*Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; and Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan.
Correspondence to Dr. Hiroshi Kawachi, Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. Phone: 81-25-227-2160; Fax: 81-25-227-0770;
ABSTRACT. Podocin is identified as a product of the gene mutatedin a patient with autosomal recessive steroid-resistant nephroticsyndrome. Although podocin is reported to be located at theslit diaphragm area, the precise role of podocin for maintainingthe barrier function of the slit diaphragm has not been clearlyelucidated. A rat homologue of podocin was cloned, and the expressionof podocin was investigated and then compared with the nephrinand the ZO-1 expressions in rat experimental proteinuric modelsand in developing glomeruli. Amino acid sequences of rat andhuman podocin are highly homologous (84.3% identity). The domainstructure of podocin is also highly conserved between rat andhuman. The mRNA expression for podocin was detected in glomeruliand the nerve tissues. The localization of podocin has closeproximity to that of nephrin in normal adult rat glomeruli.Podocin staining was restricted to the basal side of the podocyteof the early developing stage, whereas nephrin staining wasdetected on the basolateral surface of podocyte. The redistributionof podocin was observed in the anti-nephrin antibody (ANA)-inducednephropathy and puromycin aminonucleoside (PAN) nephropathy.The redistribution of podocin paralleled with nephrin in ANAnephropathy but not in PAN nephropathy. Podocin is observedat the site of tight junction newly formed in proteinuric statein PAN nephropathy. It is postulated that podocin is one ofthe critical components of a slit diaphragm for maintainingthe barrier function of the glomerular capillary wall. E-mail:kawachi@med.niigata-u.ac.jp
The primary barrier for ultrafiltration of plasma in a glomerularcapillary wall comprises three layers: a fenestrated endothelialcell, a glomerular basement membrane (GBM), and a glomerularepithelial cell (podocyte). Although the role of the GBM inrestricting the presence of macromolecules has been emphasizedfor nearly two decades (1,2), several recent studies have shownthat slit diaphragms located between the foot processes playthe critical role of a barrier to retain macromolecules (3–7).In the past several years, some molecules were reported to beassociated with the slit diaphragm (8–11). Nephrin isconsidered to be a critical component of the slit diaphragmfor maintaining the barrier function (9,12–15). CD2APis reported to be associated with nephrin (16). Boute et al.(17) cloned a novel gene, NPHS2, mutated in patients with autosomalrecessive steroid-resistant nephrotic syndrome and reportedthat this gene product, podocin, is located at the slit diaphragmarea (18). Podocin is an integral membrane protein of 383 aminoacids, with a single membrane domain forming a hairpin-likestructure and with both N- and C-terminal domains in the cytosol.Recent reports indicate that podocin interacts with CD2AP andnephrin (19,20); however, the precise role of podocin for maintainingthe barrier function of the slit diaphragm is not yet clarified.To address this, more studies using experimental models wouldbe necessary. We cloned a rat homologue of podocin and producedan anti-rat podocin antibody in rabbits, because several experimentalproteinuric models were established in rats. Then, we investigatedthat the expression of podocin and compared it with the nephrinexpression in proteinuric states and during the maturation ofglomeruli.
The results showed that the deduced amino acid sequence of ratpodocin showed 84.3% identity to human podocin. Podocin wasredistributed in both proteinuric states caused by puromycinaminonucleoside (PAN) and by anti-nephrin antibody (ANA). Itis postulated that podocin is one of the critical componentsof the slit diaphragm for maintaining the barrier function ofthe glomerular capillary wall.
Cloning of Rat Podocin
To clone a rat homologue of podocin, we employed a PCR withthe primers designed from a human podocin sequence. PCR cloningwas performed basically according to the method described previously(14). Total RNA was prepared from normal Wistar rat glomeruliwith TRI ZOL (Life Technologies BRL, Gaithersburg, MD), whichwas utilized to synthesize cDNA with SuperScript II (Life TechnologiesBRL) according to the manufacturer’s protocol. SynthesizedcDNA was used as a template. Templates were amplified in a DNAwith PROGRAM TEMP CONTROL SYSTEM PC-800 (ASTEC, Fukuoka, Japan)for 35 to 50 cycles at 48°C to 55°C of annealing temperature.The PCR products that were close to an expected size were clonedinto a Topo Vector (Invitrogen, Carlsbad, CA), and DNA sequenceswere determined by an automated DNA sequencer (ABI 310; Perkin-ElmerJapan, Urayasu, Japan). From a partial clone of rat podocin,gene-specific primers were designated and PCR was performedagain. To obtain the sequences containing the initiation codonof rat podocin, we performed a 5'-RACE with a 5'-RACE Version2.0 (Life Technologies BRL). To obtain the stop codon, a 3'-RACEwas performed. The 5'-RACE and 3'-RACE products were clonedinto a Topo Vector and sequenced.
Antibodies
To produce a specific antibody for rat podocin, we chose a peptideof 21 amino acids, RGNRGRARPDAGVERQSAGRM, as immunogen. Tworabbits were immunized with 1.0 mg of peptide conjugated withthe carrier protein KLH and boosted twice with 0.5 mg of antigen.The rabbits were bled 2 wk after the last immunization. Thespecificity of the anti-podocin antibody was confirmed by theWestern blot analysis. Mouse monoclonal antibody to rat nephrin(mAb 5-1-6), and rabbit polyclonal anti-rat nephrin antibodywere prepared as described previously (6,14). Mouse monoclonalanti-ZO-1 antibody, anti-glial fibrillary acidic protein (GFAP),and anti-rat endothelial cell antigen 1 (RECA1) antibodies werepurchased from Zymed Laboratory Inc. (San Francisco, CA), ChemiconInternational (Temeculla, CA), and Serotec Ltd (Oxford, UK),respectively.
