Glomerular Permeability Is Altered by Loss of P0, a Myelin Protein Expressed in Glomerular Epithelial Cells
Emmanuelle Plaisier*,
Béatrice Mougenot*,
Marie Christine Verpont*,
Chantal Jouanneau*,
Joan J. Archelos,
Rudolf Martini,
Dontscho Kerjaschki and
Pierre Ronco*
* Department of Nephrology, INSERM Unit 702, Tenon Hospital (AP-HP), University Pierre et Marie Curie, Paris, France; Department of Neurology, University of Graz, Graz, Austria; Department of Developmental Neurobiology, University of Würzburg, Würzburg, Germany; and Department of Clinical Pathology, University of Vienna, Vienna, Austria
Address correspondence to: Dr. Emmanuelle Plaisier, Department of Nephrology and INSERM Unit 702, Hôpital Tenon, 4 Rue de la Chine, Paris, 75020 France. Phone: +33-1-56-01-66-39; Fax: +33-1-56-01-69-99; emmanuelle.plaisier{at}tnn.ap-hop-paris.fr
Received for publication May 16, 2005.
Accepted for publication July 20, 2005.
The myelin protein 0 (MPZ or P0) is a transmembrane glycoproteinthat represents the most abundant myelin component. Mutationsin the P0 gene are associated with one form of autosomal dominantdemyelinating peripheral neuropathy, Charcot-Marie-Tooth diseasetype 1B (CMT1B). Because CMT1 may be associated with renal involvement,mostly focal segmental glomerulosclerosis, we hypothesized thatP0 could be expressed in the kidney. P0 mRNA was detected byreverse transcriptase–PCR in the human and mouse renalcortex. P0 transcripts were identified by in situ hybridizationat different stages of the mouse kidney development, especiallyin embryonic structures that give rise to the glomerulus. P0protein was also detected by Western blot in human and rat glomerularextracts and in a human podocyte cell line using a monoclonalanti-P0 antibody. Immunofluorescence studies on human kidneysections showed that the podocytes were intensely labeled. Immunogoldelectron microscopy disclosed a predominant staining of themembranes of intracellular vesicles in podocytes. P0 was alsodetected in the podocyte cell membrane, including at the footprocesses. P0–/– mice exhibited mild growth retardationand demyelinating neuropathy similar to the one observed inpatients with CMT1B. They also presented mild albuminuria, withoutsignificant ultrastructural change of the glomerular basementmembrane or the podocytes. These results demonstrate that P0,the major myelin protein, is also expressed during nephrogenesisand in mature kidney, mostly in podocytes. They suggest thatP0 gene mutations might be involved in renal diseases.
Glomerular epithelial cells, podocytes, are highly specializedcells that participate in the glomerular filtration barrierthrough the filtration slits bounded by the interdigitated footprocesses and the synthesis of glomerular basement membrane(GBM) components. In the past few years, genetic studies offamilial forms of nephrotic syndrome in human have expandedour knowledge on the cell biology of podocytes. They led tothe identification of proteins that are functionally importantin maintaining permselectivity of the glomerulus. These proteinsinclude nephrin and podocin located at the slit diaphragm andthe cytoskeleton-associated -actinin 4 protein (1–3).In vitro experimental studies and knockout mouse models havefurther identified additional podocyte proteins, interactingmostly with nephrin and podocin for the maintenance of the slitdiaphragm integrity. It is interesting that some of the podocyteproteins are electively coexpressed in the central nervous system.These include the cytoskeletal component synaptopodin, nephrin,densin, glomerular epithelial protein 1, the synaptic vesiclemolecule Rab3A and its effector Rabphilin-3a, and the neuronalamino acid transporters CAT3 and EAAT2 (4–9). However,none of these proteins has been found in the myelinated peripheralnervous system, although focal segmental glomerulosclerosis(FSGS) has been reported in the most common familial form ofdemyelinating neuropathy, Charcot-Marie-Tooth (CMT) disease(10–12).
