Murine Model Of Lupus Nephritis Research Articles

Renal Tubular Cell Ferroptosis: A New Player in Pathogenesis of Lupus Nephritis

Abstract Background New research indicates that the injury to the renal tubular epithelial cells is a better predictor of renal outcomes in lupus nephritis (LN) as compared to glomerular injury. Iron accumulates mostly in the renal tubular cells of LN patients and murine models of LN. Ferroptosis is an incompletely understood form of cell death involving iron mediated lipid peroxidation, but its role in LN is not well established. Experiment LN and control patient biopsies were stained for markers of ferroptosis. Kidneys of female (MRL/lpr) and male (NZW X BXSB) F1, lupus mice were analyzed for markers of ferroptosis. Human proximal tubular cells (PTEC) were treated with LN serum with or without Liporxstatin-2, a novel ferroptosis inhibitor. Results Compared to controls, LN patients expressed higher levels of ACSL4 and 4HNE, the ferroptosis core proteins in the renal tubules. Compared to non-nephritic mice, nephritic mice had significantly higher gene expression of Aifm2, Acsl4, and Gpx1 as well as protein levels of Acsl4. Nephritic mice had an impaired renal glutathione synthesis pathway. Glutathione is an essential positive regulator of glutathione peroxidase 4 (Gpx4: ferroptosis inhibitor) which resulted in lower protein expression of Gpx4. Collectively, LN kidneys displayed a ferroptosis signature and was associated with an increase in Ngal and Kim1, PTEC injury markers. LN patients’ serum induced PTEC ferroptosis and associated pathology were significantly reduced by Liproxstatin-2. Conclusion Our data identify occurrence ferroptosis in renal tubular cell which can contribute to the pathogenesis of LN. Liproxstatin-2 mitigates human LN serum induced PTEC pathology and holds promise as an adjunct therapy to alleviate LN severity. Supported by grants from Vifor Pharma

Effect of Combined Mycophenolate and Rapamycin Treatment on Kidney Fibrosis in Murine Lupus Nephritis.

Background: A significant proportion of lupus nephritis patients develop chronic kidney disease (CKD) and progressive kidney fibrosis, for which there is no specific treatment. We previously reported that mycophenolate or rapamycin monotherapy showed comparable efficacy in suppressing kidney fibrosis in a murine model of lupus nephritis through their direct action on mesangial cells. We extended our study to investigate the effect of combined mycophenolate and rapamycin treatment (MR) on kidney fibrosis in NZBWF1/J mice. Methods: Female NZBWF1/J mice with active nephritis were randomized to receive vehicle or treatment with mycophenolate (50 mg/kg/day) and rapamycin (1.5 mg/kg/day) (MR) for up to 12 weeks, and the effect of treatment on clinical parameters, kidney histology, and fibrotic processes was investigated. Results: Progression of nephritis in untreated mice was accompanied by mesangial proliferation, glomerulosclerosis, tubular atrophy, protein cast formation, increased mTOR and ERK phosphorylation, and induction of TGF-β1, IL-6, α-smooth muscle actin, fibronectin, and collagen expression. Combined MR treatment prolonged survival, improved kidney function, decreased anti-dsDNA antibody level, and ameliorated histopathological changes. The effect of combined MR treatment on kidney histology and function was comparable to that of mycophenolate or rapamycin monotherapy. In vitro studies in human mesangial cells showed that exogenous TGF-β1 and IL-6 both induced mTOR and ERK phosphorylation and downstream fibrotic processes. Both mycophenolic acid and rapamycin inhibited inflammatory and fibrotic processes induced by TGF-β1 or IL-6 by downregulating mTOR and ERK phosphorylation. Conclusions: Our findings indicate that combined mycophenolate and rapamycin, at reduced dose, improves kidney fibrosis in murine lupus nephritis through their distinct effect on mTOR and ERK signaling in mesangial cells.

Pathological manifestation of autoimmune myocarditis is detected prior to glomerulonephritis in a murine model of lupus nephritis.

