Faculty Profiles - SAITO Akihiko

Papers - SAITO Akihiko

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The prevention of glomerulosclerosis in rats using traditional Chinese medicine, Sairei-to

Ping Li, Hiroshi Kawachi, Yasuhito Suzuki, Akihiko Saito, Michiaki Orikasa, Zhen Sheng Shi, Fujio Shimizu

Nephrology 5 ( 1-2 ) 83 - 89 2000

Language：English Publishing type：Research paper (scientific journal)

The effects of Sairei-to on chronic irreversible lesions were examined by the administration of drugs after the initiation of irreversible renal lesions by uninephrectomy and anti-thy-1 antibody injection. Twenty-one female Wistar rats were divided into three groups. Group 1 was treated with phosphate-buffered saline (PBS) as a control and groups 2 and 3 were treated with Sairei-to 400 mg/kg bodyweight per day on a daily basis beginning on day 1 (group 2) and day 7 (group 3) to day 42 after the intravenous administration of 500 μg anti-thy-1 monoclonal antibody (MoAb) into uninephrectomized rats, respectively. Statistically significant effects of Sairei-to on proteinuria were observed on days 3, 7, 14, and 21 in group 2 and on day 21 in group 3 as compared with the PBS controls. On day 42, light microscopy showed that Sairei-to ameliorated morphological lesions (Matrix score: 133.7±50.35 for group 1 vs 40.02±24.0 for group 2, P&lt
0.05). The weight of the kidneys in groups given Sairei-to was also lower than that in the PBS control. In addition, immunofluorescent findings showed that Sairei- to suppressed the expression of transforming growth factor-beta (TGF-β), alpha-smooth muscle actin (α-SMA) and collagen type I in glomeruli and decreased the number of ED1- and OX8-positive cells in tubulo-interstitium as well as in glomeruli. In conclusion, Sairei-to blocked the progression of renal lesions in this model even when it was administered after the initiation of the disease. In addition, earlier treatment more effectively prevented the progression of the renal lesions.

DOI： 10.1046/j.1440-1797.2000.00503.x

Severe proteinuria, sustained for 6 months, induces tubular epithelial cell injury and cell infiltration in rats but not progressive interstitial fibrosis

Hiroshi Kikuchi, Hiroshi Kawachi, Yumi Ito, Katsuyuki Matsui, Hitonari Nosaka, Akihiko Saito, Michiaki Orikasa, Masaaki Arakawa, Fujio Shimizu

Nephrology Dialysis Transplantation 15 ( 6 ) 799 - 810 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press

Background. Sustained proteinuria is reported to be very harmful to the tubulointerstitium, leading to severe interstitial injury. However, it remains unclear whether sustained proteinuria itself is responsible for severe interstitial injury because, in the previously reported models, the development of factors other than proteinuria in tubulointerstitial lesions could not be excluded completely. Methods. After treatment to induce immune tolerance to mouse immunoglobulin, 20 rats were injected with anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6 twice a week for 6 months and were then sacrificed. Results. mAb 5-1-6 induced massive proteinuria in 11 rats. In nine rats with mild proteinuria, no histological alteration could be detected with light microscopy and immunofluorescence. In nephrotic rats, light microscopy showed minor glomerular abnormalities, with interstitial oedema, tubular epithelial cell degeneration and interstitial cell infiltration. Immunofluorescence revealed increased expression of vimentin and an increased number of OX1-, OX19- and ED1-positive cells. However, we could not detect any accumulation of type I and IV collagen or laminin in the tubulointerstitium. RT-PCR showed that the expression of mRNA for type I collagen was not increased, compared with that in control rats. Conclusions. We succeeded in developing a model of persistent nephrosis without severe glomerular abnormalities, nephrectomy or other manoeuvres known to induce disturbed haemodynamics, using an agent without tubulointerstitial toxicity, and considered it to be suitable for investigating the direct toxicity of proteinuria. In this model, isolated massive proteinuria induced interstitial injury. However, the degree of injury was suggested to be much less than that observed in other previously developed models.

