NEPH2 Is Located at the Glomerular Slit Diaphragm, Interacts with Nephrin and Is Cleaved from Podocytes by Metalloproteinases
Peter Gerke*,
Lorenz Sellin,
Oliver Kretz,
Daniel Petraschka*,
Hanswalter Zentgraf,
Thomas Benzing* and
Gerd Walz*
* Renal Division and Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg; Marienhospital Herne, University of Bochum, Bochum; and German Cancer Research Center, Heidelberg, Germany
Address correspondence to: Dr. Gerd Walz, Renal Division, University Hospital Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany. Phone: 49-761-270-3250; Fax: 49-761-270-3245; E-mail: walz{at}med1.ukl.uni-freiburg.de
Received for publication June 4, 2004.
Accepted for publication February 26, 2005.
The NEPH family comprises three transmembrane proteins of theIg superfamily interacting with the glomerular slit diaphragmproteins podocin and ZO-1. NEPH1 binds to nephrin, another componentof the slit diaphragm, and loss of either partner causes heavyproteinuria. NEPH2, which is strongly conserved among a largenumber of species, is also expressed in the kidney; however,its function is unknown. The authors raised NEPH2 antisera todemonstrate NEPH2 expression in a variety of mouse tissues,including the kidney and a podocyte cell line. The authors localizedthe expression of NEPH2 to the glomerular slit diaphragm byelectron microscopy and show NEPH2 homodimerization and specificinteractions with the extracellular domain of nephrin in vitroand in vivo. NEPH1, however, failed to interact with NEPH2.The authors detected immunoreactive NEPH2 in urine of healthysubjects, suggesting that the extracellular domain is cleavedunder physiologic conditions. These findings were confirmedin vitro in podocyte cell culture. Shedding is increased bytyrosine phosphatase inhibitors and diminished by GM6001, aninhibitor of metalloproteinases. Overexpression experimentsindicate an involvement of the MT1-matrix metalloproteinase.The results suggest a role for NEPH2 in the organization and/ormaintenance of the glomerular slit diaphragm that may differfrom the functions of NEPH1 and nephrin.
Renal filtration of small solutes and water without loss oflarger molecules is intimately linked to the glomerular basementmembrane and the slit diaphragm between interdigitating podocytes.Alterations of these structures, either acquired or hereditary,commonly lead to proteinuria. Hereditary nephrotic syndromeis a heterogeneous disease, displaying severe proteinuria andrenal failure. Best characterized is the congenital nephroticsyndrome of the Finnish type, caused by mutations in NPHS1,the gene encoding nephrin. Affected individuals exhibit massiveproteinuria in utero and nephrosis at birth (1).
Nephrin is an integral membrane protein of the Ig superfamilylocated at adjacent sites of secondary foot processes of podocytes(2). The precise function of nephrin is unknown; however, nephrinis a critical structural component of the slit diaphragm, anultra-thin zipper-like structure that bridges the approximately40-nm-wide gap between interdigitating podocyte foot processes(3).
We have recently demonstrated that NEPH1, a transmembrane proteinof the Ig superfamily, is an extracellular ligand for nephrin(4). Like nephrin, it is expressed on podocytes and localizesto the slit diaphragm by electron microscopy. In mice, the deletionof NEPH1 causes severe proteinuria and perinatal death (5).Injection of antibodies directed against nephrin that couldpotentially disrupt the nephrin-NEPH1 interaction leads to severenephrotic syndrome in mice (6). We found that NEPH1 belongsto a family of three closely related proteins termed NEPH1,NEPH2, and NEPH3, which are all expressed in the renal cortexas determined by reverse-transcriptase PCR (7). Furthermore,we showed direct interactions of their intracellular domainswith podocin and ZO-1, two other components of the glomerularslit diaphragm (7,8). Although NEPH3, also termed Kirrel2, hasbeen associated with pancreatic beta cell function, NEPH2 hasbeen detected in bone marrow stromal cells (9,10), heart, andactivated spleen in mice (unigene mm. 258989). The extracellulardomain of mouse NEPH2, also known as mKirre, can be shed byunknown mechanisms and seems to facilitate hematopoietic stemcell support. These findings prompted us to explore the roleof NEPH2 in the kidney.
We raised antisera directed against the extracellular and theintracellular domains of NEPH2, respectively, to demonstrateNEPH2 expression in a wide variety of mouse tissues, includingthe kidney and an immortalized podocyte cell line. Using electronmicroscopy, we localized the expression of Neph2 to the glomerularslit diaphragm. NEPH2 forms homodimers and specifically interactswith the extracellular domain of nephrin in vitro and in vivo.NEPH1, however, failed to interact with NEPH2. Furthermore,we show that NEPH2 is cleaved by metalloproteinases in podocytesin a tyrosine-phosphorylation-dependent manner. Finally, thedetection of shed NEPH2 in urine samples suggests a role forNEPH2 shedding in vivo.
