Faculty Profiles - WADA Jun

Papers - WADA Jun

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Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropathy: aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide synthase in diabetic rat glomeruli Reviewed

H Sugimoto, K Shikata, J Wada, S Horiuchi, H Makino

DIABETOLOGIA 42 ( 7 ) 878 - 886 1999.7

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

Aims/hypothesis. Advanced glycation end products are believed to contribute to diabetic microvascular complications by inducing glomerular damage but their role has not been fully clarified. In this study, we explain their central role in the induction of inducible nitric oxide synthase and production of nitric oxide (NO) in streptozotocin-induced diabetic rat glomeruli.
Methods. Localization of carboxymethyllysine, which is one of the chemical components of advanced glycation end products, glomerular expression of inducible nitric oxide synthase and urinary excretion and glomerular production of NO2-/NO3- were examined at 0, 26, 51, and 52 weeks after the induction of diabetes. Therapeutic effects of aminoguanidine were also examined.
Results. Carboxymethyllysine was detected in the mesangial area in glomeruli and it progressively accumulated during 52 weeks of observation. Immunohistochemistry and hybridization studies in situ showed that the number of inducible nitric oxide synthase-positive cells was notably increased in diabetic rat glomeruli at 52 weeks. Further, this augmented expression paralleled intraglomerular expression of TNF-alpha and NO2-/NO3- in diabetic rat glomeruli. Treatment with aminoguanidine reduced the expression of TNF-alpha, inducible nitric oxide synthase and intraglomerular NO2-/NO3- production. It also ameliorated proteinuria in diabetic rats.
Conclusion/interpretation. This study showed that carboxymethyllysine possibly enhances the expression of inducible nitric oxide synthase by stimulating the expression of TNF-alpha in diabetic rat glomeruli. The carboxymethyllysine-cytokine-NO sequence pathway could be one of the major mechanisms in the development of diabetic nephropathy.

DOI： 10.1007/s001250051241

L-selectin and its ligands mediate infiltration of mononuclear cells into kidney interstitium after ureteric obstruction Reviewed

K Shikata, Y Suzuki, J Wada, K Hirata, M Matsuda, H Kawashima, T Suzuki, M Iizuka, H Makino, M Miyasaka

JOURNAL OF PATHOLOGY 188 ( 1 ) 93 - 99 1999.5

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

It was previously reported that the L-selectin ligands detected by a rat L-selectin and human IgG chimeric molecule (rLEC-IgG) are expressed in the distal tubules of the kidney, where no leukocyte traffic is seen under physiological conditions. In the present study, the role of L-selectin Ligands in leukocyte infiltration into the kidney interstitium was investigated using a rat ureteric obstruction model. After ligation of the ureter, ligands for L-selectin rapidly disappeared from tubular epithelial cells and were relocated to the interstitium and peritubular capillary walls, where infiltration of monocytes and CD8(+) T cells subsequently occurred, Mononuclear cell infiltration was significantly inhibited by intravenous injection of a neutralizing monoclonal antibody (MAb) against L-selectin, indicating the possible involvement of an L-selectin-mediated pathway, Interestingly, immunohistochemical studies with a MAb against sulphatide showed that the distribution of sulphatide, known to be one of the candidates of L-selectin ligand, was almost indistinguishable from the staining pattern of rLEC-IgG in both normal and ureteric obstructed kidneys, suggesting that sulphatide and/or related molecule(s) relocated to the renal interstitium were recognized by leukocyte L-selectin, leading to interstitial leukocyte infiltration, In line with this notion, intravenous injection of sulphatide markedly inhibited leukocyte infiltration, suggesting that L-selectin-sulphatide interaction may play a pivotal role in interstitial leukocyte infiltration in the kidney following ureteric obstruction. Copyright (C) 1999 John Wiley & Sons, Ltd.