DOI： 10.1093/ndt/15.6.799

Down-regulation of interferon-γ signaling by gene transfer of Stat1 mutant in mesangial cells

Minoru Sakatsume, Ichiei Narita, Hajime Yamazaki, Akihiko Saito, Yoichi Nakagawa, Hideyuki Kuriyama, Ryozo Kuwano, Fumitake Gejyo, Masaaki Arakawa

Kidney International 57 ( 2 ) 455 - 463 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. Interferon-γ (IFN-γ) is secreted by T lymphocytes and natural killer (NK) cells in the cellular immunity-mediated inflammatory lesion, including endocapillary or extracapillary proliferative glomerulonephritis. It induces and/or enhances multiple histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and Fc receptor expression in renal resident cells, resulting in the initiation and promotion of inflammation. Recently, the signaling mechanism of IFN-γ has been investigated, and it appears that Stat1α is essential for signaling. We investigated Stat1α activation by IFN-γ in mesangial cells and attempted to regulate the signal transduction by gene transfer. Methods. Western blot with anti-Stat1 and antiphosphotyrosine after immunoprecipitation of Stat1 and Northern blot for detection of Stat1 mRNA were performed. The dominant negative form of Flag- tagged Stat1 was expressed in cultured rat mesangial cells. Flag was immunostained in transfectants, and luciferase reporter assay was carried out to measure the transcriptional activity of Stat1α. The expression of IFN-γ- inducible genes such as MHC class II (Ia-Aα) and MHC class II transactivator (CIITA) was detected by reverse transcriptase-polymerase chain reaction (RT- PCR). Results. Stat1α was tyrosine phosphorylated and activated by IFN-γ in mesangial cells, and the mRNA and protein level of Stat1α increased upon stimulation by IFN-γ. Overexpression of Stat1-mutant lacking 35 C-terminal amino acids strongly suppressed the IFN-γ-induced signal transduction and inhibited the expression of MHC class II and CIITA genes in mesangial cells. Conclusions. Stat1α is a critical molecule in the signal transduction of IFN-γ in mesangial cells. The inhibition of an endogenous function of Stat1α by gene transfer of the Stat1 mutant may be a new method to reduce the inflammatory effects of IFN-γ in localized inflammation of the kidney.

DOI： 10.1046/j.1523-1755.2000.00865.x

Ultrastructural localization of megalin in the rat cochlear duct

K Mizuta, A Saito, T Watanabe, M Nagura, M Arakawa, F Shimizu, T Hoshino

HEARING RESEARCH 129 ( 1-2 ) 83 - 91 1999.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Megalin is an endocytic receptor predominantly expressed in the kidney proximal tubule cells. In the present study, localization of megalin was examined using a post-embedding immunogold method in the rat cochlear duct. Marginal cells of the stria vascularis were labeled on the apical surface, but not on the basolateral surface. This localization pattern resembles kidney proximal tubule cells. Immunoreactivity was also detected on various other cells, including epithelial cells of the spiral prominence and epithelial cells of Reissner's membrane. In contrast, virtually no gold particles were seen on intermediate cells and basal cells of the stria vascularis, mesothelial cells of Reissner's membrane or fibrocytes in the lateral wall. Also unlabeled were cells in the tympanic wall of the cochlear duct, including sensory cells and supporting cells of the organ of Corti. The present findings show the involvement of megalin in endocytosis of marginal cells and are suggestive of different uptake mechanisms for aminoglycosides in the kidney proximal tubule cells and in the cochlear sensory cells. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(98)00221-4

All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann nephritis

H Yamazaki, R Ullrich, M Exner, A Saito, RA Orlando, D Kerjaschki, MG Farquhar

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 9 ( 9 ) 1638 - 1644 1998.9

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD)I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LED I, III, or IV was similar to that obtained with antibodies to LED II. It is concluded that passive HN can be induced with antibodies against LED I, III, and IV, as well as LED II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.