Plasmids and Antibodies
Full-length nephrin, NEPH1, and NEPH2 cDNAs were recently described(4,7). The Fc fusion proteins contain the NEPH1, NEPH2, or nephrinsequences as indicated, followed by the CH2 and CH3 domain ofhuman IgG. All truncations were generated by PCR and standardcloning procedures, and verified by automated sequencing. Thedomain structures of nephrin, NEPH1, and NEPH2 were predictedusing SMART (http://smart.embl-heidelberg.de) and motif scanner(http://scansite.mit.edu). MT1-matrix metalloproteinase (MT1-MMP)cDNA was a kind gift from J. Weiss, University of Michigan (AnnArbor, MI). The plasmids pMstGV (pGal4/VP16), pMstGV-APP695(pAPP-Gal4/VP16), and pMstGV-LDL receptor (pLDLR-Gal4/VP16),and the pG5E1B-Luc plasmid, which contains five Gal4 bindingsites and the E1B minimal promoter preceding the luciferasefirefly gene, were generously provided by T. Südhof, Universityof Texas Southwestern Medical Center, (Dallas, TX). For constructionof neph2-Gal4/VP16 and neph2-cyt-Gal4/VP16, the human full-lengthneph2 cDNA without stop codon or the cytoplasmic tail containingKozak and first ATG 5' of the sequence were PCR-amplified andcloned into the BglII and NheI sites of pMstGV as describedpreviously (11). The resulting constructs encode fusion proteinscontaining the neph2 full-length protein or the cytoplasmictail with a VP16 transactivation domain, a nuclear localizationsignal, and a Gal4 binding region. The human fetal librarieswere purchased from Edge BioSystems (Gaithersburg, MD). Fordetection of human NEPH2, the 5'-oligonucleotide 5'-CCAACATCTACAGCACCCTGAGCGGCCand 3'-oligonucleotide 5'-TTAGACGTGAGTCTGCATCCG were used toyield a 311-bp PCR fragment. Polyclonal NEPH2 and nephrin antiserawere generated by immunization of rabbits (Eurogentec, Herstal,Belgium). The following peptides were used: NEPH2.ec, MAKDKFRRMNEGQVY(human, mouse: aa34-48); and NEPH2.ic, SDPSRPLQRRMQTHV (human:aa764-778, mouse: aa752-766). The peptide used for nephrin wasKDSRTVTESRLPQES (human: aa480-494). All antisera were affinity-purifiedagainst the peptides. Monoclonal anti-NEPH2 antibody was raisedagainst the intracellular fragment of human NEPH2 (aa 593-778)and used for immunohistochemistry at 1:20 and for Western blotat 1:1000. M2 anti-FLAG antiserum was obtained from Sigma-Aldrich(Taufkirchen, Germany), and protein G-sepharose was obtainedfrom Amersham Pharmacia Biotech (Freiburg, Germany). Anti-MT1-MMPwas obtained from Chemicon International (Temecula, CA), andthe secondary antisera were obtained from Jackson Immuno ResearchLaboratories (West Grove, PA).
Reporter Gene Assays
Reporter assays were performed as described previously (11).Luciferase gene expression was analyzed using the LuciferaseAssay System (Promega, Mannheim, Germany). A 10-s integral measurementof light emission from each reaction mixture was recorded ina plate luminometer and normalized for -galactosidase activity.All transfections for reporter gene assays were performed intriplicate and repeated in at least two independent experiments.
Cell Culture
Conditionally immortalized mouse podocytes were generated asdescribed previously (12) and grown at permissive temperaturein the presence of gamma IFN at 10 U/ml. To induce differentiation,the cells were maintained on type I collagen at 37°C withoutgamma IFN for at least 14 d. For shedding experiments, differentiatedpodocytes were incubated in medium containing the agents asdepicted for 60 min at 37°C. GM6001 was obtained from ChemiconInternational; pervanadate was freshly prepared from sodiumvanadate and H2O2 immediately before use.
Co-Immunoprecipitation
Co-immunoprecipitations were performed as described (13). Briefly,HEK 293T cells were transiently transfected using the calciumphosphate method. After incubation for 24 h, cells were washedtwice and lysed in a 1% Triton X-100 lysis buffer (20 mM Tris-HCl,pH 7.5; 1% Triton X-100; 50 mM NaCl; 50 mM NaF; 15 mM Na4P2O7;0.1 mM EDTA; 2 mM Na3VO4; and protease inhibitors). After centrifugationat 15,000 x g (15 min, 4°C) and ultracentrifugation at 100,000x g (30 min, 4°C), cell lysates containing equal amountsof total protein were precleared with protein G-sepharose, andthen incubated for 1 h at 4°C with the appropriate antibody,followed by incubation with 30 µl of protein G-sepharosebeads for 3 h. The beads were washed extensively with lysisbuffer, and bound proteins were resolved by 10% SDS-PAGE. Forendogenous interaction, eight mouse kidneys were homogenizedusing a glass potter, cleared by centrifugation, and solubilizedin lysis buffer supplemented with 20 mM CHAPS and 3 mM ATP.Before immunoprecipitation, cellular lysates were further preclearedby ultracentrifugation and absorption to protein G beads. Alltissues were freshly prepared and perfused in situ with ice-coldPBS before lysis. Control samples were incubated with equalamounts of rabbit IgG followed by protein A beads. Human kidneylysates were prepared from the healthy pole of tumor nephrectomies.
