Glomerular Expression of Type IV Collagen Chains in Normal and X-Linked Alport Syndrome Kidneys
2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65063-8
ISSN1525-2191
AutoresLaurence Heidet, Yi Cai, Liliane Guicharnaud, Corinne Antignac, Marie‐Claire Gubler,
Tópico(s)Renal and related cancers
ResumoAlport syndrome is an inherited nephropathy characterized by alterations of the glomerular basement membrane because of mutations in type IV collagen genes. COL4A5 mutations, causing X-linked Alport syndrome, frequently result in the loss of the α5 chains of type IV collagen in basement membranes. This is associated with the absence of the α3(IV) and α4(IV) chains and increased amounts of α1(IV) and α2(IV) in glomerular basement membranes. The mechanisms resulting in such a configuration are still controversial and are of fundamental importance for understanding the pathology of the disease and for considering gene therapy. In this article we studied, for the first time, type IV collagen expression in kidneys from X-linked Alport syndrome patients, using in situ hybridization and immunohistochemistry. We show that, independent of the type of mutation and of the level of COL4A5 transcription, both COL4A3 and COL4A4 genes are actively transcribed in podocytes. Moreover, using immunofluorescence amplification, we were able to demonstrate that the α3 chain of type IV collagen was present in the podocytes of all patients. Finally, the α1(IV. chain, which accumulates within glomerular basement membranes, was found to be synthesized by mesangial/endothelial cells. These results strongly suggest that, contrary to what has been found in dogs affected with X-linked Alport syndrome, there is no transcriptional co-regulation of COL4A3, COL4A4, and COL4A5 genes in humans, and that the absence of α3(IV) to α5(IV) in glomerular basement membranes in the patients results from events downstream of transcription, RNA processing, and protein synthesis. Alport syndrome is an inherited nephropathy characterized by alterations of the glomerular basement membrane because of mutations in type IV collagen genes. COL4A5 mutations, causing X-linked Alport syndrome, frequently result in the loss of the α5 chains of type IV collagen in basement membranes. This is associated with the absence of the α3(IV) and α4(IV) chains and increased amounts of α1(IV) and α2(IV) in glomerular basement membranes. The mechanisms resulting in such a configuration are still controversial and are of fundamental importance for understanding the pathology of the disease and for considering gene therapy. In this article we studied, for the first time, type IV collagen expression in kidneys from X-linked Alport syndrome patients, using in situ hybridization and immunohistochemistry. We show that, independent of the type of mutation and of the level of COL4A5 transcription, both COL4A3 and COL4A4 genes are actively transcribed in podocytes. Moreover, using immunofluorescence amplification, we were able to demonstrate that the α3 chain of type IV collagen was present in the podocytes of all patients. Finally, the α1(IV. chain, which accumulates within glomerular basement membranes, was found to be synthesized by mesangial/endothelial cells. These results strongly suggest that, contrary to what has been found in dogs affected with X-linked Alport syndrome, there is no transcriptional co-regulation of COL4A3, COL4A4, and COL4A5 genes in humans, and that the absence of α3(IV) to α5(IV) in glomerular basement membranes in the patients results from events downstream of transcription, RNA processing, and protein synthesis. Alport syndrome (AS) is an inherited disorder of the glomerular basement membrane (GBM) characterized by hematuria, progressive renal failure, and sensorineural hearing loss, frequently associated with ocular abnormalities such as lenticonus and retinal anomalies.1Alport AC Hereditary familial congenital haemorrhagic nephritis.BMJ. 1927; 1: 504-506Crossref PubMed Scopus (501) Google Scholar, 2Atkin CL Gregory MC Border WA Alport syndrome.in: Schrier RW Gottschalk CW Diseases of the Kidney. ed 4. 