Upregulation of FoxO3a expression through PI3K/Akt pathway attenuates the progression of lupus nephritis in MRL/lpr mice.

FoxO3a plays key roles in inflammation and autoimmunity, and the PI3K-Akt-FoxO3a pathway has been proposed to modulate diverse biological processes. The aim of the present study, using lupus murine models, was to investigate whether FoxO3a contributes to the pathogenesis of lupus nephritis. LY294002 was used as an inhibitor of PI3K/AKT signaling pathway. FoxO3a-targeted small interfering RNA (siRNA) was also used for in vivo intervention. Female MRL/lpr mice were separately injected with LY294002, LY294002+siFoxO3a, and LY294002+siControl for 8 weeks. C57BL/6 mice were normal controls. Disease development, including serum creatinine (CRE), blood urea nitrogen (BUN), proteinuria, and renal pathological changes, was monitored. Levels of anti-dsDNA antibodies and immune complex (IC) deposition in the kidney were also measured. The expression of proteins was evaluated. We found that significant downregulation of FoxO3a was detected in the kidney of MRL/lpr mice as compared with normal control mice. Blockade of p-FoxO3a activation by LY294002 suppressed PI3K/Akt/FoxO3a pathway and the subsequent upregulation of FoxO3a in the nucleus resulting in the severity of inflammation and fibrosis in the kidney of MRL/lpr mice. Also, improved kidney function and decreased circulating anti-dsDNA antibodies were due to the upregulation of FoxO3a. Opposite results were obtained by specific siRNA silencing of Foxo3a in vivo. In conclusion, our research demonstrated that the upregulation of FoxO3a expression through inhibiting PI3K/Akt pathway attenuates murine lupus nephritis (LN). Thus, our results suggest that targeting of FoxO3a can be considered as a novel strategy for the treatment of LN.

Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice.

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

Calcium/Calmodulin Kinase IV Controls the Function of Both T Cells and Kidney Resident Cells.

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4+ T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.

CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease.

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3ß, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.

Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression.

Renal DNase I is lost in advanced stages of lupus nephritis. Here, we determined if loss of renal DNase I reflects a concurrent loss of urinary DNase I, and whether absence of urinary DNase I predicts disease progression. Mouse and human DNase I protein and DNase I endonuclease activity levels were determined by western blot, gel, and radial activity assays at different stages of the murine and human forms of the disease. Cellular localization of DNase I was analyzed by immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy. We further compared DNase I levels in human native and transplanted kidneys to determine if the disease depended on autologous renal genes, or whether the nephritic process proceeded also in transplanted kidneys. The data indicate that reduced renal DNase I expression level relates to serious progression of lupus nephritis in murine, human native, and transplanted kidneys. Notably, silencing of renal DNase I correlated with loss of DNase I endonuclease activity in the urine samples. Thus, urinary DNase I levels may therefore be used as a marker of lupus nephritis disease progression and reduce the need for renal biopsies.

Neuraminidase activity mediates IL-6 production by activated lupus-prone mesangial cells.

The development of nephritis is a leading cause of morbidity and mortality in lupus patients. Although the general pathophysiological progression of lupus nephritis is known, the molecular mediators and mechanisms are incompletely understood. Previously, we demonstrated that the glycosphingolipid (GSL) catabolic pathway is elevated in the kidneys of MRL/lpr lupus mice and human lupus patients with nephritis. Specifically, the activity of neuraminidase (NEU) and expression of Neu1, an enzyme in the GSL catabolic pathway is significantly increased. To better understand the role and mechanisms by which this pathway contributes to the progression of LN, we analyzed the expression and effects of NEU activity on the function of MRL/lpr lupus-prone mesangial cells (MCs). We demonstrate that NEU1 and NEU3 promote IL-6 production in MES13 MCs. Neu1 expression, NEU activity, and IL-6 production are significantly increased in stimulated primary MRL/lpr lupus-prone MCs, and blocking NEU activity inhibits IL-6 production. NEU1 and NEU3 expression overlaps IgG deposits in MCs in vitro and in renal sections from nephritic MRL/lpr mice. Together, our results suggest that NEU activity mediates IL-6 production in lupus-prone MCs possibly through an IgG-receptor complex signaling pathway.

Association of angiotensin-converting enzyme insertion/deletion polymorphism with susceptibility to systemic lupus erythematosus: a meta-analysis.