Western Blot Analysis
Normal rat glomeruli are isolated with PBS-protease inhibitors(PI) and sequentially solubilized with 1% Triton X-100, RIPA-buffer(0.1% SDS, 1% DOC, 1% Triton X-100, 150 mM NaCl, 10 mM EDTAin 25 mM Tris-HCl, pH 7.2) with PI, and separated into TritonX-100–soluble (TXS), RIPA-soluble (RIPAS), and RIPA-insoluble(RIPAI) fractions. RIPAI fraction was solubilized with SDS-PAGEsample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol in62.5 mM Tris-HCl, pH 6.8). Solubilized material was subjectedto SDS-PAGE with 10% or 7.5% acrylamide gel according to themethod of Laemmli et al. (21) and transferred to a nitrocellulosemembrane (Bio-Rad, Hercules, CA) by electrophoretic transblottingfor 30 min using Trans-Blot SD (Bio-Rad). After blocking withBSA, the strips of membranes were exposed to one of the following:anti-rat podocin, anti-rat podocin pre-absorbed with rat podocinpeptide used for immunogen, pre-immune rabbit serum, or anti-ratnephrin antibody. They were then washed and incubated with alkalinephosphatase-conjugated anti-rabbit IgG (Bio Source International,Tago Immunologicals, Camarillo, CA). The reaction was developedwith an alkaline phosphatase chromogen kit (5-bromo-4-chloro-3-indolilphosphate p-toluidine salt/nitro blue tetrazolium; Biomedica,Foster City, CA). For semiquantitative Western blot analysis,glomeruli from normal or proteinuric rats were solubilized withSDS-PAGE sample buffer. The protein concentration was measuredby the bicinchoninic acid method (Pierce Chemical, Rockford,IL), and 75 µg were subjected to SDS-PAGE and transferredto nitrocellulose membrane. The stripes were exposed to anti-podocinantibody and then incubated with alkaline phosphatase-conjugatedanti-rabbit IgG. Color development was performed as describedabove, and the density of the positive bands was quantitatedby Densitograph (ATTO, Tokyo, Japan). To analyze the subcellularlocalization of podocin, 10,000 glomeruli were sequentiallysolubilized with 1% Triton X-100, RIPA-buffer, and SDS samplebuffer as described above. Equal volumes of fractions sequentiallysolubilized were subjected to SDS-PAGE with 10% acrylamide gel,and Western blot analysis was performed with anti-podocin antibodyas described above.
Organ Specificity Analysis
Six-week-old normal Wistar rats were sacrificed, and the liver,lung, heart, spleen, stomach, small intestine, large intestine,skeletal muscles, cerebrum, cerebellum, medulla oblongata, testes,ovaries, and kidneys were removed from them. Glomeruli wereisolated by the sieving method. Total RNA was prepared fromthese organs or glomeruli or cortex of the kidneys with TRIZOL Reagent (Life Technologies BRL). mRNA expressions for nephrinwere analyzed by RT-PCR. Five milligrams of total RNA preparedwere utilized to synthesize cDNA with SuperScript II (Life TechnologiesBRL) according to the manufacturer’s protocol. Three microlitersof the reverse transcription mixture were utilized for the subsequentPCR. As primers for rat podocin, we employed sense primer CCTGTG AGT GGC TTC TTG TCC TC and antisense primer GGA GAC GCTTCA TAG TGG TTT GCA. The PCR product corresponds to 378 bp.The PCR was performed for 30 to 35 cycles of denaturation at94°C for 30 s, annealed at 58°C for 30 s, and extendedat 72°C for 1 min. The PCR products were subjected to electrophoresisthrough 1.5% agarose gel and are stained with ethidium bromide.The PCR product was subcloned into a TopoVector (Invitrogen),sequenced, and confirmed to be a specific product of rat podocin.The amount of mRNA in the sample was monitored by the mRNA expressionof the internal control glyceraldehyde-3-phosphate dehydrogenase(GAPDH). The sequences of primers for rat GAPDH are CTC TACCCA CGG CAA GTT CAA and GGA TGA CCT TGC CCA CAG C.
Immunofluorescence Microscopy
An immunofluorescence study was performed basically accordingto the method described previously (22). Cryostat sections ofadult and neonatal rat kidney, cerebrum cerebellum, and themedulla oblongata were fixed with acetone for 1 min, incubatedwith the primary antibodies described above, stained with FITC-conjugatedanti-rabbit IgG (DAKO, Glostrup, Denmark) (for anti-podocin)or TRITC-conjugated anti-mouse IgG1 (Southern Bio-technologyAssociates, Inc., Birmingham, AL) (for anti-nephrin mAb 5-1-6),and observed with an immunofluorescence microscope (BX50; Olympus,Tokyo, Japan). To compare the localization of podocin with thatof nephrin, ZO-1, GFAP, or RECA1, dual immunofluorescent labelingwas carried out. The cryostat sections were incubated with rabbitanti-rat podocin, FITC-conjugated anti-rabbit IgG, murine monoclonalantibodies (anti-nephrin mAb 5-1-6, anti-ZO-1, anti-GFAP, anti-RECA1),and then TRITC-conjugated anti-mouse IgG1 in this order.
