endothelin-1 and Glomerulonephritis--Membranoproliferative

The guanine nucleotide exchange factor C3G is one of the mediators of endothelin-1 (ET-1) intracellular signaling cascades and is vital for kidney development and homeostasis. The aim of the current study was to analyze the specificity of ET-1-induced signaling via C3G in rat glomerular mesangial cells (GMC) and to investigate the biological significance of C3G during mesangioproliferative glomerulonephritis. In GMC, C3G expression was increased (1) in vivo after induction of the anti-Thy1 model of glomerulonephritis and (2) in cell culture experiments after fetal bovine serum incubation. To examine the consequences of C3G up-regulation, adenovirus-mediated gene transfer of C3G into cultured glomerular cells was done, and the GTP loading of the small G proteins Rap1 and R-Ras was analyzed. Overexpression of C3G in mesangial cells resulted in enhanced activation of Rap1, but failed to affect the GTP-bound status of R-Ras in ET-1-stimulated cells. C3G overexpression led to significant changes in GMC spreading and migration patterns in response to ET-1 stimulation and increased stress fiber formation, which was mimicked by Rap1A overexpression. Together, these findings suggest (1) the existence of regulatory mechanisms resulting in disease-related up-regulation of C3G in GMC and (2) that an increase in the C3G protein level may contribute to the resolution stage of mesangioproliferative glomerulonephritis by reducing GMC sensitivity to ET-1, modulating cellular motility, and actin dynamics.

Topics: Actin Cytoskeleton; Animals; Antibodies, Monoclonal; Cell Movement; Cell Shape; Cell Size; Cells, Cultured; Disease Models, Animal; Endothelin-1; Gene Expression; Glomerulonephritis, Membranoproliferative; Guanine Nucleotide-Releasing Factor 2; Guanosine Triphosphate; Kidney Glomerulus; Male; Mesangial Cells; rap1 GTP-Binding Proteins; ras Proteins; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Serum; Signal Transduction; Thy-1 Antigens; Transduction, Genetic; Wound Healing

Ghrelin is a novel 28 amino acid growth hormone-releasing peptide hormone that has been shown to inhibit cell proliferation and to decrease the production of proinflammatory cytokines by monocytes/macrophages. Moreover it decreases the release of endothelin-1 (ET-1), as well as mononuclear cell binding.. Seventeen patients with proliferative glomerulopathies (PG) and 15 patients with non-proliferative glomerulopathies (NPG) were examined by percutaneous renal biopsy. As a control 11 biopsy specimens of the kidneys removed because of trauma were used. The immunoexpression of ghrelin and ET-1 was assessed semiquantitatively whereas the interstitial monocytes/macrophages and interstitial area were evaluated quantitatively.. The mean value of the immunoexpression of ghrelin was significantly diminished in PG patients as compared to both NPG group and controls while the mean values of ET-1, interstitial CD68+ cells, as well as interstitial area were in PG group increased in comparison with controls and NPG patients, most of them significantly. In all groups there were significant negative correlations between immunostaining of ghrelin and ET-1, whereas negative correlation between immunostaining of ghrelin and CD68+ cells was significant only in PG group.. We can confirm the presence of ghrelin in tubular epithelial cells in normal and diseased human kidneys. Lack or low level of this protein in proliferative glomerulopathies may be, in part, responsible for interstitial accumulation of monocytes/macrophages in these cases.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Endothelin-1; Epithelial Cells; Female; Fibrosis; Ghrelin; Glomerulonephritis; Glomerulonephritis, Membranoproliferative; Humans; Immunoenzyme Techniques; Kidney Tubules; Macrophages; Male; Middle Aged; Monocytes; Nephritis, Interstitial; Young Adult

