A vitamin D analog ameliorates glomerular injury on rat glomerulonephritis.

OCT (22-oxa-calcitriol), a vitamin D analog, has been reported to show strong inhibitory effects on mesangial cell proliferation in vitro. In the present study, we report a study of the effect of OCT on anti-thy-1 glomerulonephritis. Both OCT and 1,25(OH)(2)D(3) significantly inhibited mesangial cell proliferation, the degree of glomerulosclerosis, and albuminuria at day 8 compared to the disease control group. The OCT-treated group showed normal calcium levels but the 1,25(OH)(2)D(3)-treated group showed higher levels. The disease control group showed a marked increase of type I and type IV collagens, and alpha-smooth muscle actin (alpha-SMA) compared to the normal group. The treatment of OCT or 1,25(OH)(2)D(3) significantly reduced the expression of these proteins. The mRNA of the glomeruli of anti-thy-1 model expressed significantly higher levels of type I and type IV collagens, and alpha-SMA at day 8 compared to normal rats. Treatment with OCT or 1,25(OH)(2)D(3) inhibited the mRNA expressions of type I and type IV collagens, as well as that of alpha-SMA. These data demonstrate that OCT inhibits mesangial cell proliferation and extracellular matrix expansion with a low calcemic activity. Disease control rats showed significantly increased levels of transforming growth factor-beta1 protein in the glomeruli, but treatment with OCT or 1,25(OH)(2)D(3) markedly reduced this expression. The levels of mRNA in glomeruli were also consistent with these protein levels. Therefore, the suppressive effect of OCT may be mediated by inhibition of transforming growth factor-beta1. The present results suggest that OCT has potential for use in therapeutic strategy for the treatment of glomerulonephritis without inducing hypercalcemia.

Regulation of mOAT-mediated organic anion transport by okadaic acid and protein kinase C in LLC-PK(1) cells.

Organic anion transporters in the kidney proximal tubule play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites, thereby preventing their potentially toxic effects within the body. We have previously cloned a cDNA encoding an organic anion transporter from mouse kidney (mOAT) (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478; Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524). In the present study, we assessed the potential for regulation of this transporter by heterologous expression of mOAT in the pig proximal tubule-like cell line, LLC-PK(1). We report here that both protein phosphatase (PP1/PP2A) inhibitor, okadaic acid, and protein kinase C (PKC) activators down-regulate mOAT-mediated transport of para-aminohippuric acid (PAH), a prototypic organic anion, in a time- and concentrationdependent manner. However their mechanisms of action for this down-regulation are distinct. Okadaic acid modulated PAH transport, at least in part, through phosphorylation/dephosphorylation of mOAT; phosphoamino acid analysis indicated this phosphorylation occurs on serine. In contrast, PKC activation induced a decrease in the maximum transport velocity (V(max)) of PAH transport without direct phosphorylation of the transporter protein. Together these results provide the first demonstration that regulation of organic anion transport by mOAT is likely to be tightly controlled directly and indirectly by phosphatase PP1/PP2A and PKC. Our results also suggest that kinases other than PKC are involved in this process.

Heterologous expression and functional characterization of a mouse renal organic anion transporter in mammalian cells.

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and post-translational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, anti-hypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

Expression of heat shock protein 47 is increased in remnant kidney and correlates with disease progression.

Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and V collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progressive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the biosynthesis and secretion of procollagen molecules. The expression of HSP47 has been reported to increase in parallel with the expression of collagens during the progression of various fibrosis models. However, it remains unclear whether an inhibition of HSP47 overexpression would suppress collagen accumulation and thus reduce the progression of fibrotic diseases. In this study, we attempted to attenuate glomerular collagen accumulation by inhibiting the overexpression of HSP47 with antisense oligodeoxynucleotides in an experimental glomerulonephritis model induced by anti-Thy-1 antibodies. The administration of antisense oligodeoxynucleotides against HSP47 at the induction of the glomerular disease markedly suppressed the increased production of collagens and attenuated the histologic manifestations of the disease. These results provide direct evidence of a pivotal role for HSP47 in the pathogenesis of glomerulonephritis.

Demonstration of DNA replication factor C in human glomerular lesions.