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They are encoded by six distinct genes, COL4A1 to COL4A6, localized pairwise on three chromosomes.6Soininen R Huotari M Hostikka SL Prockop DJ Tryggvason K The structural genes for alpha1 and alpha2 chains of human type IV collagen are divergently encoded on opposite DNA strands and have an overlapping promoter region.J Biol Chem. 1988; 263: 17217-17220Abstract Full Text PDF PubMed Google Scholar, 7Hostikka SL Eddy RL Byers MG Hoyhtya M Shows TB Tryggvason K Identification of a distinct type IV collagen a chain with restricted kidney distribution and assignment of its gene to the locus of X-linked Alport syndrome.Proc Natl Acad Sci USA. 1990; 87: 1606-1610Crossref PubMed Scopus (322) Google Scholar, 8Mariyama M Zheng KG Yang FT Reeders ST Colocalization of the genes for the alpha3(IV) and alpha4(IV) chains of type-IV collagen to chromosome 2 bands 2-q35–q37.Genomics. 1992; 13: 809-813Crossref PubMed Scopus (92) Google Scholar, 9Hudson BG Reeders ST Tryggvason K Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 10Zhou J Ding M Zhao Z Reeders ST Complete primary structure of the sixth chain of human basement membrane collagen α6(IV). Isolation of the cDNAs for α6(IV) and comparison with five other type IV collagen chains.J Biol Chem. 1994; 269: 13193-13199Abstract Full Text PDF PubMed Google Scholar, 11Sugimoto M Oohashi T Ninomiya Y The genes COL4A5 and COL4A6, coding for basement membrane collagen chains α5(IV) and α6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters.Proc Natl Acad Sci USA. 1994; 91: 11679-11683Crossref PubMed Scopus (79) Google Scholar COL4A5 mutations lead to the most common form of AS which is X-linked, whereas COL4A3 and COL4A4 mutations are responsible for the autosomal recessive forms.12Antignac C Knebelmann B Drouot L Gros F Deschênes G Hors-Cayla MC Zhou J Tryggvason K Grünfeld JP Broyer M Gubler MC Deletions in the COL4A5 collagen gene in X-linked Alport syndrome: characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.J Clin Invest. 1994; 93: 1195-1207Crossref PubMed Google Scholar, 13Knebelmann B Breillat C Forestier L Arondel C Jacassier D Giatras I Drouot L Deschênes G Grünfeld JP Broyer M Gubler MC Antignac C Spectrum of mutations in the COL4A5 gene in X-linked Alport syndrome.Am J Hum Genet. 1996; 59: 1221-1232PubMed Google Scholar, 14Lemmink HH Schroder CH Monnens LAH Smeets HJM The clinical spectrum of type IV collagen mutations.Hum Mutat. 1997; 9: 477-499Crossref PubMed Scopus (145) Google Scholar, 15Renieri A Bruttini M Galli L Zanelli P Neri T Rossetti S Turco A Heiskari N Zhou J Gusmano R Massella L Banfi G Scolari F Sessa A Rizzoni G Tryggvason K Pignatti PF Savi M Ballabio A De Marchi M X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.Am J Hum Genet. 1996; 58: 1192-1204PubMed Google Scholar, 16Tryggvason K Mutations in type IV collagen genes and Alport phenotypes. Contributions to Nephrology: Molecular Pathology and Genetics of Alport Syndrome, vol 117. Karger, Basel1996: 154-171Google Scholar, 17Kawai S Nomura S Harano T Harano K Fukushima T Osawa G the Japanese Alport Network The COL4A5 gene in Japanese Alport syndrome patients. Spectrum of mutations in all exons.Kidney Int. 1996; 49: 814-822Crossref PubMed Scopus (56) Google Scholar, 18Boye E Mollet G Forestier L Cohen-Solal L Heidet L Cochat P Grünfeld JP Palcoux JB Gubler MC Antignac C Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.Am J Hum Genet. 1996; 63: 1329-1340Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 19Plant KE Green PM Vetrie D Flinter FD Detection of mutations in COL4A5 in patients with Alport syndrome.Hum Mutat. 