Faculty Profiles - WADA Jun

Papers - WADA Jun

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Developmental regulation, expression, and apoptotic potential of galectin-9, a beta-galactoside binding lectin Reviewed

J Wada, K Ota, A Kumar, EI Wallner, YS Kanwar

JOURNAL OF CLINICAL INVESTIGATION 99 ( 10 ) 2452 - 2461 1997.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ROCKEFELLER UNIV PRESS

Galectin-9, a beta-galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 mu M concentration, induced apoptosis in similar to 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed similar to 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus.

DOI： 10.1172/jci119429

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Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin Reviewed

J Wada, YS Kanwar

JOURNAL OF BIOLOGICAL CHEMISTRY 272 ( 9 ) 6078 - 6086 1997.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in Liver, small intestine, thymus > kidney, spleen, lung cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.

DOI： 10.1074/jbc.272.9.6078

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D-glucose-induced dysmorphogenesis of embryonic kidney Reviewed

YS Kanwar, ZZ Liu, A Kumar, MI Usman, J Wada, EI Wallner

JOURNAL OF CLINICAL INVESTIGATION 98 ( 11 ) 2478 - 2488 1996.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ROCKEFELLER UNIV PRESS

An organ culture system was used to study the effect of D-glucose on embryonic kidneys, and to delineate the mechanism(s) relevant to their dysmorphogenesis. Metanephroi were cultured in the presence of 30 mM D-glucose. A notable reduction in the size and population of nephrons was observed. Ureteric bud branches were rudimentary and the acuteness of their tips, the site of nascent nephron formation, was lost. Metanephric mesenchyme was atrophic, had reduced cell replication, and contained numerous apoptotic cells. Competitive reverse transcriptase-PCR analyses and immunoprecipitation studies indicated a decrease in expression of heparan sulfate proteoglycan (perlecan). Status of activated protein-2 was evaluated since its binding moths are present in the promoter region of the perlecan gene. Decreased binding activity of activated protein-5 related to its phosphorylation, was observed. D-glucose-treated explants also had reduced levels of cellular ATP. Exogenous administration of ATP restored the altered metanephric morphology and reduced [S-35]sulfate-incorporated radioactivity associated with perlecan. The data suggest that D-glucose adversely affects the metanephrogenesis by perturbing various cellular phosphorylation events involved in the transcriptional and translational regulation of perlecan. Since perlecan modulates epithelial/mesenchymal interactions, its deficiency may have led to the metanephric dysmorphogenesis and consequential atrophy of the mesenchyme exhibiting accelerated apoptosis.

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Comparative role of phosphotyrosine kinase domains of c-ros and c-ret protooncogenes in metanephric development with respect to growth factors and matrix morphogens Reviewed

ZZ Liu, J Wada, A Kumar, FA Carone, M Takahashi, YS Kanwar

DEVELOPMENTAL BIOLOGY 178 ( 1 ) 133 - 148 1996.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

Receptor-like protooncogenes, with tyrosine kinase catalytic domains, are expressed in neoplastic and fetal tissues and potentially have a role in embryonic development. Which protooncogene may have the dominant role in embryonic renal development during the ''postinductive'' period, i.e., Day 10 onward, was addressed in this study by utilizing an in vitro organ culture system. The role of various receptor-like protooncogenes, with the emphasis on c-ros and c-rer, was investigated by antisense-oligodeoxynucleotide (ODN) gene-targeting strategies at a point in metanephric development when reciprocal-inductive interactions between the epithelium and mesenchyme have already been initiated and are rampant. Also, their relationship with other morphogens, like extracellular matrix (ECM) proteins and growth factors, was studied. Initial in situ hybridization and RT-PCR analyses revealed a similar spatiotemporal expression for both c-ros and c-ret in the embryonic kidneys. At Day 13, they were mainly expressed in the developing nephrons in the nephrogenic zone and ureteric bud branches, where the signals from the mesenchymal ligands are transduced to the epithelial cell surface receptors. Minimal expression was observed in the newborn kidneys. Inclusion of antisense ODNs, derived from the phosphotyrosine kinase domains, inhibited metanephric growth in the organ culture; the most dramatic effects were observed with the c-ret antisense ODN. The c-ret-induced dysmorphogenetic effects were characterized as a decrease in the population of nephrons, atrophy of the mesenchymal cells, and loss of acuteness of the tips of ureteric bud branches. Interestingly, the ureteric bud branches continue to grow in the atrophic mesenchyme. Both c-ros and c-ret antisense ODNs reduced the gene expression and biosynthesis of various ECM proteins. The proteoglycans, expressed at the epithelial:mesenchymal interface, were most adversely affected, especially by the c-ret antisense. The treatment of metanephric explants with c-ret did not affect the gene expression of c-ros and vice versa. The specificity of the effects of c-ret antisense was also reflected by a decrease of anti-Ret protein immunoreactivity. The studies were extended to establish a relationship between c-ret protooncogene and some of a the growth factors which are known to influence renal development via their tyrosine kinase-like receptors localized in the ureteric bud branches, the site apparently where c-ret is also expressed. Among the various growth factors examined, transforming growth factor-alpha (TGF-alpha) and insulin like growth factor-I (IGF-I) had the most notable trophic effects on metanephric explants and caused maximal phosphorylation of Ret protein. In addition, concurrent exposure of TGF-alpha or IGF-I and c-ret antisense ODN explants caused partial recovery from the c-ret-induced dysmorphogenetic effects in the metanephroi. The data suggest that, although a number of protooncogenes share similar catalytic domains, c-ret plays a major role during a the ''postinductive'' period of metanephric development by perturbing the growth factor-dependent expression of ECM morphogenetic macromolecules, notably that of the proteoglycans, and also by affecting certain yet undefined growth factor-mediated phosphorylation mechanism(s) involving c-ret. (C) 1996 Academic Press, Inc.

