Human Mesangial Proliferative Glomerulonephritis Research Articles

Modulation of IL-6 Expression by KLF4-Mediated Transactivation and PCAF-Mediated Acetylation in Sublytic C5b-9-Induced Rat Glomerular Mesangial Cells.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

KLF6 Acetylation Promotes Sublytic C5b-9-Induced Production of MCP-1 and RANTES in Experimental Mesangial Proliferative Glomerulonephritis

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-κB and Smad2/3 Signaling.

Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an IκB kinase inhibitor), SB431542 (an activin receptor-like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β/activins mRNAs and activation of nuclear factor (NF)-κB p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-κB p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-α and TGF-β/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-κB and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.

Glomerular Expression of Hydrogen Peroxide-Inducible Clone-5 in Human and Rat Progressive Mesangial Proliferative Glomerulonephritis

Background/Aims: Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor-β₁ (TGF-β₁)- and hydrogen peroxide (H₂O₂)-inducible focal adhesion protein that may be necessary for maintaining the myofibroblastic phenotype in pathological scar formation. To investigate the involvement of Hic-5 in the pathogenesis of glomerulonephritis (GN), we examined the glomerular expression of Hic-5 in human and rat GN as well as the regulation of Hic-5 by TGF-β₁ in vitro. Methods and Results: Immunohistochemical analyses showed that the expression of Hic-5 was increased in mesangial cells (MCs) in human mesangial proliferative GN. Hic-5 expression was significantly correlated not only with the levels of α-smooth muscle actin (α-SMA) and TGF-β₁, the accumulation of extracellular matrix, and the number of glomerular cells, but also with the urinary protein level in patients with GN. Glomerular Hic-5 expression increased in parallel with α-SMA expression in a rat model of mesangial proliferative GN. Combined therapy with an angiotensin type I receptor blocker and an antioxidant in this model improved the histology and the expression of Hic-5 and α-SMA. TGF-β₁ upregulated Hic-5 and α-SMA protein levels in human cultured MCs. Conclusion: Our findings suggest that Hic-5 is involved in changes in the MC phenotype to produce abnormal extracellular matrix remodeling in GN.

PPARα Activation Protects against Anti-Thy1 Nephritis by Suppressing Glomerular NF-κB Signaling

The vast increase of chronic kidney disease (CKD) has attracted considerable attention worldwide, and the development of a novel therapeutic option against a representative kidney disease that leads to CKD, mesangial proliferative glomerulonephritis (MsPGN) would be significant. Peroxisome proliferator-activated receptor α (PPARα), a member of the steroid/nuclear receptor superfamily, is known to perform various physiological functions. Recently, we reported that PPARα in activated mesangial cells exerted anti-inflammatory effects and that the deficiency of PPARα resulted in high susceptibility to glomerulonephritis. To investigate whether PPARα activation improves the disease activity of MsPGN, we examined the protective effects of a PPARα agonist, clofibrate, in a well-established model of human MsPGN, anti-Thy1 nephritis, for the first time. This study demonstrated that pretreatment with clofibrate (via a 0.02% or 0.1% clofibrate-containing diet) continuously activated the glomerular PPARα, which outweighed the PPARα deterioration associated with the nephritic process. The PPARα activation appeared to suppress the NF-κB signaling pathway in glomeruli by the induction of IκBα, resulting in the reduction of proteinuria and the amelioration of the active inflammatory pathologic glomerular changes. These findings suggest the antinephritic potential of PPARα-related medicines against MsPGN. PPARα-related medicines might be useful as a treatment option for CKD.

Mesangial sclerotic change with persistent proteinuria in rats after two consecutive injections of monoclonal antibody 1-22-3.

Irreversible mesangial changes with persistent proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1-22-3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophage-like cells in glomeruli observed at 1 week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present 1 week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ultrastructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulonephritis.

Agmatine Suppresses Mesangial Cell Proliferation by Modulating Polyamine Metabolism

Polyamines play an essential role in the growth and differentiation of mammalian cells. The depletion of intracellular polyamines results in the suppression of growth. Proliferation of glomerular mesangial cells (MC) is the most common pathologic change in many forms of glomerulonephritis. Agmatine is a metabolite of arginine via arginine decarboxylase (ADC), highly expressed in the kidney, and unique in its capacity to suppress intracellular polyamine levels required for proliferation. As agmatine enters mammalian cells via the polyamine transport system, its antiproliferative effects may preferentially target cells with increased proliferative kinetics. In the present study, we evaluated the antiproliferative effects of agmatine on human MC in vitro. MC proliferation was stimulated with 20% fetal bovine serum (FBS) or platelet-derived growth factor (PDGF-BB, 20 ng/ml). Cell proliferation was measured using the (4.3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) proliferation assay. Intracellular polyamine levels were assayed by high performance liquid chromatography, and cell death was assessed by cellular DNA fragmentation enzyme-linked immunosorbent assay. The MTT proliferation assay showed that agmatine significantly suppressed proliferation of human MC treated with 20% FBS or 5% FBS + PDGF as compared to human MC treated with 5% FBS. Polyamine levels were markedly lower in cells treated with agmatine, and proliferation was rescued by administration of putrescine. The fragmented DNA was hardly detected in agmatine-treated human MC. In summary, human MC stimulated to increase their proliferative kinetics are significantly more sensitive to the antiproliferative effects of agmatine than normally cultured cells. Suppressed proliferation of the agmatine-treated human MC is not due to increased cell death. These results suggest that agmatine is a promising drug candidate for the treatment of human mesangial proliferative glomerulonephritis.