1999; 13: 124-132Crossref PubMed Scopus (52) Google Scholar The primary structure of the six α(IV) chains is very similar. Each is characterized by an ∼25-residue noncollagenous domain at the amino terminus, an ∼1400 residue collagenous domain of Gly-X-Y repeats (in which X is frequently proline and Y is frequently hydroxyproline), that forms, in association with two other chains, the triple helix, and an ∼230-residue noncollagenous (NC1) domain at the carboxyl terminus.9Hudson BG Reeders ST Tryggvason K Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 20Heikkilä P Soininen R The type IV collagen gene family.in: Tryggvason K Contributions to Nephrology: Molecular Pathology and Genetics of Alport Syndrome. vol 117. Karger, Basel1996: 105-129Google Scholar, 21Zhou J Reeders ST The α chains of type IV collagen.in: Tryggvason K Contributions to Nephrology: Molecular Pathology and Genetics of Alport Syndrome. vol 117. Karger, Basel1996: 80-104Google Scholar The amino terminus of the collagenous domain is involved in the tetramerization of triple helical molecules, whereas the NC1 domain is involved in their dimerization. This organization eventually leads to the formation of a three-dimensional tight network that forms the scaffold of the basement membrane. The expression of the six α(IV) chain proteins and mRNA varies from one tissue to another. The α1(IV) and α2(IV) chains are expressed in all basement membranes, mainly in the form of the [α1(IV)]2-α2(IV)] trimer, whereas the α3(IV) to α6(IV. chains have a tissue-restricted distribution. In the human and rodent kidney, immunohistochemical studies have shown a low-level expression of α1(IV) to α2(IV) in mature GBM whereas the α3(IV) to α5(IV) chains are highly expressed.22Butkowski RJ Wieslander J Kleppel M Michael AF Fish AJ Basement membrane collagen distribution in the kidney: regional localization of novel chains related to collagen IV.Kidney Int. 1989; 35: 1195-1202Crossref PubMed Scopus (136) Google Scholar, 23Ninomiya Y Kagawa M Iyama K Naito I Kishiro Y Seyer JM Sugimoto M Oohashi T Sado Y Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.J Cell Biol. 1994; 130: 1219-1229Crossref Scopus (266) Google Scholar, 24Yoshioka K Hino S Takemura T Maki S Wieslander J Takekoshi Y Makino H Kagawa M Sado Y Kashtan CE Type IV collagen α5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibodies.Am J Pathol. 1994; 144: 986-996PubMed Google Scholar, 25Peissel B Geng L Kalluri R Kashtan C Rennke HG Gallo GR Yoshioka K Sun MJ Hudson BG Neilson EG Zhou J Comparative distribution of the α1(IV), α5(IV) and α6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.J Clin Invest. 1995; 96: 1948-1957Crossref PubMed Scopus (121) Google Scholar, 26Kalluri R Shield CF Todd P Hudson BG Neilson EG Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increases susceptibility of renal basement membrane to endoproteolysis.J Clin Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (264) Google Scholar, 27Gubler MC Heidet L Antignac C Alport's syndrome, thin basement membrane nephropathy and nail-patella syndrome.in: Jennette JC Olson JL Schwartz MM Silva FG Heptinstall's Pathology of the Kidney. ed 5. Little Brown and Company, Boston1998: 1207-1230Google Scholar Little is known about the different isoforms of triple-helical type IV collagen molecules,5Timpl R Structure and biochemical activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (814) Google Scholar, 28Johansson C Butkowski R Wieslander J The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.J Biol Chem. 1992; 267: 24533-24537Abstract Full Text PDF PubMed Google Scholar and their supramolecular organization in the different basement membranes. However, different subpopulations of NC1 hexamers, which reflect the association of two triple-helical molecules within the type IV collagen network, have been described recently in GBM as well as in other basement membranes.28Johansson C Butkowski R Wieslander J The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.J