Activated intrarenal reactive oxygen species and renin angiotensin system in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is recognized worldwide as the most common primary glomerulopathy. Although the mechanisms underlying the development of IgA nephropathy are gradually being clarified, their details remain unclear, and a radical cure for this condition has not yet been established. It has been clinically demonstrated that the immunoreactivities of intrarenal heme oxygenase-1 (HO-1) and 4-hydroxy-2-nonenal (4-HNE) markers of reactive oxygen species (ROS) and those of intrarenal angiotensinogen (AGT) and angiotensin II (Ang II) markers of renin angiotensin system (RAS) in IgA nephropathy patients were significantly increased as compared to those of control subjects. In an animal study, high IgA of ddY (HIGA) mice were used as an IgA nephropathy model and compared with BALB/c mice, which served as the control. The levels of markers for ROS (urinary 8-isoprostane and intrarenal 4-HNE), RAS (intrarenal AGT and Ang II), and renal damage in the HIGA mice were significantly increased as compared to those in the BALB/c mice. Moreover, an interventional study using HIGA mice demonstrated that the expressions of 2 lines of intrarenal ROS markers (4-HNE and HO-1), 2 lines of intrarenal RAS markers (AGT and Ang II) and renal damage decreased significantly in HIGA mice receiving treatment with the Ang II receptor blocker olmesartan but not in HIGA mice receiving treatment with RAS-independent antihypertensive drugs (hydralazine, reserpine, and hydrochlorothiazide) when compared with HIGA mice that were not treated. These data suggest that intrarenal ROS and RAS activation plays a pivotal role in the development of IgA nephropathy.

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells.

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.

Role of beta isoform-specific insertions of Ca2+/calmodulin-dependent protein kinase II.

Alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II (alpha and beta CaM kinase II, respectively) are highly conserved except for beta-specific insertions 1 and 2, located at amino acids 316-340 and 354-392, respectively. To investigate the role of these beta-specific insertions, we prepared the deletion mutants betaDelta1, betaDelta2 and betaDelta1/2, which lacked insertions 1, 2 and both, respectively. These mutant DNAs were expressed in neuroblastoma cells and compared with the wild-type enzyme. Green fluorescent protein tagged CaM kinase II was used to further explore the distribution of the kinase in living cells. Most (80%) of wild-type beta and mutant betaDelta1 were located in the particulate fraction, and distributed in the cell body and neurites, forming punctate or spot-like structures in the neurites. Mutants betaDelta2 and betaDelta1/2 were distributed in almost equal amounts in the soluble and particulate fractions. They were concentrated in the base of neurites and only partlially distributed throughout neurites, indicating that their transport to neurites was impaired. Beta(1-410), a deletion mutant of the association domain with a monomeric form, was located primarily in the soluble fraction. These results indicate that insertion 2, the association domain, and the oligomeric form of beta CaM kinase II play an important role in the cellular distribution of beta CaM kinase II.

Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells.

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.

Involvement of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth induced by cAMP treatment and serum deprivation in a central nervous system cell line, CAD derived from rat brain.

A central nervous system (CNS) cell line, CAD, is known to differentiate in the absence of serum. This cell line was found to differentiate by the treatment of cAMP. Expression of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) was induced to about 2-fold or more on day 1, and was continued at a high level for 5 days after the exposure to differentiating conditions. Neurite extension was stimulated from day 1 and continued for 5 days, suggesting that CaM kinase II activity is correlated with neurite outgrowth. Of the four distinct isoforms (alpha, beta, gamma, and delta) of the kinase, the delta isoform was the major isoform in CAD cells. The splicing pattern of this isoform in the differentiated cells differed from that in undifferentiated cells, suggesting that expression of CaM kinase II is regulated during neural differentiation.

Overexpression of alpha1beta1 integrin directly affects rat mesangial cell behavior.