Immunoelectron Microscopy
Normal and proteinuric rats were perfused with 1.0% paraformaldehydefixative buffered with 0.1 mol/L sodium phosphate buffer (PB;pH 7.4) and immersed in the same fixative for 1 h at 4°C.The samples were rinsed three times with 10% sucrose for 20min. The tissue samples were then infiltrated with 40% polyvinylpyrrolidone(Sigma)/2.3 mol/L sucrose buffered with 0.1 mol/L PB, embedded,and frozen quickly. Ultrathin cryosections of the samples weremounted on Formnar/carbon grid. After quenching free aldehydewith phosphate buffered saline (PBS)–0.01 mol/L glycine,sections were incubated with anti-podocin antibody. They werethen incubated with 10 nm of gold-conjugated anti-rabbit IgG(British BioCell, Cardiff, UK). After immunostaining, they werefixed with 2.5% glutaraldehyde in 0.1 mol/L PB (pH 7.4). Thesections were then contrasted with 2% uranyl acetate for 20min, and adsorption stained with 3% polyvinyl alcohol containing0.2% uranyl acetate for 10 min.
Semiquantitative Reverse Transcription (RT)-PCR
Five micrograms of total RNA prepared from glomeruli were utilizedto synthesize cDNA. Three microliters of the reverse transcriptionmixture were utilized for the subsequent PCR. Primers used forrat podocin and GAPDH were described above. The PCR productcorresponds to 235 bp, and the target sequence is located inthe putative extracellular domain close to the membrane-spanningregion. The PCR was performed for 20 to 30 cycles of denaturationat 94°C for 30 s, annealed at 58°C for 30 s, and extendedat 72°C for 1 min. The optimal cycle number was determinedin a preliminary trial to be in the linear phase of amplification.The PCR products were subjected to electrophoresis with 1.5%agarose gel and stained with ethidium bromide. The band intensitywas determined by an image analysis using a Macintosh computerand densitometry software (Densitograph; ATTO, Tokyo, Japan).All results were corrected for the amount of mRNA in the sampleby dividing by the intensity of the internal control, GAPDH.
Real-Time RT-PCR
Real-time PCR was performed basically according to the manufacturer’smanual. cDNA was synthesized as described above. cDNA, specificprimers, and SYBR Green (Takara, Otsu, Japan) were mixed witha Takara Ex Taq R-PCR Version For Real Time PCR kit (Takara).PCR reactions were run on a Smart Cycler System (Takara). Thesequences of the primers of podocin and GAPDH were describedabove. The reactions and run for all samples were performedin duplicate.
Induction of Proteinuric State ANA Nephropathy.
A total of 20 rats were intravenously injected with 6 mg ofanti-rat nephrin mAb 5-1-6, and five rats each were sacrificedjust before injection, 1 h, 24 h, or 5 d after injection. Smallpieces of the rat’s kidney tissues were snap-frozen andused for immunofluorescence study. Glomerular RNA was preparedfrom the remaining kidney tissues pooled from five rats forsemiquantitative RT-PCR and real-time PCR analysis. Twenty-four–hoururine samples were collected, and their protein concentrationswere measured by colorimetric assay with a Bio-Rad Protein AssayReagent (Bio-Rad) using BSA as a standard. For Western blotanalysis, five rats were intravenously injected with 6 mg ofanti-nephrin mAb 5-1-6 and sacrificed on day 5. Glomeruli wereisolated from pooled kidneys from five rats of each group andsolubilized directly with SDS-PAGE sample buffer or sequentiallysolubilized. For immunoelectron microscopic study, three ratswere treated with anti-nephrin mAb 5-1-6 and sacrificed on day5.
Puromycin Aminonucleoside (PAN) Nephropathy.
A total of 20 rats were intravenously injected with 10 mg/100g body weight of PAN, and five rats each were sacrificed justbefore injection and 1 h, 24 h, or 9 d after injection. Thecryostat sections for the immunofluorescence study and totalRNA for RT-PCR were prepared as described above. The amountof urinary protein excretion/24 h was determined as describedabove. For Western blot analysis, five rats were intravenouslyinjected with 10 mg/100 g body wt of PAN and sacrificed on day9 as described above. For immunoelectron microscopic study,three rats were treated with PAN and sacrificed on day 9.
Cloning of Rat Podocin cDNA
The complete nucleotide sequences of rat homologue of podocinand its deduced amino acid sequences are shown in Figure 1.The nucleotide sequencing analysis revealed an open readingframe of 1149 nucleotides coding for a predicted protein of383 amino acids, which is the same as human podocin (deducedmolecular weight, 42 kd). The deduced amino acid sequence ofrat podocin showed 84.3% identity and 97.1% similarity to humanpodocin (Figure 2). N-terminal beyond the stomatin-like sequencesof rat podocin (97 amino acids) has 63.9% identity to that ofhuman podocin. The ExPASy search showed that the rat podocinhas a single transmembrane site, which is the same as humanpodocin. The sequence of rat podocin showed 92.7% identity and98.9% similarity to mouse podocin. The sequence of rat podocinfor N-terminal 97 amino acids showed 82.8% identity to thatof mouse podocin.
Figure 1. Nucleotide sequences of rat podocin cDNA and its deduced amino acid sequence. Reading frame of this sequence starts at nucleotide 62 and extends until nucleotide 1213, thereby coding protein of 383 amino acids. The sequence data are available from GenBank under accession number (AY039651).
Figure 2. Comparison of amino acid sequence of podocin of rat and human. Gray box indicates the conserved amino acids shared by rat and human. The identity of amino acid sequence of rat and human is 84.3%.