DOI： 10.1006/dbio.1996.0204

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The critical role of intercellular adhesion molecule-1 in Masugi nephritis in rats Reviewed

J Wada, K Shikata, H Makino, S Morioka, K Hirata, K Ota, T Tamatani, M Miyasaka, T Horiuchi, S Noji, K Nishikawa, F Myokai, S Taniguchi, YS Kanwar, Z Ota

NEPHRON 73 ( 2 ) 264 - 272 1996.6

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

Intercellular adhesion molecule-1 (ICAM-1, CD54), an adhesion molecule of the immunoglobulin superfamily, is an endothelial cell surface ligand for such leukocyte integrins as lymphocyte-function-associated molecule 1 (LFA-1, CD11a/CD18), Mac-1 (CD11b/CD18) and CD43. These molecules mediate adhesive interactions between leukocytes and endothelial cells and are critically involved in infiltration of leukocytes into inflammatory lesions, We examined time expression of ICAM-1 in renal tissues of Masugi nephritis rats and directly examined time role of ICAM-1 by administration of neutralizing monoclonal antibodies (MAbs) to rat ICAM-1, LFA-1 alpha-subunit (LFA-1 alpha), beta-subunit (LFA-1 beta) and Mac-1 alpha-subunit (Mac-1 alpha). Within 3 h after injection of nephrotoxic serum, increased expression of ICAM-1 was detected in the glomeruli by in situ hybridization and an immunofluorescence study, Proteinuria was significantly suppressed by the MAbs against ICAM-1, Mac-1 alpha and LFA-1 beta. Neutrophil in filtration into the glomeruli was significantly prevented by injection of the MAbs against ICAM-1, LFA-1 alpha and LFA-1 beta. These results indicate both ICAM-1/LFA-1 and ICAM-1/Mac-1 pathways are involved in neutrophil infiltration into the glomeruli. On the other hand, monocytic infiltration was prevented by the MAbs against ICAM-1, LFA-1 alpha and LFA-1 beta but not hy anti-Mac-1 alpha MAb. Due to these results, ICAM-1 is considered to be a critical molecule involved in the pathogenesis of the leukocyte infiltration into the glomeruli in the heterologous phase of Masugi nephritis. Anti-ICAM-1 antibody may be beneficial in the treatment of leukocyte-mediated glomerular diseases.

