Expression and Subcellular Distribution of Novel Glomerulus-Associated Proteins Dendrin, Ehd3, Sh2d4a, Plekhh2, and 2310066E14Rik
Jaakko Patrakka*,,
Zhijie Xiao*,
Masatoshi Nukui*,
Minoru Takemoto*,
Liqun He*,
Asmundur Oddsson*,
Ljubica Perisic*,
Anne Kaukinen,
Cristina Al-Khalili Szigyarto,
Mathias Uhlén,
Hannu Jalanko,
Christer Betsholtz*,|| and
Karl Tryggvason*
* Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, and || Department of Medicine, Karolinska Institute, and Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; and Electron Microscopy Unit, Institute of Biotechnology, and Hospital for Children and Adolescents and Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
Address correspondence to: Dr. Karl Tryggvason, Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden. Phone: +46-8-5248-7720; Fax: +46-8-316-165; E-mail: karl.tryggvason{at}ki.se
Received for publication June 29, 2006.
Accepted for publication December 7, 2006.
The glomerular capillary tuft is a highly specialized microcapillarythat is dedicated to function as a sophisticated molecular sieve.The glomerulus filter has a unique molecular composition, andseveral essential glomerular proteins are expressed in the kidneyexclusively by glomerular podocytes. A catalog of >300 glomerulus-upregulatedtranscripts that were identified using expressed sequence tagprofiling and microarray analysis was published recently. Thisstudy characterized the expression profile of five glomerulus-upregulatedtranscripts/proteins (ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik)in detail. The expression pattern of these novel glomerulartranscripts in various mouse tissues was studied using reversetranscriptase–PCR, Northern blotting, and in situ hybridization.For studying the distribution of corresponding proteins, polyclonalantibodies were raised against the gene products, and Westernblotting, immunofluorescence, and immunoelectron microscopicanalyses were performed. Remarkably, it was discovered thatall five transcripts/proteins were expressed in the kidney exclusivelyby glomerular cells. Ehd3 was expressed only by glomerular endothelialcells. Importantly, ehd3 is the first gene ever shown to beexpressed exclusively by glomerular endothelial cells and notby other endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2,and 2310066E14Rik, however, were transcribed solely by podocytes.With the use of polyclonal antibodies, dendrin, sh2d4a, andplekhh2 proteins were localized to the slit diaphragm and thefoot process, whereas 2310066E14Rik protein was localized tothe podocyte major processes and cell body. This study providesfresh insights into glomerular biology and uncovers new possibilitiesto explore the role of these novel proteins in the glomerularphysiology and pathology.
The glomerular capillary tuft of the kidney is a unique micro-organthat is specialized to operate as a sophisticated molecularsieve (1). The filtration barrier is composed of the glomerularendothelial cells (GEC), the glomerular basement membrane (GBM),and the podocyte cells. GEC differ from most other endothelialcells in that they are extraordinarily flattened and fenestrated(2). The fenestrated structure of GEC depends on podocyte-derivedvascular endothelial growth factor A (3), but the mechanismsbehind their unique structure largely are unknown. The GBM,like all basement membranes (BM), is composed of interconnectedcollagen type IV and laminin networks, where proteoglycans andother matrix components are attached. The GBM, however, is distinctfrom most other BM of the body, because it contains a uniquecomposition of collagen type IV and laminin isoforms. The importanceof these components is highlighted by the fact that defectsin GBM-specific type IV collagen or laminin lead to Alport syndromeand Pierson syndrome, respectively (4–6).
The podocyte foot processes and the slit diaphragm serve asa final filtration barrier in the glomerulus. This filtrationmachinery contains a number of unique molecular components.Two fundamental molecules of the slit diaphragm, nephrin andpodocin, were discovered through studies in human proteinurickidney diseases (7,8). Absence of NEPH1 and FAT1, two othermembrane-spanning proteins of the slit diaphragm, result inmassive proteinuria in mice (9,10). Intracellularly, the slitdiaphragm is connected to the actin cytoskeleton via nck proteinsand CD2-associated protein (CD2AP) (11,12). Mice that lack nckproteins from the podocytes and CD2AP knockout mice developmassive proteinuria (12,13). In addition, studies that werecarried out in human proteinuric kidney diseases and knockoutmice showed the critical importance of podocyte-associated proteins-actinin-4 (ACTN4), synaptopodin, podocalyxin, and glomerularepithelial protein 1 for the glomerular filtration barrier (14–17).Importantly, many of these essential proteins are highly specificfor the glomerulus and the podocyte. Nephrin, podocin, NEPH1,FAT1, synaptopodin, -actinin-4, and glomerular epithelial protein1, for example, are expressed in the kidney only by podocytes(7,17–21). This emphasizes the unique function and molecularcomposition of the podocyte foot process and the slit diaphragm.