Western Blot Finding of Podocin in Normal Glomerular Protein
An antibody to the peptide of 21 amino acids of the rat podocinsequence was detected to bind to a single band with molecularweight of approximately 42 kd in an extract of normal rat glomeruli.The calculated molecular weight of a 383–amino acid proteinof rat podocin is 42 kd. The ExPASy search showed that rat podocinhas only one putative N-glycosylation site. The 42-kd band iscompatible for the specific band of rat podocin. A weak bandof podocin was detected in the Triton X-100–solubilizedfraction, and the stronger bands were detected in the RIPA-solubilizedand RIPA-insolubilized fractions (Figure 3A). In contrast, aclearer band of nephrin was detected in the Triton X-100–solubilizedfraction. No positive band was detected with the anti-podocinantibody pre-absorbed with the podocin peptide used for immunizationor pre-immune rabbit serum.
Figure 3. Western blot analyses of the sequentially solubilized glomerular proteins with anti-podocin antibody. Normal rat glomeruli were solubilized sequentially with 0.5% Triton X-100 and RIPA lysis buffer and separated into Triton-X-100 soluble (TXS), RIPA-soluble (RIPAS), and RIPA-insoluble (RIPAI) fractions. (A) The fractions of TXS (lanes 1 and 4), RIPAS (lanes 2 and 5), and RIPAI (lanes 3 and 6) were loaded on a 7.5% polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The stripes were stained with alkaline phosphatase-conjugated anti-rabbit IgG after exposure with anti-rat podocin antibody (lanes 1 to 3) or anti-rat podocin antibody preabsorbed with the peptide used for immunization. Approximately 42-kd bands were detected by anti-rat podocin (lanes 1 to 3), although no bands were detected by the pre-absorbed antibody (lanes 4 to 6). The strong bands of podocin were detected in RIPAS and RIPAI fractions, and a weak band of podocin was detected in TXS. (B) The fractions of TXS (lanes 1 and 4), RIPAS (lanes 2 and 5), and RIPAI (lanes 3 and 6) were loaded on a 10% polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The stripes were exposed with anti-rat nephrin antibody (lanes 1 to 3) or pre-immune normal rabbit serum. The strong bands of nephrin were detected in the TXS fraction. No bands were detected by the pre-immune normal rabbit serum (lanes 4 to 6).
Organ-Specific Expression of Rat Podocin
The mRNA expression for podocin was detected in the kidney andin the nervous tissues (Figure 4). A clear specific band ofPCR product of podocin was detected in the glomerulus and cortex,whereas a faint signal was detected in the whole kidney. A positiveband was also detected in the cerebrum, cerebellum, and themedulla oblongata.
Figure 4. RT-PCR finding of podocin mRNA expression in several organs and tissues. A distinct expression of podocin was detected in the glomeruli (lane 1) and cortex (lane 2) of the kidney. A faint signal was detected in the whole kidney (lane 3). Positive signals were detected in the cerebrum (lane 12), cerebellum (lane 13), and the medulla oblongata (lane 14). No signal could be detected in the liver (lane 4), lung (lane 5), heart (lane 6), spleen (lane 7), stomach (lane 8), small intestine (lane 9), large intestine (lane 10), skeletal muscle (lane 11), testes (lane 15), and the ovaries (lane 16).
Expression of Podocin in Glomeruli of Normal and Proteinuric Rats Dual-Labeling Immunofluorescent Study.
Podocin staining is detected as the very fine linear-like patternalong the capillary loop in a normal adult section of a rat(Figure 5A). Dual-labeled immunofluorescent study of podocinand nephrin with a normal adult section of a rat showed theclose proximity of these molecules (Figure 5, A through C),although some non-overlapping staining is detected. In ANA nephropathy,the staining pattern of podocin shifted to a coarse granularpattern on day 5, when severe proteinuria was detected (89.1± 11.5 mg/24 h) (Figure 5D). In PAN nephropathy, thestaining pattern of podocin also shifted to a coarse granularpattern, and its intensity decreased on day 9, when massiveproteinuria was detected (374.4 ± 57.9 mg/24 h) (Figure 5G).The shift of podocin staining was paralleled with nephrinstaining in ANA nephropathy (Figure 5, D through F) but notin PAN nephropathy (Figure 5, G through I). Dual-labeling studywith podocin and ZO-1 showed that these molecules were closelylocalized (Figure 5J). In ANA nephropathy, ZO-1 staining (red)was shifted similarly to podocin (Figure 5K). In PAN nephropathy,although the intensity of podocin staining (green) was dramaticallydecreased, that of ZO-1 (red) was preserved (Figure 5L).
Figure 5. Localization of podocin, nephrin, and ZO-1 in glomeruli of proteinuric states. For dual-labeling immunofluorescence, cryostat sections were incubated with rabbit anti-rat podocin, FITC-conjugated anti-rabbit IgG, anti-rat nephrin mAb 5-1-6, and then TRITC-conjugated anti-mouse IgG1, respectively. The localization of podocin (green staining: A, D, G), nephrin (red staining: C, F, I), and double exposure findings (B, E, H) were shown. Close proximity of podocin and nephrin localization is observed in the normal rat glomeruli, although some nonoverlapping staining is detected. The staining pattern of rat podocin and nephrin in normal rat glomeruli was the linear-like pattern of the continuous fine granules (A through C). Podocin staining shifted a discontinuous coarse granular pattern in the proteinuric state caused by ANA (D). Nephrin staining shifted concomitantly with podocin (E and F). In PAN nephropathy (G through I), podocin and nephrin staining shifted in a discontinuous pattern and intensity of staining was dramatically decreased. It should be noted that nephrin staining was not always coincided with podocin as indicated by the arrows (H). Double-exposure findings of podocin (green staining) and ZO-1 (red staining) are shown (J through L). Close localization of podocin and ZO-1 was observed in normal rat glomeruli (J). In ANA nephropathy, the localization of podocin shifted concomitantly with ZO-1 (K). In PAN nephropathy, although the intensity of podocin staining (green staining) was dramatically decreased, that of ZO-1 (red staining) was preserved (L).