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Cloning of mouse integrin alpha(V) cDNA and role of the alpha(V)-related matrix receptors in metanephric development Reviewed

J Wada, A Kumar, Z Liu, E Ruoslahti, L Reichardt, J Marvaldi, YS Kanwar

JOURNAL OF CELL BIOLOGY 132 ( 6 ) 1161 - 1176 1996.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ROCKEFELLER UNIV PRESS

Metanephrogenesis has been a long-standing model to study cell-matrix interactions. A number of adhesion molecules, including matrix receptors (i.e., integrins), are believed to be involved in such interactions. The integrins contain alpha and beta subunits and are present in various tissues in different heterodimeric forms. In this study, one of the members of the integrin superfamily, alpha(v), was characterized, and its relevance in murine nephrogenesis was investigated. Mouse embryonic renal cDNA libraries were prepared and screened for alpha(v), and multiple clones were isolated and sequenced. The deduced amino acid sequence of the alpha(v) cDNA clones and hydropathic analysis revealed that it has a typical signal sequence and extracellular, transmembrane, and cytoplasmic domains, with multiple Ca2+ binding sites. No A(U)(n)A mRNA instability motifs were present. Conformational analysis revealed no rigid long-range-ordered structure in murine alpha v. The alpha(v) was expressed in the embryonic kidney at day 13 of the gestation, with a transcript size of similar to 7 kb. Its expression increased progressively during the later gestational stages and in the neonatal period. It was distributed in the epithelial elements of developing nephrons and was absent in the uninduced mesenchyme. In mature metanephroi, the expression was relatively high in the glomeruli and blood vessels, as compared to the tubules. Various heterodimeric associations of alpha(v), i.e., with beta(1), beta(3), beta(5), and beta(6), were observed in metanephric tissues. Inelusion of alpha(v)-antisense-oligodeoxynucleotide or -antibody in metanephric culture induced dysmorphogenesis of the kidney with reduced population of the nephrons, disorganization of the ureteric bud branches, and reduction of mRNA and protein expressions of alpha(v). The expressions of integrin beta(3), beta(5), and beta(6) were unaltered. These findings suggest that the integrin alpha(v) is developmentally regulated, has a distinct spatio-temporal expression, and is relevant in the mammalian organogenesis.

DOI： 10.1083/jcb.132.6.1161

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The Critical Role of Intercellular Adhesion Molecule-1 in Masugi Nephritis in Rats

Jun Wada

Nephron Clinical Practice 73 ( 2 ) 1996

Erratum: Cloning of mouse c-ros renal cDNA, its role in development and relationship to extracellular matrix glycoproteins (Kidney Int (48: 1646-1659))

Kanwar, Y.S., Liu, Z.Z., Kumar, A., Wada, J., Carone, F.A.

Kidney International 49 ( 1 ) 306 - 306 1996

CLONING OF MOUSE C-ROS RENAL CDNA, ITS ROLE IN DEVELOPMENT AND RELATIONSHIP TO EXTRACELLULAR-MATRIX GLYCOPROTEINS Reviewed

YS KANWAR, ZZ LIU, A KUMAR, J WADA, FA CARONE

KIDNEY INTERNATIONAL 48 ( 5 ) 1646 - 1659 1995.11

Language：English Publisher：BLACKWELL SCIENCE PUBL INC CAMBRIDGE

Renal organogenesis ensues following reciprocal interactions between the uninduced metanephric mesenchyme and the ureteric bud. Conceivably, the presence of ligands or growth factors on a given cell type, and expression of receptors, including receptor proto-oncogenes, on the other cell type of different lineage would facilitate such epithelial-mesenchymal interactions. During these interactions, other macromolecules, such as extracellular matrix (ECM) proteins, present at the epithelial-mesenchymal surface, also play a role in the kidney morphogenesis. In this study the proto-oncogene, c-ros, was cloned and sequenced; its role in the metanephric development was examined, and correlated with the changes in the expression of ECM proteins. The mouse c-ros renal cDNA, belonging to phosphotyrosine kinase (PTK) receptor family, had a translation product of 2340 amino acids. The extracellular domain had 32 N-linked glycosylation sites acid 30 cysteine residues. The transmembrane segment had a hydrophobicity approaching similar to 3.5. Multiple phosphorylation sites, typical of a PTK catalytic unit, were present in the cytoplasmic domain. The 3' noncoding region did not contain any A(U)(n)A mRNA instability motifs. The c-ros mRNA was highly expressed on the ureteric bud branches and their tips and on the developing glomeruli. Competitive RT-PCR analyses revealed the c-ros expression was the highest at 13th day of gestation, and it declined to very low levels during the neonatal period. Exposure of metanephric kidneys to c-los antisense-oligonucleotide, derived from the PTK domain, caused dysmorphogenesis of the kidney and loss of c-ros expression on the ureteric bud branches. Concomitant with the reduced c-ros gene expression, a decreased expression of ECM glycoproteins, in particular the proteoglycans, was observed. These findings suggest that the c-los plays a role in the metanephric development, and its effects may be modulated by the ECM macromolecules present at the epithelial-mesenchymal interface.