Molecular analysis of pathologic changes in glomeruli requires methods allowing rapid and exact detection of alterations in gene expression. Here, we analyzed endothelin-1 (ET-1) mRNA expression in mesangiolytic glomeruli during the course of a rat and murine model of mesangioproliferative glomerulonephritis (GN). A novel method combining laser capture microdissection (LCM), which permits the precise removal of selected mesangiolytic glomeruli, with a highly sensitive real-time RT-PCR technique was used. Anti-Thy 1.1. GN was introduced in male Sprague-Dawley rats (1.0 mg/kg body weight of OX-7 IV) and Habu Snake Venom GN was introduced in C57BL6 mice (habu snake venom toxin 6 mg/kg body weight IV). The degree of mesangiolysis during both GNs was analyzed using a semiquantitative scoring system. Mesangiolytic glomeruli were microdissected at different days of the diseases (day 2, 6, and 12 in anti-Thy 1.1 GN and days 1, 3, 7, and 14 in Habu Snake Venom GN) and from normal control animals. After RNA extraction and cDNA synthesis, ET-1 gene expression was measured by real-time RT-PCR. In parallel, in anti-Thy 1.1. GN ET-1 mRNA expression was analyzed using semiquantitative nonradioactive in situ hybridization; ET-1 protein expression was investigated by immunohistochemistry. Mesangiolysis peaked at day 6 in anti-Thy1.1 GN and at day 1 in Habu Snake Venom GN. Mesangiolytic glomeruli were easily microdissected on cryostat sections in both models; quantification of mRNA with RT-PCR was reliable and reproducible. Glomerular ET-1 mRNA expression increased during the course of anti-Thy 1.1 GN and Habu Snake Venom GN peaked when mesangiolysis was most pronounced. This was seen by RT-PCR after glomerular LCM and by in situ hybridization; in parallel, glomerular ET-1 protein expression was increased. Combination of LCM and RT-PCR is a reliable method for quantification of localized gene expression in isolated renal structures. The above data argue for an important role of ET-1 in pathogenesis and/or repair of mesangiolysis in experimental mesangioproliferative GN.

Topics: Animals; Antibodies, Monoclonal; Crotalid Venoms; Disease Models, Animal; Endothelin-1; Gene Expression; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Laser Therapy; Male; Mice; Mice, Inbred C57BL; Microdissection; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thy-1 Antigens

To investigate the effect of endothelin-1 (ET-1) on mesangial cells(MC) proliferation and mesangial matrix expansion in vivo.. Antisense oligodeoxynucleotide(As-ODN) and its control sequences, sense oligodeoxynucleotide(Se-ODN) and mismatch oligodeoxynucleotide(Mis-ODN), targeting pre-proendothelin-1 mRNA (ppET-1) were delivered into the kidney of rats with mesangioproliferative glomerulonephritis (MsPGN) model respectively at day 2 by lipofectin-mediated gene transfer method. The efficiency of transfer of ODNs into MC was examined with biotinylated As-ODN staining. Four days after the rats were sacrificed, the influence of ODNs on ET-1 production by glomeruli was tested with radioimmunoassay(RIA). The action of ODNs on MC proliferation and mesangial matrix expansion was evaluated with quantitative pathologic analysis.. ODNs were transferred into MC in vivo. The glomerular ET-1 production was decreased by As-ODN [(32 +/- 19) ng/g, P < 0.05] with reduction of mesangial cell proliferation [(0.82 +/- 0.04)/100 micron 2, P < 0.05] and mesangial matrix expansion[(12.8 +/- 1.6)%, P < 0.05], where Se-ODN and Mis-ODN did not show any effect on all of these.. Specific inhibition of ET-1 gene expression leads to reduction of proliferation of mesangial cell and expansion of mesangial matrix in rats with MsPGN model. ET-1 is one of the important inflammatory mediators which can stimulate MC proliferation and mesangial matrix expansion in vivo.

Topics: Animals; Endothelin-1; Endothelins; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Male; Oligodeoxyribonucleotides, Antisense; Protein Precursors; Rats; RNA, Messenger

Recent studies have revealed that endothelin-1 (ET-1) is a potent mitogen for mesangial cells in vitro. To determine whether ET-1 exerts the mitogenic action on mesangial cells in vivo, we examined the glomerular expression of ET-1 and its receptors in a rat model of mesangial proliferative glomerulonephritis and assessed the effect of a specific endothelin A (ET(A)) receptor antagonist, FR139317, on mesangial cell proliferation in this model. The levels of preproET-1 mRNA expression and ET-1 protein production in glomeruli increased markedly on days 4 and 7 after disease induction, and the levels changed in concordance with the glomerular cell proliferation. In contrast, the level of ET(A) receptor mRNA initially decreased on day 1, and thereafter increased on days 4 and 7. Administration of FR139317 to rats with experimental glomerulonephritis induced a significant reduction in mesangial cell proliferation. In addition, in situ hybridization of preproET-1 mRNA and double-immunolabeling of ED-1 and OX-7 in a mirror image section revealed that the principal cell expressing ET-1 in glomeruli were infiltrating macrophages on day 1, and they were replaced by mesangial cells on day 4. These findings indicate that ET-1 functions as a potent mitogen for mesangial cells in vivo in an autocrine or paracrine fashion.