Immunohistochemistry
Mouse kidneys were subject to retrograde perfusion fixationusing 3% glutaraldehyde and embedded in paraffin; 4-µmsections were cut and deparaffinized. After standard immunohistologicalprocedures, including antigen retrieval with proteinase K, thesections were incubated with anti-NEPH2 antiserum or mouse serum(1:1000), followed by goat anti-mouse rhodamine red. For injectionstudies, sera were heat-inactivated at 56°C for 30 min.Adult male C57Bl/6 mice were injected with 200 µl intravenouslyof either anti-NEPH2 antiserum or pre-immune serum and euthanized24 h later. Kidneys were fixed and embedded as described, andsections were incubated with goat anti-rabbit rhodamine red.An Axiophot 2 microscope (Zeiss, Jena, Germany) equipped witha CCD camera was used for image documentation.
Electron Microscopy
Adult mice were anesthetized with sodium pentobarbital and transcardiallyperfused using 4% paraformaldehyde and 0.1% glutaraldehyde inPBS. Kidneys were removed and postfixed in the same fixative(overnight, 4°C). Tissues were washed in PBS, and then horizontalsections (50 µm) were cut on a vibratome and cryoprotectedin a solution containing 25% sucrose and 10% glycerol in 50mM PBS. The sections were freeze-thawed and incubated in blockingsolution containing 20% normal goat serum in 50 mM Tris-bufferedsaline for 1 h, followed by incubation with an anti-Neph2.ecantiserum (overnight, 4°C). After washing, the sectionswere with incubated with 1.4-nm gold-coupled goat anti-rabbitsecondary antibody (1:100, Nanogold; Nanoprobes, Stony Brook,NY) for immunogold reaction. Immunogold labeling was then enhancedwith HQ silver kit (Nanoprobes). After treatment with OsO4,the sections were stained with uranyl acetate, dehydrated, andflat-embedded in epoxy resin (Durcupan ACM, Fluka; Sigma-Aldrich,Gillingham, UK). Ultra-thin sections were cut and analyzed ina Philipps CM 100 electron microscope.
Urine Preparation
Eight ml urine per sample were cleared from cell debris andcrystals by centrifugation and diluted in acetone to achievean 80% acetone solution. After incubation o.n. at –20°C,the solutions were centrifuged at 10,000 x g (10 min, 4°C).Supernatants were removed and pallets washed with 100% acetone(–20°C). Dry pallets were weighted and resuspendedin Laemmli buffer at 1 µg/µl.
Software
Alignments and phylogenetic trees were created using Lasergene5.51 (DNASTAR Inc, Madison, WI).
Sequence Comparison of the NEPH Gene Family
Human NEPH proteins are type 1 transmembrane proteins characterizedby five extracellular Ig domains. Searching GenBank, we foundorthologs in a variety of species (Figure 1A). These includedwell-characterized Drosophila proteins as well as predictedunnamed proteins in zebrafish or Xenopus. Phylogenetic analysisreveals a close relation between the mammalian NEPHs, with >97%identity of human and mouse NEPH2 (Figure 1B). We thereforedecided to use peptide sequences unique to NEPH2 but commonto the human and the mouse protein as epitopes for the generationof polyclonal antisera. The same approach was used for anti-nephrinantiserum. Fusion proteins used for NEPH2 protein interactionstudies are shown in Figure 1C.
Figure 1. (A) Phylogenetic tree of NEPH proteins. GenBank was searched for NEPH2-related proteins. We use the term NEPH for unnamed proteins with high homology to human NEPHs. GenBank accession numbers are: human NEPH1, CAH73881, human NEPH2, NP_115920; human NEPH3, NP_954649; mouse NEPH1, AAN73043; mouse NEPH2, BAC32333; mouse NEPH3, NP_766486; rat NEPH1, AAP78673; rat NEPH2, XP_235986; rat NEPH3, XP_218486; Drosophila melanogaster kirre, AAF86308; Drosophila melanogaster irreC-rst, Q08180; anopheles gambiae kirre, XP_310916; Xenopus laevis NEPH, AAH57728; SYG-1, AAC47047; danio rerio NEPH, AAH66766. (B) Homology with human NEPH2 of selected Ig family proteins. Data were obtained using Clustal V (Megalign, DNASTAR). A high degree of similarity is seen among mammalian NEPH2 orthologs and between NEPH2 and NEPH1. (C) NEPH2 fusion proteins used for interaction studies. The Ig domains were predicted using the SMART prediction program (http://smart.EMBL-Heidelberg.de/). All constructs include the authentic signal peptide (SP). NEPH2.ec.Fc contains the entire extracellular (ec) domain of NEPH2 fused to the carboxy-terminus of the CH2 and CH3 domain of human IgG (Fc).
