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Objective: To explore the role of sublytic C5b -9 upregulating the transcription factor Krüppel -like factor 5 (KLF5) in promoting the production of the inflammatory cytokine interleukin-23 (IL-23) in glomerular mesangial cells (GMCs) of rats with Thy-1 nephritis (Thy-1N) . Methods: ①A rat model of Thy-1N was established and rat GMCs were cultured in vitro. The expression levels of KLF5 and IL-23 in the renal tissues of Thy-1N rats and in GMCs stimulated by sublytic C5b-9 were detected by using Western blot (WB) . ②The levels of mRNA and protein of KLF5 and IL -23 in the GMCs transfected with either a KLF5 overexpressing plasmid (pIRES2 - KLF5) or a KLF5 small interfering plasmid (shKLF5) were examined by RT - qPCR and WB. ③The full - length IL - 23 promoter luciferase reporter gene plasmid (pGL3-IL-23-FL) was transfected into GMCs, followed by stimulation with sublytic C5b-9. Alternatively, pGL3- IL -23-FL was co -transfected with either pIRES2-KLF5 or shKLF5 into GMCs, and changes in IL -23 promoter activity were measured using a luciferase reporter gene assay. ④The LV-shKLF5 and LV-shCTR lentivirus vectors were respectively perfused into rat renal tissues via the artery perfusion. After confirming that LV - shCTR could enrich in rat kidney through animal imaging system and frozen section, the Thy-1N was reproduced, and the KLF5 and IL-23 expression in the renal tissues were measured by WB. Results: ①The expressions of KLF5 and IL -23 were significantly increased in the renal tissues of Thy -1N rats and in GMCs stimulated by sublytic C5b-9, with KLF5 expression peaking slightly earlier than IL-23. ②Overexpression or knockdown of KLF5 in GMCs led to an increase or decrease in IL - 23 expression, respectively. ③ Sublytic C5b - 9 stimulation or KLF5 overexpression upregulated the activity of IL-23 promoter, while KLF5 knockdown markedly reduced the IL-23 promoter activity induced by sublytic C5b-9. ④IL-23 expression in the renal tissues of the rats treated by knocking down of renal KLF5 gene was significantly downregulated. Conclusion: In the early stage of Thy-1N in rats, sublytic C5b-9 stimulates GMCs to promote the transcription and expression of the IL-23 gene by upregulating KLF5. [ABSTRACT FROM AUTHOR]

Rat Thy‐1 nephritis (Thy‐1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b‐9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG‐BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet‐derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy‐1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b‐9 stimulation. Then, we not only discovered that sublytic C5b‐9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy‐1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b‐9‐triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy‐1N and MsPGN. [ABSTRACT FROM AUTHOR]

Background/Aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Background: T-type calcium channels (TTCC) are involved in mesangial cell proliferation. In acute thy-1 nephritis in the rat TTCC inhibition reduces glomerular damage and cell proliferation. This work is extended here by a study of the non-selective TTCC inhibitor TH1177 in a chronic model of proliferative glomerulonephritis (GN) including late treatment starting after the initial inflammation has resolved. The objective was to determine the effects of TH1177 in a model of chronic mesangioproliferative renal disease.Methods: Chronic GN was induced in WKY rats by unilateral nephrectomy (day - 7) followed by day 0 injection of Ox7 thy-1 mAb. Treatment with TH1177 (10-20 mg/Kg daily IP) was started on day 2 (early treatment) or on day 14 (late treatment) and compared to vehicle-treated controls until sacrifice at day 42. Glomerular disease was assessed with a damage score, fibrosis assay, cellular counts and renal function measured by serum creatinine.Results: Treatment with TH11777 was associated with reduced serum creatinine, less glomerular damage, reduced fibrosis and reduced glomerular cellularity. The results for early and late TH1177 treatments were essentially the same and differed significantly from vehicle.Conclusions: The ion-channel modulator TH1177 is capable of improving glomerular outcome in chronic rat GN even when treatment starts 14 days after initiation of the disease. These data are discussed in the context of the possible targets of TH1177 including TTCC, TRP family, Stim/Orai group and other cation channels. The work supports the use of genetic models to examine the roles of individual cation channels in progressive glomerulonephritis to further define the targets of TH1177. [ABSTRACT FROM AUTHOR]

