfasc overlaps with that of vimentin at the basal aspects and the apical membrane of developing podocytes. (g, h) During podocyte development, Olfml2a is first detected at the capillary stage, wherein Olfml2a is observed at the basal aspects of immature podocyte. The staining for Olfml2a overlaps with that of vimentin. (i) In maturing glomeruli, Olfml2a expression is detected at the basal aspects and the apical surface of podocytes, and it colocalizes with vimentin. (a–i) Original magnification 500. bs, Bowman’s space. may have another role than promoting glutamate signaling in in neurons.20 In line with this, Nfasc knockout mice die soon podocytes. after birth and display severe defects in the formation of Nfasc is a member of the L1 cell adhesion transmembrane axons.21 Importantly, Nfasc has previously been thought to molecules.18 The characteristic of L1 cell adhesion trans- be specific for the neuronal tissue, where three major membrane molecules is an extracellular domain composed isoforms, NF155, NF166, and NF186 (named after the sizes of repetitive immunoglobulin-like and fibronectin-like detected in sodium dodecyl sulfate–page gel), have been domains. The cytoplasmic domain binds ankyrin and a identified.21 In this report, we have identified Nfasc as a novel member of ezrin–radixin–moesin proteins, and through component of major processes in podocytes. In western these, L1 cell adhesion transmembrane molecules are linked blotting, the size of glomerular Nfasc is B185 kD, which to the actin cytoskeleton.18 In addition, the cytoplasmic tail probably represents the NF186 isoform. The restricted of Nfasc binds doublecortin, a microtubule-binding/stabiliz- expression of Nfasc in podocyte major processes is likely ing protein.19 This interaction may have an important role in linked to the presence of an extensive microtubular system in microtubular dynamics, because a mutation in the droso- these cellular processes. It is possible that Nfasc has a role in phila ortholog of Nfasc impairs the assembly of microtubules the microtubular dynamics of major processes, similar to that

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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 Hip1 Hip1 nodes of Ranvier, binds to neurofascin, and this interaction stabilizes these structures.22 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 Olfml2a Olfml2a 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 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 Pdlim2 Pdlim2 remained unclear.10 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.9,23,24 However, data reporting the presence of podocytes in these structures have emerged.25,26 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 In this study, our data Synaptopodin Synaptopodin highlight the presence of podocytes in the glomerular crescents of human patients. In the renal biopsies examined, Figure 6 | Expression of podocyte markers Huntingtin Hip1 and Olfml2a (identified as major processes markers), interacting protein 1 (Hip1), olfactomedin-like 2a (Olfml2a), Pdlim2, and synaptopodin in glomerular crescents using as well as pdlim2 and synaptopodin (foot processes markers), immunoperoxidase staining. (a, b) Staining for Hip1 is detected were clearly detected in the cellular components of in the cellular components of glomerular crescents (arrow). The glomerular crescents. The presence of these markers in these staining is observed diffusely in the cytoplasm. (c, d) Olfml2a immunoreactivity is detected in cells between the capillary tuft lesions suggests the participation of podocytes in the and Bowman’s capsule (arrow). These cells are part of the pathological cascade of the glomerular crescent formation. crescent. (e, f) Some cells in glomerular crescents show positivity It is noteworthy that we also detected another subpopulation for Pdllim2 (arrow). Staining is observed diffusely in the of cells in crescents that were positive for CK8, a marker for cytoplasm. (g, h) Staining for synaptopodin is detected in glomerular crescents (arrow). Original magnifications: activated parietal epithelial cells, supporting the idea that (a, c, e, g) 60; (b, d, f, h) 300. 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 seen in drosophila.20 In fact, we have identified Nfasc-bind- reason for the contradictory results is that podocytes ing protein doublecortin in major processes (L Sistani et al., dedifferentiate or transdifferentiate and lose their podocyte- unpublished data), which could link Nfasc to microtubules. specific markers in parallel with the progression of disease. At present, we are generating a podocyte-specific knockout Consequently, the antigens are not detected by the traditional

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immunohistochemistry approach in human material. This Immunoperoxidase staining explains the fact that only some crescents were positive Immunoperoxidase staining was performed using standard proce- for podocyte markers in our study. These positive crescents dures on renal biopsies from patients with crescentic glomerulo- were all fresh crescents, which are likely to contain podocyte nephritis. The underlying diagnosis in all cases was ANCA- cells that have maintained their differentiated phenotype. positive vasculitis. Additional antibodies used in immunoperoxidase stainings were as follows: pdlim2 (described previously in ref. 11), In contrast to this, the cells in more chronic lesions were synaptopodin (Progen), and CK8 antibody (Sigma). negative for podocyte markers, which probably reflects the progressive dedifferentiation of podocytes. In summary, we have identified three novel podocyte DISCLOSURE All the authors declared no competing interests. 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 ACKNOWLEDGMENTS processes. Furthermore, by using new podocyte markers, We are thankful to Ms Anneli Hansson for skillful technical assistance. we show that podocytes are present in glomerular crescents. Our findings provide novel insights into the molecular SUPPLEMENTARY MATERIAL composition of major processes and show that podocytes Figure S1. Expression of cytokeratin 8 (CK8) and synaptopodin in crescentic glomerulonephritis. contribute to the formation of glomerular crescents. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki MATERIALS AND METHODS RT–PCR REFERENCES The expression of transcripts was analyzed in the mouse glomerulus 1. 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