Mouse Model Of Lupus Nephritis Research Articles

Multiscale topology classifies cells in subcellular spatial transcriptomics

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3–6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7–9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

#525 SGLT2 inhibition in non-diabetic CKD: results in experimental lupus nephritis

Abstract Background and Aims Sodium glucose cotransporter type 2 inhibitors (SGLT2i) potently attenuate cardiovascular morbidity and progression of CKD in patients with type 2 diabetes. It has been proposed that SGLT2 inhibition may also elicit renoprotective effects in non-diabetic CKD. The aim of this work is to evaluate whether the treatment with an SGLT2i improves kidney disease progression in a mouse model of lupus nephritis (LN). Method We performed a single center randomized controlled preclinical trial using female MRLlpr/lpr mice. The animals were randomly assigned to (1) vehicle or (2) empagliflozin (EMP, 10 mg/kg/day) treatment once signs of active systemic lupus erythematosus (SLE) appeared: proteinuria >100 mg/dL (++ in reactive strips) or lymphadenopathy in minimum 2 bilateral sites with proteinuria >30 mg/dL (+ in reactive strips). We included 13 mice per group that were treated during 4 weeks. Empagliflozin was given as an admix into standard chow diet. Reduction of urinary albumin to creatine ratio (UACR) was considered the primary endpoint whereas glomerular filtration rate (GFR), renal histology (LN activity/chronicity index, glomerulosclerosis, interstitial fibrosis) and SLE markers (total serum IgG, anti-dsDNA IgG, glomerular IgG deposits, lymph node weight, spleen weight) were considered secondary endpoints. Results After 4 weeks of treatment, no significant differences in fasting blood glucose levels were found between the experimental groups. As expected, empagliflozin administration notably inhibited SGLT2 in the tubular cells demonstrated by a significant increase of urinary glucose (p < 0.0001). This is consistent with the increased water intake observed in MRLlpr/lpr mice treated with empagliflozin (p < 0.0001). At the end of the experiment, vehicle treated MRLlpr/lpr mice showed a significant increase in UACR (p = 0.0012) consistent with LN progression that was not attenuated by empagliflozin. In line, empagliflozin did not modify GFR and LN activity/chronicity indexes, glomerulosclerosis or interstitial fibrosis in kidney. Finally, regarding SLE markers, 4-weeks empagliflozin treatment was not able to decrease total serum IgG, anti-dsDNA IgG, glomerular IgG deposits or lymph node weight. Only spleen weight was significantly decreased in empagliflozin treated MRLlpr/lpr mice (p = 0.003). Conclusion In our experimental setting, empagliflozin treatment reduced spleen weight suggesting a protective role in SLE in MRLlpr/lpr mice. However, empagliflozin did not modify LN progression in MRLlpr/lpr mice probably because the acute LN phase. It is expected that the positive effect in terms of reducing proteinuria will be observed in patients with LN and residual proteinuria

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model.

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target.

Smith-specific regulatory T cells halt the progression of lupus nephritis

Antigen-specific regulatory T cells (Tregs) suppress pathogenic autoreactivity and are potential therapeutic candidates for autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis is associated with autoreactivity to the Smith (Sm) autoantigen and the human leucocyte antigen (HLA)-DR15 haplotype; hence, we investigated the potential of Sm-specific Tregs (Sm-Tregs) to suppress disease. Here we identify a HLA-DR15 restricted immunodominant Sm T cell epitope using biophysical affinity binding assays, then identify high-affinity Sm-specific T cell receptors (TCRs) using high-throughput single-cell sequencing. Using lentiviral vectors, we transduce our lead Sm-specific TCR into Tregs derived from patients with SLE who are anti-Sm and HLA-DR15 positive. Compared with polyclonal mock-transduced Tregs, Sm-Tregs potently suppress Sm-specific pro-inflammatory responses in vitro and suppress disease progression in a humanized mouse model of lupus nephritis. These results show that Sm-Tregs are a promising therapy for SLE.