Background/Aims: To determine whether an aqueous extract of Trametes robiniophila Murr. (Huaier) suppresses anti-Thy-1 mesangial proliferative glomerulonephritis (MsPGN) in vivo and platelet-derived growth factor (PDGF)-BB–induced mesangial cell proliferation in vitro. Methods: Male Wistar rats were randomly categorized into 5 groups: Sham, Thy-1, and 3 Huaier-treated groups (low, medium, and high dose). Two weeks after treatment, urinary proteins were quantified and renal pathological changes were examined. MAX interactor 1 (Mxi-1) and proliferating cell nuclear antigen (PCNA) expression levels in isolated glomeruli, rat mesangial cell viability, cell-cycle distribution, and cell-cycle pathways were assessed. Results: Huaier diminished the proliferative damages and urinary protein secretion in Thy-1 rats. PCNA was downregulated, whereas Mxi-1 was upregulated in the isolated glomeruli of Huaier-treated groups compared with the Thy-1 group. Huaier inhibited PDGF-BB– stimulated proliferation of rat mesangial cells in a time- and dose-dependent manner (50% inhibitory concentration = 6.19 mg/mL) and induced G2 cell-cycle arrest. Cell-cycle pathway proteins were downregulated, whereas Mxi-1 was upregulated in Huaier-treated mesangial cells compared with PDGF-BB–stimulated cells. Conclusion: Huaier reduces urinary protein excretion and relieves hyperplasia in mesangial cells in anti-Thy-1 MsPGN as well as inhibits PDGF-BB–stimulated proliferation and DNA synthesis of rat mesangial cells in vitro, suggesting its novel therapeutic potential in MsPGN.

• KLF5, TRAF6 and TIMP3 were up-regulated in the renal tissue of Thy-1N rats and in sublytic C5b-9-stimulated GMC. • Sublytic C5b-9-elevated KLF5 and TRAF6 could enhance TIMP3 gene transcription. • KLF5 and TRAF6 as a complex bound to −1801nt to −1554nt and −228nt to −46nt of TIMP3 promoter. • TRAF6 promoted KLF5 nuclear localization and TIMP3 transcription via K63-linked ubiquitination at KLF5-K99 and -K100. • Renal KLF5, TRAF6 or TIMP3 gene knockdown could lessen TIMP3 expression, ECM accumulation and other lesions of Thy-1N rats. Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., −1801nt to −1554nt (GGGGAGGGGC) and −228nt to −46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN. [ABSTRACT FROM AUTHOR]

Background/Aims: Immunologically and hemodynamically mediated the destruction of glomerular architecture is thought to be the major causes of end-stage renal failure. The purpose of this study is to evaluate the effect of glomerular hypertension on glomerular injury and the progression of glomerular sclerosis after Thy-1 nephritis was induced. Method: Thy-1 nephritis was induced in the stroke-prone spontaneously hypertensive rat strain (SHR-SP) (group SP) and in age-matched Wistar-Kyoto (WKY) (group WKY) rats, following unilateral nephrectomy (UNX), and a vehicle was injected alone in UNX SHR-SP as control (group SC). Result: The degree of glomerular damage in response to a single dose of anti-thy-1 antibody, and its functional consequences (eg. proteinuria, diminished GFR) are more pronounced in group SP than normotensive group WKY and hypertensive group SC without mesangial cell injury. While normotensive group WKY rats recovered completely from mesangial cell injury on day 28-42, glomeruli in group SP kept on persistent macrophage infiltration, α-SMA expression on day 42-56. In addition, glomerular capillary repair with the GECs was rarely seen in pronouncedly proliferative and sclerostic areas. The incidence of glomerular sclerosis and the level of proteinuria were markedly increased by day 56 in the group SP. Conclusions: Our results demonstrate that glomerular hypertension aggravate glomerular damage and glomerulosclerosis in this model of Thy 1 nephritis.

