Cloning and Expression of the Rat Nephrin Homolog
1999; Elsevier BV; Volume: 155; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65190-5
ISSN1525-2191
AutoresHeikki Ahola, Shixuan Wang, Pauliina Luimula, Marja‐Liisa Solin, Lawrence B. Holzman, Harry Holthöfer,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoDespite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat. Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat. The molecular pathogenesis of diseases that result in abnormalities of glomerular filtration has remained poorly understood. Recent results indicate that podocytes play an important role in regulating the passage of macromolecules and have several structural properties suitable for allowing the rapid modulation of the permeability barrier.1Rennke HG How does glomerular epithelial cell injury contribute to progressive glomerular damage?.Kidney Int. 1994; 45: S58-S63Crossref PubMed Scopus (39) Google Scholar, 2Kerjaschki D Dysfunctions of cell biological mechanisms of visceral epithelial cells (podocytes) in glomerular diseases.Kidney Int. 1994; 45: 300-313Crossref PubMed Scopus (104) Google Scholar, 3Mundel P Kriz W Structure and function of podocytes: an update.Anat Embryol. 1997; 192: 385-397Google Scholar, 4Smoyer WE Mundel P Regulation of podocyte structure during the development of nephrotic syndrome.J Mol Med. 1998; 76: 172-183Crossref PubMed Scopus (141) Google Scholar Detailed studies of the recently discovered podocyte-specific proteins, including podocalyxin,5Kerjaschki D Sharkey DJ Farquhar MG Identification and characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell.J Cell Biol. 1984; 98: 1591-1596Crossref PubMed Scopus (403) Google Scholar GLEPP-1,6Thomas PE Wharram BL Goyal M Wiggins JE Holzman LB Wiggins RC GLEPP1, a renal glomerular epithelial cell (podocyte) membrane protein-tyrosine phosphatase. Identification, molecular cloning, and characterization in rabbit.J Biol Chem. 1994; 269: 19953-19962Abstract Full Text PDF PubMed Google Scholar synaptopodin,7Mundel 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. 1997; 139: 193-204Crossref PubMed Scopus (502) Google Scholar and lipids, including 0-acetylGD3 ganglioside,8Reivinen J Holthöfer H Miettinen A A cell-type specific ganglioside of glomerular podocytes in rat kidney: an O-acetylated GD3.Kidney Int. 1992; 42: 624-631Crossref PubMed Scopus (48) Google Scholar, 9Holthöfer H Reivinen J Miettinen A Nephron segment and cell-type specific expression of gangliosides in the developing and adult kidney.Kidney Int. 1994; 45: 123-130Crossref PubMed Scopus (32) Google Scholar should shed new light on the glomerular filter. However, the functional characterization of many of these is still under way. Experimental models of glomerular disease are important tools for investigating the functional significance of the novel molecules by providing a means to effectively modulate the basic glomerular functions. Thus, even if transgenic and knockout mouse models are extremely useful in the targeted analysis of molecules, the well-established experimental models in the rat remain best characterized and most widely used for glomerular pathophysiology. In particular, the classic models of Heyman nephritis, which mimicks membranous nephropathy, and anti-GBM nephritis and puromycin aminonucleoside (PAN) nephrosis of the rat, which models minimal change nephropathy, are still widely used.10Heymann W Hackel DB Harwood S Wilson SGF Hunter JLP Production of nephritic syndrome in rats by Freunds adjuvant and rat kidney suspension.Proc Soc Exp Biol Med. 1959; 100: 660-664Crossref PubMed Scopus (461) Google Scholar, 11Krakower CA Greenspon SA Localization of the nephrotoxic antigen within the isolated glomerulus.Arch Pathol Lab Med. 1951; 51: 629-639Google Scholar, 12Michael AF Blau E Vernier RL Glomerular polyanion: alteration in aminonucleoside nephrosis.Lab Invest. 1970; 23: 649-657PubMed Google Scholar, 13Groggel GC Hovingh P Border WA Linker A Changes in glomerular heparan sulphate in puromycin aminonucleoside nephrosis.Am J Pathol. 1987; 128: 521-527PubMed Google Scholar Similar modeling is often impossible in the mouse. Kestilä et al14Kestilä M Lenkkeri U Mannikko M Lamerdin J McCready P Putaala H Ruotsalainen V Morita 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.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar recently cloned a new gene, NPHS1, which is mutated in the congenital nephrotic syndrome of the Finnish type (CNF). The current treatment of CNF with early nephrectomy and final renal transplantation appears to cure all symptoms.15Holmberg C Antikainen M Rönnholm K Ala-Houhala M Jalanko H Management of congenital nephrotic syndrome of the Finnish type.Ped Nephrol. 