Identification of genes specifically expressed in chronic and progressive glomerulosclerosis Reviewed

Narita, I, H Nakayama, S Goto, T Takeda, M Sakatsume, A Saito, Y Nakagawa, M Arakawa

KIDNEY INTERNATIONAL S215 - S217 1997.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE INC

To identify genes expressed predominantly in the kidney with chronic and progressive glomerulosclerosis but not in acute and transient form of glomerulonephritis, we induced progressive glomerulosclerosis in rats by applying unilateral nephrectomy prior to injection of monoclonal anti-Thy1.1 antibody (OX-7), which cause acute and transient glomerulonephritis with single injection. In rats with nephrectomy and OX-7 injection (Nx group), proteinuria increased with time and mesangial expansion accompanied with interstitial fibrosis was recognized, whereas transient proteinuria and mesangiolysis followed by mesangial hypercellularity were seen in rats with sham operation and OX-7 injection (Sham group). Four weeks after the induction of glomerulonephritis, mRNAs were isolated from kidney cortex of both groups and used for cDNA synthesis. By subtraction hybridization of cDNAs from Nx with excess amount of those from Sham, we isolated and sequenced several genes expressed specifically in the Nx group. These included genes, which contain identical sequences with serine protease inhibitors, cytokine receptors, osteopontin as well as genes with unknown function. These genes may play important roles in the process which promotes acute glomerular damage advance to chronic and progressive glomerulosclerosis.

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions

RA Orlando, M Exner, RP Czekay, H Yamazaki, A Saito, R Ullrich, D Kerjaschki, MG Farquhar

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 ( 6 ) 2368 - 2373 1997.3

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families, To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor, RAP is known to inhibit the binding of all ligands to megalin, We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb, mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein Lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

DOI： 10.1073/pnas.94.6.2368

Identification of genes specifically expressed in chronic and progressive glomerulosclerosis

I. Narita, H. Nakayama, S. Goto, T. Takeda, M. Sakatsume, A. Saito, Y. Nakagawa, M. Arakawa

Kidney International, Supplement 51 ( 63 ) S215 - S217 1997

Language：English Publishing type：Research paper (international conference proceedings)

To identify genes expressed predominantly in the kidney with chronic and progressive glomerulosclerosis but not in acute and transient form of glomerulonephritis, we induced progressive glomerulosclerosis in rats by applying unilateral nephrectomy prior to injection of monoclonal anti-Thy1.1 antibody (OX-7), which cause acute and transient glomerulonephritis with single injection. In rats with nephrectomy and OX-7 injection (Nx group), proteinuria increased with time and mesangial expansion accompanied with interstitial fibrosis was recognized, whereas transient proteinuria and mesangiolysis followed by mesangial hypercellularity were seen in rats with sham operation and OX-7 injection (Sham group). Four weeks after the induction of glomerulonephritis, mRNAs were isolated from kidney cortex of both groups and used for cDNA synthesis. By subtraction hybridization of cDNAs from Nx with excess amount of those from Sham, we isolated and sequenced several genes expressed specifically in the Nx group. These included genes, which contain identical sequences with serine protease inhibitors, cytokine receptors, osteopontin as well as genes with unknown function. These genes may play important roles in the process which promotes acute glomerular damage advance to chronic and progressive glomerulosclerosis.

Molecular Genetics of Renal Diseases

Akihiko Saito, Hajime Yamazaki, Yoichi Nakagawa, Masaaki Arakawa

Internal Medicine 36 ( 2 ) 81 - 86 1997

Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Internal Medicine

Recent progress in molecular genetics has strongly influenced nephrology. Gene mutations have been identified which cause various monogenic hereditary renal diseases including Alport syndrome, autosomal dominant polycystic kidney disease, and tubular transporter disorders. The data obtained will be useful not only to develop new methods of diagnosis and treatment of such particular diseases but also to expand the knowledge of renal physiology and pathophysiology. In addition, genetic factors have been investigated which are involved in the development and progression of more common renal diseases such as IgA nephropathy and diabetic nephropathy. There have also been great advances in molecular studies of experimental renal diseases such as Heymann nephritis and in the use of transgenic and knockout mice. In this review we focused on the important achievements made recently in the field of molecular nephrology.