Biol Chem. 1992; 267: 24533-24537Abstract Full Text PDF PubMed Google Scholar, 29Kleppel MM Fan WW Cheong HI Michael AF Evidence for separate networks of classical and novel basement membrane collagen: characterization of α3(IV)-Alport antigen heterodimer.J Biol Chem. 1992; 267: 4137-4142Abstract Full Text PDF PubMed Google Scholar, 30Gunwar S Ballester F Noelken ME Sado Y Ninomiya Y Hudson BG Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4 and alpha5 chains of type IV collagen and its implication for the pathogenesis of Alport syndrome.J Biol Chem. 1998; 273: 8767-8775Crossref PubMed Scopus (176) Google Scholar, 31Kasai TZ Enders GC Gunwar S Brunmark C Wieslander J Kalluri R Zhou J Noelken ME Hudson BG Seminiferous tubule basement membrane. Compositions and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains.J Biol Chem. 1997; 272: 17023-17032Crossref PubMed Scopus (76) Google Scholar The presence of cysteine-rich α3(IV. and α4(IV) chains, forming with α5(IV) a network containing loops and supercoiled triple helices stabilized by disulfide bonds between the chains, seems to be important with regards to the long-term stability of the GBM and its role as a filter.26Kalluri R Shield CF Todd P Hudson BG Neilson EG Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increases susceptibility of renal basement membrane to endoproteolysis.J Clin Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (264) Google Scholar, 32Harvey SJ Zheng K Sado Y Naito I Ninomiya Y Jacobs RM Hudson BG Thorner PS Role of distinct type IV collagen networks in glomerular development and function.Kidney Int. 1998; 54: 1857-1866Crossref PubMed Scopus (97) Google Scholar Despite the increasing number of AS mutations reported in the literature12Antignac C Knebelmann B Drouot L Gros F Deschênes G Hors-Cayla MC Zhou J Tryggvason K Grünfeld JP Broyer M Gubler MC Deletions in the COL4A5 collagen gene in X-linked Alport syndrome: characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.J Clin Invest. 1994; 93: 1195-1207Crossref PubMed Google Scholar, 13Knebelmann B Breillat C Forestier L Arondel C Jacassier D Giatras I Drouot L Deschênes G Grünfeld JP Broyer M Gubler MC Antignac C Spectrum of mutations in the COL4A5 gene in X-linked Alport syndrome.Am J Hum Genet. 1996; 59: 1221-1232PubMed Google Scholar, 14Lemmink HH Schroder CH Monnens LAH Smeets HJM The clinical spectrum of type IV collagen mutations.Hum Mutat. 1997; 9: 477-499Crossref PubMed Scopus (145) Google Scholar, 15Renieri A Bruttini M Galli L Zanelli P Neri T Rossetti S Turco A Heiskari N Zhou J Gusmano R Massella L Banfi G Scolari F Sessa A Rizzoni G Tryggvason K Pignatti PF Savi M Ballabio A De Marchi M X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.Am J Hum Genet. 1996; 58: 1192-1204PubMed Google Scholar, 16Tryggvason K Mutations in type IV collagen genes and Alport phenotypes. Contributions to Nephrology: Molecular Pathology and Genetics of Alport Syndrome, vol 117. Karger, Basel1996: 154-171Google Scholar, 17Kawai S Nomura S Harano T Harano K Fukushima T Osawa G the Japanese Alport Network The COL4A5 gene in Japanese Alport syndrome patients. Spectrum of mutations in all exons.Kidney Int. 1996; 49: 814-822Crossref PubMed Scopus (56) Google Scholar, 18Boye E Mollet G Forestier L Cohen-Solal L Heidet L Cochat P Grünfeld JP Palcoux JB Gubler MC Antignac C Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.Am J Hum Genet. 1996; 63: 1329-1340Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 19Plant KE Green PM Vetrie D Flinter FD Detection of mutations in COL4A5 in patients with Alport syndrome.Hum Mutat. 