Recently, we identified >300 glomerulus-upregulated transcriptsthrough large-scale sequencing and microarray profiling of theglomerular transcriptome (22). In our approach, we chose fivenovel transcripts that were identified in the microarray analysesand characterized their expression profile in detail. We showthat four of the transcripts/proteins are expressed exclusivelyin the kidney by podocytes. Using immunoelectron microscopy,we localize two of these proteins to the slit diaphragm andthe foot process. In addition, we show that one of the proteinsis expressed in the kidney only by the GEC and, remarkably,not by other endothelial cells. The identification of thesenovel molecular components provides fresh insights into glomerularbiology.
Reverse Transcriptase–PCR and Northern Blot
The expression of glomerular-enriched transcripts in a varietyof mouse tissues was studied using reverse transcriptase–PCR(RT-PCR) and Northern Blot. Gene-specific oligonucleotides forPCR analysis were designed according to the predicted cDNA sequences(http://www.ensembl.org). Primer sequences and sizes of expectedPCR products were as follows: Dendrin, left 5'-AATGGAGAGGCCTTGAACCT-3',right 3'-GGGAAGGCCTAAAAGTGTCC-5', 502 bp; EHD3, right 5'-CAAGAGCAGGGTTAGGCACT-3',5'-CTAATGAACGGGAGGCTGAG-3', 498 bp; sh2d4a, left 5'-TCTGGGCTGGTCAAGTCTCT-3',right 5'-GAAGCTGCTTTCCTGGTGAC-3', 573 bp; plekhh2, left 5'-CTGGATTCGCACTCTTCACA-3',right 5'-CGCTGGAGTTGAGAAAGGTC-3', 353 bp; and 2310066E14Rik,left 5'-GGAGCAGTTCACCATCTTCC-3', right 5'-TGTCGATGAGCAGACTGTCC-3',500 bp). As a template for PCR analysis, we used cDNA librariesthat were generated from various adult mouse tissues (MouseMultiple Tissue cDNA Panel I; Clontech Laboratories, Palo Alto,CA). PCR amplification was carried out with TaqDNA polymerase(Invitrogen, Carlsbad, CA), and the amplified fragments wereanalyzed on 1.5% agarose gel.
For Northern blots, we used cDNA probes that were obtained fromthe amplification of cDNA libraries (see previous paragraph).The probes were 32P-labeled using the Rediprime II random primerlabeling system (Amersham Pharmacia Biotech, London, UK), andthe probes were hybridized to the blots that contained RNA thatwere isolated from various mouse organs (Mouse MTN Blot; ClontechLaboratories). Hybridizations also were performed on blots thatcontained mRNA that was isolated from mouse kidney fractionsthat contained either only glomerular tufts or the kidney excludingglomeruli. Glomerular fractions were isolated as described previously(23). The hybridizations were performed according to the standardprocedures. As a positive loading control, we used glyceraldehyde-3-phosphatedehydrogenase probe (Clontech Laboratories).
In Situ Hybridization
The probes for in situ hybridizations were synthesized by subcloningof the PCR products that were obtained from RT-PCR analyses(see previous section) into the pCR II-TOPO Dual Promoter Vector(Invitrogen). Antisense and sense RNA were prepared by usingT7 or SP6 polymerases. In situ hybridization experiments with35S-labeled probes were performed on snap-frozen tissue sectionsthat were collected from newborn mouse kidneys as describedpreviously (24).