Topics: Animals; Azepines; Base Sequence; Cell Division; Disease Models, Animal; DNA Primers; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; In Situ Hybridization; Indoles; Macrophages; Male; Mitogens; Molecular Sequence Data; Monocytes; Protein Precursors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptors, Endothelin; RNA, Messenger

The proliferation of mesangial cells and the extracellular matrix expansion constitute the most outstanding morphological aspects of the majority of progressive glomerular diseases. In vitro, endothelin-1 (ET-1) is mitogenic for mesangial cells and induces matrix protein synthesis. We studied the possible participation of ET-1 in the pathogenesis of renal damage in a normotensive model of proliferative nephritis. Coincidentally with maximal proteinuria and glomerular lesions, an increase was found in the glomerular mRNA expression of preproET-1 and the ETA receptor (10 and 6 times compared to controls, respectively), but not of the ETB receptor, and in ET-1 urinary excretion (217 +/- 33 vs. 84 +/- 4 pg ET-1/24 hr, N = 4 to 5, P < 0.05). By in situ hybridization, an increase in preproET-1 mRNA expression in glomerular endothelial, epithelial and mesangial cells, and in come tubular cells was observed. The administration of bosentan, an ETA/ETB receptor antagonist, had a beneficial effect on the evolution of nephritis preventing the appearance of intense proteinuria (76 +/- 35 vs. 380 +/- 77 mg/24 hr, N = 4 to 5, P < 0.05), the morphological lesions and the renal function impairment (creatinine clearance 367 +/- 46 vs. 268 +/- 33 microliters/min/100 g, N = 4 to 5). Simultaneously, there was a decrease in ET-1 urinary excretion (88 +/- 14 vs. 217 +/- 33 pgET-1/24 hr, N = 4,5, P < 0.05) and in the renal preproET-1 mRNA expression. The mean systolic blood pressures remained in the normal range in all animals. These data indicate that ET-1 participates in the pathogenesis of proteinuria and glomerular injury in a model of proliferative nephritis. The nonpeptidic orally active ETA/ETB receptor antagonists could be useful in the treatment of some human nephritis.

Topics: Animals; Antibody Specificity; Blood Pressure; Bosentan; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Female; Glomerulonephritis, Membranoproliferative; Immunohistochemistry; In Situ Hybridization; Kidney; Kidney Function Tests; Protein Precursors; Proteinuria; Rats; Rats, Wistar; Receptor, Endothelin A; Receptors, Endothelin; RNA, Messenger; Sulfonamides

We studied whether endothelin-1 (ET-1) and its receptor subtypes (ETAR, endothelin A type receptor; and ETBR, B type receptor) were up-regulated in the glomerulus of a rat model of mesangial proliferative glomerulonephritis induced by anti-thymocyte serum (anti-Thy-1 GN). A marked increase in preproET-1 mRNA could be demonstrated in glomerular RNA 3 and six days after disease induction (4.1- and 4.9-fold vs. day 0, respectively), corresponding to the time of mesangial cell proliferation, to the time of macrophage infiltration into glomeruli, and also to the time of increase in glomerular PDGF B-chain mRNA expression. The localization of ET-1 protein in the mesangial area and along the inner aspect of the glomerular capillary wall was also demonstrated by immunohistochemistry from day 3 and maximal at day 6. The major source of the cells expressing ET-1 in glomeruli appeared to be mesangial cells, glomerular endothelial cells and monocyte/macrophages. Furthermore, both gene and protein expression of ET-1 were associated with increased urinary excretion of ET-1. There was no increase in the plasma ET-1 immunoreactivity. Glomerular expression of ETBR mRNA increased in anti-Thy-1 GN (1.5-fold vs. day 0 at day 3 after disease induction, 3.6-fold at day 6 and 2.7-fold at day 10), but there was minimal change in ETAR mRNA expression. These results suggest that preproET-1 mRNA, which is induced in anti-Thy-1 GN, is linked primarily with ETBR mRNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Base Sequence; Cell Division; Disease Models, Animal; DNA, Complementary; Endothelin-1; Endothelins; Gene Expression; Glomerulonephritis, Membranoproliferative; Kidney Glomerulus; Macrophages; Male; Molecular Sequence Data; Monocytes; Platelet-Derived Growth Factor; Protein Precursors; Rats; Rats, Wistar; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Tissue Distribution