Immunoelectron Microscopic Study.
In normal rat glomeruli, gold particles indicating the localizationof podocin are detected on the cytoplasmic face close to thepoint of slit diaphragm (Figure 6, A and B). In ANA nephropathy,structural integrity of slit diaphragm is largely unaffected,although foot process effacement is partially observed. Goldparticles for podocin are detected on the cytoplasmic face atthe preserved slit diaphragm (Figure 6C). The finding in thepreserved glomeruli in ANA nephropathy is very similar to thatin normal rat glomeruli. In ANA nephropathy, gold particlesare also observed diffusely in the cytoplasm at the area offoot process effacement (Figure 6D). In PAN nephropathy, effacementof foot processes was broadly observed. Gold particles are observedat the site where tight junction is newly formed in proteinuricstates in PAN nephropathy (Figure 6, E and F).
Figure 6. Localization of podocin in normal and proteinuric rat glomeruli. In normal rat glomeruli, gold particles showing the localization of anti-N terminal region of podocin antibody are detected on the cytoplasmic face close to the point of slit diaphragm (A and B). In anti-nephrin antibody (ANA)–induced nephropathy, structural integrity of slit diaphragm is largely unaffected, although foot process effacement is partially observed. Gold particles for podocin are detected on the cytoplasmic face at the preserved slit diaphragm (C). The finding in the preserved glomeruli in ANA nephropathy is very similar to that in normal rat glomeruli. In ANA nephropathy, gold particles are also observed diffusely in the cytoplasm at the area where foot process effacement is observed (D, arrows). In PAN nephropathy, gold particles are observed at the site of tight junction newly formed in proteinuric states (E and F, arrows).
Semiquantitative Western blot Analysis.
To determine whether the reduced staining of podocin in proteinuricstates represents a loss of immunoreactive protein or simplyredistribution, Western blot analysis was performed using wholeglomerular lysate solubilized with SDS-PAGE sample buffer. Asshown in Figure 7A, intensity of podocin bands was significantlyreduced in PAN nephropathy (6.35 ± 4.60%), but that wasunchanged in ANA nephropathy. To further analyze whether thepattern of distribution of podocin is changed in proteinuricstates, Western blot analysis with sequentially solubilizedmaterials was performed (Figure 7B). The pattern of distributionof podocin in three fragments is unchanged between normal andANA nephropathy.
Figure 7. Western blot analyses of podocin in glomerular lysate from normal and proteinuric rat. Isolated glomeruli from rats of normal, ANA nephropathy, and PAN nephropathy were solubilized with SDS-PAGE sample buffer (A) or sequentially solubilized with Triton X-100 and RIPA buffer (B). (A) 75 µg of protein from each sample were loaded on 10% polyacrylamide gel, electrophoresed, and then transferred to a nitrocellulose membrane. Membrane was incubated with anti-podocin antibody, followed by alkaline phosphatase-conjugated anti-rabbit IgG. Intensity of podocin bands was significantly reduced in PAN nephropathy (6.35 ± 4.60%), but that was unchanged in ANA nephropathy. (B) Equal volumes of fractions sequentially solubilized were loaded on polyacrylamide gel, electrophoresed, and then transferred to a nitrocellulose membrane. Membrane was treated as described above. The pattern of distribution of podocin in three fragments of Triton, RIPAS, and RIPAI is unchanged in ANA nephropathy. The intensity of podocin band was significantly decreased in all fractions in PAN nephropathy.
Semiquantitative RT-PCR and Real-Time PCR Analyses.
Semi-quantitative RT-PCR study showed that decreased expressionof podocin mRNA was observed in the glomeruli of a rat 24 hafter induction of ANA nephropathy (Figure 8A). In contrast,decreased expression of podocin was not observed in PAN nephropathyat any point in time during the examination (Figure 8B). Real-timeRT-PCR finding also indicated that the mRNA expression for podocinwas decreased in rats 24 h after the induction of ANA nephropathy(Figure 9).
Figure 8. Semiquantitative RT-PCR analysis of rat podocin in ANA-induced nephropathy (A) and PAN nephropathy (B). The characteristic agarose gel electrophoretic pattern from one of three independent experiments is shown (top). Ratios of the densitometric signal of podocin to that of the internal control (GAPDH) were analyzed (bottom). The data are shown as ratios relative to normal findings and expressed as mean ± SD of three independent experiments. The mRNA expression for podocin was decreased 24 h after ANA injection (A). In PAN nephropathy, a decreased expression for podocin was not observed at any point in time of examination (B).
Figure 9. Real-time RT-PCR analysis of podocin in rats 24 h after ANA injection. The number of PCR cycles was shown on the x-axis, and the difference between measured fluorescence and the baseline was shown on the y-axis. A decreased expression of podocin was detected in rats of ANA nephropathy.
Developmental Expression of Rat Podocin
Podocin staining was first detected at the early capillary loopstage. The staining of podocin was restricted in the basal surfaceof the podocyte. No podocin signal was detected in the lateralsurface of the podocyte, whereas nephrin staining was detectedas a basolateral surface of podocyte in the early developingstage (Figure 10, A through C). Dual-labeling study of podocinwith ZO-1 (Figure 10, D through F) also showed that podocinstaining was restricted in the basal surface of the podocyte.