Production of Polyclonal Antibodies Directed against Novel Glomerular Proteins
We raised antisera directed against novel glomerular proteinsby purifying recombinant proteins with affinity tags and byimmunizing NZW rabbits with these antigens using standard protocols(SVA, Uppsala, Sweden; KTH, Stockholm, Sweden). The generationof antigens is described briefly here.
For the production of dendrin and ehd3 antigens, we generatedmouse recombinant proteins. Dendrin residues 55 to 384 werecloned into the pET-28a(+) expression vector (Novagen, Madison,WI), whereas ehd3 residues 260 to 424 were cloned into the pGEX4T-3 vector (Amersham Biosciences). The his-tagged dendrin andthe GST-tagged ehd3 recombinant proteins were solubilized frominclusion bodies in 8 M urea. Then, dendrin antigen was purifiedusing sequential S-Sepharose ion exchange and Sephadex S-200gel filtration columns (Amersham Biosciences), whereas his-taggedehd3 antigen was purified using G-sepharose 4B ion exchangecolumn (Amersham Biosciences). Finally, two NZW rabbits wereimmunized with each prepared antigen.
Human recombinant proteins were generated for production ofsh2d4a, plekhh2, and 2310066E14Rik antigens. Two sequences wereselected for sh2d4a suitable for antigen production: Residues27 to 150 and 327 to 443. For plekhh2 and 2310066E14Rik, selectedsequences comprised amino acids 223 to 357 and 845 to 961, respectively.The protein fragments were expressed as recombinant proteinswith a dual tag: A hexahistidine tag that enabled purificationof the expressed antigen by immobilized metal ion affinity chromatographyand an albumin-binding protein fragment of Streptococcus proteinG with immunopotentiating capabilities (25). Antibodies wereraised by immunization of NZW rabbits, and obtained sera wereused for purification of monospecific antibodies. Purificationwas performed using a two-step purification procedure includingdepletion of tag-specific antibodies and subsequent affinitypurification as described previously (26).
Western Blotting
In Western blotting, we compared the extracts of glomerulartufts and kidneys that lacked glomeruli. The glomeruli wereisolated from 8- to 12-wk-old adult mice and from human cadaverkidneys that were unsuitable for transplantation (from the IVDepartment of Surgery of Helsinki, Finland). The isolation methodshave been described previously (23,27). The Western blottingwas done following standard procedures using polyvinyl difluoridemembrane and horseradish peroxidase–conjugated secondaryantibody (Amersham Biosciences). As a positive loading control,we used polyclonal anti–-actin antibody (Abcam, Cambridge,UK).
Immunohistochemistry
For immunofluorescence stainings, kidney samples were collectedfrom either 4- to 12 wk-old mouse kidneys or adult human cadaverkidneys that were unsuitable for transplantation (from the IVDepartment of Surgery of Helsinki, Finland). The samples weresnap-frozen, and the cryosections (10 µm) were postfixedwith cold acetone (–20°C) followed by blocking in5% normal goat serum. The primary antibodies were incubatedovernight at 4°C, followed by a 1-h incubation with thesecondary antibody. For double-labeling experiments, the incubationswere performed sequentially.
The primary antibodies used are described above in Productionof Polyclonal Antibodies Directed against Novel Glomerular Proteins.For double-labeling experiments, we used anti-mouse synaptopodin(Progen, Heidelberg, Germany), anti-mouse CD31 (Pharmingen Int.,San Diego, CA), anti-mouse collagen type IV 5 (H53; gift fromDr. Sado, Shigei Medical Research Institute, Yamada, Japan),anti-human vimentin (Zymed Laboratories, San Francisco, CA),and anti-human nephrin 50A9 (28) antibodies. DAPI reagent waspurchased from Molecular Probe (Eugene, OR). Secondary antibodieswere purchased from Jackson ImmunoResearch Laboratories (WestGrove, PA). Microscopy was performed with standard Leica confocallaser scanning microscope.
Immunoelectron Microscopy
For immunoelectron microscopy, the samples from mouse and humanrenal cortexes were fixed in a solution that contained phosphate-buffered3.5% paraformaldehyde and 0.02% glutaraldehyde. After fixation,the samples were embedded in 10% gelatin, infiltrated with 2.3M sucrose in PBS, and frozen in liquid nitrogen. Immunolabelingexperiments were done as described previously (29).