Since enhanced cardiac magnetic resonance imaging (cMRI) signals have been associated with lupus disease activity in humans prior to renal failure and novel, cardiac-focused therapeutic strategies could be investigated with an associated animal model, autoimmune myocarditis was characterized in murine lupus nephritis (NZM2410). Weekly blood urea nitrogen (BUN) levels and weights were recorded. Cardiac function was assessed by echocardiogram. Myocardial edema was measured with quantitative T2 cMRI mapping. Endpoint serum and cardiac tissue were collected for histopathological analysis and cytokine measurements. Despite showing no signs of significant renal disease, mice displayed evidence of myocarditis and fibrosis histologically at 30-35 weeks. Moreover, T2 cMRI mapping displayed robust signals and analysis of sagittal heart sections showed significant myocardium thickening. Cytokine expression levels of IL-2, IL-10, TNF-α, CXCL1, and IL-6 were significantly enhanced in serum. Echocardiograms demonstrated significantly increased ventricular diameters and reduced ejection fractions, while immunohistochemical staining identified CD4+ and CD8+ T cells, and IL-17 in cardiac infiltrates. Human lupus cardiac tissue showed similar histopathology with enhanced infiltrates by H&E, fibrosis, and CD4+ detection. Histopathology, functional abnormalities, and enhanced cMRI signals indicative of myocarditis are detected in NZM2410 mice without glomerulonephritis, which supports the primary pathological role of autoimmune-mediated, cardiac-targeted inflammation in lupus.

CD6 modulation ameliorates immune complex-mediated glomerulonephritis

Abstract Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus (SLE) in which T cells are thought to play an essential role. CD6 is a co-stimulatory receptor on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation and trafficking, and increased levels of CD6 are associated with pathogenic T cell responses. To assess the role of the CD6-ALCAM pathway in LN pathogenesis, we tested a monoclonal antibody against CD6 in a short-term, validated, inducible murine model of lupus nephritis known as nephrotoxic serum nephritis (NTN). NTN mice were treated 3× per week with an anti-CD6 mAb (10D12, 60ug/dose, n=23) or with vehicle control (n=23). Healthy mice were also included as a control (n=12). Mice treated with the anti-CD6 mAb displayed decreased levels of proteinuria (p<0.001) and significantly improved BUN levels (p<0.01) compared to vehicle control mice. Histology also significantly improved with anti-CD6 treatment (p<0.05). RT-PCR revealed significantly decreased levels of VCAM and RANTES in the kidneys of treated mice, while anti-inflammatory IL-10 was increased, compared to vehicle control mice. Flow cytometry analysis indicated decreased accumulation of both renal-infiltrating activated T cells (CD4+CD25+CD69+, p <0.01) inflammatory macrophages (p<0.05). Overall, these results indicate that the CD6-ALCAM pathway is an important driver of inflammation and pathology in LN and, thus, a promising therapeutic option that is more selective than the immunosuppressive therapies currently offered.

Immunization with Pentraxin3 prevents transition from subclinical to clinical lupus nephritis in lupus-prone mice: Insights from renal ultrastructural findings

BackgroundPentraxin3 (PTX3) is an emerging player in lupus nephritis (LN). Anti-PTX3 antibodies showed to delay LN occurrence in vivo. AimTo evaluate renal changes following immunization with PTX3 in a murine model of LN. Materials and methodsTwenty-two lupus-prone New Zealand Black/White (NZB/W)F1 mice were divided into two groups (n = 11) and subcutaneously injected with human recombinant (hr)PTX3 100 μg or phosphate buffer saline (PBS) 200 μl, three times 3 weeks apart, starting before development of proteinuria. Five mice from each group were scheduled for sacrifice at week 22 and 6 from each group at week 29. Renal lesions included electron-dense deposits (EDD), glomerular deposition of IgG, complement and PTX3 as markers of renal inflammation. They were evaluated by immunofluorescence (IF), confocal and immunoelectron microscopy (IEM). Validated semiquantitative scores were used when available to score renal lesions. Chi-squared test with Fisher exact test was used for comparison. ResultsNineteen out of 22 mice were sacrificed as scheduled. Only hrPTX3-immunized mice developed anti-PTX3 antibodies. Compared to PBS-injected mice, they displayed a dramatic decrease in glomerular deposits of IgG, C1q and PTX3, as well as in the amount of EDD (p = 0.006) and podocyte effacement (p = 0.043). Importantly, PTX3 was pinpointed inside the EDD and co-localized with nuclear material. ConclusionsImmunization with PTX3 prevented progression from the preclinical to the clinical stage of LN, inciting anti-PTX3 antibodies and preventing renal PTX3 deposition. PTX3 is a novel component of EDD, submitting it as one initiating autoantigen in LN and as potential target for early treatment.

TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice

Murine models of lupus, both spontaneous and inducible, are valuable instruments to study SLE pathogenesis. Accelerants such as Type I IFN are often used to trigger earlier disease onset. We used a topical TLR7 agonist, previously reported to induce lupus-like disease in WT mice within weeks, to validate this data in C57BL/6j mice, and to test TLR7 agonism as an accelerant in lupus-prone NZM2410 mice. We found that TLR7-stimulated B6 and NZM2410 mice had significantly reduced survival and exhibited profound splenomegaly with significantly reduced B cells (4 vs. 40%), and T cells (8 vs. 31%). Spleen pathology and IHC revealed massive expansion of F4/80+ cells in TLR7-treated mice consistent with histiocytosis. While resiqimod treatment caused mild autoimmunity in B6 mice and accelerated autoimmunity in NZM2410 mice, it did not cause significant nephritis or proteinuria in either strain (renal function intact at death). Given the macrophage expansion, cytopenias, and disruption of normal splenic lymphoid follicle architecture, histiocytic sarcoma is favored as the cause of death. An alternative etiology is a macrophage activation syndrome (MAS)-like syndrome, since the mice also had a transaminitis and histologic hemophagocytosis in the setting of their rapid mortality. For investigators who are focused on murine models of lupus nephritis, this model is not ideal when utilizing B6 mice, however topical resiqimod may prove useful to accelerate autoimmunity and nephritis in NZM2410 mice, or potentially to investigate secondary complications of lupus such as histiocytic diseases or macrophage activation like syndromes.

FKN Facilitates HK-2 Cell EMT and Tubulointerstitial Lesions via the Wnt/β-Catenin Pathway in a Murine Model of Lupus Nephritis.

Fractalkine (FKN), also known as chemokine (C-X3-C motif) ligand 1, constitutes an intriguing chemokine with a documented role in the development of numerous inflammatory diseases including autoimmune disease. Specifically, it has been reported that FKN is involved in the disease progression of lupus nephritis (LN). The epithelial-mesenchymal transition (EMT) plays a significant role in the formation of tubulointerstitial lesions (TIL), which are increasingly recognized as a hallmark of tissue fibrogenesis after injury. However, the correlation between FKN and EMT or TIL in LN has not been determined. To investigate the potential role of FKN in EMT and TIL, MRL lymphoproliferation (MRL/lpr) strain mice were treated with an anti-FKN antibody, recombinant-FKN chemokine domain, or isotype antibody. Our results revealed that treatment with the anti-FKN antibody improved EMT, TIL, and renal function in MRL/lpr mice, along with inhibiting activation of the Wnt/β-catenin signaling pathway. In contrast, administration of the recombinant-FKN chemokine domain had the opposite effect. Furthermore, to further explore the roles of FKN in EMT, we assessed the levels of EMT markers in FKN-depleted or overexpressing human proximal tubule epithelial HK-2 cells. Our results provide the first evidence that the E-cadherin level was upregulated, whereas α-SMA and vimentin expression was downregulated in FKN-depleted HK-2 cells. In contrast, overexpression of FKN in HK-2 cells enhanced EMT. In addition, inhibition of the Wnt/β-catenin pathway by XAV939 negated the effect of FKN overexpression, whereas activation of the Wnt/β-catenin pathway by Ang II impaired the effect of the FKN knockout on EMT in HK-2 cells. Together, our data indicate that FKN plays essential roles in the EMT progression and development of TIL in MRL/lpr mice, most likely through activation of the Wnt/β-catenin signaling pathway.