Background/Aims: To determine whether an aqueous extract of Trametes robiniophila Murr. (Huaier) suppresses anti-Thy-1 mesangial proliferative glomerulonephritis (MsPGN) in vivo and platelet-derived growth factor (PDGF)-BB-induced mesangial cell proliferation in vitro. Methods: Male Wistar rats were randomly categorized into 5 groups: Sham, Thy-1, and 3 Huaier-treated groups (low, medium, and high dose). Two weeks after treatment, urinary proteins were quantified and renal pathological changes were examined. MAX interactor 1 (Mxi-1) and proliferating cell nuclear antigen (PCNA) expression levels in isolated glomeruli, rat mesangial cell viability, cell-cycle distribution, and cell-cycle pathways were assessed. Results: Huaier diminished the proliferative damages and urinary protein secretion in Thy-1 rats. PCNA was downregulated, whereas Mxi-1 was upregulated in the isolated glomeruli of Huaier-treated groups compared with the Thy-1 group. Huaier inhibited PDGF-BB-stimulated proliferation of rat mesangial cells in a time- and dose-dependent manner (50% inhibitory concentration = 6.19 mg/mL) and induced G2 cell-cycle arrest. Cell-cycle pathway proteins were downregulated, whereas Mxi-1 was upregulated in Huaier-treated mesangial cells compared with PDGF-BB-stimulated cells. Conclusion: Huaier reduces urinary protein excretion and relieves hyperplasia in mesangial cells in anti-Thy-1 MsPGN as well as inhibits PDGF-BB-stimulated proliferation and DNA synthesis of rat mesangial cells in vitro, suggesting its novel therapeutic potential in MsPGN. [ABSTRACT FROM AUTHOR]

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis (Thy-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Thy-1N rats (in vivo). The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the effective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identified. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ significantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Thy-1N rats. Together, these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300–ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats. [ABSTRACT FROM AUTHOR]

Background/Aims: Immunologically and hemodynamically mediated the destruction of glomerular architecture is thought to be the major causes of end-stage renal failure. The purpose of this study is to evaluate the effect of glomerular hypertension on glomerular injury and the progression of glomerular sclerosis after Thy-1 nephritis was induced. Method: Thy-1 nephritis was induced in the stroke-prone spontaneously hypertensive rat strain (SHR-SP) (group SP) and in age-matched Wistar-Kyoto (WKY) (group WKY) rats, following unilateral nephrectomy (UNX), and a vehicle was injected alone in UNX SHR-SP as control (group SC). Result: The degree of glomerular damage in response to a single dose of anti-thy-1 antibody, and its functional consequences (eg. proteinuria, diminished GFR) are more pronounced in group SP than normotensive group WKY and hypertensive group SC without mesangial cell injury. While normotensive group WKY rats recovered completely from mesangial cell injury on day 28-42, glomeruli in group SP kept on persistent macrophage infiltration, α-SMA expression on day 42-56. In addition, glomerular capillary repair with the GECs was rarely seen in pronouncedly proliferative and sclerostic areas. The incidence of glomerular sclerosis and the level of proteinuria were markedly increased by day 56 in the group SP. Conclusions: Our results demonstrate that glomerular hypertension aggravate glomerular damage and glomerulosclerosis in this model of Thy 1 nephritis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored for many years; however, the molecular mechanisms of GMC proliferation and ECM production remain unclear. Our previous studies have demonstrated that the thrombospondin-1 (TSP-1) gene was involved in mediating rat GMC proliferation and ECM synthesis induced by sublytic C5b-9 in vitro. In the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-1N rats were determined by using TSP-1 small hairpin RNA, and the results revealed that silencing of the TSP-1 gene in rat renal tissues could diminish GMC proliferation (P < 0.01) and ECM secretion (P < 0.01) as well as urinary protein secretion (P < 0.05) in Thy-1N rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-1N rats. [Copyright &y& Elsevier]