BACKGROUND: The aim of this study was to determine whether the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism confers susceptibility to systemic lupus erythematosus (SLE)/lupus nephritis (LN). METHODS: A meta-analysis was conducted on the association between the ACE I/D polymorphism and SLE/LN (when available) using: (i) the allelic contrast; (ii) the recessive; (iii) the dominant; and (iv) the additive models. RESULTS: A total of 27 relevant comparisons meeting the inclusion criteria were identified, involving 2718 SLE patients and 3655 controls. Meta-analysis showed a significant association between SLE and the allele D in overall populations (odds ratio [OR] = 1.25, 95% CI: 1.07-1.48, P = 0.004). Stratification by ethnicity indicated a strong association between the allele D and SLE in Asians (OR = 1.36, 95% CI: 1.05-1.75, P = 0.019). Meta-analysis also showed a significant association between SLE and the DD genotype in overall populations (additive model) (OR = 1.38, 95% CI: 1.05-1.83, P = 0.022). In addition, we found significant associations between the recessive model and SLE in overall populations, Asians and Europeans (OR = 1.44, 95% CI: 1.11-1.88, P = 0.007; OR = 1.69, 95% CI: 1.07-2.68, P = 0.024; and OR = 1.31, 95% CI: 1.06-1.62, P = 0.013, respectively). With respect to the association between ACE I/D gene polymorphism and LN risk, there was no significant association in either the overall populations or subpopulations. CONCLUSION: The present study might suggest that ACE I/D polymorphism may be a genetic molecular marker to predict SLE, while this polymorphism may not correlate with LN susceptibility.

Treatment with a selective histone deacetylase 6 inhibitor decreases lupus nephritis in NZB/W mice.

To date, there are 18 histone deacetylase (HDAC) enzymes, divided into four classes, which alter protein function by removing acetyl groups from lysine residues. Prior studies report that non-selective HDAC inhibitors decrease disease in lupus mouse models. Concern for adverse side effects of non-selective HDAC inhibition supports investigation of selective-HDAC inhibition. We hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease in a mouse model of lupus by increasing acetylation of alpha-tubulin. Intraperitoneal injections of the selective HDAC6i ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13 weeks. Disease progression was evaluated by proteinuria, serum levels of anti-dsDNA antibody, cytokines and immunoglobulins, and post mortem evaluation of nephritis and T cell populations in the spleen. HDAC6i treatment decreased proteinuria, glomerular histopathology, IgG, and C3 scores when compared to vehicle-treated mice. Within glomeruli of HDAC6i-treated mice, there was increased acetylation of alpha-tubulin and decreased NF-κB. Additionally, HDAC6i decreased serum IL-12/IL-23 and Th17 cells in the spleen. Taken together, these results suggest HDAC-6 inhibition may decrease lupus nephritis in NZB/W mice via mechanisms involving acetylation of alpha-tubulin and decreased NF-κB in glomeruli as well as inhibition of Th17 cells.

Glutathione S Transferases Polymorphisms Are Independent Prognostic Factors in Lupus Nephritis Treated with Cyclophosphamide.

OBJECTIVE: To investigate association between genetic polymorphisms of GST, CYP and renal outcome or occurrence of adverse drug reactions (ADRs) in lupus nephritis (LN) treated with cyclophosphamide (CYC). CYC, as a pro-drug, requires bioactivation through multiple hepatic cytochrome P450s and glutathione S transferases (GST). METHODS: We carried out a multicentric retrospective study including 70 patients with proliferative LN treated with CYC. Patients were genotyped for polymorphisms of the CYP2B6, CYP2C19, GSTP1, GSTM1 and GSTT1 genes. Complete remission (CR) was defined as proteinuria ≤0.33g/day and serum creatinine ≤124 µmol/l. Partial remission (PR) was defined as proteinuria ≤1.5g/day with a 50% decrease of the baseline proteinuria value and serum creatinine no greater than 25% above baseline. RESULTS: Most patients were women (84%) and 77% were Caucasian. The mean age at LN diagnosis was 41 ± 10 years. The frequency of patients carrying the GST null genotype GSTT1-, GSTM1-, and the Ile→105Val GSTP1 genotype were respectively 38%, 60% and 44%. In multivariate analysis, the Ile→105Val GSTP1 genotype was an independent factor of poor renal outcome (achievement of CR or PR) (OR = 5.01 95% CI [1.02-24.51]) and the sole factor that influenced occurrence of ADRs was the GSTM1 null genotype (OR = 3.34 95% CI [1.064-10.58]). No association between polymorphisms of cytochrome P450s gene and efficacy or ADRs was observed. CONCLUSION: This study suggests that GST polymorphisms highly impact renal outcome and occurrence of ADRs related to CYC in LN patients.