A Rationally Engineered Hyperactive Actin‐Resistant DNase1‐Fc Fusion Protein Ameliorates Autoimmune Glomerulonephritis

Loss of B cell tolerance and generation of autoreactive anti‐nuclear antibodies are hallmarks of systemic lupus erythematosus (SLE) and lupus nephritis. Lupus nephritis is characterized by glomerular and tubulointerstitial inflammation often initiated by the renal glomerular deposition of anti‐nuclear immune complexes which trigger subsequent activation of complement, macrophages/monocytes and other innate inflammatory cells. The mechanism of anti‐nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and in human lupus nephritis biopsies rapidly reduces DNase1 expression in renal cortex proximal tubular cells. To overcome the loss of endogenous DNase1, we treated lupus‐prone mice with a hyperactive actin resistant variant of DNase1 with improved catalytic activity against nucleic acid‐IgG immune complexes and acceptable in vivo pharmacokinetics. Hyperactive DNase1‐Fc fusion protein ameliorates nephritis in a murine model of lupus nephritis and reduces immune complex deposition/complement fixation. Taken together, our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti‐nuclear immune complex deposits in the kidney to promote nephritis progression. Our findings provide a therapeutic rationale for using an engineered DNase1‐Fc as a potential therapeutic approach in lupus nephritis.Support or Funding InformationAll authors are current or previous employees of Genentech, Inc.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective effects of quercetin treatment in a pristane-induced mouse model of lupus nephritis

Introduction: Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus. As murine models of LN are valuable tools to better understand its pathophysiology and to search for new effective treatments, we investigated the effects of the bioflavonoid quercetin on pristane-induced LN mice through histomorphological analyses.Methods: Immunofluorescence and biochemical assays were used to evaluate the expression of markers of inflammation (interleukin-6, IL-6; tumour necrosis factor-α, TNF-α), oxidative stress (catalase, CAT; superoxide dismutase 1, SOD1; thiobarbituric acid reactive substances, TBARS), apoptosis (Bax), and fibrosis (transforming growth factor-β1, TGF-β1). Glomerular and tubular ultrastructure was analysed, and tissue messenger RNA of podocin, podoplanin and α3β1-integrin were quantified using the real-time polymerase chain reaction.Results: Pristane-induced LN mice showed severe kidney injury, characterized by increased proteinuria, glomerular mesangial expansion and inflammation, high expression of the pro-fibrotic, apoptotic and prooxidant markers and reduction of antioxidants. In the kidney ultrastructure, foot process (FP) effacement, apoptotic mesangial cells and abnormal mitochondria with disrupted cristae were observed, along with suppressed tissue mRNA of podocin, podoplanin and α3β1-integrin. Treatment with quercetin in the pristane-induced LN mice model was nephroprotective, decreasing proteinuria levels and significantly lowering tissue expression of IL-6, TNF-α, TGF-β1, Bax and TBARS. Simultaneously, quercetin significantly increased CAT and SOD1 expressions in these mice. In addition, it was observed improvement of the kidney ultrastructure, and tissue mRNA of podocin, but not podoplanin and α3β1-integrin, was restored to the levels found in the control mice.Conclusion: In conclusion, these findings provide experimental evidence of the renoprotective effects of quercetin in the pristane-induced LN mice model. We suggest that quercetin effectively ameliorates the kidney damage caused by pristane, a bioflavonoid to be further evaluated as a new therapeutic strategy in this disease.

Anti-OSM Antibody Inhibits Tubulointerstitial Lesion in a Murine Model of Lupus Nephritis.