In this study, we investigated the renal expression and roleof the myelin protein 0 (MPZ or P0). P0 is the most abundantprotein of the peripheral nervous system (PNS), produced bySchwann cells. P0 is a small, integral membrane glycoproteinthat has a single Ig-like extracellular domain and is responsiblefor membrane adhesion and compaction of the myelin membranes(13,14). Mutations in the P0 gene have been associated withhereditary peripheral demyelinating neuropathy, including CMT1B,Dejerine-Sottas disease, and congenital hypomyelination neuropathy(15). Expression of P0 outside the PNS has not been reportedso far, except for thymic stromal cells (16). Here, we demonstratethat P0 is synthesized in the kidney and mostly localized inthe podocytes from earlier stages of glomerulogenesis. Furthermore,knockout mice, whose neurologic phenotype is closely similarto the human disease (17), show increased glomerular permeabilityto albumin, suggesting a role for P0 in the control of glomerularpermselectivity and its possible implication in CMT-associatedrenal disease.
Reverse Transcriptase–PCR
Total RNA was extracted from human adult kidney cortex (normalpole of cancer kidney) and mouse kidney using RNAwiz (AmbionInc., Austin, TX). After a 1-h incubation at 37°C with DNAse(Ambion Inc.), cDNA synthesis was performed using SuperscriptFirst Strand synthesis System for reverse transcriptase–PCR(RT-PCR; Invitrogen, Carlsbad, CA). PCR then was carried outusing different sets of specific primers (Table 1). Amplificationproducts were subcloned using the pGEM-T Easy vector systemII (Promega, Madison, WI), and the corresponding clones weresequenced.
Table 1. Primer sequences and predicted product size
Western Blot
Proteins from human and rat isolated glomeruli were extractedby lysis using a detergent-based buffer and protease inhibitors(Complete Mini; Roche Diagnostic Corp., Indianapolis, IN). Proteinextracts were also prepared according to the same procedure,from the human podocyte cell line established in our laboratory(18). Total protein extracts were run on a 12% SDS-PAGE geland transferred to a nitrocellulose membrane (Immobilon-P; Millipore,Bedford, MA) by electroblotting. After blocking unspecific bindingin 10% skimmed milk PBS, membranes were incubated overnightwith anti-P0 mAb (1:1000 in 5% skimmed milk TBS/0.5% Tween)(19) at 4°C, washed in 0.5% Tween/TBS, and then incubatedfor 2 h with alkaline phophatase–conjugated goat anti-mouseIg (Chemicon International, Temecula, CA). The anti-P0 mAb wasomitted in controls. Blots were developed using nitro blue tetrazoliumand 5-bromo-4-chloro-indolyl phosphate reagents (Sigma-Aldrich,Steinhiem, Germany).
Immunofluorescence Staining
Immunofluorescence was performed on 3-µm frozen sectionsfrom human adult kidney fixed 3 min in acetone. After washingthree times in PBS, sections were incubated for 1 h with 10%FCS-PBS and incubated overnight with anti-P0 mAb (1:100 dilutedin 10% FCS-PBS) at 4°C. After washes in PBS, sections thenwere incubated with FITC-conjugated goat anti-mouse Ig antibody(1:200 diluted in 10% FCS-PBS; Biovalley, Marne La Vallée,France). Microscopy was performed with a Zeiss Axiophot2 microscope(Carl Zeiss Jena Gmbh, Jena, Germany).
Double Immunofluorescence Labeling and Confocal Microscopy
For dual fluorochrome labeling, the slides were incubated simultaneouslywith anti-P0 mAb (1:100 diluted in 10% FCS-PBS) and rabbit anti-podocinantibody (1:1000 in 10% FCS/PBS; a gift from C. Antignac; INSERMUnit 574, Paris, France) (20) or rabbit anti-nephrin antibody(1:50 in 10% FCS/PBS; a gift from F. Shimizu, Niigata, Japan)(21). After washing in PBS, the slides were incubated with FITC-conjugatedgoat anti-mouse Ig antibody (1:200 diluted in 10% FCS-PBS; Sigma)and TRITC-conjugated goat anti-rabbit IgG (1:200; Sigma). Sectionswere examined with a Zeiss confocal microscope (Carl Zeiss Microscopy).
Immunogold Electron Microscopy
Immunogold electron microscopy was performed on ultrathin frozensections of kidney biopsies from healthy subjects. Briefly,pieces of biopsies were fixed in 4% freshly prepared formaldehyde(by depolymerization of paraformaldehyde) and 0.1% distilledglutaraldehyde (Merk, Darmstadt, Germany) in 100 mM phosphatebuffer (pH 7.2) for 6 to 12 h at 4°C, soaked in sucrose,and frozen and stored in liquid nitrogen. Ultrathin frozen sectionswere processed for indirect immunogold labeling, as described(22). A control experiment in which the first antibody was omittedwas performed.