Angiotensin-converting enzyme gene polymorphism in Thai patients with systemic lupus erythematosus.

OBJECTIVE: To determine the association between angiotensin-converting enzyme (ACE) I/D polymorphism and the presence of systemic lupus erythematosus (SLE) disease and lupus nephritis (LN), including the association with disease severity in a Thai population. METHOD: In this retrospective study, 187 SLE patients followed up for (at least) 7 years in a rheumatology clinic of an academic hospital were enrolled. Disease severity and damage score at diagnosis and every 6 months, including treatment outcome of the first episode of LN were retrieved from medical records. The ACE genotype of SLE patients were determined by polymerase chain reaction and compared with ACE genotype in 687 controls from a database of a Thai surveillance cohort. RESULT: There was an association between ACE I/D polymorphism and the presence of SLE disease and LN (P < 0.001). Unexpectedly, the prevalence of DD genotype in SLE patients was lower than controls (OR 0.44 [95% CI 0.23-0.84], P = 0.013). The prevalence of ID genotype was not different between SLE patients and controls (OR 1.44 [95%CI 0.93-2.24], P = 0.102), but was higher in LN patients compared to controls (OR 1.77 [95% CI 1.14-2.72], P = 0.01). However, the ACE I/D polymorphism is not associated with SLE disease severity, either in patients with or without nephritis. CONCLUSION: The DD genotype could not be used as a poor prognostic marker for SLE and LN susceptibility in a Thai population. However, ID genotype may be associated with risk to develop LN.

Renal interstitial mast cell count is significantly higher in membranoproliferative glomerulonephritis than in class IV lupus nephritis.

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus; in LN class IV morphologic lesions may be similar to the lesions in primary membranoproliferative glomerulonephritis (MPGN). The aim of the study was to compare the counts of tryptase-positive and chymase-positive mast cells between LN class IV and MPGN. The material consisted of 61 renal biopsies: 32 with lupus nephritis class IV, and 29 with membranoproliferative glomerulonephritis. Chymase- and tryptase-positive cells were stained by immunohistochemistry and subsequently counted. The mean count of chymase-positive mast cells was 21.94 for the whole group, 12.66 for LN class IV and 32.18 for MPGN. The mean count of tryptase-positive cells was 34.94 hpf for the entire group, 22.98 for LN class IV and 48. 13 for MPGN. The differences between lupus nephritis and membranoproliferative glomerulonephritis were significant both for chymase- and tryptase-positive cells. Both chymase-positive MC counts and tryptase-positive MC counts correlated with relative interstitial volume (RIV) (R=0.35 and R=0.28, respectively) and with creatinine level (R=0.35 and R=0.43, respectively). There was also a significant correlation between age, creatinine level and RIV (R=0.28 and R=0.26, respectively).

Urinary prostaglandin D synthase as biomarker in lupus nephritis: a longitudinal study.

OBJECTIVES: Urinary prostaglandin D synthase (uPGDS) has been identified as a biomarker in lupus nephritis (LN) mice model as well as in humans. We studied the effect of therapy for LN on its levels in a longitudinal study and its ability to differentiate between active systemic lupus erythematosus (SLE) patients with and without nephritis. METHODS: Twenty-eight SLE patients with active LN, 6 patients with inactive disease, 12 patients with active non-renal disease and 19 healthy individuals were enrolled. Urine and serum samples were collected at baseline from all patients and at a 3-monthly follow-up from 25 patients in active nephritis group. uPGDS was measured by ELISA and normalised to urinary creatinine excretion. RESULTS: In the cross-sectional study, median uPGDS was higher in patients with active nephritis (618.5 ng/mg) as compared to healthy controls (141.7ng/mg; p<0.001), active non-renal (130.1ng/mg; p=0.008) and inactive disease (56.2 ng/mg; p=0.002) patients and had modest correlation with urinary protein / creatinine ratio (r=0.39; p=0.014). In the longitudinal study, median uPDGS reduced from 618.5 ng/mg at baseline (n=28) to 91.9 at 6 months (n=25), 73.3 at 9 months (n=20) and 81.7 ng/mg at 12 months (n=13). uPGDS remained persistently elevated in a patient who developed CKD and showed an increase 2 months before the clinical relapse in another patient with relapse of LN. CONCLUSIONS: Given that uPGDS levels fall after treatment of LN, uPGDS may be used to monitor the efficacy of therapy. It can also differentiate patients with active nephritis and active non renal lupus.