DOI： 10.1002/(SICI)1096-9896(199905)188:1<93::AID-PATH305>3.0.CO;2-#

Signaling transduction pathway of angiotensin II in human mesangial cells: Mediation of focal adhesion and GTPase activating proteins Reviewed

Y Shikata, K Shikata, M Masuda, H Sugimoto, J Wada, H Makino

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 257 ( 1 ) 234 - 238 1999.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Human mesangial cells (HMCs) respond to angiotensin II stimulation, which modulates their physiological activities, i.e., contraction and proliferation. It has been revealed that focal adhesion kinase (FAK) and paxillin participate in the angiotensin Ii-mediated signaling and cytoskeletal rearrangements at focal adhesion. We investigated the influences of cell adhesion upon angiotensin LI effects in HMCs. In adherent cells, both FAR and paxillin were tyrosine phosphorylated by angiotensin II, while the cell detachment completely inhibited the tyrosine phosphorylation of paxillin. Activation of p44/42 mitogen-activated protein (MAP) kinase by angiotensin II was accentuated in suspended cells. Moreover, p190, a member of Rho GTPase activating protein (GAP), and RasGAP were coprecipitated with paxillin in adherent cells and angiotensin II stimulation reduced the formation of paxillin-p190 and paxillin-RasGAP complexes. These results suggest that the formation of focal adhesion complexes accelerated by accumulation of mesangial matrices may inhibit the proliferation of HMCs by modulating MAP kinase activity and be related to mesangial cell depletion. (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.0441

Differential distribution of insulin-like growth factor-1 and insulin-like growth factor binding proteins in experimental diabetic rat kidney Reviewed

N Miyatake, K Shikata, J Wada, H Sugimoto, S Takahashi, H Makino

NEPHRON 81 ( 3 ) 317 - 323 1999.3

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

Insulin-like growth factor-1 (IGF-1) is a peptide growth factor, and its activity is modulated by interaction with the family of IGF binding proteins (IGFBP-1 to 6). IGF-1 is detected in rat kidney and has metabolic and growth effects. To explore the possible involvement of IGFBPs in glomerular hypertrophy in streptozotocin (STZ)-induced diabetic rat, the immunolocalization of IGF-1 and IGFBPs were investigated. IGF-1 was gradually increased in the glomeruli of diabetic rats and correlated with glomerular hypertrophy. IGFBP-1 was transiently increased at 1 week after the STZ injection and declined to control level during the following period. In contrast, IGFBP-4 was increased in the diabetic glomeruli throughout the observation period. With insulin treatment, the levels of IGF-1, IGFBP-1 and 4 were normalized and glomerular hypertrophy was prevented. Initial glomerular hypertrophy of diabetic nephropathy is a related IGF-1 action, which may be modulated by IGFBP-1 and 4.

Increased urinary excretion of macrophage-colony-stimulating factor (M-CSF) in patients with IgA nephropathy: Tonsil stimulation enhances urinary M-CSF excretion Reviewed

M Matsuda, K Shikata, J Wada, H Yamaji, Y Shikata, A Doi, M Kosaka, H Akagi, Y Masuda, Y Ohmoto, H Makino