Characterization of Anti-NEPH2 Antisera
Antisera directed against the peptides NEPH2.ec and NEPH2.icwere tested in HEK 293T cells. The localization of the peptidesequences within the NEPH2 molecule is depicted in Figure 2A.We used lysates from HEK 293T cells transiently overexpressinghuman NEPH2 and nephrin, respectively, to test the antiseraby Western blot analysis. Cells overexpressing NEPH1 servedas negative control. As demonstrated in Figure 2B, both anti-NEPH2antisera recognized a single band at approximately 95 kD, whichwas not detected in the control lysates. Similarly, anti-nephrindetected a band at 175 kD, the expected size of nephrin withoutapparent cross-reactivity. Next, we tested if the antisera weresuitable for immunoprecipitation. Flag-tagged NEPH2 and nephrin,respectively, were transiently transfected. Five µg ofeach antibody coupled to protein A beads precipitated the overexpressedproteins as detected by anti-flag on Western blot (Figure 2, C and D).In addition, we raised a monoclonal antibody againstthe intracellular domain of NEPH2. On Western blot, the antibodyspecifically detected the flag-tagged NEPH2 C-terminus in lysatesfrom overexpressing HEK 293T cells (Figure 2E).
Figure 2. Generation of antisera against NEPH2 and nephrin. (A) Amino acid sequences and location within the NEPH2 protein of peptides used for rabbit immunization. NEPH2.ec is located near the N-terminal signaling peptide, and NEPH2.ic is close to the C-terminal PDZ binding domain. (B) NEPH2, NEPH1, and nephrin were transiently overexpressed in HEK 293T cells. Lysates were separated on SDS-PAGE and Western blot analysis performed using affinity purified rabbit antisera directed against NEPH2.ec, NEPH2.ic, and the N-terminus of nephrin (anti-nephrin.ec). Bands in the expected region are visible in the NEPH2 and nephrin lanes, but not in the NEPH1 lanes (negative control). (C) Anti-NEPH2.ic and anti-NEPH2.ec both precipitate NEPH2.F from transiently transfected HEK 293T cells. Lysates were subject to immunoprecipitation with 5 µg anti-NEPH2.ec and anti-NEPH2.ic, respectively, Western blot analysis with anti-M2. Note that NEPH2.F shows up as a double band on Western blot, possibly a result of differential posttranslational modifications. (D) Anti-nephrin.ec immobilizes nephrin from transiently transfected HEK 293T cells. 5 µg anti-nephrin.ec antibody bound to protein A beads were used followed by Western blot analysis performed with anti-M2. (E) Characterization of anti-NEPH2 mAb. The flag-tagged C-terminal domain of NEPH2 (F.NEPH2 cyt) was transiently overexpressed in HEK 293T cells. Lysated were separated on SDS-PAGE. Anti-NEPH2 mAb recognizes a band in the correct region (right lane).
NEPH2 Is Expressed in Various Mouse Tissues
Western blot technique was used to evaluate the tissue expressionof mouse NEPH2. Both anti-NEPH2.ic and anti-NEPH2.ec antiserumdetected a band at approximately 125 kD in mouse podocyte lysates(Figure 3A). This band was not seen in Western blots reprobedwith pre-immune serum. Because no significant immunoreactivitywas detectable in mouse tissue lysates, we immunoprecipitatedNEPH2 using anti-NEPH2.ic for immobilization and anti-NEPH2.ecfor immunoblotting. Whereas the predicted size of NEPH2 is 85.4kD, tissues from heart, lung, liver, kidney, testis, brain,and muscle showed positive bands at approximately 100 kD (Figure 3B).Some tissues expressed an additional band at approximately125 kD (heart, testis, kidney, lung). The strongest expressionwas seen in muscle and brain. These data suggest that NEPH2is alternatively spliced, partially degraded, or differentiallyglycosylated in some tissues. To test for earlier expression,we performed NEPH2 PCR from human fetal libraries. As demonstratedin Figure 3C, there is moderate expression of NEPH2 mRNA infetal human kidneys and high expression in fetal human brains.
