Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-?B and Smad2/3 Signaling.
ABSTRACT: 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 I?B 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)-? and transforming growth factor (TGF)-?/activins mRNAs and activation of nuclear factor (NF)-?B 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-?B 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-? and TGF-?/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-?B and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.
Project description:T cells play a key role in immune-mediated glomerulonephritis, but how cytotoxic T cells interact with podocytes remains unclear. To address this, we injected EGFP-specific CD8+ T cells from just EGFP death inducing (Jedi) mice into transgenic mice with podocyte-specific expression of EGFP. In healthy mice, Jedi T cells could not access EGFP+ podocytes. Conversely, when we induced nephrotoxic serum nephritis (NTSN) and injected Jedi T cells, EGFP+ podocyte transgenic mice showed enhanced proteinuria and higher blood urea levels. Morphometric analysis showed greater loss of EGFP+ podocytes, which was associated with severe crescentic and necrotizing glomerulonephritis. Notably, only glomeruli with disrupted Bowman's capsule displayed massive CD8+ T cell infiltrates that were in direct contact with EGFP+ podocytes, causing their apoptosis. Thus, under control conditions with intact Bowman's capsule, podocytes are not accessible to CD8+ T cells. However, breaches in Bowman's capsule, as also noted in human crescentic glomerulonephritis, allow access of CD8+ T cells to the glomerular tuft and podocytes, resulting in their destruction. Through these mechanisms, a potentially reversible glomerulonephritis undergoes an augmentation process to a rapidly progressive glomerulonephritis, leading to end-stage kidney disease. Translating these mechanistic insights to human crescentic nephritis should direct future therapeutic interventions at blocking CD8+ T cells, especially in progressive stages of rapidly progressive glomerulonephritis.
Project description:<h4>Background</h4>Interleukin-17 (IL-17) is a new pro-inflammatory cytokine involved in immune response and inflammatory disease. The main source of IL-17 is a subset of CD4+ T-helper cells, but is also secreted by non-immune cells. The present study analyzes expression of IL-17 in the time course of acute anti-thy1 glomerulonephritis and the role of IL-17 as a potential link between inflammation and fibrosis.<h4>Methods</h4>Anti-thy1 glomerulonephritis was induced into male Wistar rats by OX-7 antibody injection. After that, samples were taken on days 1, 5, 10 (matrix expansion phase), 15 and 20 (resolution phase). PBS-injected animals served as controls. Proteinuria and histological matrixes score served as the main markers for disease severity. In in vitro experiments, NRK-52E cells were used. For cytokine expressions, mRNA and protein levels were analyzed by utilizing RT-PCR, in situ hybridization and immunofluorescence.<h4>Results</h4>Highest IL-17 mRNA-expression (6.50-fold vs. con; p<0.05) was found on day 5 after induction of anti-thy1 glomerulonephritis along the maximum levels of proteinuria (113 ± 13 mg/d; p<0.001), histological glomerular-matrix accumulation (82%; p<0.001) and TGF-β1 (2.2-fold; p<0.05), IL-6 mRNA expression (36-fold; p<0.05). IL-17 protein expression co-localized with the endothelial cell marker PECAM in immunofluorescence. In NRK-52E cells, co-administration of TGF-β1 and IL-6 synergistically up-regulated IL-17 mRNA 4986-fold (p<0.001).<h4>Conclusions</h4>The pro-inflammatory cytokine IL-17 is up-regulated in endothelial cells during the time course of acute anti-thy1 glomerulonephritis. In vitro, NRK-52E cells secrete IL-17 under pro-fibrotic and pro-inflammatory conditions.
Project description:<h4>Background</h4>Podocyte depletion causes glomerulosclerosis, with persistent podocyte loss being a major factor driving disease progression. Urinary podocyte mRNA is potentially useful for monitoring disease progression in both animal models and in humans. To determine whether the same principles apply to crescentic glomerular injury, a rat model of anti-glomerular basement membrane (anti-GBM) nephritis was studied in parallel with a patient with anti-GBM nephritis.<h4>Methods</h4>Podocyte loss was measured by Wilms' Tumor 1-positive podocyte nuclear counting and density, glomerular epithelial protein 1 or synaptopodin-positive podocyte tuft area and urinary podocyte mRNA excretion rate. Glomerulosclerosis was evaluated by Azan staining and urinary transforming growth factor (TGF)-?1 mRNA excretion rate.<h4>Results</h4>In the rat model, sequential kidney biopsies revealed that after a threshold of 30% podocyte loss, the degree of glomerulosclerosis was linearly associated with the degree of podocyte depletion, compatible with podocyte depletion driving the sclerotic process. Urinary podocyte mRNA correlated with the rate of glomerular podocyte loss. In treatment studies, steroids prevented glomerulosclerosis in the anti-GBM model in contrast to angiotensin II inhibition, which lacked a protective effect, and urinary podocyte and TGF-?1 mRNA markers more accurately reflected both the amount of podocyte depletion and the degree of glomerulosclerosis compared with proteinuria under both scenarios. In a patient successfully treated for anti-GBM nephritis, urinary podocyte and TGB-?1 mRNA reflected treatment efficacy.<h4>Conclusion</h4>These results emphasize the role of podocyte depletion in anti-GBM nephritis and suggest that urinary podocyte and TGF-?1 mRNA could serve as markers of disease progression and treatment efficacy.