NEPHRON 81 ( 3 ) 264 - 270 1999.3

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

Upper respiratory tract infection including chronic tonsillitis is considered to be involved in the onset and/or the progression of IgA nephropathy. It is well known that deterioration of urinary findings occurs after episodes of upper respiratory tract infection in patients with IgA nephropathy. We previously showed that the expression of macrophage-colony-stimulating factor (M-CSF) is increased in the glomeruli of patients with IgA nephropathy and correlated with glomerular mesangial proliferation, suggesting that M-CSF plays an important role in the progression of IgA nephropathy. In the present study, we measured the serum and urinary concentrations of M-CSF in patients with IgA nephropathy associated with chronic tonsillitis. Furthermore, we evaluated the effects of the local provocation test of tonsils (mechanical tonsil stimulation) on the serum and urinary concentrations of M-CSF in the following three groups: (1) IgA nephropathy with severe mesangial proliferation, (2) IgA nephropathy with mild mesangial proliferation, and (3) patients with chronic tonsillitis without renal disease. The serum and urinary levels of M-CSF in the groups with severe and mild IgA nephropathy were significantly higher than those in the chronic tonsillitis group. The urinary M-CSF level but not the serum M-CSF level was positively correlated with the degrees of mesangial proliferation and glomerular M-CSF expression in the renal biopsy specimens. The urinary M-CSF concentration was significantly increased after tonsillitis stimulation in both mild and severe IgA nephropathy groups. Enhanced urinary excretion of M-CSF prolonged for 7 days after tonsil stimulation in the severe IgA nephropathy group; in contrast, the urinay M-CSF level was increased for only 2 days after tonsil stimulation in the mild IgA nephropathy group. The urinary M-CSF level was not changed in the chronic tonsillitis group after tonsil stimulation. The serum concentrations of M-CSF were not changed after tonsil stimulation in these three groups. Our present results suggest that tonsil stimulation contributes to the progression of IgA nephropathy via enhancement of glomerular production of M-CSF. The urinary excretion of M-CSF may be a useful predictor to evaluate the relevance of chronic tonsillitis to the disease and the indication of tonsillectomy in patients with IgA nephropathy.

Renal organogenesis and extracellular matrix - Knockout mice are telling the truth? -

Wada, J., Ota, K., Kashihara, N., Kanwar, Y.S., Makino, H.

Connective Tissue 31 ( 1 ) 1999

Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy Reviewed

H Sugimoto, K Shikata, M Matsuda, M Kushiro, Y Hayashi, K Hiragushi, J Wada, H Makino

DIABETOLOGIA 41 ( 12 ) 1426 - 1434 1998.12

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

The overproduction of nitric oxide (NO) is reported in the diabetic kidney and considered to be involved in glomerular hyperfiltration. The precise mechanism of NO production in the diabetic kidney is, however, not known. In this report, we compare the localization of endothelial cell nitric oxide synthase (ecNOS) isoform expression in the kidney tissue of streptozotocin (STZ)-induced diabetic rats and 5/6 nephrectomized rats and clarify the pivotal role of ecNOS for the glomerular hyperfiltration in the early stages of diabetic nephropathy. In diabetic rats, the diameters of afferent arterioles, the glomerular volume, creatinine clearance, and urinary NO2/NO3 were increased after the induction of diabetes. Efferent arterioles were, however, not altered. Insulin or L-NAME treatment returned the diameters of afferent arterioles, glomerular volume, creatinine clearance, and urinary NO2/NO3 to normal. The expression of ecNOS in afferent arterioles and glomeruli of diabetic rats increased during the early stages of the disease, but was not altered in efferent arterioles. Treatment with either insulin or L-NAME decreased ecNOS expression in afferent arterioles and in glomeruli. In contrast, the ecNOS expression was upregulated in both afferent and efferent arterioles and in the glomeruli of 5/6 nephrectomized rats, where the dilatation of afferent and efferent arterioles and glomerular enlargement were observed. Treatment with L-NAME ameliorated the ecNOS expression and dilatation of arterioles. We conclude that enhanced NO synthesis by ecNOS in afferent arterioles and glomerular endothelial cells in response to the hyperglycaemic state could cause preferential dilatation of afferent arterioles. which ultimately induces glomerular enlargement and glomerular hyperfiltration.