1995; 9: 87-93Crossref PubMed Scopus (177) Google Scholar After transplantation, no new symptoms are reported after several years of follow-up. The results of Kestilä et al14Kestilä M Lenkkeri U Mannikko M Lamerdin J McCready P Putaala H Ruotsalainen V Morita 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.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar showed that NPHS1 is only expressed in the kidney glomerulus. However, little is known thus far of the regulation of this gene of apparently great functional significance in various proteinuria-associated diseases. Here we report the molecular cloning and characterization of rat nephrin cDNA. Male Sprague-Dawley rats weighing 175–190 g, with free access to tapwater and standard rat chow throughout the study, were used. To induce PAN nephrosis, puromycin aminonucleoside (20 mg/100 g; Sigma Chemical Company, St Louis, MO) in phosphate-buffered saline (PBS) was given by a single intraperitoneal injection to 10 rats, as described earlier.16Holthöfer H Reivinen J Miettinen A Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.Am J Pathol. 1996; 149: 1009-1015PubMed Google Scholar All injected animals developed significant albuminuria (>30 mg/24 hours) by day 5 after injection, as measured by nephelometry (Behring Nephelometer 100 Analyzer; Behringwerke, Marburg, Germany). Five animals were sacrificed at day 3 before the onset of albuminuria ( 100 mg/24 hours). One kidney from each animal was used for immediate isolation of cortical RNA. For immunofluorescence studies the remaining kidney was frozen in isopentane and stored appropriately at −70°C until used. For the cloning of the rat nephrin cDNA, a rat glomerular cDNA library was prepared and used as previously described.17Miettinen A Solin M-L Reivinen J Juvonen E Väisänen R Holthöfer H Podocalyxin in rat platelets and megakaryocytes.Am J Pathol. 1999; 154: 813-822Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Briefly, the library was constructed using total RNA isolated from glomeruli of 1-month-old rats by the isothiocyanate/CsCl− method.18Chirgwin JM Przybyla AC MacDonald RJ Rutter WJ Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry. 1979; 8: 5294-5295Crossref Scopus (19087) Google Scholar Furthermore, poly(A+) mRNA was isolated using a PolyATrac mRNA isolation System III kit (Promega, Madison, WI). mRNA was used to construct a poly(T)-primed directional glomerular library, using a ZAP Express cDNA Synthesis kit and a ZAP Express cDNA Gigapack II Cloning kit (Stratagene, La Jolla, CA). For the library screening 50 × 103 plaque-forming units of the library per 13-cm plate was transferred to a nylon membrane (Hybond N+; Amersham Life Sciences, Buckinghamshire, England), according to the instructions of the manufacturer. The prehybridization and further hybridization were performed as described previously.17Miettinen A Solin M-L Reivinen J Juvonen E Väisänen R Holthöfer H Podocalyxin in rat platelets and megakaryocytes.Am J Pathol. 1999; 154: 813-822Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Briefly, the filter was prehybridized at 42°C and further hybridized in the hybridization solution containing the 32P probe (107Mundel 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. 1997; 139: 193-204Crossref PubMed Scopus (502) Google Scholar cpm; see below) labeled with [α-32P]CTP (3000 Ci/mmol, 10 mCi/ml; Amersham), using a Random priming kit (Boehringer Mannheim, Germany). After an overnight hybridization the filter was washed twice with low-stringency buffer at 42°C, twice with high-stringency buffer at 50°C, and exposed to the film (Fuji Photo Film, Japan) with intensifying screens at −70°C. The positive plaques were picked and used for successive rounds of screening. After single-clone excision protocol (ZAP Express cDNA Gigapack II Cloning kit; Stratagene), the DNA of the pBK-CMV phagemid clone was isolated (Quiagen plasmid Mini Kit; Quiagen, Hilden, Germany. and sequenced from three clones along both strands (ABIPrism 310. Perkin-Elmer Applied Biosystems, Foster City, CA), and the sequences were screened for homology with database sequences, using the BLAST search algorithm at the National Center for Biotechnology (Washington, DC) via the Internet as described earlier.19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar For screening of the rat glomerular cDNA library the probe (HN3′. was constructed by polymerase chain reaction (PCR) amplification of normal human kidney cortex cDNA, using primer pair (numbering according