DOI： 10.2169/internalmedicine.36.81

Mapping rat megalin: The second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis

A Saito, H Yamazaki, K Rader, A Nakatani, R Ullrich, D Kerjaschki, RA Orlando, MG Farquhar

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 93 ( 16 ) 8601 - 8605 1996.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

DOI： 10.1073/pnas.93.16.8601

THE HEYMANN NEPHRITIS ANTIGENIC COMPLEX - MEGALIN (GP330) AND RAP

MG FARQUHAR, A SAITO, D KERJASCHKI, RA ORLANDO

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 6 ( 1 ) 35 - 47 1995.7

Language：English Publisher：WILLIAMS & WILKINS

Heymann nephritis (HN) has been extensively studied as a model of human membranous nephropathy since it was first described by Heymann in 1959, HN was induced in active form by the immunization of rats with antigens derived from the proximal tubule brush border, resulting in subepithelial glomerular immune deposits. HN was also induced passively by the injection of antibrush border antibodies into normal rats, A breakthrough in the understanding of the pathogenesis of HN was made in the 1970s, when it was established that the disease was due to the binding of circulating antibodies to glomerular components. This in turn led to a search to identify the endogenous antigen(s), In 1982, gp330 (now called megalin), a glycoprotein located in clathrin-coated pits of glomerular and proximal tubular epithelia, was identified as a target antigen, In 1990, a second protein (44 kd), now known as RAP (for receptor associated protein), that binds to megalin was also shown to be a target antigen. Both molecules have been cloned and sequenced, and their role in normal epithelial cells has been explored. It has come to light that megalin (gp330) is a member of the low-density lipoprotein receptor gene family and functions as a multiligand receptor for the uptake of a variety of macromolecules (plasminogen, protease: protease inhibitor complexes, apolipoprotein E-enriched very low-density lipoproteins, lactoferrin, among others). RAP associates with megalin and appears to function as a chaperone assisting in the folding of megalin in the endoplasmic reticulum and its transport to the cell surface. This review considers what is now known about the structure, function, and trafficking of megalin and RAP and the role of these two molecules in the pathogenesis of HN.

No linkage to the COL4A3 gene locus in Japanese thin basement membrane disease families

Hajime YAMAZAKI, Yoichi NAKAGAWA, Akihiko SAITO, Shinichi NISHI, Shiminoru SAKATSUME, Tetsuro TAKEDA, Yuichiro MARUYAMA, Sojiro OGINO, Shiro MARUYAMA, Toshio MOCHIZUKI, Stephen T REEDERS, Masaaki ARAKAWA

Nephrology 1 ( 4 ) 315 - 321 1995

Language：English Publishing type：Research paper (scientific journal)

Summary: Patients with thin basement membrane disease (TBMD) exhibit persistent haematuria with a diffuse thinning of the glomerular basement membrane (GBM), especially of the lamina densa. It appears to be an autosomal dominant trait. It has been reported that the Goodpasture epitope, which is located in the non‐collagenous domain of type IV collagen α 3 chain, may be reduced in patients with TBMD. We speculated that the candidate gene for TBMD could be the type IV collagen α 3 chain gene (COL4A3), which is present closely to type IV collagen α 4 chain gene (COL4A4) on chromosome 2q35–37. We conducted a linkage analysis to investigate the relationship between familial TBMD and COL4A3 gene, using COL4A3 cDNA polymorphism and a (CA)n microsatellite marker located in the COL4A3 gene. We examined 32 individuals from four Japanese families with TBMD. There were no associations between the patients with haematuria and certain alleles of the two markers in the pedigrees of three families. It has been reported that type IV collagen α 1 chain gene (COL4A1) and α 2 chain gene (COL4A2) are not involved in TBMD, and that α 5 chain gene (COL4A5) and a 6 chain gene (COL4A6) map to chromosome X. In conclusion, our findings suggested that familial TBMD is not caused by the genetic abnormalities of type IV collagen genes isolated thus far. Copyright © 1995, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1440-1797.1995.tb00046.x

Mesangial cell-matrix interactions: Modulation of matrix expression in mesangial cells by extracellular matrices

M. Sakatsume, A. Saito, T. Takeda, H. Yamazaki, I. Narita, S. I. Nishi, Y. Nakagawa, M. Arakawa

Experimental Nephrology 3 ( 6 ) 362 - 368 1995

Language：English Publishing type：Research paper (scientific journal)