DOI： 10.1007/s001250051088

Deposition of mannan binding protein and mannan binding protein-mediated complement activation in the glomeruli of patients with IgA nephropathy Reviewed

M Matsuda, K Shikata, J Wada, H Sugimoto, Y Shikata, T Kawasaki, H Makino

NEPHRON 80 ( 4 ) 408 - 413 1998.12

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

Mannan binding protein (MBP) is a C-type lectin and has a high affinity to mannose and N-acetyl glucosamine. It is also known to activate C4 and C2 without C1 component, which is called 'lectin pathway'. We now report the presence of MBP and MBP-mediated complement activation in renal glomeruli of IgA nephropathy patients using an immunohistochemical method. In 7 of 42 cases with IgA nephropathy, MBP was detected in the glomerular mesangial area and colocalized with IgA1. In 19 cases with other types of IC-mediated glomerulonephritis including lupus nephritis and membranous nephropathy or without nephritis, MBP was not detected in the glomerulus. The C2- and/or C4-positive rate was 87.5% in the MBP-positive group and 20% in the MBP-negative group of IgA nephropathy. In addition, MBP-positive cases showed marked mesangial cell proliferation, lower creatinine clearance (53.4 +/- 10.0 vs. 77.8 +/- 4.7 ml/min) and higher urinary protein excretion (2.5 +/- 0.9 vs. 0.9 +/- 0.2 g/day) compared with MBP-negative cases. These findings suggested that MBP was involved in glomerular complement activation through the lectin pathway and thus induced glomerular injury of IgA nephropathy. Since oligosaccharide chain alterations such as reduced sialic acid and galactose of IgA1 molecule have been reported in IgA nephropathy patients, MBP might bind to the IgA1 molecule via interaction between MBP and sugar chain.

Isolation of rat fibrillin-1 cDNA and its relevance in metanephric development Reviewed

YS Kanwar, K Ota, QW Yang, A Kumar, J Wada, N Kashihara, DR Peterson

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 275 ( 5 ) F710 - F723 1998.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

The role of fibrillin-1 in metanephrogenesis was investigated. Fibrillin-1 cDNA was isolated from the rat kidney cDNA Library and sequenced, and its spatiotemporal expression was studied. It had similar to 88% homology with human fibrillin-1 and had Ca(2+) binding epidermal growth factor-like domains, transforming growth factor-p binding protein motifs, and an RGD binding site. Northern blot analysis revealed an similar to 10-kb transcript, and fibrillin-1 expression was developmentally regulated. In situ hybridization and immunofluorescence studies indicated that at day 15 of gestation, fibrillin-1 is expressed in the metanephric mesenchyme. At day 18, its expression was confined to nascent blood vessels and glomeruli, and it increased in the newborn and neonatal kidneys. Immunoprecipitation revealed an similar to 300-kDa band by SDS-PAGE. Treatment with fibrillin-1 antisense oligodeoxynucleotide induced marked dysmorphogenesis of the embryonic metanephroi. Concomitantly, the fibrillin-1 mRNA, antibody reactivity in the metanephroi, and fibrillin-1-specific radioincorporation were reduced. These data indicate that, like alpha(v)beta(3) integrin, a known morphogen and a putative receptor of fibrillin-1, the fibrillin-1 modulates events related to early organogenesis and possibly also the vascularization of the rat kidney.

Relevance of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis Reviewed

EI Wallner, QW Yang, DR Peterson, J Wada, YS Kanwar

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 275 ( 4 ) F467 - F477 1998.10

Language：English Publisher：AMER PHYSIOLOGICAL SOC

Mammalian nephrogenesis begins by the reciprocal interaction of the ureteric bud with the undifferentiated mesenchyme. The mesenchyme differentiates into an epithelial phenotype with the development of the glomerulus and proximal and distal tubules. At the same time, the mesenchyme stimulates the branching morphogenesis of the ureteric bud that differentiates into the collecting ducts. These inductive interactions and differentiation events are modulated by a number of macromolecules, including the extracellular matrix (ECM), integrin receptors, and cell adhesion molecules. Many of these macromolecules exhibit spatiotemporal developmental regulation in the metanephros. Some are expressed in the mesenchyme, whereas others appear in the ureteric bud, epithelia. The molecules expressed in the mesenchyme or at the epithelial:mesenchymal interface may serve as ligands while those in the epithelia serve as the receptors. In such a scenario the ligand and the receptor would be ideally suited for epithelial: mesenchymal paracrine/juxtacrine interactions that are also influenced by RGD sequences and Ca(2+) binding domains of the ECM proteins and their receptors. This review addresses the role of such interactions in metanephric development.