Mesangioproliferative glomerulonephritis (MsPGN) is a disease of high incidence in humans. Rats with Thy-1 nephritis (Thy-1 N) are used as an animal model for studying MsPGN. Although several studies have demonstrated that many pathological factors could cause the injury of glomerular mesangial cells (GMCs) in Thy-1 N, changes of profile and the molecular mechanism of the disease (i.e. the role of transcription factors) at intervals remain unclear. The purpose of this study was to identify the changes in gene expression profile and to observe the role of nuclear factor kappa B (NF-κB) on the pathological change of renal tissue in Thy-1 N rats. Our results showed that the pathological changes of GMCs in Thy-1 N included three phases: apoptosis (40 min), necrosis (24 h) and proliferation (7 days). Concomitantly, at 40 min and on day 7, the up-regulation of 341 genes and 250 genes were observed, while 392 genes and 119 genes were down-regulated in Thy-1 N. Expression of interleukin (IL)-1β, IL-6, proliferating cell nuclear antigen, α-smooth muscle actin, collagen type IV and excretion of urinary protein was increased in rats with Thy-1 N and decreased in pyrrolidine dithiocarbamate-treated rats with Thy-1 N. These data indicated that the significant changes in the gene profile were coupled with the pathological changes of Thy-1 N, and activation of NF-κB may contribute to the pathogenesis of GMCs apoptosis, proliferation, extracelluar matrix accumulation and proteinuria in Thy-1 N. [ABSTRACT FROM AUTHOR]

Mesangioproliferative glomerulonephritis (MPGN) is a disease of high incidence in humans. Rat Thy-1 nephritis (Thy-1 N), namely, anti-thymocyte serum (ATS)-induced nephritis, is considered to be an animal model for studying MPGN. Although previous studies have demonstrated that glomerular mesangial cell (GMCs) injury might be a feature of Thy-1 N, the mechanism of the disease (i.e., GMC apoptosis) remains unclear. We have examined the pathologic changes of GMCs and the gene expression profile of renal tissues in Thy-1 N. The pathologic changes of Thy-1 N include three phages: GMC apoptosis (40 min), necrosis (2 h), and proliferation (5 days). Many TUNEL-positive cells are found 40 min after administration of ATS. Concomitantly, 341 genes are up-regulated, whereas 392 genes are down-regulated, as shown by microarrays analysis. The mRNA and protein of two of the up-regulated genes (nerve growth factor induced protein I-B, NGFI-B; growth arrest- and DNA-damage-inducible protein 45 gamma, Gadd 45 γ) in the GMC apoptotic phase of Thy-1 N are markedly elevated, as observed by real-time polymerase chain reaction and immunohistochemistry. Our data indicate that pathologic changes of Thy-1 N are involved in the abnormal gene profile. The overexpression of the NGFI-B and Gadd 45 γ genes may be associated with GMC apoptosis of Thy-1 N. [ABSTRACT FROM AUTHOR]