Figure 10. Localization of podocin in developing glomeruli (A through F) and cerebrum (G through I). Dual-labeling immunofluorescence findings of podocin (green staining) with nephrin (red staining) in early capillary loop stage were shown (A, podocin; B, merge; C, nephrin). Clear staining of rat podocin was detected in the basal side of podocyte of developing glomeruli (A). Nephrin staining was detected not only in the basal side but also in the lateral side of the podocyte (arrow) (C). Dual-labeling immunofluorescence findings of podocin (green staining) with ZO-1 (red staining) in late capillary loop stage are shown (D, podocin; E, merge; F, ZO-1). Podocin staining is restricted in the basal side of podocyte (D). Close localization of podocin and ZO-1 in glomeruli was observed as yellow (E). ZO-1 staining is observed not only in glomeruli but also in junctions between tubular epithelial cells. Double-exposure finding of podocin (green staining) with GFAP (G), RECA1 (H), and nephrin (I) in cerebrum is shown. Podocin staining was observed at the GFAP-positive cells (G). Clear podocin staining was observed at a side of GFAP-positive cells (arrow). Dual-labeling study with RECA1 showed that podocin was localized outer area of vessels (H, arrow). Close proximity of podocin and nephrin was also observed in cerebrum sections (I), although some nephrin staining that was not accompanied with podocin was observed.
Podocin Staining in Nerve Tissues
Positive immunostaining of podocin was detected in the cerebrum,cerebellum, and the medulla oblongata. Staining of podocin inthe cerebrum is shown in Figures 10G through 10I). Dual labelingstudy with anti-GFAP antibody showed that podocin staining wasobserved at the GFAP-positive cells, but podocin staining isrestricted more than GFAP staining. Clear podocin staining wasobserved at a side of GFAP-positive cells (Figure 10G). Dual-labelingstudy with RECA1 showed that podocin was localized just in theouter area of vessels (Figure 10H). Close proximity of podocinand nephrin was also observed in cerebrum sections (Figure 10-I),although some nephrin staining that was not accompanied withpodocin was observed.
In this study, we cloned the rat homologue of podocin to investigatethe expression of podocin in proteinuric models of rat. We comparedthe expression of podocin with that of nephrin and ZO-1 in normalmatured and developing glomeruli and the glomeruli of proteinuricstates using experimental models of rat. The sequencing of ratpodocin revealed that the deduced amino acid sequence of ratnephrin shows 84.3% identity to human podocin. Recently, Schwarzet al. (20) cloned the mouse homologue of podocin. The sequenceof rat podocin has 92.7% identity to mouse podocin. The domainstructure of podocin is also highly conserved between rat, mouse,and human. Podocin presents similarities with proteins of thestomatin family and is considered to have a similar molecularfunction (17). It is suggested that MEC-2, a member of the stomatinprotein family plays a role in linking ion channels to the cytoskeletonin the primary sensory neuron. Although the recent reports showedthat podocin has interaction with nephrin and CD2AP (19,20),the precise function of podocin has not been clarified. Westernblot analysis with sequentially solubilized material showedthat the fractions of RIPA-soluble and RIPA-insoluble materialswere rich in podocin, although the Triton X-100 solubilizedfraction was rich in nephrin. It was suggested that podocinis resistant to solubilization with mild detergent (20). Thesefindings might suggest that podocin has a connection with thecytoskeletal component.
The podocin staining was observed as a linear-like pattern withfine granule along the capillary loop in normal adult rat glomeruli.The localization of podocin is very close to that of nephrin(Figure 5, A through C) and ZO-1 (Figure 5G). Immunoelectronmicroscopic study showed that podocin is localized on the cytoplasmicface close to the point of slit diaphragm (Figure 6, A through B).These findings indicate that podocin is a slit diaphragmassociated protein. Anti-podocin antibody used in this studyrecognizes a peptide of 21 amino acids located at N-terminalregion of rat podocin. Immunoelectron microscopic findings inthis study indicated that N-terminal region of rat podocin locatesat cytoplasm as same as human podocin (18).
In this study, we analyzed the expression of podocin in proteinuricstates. In ANA nephropathy, the staining intensity of podocinclearly decreased and its staining pattern shifted to the coarsegranular pattern (Figure 5, D through F). In ANA nephropathy,structural integrity of slit diaphragm is largely unaffected,although foot process effacement is partially observed. Immunogoldparticles for podocin are observed at the cytoplasm at the preservedslit diaphragm and also observed diffusely in the cytoplasmat the site where foot processes effacement was observed (Figure 6, C and D).We could not detect the reduced intensity of podocinband by Western blot analysis using glomerular lysate from ratsof ANA nephropathy. Although the decreased expression of mRNAof podocin was detected at the early phase of ANA nephropathy,mRNA expression of podocin recovered on day 5, when the amountof proteinuria peaked. Although the precise mechanism of proteinuriacaused by ANA remains unclear, we have previously reported thatANA injected intravenously binds the extracellular site of nephrinand alters the molecular composition of the slit diaphragm,thereby causing proteinuria (7,23). We showed here that podocinis redistributed concomitantly with nephrin in ANA nephropathy.All these findings suggest that altered localization of podocinis concerned with the development of proteinuria in ANA nephropathy.In PAN nephropathy, the shift of the localization of podocinand the decreased intensity of podocin band in Western blotanalysis were observed, although the decreased mRNA expressionof podocin was not detected at any time points examined. Thefindings suggest that podocin is actively degradated and/orsecreted into urine in proteinuric state. We have previouslyreported that nephrin staining shifted to a discontinuous granularpattern in PAN nephropathy (14). It should be noted that theredistribution of podocin was not concomitant with nephrin inPAN nephropathy (Figure 5, G through I). The dual-labeling immunofluorescentfindings clearly showed that podocin was localized apart fromnephrin. Immunoelectron microscopic findings showed that immunogoldparticles for podocin lay at the site where tight junction wasformed in proteinuric state of PAN nephropathy (Figure 6, E and F).Although PAN nephropathy is widely used as a model ofminimal change nephrotic syndrome, the pathogenesis of proteinuriais not clearly explained. It is reported that oxygen radicalsthat are produced during the metabolism of PAN contribute tothe podocyte injury (24). In PAN nephropathy, some pathogenicfactors might cause the disassembling of nephrin with podocin.The disconnection of nephrin and podocin, as well as the decreasedexpression of these proteins, might result in the dysfunctionof the slit diaphragm.