Figure 3. Expression of NEPH2. (A) Mouse podocytes express NEPH2. Anti-NEPH2.ic antiserum or anti-NEPH2.ec antiserum were used to detect endogenous NEPH2. NEPH2 immunoreactivity is detected at approximately 125 kD by both antisera, but not by pre-immune serum. (B) NEPH2 is widely expressed in mouse tissues. Lysates were prepared from freshly explanted mouse organs and separated on SDS-PAGE. Immunoprecipitations were performed using the NEPH2.ic antibody coupled with protein A beads. NEPH2 expression was determined using the NEPH2.ec polyclonal antibody. Strong immunoreactivity is detected at approximately 100 kD in all tissues tested with strongest expression in muscle and brain. Note the additional bands at approximately 125 kD in some tissues. Specificity was confirmed by re-incubation of the blot with pre-immune serum (lower panel). (C) NEPH2 is expressed in fetal tissues. cDNA library PCR with intron-spanning primers detects a specific transcript for NEPH2 (311 bp) in human fetal kidney and human fetal brain.
NEPH2 Forms Homodimers and Heterodimers with Nephrin
Candidate extracellular ligands for NEPH2 were detected by immunoprecipitationin overexpressing HEK 293T cells. As depicted in Figure 4A,nephrin.ec.Fc, a construct containing the entire extracellulardomain of nephrin, precipitated NEPH2. The NEPH1 extracellulardomain, or a control construct containing parts of the PKD1extracellular domain (PKD.Fc), however, did not precipitateNEPH2. As reported for the nephrin-NEPH1 interaction, the interactionbetween NEPH2 and nephrin was not confined to single Ig domains(Figure 4B). Several nephrin truncations precipitated NEPH2,whereas the control construct did not. Figure 4C demonstratesthat NEPH2 forms homodimers; NEPH2.F is precipitated by theNEPH2 extracellular domain, but not by the control construct.
Figure 4. Homodimeric and heterodimeric interactions of NEPH2. (A) NEPH2 precipitates nephrin but not NEPH1. Fc-tagged extracellular domains of nephrin, NEPH1, or Ig domains of PKD1 (PKD.Fc) were co-expressed with NEPH2 in transiently transfected HEK 293T cells. Cellular lysates were incubated with protein G to precipitate the Fc fusion proteins. NEPH2, bound to nephrin.Fc, was detected by Western blot analysis, using the NEPH2.ec polyclonal antibody. In contrast, NEPH1.Fc and control protein PKD.Fc failed to immobilize NEPH2. (B) Nephrin fusion proteins precipitate NEPH2. (B) Nephrin fusion proteins containing either the entire extracellular domain of nephrin (nephrin.Fc), the nephrin Ig domains 1 to 3 (nephrin.Ig1-3.Fc), or Ig domains 5 and 6 (sFc.nephrin.Ig5 + 6) fused with Fc precipitated NEPH2, whereas PKD.Fc did not. Western blot analysis was performed with anti-NEPH2.ec. (C) NEPH2 fusion protein precipitates NEPH2. Co-expression of NEPH2.Fc, containing the entire extracellular domain of NEPH2, but not PKD.Fc immunoprecipitated flag-tagged NEPH2 (NEPH2.F). NEPH2.F, bound to NEPH2.ec.Fc, was detected by Western blot analysis, using the M2 anti-flag monoclonal antibody. (D) Interaction between NEPH2 and nephrin in vivo. Lysates prepared from human kidneys were incubated with anti-nephrin antiserum or rabbit IgG and subsequently precipitated with protein A. Immobilized NEPH2 was detected by Western blot analysis using anti-NEPH2.ec antiserum. Nephrin but not rabbit IgG immobilizes NEPH2, suggesting that nephrin and NEPH2 interact in vivo. Immunoprecipitation with anti-NEPH2.ec antiserum served as size control (right lane).
The in vivo interaction between nephrin and NEPH2 is shown inFigure 4D. Nephrin co-precipitated NEPH2 in human kidney lysates.Rabbit IgG, used as negative control, did not precipitate eitherprotein.
NEPH2 Is Located at the Glomerular Slit Diaphragm
The interaction between NEPH2 and nephrin suggests that NEPH2is expressed on podocytes. Accordingly, our monoclonal anti-NEPH2antibody detected NEPH2 immunoreactivity in a glomerular stainingpattern (Figure 5, A and B). Injection of anti-NEPH2.ec butnot pre-immune serum showed staining in the glomerular basementmembrane region (Figure 5C, D). Finally, immunogold electronmicroscopy revealed that NEPH2 is located at the glomerularslit diaphragm (Figure 5, E and F). Only minor additional immunoreactivitywas detected at other locations of the podocyte foot processes.