An anti-CD5 monoclonal antibody ameliorates proteinuria and glomerular lesions in rat mesangioproliferative glomerulonephritis

Increased numbers of lymphocytes have been identified in biopsy specimens of human mesangial proliferative glomerulonephritis (GN). However, the causal relationship between infiltrating T lymphocytes and mesangial changes in mesangial proliferative GN has not been previously evaluated. In this study, we elucidated the role of lymphocytes in the development of mesangial proliferative GN. Immunohistological and flow cytometric analyses as well as a reverse transcription-polymerase chain reaction (RT-PCR) studies were performed in monoclonal antibody (mAb) 1-22-3-induced Thy 1.1 GN. To elucidate the role of these lymphocytes, depletion studies were carried out using anti-CD8 mAb (OX-8), which depletes both CD8+ T lymphocytes and natural killer (NK) cells and anti-CD5 mAb (OX-19), which depletes both CD4+ and CD8+ T lymphocytes. Immunofluorescence (IF) studies revealed that NK cells and CD4+ T lymphocytes were recruited into glomeruli. Glomerular mRNA expression for interferon-gamma, interleukin-2 (IL-2), IL-10, and perforin increased after induction of GN. Increased expressions of several chemokines, which have the potential to attract lymphocytes, were also detected. Anti-CD8 mAb treatment completely prevented the recruitment of NK cells; however, it had no protective effect on proteinuria and mesangial injury. By contrast, anti-CD5 mAb treatment suppressed the recruitment of CD4+ T lymphocytes into glomeruli and reduced proteinuria (60.4 +/- 25.7 vs. 120.0 +/- 32.3 mg/day, P < 0.05) and mesangial changes evaluated by total number of cells in glomeruli (63.2 +/- 6.0 vs. 81.4 +/- 5.9, P < 0.01) and alpha-smooth muscle actin staining score (1.4 +/- 0.2 vs. 2.2 +/- 0. 4, cf2eth P < 0.01) on day 14 after induction of GN. mRNA expression for IL-2 was significantly reduced by OX-19 treatment. T lymphocytes participate in the development of mesangial proliferative GN.

Gene expression of PDGF and PDGF receptor in various forms of glomerulonephritis.

Platelet-derived growth factor (PDGF) is an important mitogenic factor for various cells. In order to elucidate the role of PDGF in the development of human glomerulonephritis, we examined the gene and protein expression of the PDGF-B chain (PDGF-B) and PDGF-beta receptor (PDGFR-beta) in renal biopsy specimens from patients with various forms of glomerulonephritis using a nonradioactive in situ hybridization and an immunohistochemical technique. The mRNA expression of PDGF-B and PDGFR-beta was significantly increased in the glomeruli of patients with mesangial proliferative glomerulonephritis (IgA nephropathy, Henoch-Schönlein purpura nephritis, and lupus nephritis) compared with those in normal glomeruli. In cases with increased protein expression of PDGF-B and PDGFR-beta, each mRNA expression was also increased. The degree of glomerular injury was positively correlated with the number of mRNA-positive cells for both PDGF and PDGF receptor. There was also a positive correlation between the number of mRNA-positive cells for PDGF-B and PDGFR-beta. PDGF-B and PDGFR-beta were also expressed on cells of the capillary wall, cellular crescents and infiltrated cells in the interstitium. The results suggest that PDGF acts as an important and common mediator for the development of various forms of human mesangial proliferative glomerulonephritis. Furthermore, PDGF may participate in crescent formation and tubulo-interstitial injury.

Induction of interleukin 6 synthesis in mouse glomeruli and cultured mesangial cells.

Interleukin 6 (IL-6) is an autocrine growth factor of cultured mesangial cells (MC) and intraglomerular IL-6 production is suggested to be closely associated with the pathogenesis of human mesangial proliferative glomerulonephritis (mesPGN). In this study, to elucidate the mechanisms regulating the intraglomerular production of IL-6, we examined what kinds of stimuli are significant in the induction of IL-6 synthesis in vitro and in vivo. Incubation of cultured mesangial cells with interleukin 1 (IL-1) or bacterial lipopolysaccharide (LPS) induced significant IL-6 production, and intravenous injection of IL-1 or LPS into normal BALB/c mice induced significant intraglomerular IL-6 mRNA expression. Furthermore, we indicated in this study that IL-6 mRNA expression was augmented in the glomeruli of mice with immune complex-mediated glomerulonephritis.