A case of membranous nephropathy associated with psoriasis vulgaris Reviewed

H Yamaji, K Shikata, J Wada, Y Hayashi, S Ikeda, H Makino

NEPHRON 80 ( 1 ) 111 - 112 1998.9

Fatal hyperammonemia in a patient with systemic lupus erythematosus Reviewed

H Ichikawa, T Amano, K Kawabata, M Kushiro, J Wada, Y Nagake, H Makino

INTERNAL MEDICINE 37 ( 8 ) 700 - 703 1998.8

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC INTERNAL MEDICINE

We treated a 31-year-old woman with systemic lupus erythematosus, renal failure with nephrotic syndrome, and a long-standing seizure disorder, who developed severe hyperammonemia with a fatal outcome. Blood chemistry examination did not indicate liver disease, and amino acid concentrations did not suggest a defect in the urea cycle. Discontinuation of anticonvulsant treatment with valproic acid (VPA) failed to bring about improvement. We speculated that hyperammonemia in this case was induced by VPA, and the existence of other underlying factors, including the administration of aspirin and cimetidine, hypoalbuminemia, and renal failure might elevate the concentration of the serum free fraction of VPA.

DOI： 10.2169/internalmedicine.37.700

Ultrastructure of mesangial type III collagen deposition in a patient with IgA nephropathy Reviewed

M Kunitomi, J Wada, N Miyatake, Y Hayashi, K Ota, H Makino

AMERICAN JOURNAL OF KIDNEY DISEASES 32 ( 1 ) 146 - 152 1998.7

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

We report a case of IgA nephropathy with focal and segmental deposition of type III collagen in mesangium, confirmed by immunohistochemical and electron microscopic methods. Tissue negative staining showed that focal and segmental fibrotic lesions in the mesangial area consisted of disarrayed or curled striated collagen fibers and striated membranous structures. Diabetes mellitus, hypertension, and advanced glomerular sclerosis were absent in this case, and mesangial cells surrounding the type III collagen showed vacuolar degeneration revealed by electron microscopy. Production of type III collagen may be the marker for phenotypic change of mesangial cells in immune-mediated glomerular diseases, (C) 1998 by the National Kidney Foundation, Inc.

DOI： 10.1053/ajkd.1998.v32.pm9669436

Cloning of murine membrane-type-1-matrix metalloproteinase (MT-1-MMP) and its metanephric developmental regulation with respect to MMP-2 and its inhibitor Reviewed

K Ota, WG Stetler-Stevenson, QW Yang, A Kumar, J Wada, N Kashihara, EI Wallner, YS Kanwar