Project description:Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFR? promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-?1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.
Project description:Glomerular damage mediated by glomerulus-infiltrating myeloid-derived cells is a key pathogenic event in lupus nephritis (LN), but the process is poorly understood. Confocal microscopy of kidney sections and flow cytometry analysis of glomerular cells from magnetic bead-purified glomeruli have identified glomerulus-infiltrating leukocyte populations in NZM2328 (NZM) lupus-prone mice with spontaneous chronic glomerulonephritis (GN) and anti-glomerular basement membrane-induced nephritis. The occurrence of a major glomerulus-infiltrating CD11b+F4/80-I-A- macrophage population exhibiting the markers programmed death ligand-1 (PD-L1), Mac-2, and macrophage mannose receptor (CD206) and producing Klf4, Il10, Retnla, Tnf, and Il6 mRNA, which are known to be expressed by alternatively activated (M2b) macrophages, correlated with proteinuria status. In NZM mice with spontaneous LN, glomerular macrophage infiltration is predominant. CD11b+F4/80-I-A- intraglomerular macrophages and polymorphonuclear neutrophils (PMN) are important in inducing GN, as anti-CD11b and -ICAM-1 mAb inhibited both proteinuria and macrophage and PMN infiltration. The predominant and high expression of PD-L1 by CD11b+F4/80-I-A- glomerular macrophages in kidneys of mice with GN and the inhibition of proteinuria by anti-PD-L1 mAb supported the pathogenic role of these macrophages but not the PD-L1- PMN in GN development and in inducing podocyte damage. In NZM mice with spontaneous chronic GN and severe proteinuria, few glomerulus-infiltrating PMN were found, leaving macrophages and, to a less extent, dendritic cells as the major infiltrating leukocytes. Taken together, these data support the important pathogenic effect of CD11b+F4/80-I-A- M2b-like glomerulus-infiltrating macrophages in LN and reinforce macrophages as a promising target for GN treatment.
Project description:A key hallmark of cancer, altered metabolism, is central to cancer pathogenesis and therapy resistance. Robust glutamine metabolism is among cellular processes regulating tumor progression and responsiveness to therapy in a number of cancers, including melanoma and breast cancer. Among mechanisms underlying the increase in glutamine metabolism in tumors is enhanced glutamine uptake mediated by the glutamine transporters, with SLC1A5 (also known as ASCT2) shown to play a predominant role. Correspondingly, increased SLC1A5 expression coincides with poorer survival in patients with breast cancer and melanoma. Therefore, we performed an image-based screen to identify small molecules that are able to prevent the localization of SLC1A5 to the plasma membrane without impacting cell shape. From 7,000 small molecules, nine were selected as hits, of which one (IMD-0354) qualified for further detailed functional assessment. IMD-0354 was confirmed as a potent inhibitor of glutamine uptake that attained sustained low intracellular glutamine levels. Concomitant with its inhibition of glutamine uptake, IMD-0354 attenuated mTOR signaling, suppressed two- and three-dimensional growth of melanoma cells, and induced cell-cycle arrest, autophagy, and apoptosis. Pronounced effect of IMD-0354 was observed in different tumor-derived cell lines, compared with nontransformed cells. RNA-sequencing analysis identified the unfolded protein response, cell cycle, and response (DNA damage response pathways) to be affected by IMD-0354. Combination of IMD-0354 with GLS1 or LDHA inhibitors enhanced melanoma cell death. <i>In vivo</i>, IMD-0354 suppressed melanoma growth in a xenograft model. As a modulator of glutamine metabolism, IMD-0354 may serve as an important therapeutic and experimental tool that deserves further examination.