Ethical Considerations
This study was approved by the ethical committees of the KarolinskaInstitute and The Hospital for Children and Adolescence of theUniversity of Helsinki.
Dendrin
Dendrin is an intracellular protein without known motifs orfunction (30,31). RT-PCR and Northern blotting experiments demonstratedthe presence of dendrin transcript in brain, kidney, and lungtissues (Figure 1). The size of the transcript (approximately3.5 kb) is in agreement with the predicted mouse dendrin cDNA(NCBI cDNA accession no. XM_912147).
Figure 1. Expression of novel glomerular transcripts in various mouse organs using reverse transcriptase–PCR (RT-PCR; A) and Northern blot (B) analysis. Dendrin mRNA (approximately 3.5 kb) is present abundantly in brain tissue, whereas weaker expression is observed in kidney tissue. In Northern analysis, dendrin probe hybridizes also a weak band in lung mRNA. Ehd3 mRNA is detected in all organs analyzed using RT-PCR, whereas Northern blot reveals the presence of ehd3 mRNA (approximately 3.6 kb) only in kidney, liver, and brain tissues. Sh2d4a mRNA is detected in kidney, muscle, lung, and heart tissues using RT-PCR, whereas in Northern blotting, sh2d4a mRNA (approximately 3.3 kb) is detectable only in kidney and heart tissues. Plekhh2 mRNA (approximately 4.5 kb) is detected only in testis tissue using RT-PCR and Northern blotting. 2310066E14Rik mRNA is detected in testis, kidney, lung, spleen, and heart tissues using RT-PCR, whereas Northern blotting shows the presence of 2310066E14Rik transcript (approximately 4.4 kb) in all organs analyzed.
Two antisera raised against the mouse dendrin protein gave similarresults in our experiments, and the specificity of the antiserawas confirmed by transfection of HEK293 cells with the full-lengthdendrin cDNA (data not shown). In the analysis of kidney fractions,both Northern and Western blotting revealed the presence ofdendrin (mRNA and protein) in glomerular tufts, whereas no mRNAor protein was detected in the kidney extract that lacked glomeruli(Figure 2). Using Western blotting, anti-dendrin antisera recognizeda protein that was approximately 80 kD (Figure 2). This is inline with the published rat dendrin protein size (81 kD) (30).
Figure 2. Expression of novel glomerular transcripts/proteins in glomerular and the rest of the kidney fractions as detected using Northern and Western blotting. All of the transcripts (dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik) analyzed with Northern blotting show strong expression in the glomerulus fraction, whereas no expression is detected in the kidney fraction that lacked glomeruli. The sizes of glomerular transcripts are as follows: Dendrin 3.5 kb, ehd3 3.6 kb, sh2d4a 3.3 kb, plekhh2 4.5 kb, and 2310066E14Rik 4.4 kb. Control hybridization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe shows strong signal in both lanes. In Western blotting, the antibodies/antisera directed against dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik give immunoreactivity in glomerular fractions, whereas the rest of the kidney fraction remains negative. The sizes of the proteins detected in Western blotting are as follow: Dendrin 81 kD, ehd3 68 kD, sh2d4a 46 kD, plekhh2 120 kD, and 2310066E14Rik 120 kD. Anti–-actin antibody, which was used as a loading control, gave similar reactivity in both lanes.
The glomerulus specificity of dendrin mRNA was verified usingin situ hybridization. An antisense probe showed signal exclusivelyin the glomeruli (Figure 3). With a higher magnification, signalcould be localized to the podocytes in newborn mouse kidney(Figure 3, inset). Sense probe gave only background signal (datanot shown). In immunofluorescence staining, anti-dendrin antiseragave strong immunoreactivity in glomeruli, whereas the restof the kidney remained negative (Figure 4). In glomerular tufts,dendrin protein was observed as a linear line around glomerularcapillary loops. The staining for dendrin was found on the urinaryside of CD31 immunoreactivity, whereas double labeling withCOL4a5 revealed nearly overlapping of the two immunoreactivities.However, staining for dendrin was localized mostly on the urinaryside of COL4a5 reactivity. Double staining with synaptopodinantibody revealed almost complete overlapping of the two proteinsat the light microscopic level. In immunoelectron microscopy,gold label for dendrin was concentrated at the cytoplasmic insertionsites of the slit diaphragms (Figure 5A).