to NPHS1; Gene Bank accession AF035835) NPHS-2606U (5′-cca aca tcg ttt tca ctt gg) and NPHS-3515L (5′-ggg aag gcc ata tcc tca t), producing a 909-bp-long PCR product, which was gel-purified after cloning to pGEM-T vector (Promega). The clone was digested by restriction enzymes ApaI and SacI, yielding a 777-bp-long fragment that was further used as a probe. For screening the long transcripts from the library, a probe for the 5′ end of rat nephrin (RN5′) was constructed by PCR amplification of rat kidney cortex cDNA. The primers were designed according to the sequence of mouse nephrin (Holzman et al., manuscript in preparation) for the regions conserved in human and mouse. PCR reaction with the primers RN-S1 (5′-cca cct cag cac ctc gag) and RN-AS1 (5′-gag aca cga gct cgg gac c) produced a PCR product of 293 bp, which was cloned to pGEM-T vector as above. The isolated insert was further used as a probe. Screening of the splicing variants of rat nephrin gene by PCR was performed by amplification of normal rat kidney cortex cDNA, using sense primer RN-S4 (5′-ctgctgcctgtgctctttgc) and antisense primer RN-AS2 (5′-ggccataggctctctccact), corresponding to nucleotides 3172–3541 (all numberings according to rat nephrin gene; Gen Bank accession number AF125521). PCR screening was also done by nested PCR, using primers (sense primer. RN-S3 5′-agcctcttgaccatcgctaa; antisense primer: RN-AS2) flanking the transmembrane region corresponding to nucleotides 2692–3541. The nested sense primer was RN-S2 (5′-tccaggtctccgtcactacc), and the antisense primer was RN-AS2, as in the first reaction. All PCR reactions were performed as before,19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar using AmpliTaq DNA polymerase (Perkin Elmer) and the following amplification program: after the initial denaturation at 94°C for 5 minutes, DNA was amplified using 30 cycles (94°C 1 minute, 55°C 1 minute, and 72°C 1 minute), followed by final elongation at 72°C for 10 minutes. In the nested PCR reactions, denaturation, annealing, and extension times were reduced to 30 seconds with 20 cycles. Twenty-five micrograms of total RNAs from rat tissues (kidney cortex, liver, heart, muscle, testis, brain, lung, and spleen) were used for Northern blottings as previously described.20Haltia A Solin M-L Jalanko H Holmberg C Miettinen A Holthöfer H Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.Pediatr Res. 1996; 40: 652-657Crossref PubMed Scopus (22) Google Scholar To reveal the transcript levels of rat nephrin, the respective cDNA probes as described above were used. To control the total RNA content and lack of degradation in the preparations, blots were hybridized with a β-actin probe.19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar For autoradiography of Northern blots the filters were exposed on Fuji Bas IIIS Imaging Plates, and the expression was recorded using a Fuji phosphoimager and accompanying MacBAS software (Fuji Photo Film Co, Tokyo, Japan). Because Northern blotting was not sensitive enough for detection of nephrin mRNA in renal cortex and other tissues mentioned above, we determined its transcript levels by reverse transcriptase-polymerase chain reaction (RT-PCR) with controls as previously used.19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar RNA-samples from isolated kidney cortex of the PAN rats were used as a starting material in this analysis.19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar, 20Haltia A Solin M-L Jalanko H Holmberg C Miettinen A Holthöfer H Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.Pediatr Res. 1996; 40: 652-657Crossref PubMed Scopus (22) Google Scholar These analyses were done in triplicate. Sequence-specific primers RN-S3 (see above) and RN-AS3 (5′-ccc agt cag cgt gaa ggt ag) yielded a 302-bp product. The semiquantitation of nephrin expression was done as previously described,19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar using serial dilutions of sample cDNAs in the linear range of amplification and normalization to the amount of β-actin amplification product. Sequence-specific polypeptides against intracellular and extracellular nephrin domains were selected over the human nephrin sequence, using the MacDNAsis and DNAStar programs as described. The sequence from amino acid 1101 to amino acid 1126 for the intracellular domain (Figure 1) was selected. This sequence was identical with the human sequence, whereas no homology to other known protein sequences could be found as screened by the PredictProtein program (European Molecular Biology Laboratory, Heidelberg, Germany). The peptides, coupled to a high-density multiple