Abstract: Thy-1 nephritis (Thy-1 N), namely, anti-Thy-1 or anti-thymocyte serum (ATS) induced nephritis (ATSN), is a typical model of human mesangioproliferative glomerulonephritis. The pathologic changes of glomerular mesangial cells (GMCs) in Thy-1 N are complement-dependent, especially C5b-9 complexes, but the role of C5b-9 in the mechanism of Thy-1 N has not been defined. Because previous studies have demonstrated that sublytic C5b-9 can increase production of several inflammatory mediators from resident glomerular cells, we utilized the isolated human membrane-bound C5b-9 complexes to stimulate the cultured rat GMCs and examined whether the GMCs can also induce the synthesis of nitric oxide (NO) in vitro. Simultaneously, the effects of antiserum against rat C5b-9 and N G-monomethyl-l-arginine (l-NMMA, NO inhibitor), including interfering with the formation of C5b-9, reducing NO production and GMCs injury were observed. The results showed that sublytic C5b-9 can increase synthesis of inducible NO from the stimulated GMCs, and that the anti-C5b-9 antiserum can obviously inhibit the pathologic changes in Thy-1 N, while l-NMMA can decrease the GMCs damage although the effect is not so significant as that of the anti-C5b-9 antiserum. These findings indicate that the synthesis of NO by GMCs can be promoted by sublytic C5b-9, and that lesions of GMCs in rats with Thy-1 N are prevented by either inhibiting C5b-9 formation or NO elevation in advance. The pathologic changes of GMCs in Thy-1 N are indeed complement C5b-9-dependent, and the glomerular injury can be mediated in part through elevation of NO from the GMCs after the sublytic C5b-9 stimulation. [Copyright &y& Elsevier]

To clarify the relationship between macrophages and development of glomerulosclerosis, the authors developed a new experimental nephritis model with macrophages persisting in Thy-1 nephritis. Methyl-cellulose was administered intraperitoneally in addition to the intravenous injection of the anti-Thy-1 antibody to Wistar rats. Foamy macrophages influxed into the lytic mesangium and stayed to form nodular aggregates. Mesangial cells proliferated with the formation of extracellular matrices around these nodular aggregates of macrophages. Immunohistochemical analyses revealed that α-smooth muscle actin (α-SMA) was expressed in the proliferative area around these nodules of foamy macrophages from day 7. Type I collagen and type IV collagen were also expressed around the foamy macrophages in correspondence with α-SMA expression from day 7. The electron microscopic study revealed that collagen fibrils were formed around the transformed mesangial cells. The expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), a marker of glomerular vasculature endothelial cells, was not found in the area occupied by the foamy macrophages, suggesting the impairment of glomerular reconstruction. Macrophages may participate in the progression of glomerulosclerosis in Thy-1 nephritis by enhancing the production of the extracellular matrix through transformed mesangial cells and preventing reconstruction of the capillary network. [ABSTRACT FROM AUTHOR]

The relationship between mesangial cells (MC) and endothelial cells (EC) in the remodeling of glomerular capillary loops was investigated in a rat model of anti-Thy-1 antibody (Ab)-induced glomerulonephritis. Immunohistochemical analysis showed that cells positive for α-smooth muscle actin (α-SMA) appeared in the mesangial stalks at day three, and had increased in number at day seven, after injection of Thy-1 Ab. Double staining for α-SMA and proliferating cell nuclear antigen (PCNA) showed that some MC expressing PCNA were negative for α-SMA at day three, but by day seven almost all PCNA-positive MC expressed α-SMA. Western blotting for α-SMA from isolated glomeruli was negative at day one after injection of Thy-1 Ab, but positive at day seven. Type III collagen appeared at day seven, followed by an increase of EC in the capillary loops, as determined by double immunofluorescent staining for rat endothelial cell antigen-1 (RECA-1) and type III collagen. RECA-1-positive cells increased rapidly in number after day seven and eventually showed the same distribution pattern as that in control rats. Both type I and type III collagens were expressed in the mesangial and the ballooning area of the glomerulus at day seven. Electron microscopy revealed that immature MC and EC forming small capillary lumina appeared in the enlarged mesangial area at day seven. In accordance with the increase of capillaries and the enlargement of the lumina, the number of MC and the amount of mesangial matrix decreased gradually, and most of the glomeruli returned to a normal structure by week 4. These data show that type I and type III collagen produced by transformed MC may be of benefit to proliferation of EC and remodeling of the capillary in Thy-1-induced nephritis. [ABSTRACT FROM AUTHOR]