Project description:Osteoporosis is caused by an imbalance between bone formation and bone resorption. Receptor activator of nuclear factor-κB ligand (RANKL) promotes the activity and differentiation of osteoclasts via activating the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. IMD 0354 is a selective molecular inhibitor of inhibitor of NF-κB kinase subunit beta (IKKβ) and effective for treatment of acute and subacute inflammatory diseases through the suppression of NF-κB activation. However, the effect of IMD 0354 on bone homeostasis is unknown. In this study, we demonstrated that IMD 0354 significantly attenuated ovariectomy-induced bone loss and inhibited osteoclastogenesis in mice, whereas bone formation was not affected. Additionally, IMD 0354 dramatically inhibited osteoclast differentiation and function induced by RANKL and macrophage colony-stimulating factor in bone marrow monocytes as verified by tartrate-resistant acid phosphatase (TRAP) staining as well as bone resorption assay in vitro. Subsequently, we found that activation of NF-κB signaling and the ERK/c-Fos axis were blunted during osteoclast formation induced by RANKL. Transcription factors nuclear factor of activated T cells c1 (NFATc1) and c-Fos were suppressed with the decreased expression of osteoclast-related genes by IMD 0354. Our findings suggest that IMD 0354 could be a potential preventive and therapeutic drug for osteoporosis.
Project description:Chronic inflammation due to the absorption of asbestos is an important cause of mesothelioma. Although the increased prevalence of mesothelioma is a serious problem, the development of effective chemotherapeutic agents remains incomplete. As the nuclear factor-κB (NF-κB) pathway contributes to malignant transformation of various types of cells, we explored NF-κB activity in three different pathological types of malignant mesothelioma cells, and evaluated the therapeutic potential of a recently reported NF-κB inhibitor, IMD-0354. NF-κB was constantly activated in MSTO-211H, NCI-H28, and NCI-H2052 cells, and the proliferation of these cell lines was inhibited by IMD-0354. D-type cyclins were effectively suppressed in mixed tissue type MSTO-211H, leading to cell cycle arrest at sub G1 /G1 phase. IMD-0354 reduced cyclin D3 in both epithelial tissue type NCI-H28 and sarcomatoid tissue type NCI-H2052. In a sphere formation assay, IMD-0354 effectively decreased the number and diameter of MSTO-211H spheres. Preincubation of MSTO-211H cells with IMD-0354 delayed tumor formation in transplanted immunodeficient mice. Furthermore, administration of IMD-0354 markedly rescued the survival rate of mice that received intrathoracic injections of MSTO-211H cells. These results indicate that a targeted drug against NF-κB might have therapeutic efficacy in the treatment of human malignant mesothelioma.
Project description: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 ? (PPAR?), a member of the steroid/nuclear receptor superfamily, is known to perform various physiological functions. Recently, we reported that PPAR? in activated mesangial cells exerted anti-inflammatory effects and that the deficiency of PPAR? resulted in high susceptibility to glomerulonephritis. To investigate whether PPAR? activation improves the disease activity of MsPGN, we examined the protective effects of a PPAR? 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 PPAR?, which outweighed the PPAR? deterioration associated with the nephritic process. The PPAR? activation appeared to suppress the NF-?B signaling pathway in glomeruli by the induction of I?B?, resulting in the reduction of proteinuria and the amelioration of the active inflammatory pathologic glomerular changes. These findings suggest the antinephritic potential of PPAR?-related medicines against MsPGN. PPAR?-related medicines might be useful as a treatment option for CKD.
Project description:<i>Rhodnius prolixus</i> is an insect vector of <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease in Latin America. Nuclear factor-<i>?</i>B (NF-<i>?</i>B) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [<i>N</i>-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of I<i>?</i>B kinases. It blocks I<i>?</i>B? phosphorylation thus preventing nuclear translocation of the NF-<i>?</i>b TF. In humans, NF-<i>?</i>B is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-<i>?</i>B TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In <i>Drosophila melanogaster</i>, the activation and regulation of NF-<i>?</i>B TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the <i>R. prolixus</i> immune response to challenge with bacteria and <i>T. cruzi</i>, as well as the impact on the gut bacterial microbiota. <i>R. prolixus</i> were fed with rabbit blood containing IMD-0354 and <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, or <i>T. cruzi</i>. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of <i>R. prolixus</i> immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of bacterial microbiota in treated insects. Insects treated with IMD-0354 and concomitantly infected with bacteria or <i>T. cruzi</i> through the blood meal had increased mortality, while the <i>T. cruzi</i> population in <i>R. prolixus</i> midgut was reduced. The inhibitory effect of IMD-0354 indicates the importance of NF-<i>?</i>B TF in the innate immune responses involved in the control of bacteria and parasite infections in the <i>R. prolixus</i> midgut.