Figure 3. Expression of novel glomerular transcripts in newborn mouse kidneys as revealed by radioactive in situ hybridization. Insets show capillary loop stage glomerulus in which podocytes are localized as one row (arrowheads). (A) Strong signal for dendrin mRNA is detected in the glomerular tufts of newborn mouse kidneys, whereas the rest of the kidney remains negative. With a higher magnification, the signal for dendrin mRNA can be localized to the podocytes (inset). (B) Probe for ehd3 gives strong signal in the glomerular tufts, and no signal is detected outside the glomeruli. With higher magnification, ehd3 mRNA can be localized to the vascular cleft in the developing glomerulus, which indicates localization to the glomerular endothelial ells (GEC). (C) Signal for 2310066E14Rik mRNA is detected in the glomeruli, whereas no signal is observed in the rest of the kidney. With higher magnification, the signal can be localized to the podocytes (inset). Magnifications: x50 in A through C; x300 in insets.
Figure 4. Expression of novel glomerular proteins in adult mouse or human kidneys as detected using double immunofluorescence staining. All antibodies give immunoreactivity exclusively in the glomerular tufts of adult mouse/human kidneys, and no significant signal is detected outside glomeruli. Double immunolabeling experiments were done with CD31, synaptopodin, vimentin, and nephrin (red). DAPI (nuclear) staining is shown in blue. Analysis with high magnification using confocal microscope reveals subcellular localization of the antigens (green) as follows. Dendrin-specific immunofluorescence (green) is found in close proximity of CD31 and COL4A5 proteins (red). The staining for dendrin is found mostly on the urinary side of these two markers. Double labeling with synaptopodin reveals almost complete overlapping (yellow) of dendrin and synaptopodin immunoreactivities. Both proteins are distributed along the glomerular capillary loops as a linear line. Ehd3 immunoreactivity is detected as a linear line in close proximity of CD31 reactivity. The staining for ehd3 is localized mostly on the urinary side of CD31 staining. The double labeling with COL4A5 and synaptopodin shows partial overlapping of ehd3 immunoreactivity with these two markers. Ehd3 immunoreactivity can be observed on both the capillary and the urinary side of the COL4A5 and synaptopodin stainings. The staining for sh2d4a is found as a line surrounding the glomerular capillaries. The immunoreactivity for sh2d4a does not overlap with vimentin or nuclear stainings. Instead, the staining for sh2d4a practically completely overlaps (yellow) with that of nephrin. Plekhh2 protein is observed as a line around the glomerular capillary loops. Double stainings with DAPI and anti-vimentin antibody do not show overlapping. Instead, the staining for nephrin mostly overlaps with plekhh2 immunoreactivity. Staining for 2310066E14 protein was localized to podocytes. Double staining with DAPI does not show co-localization, although diffuse cytoplasmic staining is observed in close proximity of nuclear staining. The staining for vimentin partially overlaps (yellow) with that of 2310066E14Rik. The staining for nephrin does not overlap with 2310066E14Rik immunoreactivity. Magnifications: x80 in overviews, x400 in single glomeruli, and x2000 in blow-ups.
Figure 5. Immunoelectron microscopic localization of dendrin, ehd3, and SH2D4A in mouse and human kidneys. (A) Gold label (10 nm) for dendrin binds to epitopes at the inner leaflet of the foot process plasma membrane in regions where they appose to form slit diaphragms (arrows). (B) Gold label for ehd3 protein is observed in the cytoplasm of endothelial cells. The gold label for ehd3 often is found at the level of endothelial foramina. (C and D) Polyclonal antibodies directed against SH2D4A protein recognize an antigen at the cytosol of podocyte foot processes. Gold label is distributed diffusely in the cytosol of the foot processes. Fp, foot process. Bar = 100 nm.