Glomerulosclerosis is a common pathological finding in many human glomerular diseases that ultimately leads to end-stage kidney disease. The regulatory mechanism that controls mesangial proliferation as well as accumulation of mesangial matrix, however, is not known. Recently, a protein factor (MSW) which binds to the specific sequence of the promoter of the alpha 1 and alpha 2 type IV collagen genes was cloned. MSW was found to be identical to a large subunit (Alp145) of DNA replication factor C. These findings suggest that MSW may have important functions in mesangial cell proliferation and type IV collagen synthesis, both of which are prominent findings in glomerulosclerosis. In the present study, we report that augmented expression of MSW protein in human glomerular diseases that exhibit glomerulosclerosis (IgA nephropathy, membranoproliferative glomerulonephritis, and focal glomerulosclerosis). Minimal expression of MSW protein was observed in human glomerular diseases that rarely show glomerulosclerosis (membranous nephropathy, and minimal change nephrotic syndrome). There was a significant correlation between the levels of MSW expression and type IV collagen expression. Elevated expressions of both proliferating cell nuclear antigen and MSW were also observed in most patients with proliferative glomerular diseases. These studies suggest that MSW protein plays a regulatory role in the development of mesangial cell proliferation and matrix expansion during progression of glomerular injuries.

A novel transcription factor is correlated with both glomerular proliferation and sclerosis in the rat renal ablation model.

Glomerular accumulation of the extracellular matrix (ECM) with subsequent sclerosis is a common finding in most progressive renal diseases. Recently MSW (Mouse South Western) protein was cloned by its ability to bind the bidirectional promoter of the collagen IV genes. This protein was also reported as the large subunit of the DNA replication complex A1, as well as the promoter binding protein of corticotropin-releasing hormone and the angiotensinogen gene. To investigate the mechanism of accumulation of the ECM as it relates to glomerular cellular events, the expression of MSW protein was studied in the remnant kidney model. Progressive expression of MSW protein was found in the glomerular sclerotic lesion at week 4 and at later time points after renal ablation. The expression of proliferating cell nuclear antigen (PCNA) and type IV collagen was also correlated with the expression of MSW protein by immunofluorescence. RNA dot blot analysis also showed that the expression of MSW mRNA was increased at week 7 in association with the augmented expression of type IV collagen. These results, taken together, suggest that MSW protein plays an important role in the regulation of type IV collagen gene expression in vivo and may contribute to glomerular cell proliferation and the development of glomerulosclerosis.

Metabolism substrate with negative myocardial uptake of iodine-123-BMIPP.

UNLABELLED: Iodine-123-BMIPP is an iodinated methyl-branched-chain fatty acid. Low uptake of BMIPP relative to thallium or other perfusion tracer indicates metabolically damaged but viable myocardium (for example, ischemic but viable myocardium). In some cases, however, negative myocardial uptake of BMIPP is observed. The main purposes of this study were to assess the frequency of such BMIPP findings and to clarify metabolism of such cases by using PET. METHODS: Among the 1258 patients who underwent BMIPP scintigraphy, 11 patients (0.9%) showed negative myocardial uptake of BMIPP. Under fasting condition, PET using [11C]palmitate, 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) and [11C]acetate was performed in nine of these 11 patients. RESULTS: Global myocardial uptake of [11C]palmitate, expressed as the standardized uptake value, was significantly lower in the patients than in control (3.62 +/- 0.44 versus 5.49 +/- 1.62; p < 0.01). However, the early phase clearance rate of [11C]palmitate and oxidative metabolism was not significantly different. In the fasting state, PET studies showed increased FDG accumulation in seven of nine patients (high group) and decreased accumulation in two patients (low group). In the high group patients, glucose metabolism in the fasting state was similar to that in the normal volunteers after glucose loading (Kcomplex: 0.050 +/- 0.016 versus 0.038 +/- 0.015; p = ns). However, low glucose metabolism was noted in the low group patients (Kcomplex: 0.007 and 0.005). CONCLUSION: Negative myocardial uptake of BMIPP is occasionally, but not often, observed. Global uptake of [11C]palmitate was decreased in these patient. The majority of these patients showed "metabolic switching" from normal free fatty acid metabolism to abnormally enhanced glucose metabolism in the fasting state. However, some patients showed decreases in both exogenous glucose utilization and free fatty acid uptake in the fasting state.

Factor IX Bm Kiryu: a Val-313-to-Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling.

Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patient's plasma by immunoaffinity chromatography with a calcium-dependent anti-factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two-chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z-Arg-p-nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single-strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T-to-A substitution at nucleotide number 31,059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val-313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patient's mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val-313 to Asp substitution altered the conformation of the substrate-binding site. These data combined with our previous findings on a Gly-311-to-Glu mutant of factor IX suggest that the loop conformation from Gly-311 to ARg-318 is important for the expression of coagulant activity.

Characterization of the enhancer region for germline transcription of the gamma 3 constant region gene of human immunoglobulin.