Figure 5. (A, B) Localization of NEPH2 to the renal glomerulus. Anti-NEPH2 mAb specifically labels glomerular cells in mouse kidney sections (A), whereas mouse control serum does not (B, original magnification 200x). (C, D) Localization of NEPH2 near the glomerular basement membrane. Anti-rabbit IgG labels the glomerular basement membrane and some glomerular cells in mice injected with anti-NEPH2.ec antiserum (C), but not in mice injected with pre-immune serum from the same rabbit (D, original magnification 400x). (E, F) Immunogold localization of Neph2 to the slit diaphragm in adult mouse glomeruli. Pre-embedding silver-enhanced immunogold labeling using rabbit anti-Neph2.ec antibody. Neph2 is mostly localized to the slit diaphragm (arrows). FP indicates foot process; GBM, glomerular basement membrane; E, endothelial cell.
Shedding of the NEPH2 Extracellular Domain
Because shedding of nephrin and NEPH2 has been reported, weincubated supernatants of murine podocytes with anti-NEPH2.ec.Western blot analysis revealed a band at approximately 95 kD(Figure 6A). Incubation of the cells with the tyrosine phosphataseinhibitor pervanadate leads to an increase in soluble NEPH2,whereas the band vanished after incubation with the MMP inhibitorGM6001. These findings were confirmed in several independentexperiments.
Figure 6. Shedding of NEPH2. Shedding of NEPH2 in vitro. (A) Immortalized murine podocytes were incubated with agents as indicated for 60 min. Supernatants were then precleared and incubated with anti-NEPH2.ec coupled to protein A beads. Anti-NEPH2.ec immunoreactivity is detectable in supernatants of pervanadate exposed cells, and to a lesser extent of untreated cells. No immunoreactivity can be found in supernatants of cells exposed to GM6001. (B) Lysates obtained from murine podocytes were tested for MT1-MMP. Using specific polyclonal antiserum, intact MT1-MMP is detected at 63 kD, its inactivated form is detected at 42 kD. (C) NEPH2 was overexpressed with or without MT1-MMP in transiently transfected HEK 293T cells. Supernatants were incubated with anti-NEPH2.ec antibody bound to protein A beads. Precipitates were analyzed on Western blot using anti-NEPH2.ec antiserum. In the presence of MT1.MMP, a band of approximately 95 kD appears in supernatants of cells expressing both constructs. This band vanishes if either construct is missing. This band is not seen when using anti-NEPH2.ic antibody for Western blot analysis. Bcl2 is used as replacement for NEPH2 as a negative control. (D) Detection of the NEPH2 cytoplasmic fragment in a gene reporter assay. All constructs contained the transcription factor Gal4-VP16 near the C-terminus and were co-transfected with the pG5E1B-Luc plasmid. Full-length NEPH2 leads to a luciferase activity of 6000-fold, suggestive of shedding and release of the cytoplasmic NEPH2 fragment. Negative controls are the LDL receptor (LDLR, lane 4), or empty vector (lane 1). Positive controls are a cytoplasmic fragment of NEPH2 (lane 2), -amyloid precursor protein (APP, lane 5) and a cytoplasmic construct (pMstGV, lane 6).
Next, we tested whether the podocyte cell line expressed theslit diaphragm protein MT1-MMP as a possible cleaving enzymefor the NEPH2 extracellular domain. Figure 6B demonstrates significantexpression of the proteinase (63 kD) and its major degradationproduct (42 kD) in the cell lysates of cultured podocytes.
To further evaluate the possible role of MT1-MMP, we transientlyoverexpressed MT1-MMP and NEPH2 in HEK 293T cells. Figure 6Cdemonstrates the presence of a positive band in the supernatantsof HEK 293 T cells when both NEPH2 and MT1-MMP were co-transfected.In the absence of MT1-MMP or expression of a control construct(Bcl2), no significant immunoreactivity was detectable. Becausethe immunoreactive band is not detectable after reprobing withanti-NEPH2.ic, our results suggest that MT1-MMP (and potentiallyother proteases) cleave and release a NEPH2 fragment that lacksthe intracellular C-terminal domain of NEPH2.
To monitor the release of the intracellular C-terminal domain,we performed a reporter gene assay that requires episomal translocationof the cleaved C-terminal NEPH2 domain. The chimeric transcriptionfactor Gal4-VP16 was inserted into NEPH2 near the C-terminus.As shown in Figure 6D, co-expression with the pG5E1B-Luc plasmidled to a strong increase in luciferase activity as comparedwith negative control. Another transmembrane Gal4-VP16 construct(LDLR) did not have these effects, suggesting cleavage of NEPH2but not LDLR.
To evaluate the significance of NEPH2 shedding in vivo, we testedhuman urine samples for anti-NEPH2.ec immunoreactivity. Urinaryprotein was precipitated and subject to SDS-PAGE followed byWestern blotting and incubation with anti-NEPH2.ec antiserum.A 95-kD band was detectable in the urine of healthy controls,suggesting that NEPH2 is secreted under physiologic conditions(Figure 7A). Additional bands were detected at 65 kD and 55kD, and more variably at 23 kD, whereas the reprobe of the Westernblots for NEPH2.ic was negative. In patients with membranousglomerulonephritis, there seems to be an increase in NEPH2 sheddingas compared with a healthy control (Figure 7B).