In Situ Hybridization
Mouse kidneys sampled at 17.5 dpc and in the neonate periodwere fixed in paraformaldehyde 4%, paraffin-embedded, and sectioned(5 µm). The mouse P0 riboprobes were synthesized froma 497-bp PCR product (spanning from 5 to 492 bp in mouse P0cDNA), then subcloned into the pGEM-Teasy vector (Promega).The antisense probe was synthesized after digestion with PstIusing the T7 polymerase, and the sense probe was synthesizedafter digestion with SphI using the Sp6 polymerase. The riboprobeswere labeled with digoxigenin-11-UTP (Roche) according to themanufacturer’s instructions. In situ hybridization wascarried out as described previously (23).
Knockout Mice Studies
The generation and neurologic phenotyping of P0–/–mice have been described previously (17). These mice are ona C57Bl/6 background. All experiments were performed accordingto the European recommendations for the care and use of laboratoryanimals.
Mouse Genotyping.
DNA was isolated from mouse tail, and mice were genotyped byPCR amplification with the following primers: mP0-Ex1(F) CCCCGCTCTCTCCACCCCACAGACAand mP0-Ex1(R) GGGCAGCCAGGATAGGGCTGGGGC. A 1250-bp fragmentwas amplified in P0–/– mice.
Serum and Urine Analysis.
Individual mice were housed overnight in a diuresis cage, andurine was collected after 24 h. Individual urine samples wereanalyzed by 10% SDS-PAGE, and gels were stained with Coomassieblue. Samples that were applied to the gel were normalized forcreatinine content. Urine albumin and creatinine concentrationswere assayed by ELISA and automated Jaffe method, respectively.Blood was collected from anesthetized mice by cardiac puncture,and serum samples were analyzed for creatinine by automatedJaffe method.
Histologic Analysis.
Kidneys were removed from killed mice, fixed in Dubosq-Brazil,dehydrated, and paraffin embedded. Sections were stained withTrichrome, silver stain, or periodic acid-Schiff. For electronmicroscopy, tissue samples from male P0+/+ and P0–/–mice were processed as described previously (8). For immunofluorescence,frozen kidney sections (3 µm) were incubated as abovewith the anti-podocin antibody.
Expression of P0 in mouse, rat, and human kidney was demonstratedby combined approaches including RT-PCR, Western blot, immunochemistry,immunoelectron microscopy, and in situ hybridization.
RT-PCR
RT-PCR analysis used different sets of primers (Table 1). Thefull-length P0 cDNA could be amplified from normal human andmouse kidney cortex, together with shorter cDNA, which encodedifferent domains of P0 (Figure 1). Sequencing of the amplifiedPCR products revealed 100% identity with the peripheral nerveP0 cDNA from human and mouse, respectively.
Figure 1. Myelin protein 0 (P0) mRNA expression in the human and mouse kidney cortex. Total RNA from the human (A) and the mouse (B) kidney cortex was used as a template in reverse transcriptase–PCR. (A) Three different combinations of primers were used to amplify the extracellular (lane 1) and intracellular/3'UTR (lane 2) domains of human P0 cDNA as well as the full-length stretch (lane 3). (B) Two combinations of primers were used to amplify the extracellular and transmembrane domains (lane 1) and the full-length stretch of the mouse P0 cDNA (lane 2), respectively.
Immunoblotting
The glomerular expression of P0 protein was detected by immunoblotusing monoclonal mouse anti-P0 antibody raised against the extracellularpart of the rat P0 protein (18). Blotting of extracts from isolatedhuman glomeruli and from a human podocyte cell line both revealeda single 30-kD band as described earlier in human peripheralnerve lysates (Figure 2A) (24). A doublet at approximately 28kD was detected in rat glomerulus protein extracts, similarto the one observed with the nerve rat protein (Figure 2B) (19).
Figure 2. (A) Immunoblot of human glomerular lysate (lane 1) and human podocyte cell line (lane 2) with mAb to the extracellular domain of P0 showed a reactive protein band at 30 kD. (B) mAb P0 immunoblotted a doublet at approximately 28 kD in rat glomerular lysate. Controls including omission of the anti-P0 mAb were negative. Molecular weight markers are shown in the left lane.