KIDNEY INTERNATIONAL 54 ( 1 ) 131 - 142 1998.7

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

Background. Extracellular matrix macromolecules regulate morphogenesis of embryonic organs, and are developmentally regulated. Their expression and turnover is regulated by matrix metalloproteinases (MMPs). Recently, an epithelial cell "membrane" associated metalloproteinase (MT-1-MMP) has been identified that acts as an activator of a "secreted" MMP-2, and is produced by mesenchymal fibroblasts. The activity of MMP-2 is inhibited by a "soluble" tissue inhibitor of MMP-2, TIMP-2. The role of MT-1-MMP in renal development is unknown.
Methods. MT-1-MMP was cloned from embryonic mouse kidney cDNA library, and its spatio-temporal distribution during development in the context of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was studied.
Results. The cloned MT-1-MMP exhibited similar to 86% nucleotide sequence homology with human MT-1-MMP, and had a catalytic domain and a zinc binding site preceded by a RRKR furin recognition motif. A similar to 4.5 Kb MT-1-MMP mRNA transcript was detected, and its expression was developmentally regulated. A parallel developmental regulation of MMP-2 mRNA expression was also observed. TIMP-2 expression was also developmentally regulated, but lagged behind MT-1-MMP and MMP-2. By in situ hybridization, MT-1-RAMP mRNA was seen to be confined to ureteric bud epithelia, and was absent in the mesenchyme, while MMP-2 was confined to the mesenchyme. MT-1-MMP protein expression was seen on ureteric bud epithelia, induced mesenchyme and nascent nephrons, and it was highest during mid gestation. Similar spatio-temporal expressions of MMP-2 and TIMP-2 proteins were observed.
Conclusions, mRNAs of MT-MMP-1 and MMP-2 are expressed in the respective epithelial and mesenchymal compartments, while their proteins are co-expressed in the epithelia suggest that MT-1-MMP and MMP-2, in conjunction with TIMP-2, may be involved in paracrine/juxtacrine epithelial:mesenchymal interactions during metanephrogenesis.

Therapeutic effects of prostacyclin analog on crescentic glomerulonephritis of rat Reviewed

M Kushiro, K Shikata, H Sugimoto, Y Shikata, N Miyatake, J Wada, M Miyasaka, H Makino

KIDNEY INTERNATIONAL 53 ( 5 ) 1314 - 1320 1998.5

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

Prostacyclin (PGI(2)) is known to have a relaxative action on vascular smooth muscle, an inhibitory action against platelet activation and neutrophil function. Previous studies showed the preventive effects of PGI(2) on lupus nephritis and Thy-1 nephritis, although the mechanism has not been clarified. Glomerular endothelial expression of intercellular adhesion molecule-1 (ICAM-1) is up-regulated in experimental and human glomerular diseases, and is known to facilitate leukocyte infiltration into the glomeruli, which ultimately induces the various glomerular injuries. In the present study, we evaluated the therapeutic effects of PGI(2) tin a rat model for crescentic glomerulonephritis and investigated its putative mechanism in relation to ICAM-1-mediated leukocyte recruitment. Wistar-Kyoto (WKY) rats were injected with nephrotoxic serum and received continuous intraperitoneal infusion of PGI(2). PGI(2) dramatically decreased proteinuria (123.0 +/- 18.8 vs. 31.6 +/- 4.5), crescent formation and deposition of fibrinogen in the glomeruli, while the deposition of rabbit IgG, rat IgG and rat C3 alone the capillary walls was not changed. Furthermore, intraglomerular expression of ICAM-1 and infiltration of macrophages were significantly suppressed by administration with PGI(2). In contrast, influx of CD4 or CD8 positive cells was not altered. The present results suggest that PGI(2) shows the preventive effects on experimental crescentic glomerulonephritis by inhibiting intraglomerular coagulation and ICAM-1-mediated macrophage-glomerular endothelial cell adhesive pathway.

