Effect of mycophenolate and rapamycin on renal fibrosis in lupus nephritis.

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor ß1 (TGF-ß1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-ß1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

miR-200c Prevents TGF-ß1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1.

Peritoneal fibrosis and loss of transport function is a common complication contributing to adverse outcomes in patients on long-term peritoneal dialysis (PD). Epithelial-to-mesenchymal transition (EMT) in mesothelial cells is a salient feature, but its triggering mechanisms remain obscure. Dysregulation of microRNA (miR) expression is implicated in EMT and tissue fibrosis. We investigated the role of miR-200c in EMT and fibrogenesis in a murine PD model and in cultured peritoneal mesothelial cells. PD-fluid-treated mice showed peritoneal miR-200c expression reduced by 76.2% compared with PBS-treated mice, and this was accompanied by increased peritoneal α-smooth muscle actin, fibronectin, and collagen expression. PD fluid and TGF-ß1 both reduced miR-200c expression in cultured mesothelial cells, accompanied by downregulation of E-cadherin and decorin, and induction of fibronectin, collagen I and III, and transcription factors related to EMT. Decorin prevented the suppression of miR-200c by TGF-ß1. Lentivirus-mediated miR-200c overexpression prevented the induction of fibronectin, collagen I, and collagen III by TGF-ß1, independent of decorin, and partially prevented E-cadherin suppression by TGF-ß1. Target genes of miR-200c were identified as ZEB2 and Notch1. Our data demonstrate that miR-200c regulates EMT and fibrogenesis in mesothelial cells, and loss of peritoneal miR-200c contributes to PD-associated peritoneal fibrosis.

Annexin II-binding immunoglobulins in patients with lupus nephritis and their correlation with disease manifestations.

Annexin II on mesangial cell surface mediates the binding of anti-dsDNA antibodies and consequent downstream inflammatory and fibrotic processes. We investigated the clinical relevance of circulating annexin II-binding immunoglobulins (Igs) in patients with severe proliferative lupus nephritis, and renal annexin II expression in relation to progression of nephritis in New Zealand Black and White F1 mice (NZBWF1/J) mice. Annexin II-binding Igs in serum were measured by ELISA. Ultrastructural localization of annexin II was determined by electron microscopy. Seropositivity rates for annexin II-binding IgG and IgM in patients with active lupus nephritis were significantly higher compared with controls (8.9%, 1.3% and 0.9% for annexin II-binding IgG and 11.1%, 4.0% and 1.9% for annexin II-binding IgM for patients with active lupus nephritis, patients with non-lupus renal disease and healthy subjects respectively). In lupus patients, annexin II-binding IgM level was higher at disease flare compared with remission. Annexin II-binding IgG and IgM levels were associated with that of anti-dsDNA and disease activity. Annexin II-binding IgG and IgM levels correlated with histological activity index in lupus nephritis biopsy samples. In NZBWF1/J mice, serum annexin II-binding IgG and IgM levels and glomerular annexin II and p11 expression increased with progression of active nephritis. Annexin II expression was present on mesangial cell surface and in the mesangial matrix, and co-localized with electron-dense deposits along the glomerular basement membrane. Our results show that circulating annexin II-binding IgG and IgM levels are associated with clinical and histological disease activity in proliferative lupus nephritis. The co-localization of annexin II and p11 expression with immune deposition in the kidney suggests pathogenic relevance.

Binding of anti-dsDNA antibodies to proximal tubular epithelial cells contributes to renal tubulointerstitial inflammation.

Immune deposits are often observed along the tubular basement membrane in patients with lupus nephritis, but the role of anti-dsDNA antibody (Ab) deposition on tubulointerstitial inflammation remains to be investigated. We examined the effect of human polyclonal anti-dsDNA Abs on inflammatory processes in cultured proximal renal tubular epithelial cells (PTEC, HK-2 cells) and their association with serum levels of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in patients. Binding of anti-dsDNA Abs to HK-2 cells was investigated by cellular ELISA, flow cytometry and immunohistochemistry. IL-6, IL-8 and MCP-1 secretion, mitogen-activated protein kinase (MAPK) activation and the effect of mycophenolic acid (MPA) were investigated by ELISAs and Western blot analysis. NZBWF1/J mice with active nephritis were randomized to receive either mycophenolate mofetil (MMF) (100 mg/kg per day) or vehicle for up to 12 weeks to study renal histopathology focusing on tubulointerstitial changes. Our results demonstrated that anti-dsDNA Abs bound to HK-2 cell surface and induced IL-6, IL-8 and MCP-1 secretion through distinct MAPK pathways. MPA inhibited anti-dsDNA Ab binding to HK-2 cells and suppressed apical and basolateral IL-6 and IL-8, but not MCP-1, secretion. Anti-dsDNA Ab level correlated with serum and tubulointerstitial expression of IL-6, IL-8 and MCP-1. MMF treatment in NZBWF1/J mice reduced anti-dsDNA Ab production and MAPK activation in the renal tubulointerstitium, together with decreased IL-6 and MCP-1 expression. Our data demonstrate that anti-dsDNA Abs contribute to inflammatory processes in the tubulointerstitium in lupus nephritis through their binding to proximal renal tubular epithelial cells and induction of pro-inflammatory mediators, and MPA ameliorates anti-dsDNA Ab induced IL-6 and IL-8 secretion in these cells.

MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes sporadic zoonotic disease and healthcare-associated outbreaks in human. MERS is often complicated by acute respiratory distress syndrome (ARDS) and multi-organ failure(1,2). The high incidence of renal failure in MERS is a unique clinical feature not often found in other human coronavirus infections(3,4). Whether MERS-CoV infects the kidney and how it triggers renal failure are not understood(5,6). Here, we demonstrated renal infection and apoptotic induction by MERS-CoV in human ex vivo organ culture and a nonhuman primate model. High-throughput analysis revealed that the cellular genes most significantly perturbed by MERS-CoV have previously been implicated in renal diseases. Furthermore, MERS-CoV induced apoptosis through upregulation of Smad7 and fibroblast growth factor 2 (FGF2) expression in both kidney and lung cells. Conversely, knockdown of Smad7 effectively inhibited MERS-CoV replication and protected cells from virus-induced cytopathic effects. We further demonstrated that hyperexpression of Smad7 or FGF2 induced a strong apoptotic response in kidney cells. Common marmosets infected by MERS-CoV developed ARDS and disseminated infection in kidneys and other organs. Smad7 and FGF2 expression were elevated in the lungs and kidneys of the infected animals. Our results provide insights into the pathogenesis of MERS-CoV and host targets for treatment.

Anti-dsDNA antibody induces soluble fibronectin secretion by proximal renal tubular epithelial cells and downstream increase of TGF-ß1 and collagen synthesis.

The level of anti-dsDNA antibodies correlates with disease activity in lupus nephritis, but their role in pathogenic mechanisms remains to be defined. We investigated the effect of anti-dsDNA antibodies isolated from lupus nephritis patients on fibronectin synthesis and downstream fibrogenesis in proximal renal tubular epithelial cells (PTEC). Kidney biopsies were obtained from patients with active severe proliferative lupus nephritis. In vitro studies with cultured PTEC were performed to investigate the effect of human polyclonal IgG anti-dsDNA antibodies and mycophenolic acid (MPA). The role of IL-6, IL-8, MCP-1, TNF-α, TGF-ß1, and MAPK and PKC signaling pathways on soluble and cell-associated fibronectin synthesis was investigated using neutralizing antibodies or specific inhibitors. The effect of exogenous endotoxin-free soluble fibronectin on downstream fibrotic processes was also examined. Fibronectin expression was markedly increased in the tubulo-interstitium of lupus nephritis renal biopsies and it co-localized with IgG deposition. Anti-dsDNA antibodies significantly increased both secreted and cell-associated fibronectin, through prior activation of ERK, p38 MAPK, JNK, PKC-α and PKC-ßII. There was downstream induction of IL-6, IL-8, MCP-1, TNF-α and TGF-ß1. MPA inhibited the induction of inflammatory and fibrotic processes by anti-dsDNA antibody. Exogenous soluble fibronectin induced TGF-ß1 secretion and type I collagen synthesis in PTEC in a dose-dependent manner. Our data demonstrate that anti-dsDNA antibody contributes to tubulo-interstitial fibrosis in lupus nephritis through its action on PTEC. Anti-dsDNA antibody induces both cell-associated and soluble fibronectin secretion in PTEC, the former adds to extracellular matrix deposition while the latter amplifies the fibrotic process through induction of TGF-ß1 and collagen type I. The pro-fibrotic effects of anti-dsDNA antibody are ameliorated by MPA.

Mediators of inflammation and their effect on resident renal cells: implications in lupus nephritis.

Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is a major cause of morbidity and mortality. It is characterized by a breakdown of immune tolerance, production of autoantibodies, and deposition of immune complexes within the kidney parenchyma, resulting in local inflammation and subsequent organ damage. To date, numerous mediators of inflammation have been implicated in the development and progression of lupus nephritis, and these include cytokines, chemokines, and glycosaminoglycans. Of these, type I interferons (IFNs) can increase both gene and protein expression of cytokines and chemokines associated with lupus susceptibility, and interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α ) and hyaluronan have been shown to elicit both pro- and anti-inflammatory effects on infiltrating and resident renal cells depending on the status of their microenvironment. Expression of IL-6, TNF- α , type I IFNs, and hyaluronan are increased in the kidneys of patients and mice with active lupus nephritis and have been shown to contribute to disease pathogenesis. There is also evidence that despite clinical remission, ongoing inflammatory processes may occur within the glomerular and tubulointerstitial compartments of the kidney, which further promote kidney injury. In this review, we provide an overview of the synthesis and putative roles of IL-6, TNF- α , IFN- α , and hyaluronan in the pathogenesis of lupus nephritis focusing on their effects on human mesangial cells and proximal renal tubular epithelial cells.