Immunofluorescence
Immunofluorescence of normal adult human kidney sections showeda glomerular positivity along the capillary wall with a patternhighly suggestive of podocyte staining (Figure 3). Extraglomerularstaining was present also in vessel, specifically in endothelialcells. No tubular and interstitium labeling was present. Doublestaining was performed with anti-P0 antibody (green) and anti-nephrinand anti-podocin antibodies (red; Figure 4). P0 tightly co-localizedwith nephrin. Dual labeling with podocin also showed a co-localizationalong with an extension of P0 positivity at the periphery ofpodocin staining.
Figure 3. Normal adult human kidney. Indirect immunofluorescence on frozen 3-µm section with monoclonal anti-P0 antibody. A podocyte labeling is observed in glomeruli, with a comma-like pattern staining on the external side of glomerular capillary wall. Staining was also observed outside the glomeruli in the endothelium of vessels (v). Magnification, x400.
Figure 4. Normal adult human kidney. Dual immunofluorescence and confocal microscopy with monoclonal anti-P0 antibody (green; a and d) and polyclonal antibodies (red) against nephrin (b) and podocin (e). The respective merge images are shown in c and f. Quantitative analysis of the signals recorded at the level of the bars shown in c and f is presented on the right-hand side of the figure. Note the superimposition of P0 and nephrin signals, whereas P0 and podocin show only partial overlap. Magnification: x400.
Immunoelectron Microscopy
The glomerular localization of P0 protein was determined preciselyby electron microscopy. Gold particles were distributed in thepodocyte cell body, especially in cytoplasmic vesicles, as wellas in the nearby actin cytoskeleton (Figure 5A). Podocyte synthesisof P0 protein was attested by the dense labeling of endoplasmicreticulum (Figure 5B). Particles were also found in foot processes,being occasionally close to the slit diaphragm (Figure 5C).Sparse reactivity of the glomerular capillary endothelia waspresent, but no significant labeling was observed in GBM. Controlexperiment, in which the first antibody was omitted, was completelynegative.
Figure 5. Normal adult human kidney. Immunoelectron microscopy with anti-P0 antibody. (A) Gold particles are localized in podocyte cell body, mostly in vesicles, sometimes in close contact with actin network. (B) Staining of the endoplasmic reticulum. (C) Staining in foot processes close to the slit diaphragm. Note the absence of particle in the slit diaphragm. GBM, glomerular basement membrane; US, urinary space; er, endoplasmic reticulum; V, vesicle.
In Situ Hybridization
To analyze developmental expression of P0/MPZ during nephrogenesis,we conducted in situ hybridization in the 17.5-dpc and neonatemouse kidney. In the developing metanephric kidney, P0 transcriptwas detected in the peripheral cortex (Figure 6A) and in primitivenephronic structures that give rise to mature glomeruli includingvesicles and S-shaped bodies (Figure 6B and insert). Antisenseriboprobe also labeled the ureteric bud and the collecting ducts,although theses structures were not stained by immunofluorescencein the adult kidney. No specific signal was observed with thesense probe (Figure 6C). Similar results were obtained in theneonate kidney (data not shown).
Figure 6. A 17.5-d postcoitum mouse kidney. In situ hybridization with P0 antisense riboprobe labeled with digoxigenin. (A) A strong signal is seen in the peripheral cortex and collecting ducts. (B) Expression of P0 transcript in primitive nephronic structure including vesicles, S-shape body (insert) and in ureteric bud and collecting ducts. (C) Absence of signal with sense probe. Magnification: x100 in A and C; x200 in B; x400 in B insert.
Renal Phenotyping of P0–/– Mice
To establish the potential role of P0 in glomerular permselectivity,we analyzed P0–/– mice that were previously establishedby one of us (17). These mice present with demyelinating neuropathyclosely resembling the neurologic phenotype of patients withCMT1B and heterozygous P0 gene mutation. In addition to neurologicsigns that appear during the first month of life, we observeda moderate growth retardation in P0–/– mice (22.4± 0.78 g; n = 7) compared with P0+/– (27.4 ±1.42 g; n = 9; P < 0.05) and P0+/+ mice (27.8 ± 1.48;n = 5; P < 0.05). As shown in Figure 7A, P0–/–mice exhibited significantly higher albuminuria/creatininuriaratio than P0+/– and P0+/+ mice, at both 4 to 6 mo and12 mo of age. Urinary albumin excretion did not increase significantlyat 12 mo, compared with earlier age. Electrophoresis of urinaryproteins confirmed that albumin was predominant (data not shown).