Ehd3
Ehd3 is predicted to be a cytosolic protein (32). RT-PCR andNorthern blotting analyses showed that ehd3 transcripts werepresent in many different mouse organs (Figure 1). Using Northernblotting, the strongest expression was observed in kidney, liver,and brain tissues. The size of ehd3 mRNA was approximately 3.6kb, which is in line with the published transcript size (32).
In the analysis of kidney fractions, both Western and Northernblotting revealed the presence of ehd3 (mRNA and protein) exclusivelyin the glomerular fractions, whereas no expression was detectedin the kidney extract excluding glomeruli (Figure 2). With theuse of Western blotting, a protein that was approximately 68kD in size was detected, which is in line with the predictedehd3 protein size (Ensembl protein ENSMUSP00000024860).
In situ hybridization of the newborn mouse kidney confirmedthe findings because strong signal for ehd3 mRNA was observedsolely in glomerular tufts (Figure 3). With higher magnification,the signal for ehd3 mRNA was localized to the GEC in newbornmouse kidney (Figure 3, inset). This is in line with our previousmicroarray results; ehd3 mRNA showed 11-times upregulation inthe nonpodocyte fraction of the glomerulus transcriptome (22).In immunofluorescence stainings, anti-ehd3 antisera gave strongreactivity in glomerular capillary tufts, whereas the rest ofthe kidney remained negative (Figure 4). In the glomerulus,the staining for ehd3 showed linear staining pattern aroundglomerular capillary loops. In double labeling with anti-CD31antibody, the immunoreactivity for ehd3 was found in close proximityof this endothelial marker. The staining for ehd3, however,was localized mostly on the urinary side of CD31 immunoreactivity.The double labeling with COL4A5 and synaptopodin showed partialoverlapping of the ehd3 protein with these two proteins. Surprising,the staining for ehd3 was observed occasionally on the urinaryside of these two markers. This probably reflects the closeproximity of the basal aspects of the GEC, the GBM, and thefoot processes. Therefore, we performed immunoelectron microscopyfor ehd3, which revealed that this protein was localized tothe GEC (Figure 5B). The gold labeling in the GEC was abundantwith only minimal background labeling of other kidney cell typesand tissue components (data not shown). In the endothelium,the label often was confined to the level of the endothelialforamina.
Sh2d4a
Sh2d4a is a previously uncharacterized cytosolic protein. Expressionprofiling using RT-PCR and Northern blotting revealed the presenceof sh2d4a transcripts in kidney, liver, and heart tissues (Figure 1).The size of the transcript (approximately 3.3 kb) is in linewith the predicted transcript size (XM_134197).
Two affinity-purified polyclonal antibodies raised against sh2d4aprotein gave similar results in our experiments (data not shown).In the analysis of kidney fractions, sh2d4a mRNA and proteinboth were detected in the glomerular fraction, whereas no signalwas observed in the kidney fraction that lacked glomeruli (Figure 2).In the Western analysis, the antibodies detected a protein sizeof approximately 46 kD.
In immunofluorescence stainings, strong sh2d4a-specific immunoreactivitywas observed in glomeruli, whereas the rest of the kidney wascompletely negative (Figure 4). Double labeling with nephrinrevealed almost complete overlapping of the two proteins inthe glomerulus at the light microscopic level (Figure 4). Nooverlapping was observed in double stainings with anti-vimentinantibody or DAPI stainings. In immunoelectron microscopy, goldlabel for sh2d4a was found diffusely distributed in the cytoplasmof podocyte foot processes (Figure 5, C and D).
Plekhh2
Plekhh2 is a previously uncharacterized intracellular protein.Using RT-PCR and Northern blotting, we detected the presenceof plekhh2 transcript only in testis and kidney tissues (Figure 1).The size of plekhh2 transcript was approximately 4.5 kb.
In the analysis of kidney fractions, both Northern and Westernblotting experiments showed the presence of plekhh2 (mRNA andprotein) in the glomerulus fraction (Figure 2). Two plekhh2transcripts were detected in the glomerulus (data not shown).No plekhh2 mRNA or protein was detected in the kidney fractionthat lacked glomeruli. Affinity-purified polyclonal antibodyrecognized a protein of approximately 120 kD.