Aberrant extracellular matrices (ECMs) observed in mesangial areas during the progression of glomerulonephropathy could be produced by mesangial cells (MCs), interacting with altered ECM components, under the influence of various mediators. We studied the interaction between MCs and ECMs, especially focusing on matrix expression of MCs in contact with ECMs. We analyzed mRNA expression and protein synthesis of ECM components by cultured rat MCs which were plated on different types of ECMs, basement membrane-type matrix (BMM), type IV collagen and type I collagen. The level of mRNA for ECM components (α1 type I collagen, α1 type IV collagen, fribronectin and laminin B2) increased in MCs on the two types of collagen compared with BMM. The relative increase of α1 type I collagen mRNA expression in MCs on type I collagen was greater than that of α1 type IV collagen expression. At the protein level, type I collagen production by MCs on coated type I collagen showed a 2.2-fold increase relative to that on BMM. MCs on type IV collagen produced it at an intermediate level. The phenomena were not associated with differences of proliferative activity, but were accompanied by minor morphological differences. These results indicate that ECMs can modulate the phenotype of MCs in terms of matrix expression and suggest that MC-matrix interaction may in part contribute to abnormal ECM accumulation in diseased glomeruli.

COMPLETE CLONING AND SEQUENCING OF RAT GP330 MEGALIN, A DISTINCTIVE MEMBER OF THE LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE FAMILY

A SAITO, S PIETROMONACO, AKC LOO, MG FARQUHAR

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 ( 21 ) 9725 - 9729 1994.10

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

We completed the cDNA cloning and sequencing of gp330, the major kidney glomerular antigen for rat Heymann nephritis. The deduced 4660-aa sequence, expected to constitute a mature protein of M(r) 516,715, consists of a probable N-terminal signal peptide sequence (25 aa), an extracellular region (4400 aa), a single transmembrane domain (22 aa), and a C-terminal cytoplasmic tail (213 aa), The extracellular region contains three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) gene family-36 LDLR ligand-binding repeats forming four clusters of putative ligand-binding domains, 16 growth factor repeats separated by 8 YWTD spacer regions, and 1 C-terminal epidermal growth factor repeat. The cytoplasmic tail contains two copies of the (FX)NPXY moth, which represents a signal for coated pit-mediated internalization and an additional similar moth. The overall structure of gp330 is similar to that of the LDLR-related protein (LRP)/alpha(2)-macroglobulin receptor and shows even greater similarity to the Caenorhabditis elegans protein, reported as a homologue of LRP. However, gp330 differs from these proteins in (i) the cysteine-rich repeat arrangements found in the extreme extracellular N- and C-terminal regions, (ii) the distribution pattern of cysteine residues in the YWTD spacer regions, (iii) the location of the RX(K/R)R consensus recognition sequence of furin, a precursor processing endoprotease, and (iv) the length and structure of the cytoplasmic tail. We suggest the name megalin (from Greek mega) for gp330, the largest plasma membrane protein identified so far in vertebrates. The cloned cDNA will be useful for studies on the physiological functions of gp330/megalin and for determining its role in Heymann nephritis.

DOI： 10.1073/pnas.91.21.9725

A DELETION MUTATION IN THE 3' END OF THE ALPHA-5(IV) COLLAGEN GENE IN JUVENILE-ONSET ALPORT SYNDROME

A SAITO, M SAKATSUME, H YAMAZAKI, F OGATA, Y HIRASAWA, M ARAKAWA

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 4 ( 9 ) 1649 - 1653 1994.3

Language：English Publishing type：Research paper (scientific journal) Publisher：WILLIAMS & WILKINS

Alport syndrome is a hereditary progressive glomerular basement membrane disorder in which juvenile-or adult-onset renal failure is often accompanied by sensorineural deafness and ocular abnormalities. Recently, mutations have been found in the type IV collagen alpha 5 chain gene in patients with X-linked Alport syndrome. This study searched for gene mutations in seven unrelated Japanese patients by the use of conventional Southern blot analysis with cDNA probes for the carboxyl-terminal noncollagenous domain that is encoded by exons 46 to 51. A deletion mutation was found in a patient who developed juvenile-onset (age 15) ESRD with typical ultrastructural glomerular basement membrane destruction and sensorineural hearing loss but no characteristic ocular abnormalities. His mother showed hematuria and proteinuria with nomal renal function, suggesting that she may be the heterozygous carrier. Exon-specific polymerase chain reaction amplified the coding sequence of exon 48 but not exons 49 to 51. Analysis with pulsed-field gel electrophoresis revealed that the deletion is approximately 10 kb in length and does not involve the CpG island, which is located in the 3' distal site of the gene. Identification of this novel deletion causing juvenile-type Alport syndrome would contribute to elucidating the mechanisms of renal failure progression in the syndrome.