Figure 7. Shedding of NEPH2 in vivo. (A) Urine from healthy individuals was subject to acetone precipitation. 25 µg were separated on SDS-PAGE. Western blot analysis with anti-NEPH2.ec antibody revealed NEPH2-shedding products. (B) Shedding of NEPH2 in vivo under disease conditions. Urine samples of a healthy individual and three patients with membranous glomerulonephritis were subjected to acetone precipitation and 15 µg and 25 µg were separated on SDS-PAGE. Western blot analysis with anti-NEPH2.ec antibody suggested that patients with membranous glomerulonephritis (urinary protein levels 1 to 2 g/d) excreted significantly more NEPH2 shedding products than a healthy control (left two lanes).
NEPH2 Expression and Interactions
NEPH2 belongs to the NEPH family of proteins, which are highlyconserved within mammalian and submammalian species. Searchingthe GenBank database, we found NEPH proteins in mice and rats,but also in Xenopus laevis, Danio rerio, and insects. NEPH2has been shown to interact with cytoplasmatic proteins of theslit diaphragm. Extracellular ligands, however, have not beenidentified so far. We now demonstrate homodimerization of extracellularNEPH2 domains as well as extracellular interactions with nephrin.Accordingly, NEPH2 was expressed at the glomerular slit diaphragm.
Similar findings have been reported for NEPH1 (4). It is expressedat the slit diaphragm and knockout mice show a renal phenotypewith podocyte foot process effacement and congenital nephroticsyndrome (5,6). NEPH1 forms homodimers and interacts with nephrin,and as in NEPH2, these interactions cannot be mapped to singleIg domains (4). Similar to NEPH1, NEPH2 was expressed in a widevariety of tissues, including fetal and adult kidneys and apodocyte cell line. Both NEPH1 and NEPH2 interact with the slitdiaphragm components podocin and ZO-1 and, as shown here, bothare expressed at the slit diaphragm and are extracellular ligandsof nephrin. These observations suggest a similar role for bothNEPH proteins in renal filtration. There are differences, however.NEPH1 expression is much reduced in adult mice as compared withembryos, whereas we found high protein levels of NEPH2 in adultanimals. Although both proteins form homodimers and interactwith nephrin, they did not interact with one another.
In Drosophila, the nephrin-like proteins hibris (Hbs) and sticksand stones (Sns), as well as the NEPH proteins kirre/dumbfounded(Duf) and irregular chiasm roughest (IrreC-rst), have been implicatedin myoblast fusion and myotube guidance (14). S2 cell aggregationassays revealed heterotypic interactions of Duf with Hbs andSns (15). Muscle precursor cells in Drosophila carry eitherNEPH proteins (founder cells) or nephrin-like proteins (fusion-competentmyoblasts). These cells will only fuse heterogeneously, i.e.,NEPH protein expressing cells will never fuse with one another(16). As a first step in muscle fusion, fusion-competent myoblastsextend filopodia directed toward founder cells. These processeswill be randomly orientated in Duf mutant embryos and they maybe misguided by ectopic expression of either Duf or IrreC-rst(15,17). These observations indicate that NEPH proteins mayguide cell processes and control their separation or fusion.Similar findings have recently been reported for Caenorhabditiselegans (18). Synaptic guidepost cells required to drive synapsesfrom the motor neuron onto adjacent target cells transientlyexpress the nephrin-like protein SYG-2 during synapse formation.SYG-2 interacts with the NEPH protein SYG-1 expressed on presynapticaxons and directs SYG-1 accumulation and synapse formation toadjacent regions of the axon. Mutants of either SYG-1 or SYG-2cause defects in synaptic specificity with ectopic synapse formationon inappropriate targets. Furthermore, SYG-2 misexpression causesaberrant accumulation of SYG-1 and synaptic markers adjacentto the SYG-2-expressing cells. NEPH2 is highly expressed inthe mammalian muscle and brain. Further studies will need toexamine whether NEPH2 controls synapse and myocyte formationduring mammalian development.
NEPH2 Shedding
A number of membrane proteins have soluble forms that are releasedinto the extracellular space. Although these soluble forms mayresult from alternative splicing, they most often derive fromproteolysis of the extracellular domain. The cleavage occursclose to the transmembrane domain, often releasing physiologicallyactive protein. In many cases, shedding can be blocked by metalloproteinaseinhibitors, suggesting the involvement of MMP.