In order to investigate the role of macrophages in glomeruli in the progression of glomerular sclerosis, methyl-cellulose (MC) was administered intraperitoneally to Wistar rats, in addition to intravenous injection of anti-thy1-1 antibody. In this group of rats (Thy-1 + MC group), many macrophages infiltrated in the lytic mesangium accompanied by rupture of capillary loops at an early stage and stayed with abundant deposition of mesangial matrices until day 35, whereas the proliferative lesions following mesangiolysis almost vanished in the rats treated with anti-thy1-1 antibody alone (Thy-1 group). In immunostaining, matrix metalloproteinase (MMP)-9 was expressed along regenerating capillaries of the Thy-1 group and in extracapillary lesions of the Thy-1 + MC group after day 7. In gelatin zymography, the gelatinolytic band for MMP-9 was expressed much more strongly in the Thy-1 + MC group than in the Thy-1 group at day 3, but it was expressed a little more strongly in the Thy-1 group than in the Thy-1 + MC group at day 7. The bands for an active form of MMP-2 were more strongly expressed in the Thy-1 + MC group than in the Thy-1 group throughout the experimental period. These results suggest that persistent accumulation of macrophages in mesangium induces glomerular sclerosis through expression and activation of MMP. [ABSTRACT FROM AUTHOR]

Mesangioproliferative glomerulonephritis (MsPGN) is a common human kidney disease. Rat Thy-1 nephritis (Thy-1N) is an animal model widely used for the study of MsPGN. Thy-1N is not only sublytic C5b-9-dependent, but also related to pro-inflammatory cytokine production and macrophage (Mφ) accumulation in rat renal tissues. In this study, we found that the expression or phosphorylation of chemokine CCL3/4, CD68 (Mφ marker), IRF-8, PKC-α and NF-κB-p65 (p65) were all up-regulated both in the renal tissues of Thy-1N rats ( in vivo ) and in the glomerular mesangial cells (GMCs) upon sublytic C5b-9 stimulation ( in vitro ). Further experiments in vitro revealed that the phosphorylated PKC-α (p-PKC-α) could promote p65 phosphorylation, and then p-p65 enhanced IRF-8 expression through binding to IRF-8 promotor (-591 ~ -582 nt and -299 ~ -290 nt). Additionally, up-regulation or silencing of IRF-8 gene promoted or reduced CCL3/4 production, and then regulated Mφ chemotaxis. The underlying mechanism involved in IRF-8 binding to CCL3 promoter (-249 ~ -236 nt), which resulted in CCL3 gene transcription. The experiments in vivo showed that knockdown of renal PKC-α, p65, IRF-8 and CCL3/4 genes could inhibit CCL3/4 production, Mφ accumulation, GMC proliferation and proteinuria of Thy-1N rats. Furthermore, p-PKC-α, p-p65, IRF-8, CCL3/4 expression and Mφ accumulation were also increased in the renal tissues of MsPGN patients. Collectively, these findings indicate that sublytic C5b-9 induces CCL3/4 production and Mφ accumulation via PKC-α/p65/IRF-8 axis, and finally aggravates the pathological changes of MsPGN., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Thy-1.1 nephritis is a widely used model of proliferative glomerulonephritis. Lesions are characterized by diffuse mesangiolysis and prominent ballooning of capillary loops, followed by severe mesangial proliferation. However, such severe lesions are not common in human mesangial proliferative nephritis. We analyzed mild-to-moderate mesangial proliferative nephritis induced by a lower dose of anti-Thy-1.1 antibody. We administered a low intravenous dose (0.02 mg/mL) of antibody to Wislar rats, and the histologic changes were observed by light and electron microscopy and by various immunopathologic methods. The serum complement level and distribution of polymorphonuclear leukocytes were also examined. Diffuse mesangial-cell lysis occurred at day 1, but mesangial-matrix lysis was mild, and ballooning of capillary loops was rare. Mesangial cell proliferation appeared at day 2 and reached its maximum at day 5, but it was milder than that seen in the usual dose (0.2 mg/mL) model. The glomerulus was repaired to almost normal structure by day 14. Residual mesangial matrices and endothelial cells seemed to prevent ballooning by making bridges with capillary basement membranes. In the healing stage, this low-dose model demonstrated mild vascular remodeling without active angiogenesis, while the usual-dose model of Thy-1 nephritis showed prominent angiogenic activity to reconstruct capillary tufts. The low-dose model of Thy-1 nephritis, which does not require prominent angiogenesis, may be more useful in analyzing the mechanism of glomerulonephritis than the usually adopted Thy-1 nephritis model, which showed drastic glomerular destruction and prominent angiogenesis. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background/aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism., Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry., Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription., Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis (Thy-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Thy-1N rats (in vivo). The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the effective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identified. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ significantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Thy-1N rats. Together, these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300-ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats., (© The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Background/Aims: Inducible nitric oxide (NO) synthase (iNOS) generated NO increases in the early phase of Thy-1 glomerulonephritis concurrently with mesangiolysis and reduction in glomerular filtration rate (GFR). Activation of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, is upregulated to allow mesangial cell proliferation which constitutes the repair phase in this model. Antiproliferative high-output NO generation inhibits proproliferative ODC activity, thereby temporally separating the early 'bactericidal' phase from the later 'growth' repair phase. Methods: Renal function, ODC protein expression, arginine, ornithine, and polyamines by high-performance liquid chromatography, and histological changes were assessed in rats after induction of Thy-1 nephritis with and without NOS inhibition. Results: Thy-1 significantly reduced the GFR relative to untreated controls. Treatment with a non-specific NOS inhibitor, but not a selective iNOS inhibitor, further decreased the GFR at day 1. This implys a protective role for constitutive NOS in the early phase of this inflammatory model. Selective iNOS inhibition abrogated increased plasma NO2/NO3 levels in Thy-1 glomerulonephritis, but did not significantly reduce mesangiolysis. However, inhibition of iNOS did result in significantly more nuclei/glomerulus during the proliferative phase, increasing the hypercellularity component of this disease model. This correlates with increased levels of polyamines, ornithine, and arginine beyond those observed with Thy-1 administration alone. Conclusions: These studies provide evidence that NO generation from different NOS isoforms can be protective in the temporal course of Thy-1 glomerulonephritis. The finding that iNOS attenuates hypercellularity in the repair phase of this inflammatory model adds cautionary insight in the therapeutic use of selective iNOS inhibition in vivo. [ABSTRACT FROM AUTHOR]