The purpose of this study was to investigate the role of oncostatin M (OSM) in tubulointerstitial lesion (TIL) in lupus nephritis (LN). We found that OSM was highly expressed in the renal tissue of LN mice. OSM is one of the interleukin-6 cytokine family members. In order to clarify the role and mechanism of OSM in LN, mice with LN were treated with anti-OSM antibody or isotype antibody. We evaluated the tubular epithelial-mesenchymal transdifferentiation (EMT) by detecting the E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin (FN) expression. We analyzed the inflammation by observing the monocyte chemotactic factor-1 (MCP-1) and intercellular adhesion molecule (ICAM-1) expression and calculated the tubulointerstitial fibrosis area by Masson staining. The results showed that anti-OSM antibody, rather than isotype antibody, improved EMT, inflammation, and tubulointerstitial fibrosis. In addition, the signal transducer and activator of transcription (STAT) 1 and STAT3 signaling was activated by tyrosine phosphorylation in LN mouse renal tissue, indicating that the phosphorylated STAT1 (p-STAT1) and p-STAT3 were involved in kidney injury. Moreover, decreased p-STAT3 instead of p-STAT1 has been observed after anti-OSM antibody injection. Thus, we concluded that OSM is associated with TIL in lupus nephritis, which may be connected with the activation of STAT3 rather than that of STAT1.

Bcl-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation.

In lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. Since conventional therapy appears ineffective for severe TII, and these patients often progress to renal failure, understanding in situ mechanisms might reveal new therapeutic targets. This study was undertaken to assess whether dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation in TII. This study utilized novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser-capture microdissection (LCM) coupled to quantitative polymerase chain reaction (qPCR). Furthermore, the consequences of dysregulated apoptotic mediator expression were explored in a murine model of lupus nephritis. Analyses of renal biopsy tissue from patients with lupus nephritis and those with mixed cellular renal allograft rejection revealed that the B cell lymphoma 2 protein (Bcl-2) was frequently expressed in infiltrating lymphocytes, whereas expression of myeloid cell leukemia 1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. Bcl-2 was also highly expressed in tubulointerstitial infiltrates in (NZB × NZW)F1 (NZB/NZW) mice. Furthermore, treatment of NZB/NZW mice with ABT-199, a selective oral inhibitor of Bcl-2, prolonged survival and prevented proteinuria and development of TII in a lupus prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 treatment, and serum anti-double-stranded DNA antibody titers were unaffected. These data demonstrate that Bcl-2 is an attractive therapeutic target in patients with lupus nephritis who manifest TII.

Lupus nephritis develops in a RIP3-independent manner for both males and females (BA4P.136)

Abstract Lupus nephritis is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis, and if untreated leads to renal failure. Although lupus occurs more often in women, both sexes develop glomerulonephritis, which in males develops earlier and is more severe than in females. Previous work by our lab found that loss of Poly [ADP-ribose] polymerase 1 (PARP-1) activity, an enzyme involved in DNA repair and necrotic cell death, resulted in milder nephritis and higher survival rates among males only. A second necrotic pathway involves Receptor-Interacting Serine-Threonine Kinase 3 (RIP3), hence in this study we aimed to investigate the impact of RIP3 on nephritis and determine if this pathway is relevant in both sexes. To this end we used three inducible murine models of lupus nephritis: nephrotoxic serum (NTS)-induced nephritis, pristane-induced lupus and chronic graft versus host (cGVH) disease. We found that absence of RIP3 imparts no positive or negative effects on the development of lupus nephritis in all three models and in both male and female mice. In addition, in vivo experiments done in absence of both RIP3 and PARP-1 did not result in any additive effects between the two pathways, conferring protection only when PARP-1 was absent. Based on these studies, we conclude that RIP3 does not appear to play a significant role in lupus nephritis, nor does it appear to interact with PARP-1 to accelerate, worsen or ameliorate the disease.

Structure and function of renal macrophages and dendritic cells from lupus-prone mice.