antigenic peptide-polylysine matrix,21Tam JP Synthetic peptide vaccine design. Synthesis and properties of a high-density multiple antigenic peptide system.Proc Natl Acad Sci USA. 1988; 85: 5409-5413Crossref PubMed Scopus (1247) Google Scholar were synthesized and purified at a local peptide synthesis unit (Haartman Institute, University of Helsinki). The polyclonal antipeptide antibodies were produced in the rabbit. Briefly, the synthetic peptides in Freund's complete adjuvant were injected into two rabbits. After three booster immunizations 4 weeks apart, peptide-specific IgG fractions were further immunoaffinity purified on CNBr-Sepharose coupled to the corresponding peptides. The specificity of the antisera was tested by immunofluorescence (IF) on kidney sections and by Western blotting of rat glomerular extracts.22Ljungberg P Haltia A Kuusela P Jalanko H Holmberg C Holthöfer H Noncollagenous matrix components of glomeruli in congenital nephrotic syndrome of the Finnish type: evidence of abnormal splitting of nidogen.Exp Nephrol. 1996; 4: 286-294PubMed Google Scholar To study the presence and tissue distribution of nephrin, frozen sections of the rat kidneys were cut at 5 μm, fixed in acetone at −20°C for 5 minutes, and washed in PBS.9Holthöfer H Reivinen J Miettinen A Nephron segment and cell-type specific expression of gangliosides in the developing and adult kidney.Kidney Int. 1994; 45: 123-130Crossref PubMed Scopus (32) Google Scholar Thereafter, the rabbit antibodies were flooded over sections for 1 hour. After washing the tissue sections were further incubated with rat anti-rabbit IgG (Boehringer Mannheim, Mannheim, Germany) coupled with fluorescein isothiocyanate (FITC). An Olympus OX50 microscope equipped with an epiilluminator and a filter system for FITC fluorescence was used for microscopy. Molecular cloning of the rat nephrin cDNA was undertaken to generate reagents necessary for studying nephrin in rat model systems. A human nephrin cDNA probe was amplified by PCR and was used to screen the rat glomerular cDNA library. Additional library screening was necessary to obtain the entire open reading frame of the rat nephrin sequence. Using the mouse nephrin cDNA sequence (Holzman, manuscript in preparation), we designed primers that allowed the amplification of 5′ rat nephrin cDNA probe. This cDNA fragment showed 93% sequence identity with mouse nephrin and was successfully employed to assemble the full-length cDNA sequence. The rat nephrin cDNA coding region of 1234 nucleotides revealed 82. sequence identity with the open reading frame of the human nephrin cDNA sequence. The predicted amino acid sequence, with a calculated molecular mass of 134 kd was 89% identical to the human sequence (see Figure 1) and the extracellular, transmembrane, and intracellular domains showed 90%, 99%, and 83% sequence identity with human nephrin. A highly conserved general structure of the extracellular region with the conserved Ig-like and fibronectin type III-like modules and signal sequence were found as in human nephrin. Putative N-glycosylation sites were also identically located in human and rat nephrin. Cysteine residues were also similarly conserved, showing similarity in location, and two additional cysteine residues in rat (positions 192 and 356) could be found. In addition to the whole coding sequence, two splicing variants (rat nephrin-α and nephrin-β; see below) could be detected with library screening (see Figure 2). Our previous data suggested the presence of splice isoforms of human nephrin. Evidence of alternatively spliced rat nephrin cDNA clones prompted further examination for alternatively spliced forms. Primers RN-S2, RN-AS2, RN-S3, and RN-S4 (see Figure 2) were particularly designed to reveal possible additional splicing of the transmembrane coding sequence in the rat. PCR with primers RN-S4 and RN-AS2 amplified the expected full-size product (370 bp) as well as a product that was about 100 bp smaller. After isolation and direct sequencing, the smaller PCR product was identified as a splicing variant rat nephrin-γ (Figure 2). Nested amplification using primers RN-S2 and RN-AS2 (first PCR reaction with primers RN-S3 and RN-AS2) showed five reaction products. In addition to the expected full-size product (460 bp) and the already identified variants nephrin-α, -β and -γ, a fourth splicing variant giving a 230-bp product (nephrin-δ) was seen and verified by direct sequencing of the PCR product. The sequences of all splicing variants translated the COOH-terminal rat nephrin in the correct reading frame. The tissue distribution of rat nephrin mRNA was further