Background/Aim: Evidence from ex vivo glomerular analysis has implicated overexpression of transforming growth factor (TGF) beta 1 in progressive renal disease. The roles of TGF-β2 and TGF-β3 are less clear. The purpose of this study was to define the temporal expression and abundance of TGF-β isoforms in both acute and progressive Thy-1 glomerulonephritis during the crucial initiation phase of these models. Methods: Acute Thy-i glomerulonephritis was induced by a single injection of OX7, while the progressive model was induced by two injections, 7 days apart. Results: Cellular infiltration of glomeruli consisted of transient increases of neutrophils and ED1+ macrophages. The distribution of TGF-β1, TGF-β2, and TGF-β3 revealed distinct differences in normal and nephritic rats. No changes in TGF-β1 staining were observed within glomeruli of either model. In marked contrast, in the one-shot model, TGF-β2 and TGF-β3 stainings increased rapidly, yet transiently, throughout affected glomeruli, followed by more sustained staining in glomerular epithelial cells. Diffuse, transient staining was absent in two-shot glomerulonephritis, but an increase in epithelial cell staining mirrored that seen in the one-shot model. Conclusion: Based on these results, we propose that the effects, formerly thought of as solely due to a single entity, TGF-β1, may be the result of an interplay between individual TGF-β isoforms. [ABSTRACT FROM AUTHOR]