Hyperbaric oxygen as an immunosuppressant in mouse model of lupus nephritis

Lupus nephritis (LN) is the main cause of morbidity and death due to the most severe complications of the kidney organ in Systemic Lupus Erythematosus (SLE). Immunosuppressive drugs are needed to control this disease, but currently available immunosuppressive drugs do not provide optimal results. This research used 21 Mus Musculus BALB/c mice, female, body weight 25 – 30 grams, 8-12 weeks old, healthy and active, divided into 3 groups. The group of normal mice (G1) was the negative control group, the group of lupus nephritis mice without Hyperbaric Oxygen (HBO) therapy (G2) was the positive control group and the group of lupus nephritis mice with HBO therapy (G3) was the treatment group. The results showed a significant reduction in anti-dsDNA Ab levels p = 0.002 (p<0.05) and proteinuria levels p = 0.005 (p<0.05) in the group given HBO (G3) compared to the group not given HBO (G2). Repair of kidney tissue damage was characterized by a decrease in inflammatory cells, a decrease in tubular constriction and a decrease in immune complex deposits in the group given HBO (G3) compared to the group not given HBO (G2). So HBO can reduce anti-dsDNA Ab levels and proteinuria, and repair damaged kidney tissue. HBO as an immunosuppressive can be used as additional therapy in lupus nephritis.

Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis.

Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.

Potential regulatory role of the Nrf2/HMGB1/TLR4/NF-κB signaling pathway in lupus nephritis

ObjectivesSystemic lupus erythematosus is an autoimmune disease that involves multiple organ systems. One of its major complications, lupus nephritis (LN), is associated with a high mortality rate, and children-onset LN have a more severe course and worse prognosis than adults. Oxidative stress and inflammatory responses are involved in LN development and pathogenesis. Thus, this study aimed to explore the role of signaling regulation of the Nrf2/HMGB1/TLR/NF-κB pathway in LN pathogenesis and unravel the expression of TLR4+CXCR4+ plasma cells subset (PCs) in LN.MethodsC57BL/6 and MRL/lpr mice were divided into four groups: control, model, vector control, and Nrf2 overexpression groups. The vector control and Nrf2 overexpression groups were injected with adenoviral vectors into the kidney in situ. Pathological changes in kidney tissues were observed by hematoxylin–eosin staining. The expression of Nrf2, HMGB1, TLR4, NF-κB, and downstream inflammatory factors in kidney samples was analyzed by quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The ratios of TLR4+CXCR4+ PC subsets in the blood and kidneys of mice were determined by flow cytometry.ResultsIn MRL/lpr mice, Nrf2 was downregulated while HMGB1/TLR4/NF-κB pathway proteins were upregulated. Nrf2 overexpression decreased the expression of HMGB1, TLR4, NF-κB, and its downstream inflammatory cytokines (IL-1β and TNFα). These cytokines were negatively correlated with an increase in Nrf2 content. PC and TLR4 + CXCR4 + PCs in the blood and kidney samples were significantly increased in MRL/lpr mice; however, they were decreased upon Nrf2 overexpression.ConclusionThis study showed severe kidney injury in an LN mouse model and an increased ratio of TLR4 + CXCR4 + PCs. Furthermore, we observed that Nrf2 regulates LN immune response through the Nrf2/HMGB1/TLR4/NF-κB pathway, which can be considered an important target for LN treatment. The clinical value of the findings of our study requires further investigation.

Gbp3 is associated with the progression of lupus nephritis by regulating cell proliferation, inflammation and pyroptosis

Lupus nephritis (LN) is a major cause death in patients with systemic lupus erythematosus. We aimed to find the differentially expressed genes (DEGs) in LN and confirm the regulatory mechanism on LN. The mouse model of LN was constructed by subcutaneous injection of pristane. RNA-seq screened 392 up-regulated and 447 down-regulated DEGs in LN mouse model, and KEGG analysis found that the top 20 DEGs were enriched in arachidonic acid metabolism, tryptophan metabolism, etc. The hub genes, Kynu, Spidr, Gbp3, Cbr1, Cyp4b1, and Cndp2 were identified, in which Gbp3 was selected for following study. Afterwards, the function of Gbp3 on the proliferation, inflammation, and pyroptosis of LN was verified by CCK-8, ELISA, and WB in vitro. The results demonstrated that si-Gbp3 promoted cell proliferation and inhibited the levels of inflammatory factors (IL-1β, TNF-α and IL-8) and pyroptosis-related proteins (GSDMD, Caspase-1 and NLRP3) in a cell model of LN. In constrast, Gbp3 overexpression played an opposite role. In summary, Gbp3 promoted the progression of LN via inhibiting cell proliferation and facilitating inflammation and pyroptosis.