studied using Northern blotting and RT-PCR of the following tissues: liver, skeletal muscle, cardiac muscle, spleen, kidney cortex, and brain. Whereas the sensitivity of Northern blotting was found to be too weak for clear detection of the nephrin specific RNA (data not shown), the RT-PCR analysis could verify expression in kidney cortex and in the spleen (Figure 3). In semiquantitative RT-PCR analysis of rat cortical kidney in the PAN nephrosis model, a distinct down-regulation of the rat nephrin homolog was constantly seen (see Figure 4). Interestingly, this down-regulation up to 60% was observed already at 3 days after the single injection of puromycin amino nucleoside and further decreased down to 20% of the level of controls at day 10 in this model. Immunohistochemical staining of cortical kidney with antipeptide antibodies to the intracellular nephrin domain showed distinct reactivity exclusively with glomeruli (Figure 5). Notably, finely granular dotted lines of reactivity resembling a podocyte-like reactivity at the elements facing the urinary space could be seen (Figure 5). In the kidneys of PAN rats a systematic decrease (from intense +++ to faint/moderate +/++) in glomerular nephrin reactivity could be seen (data not shown). In Western blotting of isolated rat glomeruli, the antipeptide antibodies gave a double band at 200 kd (Figure 6). Here we report the cloning and characterization of the rat nephrin cDNA and the characteristic splicing of the respective mRNA. Distinct reactivity exclusively in the kidney glomerulus was observed with antipeptide antibodies to the intracellular nephrin-specific domain. Initial results showed down-regulation of its glomerular expression in the puromycin amino nucleoside nephrosis model. The characteristic massive proteinuria of the congenital nephrotic syndrome of the Finnish type has long been considered a unique model disease of the perturbed glomerular filtration function.15Holmberg C Antikainen M Rönnholm K Ala-Houhala M Jalanko H Management of congenital nephrotic syndrome of the Finnish type.Ped Nephrol. 1995; 9: 87-93Crossref PubMed Scopus (177) Google Scholar, 19Holthöfer H Kretzler M Haltia A Solin M-L Taanman J-W Schägger H Kriz W Kerjaschki D Schlöndorff D Altered gene expression and functions of mitochondria in human nephrotic syndrome.FASEB J. 1999; 13: 523-532PubMed Google Scholar, 20Haltia A Solin M-L Jalanko H Holmberg C Miettinen A Holthöfer H Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.Pediatr Res. 1996; 40: 652-657Crossref PubMed Scopus (22) Google Scholar, 23Rapola J Congenital nephrotic syndrome.Ped Nephrol. 1987; 1: 441-446Crossref PubMed Scopus (41) Google Scholar Thus, the recent identification of the NPHS1 gene14Kestilä M Lenkkeri U Mannikko M Lamerdin J McCready P Putaala H Ruotsalainen V Morita 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.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar responsible for this disease phenotype has suggested that this gene and the respective protein product nephrin play some key roles in the maintenance of the glomerular filtration barrier. Kestilä et al14Kestilä M Lenkkeri U Mannikko M Lamerdin J McCready P Putaala H Ruotsalainen V Morita 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.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar showed that NPHS1 encodes a putative transmembrane protein with sequence similarity to that of cell adhesion molecules of the immunoglobulin superfamily and with expression in the kidney but not in other tissues. Here we show that in addition to the kidney, nephrin can be found in the spleen of the rat. The significance of this additional expression remains to be studied in detail. The cDNA sequence cloned from the rat glomerular cDNA library shows 82% identity with the respective human cDNA sequence. No other related sequences were obtained in similarity searches. Furthermore, the rat nephrin cDNA bears high sequence similarity to human14Kestilä M Lenkkeri U Mannikko M Lamerdin J McCready P Putaala H Ruotsalainen V Morita 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.