1999; 13: 124-132Crossref PubMed Scopus (52) Google Scholar and the existence of AS animal models,33Zheng K Thorner P Marrano P Baumal R McInnes RR Canine X chromosome-linked hereditary nephritis: a genetic model for X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV.Proc Natl Acad Sci USA. 1994; 91: 3989-3993Crossref PubMed Scopus (103) Google Scholar, 34Hood JC Savige J Hendtlass A Kleppel MM Huxtable CR Robinson WF Bull terrier hereditary nephritis: a model for autosomal dominant Alport syndrome.Kidney Int. 1995; 47: 758-765Crossref PubMed Scopus (46) Google Scholar, 35Miner JH Sanes JR Molecular and functional defects in kidneys of mice lacking collagen α3(IV): implications for Alport syndrome.J Cell Biol. 1996; 135: 1403-1413Crossref PubMed Scopus (255) Google Scholar, 36Cosgrove D Meehan DT Grunkemeyer JA Kornak JM Sayers R Hunter WJ Samuelson GC Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (297) Google Scholar, 37Lees GE Helman RG Kashtan CE Michael AF Homco LD Millichamp NJ Ninomiya Y Sado Y Naito I Kim Y A model of autosomal recessive Alport syndrome in English cocker spaniel dogs.Kidney Int. 1998; 54: 706-719Crossref PubMed Scopus (57) Google Scholar several questions regarding the consequences of AS mutations on the collagen organization within the GBM and the mechanisms responsible for the progressive development of AS nephropathy remain unanswered. A striking feature observed in the majority of AS is the absence of all three α3(IV), α4(IV), and α5(IV) chains within the GBM although only one of these chains is actually mutated.13Knebelmann B Breillat C Forestier L Arondel C Jacassier D Giatras I Drouot L Deschênes G Grünfeld JP Broyer M Gubler MC Antignac C Spectrum of mutations in the COL4A5 gene in X-linked Alport syndrome.Am J Hum Genet. 1996; 59: 1221-1232PubMed Google Scholar, 24Yoshioka K Hino S Takemura T Maki S Wieslander J Takekoshi Y Makino H Kagawa M Sado Y Kashtan CE Type IV collagen α5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibodies.Am J Pathol. 1994; 144: 986-996PubMed Google Scholar, 25Peissel B Geng L Kalluri R Kashtan C Rennke HG Gallo GR Yoshioka K Sun MJ Hudson BG Neilson EG Zhou J Comparative distribution of the α1(IV), α5(IV) and α6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.J Clin Invest. 1995; 96: 1948-1957Crossref PubMed Scopus (121) Google Scholar, 26Kalluri R Shield CF Todd P Hudson BG Neilson EG Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increases susceptibility of renal basement membrane to endoproteolysis.J Clin Invest. 1997; 99: 2470-2478Crossref PubMed Scopus (264) Google Scholar, 27Gubler MC Heidet L Antignac C Alport's syndrome, thin basement membrane nephropathy and nail-patella syndrome.in: Jennette JC Olson JL Schwartz MM Silva FG Heptinstall's Pathology of the Kidney. ed 5. Little Brown and Company, Boston1998: 1207-1230Google Scholar, 32Harvey SJ Zheng K Sado Y Naito I Ninomiya Y Jacobs RM Hudson BG Thorner PS Role of distinct type IV collagen networks in glomerular development and function.Kidney Int. 1998; 54: 1857-1866Crossref PubMed Scopus (97) Google Scholar, 38Kashtan CE Kleppel MM Gubler MC Immunohistochemical findings in Alport syndrome.in: Tryggvason K Contributions to Nephrology: Molecular Pathology and Genetics of Alport Syndrome. vol 117. Karger, Basel1996: 142-153Google Scholar, 39Gubler MC Knebelmann B Beziau A Broyer M Pirson Y Haddoum F Kleppel MM Antignac C Autosomal recessive Alport syndrome. Immunohistochemical study of type IV collagen chain distribution.Kidney Int. 1995; 47: 1142-1147Crossref PubMed Scopus (167) Google Scholar, 40Mazzucco G Barsotti P Muda AO Fortunato M Mihatsch M Torri-Tarelli L Renieri A Faraggiana T De Marchi M Monga G Ultrastructural and immunohistochemical findings in Alport's syndrome: a study of 108 patients from 97 Italian families with particular emphasis on COL4A5 gene mutation correlations.J Am Soc Nephrol. 