[Effects of fluvastatin on expression of p38 mitogen-activated protein kinase in renal tissue of chronic graft versus host disease lupus nephritis in mice].

OBJECTIVE: To explore the effects of fluvastatin on expression of p38 mitogen-activated protein kinase (p38MAPK) in renal tissue of chronic graft versus host disease (cGVHD) lupus nephritis in mice. METHODS: Mice were randomly divided into 4 groups of model, low-dose fluvastatin intervention (5 mg×kg(-1)×d(-1)), high-dose fluvastatin intervention (10 mg×kg(-1)×d(-1)) and normal control. The 24 h total amount of urine protein was evaluated by biuret reaction colorimetric assay. And the protein and mRNA expression levels of p38MAPK, p-p38MAPK and TGF-ß1 in renal tissue were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. RESULTS: Compared with the normal control group, 24 h total amount of urine protein, protein and mRNA expression levels of p38MAPK, p-p38MAPK and TGF-ß1 increased significantly in the model group respectively ((7.84 ± 0.36) vs (1.15 ± 0.15) mg/24 h, 0.81 ± 0.03 vs 0.50 ± 0.01, 0.69 ± 0.02 vs 0.10 ± 0.01, 0.76 ± 0.02 vs 0.28 ± 0.02, 0.84 ± 0.04 vs 0.25 ± 0.02, 0.82 ± 0.04 vs 0.12 ± 0.02, all P < 0.01). Fluvastatin treatment suppressed markedly their increased expressions (P < 0.05). And high-dose fluvastatin treatment suppressed more significantly than low-dose group (P < 0.05). CONCLUSION: The renal protection effect of fluvastatin may be achieved by inhibiting the signal pathway of p38MAPK.

B cell-specific loss of Lyn kinase leads to autoimmunity.

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

NADPH oxidase and nitric oxide synthase-dependent superoxide production is increased in proliferative lupus nephritis.

OBJECTIVE: Lupus nephritis (LN) is an immune complex-mediated glomerulonephritis. Proliferative LN (PLN, ISN/RPS classes III and IV)) often leads to renal injury or failure despite traditional induction and maintenance therapy. Successful targeted therapeutic development requires insight into mediators of inflammation in PLN. Superoxide (SO) and its metabolites are mediators of the innate immune response through their ability to mediate reduction-oxidation signaling. Endothelial nitric oxide synthase (eNOS) modulates inflammatory responses in endothelial cells. We hypothesized that markers of SO production would be increased in active PLN and that SO production would be dependent on the activity of select enzymes in the renal cortex. METHODS: Patients with systemic lupus erythematosus were enrolled at the time of renal biopsy for active LN of all classes. Serum collected at baseline was analyzed by HPLC with electrochemical detection for markers of SO production (durable modifications of serum protein Tyr ultimately requiring SO as a substrate). Renal cortex from MRL/MpJ-FAS(lpr) (MRL/lpr) mice with and without functional eNOS was analyzed during active disease for superoxide (SO) production with and without inhibitors of SO-producing enzymes. RESULTS: Serum protein modifications indicative of total SO production were significantly higher in patients with PLN. These markers were increased in association with more active, inflammatory PLN. Mice lacking functional eNOS had 80% higher levels of renal cortical SO during active disease, and inhibitors of nitric oxide synthase and NADPH oxidase reduced these levels by 60% and 77%, respectively. CONCLUSION: These studies demonstrate that SO production is unique to active PLN in a NOS and NADPH oxidase-dependent fashion. These findings suggest the emulating or augmenting eNOS activity or inhibiting NADPH oxidase SO production may be targets of therapy in patients with PLN. The markers of SO production used in this study could rationally be used to select SO-modulating therapies and serve as pharmacodynamic indicators for dose titration.

Endothelial nitric oxide synthase reduces crescentic and necrotic glomerular lesions, reactive oxygen production, and MCP1 production in murine lupus nephritis.