In this study, we also analyzed the expression of podocin indeveloping glomeruli. Immunohistochemical studies with a neonatalrat kidney section showed that podocin first appeared in theearly capillary loop stage, later than the nephrin expression.Podocin expression was restricted to the basal side of the podocyteof the developing glomeruli. No positive staining of podocinwas observed on the lateral side of the podocyte, whereas thenephrin expression was detected not only in the basal surfacebut also in the lateral surface of podocyte. The findings showedthat nephrin was not accompanied with podocin at the early developingstage. Previously, we reported that nephrin was first expressedas the membrane protein and became to be concentrated at theslit pore (25). The findings obtained here suggest that nephrinand podocin originate from a different part of the cell andare assembled at the slit pore. It is conceivable that the connectedlocalization of nephrin and podocin might be necessary for thematuration of the slit diaphragm.
We show here that the expression of podocin is restricted inglomerular podocyte in the kidney. However, RT-PCR analysisindicates that mRNA of podocin was expressed not only in thekidney but also in the nerve tissues such as the cerebrum, cerebellum,and the medulla oblongata. The expression of some podocyte-associatedmolecules, such as synaptopodin and nephrin, was also restrictedto podocyte and nerve tissues (26,27). These findings suggestthat podocyte shared its specific function with the nerve tissues.Galloway and Mowat reported the syndrome that was characterizedby nephrotic syndrome associated with central nerve system anomalies(28). Some reports postulated that the mutation of a moleculethat is expressed both in the podocyte and in nerve tissue isinvolved in this syndrome (29,30). The dual-labeling immunofluorescencestudy of podocin with anti-GFAP antibody, a marker of astrocyte(31), showed that podocin staining was detected at GFAP-positivecells. Podocin staining was observed at the end foot processesand at a side of astrocyte facing vessels (Figure 10G). Thedual-labeling study with RECA1, a marker of endothelial cell(32), shows that podocin is localized just outer area of vessels(Figure 10H). It is conceivable that clear podocin stainingis at the side of astrocyte-facing vessels. We also observedhere that podocin and nephrin are closely localized in nervetissues as well as glomerular podocyte (Figure 10I). Astrocytesurrounds neuron or vessels. Recently, astrocyte is reportedto play a critical role for maintaining the barrier functionof blood-brain barrier (33,34). These findings might suggestthat podocin and nephrin play an important role for maintainingthe barrier function not only in glomerular capillary wall butalso in blood-brain barrier.
In conclusion, the deduced amino acid sequence of rat podocinshowed 84.3% identity to human podocin. Podocin expression wasdetected not only in the kidney but also in the nerve tissues.Podocin expression was first detected in the early capillaryloop stage. Podocin expression was restricted at the basal surfaceof podocyte of the developing glomeruli. Podocin localizationwas very closed in nephrin in normal glomeruli. Podocin wasredistributed in both proteinuric states caused by PAN and ANA.It is postulated that podocin is one of critical componentsof the slit diaphragm for maintaining the barrier function ofthe glomerular capillary wall.
Acknowledgments
This work was supported by Grant-Aids for Scientific Research(B) (13557084 to H. Kawachi), Grant-Aids for Scientific Research(B) (14370317 to H. Kawachi), and Grant-Aids for ScientificResearch (B) (08457286 to F. Shimizu) from Ministry of Education,Science, Culture and Sports of Japan. We wish to thank Ms. M.Kayaba and Ms. C. Nagasawa for their excellent technical assistance.We wish to thank Dr. Hitoshi Takahashi (Department of Pathology,Brain Research Institute, Niigata University) for his helpfuladvice. A portion of this study was presented at the ASN/ISNWorld Congress, San Francisco, California, October 2001, andwas published in abstract form (J Am Soc Nephrol 12: 680A, 2001).