Cardiovascular changes in the NZB/W F1 mouse model of lupus nephritis.

Patients with systemic lupus erythematosus (SLE), an autoimmune disease, have a higher risk of cardiovascular (CV) disease and death. In addition, up to 40%-50% of SLE patients develop lupus nephritis (LN) and chronic kidney disease, which is an additional CV risk factor. Thus, the individual contributions of LN and other SLE-specific factors to CV events are unclear. In this study, we investigated the effect of LN on the development of CV changes using the female NZBxNZW F1 (NZB/W) mouse model of lupus-like disease, with female NZW mice as controls. Standard serologic, morphologic, immunohistologic, and molecular analyses were performed. In a separate group of NZB/W mice, systolic blood pressure (BP) was measured during the course of the disease using tail plethysmography. Our data show marked CV changes in NZB/W mice, i.e., increased heart weight, hypertrophy of the left ventricle (LV) and septum, and increased wall thickness of the intramyocardial arteries and the aorta, which correlated with the progression of renal damage, but not with the age of the mice. In addition, systolic BP was increased in NZB/W mice only when kidney damage progressed and proteinuria was present. Pathway analysis based on gene expression data revealed a significant upregulation of the response to interferon beta in NZB/W mice with moderate kidney injury compared with NZB mice. Furthermore, IFI202b and IL-6 mRNA expression is correlated with CV changes. Multiple linear regression analysis demonstrated serum urea as a surrogate marker of kidney function and IFI202b expression as an independent predictor for LV wall thickness. In addition, deposition of complement factors CFD and C3c in hearts from NZB/W mice was seen, which correlated with the severity of kidney disease. Thus, we postulate that the pathogenesis of CV disease in SLE is affected by renal impairment, i.e., LN, but it can also be partly influenced by lupus-specific cardiac expression of pro-inflammatory factors and complement deposition.

Identification of T cell Autoantigens in a mouse model of Lupus Nephritis

Abstract Lupus nephritis (LN) is the most common severe organ manifestation of Systemic lupus erythematosus (SLE). Autoreactive CD4+ T cells play a key role in SLE, but the mechanism of loss of self-tolerance and antigen specificity of CD4+ T cells is unclear. CD4+ αβ T cells use their T cell receptor (TCR) to recognize 12–20 residue-long epitopes presented on class II MHC molecules. Identifying CD4+ T cell epitopes has been challenging due to lack of efficient and easy-to-implement methods for epitope discovery. We developed a robust T cell antigen discovery technology that uses chimeric receptors called SABR-II (Signaling and Antigen-presenting Bifunctional Receptors) to express large peptide antigen libraries in the context of MHC molecules. Using SABR-IIs, we sought to identify the autoantigens recognized by kidney-infiltrating CD4+ T cells in a mouse mode of LN – MRL/lpr mice. We used a high-throughput single cell TCR cloning to clone and express 93 TCRs from CD4+ T cells sorted from kidneys of 26-week-old MRL/lpr mice. For antigen discovery, we constructed SABR-IIs with I-Ek and I-Ak (class II MHC allele of MRL/lpr mice) and confirmed their functionality. We built libraries of ~25,000 epitopes derived from nuclear proteins and validated them using a known antigen-specific TCR. We have identified novel putative autoantigens, which will be validated in vitro. This study will lead to identification of novel self-antigens, providing the groundwork for understanding the biology of LN and for development of preventative and therapeutic approaches in SLE.