Figure 7. Renal phenotyping of P0–/– mice. (A) Albuminuria/urine creatinine ratio in P0+/+, P0+/–, and P0–/– mice at 4 to 6 mo and at 12 mo. Values are the mean ± SEM of n mice. *P < 0.05 P0–/–versus P0+/– and P0–/–versus P0+/+. (B and C) Electron microscopy study of 12-mo male P0+/+ and P0–/– mice from the same litter. Focal areas of GBM thickening () are observed both in P0–/– and in control mice without alteration of foot processes.
For analyzing histologic lesions underlying increased urinaryalbumin excretion in P0–/– mice, mice were killedat 6 and 12 mo of age. Serum creatinine/body weight ratio wasnot significantly different between P0–/– (1.33± 0.18 µmol/L per g body wt; n = 7), P0+/–(1.05 ± 0.39 µmol/L per g body wt; n = 4), andP0+/+ mice (1.41 ± 0.17 µmol/L per g body wt; n= 7) at 6 mo. Kidney weight/body weight ratio was similar inP0–/–, P0+/–, and P0+/+ mice (0.0191, 0.0195,and 0.0183, respectively). No significant glomerular, vascular,or interstitial abnormality was noted by histologic examinationof P0–/– kidney sections that were stained withhematoxylin eosin and Trichrome (data not shown). Immunofluorescenceexamination with anti-podocin antibodies of kidneys from P0–/–mice and age- and gender-matched P0+/+ littermates showed normalexpression of the protein (data not shown). Electron microscopythat was performed at 4 and 12 mo of age showed no significantabnormality, including no foot process effacement, both in P0–/–mice and in their controls (Figure 7, B and C). These resultsare compatible with the entity referred to as "no change disease"previously described in experimental models and in cases ofhuman nephrotic syndrome (25–27).
In this work, we demonstrate the presence of P0 protein in human,rat, and mouse podocytes and increased urinary albumin excretionin mice that are deficient in this protein. These results wereunexpected because P0 protein expression was thought to be limitedto the peripheral myelinating Schwann cells (13). In the peripheralnerves, this transmembrane glycoprotein induces and maintainsmyelin compaction around nerve fibers through homophilic interactionsof its Ig-like extracellular domains between opposed P0 proteins.Heterozygous P0 gene mutations in human define CMT1B. This demyelinatingperipheral neuropathy is a consequence of uncompacted myelinsheaths, responsible for slowed nerve conduction velocity. Inaddition, P0 was described recently in thymic stroma cells,where its role remains elusive (16). Thymic expression may accountfor P0/MPZ central tolerance induction, as described for othertissue-specific antigens.
In addition to Schwann cells and thymic stroma cells, we showthat full-length P0 mRNA transcripts are expressed in mouseand human kidney cortex and that expression of intact P0 proteincan be detected by Western blot in human and rat glomeruli andin human cultured podocytes. Immunofluorescence study identifieda glomerular staining with a pattern highly suggestive of podocytelabeling, as attested by partial and complete co-localizationwith podocin and nephrin, respectively. Extraglomerular labelingwas also present in vessel endothelium but absent in tubulesegments. Our immunogold results confirmed P0 synthesis in theendoplasmic reticulum and further highlighted P0 subcellulardistribution within the podocyte cell body, mainly in vesicularstructures and in foot processes, sometimes close to slit diaphragm.In situ hybridization in 17.5-dpc and neonate mouse kidneysdisclosed P0 transcript in embryonic structures that give riseto mature glomerulus, including vesicles and S-shaped bodies,and also in the collecting duct. These results indicate thatP0 is synthesized from the early stages of glomerular developmentand also transiently in ureteric bud–derived structures.
The identification of the myelin protein P0 in podocyte extendsthe range of proteins whose expression is shared by nervouscells and the glomerular epithelial cell. However, P0 to dateis the only podocyte protein to be expressed in the PNS.