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DISTRIBUTION OF EXTRACELLULAR-MATRIX RECEPTORS IN VARIOUS FORMS OF GLOMERULONEPHRITIS Reviewed

K SHIKATA, H MAKINO, S MORIOKA, T KASHITANI, K HIRATA, Z OTA, J WADA, YS KANWAR

AMERICAN JOURNAL OF KIDNEY DISEASES 25 ( 5 ) 680 - 688 1995.5

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO

Integrins are heterodimeric transmembrane receptor glycoproteins consisting of alpha and beta subunits that mediate adhesion and interactions between cells and extracellular matrix. Such interactions may be perturbed in various pathologic states, resulting in the altered phenotypic expressions of the integrins in affected tissues. To ascertain the alterations in integrins in various renal diseases, their distribution was investigated in different forms of glomerulonephritis by indirect immunofluorescence and immunoelectron microscopy using specific antibodies directed against beta(1) integrins and integrin alpha(v) beta(3) (vitronectin receptor). In addition, the distribution of certain extracellular matrix components (ie, fibronectin, vitronectin, and type IV collagen) was examined. Integrin beta(1) and alpha(v) beta(3) were highly expressed in proliferating mesangial cells in immunoglobulin A nephropathy, membranoproliferative glomerulonephritis type I and diffuse proliferative lupus nephritis. Their putative ligands (ie, fibronectin, vitronectin, and type IV collagen) also were increased in the expanded mesangial regions. In immunoglobulin A nephropathy, integrin beta(1) and alpha(v) beta 3 were seen by immunoelectron microscopy to be localized to the mesangial cell membranes in close proximity to the immune complex deposits; however, fibronectin and vitronectin immunoreactivities were observed in the mesangial immune complex deposits. Similarly, vitronectin also was detected in the immune complex deposits of other forms of proliferative nephritis, ie, membranoproliferative glomerulonephritis type I and diffuse proliferative lupus nephritis. In diffuse proliferative lupus nephritis, the cellular crescents displayed immunoreactivity toward integrin alpha(v) beta(3) and vitronectin. In nonimmune complex glomerular disease associated with nephrotic syndrome (ie, minimal change nephrotic syndrome), integrin alpha(3) beta(1) which normally has a linear capillary distribution, was decreased. In immune complex nephritis associated with nephrotic syndrome (ie, membranous nephropathy), integrin alpha(3) beta(1) immunoreactivity was focally disrupted and capillary loops displayed a discontinuous linear distribution. Finally, in membranous nephropathy, immunoreactivity toward vitronectin was accentuated in immune complex deposits. The differential altered distributions of the integrins and of their ligands may be reflective of mesangial cell proliferative activity in certain forms of glomerulonephritis, while in other forms of glomerular diseases, the decreased immunoreactivity of integrin alpha(3) beta(1) may be related to the glomerular capillary wall alterations associated with proteinuric states. (C) 1995 by the National Kidney Foundation, Inc.

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A case of systemic lupus erythematosus associated with severe fibrinoid necrosis located mainly in the glomerular afferent arteriole

Morioka, S., Makino, H., Wada, J., Shikata, K., Yamasaki, Y., Ogura, T., Amano, T., Asaumi, A., Okada, S., Ota, Z.

The Japanese Journal of Nephrology 37 ( 1 ) 1995

Activation of complement during hemodialysis

Nagake, Y., Amano, T., Wada, J., Sugimoto, H., Kawabata, K., Shikata, K., Makino, H., Ota, Z.

Japanese Journal of Clinical Immunology 18 ( 2 ) 1995

TROPHIC EFFECT OF INSULIN-LIKE GROWTH-FACTOR-I ON METANEPHRIC DEVELOPMENT - RELATIONSHIP TO PROTEOGLYCANS Reviewed

ZZ LIU, A KUMAR, EI WALLNER, J WADA, FA CARONE, YS KANWAR

EUROPEAN JOURNAL OF CELL BIOLOGY 65 ( 2 ) 378 - 391 1994.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WISSENSCHAFTLICHE VERLAG GMBH