We found NEPH2 immunoreactivity in the supernatants of culturedmouse podocytes using Western blot analysis. There was a strongincrease in soluble NEPH2 after incubating cells with pervanadate,an inhibitor of tyrosine phosphatases. In contrast, solubleNEPH2 disappeared from the supernatants in the presence of GM6001,an inhibitor of MMP. These findings prompted us to suspect thata cleaving enzyme of the MMP family is responsible for NEPH2shedding. A prominent candidate was MT1-MMP, also known as MMP14,a membrane-bound calcium-dependent MMP. MT1-MMP cleaves severalother membrane proteins, e.g., betaglycan, in a tyrosine-phosphorylation-dependentmanner (19). MT1-MMP can be blocked by GM6001 in MCF7 cellsand is activated by furin (20,21). Importantly, both MT1-MMPand furin have recently been shown by electron microscopy toco-localize at the glomerular slit diaphragm (22). We foundthat supernatants of HEK 293T cells, expressing both NEPH2 andMT1-MMP, contained NEPH2. HEK 239T cells express endogenousNEPH2, yet no immunoreactivity was detectable in the absenceof MT1-MMP. A faint band appeared in cells transfected withMT1-MMP and a control vector (Bcl2), probably as a result ofshed endogenous NEPH2. Using a gene reporter assay, we wereable to demonstrate the release of the cytoplasmic tail of NEPH2from the plasma membrane. Further studies will be required todetermine potential signaling capacities of this fragment.
Shedding appears to occur under physiologic conditions. Examiningurine of healthy subjects with antiserum directed against theextracellular NEPH2 domain, we detected several NEPH2 fragments,ranging from approximately 95 kD to 23 kD.
COS7 cells transiently overexpressing mouse NEPH2 shed a proteinof approximately 70 kD (10), suggesting that the size of fullyglycosylated NEPH2 in podocytes might be larger than the reported97 kD. The calculated mass of the NEPH2 extracellular domainis approximately 60 kD, but it contains five predicted N-glycosylationsites. Transient overexpression of human NEPH2 cDNA in HEK 293Tcells results in a band of approximately 100 kD. However, mousepodocytes express a 125-kD protein, suggesting cell-specificdifferences in posttranslational modification and/or splicing.The presence of multiple tissue-specific forms is supportedby the presence of several splice variants of the closely relatedNEPH3 (9). Soluble NEPH2 could also represent a splice variantlacking the transmembrane domain; this has been reported fornephrin (23). However, antiserum directed against the intracellularNEPH2 domain failed to react with urine proteins, arguing againstthis possibility.
Doublier et al. found rapid loss of nephrin immunoreactivityon the surface of cultured glomerular epithelial cells in responseto aggregated human IgG4 and puromycin, suggesting an involvementof the cytoskeleton in nephrin shedding (24). Interestingly,nephrin immunoreactivity was rapidly lost when incubating thecells with TNF-, a strong inducer of MT1-MMP expression (25).Shedding of nephrin has also been reported in rats with streptozotocin-induceddiabetes mellitus (26). More recently, nephrinuria was foundin 19% to 35% of type 1 diabetic patients using Western blottingtechniques (27). Although nephrinuria was only detectable indiabetic patients, NEPH2 was clearly detectable in urine fromhealthy volunteers. In contrast to nephrin, shedding of NEPH2may therefore represent an integral part of its physiologicfunction. In addition, we observed increased NEPH2 sheddingin patients with membranous glomerulonephritis and proteinuria.Thus, urinary NEPH2 might also serve as a novel biomarker ofglomerular disease activity and/or efficacy of anti-proteinurictreatment.
We propose that the network created by podocyte foot processesrequires dynamic remodeling in response to changes in intraglomerularpressure and fluid composition. NEPH proteins and nephrin mayplay an important role in the guidance of podocyte foot processesto establish the complex three-dimensional structure that characterizesthese epithelial cells. Shedding of the extracellular domainsmay regulate turnover and regeneration, and can be controlledby environmental changes that stimulate metalloproteinases.Further work will be required to test this hypothesis.
Acknowledgments
We thank Petra Stunz, Temel Kilic, and Barbara Joch for excellenttechnical assistance. This work was supported by a grant ofthe Deutsche Forschungsgemeinschaft (DFG Wa597).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication June 4, 2004.
Accepted for publication February 26, 2005.
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Top
Abstract
Introduction
-galactosidase activity. All transfections for reporter gene assays were performed in triplicate and repeated in at least two independent experiments. 
, a strong inducer of MT1-MMP expression (25). Shedding of nephrin has also been reported in rats with streptozotocin-induced diabetes mellitus (26). More recently, nephrinuria was found in 19% to 35% of type 1 diabetic patients using Western blotting techniques (27). Although nephrinuria was only detectable in diabetic patients, NEPH2 was clearly detectable in urine from healthy volunteers. In contrast to nephrin, shedding of NEPH2 may therefore represent an integral part of its physiologic function. In addition, we observed increased NEPH2 shedding in patients with membranous glomerulonephritis and proteinuria. Thus, urinary NEPH2 might also serve as a novel biomarker of glomerular disease activity and/or efficacy of anti-proteinuric treatment. 