Anti-dsDNA antibodies bind to mesangial annexin II in lupus nephritis.

Production of anti-dsDNA antibodies is a hallmark of lupus nephritis, but how these antibodies deposit in organs and elicit inflammatory damage remains unknown. In this study, we sought to identify antigens on the surface of human mesangial cells (HMC) that mediate the binding of human anti-dsDNA antibodies and the subsequent pathogenic processes. We isolated anti-dsDNA antibodies from patients with lupus nephritis by affinity chromatography. We used multiple methods to identify and characterize antigens from the plasma membrane fraction of mesangial cells that crossreacted with the anti-dsDNA antibodies. We found that annexin II mediated the binding of anti-dsDNA antibodies to HMC. After binding to the mesangial cell surface, anti-dsDNA antibodies were internalized into the cytoplasm and nucleus. This also led to induction of IL-6 secretion and annexin II synthesis, mediated through activation of p38 MAPK, JNK, and AKT. Binding of anti-dsDNA antibodies to annexin II correlated with disease activity in human lupus nephritis. Glomerular expression of annexin II correlated with the severity of nephritis, and annexin II colocalized with IgG and C3 deposits in both human and murine lupus nephritis. Gene silencing of annexin II in HMC reduced binding of anti-dsDNA antibody and partially decreased IL-6 secretion. In summary, our data demonstrate that annexin II mediates the binding of anti-dsDNA antibodies to mesangial cells, contributing to the pathogenesis of lupus nephritis. This interaction provides a potential target for therapeutic intervention.

Chinese medicines as a resource for liver fibrosis treatment.

Liver fibrosis is a condition of abnormal proliferation of connective tissue due to various types of chronic liver injury often caused by viral infection and chemicals. Effective therapies against liver fibrosis are still limited. In this review, we focus on research on Chinese medicines against liver fibrosis in three categories, namely pure compounds, composite formulae and combination treatment using single compounds with composite formulae or conventional medicines. Action mechanisms of the anti-fibrosis Chinese medicines, clinical application, herbal adverse events and quality control are also reviewed. Evidence indicates that some Chinese medicines are clinically effective on liver fibrosis. Strict quality control such as research to identify and monitor the manufacturing of Chinese medicines enables reliable pharmacological, clinical and in-depth mechanism studies. Further experiments and clinical trials should be carried out on the platforms that conform to international standards.

Hepatic stellate cell-targeted delivery of M6P-HSA-glycyrrhetinic acid attenuates hepatic fibrogenesis in a bile duct ligation rat model.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) play a key role in fibrogenesis. Here, we used mannose-6-phosphate-modified human serum albumin (M6P(26)-HSA) as a selective carrier to deliver antifibrotic drug 18beta-glycyrrhetinic acid (18beta-GA) in experimental fibrosis animals, and tested its effect in injured liver tissues. METHODS: Bile duct ligation (BDL) was performed to induce liver damage in rats. Masson's stain and immunocytochemistry were used to assess hepatic collagen deposits and uptakes of M6P(26)-HSA-GA in HSCs in rat livers. Gene expression profiles of procollagen type I alpha2, smooth muscle actin (SMA), and transforming growth factor-beta1 (TGF-beta1) were analysed by TaqMan and quantitative polymerase chain reaction assays. The depositions of M6P(26)-HSA-GA in the HSC-T6 cell line and primary HSCs were assessed by immunofluorescent staining. RESULTS: Treatment with M6P(26)-HSA-GA at 10 mg/kg (three times/week for 2 weeks), but not the equivalent doses of free 18beta-GA and M6P(26)-HSA carrier alone, could significantly attenuate collagen deposits in BDL rat liver. Masson's stain and TaqMan assay revealed significant modulation of procollagen type I alpha2 in the BDL-injured liver. The depositions of M6P(26)-HSA-GA in HSCs were revealed by immunostaining with HSA and SMA markers. M6P(26)-HSA bound activated HSCs in vitro and the immunoreactivity of M6P(26)-HSA-GA was detected in the cytoplasm and cell surface of HSCs and HSC-T6 cells. The gene transcript levels of SMA and TGF-beta1 were modulated in HSC-T6 cells treated with M6P(26)-HSA-GA. CONCLUSIONS: The M6P(26)-HSA holds promise as a targeting carrier for the liver or HSCs, which may be used to deliver 18beta-GA as a therapeutic agent to treat liver fibrosis.