1998; 9: 1023-1031PubMed Google Scholar, 41Naito I Kawai S Nomura S Sado Y Osawa G the Japanese Alport Network Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome.Kidney Int. 1996; 50: 304-311Crossref PubMed Scopus (76) Google Scholar, 42Nakanishi K Yoshikawa N Iijima K Kitagawa K Nakamura H Ito H Yoshioka K Kagawa M Sado Y Immunohistochemical study of α1–5 chains of type IV collagen in hereditary nephritis.Kidney Int. 1994; 46: 1413-1421Crossref PubMed Scopus (118) Google Scholar This suggests that transcriptional, translational, and/or posttranslational events link the expression of the different type IV collagen chains. Furthermore, the α1 and α2 chains, which are normally confined to the subendothelial aspect of the GBM, and presumed to be synthesized by mesangial/endothelial cells in the normal kidney, are strongly expressed across the entire width of the GBM in AS patients.43Kashtan C Kim Y Distribution of the α1 and α2 chains of collagen IV and of collagens V and VI in Alport syndrome.Kidney Int. 1992; 42: 115-126Crossref PubMed Scopus (103) Google Scholar The cellular origin, whether mesangial-subendothelial or epithelial, of these two chains in AS GBM, remains to be elucidated. To address these questions, we analyzed the expression of type IV collagen chains in glomeruli from normal controls and patients with X-linked AS, both at the transcriptional and at the protein level. Renal specimens from six unrelated AS male patients previously shown to be lacking α3(IV) to α5(IV) isoforms within their GBM were used for this study. Clinical, morphological, and genetic data are presented in Table 1. All patients were affected with a severe (juvenile) disease. X-linked transmission was demonstrated on the basis of pedigree structure (5 patients), linkage analysis (3 patients), and/or the demonstration of a COL4A5 mutation (4 patients) which has been previously reported.12Antignac C Knebelmann B Drouot L Gros F Deschênes G Hors-Cayla MC Zhou J Tryggvason K Grünfeld JP Broyer M Gubler MC Deletions in the COL4A5 collagen gene in X-linked Alport syndrome: characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.J Clin Invest. 1994; 93: 1195-1207Crossref PubMed Google Scholar, 13Knebelmann B Breillat C Forestier L Arondel C Jacassier D Giatras I Drouot L Deschênes G Grünfeld JP Broyer M Gubler MC Antignac C Spectrum of mutations in the COL4A5 gene in X-linked Alport syndrome.Am J Hum Genet. 1996; 59: 1221-1232PubMed Google ScholarTable 1Clinical, Morphological, and Genetic Data in the Six X-Linked Alport Syndrome PatientsPatientsFamily historyAge at ESRDHearing lossOcular lesionsGBM changesGBM antigeniticyCOL4A5 mutationsEffect on coding sequenceExon (Reference)1+−*Chronic renal failure was observed in patient 1 at 23 years and in patient 6 at 26 years.++ (L)T+t−DeletionFrameshift3 → 41 (12)2+14++ (L)nd−del of G at 890-1/891Frameshift13 (13)3[−]14+−T+t−A → T at 3807-33′ splice41 (13)4+13+−nd−nd//5+11+−T+t−nd//6+−*Chronic renal failure was observed in patient 1 at 23 years and in patient 6 at 26 years.++ (M)T−G → C at 3398Gly → Arg at 106636 (13)L, lenticonus; M, maculopathy; T, thickening and splitting of the GBM; T+t, alternatively thick and thin GBM; nd, not determined.* Chronic renal failure was observed in patient 1 at 23 years and in patient 6 at 26 years. Open table in a new tab L, lenticonus; M, maculopathy; T, thickening and splitting of the GBM; T+t, alternatively thick and thin GBM; nd, not determined. Commercially available affinity-purified antibodies raised against pepsin-digested human placenta type IV collagen were obtained from Pasteur-Lyon (Lyon, France). They recognize the collagenous domain of the [α1(IV)2 α2(IV)] collagen protomer. Monoclonal antibodies recognizing the NC1 domain of, respectively, the α1 (mAb1), α3 (mAb3), and α5 (mAb 5) chains of type IV collagen were from Wieslab (Lund, Sweden). Monoclonal antibodies against the NC1 domain of the α4 chain of type IV collagen (mAb 85) (22) were a gift from MM Kleppel, (22) and monoclonal antibodies against the NC1 domain of the α6(IV) chain (H63) were from Y Sado.23Ninomiya Y Kagawa M Iyama K Naito I Kishiro Y Seyer JM Sugimoto M Oohashi T Sado Y Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.J