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1586) Google Scholar and mouse cDNA (Holzman et al, manuscript in preparation) nephrin sequences. Similarly, the rat nephrin cDNA sequence translates into a closely related amino acid sequence of 89% homology to human nephrin. Indeed, the translated open reading frame includes conserved extra-, intra-, and transmembrane domains, identically located cysteines, signal sequence, and glycosylation sites. Together this suggests that our rat nephrin cDNA is a true homolog of the respective human nephrin cDNA. Our results have indicated (Holthöfer et al, manuscript submitted for publication) that NPHS1-specific mRNA is spliced in the human kidney glomeruli with a variant lacking exon 24 covering the whole transmembrane domain. Interestingly, the splicing at this apparently important functional domain appears to be more complex in the rat with all of the splicing variants identified here. The unspliced form appears as the dominant one, whereas the less dominant forms lacking human exons 24–27 are found in the order of nephrin-α > β > γ > δ. Kendall and Thomas have shown suppression of endothelial cell growth factor activity by a secreted splice variant of vascular endothelial cell growth factor receptor lacking the transmembrane domain.24Kendall RL Thomas KA Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.Proc Natl Acad Sci USA. 1993; 90: 10705-10709Crossref PubMed Scopus (1217) Google Scholar In addition, similar transmembrane-negative modifications generated by alternative splicing have been reported, e.g., for the T-cell receptor,25Takase K Okazaki Y Wakizaka K Shevchenko A Mann M Saito T Molecular cloning of pTAC12 an alternative splicing product of the CD3uchain as a component of the pre-T cell antigen receptor complex.J Biol Chem. 1998; 273: 30675-30679Crossref PubMed Scopus (9) Google Scholar angiotensin-converting enzyme,26Sugimura K Tian X-l Hoffmann S Ganten D Bader M Alternative splicing of the mRNA coding for the human endothelial angiotensin converting enzyme: a new mechanism for solubilization.Biochem Biophys Res Commun. 1998; 247: 466-472Crossref PubMed Scopus (28) Google Scholar and interleukin-6 receptor.27Säily M Koistinen P Pulkki K Zheng A Savolainen E-R Acute myeloblastic leukemia cells produce soluble interleukin 6 receptor by a mechanism of alternative splicing.Cytokine. 1998; 10: 860-867Crossref PubMed Scopus (17) Google Scholar Furthermore, the distinct functional role of such splicing variants was recently shown for the soluble receptor of advanced glycosylation,28Park L Raman KG Lee KJ Lu Y Ferran LJ Chow WS Stern D Schmidt AM Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts.Nature Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1044) Google Scholar which appears to be an important physiological mechanism regulating atherogenesis in hyperglycemia.28Park L Raman KG Lee KJ Lu Y Ferran LJ Chow WS Stern D Schmidt AM Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts.Nature Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1044) Google Scholar What is the significance of the splicing variants of nephrin for glomerular functions and whether their mutual ratios are changing in experimental glomerular diseases is currently being studied in detail. PAN nephrosis in rats is a model for minimal change nephropathy in humans. Interestingly, our initial results on PAN nephrosis show distinct down-regulation of rat nephrin gene-specific mRNA to 60% of the level of controls at day 3 before peak proteinuria, and an additional drop to only 20% at day 10 of the controls at the highest proteinuria. This was supported by findings from immunofluorescence microscopy in which weaker fluorescence was observed in the PAN rat kidney glomeruli, especially at day 10, although immunofluorescence can be considered semiquantitative at best. Currently nothing is known of the metabolic rates of nephrin, but our results suggest a relatively slow degradation. Further studies are in progress to accurately study the decrease in the specific mRNA and the respective nephrin protein in various experimental models of glomerular disease in the rat. A double band in immunoblotting at 200 kd, the molecular mass also found for human nephrin (Holthöfer et al, manuscript submitted for publication), suggests that two protein transcripts of nephrin are actively produced in the normal rat kidney. The calculated molecular mass of nephrin is 134 kd; the difference from the observed molecular mass is most likely explained by glycosylation at the multiple glycosylation sites of the nephrin sequence. Taken together, the data presented here show the close identity of rat nephrin cDNA with the respective human cDNA. The observed characteristic alternative splicing suggests that the splicing variants may be important for nephrin functions. The widely used experimental models of glomerular damage in the rat may now be fully exploited to study the functions of nephrin. (Review started on January 22, 1999). The expert technical assistance of Ms Riitta Väisänen and Ms Liisa Pirinen is gratefully acknowledged.
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