The Role of Glutathione Peroxidase 4 in Regulating Glomerular Permeability in a Mouse Model of Lupus Nephritis

Background/hypothesis of the study: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that often results in kidney damage (lupus nephritis). Glutathione peroxidase 4 (GPx4) is a key defensive player in clearing reactive oxygen species and preventing cell death using glutathione. Reductions of neutrophil GPx4 activity has recently been implicated in autoimmunity in SLE, and reduced renal expression of GPx4 was previously reported in a mouse model of SLE. Whether loss of GPx4 activity has a direct role in glomerular injury in SLE is currently unknown. Therefore, the present study aimed to investigate the role of GPx4 in regulating glomerular permeability and whether deficits in GPx4 may contribute to albuminuria in lupus nephritis. Methods and Results: Experiments utilized female (New Zealand Black x New Zealand White) F1 (NZBWF1) mice, a well-established mouse model of lupus nephritis that has a female sex bias, similar to patients. In our hands, these mice typically develop albuminuria around 34 weeks of age. Glomeruli were isolated on ice by graded sieving and underwent functional and biochemical analysis. Glomerular GPx4 expression was compared between age- and sex-matched albuminuric NZBWF1 mice (albuminuria status determined by dipstick) and healthy control New Zealand White (NZW) mice by western blot. Protein expression of GPx4 in glomeruli isolated from albuminuric 34-week-old female NZBWF1 mice was reduced to approximately 30% of the level found in NZW mice (P<0.01; n=4 mice each group). For functional analysis, glomeruli were suspended in a 5% albumin solution, and the change in volume in response to a decrease in external oncotic pressure caused by switching to a 1% albumin solution was used to determine the albumin reflection coefficient and calculate relative glomerular permeability to albumin (Palb). Experiments measured (i) Palb of glomeruli from healthy young 8-week-old NZBWF1 compared to 34-week-old albuminuric NZBWF1, and (ii) Palb of glomeruli from 8-week-old NZBWF1 mice following ex vivo treatment of glomeruli with the GPx4 inhibitor RSL3 (3μM) for 30 minutes compared to untreated glomeruli from the same mice. At least 5-8 glomeruli/mouse/treatment or age were analyzed, and the data from each mouse was averaged. As expected, 34-week-old albuminuric NZBWF1 mice showed significantly increased Palb compared with 8-week-old mice (P<0.001; n=5 mice per group). Glomeruli isolated from 8-week-old NZBWF1 mice showed increased Palb after 30 min incubation with 3μM RSL3 when compared to non-treated glomeruli (P<0.001; n=4 mice per group). Conclusion: Our data suggest that the development of albuminuria in NZBWF1 mice is associated with a reduction in glomerular expression of GPx4, and impairment of GPx4 activity can promote increased Palb. A loss of GPx4 activity may contribute to glomerular filtration barrier dysfunction and albuminuria in SLE. This work was supported by the National Institutes of Health (grant number 1R01DK119337-01A1) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.

TRAF3 plays a role in lupus nephritis by regulating Th17 cell and Treg cell balance as well as NF-κB signaling pathway in mice

Lupus nephritis (LN) occurs with inflammatory lesion in patients suffering from systemic lupus erythematosus (SLE). Tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) is an important mediator in inflammation. To explore the roles of TRAF3 in LN, the LN mouse model was firstly established with intraperitoneal (i.p.) injection of pristine. Our results found that the amount of urinary protein was increased evidently at day 28, and renal damage occurred in the LN mouse model, but the TRAF3 knockdown reduced the urinary protein and alleviated the inflammatory lesion. The proinflammatory cytokines TNF-α, IL-1β, IL-17a, IFN-γ and IgM, IgG antibody were enriched, but there was little amount of IL-10 in the LN mouse model. Moreover, the amount of CD40+ B cells, CD4+ T cells sub-type, Th17 cells were abundant, and the proteins TRAF3, TRAF2, NF-κBp52, IKKα, ICAM1 in the kidney were highly expressed in the LN mouse model. However, TRAF3 knockdown enhanced the production of IL-10 and reduced the amount of pro-inflammatory cytokines, immunoglobulin, and the protein expressions of TRAF3, TRAF2, NF-κBp52, IKKα, ICAM1. In conclusion, TRAF3 plays a role in LN by regulating Th17 cell and Treg cell balance as well as NF-κB signaling pathway in mice.