Cell Biol. 1994; 130: 1219-1229Crossref Scopus (266) Google Scholar Affinity-purified fluorescein isothiocyanate-conjugated sheep immunoglobulin G anti-rabbit and anti-mouse immunoglobulins were from Silenius (Victoria, Australia). Fluorescein-conjugated affinity-purified donkey anti-sheep immunoglobulin G (H+L) antibodies were from Jackson Laboratories (West Grove, PA). Renal tissues from the six AS patients were snap-frozen in liquid nitrogen using cryo-M-bed (Bright Instrument Co., Huntingdon, UK). Cryostat sections (3-μm thick) were air dried and fixed in acetone for 10 minutes. Sections to be stained with mAb 85 and mAb A7 were pretreated with 0.1 mol/L glycine, 6 mol/L urea, pH 3.5, for 10 minutes, then rinsed with distilled water, as previously described.39Gubler MC Knebelmann B Beziau A Broyer M Pirson Y Haddoum F Kleppel MM Antignac C Autosomal recessive Alport syndrome. Immunohistochemical study of type IV collagen chain distribution.Kidney Int. 1995; 47: 1142-1147Crossref PubMed Scopus (167) Google Scholar After washing in fresh buffer (0.01 mol/L phosphate-buffered saline, pH 7.4), sections were incubated in a moist chamber with the appropriate dilution of monoclonal antibodies against the α1, α3, α4, α5, and α6 chains of type IV collagen before incubation with fluorescein isothiocyanate sheep anti-mouse antibodies (1/20). For amplification of the signal, a further incubation with fluorescein isothiocyanate donkey-anti-sheep antibodies (1/50) was performed. A mounting media containing p-phenylenediamine was used to delay fluorescence quenching. Labeling was examined with a Leitz Orthoplan microscope (Leica Microscopic Systems, Heezbrugg, Switzerland) equipped with appropriate filters. Tissue sections directly incubated with secondary antibodies served as a control for nonspecific binding, and negative results were obtained in all cases. Four normal kidneys (two nontransplanted kidneys and normal renal tissue adjacent to renal cell carcinoma in two patients) and 12 renal biopsy specimens of patients presenting various types of acquired glomerulopathies were tested as controls. Recombinant plasmids for type α(IV) collagen chain cDNA, restriction enzymes for linearization, and RNA polymerase used for in vitro transcription to generate anti-sense and sense cRNA probes are presented in Table 2.Table 2Recombinant Plasmids, Restriction Enzymes Used for Linearization, RNA Polymerases Used to Generate Antisense and Sense mRNA ProbesAnti-sense probeSense probeChainInsert length (bp)PlasmidEnzymePromotorEnzymePromotorLocation in cDNA (reference)α1533PCRTMIIHindIIIT7EcoRVSp6NC1 domain(1–533 of NC domain)(44)α3462pBSII SKEcoRVT3EagIT7NC1 domain (4357–4819)(45)α4760pBSII KSEcoRVT7EagISp6NC1 domain (4521–5281)(46)α5579pBSII SKEcoRVT3EagIT7Collagenous domain(1921–2500) (47) Open table in a new tab Labeled RNA probes were synthesized with Sp6, T7, or T3 RNA polymerases (Boehringer-Mannheim, Mannheim, Germany) using 35S-UTP (Amersham, Brauschweig, United Kingdom) according to the manufacturer's instructions. They were DNaseI-treated (Boehringer-Mannheim) immediately after the RNA polymerase reaction. After purification by ammonium acetate-ethanol precipitation, the probes were dissolved in 10 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid, 20 mmol/L dithiothreitol, and stored at −80°C. Anti-sense and sense strands were synthesized for all riboprobes. In patients 1 to 3, nephrectomy specimens obtained at the time of renal transplantation were used for in situ hybridization techniques. In all specimens preserved glomeruli were focally present between sclerotic areas. For technical reasons (too long of a time between nephrectomy and fixation revealed by the absence of labeling with COL4A1 anti-sense probes), in situ hybridization studies could not be performed in patients 4 and 5. Tissue from the smal
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