In immunofluorescence stainings, anti-plekhh2 antibody gaveglomerulus immunoreactivity, and no significant signal was observedoutside glomeruli (Figure 4). With higher magnification, plekhh2was detected mainly as a linear line around glomerular capillaryloops, indicating localization to the podocyte foot processes.At the light microscopic level, plekhh2 seemed to co-localizepartially with nephrin. No overlapping was observed in doublestainings with anti-vimentin antibody or DAPI stainings (Figure 4).
2310066E14Rik
2310066E14Rik transcript codes for an unknown intracellularprotein. Using RT-PCR and Northern blotting, we observed wideexpression of 2310066E14Rik transcript in mouse tissues (Figure 1).In Northern blotting, the probe for 2310066E14R hybridized anmRNA of approximately 4.4 kb.
In the analysis of kidney fractions, both Northern and Westernanalyses revealed the presence of 2310066E14Rik (mRNA and protein)in the glomerulus fraction, whereas no mRNA or protein was observedin the kidney fraction that lacked glomeruli (Figure 2). Polyclonalantibodies raised against this novel protein detected a proteinof approximately 125 kD.
The exclusive glomerulus expression was confirmed using in situhybridization; antisense probe gave strong signal in the glomeruli,and no signal was observed outside glomeruli (Figure 3). Witha higher magnification, signal for 2310066E14Rik could be localizedto the podocytes in newborn mouse kidney (Figure 3, inset).Sense probe showed only background signal (data not shown).In immunofluorescence stainings, strong immunoreactivity wasobserved in the glomerular tufts, and no staining was observedoutside glomeruli (Figure 4). Immunoreactivity was observedon the outer aspects of the glomerular tufts in which diffusecytoplasmic staining mainly was observed. Double labeling withnephrin antibody showed that the product of 2310066E14Rik genewas localized on the urinary side of nephrin reactivity. Doublelabeling with anti-vimentin antibody showed partial overlappingreactivity. No overlapping was observed with DAPI stainings,although staining for 2310066E14Rik protein often was localizedin close proximity of the nuclear staining. To conclude, thestaining results for 2310066E14Rik protein suggest stronglythat this protein localizes to the cytoplasm of the podocytecell bodies and major processes.
Recently, we generated a comprehensive catalogue of glomerulus-expressedtranscripts (22). Through large-scale expressed sequence tagsequencing and microarray analyses, we characterized the transcriptomeof the mouse glomerulus and identified >300 glomerulus-enrichedtranscripts. One of the transcripts discovered, a podocyte transcriptionfactor Foxc2, was investigated further through analysis of Fox2knockout mice. The glomerulogenesis in the mice was arrestedbefore the capillary loop stage of glomerular development, indicatingthat Fox2 is essential for podocyte development. This exampledemonstrated that our large-scale approach was a feasible methodto discover genes that are critical for normal glomerular function.In this report, we chose five other glomerulus-upregulated transcriptsand performed a detailed expression analysis. It is interestingthat we discovered that all five transcripts and their proteinproducts were expressed in the kidney only by glomerular cells.Therefore, our results further emphasize the importance of thelarge-scale transcript profiling effort from the standpointof nephrology.
The glomerulus filtration apparatus is an extremely specializedmicro-organ that is dedicated to function as a sophisticatedmolecular sieve. The filter is composed of a variety of uniquemolecules that assist in the orchestration of its specializedfunction. Therefore, several critical glomerular proteins, suchas nephrin, podocin, NEPH1, and FAT1, have very restricted expressionpattern. In the kidney, these crucial proteins are expressedexclusively by podocytes. The proteins discovered in this studyshare this limited expression pattern. This suggests that thenovel proteins identified in our study also may have a highlyspecific role in the glomerulus.