Alternative splicing in the α5(IV) collagen gene in human kidney and skin tissues

Akihiko Saito, Minoru Sakatsume, Hajime Yamazaki, Masaaki Arakawa

The Japanese Journal of Nephrology 36 ( 1 ) 19 - 24 1994

Language：English Publishing type：Research paper (scientific journal)

Type IV collagen is a complex molecule composed of three α chains that can be of types αl(IV) to α5(IV). Each α chain contains an amino-terminal collagenous domain and a carboxyl-terminal noncol-lagenous (NC) domain consisting of 229 amino acids, which is involved in intermolecular cross-linking to construct the basement membrane network. Specific localization of α5(IV) collagen in glomerular basement membranes (GBM) in the kidney indicates its crucial role for that construction. In human kidney cortex and skin RNA, we found an alternative splicing transcript which skips exon 50 of the α5(IV) collagen gene using reverse transcription-polymerase chain reaction and direct sequencing of the PCR products. This alternative splicing introduces a stop codon at the first codon of exon 51, resulting in an mRNA encoding a protein lacking the 84 NC domain carboxylterminal amino acid residues encoded by exons 50 and 51. Recently, gene mutations affecting the α5(IV) collagen NC domain have been reported in patients with X-linked Alport syndrome who display GBM destruction and progressive renal failure, which are usually milder in female patients. Therefore, the alternative splicing, if its truncated products are dominantly expressed in glomeruli, may cause GBM impairment and renal failure progression. © 1994, Japanese Society of Nephrology. All rights reserved.

DOI： 10.14842/jpnjnephrol1959.36.19

Clinical Significance of Urinary Methylguanidine in Non-Dialyzed Patients with Chronic Renal Failure —Prediction of Progression Rate of Renal Dysfunction—

Yutaka Koda, Akihiko Saito, Katsunobu Shinohara, Ran Ei, Shigeru Miyazaki, Yasuko Yuasa, Shinji Sakai, Masashi Suzuki, Sachio Takahashi, Yoshihei Hirasawa, Motoo Nakajima

Japanese Journal of Nephrology 35 ( 3 ) 203 - 208 1993

Language：English Publishing type：Research paper (scientific journal)

To clarify a clinical significance of urinary methylguanidine (U-MG, dl) in non-dialyzed patients with chronic renal failure, we measured U-MG, urinry creatinine (U-Cr, mg/ dl) and serum creatinine (S-Cr, mg/dl), concurrently and continually in 36 out-patients whose S-Cr was over 4. 0 mg/dl. Fresh urine sample was obtained and U-MG was measured by an enzymic method. © 1993, Japanese Society of Nephrology. All rights reserved.

DOI： 10.14842/jpnjnephrol1959.35.203

Molecular genetics of Alport syndrome

H. Yamazaki, A. Saito, Y. Nakagawa, M. Arakawa

Nippon rinsho. Japanese journal of clinical medicine 50 3021 - 3026 1992.12

Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by sensorineural deafness. Ultrastructural studies in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered GBM protein structure as the cause of nephritis. The product of COL4A5, the alpha 5 (IV) collagen chain, is a specific component of GBM of the kidney. Various mutations in the COL4A5 gene have been identified in X-linked dominant Alport syndrome, and these aberrations of the alpha 5 (IV) can account for at least a part of X-linked Alport syndrome.

LONG-DISTANCE RESTRICTION MAPPING OF THE PROXIMAL LONG ARM OF HUMAN CHROMOSOME-21 WITH NOT-I LINKING CLONES

H ICHIKAWA, K SHIMIZU, A SAITO, D WANG, R OLIVA, H KOBAYASHI, Y KANEKO, H MIYOSHI, CL SMITH, CR CANTOR, M OHKI

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 89 ( 1 ) 23 - 27 1992.1

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD PRESS

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

DOI： 10.1073/pnas.89.1.23

Single dialysis system

T. Kawasaki, A. Saito, H. Yamazaki, H. Ichikawa

Nippon rinsho. Japanese journal of clinical medicine 49 Suppl 196 - 204 1991.12

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