DOI： 10.1046/j.1523-1755.1998.00881.x

Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney Reviewed

J Wada, YS Kanwar

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95 ( 1 ) 144 - 149 1998.1

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

Mammalian translocase of mitochondrial inner membrane (mTim44) was isolated during representational difference analysis of cDNA from diabetic mouse kidney. Streptozotocin-induced diabetic mouse kidney cDNA was prepared and subtracted by normal mouse kidney cDNA. By using one of the isolated cDNA fragments as a screening probe, full length cDNA of mTim44 was isolated from lambda ZAP kidney cDNA library. At the nucleotide level, mTim44 did not exhibit significant homology with any known genes; however, at the amino acid level, it had 50% similarity and 29% identity with yeast Tim44. C-terminal FLAG epitope tagged mTim44 fusion protein was transiently expressed in COS7 cells. By using anti-FLAG epitope M2 monoclonal antibody, mTim44 was found to have its subcellular localization associated with mitochondria. By immunoelectron microscopy, mTim44 was seen in the paracrystalline structures within the mitochondria, as well as in their cristae. Mitochondrial import assay of in vitro translated mTim44 indicated that its precursor product (approximate to 50 kDa) was imported and proteolytically processed to a mature approximate to 44-kDa protein, and its translocation was inner membrane potential (Delta Psi)-dependent. Imported mTim44 was protected from protease digestion in which outer membranes were selectively permeabilized with digitonin. The mature form of mTim44 could be recovered in the supernatant of sonicated mitochondrial membrane fraction treated with 0.1 M Na2CO3, pH 11.5. The data indicate that mTim44 is a mitochondrial inner membrane protein, one of the members of the mammalian TIM complex and up-regulated in hyperglycemic states.

DOI： 10.1073/pnas.95.1.144

Bcl-2 expression and apoptosis in nephrotoxic nephritis Reviewed

H Sugiyama, N Kashihara, T Onbe, Y Yamasaki, J Wada, T Sekikawa, K Okamoto, K Kanao, Y Maeshima, H Makino

EXPERIMENTAL NEPHROLOGY 5 ( 6 ) 481 - 489 1997.11

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

The product of the Bcl-2 proto-oncogene has been shown to prolong cell survival by preventing apoptosis in several cell lineages. To investigate the regulatory mechanisms of apoptosis in glomerulonephritis, we examined the expression pattern of the Bcl-2 protein together with cellular events in rat nephrotoxic nephritis. Bcl-2 protein and proliferating cell nuclear antigen were detected in glomeruli by immunohistochemistry. Morphologic changes of apoptosis were identified by electron and light microscopy and an in situ DNA nick end labeling method. The first (heterologous) phase began with significant neutrophil infiltration shortly after the injection of nephrotoxic serum. Both Bcl-2 expression and the number of proliferating cells in the glomeruli were at maximum at 24 h in the heterologous phase. Glomerular hypercellularity with an influx of macrophages and the number of apoptotic glomerular cells peaked on day 14 in the second (autologous) phase. Glomerulonephritis resolved after that. These results suggest that overexpression of the Bcl-2 protein may play a role in glomerular cell survival and exacerbation of glomerulonephritis. Apoptosis may occur as an active mechanism in the resolution of the autologous phase in nephrotoxic nephritis.

Role of extracellular matrix, growth factors and proto-oncogenes in metanephric development Reviewed

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

KIDNEY INTERNATIONAL 52 ( 3 ) 589 - 606 1997.9

Developmental regulation and partial-length cloning of tubulointerstitial nephritis antigen of murine metanephros Reviewed