To characterize renal macrophages and dendritic cells (DCs) in 2 murine models of lupus nephritis. We used a bead-based enrichment step followed by cell sorting to isolate populations of interest from young mice and nephritic mice. Cell morphology was examined by microscopy. Arginase and nitrite production was examined using biochemical assays. The antigen-presenting functions of the cells were determined using mixed lymphocyte reactions. Selected cytokine, chemokine, and Toll-like receptor (TLR) profiles were examined using real-time quantitative polymerase chain reaction. We identified 2 populations of macrophages and 3 populations of DCs in both of our murine models of lupus (NZB/NZW and [NZW × BXSB]F1 mice). F4/80(high) macrophages, which were resident in normal kidneys and found to be increased in number during nephritis, did not produce either arginase or nitrite upon cytokine stimulation and acquired a mixed proinflammatory and antiinflammatory functional phenotype during nephritis that resembles the constitutively activated phenotype of gut F4/80(high) macrophages. The various cell types differed in their expression of chemokine receptors and TLRs, consistent with variability in their renal location. Resident renal CD103+ DCs were the best antigen-presenting cells and could easily be distinguished from CD11c(high) myeloid DCs that accumulated in large numbers during nephritis. Our study highlights the heterogeneity of the macrophage/DC infiltrate in chronic lupus nephritis and provides an initial phenotypic and functional analysis of the different cellular components that can now be used to define the role of each cell subset in nephritis progression or amelioration. Of note, the dominant macrophage population that accumulates during nephritis has an acquired phenotype that is neither M1 nor M2 and may reflect failure of resolution of inflammation.

OP0085 Therapeutic Inhibition of Anti-Apoptotic BCL-2 Family Proteins in a Murine Model of Lupus Nephritis

BackgroundApoptosis is both a conserved and highly regulated process that is essential for normal development and tissue homeostasis. This process, also known as programmed cell death, is tightly regulated by...

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.

Thinking toward improved treatments of systemic lupus erythematosus

In 2012, recommended therapies for SLE include antimalarials, glucocorticoids, azathioprine, mycophenolate mofetil (or myfortic acid), cyclophosphamide, and other immunosuppressants. Belimumab has been added recently. Most are targeted toward adaptive immune responses. We now suspect that several pathways in innate immunity are also critical to driving SLE, including dendritic cells (major source of IFNα), and monocyte/macrophages that appear central in the damage that occurs to renal tissue in lupus nephritis. Abnormalities in neutrophils may also drive IFNα production and damage to endothelial cells. Therefore, treatments that modify these innate immune cells are of great interest. Antimalarials primarily suppress antigen-presenting cells (APC), including TLR activation; clinically they suppress disease activity and damage, but not strongly. Glucocorticoids suppress DC, monocytes and lymphocytes, with reduction of trafficking of proinflammatory cells to target tissues, but they are quite toxic. Belimumab is directed primarily at prevention of B-cell maturation and has clinical benefits that are not large when added to standard therapies. Among the potential new therapies that influence APC is Laquinimod, a quinoline derivative administered orally, which has recently been shown to reduce the number of new MRI lesions and disability in patients with multiple sclerosis. A recent study in murine EAE shows that Laquinimod suppresses activity of DC, and prevents monocyte/macrophages from accessing the CNS. The suppressed APC functions result in reduced number of effector T cells (Th1, Th17) in target tissue. In addition, Laquinimod induces regulatory T cells and myeloid regulatory CD16+LyC6+ cells that on adoptive transfer suppress clinical disease. We have recent data, submitted for the 2012 ACR meeting, showing similar immune alterations in a murine model of lupus nephritis, including dramatic benefits on protection of young mice from clinical disease, and regression of disease in mice started on treatment after developing heavy proteinuria. Renal damage is minimal. Downregulation of innate immune cells to minimize their activation of adaptive immunity, and their ability to invade and initiate damage target tissues should result in not only less active acute disease but also less future damage. This approach might be discussed at the Whistler meeting.