Systemic lupus erythematosus, in both animal models and in humans, is characterized by autoantibody production followed by immune complex deposition in target tissues. Ensuing target organ damage is modulated by reactive intermediates, including reactive nitrogen and oxygen species, through as of now incompletely understood mechanisms. Endothelial nitric oxide synthase is known to impact vascular reactivity; however its impact on reactive intermediate production and inflammatory renal disease is less well defined. In this study, we assessed the impact of endothelial nitric oxide synthase (eNOS) on disease in lupus prone MRL/lpr mice. Mice lacking eNOS developed earlier more severe disease with decreased survival. eNOS deficient mice died sooner and developed significantly more glomerular crescents, necrosis, inflammatory infiltrates and vasculitis, indicating a role for eNOS in modulating these renal lesions. Immune complex deposition was similar between groups, indicating the impact of eNOS is distal to antibody/complement glomerular deposition. Urinary nitric oxide production was decreased in the eNOS deficient mice, while proteinuria was increased. Urinary monocyte chemotactic protein-1 was also increased in the knockout mice. CD4+ T cells from MRL/lpr mice demonstrated mitochondrial hyperpolarization, increased nitric oxide and superoxide production and increased calcium flux compared to B6 control mice. Deficiency of eNOS resulted in decreased nitric oxide and mitochondrial calcium levels but had no effect on mitochondrial hyperpolarization. Renal cortices from MRL/lpr mice that are eNOS deficient demonstrated increased superoxide production, which was blocked by both nitric oxide synthase and NADPH oxidase inhibitors. These studies thus demonstrate a key role for eNOS in modulating renal disease in lupus prone MRL/lpr mice. The impact appears to be mediated by effects on superoxide production in the kidney, impacting downstream mediators such as monocyte chemotactic protein-1. These results suggest that modulation of eNOS may be a novel therapeutic approach to treating lupus nephritis.

Inhibition of sphingosine kinase-2 in a murine model of lupus nephritis.

Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.

Impact of the tumor necrosis factor receptor-associated protein 1 (Trap1) on renal DNaseI shutdown and on progression of murine and human lupus nephritis.

Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by real-time quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.

Association between the angiotensin-converting enzyme insertion/deletion polymorphism and susceptibility to systemic lupus erythematosus: a meta-analysis.

OBJECTIVE: The aim of this study was to determine whether the insertion (I) /deletion (D) polymorphism within intron 16 of angiotensin-converting enzyme (ACE) confers susceptibility to systemic lupus erythematosus (SLE) and lupus nephritis (LN). METHODS: A meta-analysis was conducted on the associations between the ACE I/D polymorphism and SLE and LN. RESULTS: A total of 1907 patients with SLE and 1842 controls in 18 comparative studies were included in this meta-analysis. The analysis showed a significant association between SLE and the DD genotype (recessive effect) of the ACE polymorphism (odds ratio [OR] 1.383, 95% confidence interval [CI] 1.065-1.797, p = 0.015). Stratification by ethnicity indicated a marginal association between the DD genotype and SLE in Europeans (OR 1.210, 95% CI 0.964-1.519, p = 0.099; I(2) = 0%). Meta-analysis also revealed a trend toward to an association between SLE and the ACE polymorphism using the allelic or additive models in Europeans. Furthermore, meta-analysis of the DD+ID genotype showed a significant association with LN (OR 0.582, 95% CI 0.425-0.797, p = 0.001; I(2) = 0%). CONCLUSION: This meta-analysis demonstrates a strong association between DD genotype and SLE in overall study and a marginal association between SLE and the ACE D/I polymorphism in Europeans, and suggests that this polymorphism might confer susceptibility to LN.

Insertion/deletion polymorphism of the angiotensin-converting enzyme gene in lupus nephritis among Mexicans.

UNLABELLED: The angiotensin (Ang)-converting enzyme (ACE) insertion/deletion (I/D) polymorphism determines Ang II levels, but its relationship with lupus nephritis (LN) in different populations is controversial. OBJECTIVE: To describe the allelic and genotypic distribution of the I/D polymorphism in Mexican mestizos with LN and assess an association with histological classes. METHODS: We included 24 patients with systemic lupus erythematosus (SLE) without nephropathy, 41 with LN, 144 healthy subjects, and 36 with primary glomerulonephritis (GMN). Three ACE I/D polymorphism genotypes-ID, DD, and II--were detected by PCR using peripheral blood genomic DNA. RESULTS: Frequencies for II, ID, and DD were 0.29, 0.46, and 0.25 in the SLE group; 0.17, 0.63, and 0.20 in the LN group; 0.14, 0.5, and 0.36 in the GMN group; and 0.26, 0.52, and 0.22 among healthy subjects. The I/D polymorphism distribution according to histological class was class II: 1 II, 3 ID, and 1 DD; class III: 2 II, 10 ID, and 1 DD; class IV: 2 II, 9 ID, and 2 DD; class V: 2 II, 3 ID, and 4 DD; and class VI, 1 II. The histological classes with at least three patients had ID genotype as the most frequent except for class V. CONCLUSION: No association was identified between I/D polymorphisms of ACE and SLE, LN, or GMN in a Mexican population.