BACKGROUND: Glomerular mesangial cell (MC) proliferation, hypertrophy, and abnormal matrix remodeling characterized by increased expression of fibronectin, laminin and collagen type IV, and neoexpression of collagen I and III are the main biological features of progressive glomerulonephritis (GN). Especially, persistent pathological matrix remodeling may lead to glomerular scar formation (glomerular scarring). We reported recently that alpha1beta1 integrin, a major collagen receptor for MCs, may be a potential adhesion molecule for MC-mediated pathological collagen matrix remodeling in GN. METHODS: To address further the direct role of alpha1beta1 integrin in MC behavior, such as cell growth and matrix remodeling, alpha1beta1 integrin was overexpressed in MCs by transfecting an expression vector containing a full-length rat alpha1 integrin cDNA. Flow cytometry and immunoprecipitation analysis were applied for selection of transfectants with a stable expression of the alpha1 integrin subunit. The effect of alpha1beta1 integrin overexpression on MC biology was examined with a 3H-thymidine incorporation assay, flow cytometric analysis of cell size and DNA content, Western blot analysis of a cyclin-dependent-kinase inhibitor, p27Kip1, alpha-smooth muscle actin expression, and a collagen gel contraction assay. RESULTS: The alpha1 transfectants displayed a dramatic inhibition of 3H-thymidine incorporation as compared with the mock transfectants. Increased expression of the alpha1 subunit inversely correlated with cell cycle progression and paralleled the expression of p27Kip1 and alpha-smooth muscle actin, as well as the cell size in MCs. In addition, the alpha1-transfectants were able to enhance collagen matrix reorganization effectively. CONCLUSION: These results indicate that MC-alpha1beta1 integrin expression is a critical determinant of MC phenotypes, including cell growth, cell size, and collagen matrix remodeling ability, and thereby contributes to scar matrix remodeling (sclerosis) in GN.

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells.

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.

Novel fluoroprostacyclin analogs with modified cycloalkylenyl chains. Highly potent and orally active anti-anginal agents.

Novel fluoroprostacyclin analogs (1a-f) have been synthesized and pharmacologically evaluated. Compounds 1a-c given intravenously or orally showed potent and long-lasting anti-anginal activities in an animal model.

Identification of probasin-related antigen as cystathionine gamma-lyase by molecular cloning.

We reported previously that a monoclonal antibody against probasin (rat prostatic secretory protein) recognizes a 40-kDa protein localized in rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissues. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression library using an affinity-purified polyclonal antibody. The amino acid sequence deduced from the determined cDNA sequence included sequences identical with those of proteolytic fragments of PRB-RA, which covered about 70% of the deduced sequence. Northern blot hybridization of poly(A)+ RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in the liver and kidney. A sequence homology search revealed that PRB-RA is almost completely identical to rat cystathionine gamma-lyase (cystathionase) and that it does not show overall homology with probasin. Three candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, neutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally related to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in liver parenchymal cells.

Conjugal transfer of hydrogen-oxidizing ability of Alcaligenes hydrogenophilus to Pseudomonas oxalaticus.

Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C. The plasmid could be transferred from A. hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H2 and CO2. Plasmid pHG21-a was maintained in P. oxalaticus OX1 and OX4 as stably as in A. hydrogenophilus.

Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus.

A gene bank of a megaplasmid encoding the hydrogen-oxidizing enzyme system (Hox) in Alcaligenes hydrogenophilus was constructed using a broad host range cosmid vector pVK102, and established in Escherichia coli. Hybrid cosmids containing hox genes were identified by transferring the bank into Pseudomonas oxalaticus OX1 and screening colonies for the ability of H2-dependent autotrophic growth. About 800 colonies were formed under autotrophic conditions. One of the Hox+ transconjugants was isolated and its hydrogenases activities were measured. Although soluble hydrogenase was not detected, the Hox+ transconjugant had four times the membrane-bound hydrogenase activity of A. hydrogenophilus.