Dendrin is a poorly characterized cytosolic protein with noknown function or homology to other proteins or protein domains.Previously, dendrin was reported to be specific for the dendriticprocesses of certain neuronal cells of the forebrain (30). Particularly,dendrin showed high expression in the regions of the brain thatare capable of high synaptic plasticity. Because dendrin isnot found in all dendritic processes, it cannot be essentialfor these structures. We identified dendrin in the kidney glomerulusand localized it to the cytoplasmic face of the podocyte slitdiaphragm. Both dendritic processes and foot processes are long,slender cellular extensions, and they share many structuralcomponents, such as the actin-rich cytoskeleton (33), and functionalfeatures such as plasticity. Because dendrin also is sharedby both structures, it could contribute to this plasticity.In the podocyte foot process, the plasticity is required towithstand the continuous filtration pressure (34). However,the localization of dendrin at the cytoplasmic face of the slitdiaphragm suggests that dendrin may act as a linker moleculebetween the slit diaphragm and the actin cytoskeleton. In supportof this idea, dendrin was shown recently to interact in vivowith two cytoskeletal components, -actinin and membrane-associatedguanylate kinase inverted (35). Recently, dendrin also was shownto interact with the CD2AP homologue CIN85 in vitro (36). Wehave not detected this interaction in the glomerulus by immunoprecipitatingglomerulus lysates with CD2AP and dendrin antibodies (data notshown).
Ehd3 belongs to the protein family of eps15 homology domain–containingproteins, which are thought to be associated with endocyticvesicles (37). With the use of Northern blotting, ehd3 mRNAwas found previously in mouse brain and kidney tissues (32).This is in line with our results because we detected ehd3 mRNAin brain, kidney, and liver tissues using Northern analysis.More exciting, we discovered that ehd3 was expressed in thekidney only by GEC. To our knowledge, this is the first proteinshown to be expressed only by GEC and not by other endothelialcells in the kidney. This is of critical importance becausethe use of the ehd3 promoter enables the generation of a GEC-specificCre mouse line. Such a mouse line could provide an importanttool for future glomerulus research.
Sh2d4a encodes a novel docking protein of SH2 signaling proteinfamily. Members of this protein family probably play a rolein intracellular signaling, but no studies have been reportedto elucidate their expression or function in mammals. Plekhh2encodes for an uncharacterized cytosolic protein with pleckstrinhomology, myth4, and band 4.1 domains. Pleckstrin homology domainsare implicated in phosphatidylinositol phospholipid signaling(38), the band 4.1 domain is a membrane localization domainthat is capable of binding integral membrane proteins (39),and the myth4 domain has no known function. However, the combinationof myth4 and band 4.1 domains is found in several unconventionalmyosin proteins (40), which often are localized to actin-richprocesses of the cells. It is interesting that we discoveredthat both sh2d4a and plekhh2 proteins had a markedly restrictedexpression pattern in mouse tissues. In the kidney, both proteinswere expressed only by podocytes, and at the subcellular level,we localized these cytosolic proteins specifically to the footprocesses. Sh2d4a protein may be involved in the slit diaphragmsignaling. The peculiar domain structure of Plekhh2 proteinsuggests, however, that it may be involved in connecting theslit diaphragm to the actin cytoskeleton.
We have identified five novel, highly glomerulus-specific proteins.It is interesting that these proteins share the similar restrictedexpression pattern with several essential podocyte proteins.Therefore, it is reasonable to speculate that several of thesenovel glomerular proteins have dedicated roles in glomerulusphysiology. This report provides new insights into the biologyof the glomerulus and creates numerous possibilities to characterizethe role of these novel proteins in the glomerular physiologyand pathology.
K.T. is co-founder of and has stock ownership in NephroGenexInc. C.B. has stock ownership in NephroGenex Inc.
Acknowledgments
This study was supported by grants from the Knut and Alice WallenbergFoundation, the Swedish Research Council, Novo Nordisk, StrategicResearch, Söderberg and Hedlund foundations, Inga-Brittand Arne Lundberg Foundation, the Juselius Foundation, the FinnishCultural Foundation, and the program for Postgenomic Researchand Technology in South-West Sweden.
We are grateful to Dadi Niu and Satu Kuure for technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-actinin-4 (ACTN4), synaptopodin, podocalyxin, and glomerular epithelial protein 1 for the glomerular filtration barrier (14–17). Importantly, many of these essential proteins are highly specific for the glomerulus and the podocyte. Nephrin, podocin, NEPH1, FAT1, synaptopodin, 
-actin antibody (Abcam, Cambridge, UK). 