To investigate the role of P0 in glomerular epithelial cellsand filtration barrier, we determined the renal functional andmorphologic phenotype of P0–/– mice. Although theirneurologic phenotype close to the human disease has been investigatedextensively, there were no data on their renal function. Whereasheterozygous mice were normal, mice that lacked the P0 proteinshowed urinary albumin loss in the absence of significant ultrastructuralalterations of podocyte foot processes or GBM. Focal areas ofthickening of the GBM were detected in P0–/– micebut also in control. The renal phenotype of P0–/–mice seems to be closely similar to the one reported in DDR1–/–mice, in which the loss of discoidin domain receptor-1 in podocytesis associated with moderate high molecular weight proteinuria.Ultrastructural alterations of DDR1–/– glomeruliremain focal, with thickening affecting <4% of the GBM andadjacent loss of slit diaphragm (28). In contrast with P0–/–mice, synaptopodin-deficient mice do not show renal abnormalitiesas judged by electron microscopy and urinary protein excretionanalysis, although podocytes normally express a large amountof synaptopodin (29). Albumin excretion rate was neither increasedin Ptpro–/– mice that lack the glomerular epithelialprotein 1, even after uninephrectomy, although scanning electronmicroscopy disclosed morphologic podocyte alterations (30).Overall, these data suggest that P0 plays a significant rolein glomerular permselectivity. The expression of P0 mostly invesicles in the podocyte cell body is compatible with a rolein cell transport as suggested for Rab3 (8). Because the P0–/–mice are on C57Bl/6 background, which is known to confer resistanceto glomerulosclerosis after nephron reduction or toxic injury(31), we are currently backcrossing the C57Bl/6–P0–/–mice to other backgrounds that are more prone to the developmentof renal diseases.
P0–/– mice may represent a new model of albuminuriawithout detectable morphologic alteration of the glomerularfiltration barrier, a condition that is more common than expectedand now referred as "no change disease." Even if most proteinuricrenal diseases are associated with massive loss of podocytefoot processes, there are indeed human and experimental modelsof nephrotic-range proteinuria in which this podocyte abnormalityis absent. In human, a familial form of nephrotic syndrome withoutpodocyte alteration has been previously reported (25), and asporadic case of massive proteinuria with only segmental effacementof foot processes affecting 20 to 30% of capillary loops hasbeen observed (26). In the rat, injection of mAb 5-1-6 directedagainst nephrin induces massive proteinuria without any footprocess damage (27). Altogether, these observations suggestthat albuminuria of glomerular origin can occur in the absenceof slit diaphragm alterations even at the electron microscopylevel.
Almost 20 cases of glomerular disease, mainly focal segmentalglomerulosclerosis, have been reported in the course of familialdemyelinating neuropathy. Genetic analysis, available in onlytwo cases of CMT with FSGS, did not show mutations in PMP22,P0, and Connexin-32 genes, which are the three genes most commonlyaltered in CMT (12). Our results prompt the search for P0 mutationin cases of CMT with kidney diseases, as well as in autosomalfamilial forms of glomerulopathies that are not linked to mostcommonly mutated genes, including ACTN4, NPHS1, and NPHS2.
In conclusion, we have first identified in the murine and humanglomerulus, P0, the major myelin protein of the PNS. Resultsthat were obtained in P0–/– mice suggest that P0plays some role in glomerular permselectivity, although thisrole remains to be defined.
Acknowledgments
This work was supported by grants from INSERM, University Paris6, and AURA (Association pour l’Utilisation du Rein Artificieland by EU contract QLG1-CT-2002-01215).
We thank Professor D. Vallat (University of Limoges, France)for helpful discussion and support and Philippe Fontanges fortechnical assistance.
This work was presented at the 38th Annual Meeting of the AmericanSociety of Nephrology, St. Louis, MO, November 2004.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication May 16, 2005.
Accepted for publication July 20, 2005.
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[Abstract][Full Text][PDF]
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Abstract
Introduction
-actinin 4 protein (1–3). In vitro experimental studies and knockout mouse models have further identified additional podocyte proteins, interacting mostly with nephrin and podocin for the maintenance of the slit diaphragm integrity. It is interesting that some of the podocyte proteins are electively coexpressed in the central nervous system. These include the cytoskeletal component synaptopodin, nephrin, densin, glomerular epithelial protein 1, the synaptic vesicle molecule Rab3A and its effector Rabphilin-3a, and the neuronal amino acid transporters CAT3 and EAAT2 (4–9). However, none of these proteins has been found in the myelinated peripheral nervous system, although focal segmental glomerulosclerosis (FSGS) has been reported in the most common familial form of demyelinating neuropathy, Charcot-Marie-Tooth (CMT) disease (10–12). 
) are observed both in P0–/– and in control mice without alteration of foot processes.