A Kumar, K Ota, J Wada, EI Wallner, AS Charonis, FA Carone, YS Kanwar

KIDNEY INTERNATIONAL 52 ( 3 ) 620 - 627 1997.9

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

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix (ECM) glycoprotein that has been recently isolated and cloned from the rabbit kidney. It is an integral component of the basal lamina, and unlike other basement membrane proteins it is exclusively expressed in the tubular basement membranes (TBMs). Since other ECM glycoproteins have been shown to regulate development of various organ systems, studies were initiated to ascertain its developmental regulation in renal tubulogenesis and glomerulogenesis. Embryonic (day-13 and -17 of gestation), newborn and one-week-old mice kidneys were harvested for expression of TIN-ag as well as cDNA cloning studies. Immunostaining with polyclonal anti-TIN-ag antibody revealed its localization to the basal lamina of ureteric bud branches and epithelial elements of developing nephrons in day-13 embryonic kidneys. Interestingly, it was heavily expressed at the tips of the ureteric bud branches, and was not expressed in the distal convolutions of the S-shaped body stage of the nephrons, the region which forms the future glomerulus. Al day-17, TIN-ag expression was less, and the immuno-reactivity was mainly localized to the cortex. In the newborn and one-week-old mice kidneys, the cortical expression of TIN-ag increased progressively, but was absent in the glomeruli. The TIN-ag expression was confined to the cortical TBMs, while absent in the medullary tubules, the latter included segments of the collecting ducts and loop of Henle. Immunoprecipitation studies on [S-35]methionine-labeled metanephroi revealed a single band of similar to 58 kDa at day-13, and the incorporated radioactivity decreased at day-17. No high molecular weight isoforms were observed. A partial-length mouse TIN-ag cDNA of similar to 530 bp PCR product was generated, and it had similar to 88% and similar to 93% nucleotide and amino acid sequence homolgy, respectively, with rabbit TIN-ag. Utilizing this cDNA, Northern blot analyses revealed a single transcript of similar to 2 Kb in fetal and postnatal mice kidneys. mRNA expression initially decreased at day-17, and then progressively increased by one week. Utilizing a mouse TIN-ag riboprobe, in situ hybridization studies revealed a generalized diffuse expression of TIN-ag in the epithelial elements of developing nephrons and ureteric bud branches at day-13. Gene expression decreased by day-17, and became confined to the renal cortex, and then progressively increased during the neo-and post-natal periods, but remained absent in the renal medulla and glomeruli. These data indicate that TIN-ag is expressed in the metanephros early in embryonic life in the absence of any detectable isoforms, and it exhibits spatio-temporal characteristics during metanephric development. Being concentrated at the tips of the ureteric bud branches, it is conceivably involved in epithelial:mesenchymal interactions which are highly prevalent during renal organogenesis, and its role in tubulogenesis diverges from glomerulogenesis at the S-shaped body stage of the nephron.

Representational difference analysis of cDNA of genes expressed in embryonic kidney Reviewed

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

KIDNEY INTERNATIONAL 51 ( 5 ) 1629 - 1638 1997.5

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

Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based differential cloning method. It involves hybridiza tion of two populations of cDNA with selective amplification of differentially expressed genes. To isolate the differentially expressed renal development, mRNAs from embryonic kidneys at day 13 (E13) and postnatal kidneys from three-week-old (P3) mice were extracted, and double stranded cDNAs prepared. Double stranded cDNAs were digested with DpnII, adaptor-ligated, and amplified by PCR, using adaptor primer to generate ''representative amplicons.'' These reflect the ''representation'' of most of the cDNA population. The term ''amplicons'' denotes amplified PCR product. Among the two populations of cDNA, E13 kidney cDNA was used as a ''tester;'' containing target genes, and P3 kidney cDNA as a ''driver,'' driving the process of subtraction, following which, they were subjected to cDNA-RDA under low stringency conditions. During the first round of cDNA-RDA, embryonic globin genes were isolated. To competitively eliminate these genes, plasmid DNAs of globin genes were supplemented into driver, and subjected to the second round of cDNA-RDA. This resulted in the isolation of four cDNA clones: H19 gene, mesoderm-specific cDNA, COL2A1 gene, and a novel cDNA. By Northern blot analyses, the H19 gene and mesoderm-specific cDNA exhibited a high degree of developmental regulation, that is, they were abundantly expressed in E13 kidney, and their expression was barely detectable in P3 kidney. The differential developmental regulation of mesoderm-specific cDNA was confirmed by tissue in situ hybridization experiments. The COL2A1 and novel cDNA were rare transcripts in the embryonic kidney. However, Southern blot analyses of representations indicated their up-regulated expressions in E13 kidneys. The novel gene was differentially expressed in 13-day embyronic lung, and Northern blot analysis revealed an similar to 10 Kb transcript. These results indicate that cDNA-RDA is a sensitive technique to identify rare transcripts:vith differential expression, and since there is a minimal chance to isolate false positive clones, cDNA-RDA may serve as a powerful tool for delineating up- or down-regulation of the genes involved in various pathological or physiological states of the kidney.

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