Serum syndecan-1, hyaluronan and thrombomodulin levels in patients with lupus nephritis.

OBJECTIVES: We investigated circulating syndecan-1, HA and thrombomodulin levels in patients with biopsy-proven Class III/IV ± V LN and their clinico-pathological associations. Patients with non-renal SLE or non-lupus chronic kidney disease, and healthy subjects served as controls. METHODS: Serum syndecan-1, HA and thrombomodulin levels were determined by ELISAs. RESULTS: Syndecan-1, HA and thrombomodulin levels were significantly higher during active LN compared with remission (P < 0.01, for all), and correlated with the level of proteinuria, estimated glomerular filtration rate, anti-dsDNA antibodies, complement 3 and serum creatinine. Longitudinal studies showed that syndecan-1 and thrombomodulin levels increased prior to clinical renal flare by 3.6 months, while HA level increased at the time of nephritic flare, and the levels decreased in parallel with treatment response. Receiver operating characteristic curve analysis showed that syndecan-1 and thrombomodulin levels distinguished patients with active LN from healthy subjects, LN patients in remission, patients with active non-renal lupus and patients with non-lupus chronic kidney disease (receiver operating characteristic area under curve of 0.98, 0.91, 0.82 and 0.95, respectively, for syndecan-1; and area under curve of 1.00, 0.84, 0.97 and 0.79, respectively, for thrombomodulin). HA level distinguished active LN from healthy subjects, LN patients in remission and non-lupus chronic kidney disease (receiver operating characteristic area under curve of 0.82, 0.71 and 0.90, respectively) but did not distinguish between renal vs non-renal lupus. Syndecan-1 and thrombomodulin levels correlated with the severity of interstitial inflammation, while HA level correlated with chronicity grading in kidney biopsies of active LN. CONCLUSION: Our findings suggest potential utility of serum syndecan-1, thrombomodulin and HA levels in clinical management, and their potential contribution to LN pathogenesis.

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.

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.

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.

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.

Distinct effects of mycophenolate mofetil and cyclophosphamide on renal fibrosis in NZBWF1/J mice.

Progression to chronic renal failure varies between patients with lupus nephritis. We compared the effects of mycophenolate mofetil (MMF) and cyclophosphamide (CTX), on renal histology and cellular pathways of fibrosis in murine lupus nephritis. Female NZBWF1/J mice were randomized to treatment with vehicle, methylprednisolone (MP) alone, MMF + MP or CTX + MP for up to 12 weeks, and the effects on clinical parameters, renal histology, and fibrotic processes were investigated. Treatment with MMF + MP or CTX + MP both improved survival, renal function, and decreased anti-dsDNA antibody level and immune complex deposition in kidneys of mice with active nephritis. Vehicle-treated mice showed progressive increase in mesangial proliferation, inflammatory cell infiltration and renal tubular atrophy, associated with PKC-α activation, increased TGF-ß1 expression and increased matrix protein deposition. MP treatment alone did not have any significant effect. MMF + MP or CTX + MP treatment for 12 weeks reduced these abnormalities. MMF + MP was more effective than CTX + MP in suppressing fibrotic mediators, histological fibrosis score and expression of TGF-ß1, fibronectin and collagen I in the kidney. Results from in vitro experiments on human mesangial cells (HMC) showed that mycophenolic acid (MPA) was more effective than CTX in suppressing PKC-α activation and TGF-ß1 secretion induced by human polyclonal anti-dsDNA antibodies. While both MPA and CTX decreased TGF-ß1- and TNF-α-induced fibronectin synthesis, only MPA decreased IL-6 induced fibronectin synthesis. MPA and CTX show distinct effects on fibrotic and inflammatory processes in NZBWF1/J murine lupus nephritis, suggesting that MMF + MP may be more effective than CTX + MP in preserving normal renal histology in lupus nephritis.

Cellular cholesterol transport proteins in diabetic nephropathy.

BACKGROUND: Lipid accumulation has been shown to accelerate renal injury, and the intracellular accumulation of lipids may be caused by alterations in synthesis as well as lipid uptake and efflux. We have investigated the role of cellular cholesterol transport proteins including adenosine triphosphate binding cassette transporter A1 (ABCA1), G1 (ABCG1) and scavenger receptor class B type I (SR-BI) in diabetic nephropathy. METHODS: Protein expression and the ability to mediate cholesterol efflux of ABCA1, ABCG1 and SR-BI was determined in human renal mesangial cells and proximal tubular epithelial cells cultured under normal or high glucose conditions. Renal expression of these cholesterol transporters was examined in a murine model of streptozotocin-induced type 1 diabetes. RESULTS: ABCA1, ABCG1 and SR-BI were expressed in both human renal mesangial cells and proximal tubular epithelial cells, and mediated cholesterol efflux to apolipoprotein AI and HDL. In vitro, hyperglycemia reduced the expression and the ability to mediate cholesterol efflux of all three cholesterol transporters (p<0.05). In vivo studies showed that intra-renal accumulation of lipids was increased in diabetic mice, particularly in mice with nephropathy. This was associated with a significant reduction in the expression of ABCA1, ABCG1 and SR-BI in the kidneys. These changes were already seen in diabetic mice without nephropathy and preceded the development of nephropathy. Diabetic mice with nephropathy had the lowest level of these cholesterol transporters. CONCLUSION: Inducing diabetes with streptozotocin significantly reduced renal expression of ABCA1, ABCG1 and SR-BI. Defects in cholesterol export pathway in renal cells could therefore promote cholesterol accumulation and might contribute to the development of diabetic nephropathy.

Sulodexide decreases albuminuria and regulates matrix protein accumulation in C57BL/6 mice with streptozotocin-induced type I diabetic nephropathy.

OBJECTIVE: Sulodexide is a mixture of glycosaminoglycans that may reduce proteinuria in diabetic nephropathy (DN), but its mechanism of action and effect on renal histology is not known. We investigated the effect of sulodexide on disease manifestations in a murine model of type I DN. METHODS: Male C57BL/6 mice were rendered diabetic with streptozotocin. After the onset of proteinuria, mice were randomized to receive sulodexide (1 mg/kg/day) or saline for up to 12 weeks and renal function, histology and fibrosis were examined. The effect of sulodexide on fibrogenesis in murine mesangial cells (MMC) was also investigated. RESULTS: Mice with DN showed progressive albuminuria and renal deterioration over time, accompanied by mesangial expansion, PKC and ERK activation, increased renal expression of TGF-ß1, fibronectin and collagen type I, III and IV, but decreased glomerular perlecan expression. Sulodexide treatment significantly reduced albuminuria, improved renal function, increased glomerular perlecan expression and reduced collagen type I and IV expression and ERK activation. Intra-glomerular PKC-α activation was not affected by sulodexide treatment whereas glomerular expression of fibronectin and collagen type III was increased. MMC stimulated with 30 mM D-glucose showed increased PKC and ERK mediated fibronectin and collagen type III synthesis. Sulodexide alone significantly increased fibronectin and collagen type III synthesis in a dose-dependent manner in MMC and this increase was further enhanced in the presence of 30 mM D-glucose. Sulodexide showed a dose-dependent inhibition of 30 mM D-glucose-induced PKC-ßII and ERK phosphorylation, but had no effect on PKC-α or PKC-ßI phosphorylation. CONCLUSIONS: Our data demonstrated that while sulodexide treatment reduced proteinuria and improved renal function, it had differential effects on signaling pathways and matrix protein synthesis in the kidney of C57BL/6 mice with DN.