Many hormones/factors influence the total body growth and development during embryonic life, and very few studies have been carried out to ascertain their effects on the individual organ system. In this study, the effect of exogenous insulin-like growth factor-I (IGF-I) on embryonic kidneys was investigated, and correlated with phenotypic and gene expression and synthesis of extracellular matrix (ECM) proteoglycans (PGs). Antisense experiments were carried out to elucidate the role of endogenous (IGF-I in metanephric development. Mouse metanephroi, harvested at 13th day of gestation, were exposed to IGF-I (100 ng/ml) in an organ culture for 7 days. An enlargement of the metanephroi with accentuation of its lobules, and increase in the nephron population and [H-3]thymidine incorporation was observed. Immunofluorescence studies and Southern blot analysis of polymerase chain reaction products indicated augmented expression of the ECM PGs. A heavy concentration of [S-35]sulfate-associated radioactivity over the tips of ureteric bud branches and ECM components of maturing glomeruli was seen. Maximal effect of radioincorporation was observed on day-4 of the culture, the period when the concentration of endogenous IGF-I is the highest. PGs synthesized had elevated proportions of chondroitin sulfate vs heparan sulfate and of free chains, and reduced charge-density characteristics. Immunoprecipitation studies of [S-35]methionine-labeled glycoproteins revealed an increased synthesis of core-peptide of the PGs. IGF-I antisense oligonucleotide caused retardation in the growth of the kidneys along with the decrease in de novo synthesis of PGs. These findings indicate that IGF-I, a polypeptide essential to the renal growth and development, has a trophic effect on the embryonic kidney during the postinductive period of metanephric development, and the observed response has a temporal relationship with the increased synthesis of the PGs,

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LOCALIZATION OF FIBRIL MICROFIBRIL AND BASEMENT-MEMBRANE COLLAGENS IN DIABETIC GLOMERULOSCLEROSIS IN TYPE-2 DIABETES Reviewed

H MAKINO, K SHIKATA, J WIESLANDER, J WADA, N KASHIHARA, K YOSHIOKA, Z OTA

DIABETIC MEDICINE 11 ( 3 ) 304 - 311 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：JOHN WILEY & SONS LTD

Collagen is one of the major components of the extracellular matrices of the kidney. Basement membrane collagen, type IV collagen, is the major component in normal glomeruli. Fibril and interstitial collagen such as type III collagen, type V collagen, and type VI collagen are minor components of glomerular extracellular matrices and are localized mainly in the interstitium. Diabetic glomerulosclerosis is characterized by the expansion of the glomerular mesangial matrix as well as by thickening of the glomerular basement membrane. In order to clarify the roles of these various types of collagen in the development of diabetic glomerulosclerosis, immunohistochemical studies were perfomed in kidney specimens from patients with Type 2 diabetes. Early glomerulosclerosis is characterized by expansion of mesangial matrix with basement membrane collagen. However, in later stages glomerulosclerosis is characterized by an increase in the minor collagen components, such as type V and type VI collagen or collagens not normally present, such as type III collagen. Mesangial cells are known to synthesize all these types of collagen. In diabetes, phenotypic change in mesangial cells might produce excess amounts of fibril and interstitial collagen such as type III, type V, and type VI collagen, thus, leading to glomerulosclerosis.

DOI： 10.1111/j.1464-5491.1994.tb00276.x

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IMMUNOREACTIVITY OF THE JK-132 MONOCLONAL-ANTIBODY DIRECTED AGAINST BASEMENT-MEMBRANE COLLAGEN IN NORMAL AND DIABETIC GLOMERULI Reviewed

H MAKINO, K SHIKATA, T HAYASHI, J WIESLANDER, T HARAMOTO, K HIRATA, J WADA, T YOSHIDA, K YOSHIOKA, Z OTA

VIRCHOWS ARCHIV-AN INTERNATIONAL JOURNAL OF PATHOLOGY 424 ( 3 ) 235 - 241 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER VERLAG

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against alpha 1 to alpha 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the alpha 1 and alpha 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the alpha 3 and alpha 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the al and alpha 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the alpha 3 and alpha 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from alpha 1 to alpha 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.

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INSULIN-LIKE GROWTH-FACTOR-I RECEPTOR IN METANEPHRIC DEVELOPMENT Reviewed

YS KANWAR, ZZ LIU, J WADA

EXTRACELLULAR MATRIX IN THE KIDNEY 107 168 - 173 1994

A case of non-IgA mesangioproliferative glomerulonephritis with huge paramesangial hemispherical deposits

Sugimoto, H., Makino, H., Wada, J., Shikata, K.-I., Ikeda, S., Ota, Z.

the japanese journal of nephrology 35 ( 9 ) 1994

Effect of heparin and low-molecular-weight heparin on proliferative glomerulonephritis

Morita, Y., Makino, H., Ota, K., Wada, J., Shikata, K., Kashihara, N., Ikeda, S., Ogura, T., Ota, Z.