A constant region gene (C) of the immunoglobulin heavy chain can be transcribed as germline transcripts from a promoter located upstream of a switch region. We have studied the structure and function of the human C gamma 3 promoter region. When the human IgM-producing cell line SSK41 is stimulated with interleukin 4 (IL-4) in the presence of phorbol myristate acetate (PMA) or Staphylococcus aureus Cowan I, expression of germline C gamma 3 transcripts was specifically augmented within 4 h. Upstream DNA fragments flanking the I gamma 3 exon were fused with a reporter gene and tested for IL-4-induced promoter/enhancer activity by transfection of SSK41 cells. The DNA fragment between 450 and 250 bp upstream of the transcription initiation site of the C gamma 3 gene was shown to be required for transcriptional up-regulation by PMA and IL-4. The upstream 514 bp fragment of the I gamma 3 flanking region was shown to contain an enhancer activity in response to PMA and IL-4.

Cloning and characterization of a protein binding to the J kappa recombination signal sequence of immunoglobulin genes.

A protein with molecular weight of 60,000 that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified from the nuclear extract of a murine pre B cell line 38B9. This binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. The cDNA clone (RBP-2) was isolated based on partial amino-acid sequence of this protein. This cDNA encodes 526 amino-acid residues, and its sequence does not show extensive overall homology with any known proteins, but displays an interesting homology to a 40-residue region that is conserved among a subset of site specific recombinase (integrase family).

A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif.

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.

First trimester prenatal diagnosis of haemophilia A using factor VIII gene probe.

Accurate first-trimester prenatal diagnosis was achieved in a Japanese haemophilia A family by the use of a restriction fragment length polymorphism (RFLP) located within the F.VIII gene. Since the pregnant woman's heterozygosity for BclI polymorphism in F.VIII/intron 18 (F8A) probe was informative, chorionic villus sampling (CVS) was performed at 9 weeks of gestation. Restriction analysis showed that the fetus was heterozygous for the BclI site and had received a normal paternal X chromosome (0.9 kb) and a normal maternal X (1.2 kb). Therefore, we concluded that the fetus was a non-carrier female. Pregnancy went to term and woman gave birth to an apparently healthy female. At one week after birth a coagulation study confirmed that the newborn infant is not a carrier. The first-trimester prenatal diagnosis of haemophilia A is possible by CVS due to a RFLP in the F.VIII gene.

Production of sterile transcripts of C gamma genes in an IgM-producing human neoplastic B cell line that switches to IgG-producing cells.

A human neoplastic B cell line SSK41 that expresses IgM on its surface switches spontaneously to IgG-producing cells. The SSK41 line contains a single immunoglobulin heavy-chain locus, the constant region (C) genes of which retain the germline configuration. The IgG-producing SSK41 line was purified by sorting, and shown to have undergone S-S recombination with deletion of the C mu gene. This line produced secretory and membrane-bound forms of gamma-chain mRNA. From cDNA libraries of a mixed population of IgM+/IgG+ SSK41 cells, we have isolated cDNA clones encoding the mature membrane-bound and secretory forms of the mu and gamma 1 heavy chains, all of which share the same variable region sequence. cDNA clones containing the mature gamma 3 chain were identified as well. We also isolated cDNA clones containing C gamma 1 and C gamma 3 sterile transcripts from the SSK41 line. These sterile transcripts contained additional exon sequences designated 'I' which were localized upstream of the C gamma 1 and C gamma 3 switch regions and homologous to murine counterparts. The I sequences were precisely spliced to the 5' ends of the corresponding C gamma exon sequences. These features of germline CH transcripts, i.e. the isotype specificity to class switching, location of exons, and sequences per se, are highly conserved between man and mouse.

Introduction and expression of the interleukin 2 receptor (Tac) gene in hematopoietic stem cells with retrovirus vectors.

Retrovirus vectors provide an efficient carrier for introducing a gene into hematopoietic stem cells although expression of the inserted gene is not always successful. We constructed and compared three retrovirus vectors which carried cDNA encoding the light chain (Tac) of the interleukin 2 receptor under the control of different promoters; long terminal repeat (LTR) of murine retroviruses, the early promoter of simian virus 40 (SV40) and the promoter of the class I antigen gene of the major histocompatibility complex. We made three constructs containing these promoters. A first construct did not contain any additional promoter but LTR. A second and a third constructs contained the SV40 and the class I antigen gene promoters, respectively, in addition to LTR. The LTR of retrovirus vectors is derived from MoMuLV except that the U3 region of the 3'LTR of the third construct is derived from myeloproliferative sarcoma virus (MPSV). The second and third constructs were used for infection of bone marrow stem cells as the first construct was less efficient in expression of the interleukin 2 receptor in fibroblasts. Hematopoietic stem cells infected with the recombinant viruses were transplanted into lethally irradiated mice, and the expression of the transduced gene in hematopoietic progenitor cells was analyzed. Analysis of RNA isolated from spleen colonies showed that substantial amounts of interleukin 2 receptor mRNA were made by the construct containing the class I gene promoter and MPSV LTR. However, we could not detect any transcripts from the constructs containing MoMuLV LTR and SV40 early region promoter.