Caulfield JP, Farquhar MG: The permeability of glomerular capillaries to graded dextrans. Identification of the basement membrane as the primary filtration barrier. J Cell Biol 63: 883–903, 1974[Abstract/Free Full Text]
Farquhar MG: The primary glomerular filtration barrier: Basement membrane or epithelial slit. Kidney Int 8: 197–211, 1975[Medline]
Graham RC, Karnovsky MJ: Glomerular permeability: Ultrastructural cytochemical studies using peroxidases as protein tracers. J Exp Med 124: 1123–1133, 1966[Abstract]
Rodewald R, Karnovsky MJ: Porous substructure of the glomerular slit diaphragm in the rat and mouse. J Cell Biol 60: 423–433, 1974[Abstract/Free Full Text]
Daniels BS, Deen WM: Glomerular permeability barrier in the rat: Functional assessment by in vitro methods. J Clin Invest 92: 929–936, 1993[Medline]
Orikasa M, Matsui T, Oite T, Shimizu F: Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J Immunol 141: 807–814, 1998
Kawachi H, Kurihara H, Topham PS, Brown D, Shia MA, Orikasa M, Shimizu F, Salant DJ: Slit diaphragm-reactive nephritogenic MAb 5–1-6 alters expression of ZO-1 in rat podocytes. Am J Physiol 273: F984–F993, 1997[Medline]
Schnabel E, Anderson JM, Farquhar MG: The tight junction protein ZO-1 is concentrated along slit diaphragm of the glomerular epithelium. J Cell Biol 111: 1255–1263, 1990[Abstract/Free Full Text]
Kestila M, Lenkkeru U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Motita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephrotic syndrome. Molecular Cell 1: 572–582, 1998
Reiser J, Kriz W, Kretzler, Mundel P: The glomerular slit diaphragm is a modified adherens junction. J Am Soc Nephrol 11: 1–8, 2000[Abstract/Free Full Text]
Inoue T, Yaoita E, Kurihara H, Shimizu F, Sakai T, Kobayashi T, Ohshiro K, Kawachi H, Okada H, Suzuki H, Kihara I, Yamamoto T: FAT is a component of the slit diaphragm. Kidney Int 59: 1003–1012, 2001[CrossRef][Medline]
Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestilae M, Jalanko H, Holmberg C, Tryggvason K: Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA 96: 7962–7967, 1999[Abstract/Free Full Text]
Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shia M, Shimizu F, and Salant DJ: Nephritogenic mAb 5–1-6 is directed at the extracellular domain of rat nephrin. J Clin Invest 104: 1559–1566, 1999[Medline]
Kawachi H, Koike H, Kurihara H, Yaoita E, Orikasa M, Shia MA, Sakai T, Yamamoto T, Salant DJ, and Shimizu F: Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states. Kidney Int 57: 1949–1961, 2000[CrossRef][Medline]
Kawachi H, Shimizu F: Molecular composition and function of the slit diaphragm: Nephrin, the molecule responsible for proteinuria. Clin Exp Nephrol 4: 161–172, 2000[CrossRef]
Shih N-Y, Li J, Karpitskii V, Nguyen A, Dustin ML, Kanagawa O, Miner JH, Shaw AS: Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 286: 312–315, 1999[Abstract/Free Full Text]
Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler M-C, Niaudet P, Antignac C: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24: 349–354, 2000[CrossRef][Medline]
Roselli S, Gribouval O, Boute N, Sich M, Benessy F, Attie T, Gubler M-C, Antignac C: Podocin localized in the kidney to the slit diaphragm area. Am J Pathol 160: 131–139, 2002[Abstract/Free Full Text]
Huber TB, Kottgen M, Schilling B, Walz G, Benzing T: Interaction with podocin facilitates nephrin signaling. J Biol Chem 276: 41543–41546, 2001[Abstract/Free Full Text]
Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, Shaw AS, Holzman LB, Mundel P: Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108: 1621–1629, 2001[CrossRef][Medline]
Laemlli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970[CrossRef][Medline]
Kawachi H, Oite T, Shimizu F: Studies on the ‘linear pattern’ in renal glomeruli demonstrated with immunofluorescence. Nephron 39: 36–39, 1985[Medline]
Kawachi H, Matsui K, Orikasa M, Morioka T, Oite T, Shimizu F: Quantitative studies of monoclonal antibody 5–1-6-induced proteinuric state in rats. Clin Exp Immunol 87: 215–219, 1992[Medline]
Nosaka K, Takahashi T, Nishi T, Imaki H, Suzuki T, Kurokawa K, Endou H: An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity. Free Radical Med Biol Med 22: 597–605, 1997
Kawachi H, Abrahamson DR, St John PL, Goldstein DJ, Shia MA, Matsui K, Shimizu F, Salant DJ: Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6. Am J Pathol 147: 823–833, 1995[Abstract]
Mundel P, Heid HW, Mundel TM, Kruger M, Reiser J, Kriz W: Synaptopodin: An actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139: 193–204, 1997[Abstract/Free Full Text]
Putaala H, Soininen R, Kilpelainen P, Wartiovaara J, Tryggvason K: The murine nephrin gene is specifically expressed in kidney, brain and pancreas: Inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet 10: 1–8, 2001[Abstract/Free Full Text]
Galloway WH, Mowat AP: Congenital microcephaly with hiatus hernia and nephrotic syndrome in two sibs. J Med Genet 5: 319–321, 1968[Free Full Text]
Srivastava T, Whiting JM, Garola RE, Dasouki MJ, Ruotsalainen V, Tryggvason K, Hamed R, Alon US: Podocyte proteins in Galloway-Mowat syndrome. Pediatr Nephrol 16: 1022–1029, 2001[CrossRef][Medline]
Nakazato H, Hattori S, Karashima S, Kawano T, Seguchi S, Kanahori M, Endo F: Another autosomal recessive form of focal glomerulosclerosis with neurological findings. Pediatr Nephrol 17: 16–19, 2002[CrossRef][Medline]
Lazarides E: Intermediate filaments as mechanical integrators of cellular space. Nature 283: 249–257, 1980[CrossRef][Medline]
Duijvestein AM, van Goor H, Klatter F, Majoor GD, van Bussel E, van Breda Vriesman PJ: Antibodies defining rat endothelial cells: RECA-1 a pan-endothelial cell specific monoclonal antibody. Lab Invest 66: 459–466, 1992[Medline]
Gaillard PJ, Voorwinden LH, Nielsen JL, Ivanov A, Atsumi R, Engman C, de Boer AG, Breimer DD: Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci 12: 215–222, 2001[CrossRef][Medline]
Guerin CJ, Nolan CC, Macroudis G, Lister T, Davidson GM, Holton JL, Ray DE: The dynamics of blood-brain barrier breakdown in an experimental model of glial cell degradation. Neuroscience 103: 873–883, 2001[CrossRef][Medline]
Received for publication March 29, 2002.
Accepted for publication August 20, 2002.
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Abstract
Introduction