Japanese Journal of Nephrology 36 ( 7 ) 1994

DISTRIBUTION AND RELEVANCE OF INSULIN-LIKE GROWTH FACTOR-I RECEPTOR IN METANEPHRIC DEVELOPMENT Reviewed

ZZ LIU, J WADA, K ALVARES, A KUMAR, EI WALLNER, YS KANWAR

KIDNEY INTERNATIONAL 44 ( 6 ) 1242 - 1250 1993.12

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

During embryogenesis, various ligand-receptor interactions take place to modulate the development and growth of various mammalian organs. During these interactions, a critical concentration of a given receptor is needed to elicit a ligand-induced biologic response at a defined gestational stage of the fetus. In this study, the distribution and the relevance of insulin-like growth factor-I receptor (IGF-IR) in metanephric development was investigated. Kidneys were harvested from mouse embryos at days 13 to 19 of fetal gestation, and maintained in a metanephric culture system. Immunofluorescence studies, using anti-IGF-IR, revealed a high expression of IGF-IR at day 13, which declined during the later stages of gestation through neonatal life. To study the relevance of IGF-IR expression in metanephric development, antisense-oligodeoxynucleotide (ODN) experiments were carried out. Antisense-ODN 43 mer probes were synthesized utilizing rat IGF-IR cDNA selected nucleotide sequences which are highly conserved in other mammalian species. Southern blot analyses of various restriction fragments of the rat and mice genomic DNA yielded similar bands when hybridized with the antisense-ODN or rat IGF-IR cDNA, suggesting a high degree of homology in the region of the gene selected for the synthesis of antisense-ODN. Also, the antisense-ODN hybridized with the appropriate murine fetal kidney mRNA species, as ascertained by S1 nuclease protection assay. Inclusion of antisense-ODN in the culture medium resulted in an inhibition of the growth of the kidney, reduction in the population of the nephrons and disorganization of the ureteric bud branches. Effectivity of the antisense-ODN was reduced during the later stages of development when the expression of IGF-IR is decreased. Immunoprecipitation studies revealed a reduction in the IGF-IR associated radioactivity, indicating a specific translational arrest. These studies suggest that IGF-IR is relevant in the modulation of various developmental events during the early midgestational period, the time when it is highly expressed in the metanephric tissues.

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CLONING OF CDNA FOR THE ALPHA-SUBUNIT OF MOUSE INSULIN-LIKE GROWTH FACTOR-I RECEPTOR AND THE ROLE OF THE RECEPTOR IN METANEPHRIC DEVELOPMENT Reviewed

J WADA, ZZ LIU, K ALVARES, A KUMAR, E WALLNER, H MAKINO, YS KANWAR

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 ( 21 ) 10360 - 10364 1993.11

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

Various growth factors influence mammalian development by binding to specific cell surface receptors. These interactions are followed by a series of intracellular transductional events leading to a wide variety of biological effects. To establish the role of insulin-like growth factor I receptor (IGF-IR) in renal development, cDNA for the alpha subunit of the mouse IGF-IR was isolated, characterized, and used in expression studies and antisense experiments in a metanephric organ culture system. A 989-bp insert, encoding the signal peptide and 299 amino acids, isolated from a newborn mouse kidney cDNA library had 99% and 91% homology with the nucleotide sequences encoding the rat and the human IGF-IR, respectively. An almost-equal-to 11-kb message was readily detected by Northern blot analysis of RNA from the developing kidney at day 13 of gestation, and it declined during the subsequent embryonal and neonatal periods. In situ hybridization revealed high levels of message over the ureteric bud and its branches. A lower level of message was seen in the neonatal kidney, confined mainly to the tubules. Antisense oligodeoxynucleotide-treated metanephric kidneys were reduced in size and had a decreased population of nephrons with marked disorganization of ureteric bud branches. Immunofluorescence studies indicated an arrest of IGF-IR translation after antisense exposure. Immunoprecipitation studies showed a marked decrease in the biosynthesis of various extracellular matrix proteins that serve as regulators of morphogenesis. These studies suggest that the nucleotide sequence encoding the alpha subunit of mouse IGF-IR is highly conserved and that the receptor might play an essential role in the organogenesis of the kidney.

DOI： 10.1073/pnas.90.21.10360

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