Selective Cyclooxygenase-2 Inhibitor Suppresses Renal Thromboxane Production but Not Proliferative Lesions in the MRL/lpr Murine Model of Lupus Nephritis

IntroductionProliferative lupus nephritis (LN) is marked by increased renal thromboxane (TX) A2 production. Targeting the TXA2 receptor or TXA2 synthase effectively improves renal function in humans with LN and improves glomerular pathology in murine LN. This study was designed to address the following hypotheses: (1) TXA2 production in the MRL/MpJ-Tnfrsf6lpr/J (MRL/lpr) model of proliferative LN is cyclooxygenase (COX)-2 dependent and (2) COX2 inhibitor therapy improves glomerular filtration rate (GFR), proteinuria, markers of innate immune response and glomerular pathology. MethodsTwenty female MRL/lpr and 20 BALB/cJ mice were divided into 2 equal treatment groups: (1) SC-236, a moderately selective COX2 inhibitor or (2) vehicle. After treatment from the age of 10 to 20 weeks, the effectiveness of inhibition of TXA2 was determined by measuring urine TXB2. Response endpoints measured at the age of 20 weeks were renal function (GFR), proteinuria, urine nitrate + nitrite (NOx) and glomerular histopathology. ResultsSC-236 therapy reduced surrogate markers of renal TXA2 production during early, active glomerulonephritis. When this pharmacodynamic endpoint was reached, therapy improved GFR. Parallel reductions in markers of the innate immune response (urine NOx) during therapy were observed. However, the beneficial effect of SC-236 therapy on GFR was only transient, and renal histopathology was not improved in late disease. ConclusionsThese data demonstrate that renal TXA2 production is COX2 dependent in murine LN and suggest that NO production is directly or indirectly COX2 dependent. However, COX2 inhibitor therapy in this model failed to improve renal pathology, making COX2 inhibition a less attractive approach for treating LN.

SiRNA Therapy for Glomerulonephritis

We are well into a new era in pharmacology in which small molecules, often kinase inhibitors, are used to target signal transduction pathways in cancer and other diseases. In addition to small molecules, antibody-based treatments have become a major part of the pharmacologic armamentarium. RNA interference (RNAi)-based treatments promise to be a third pillar in this new frontier. Indeed, several biotech firms have been established in the past decade to develop RNAi-based therapies. In this issue of JASN , Shimizu et al. 1 bring RNAi-based therapies to bear on kidney disease by demonstrating that small interfering RNAs (siRNAs) to a mitogen-activated protein kinase (MAPK) prevent glomerular disease in a murine model of lupus nephritis. siRNAs are short, double-stranded RNAi molecules that contain complementary sequences to specific mRNAs encoding proteins. By interacting with mRNAs through these complementary sequences, they act similarly to naturally occurring microRNAs to cause degradation of the mRNA or to inhibit translation.2 Interruption of microRNA processing causes dramatic glomerulotubular …

Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.

Bone morphogenetic protein-7: an anti-fibrotic morphogenetic protein with therapeutic importance in renal disease.

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily of cytokines and consist of a group of at least 15 morphogens involved in intracellular messaging through complex bone morphogenetic protein receptor mediated Smad signaling. Bone morphogenetic protein-7 knockout mice die shortly after birth due to uremia, demonstrating that this morphogenetic protein is essential for renal development. Recent investigations have characterized renal bone morphogenetic protein-7 receptors, shown exogenous bone morphogenetic protein-7 to prevent fibrogenesis associated with ureteral obstruction, indicated a loss of renal bone morphogenetic protein-7 associated with diabetic nephropathy, and an improvement in glomerular pathology in rodent streptozocin-induced diabetes with bone morphogenetic protein-7 treatment. In addition, this morphogenetic protein has been shown to reduce glomerulonephritis and tubulointerstitial fibrosis in a murine model of lupus nephritis as well as decrease the peritrabecular fibrosis associated with murine high turnover renal osteodystrophy. Finally, we review the effects of bone morphogenetic protein-7 on vascular calcification in an animal model, a potential complication of this therapy given its osseous morphogenetic effect.