Ameliorated Renal Pathological Feature in MRL/MpJ-Faslpr/lpr Background Interleukin-36 Receptor-Deficient Mice.

Systemic autoimmune diseases frequently induce lupus nephritis, causing altered balance and expression of interleukin 36 receptor (IL-36R) ligands, including agonists (IL-36α, β, γ) and antagonists (IL-36Ra, IL-38), in kidneys. Here, we established and analyzed a mouse model of lupus nephritis, MRL/MpJ-Faslpr/lpr with IL-36R-knockout (KO), compared to wild-type (WT) mice. In both genotypes, indices for immune abnormalities and renal functions were comparable, although female WT mice showed higher serum autoantibody levels than males. IL-36R ligand expression did not differ significantly between genotypes at the mRNA level or in IL-36α and IL-38 scores. However, glomerular lesions, especially mesangial matrix expansion, were significantly ameliorated in both sexes of IL-36R-KO mice compared to WT mice. Cell infiltration into the tubulointerstitium with the development of tertiary lymphoid structures was comparable between genotypes. However, the positive correlation with the IL-36α score in WT mice was not evident in IL-36R-KO mice. Fibrosis was less in female IL-36R-KO mice than in WT mice. Importantly, some IL-36α+ nuclei co-localized with acetylated lysine and GCN5 histone acetyltransferase, in both genotypes. Therefore, IL-36R ligands, especially IL-36α, contribute to the progression of renal pathology in lupus nephritis via IL-36R-dependent and IL-36R-independent pathways.

Multifunctional Polydopamine-Based Nanoparticles for Dual-Mode Imaging Guided Targeted Therapy of Lupus Nephritis.

Lupus nephritis (LN) is a common and refractory inflammation of the kidneys caused by systemic lupus erythematosus. Diagnosis and therapies at this stage are inefficient or have severe side effects. In recent years, nanomedicines show great potential for imaging diagnosis and controlled drug release. Herein, we developed a polydopamine (PDA)-based nanocarrier modified with Fe3O4 and Pt nanoparticles and loaded with necrostatin-1 (Nec-1) for the bimodal imaging and therapy of LN. Results demonstrate that Nec-1/PDA@Pt-Fe3O4 nanocarrier exhibits good biocompatibility. Nec-1, as an inhibitor of receptor-interacting protein 1 kinase, can be used to inhibit receptor-interacting protein 1 kinase activity and then reduces inflammation due to LN. Experiments in vitro and in the LN mouse model confirmed that the nanocarrier can reduce neutrophil extracellular traps (NETs) production by RIPK1 and alleviate the progression of inflammation. Previous studies proved that Pt nanoparticles can catalyze H2O2 to produce oxygen. A blood oxygen graph of mouse photoacoustic tomography confirmed that Nec-1/PDA@Pt-Fe3O4 can generate oxygen to fight against the hypoxic microenvironment of LN. PDA and Fe3O4 are used as photographic developers for photoacoustic or magnetic resonance imaging. The preliminary imaging results support Nec-1/PDA@Pt-Fe3O4 potential for photoacoustic/magnetic resonance dual-mode imaging, which can accurately and non-invasively monitor microscopic changes due to diseases. Nec-1/PDA@Pt-Fe3O4 combining these advantages exhibited outstanding performance in LN imaging and therapy. This work offers valuable insights into LN diagnosis and therapy.

Polymeric dexamethasone prodrugs attenuate lupus nephritis in MRL/lpr mice with reduced glucocorticoid toxicity

Due to their potent immunosuppressive and anti-inflammatory effects, glucocorticoids (GCs) are the most widely used medications in treating lupus nephritis (LN). Long-term use of GCs, however, is associated with numerous off-target adverse effects. To reduce GCs' adverse effects, we previously developed two polymeric dexamethasone prodrug nanomedicines: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based dexamethasone prodrug (P-Dex), and micelle-forming polyethylene glycol (PEG)-based dexamethasone prodrug (ZSJ-0228). Both P-Dex and ZSJ-0228 provided sustained amelioration of LN in lupus-prone NZB/W F1 mice with reduced GC-associated adverse effects. Here, we have extended our investigation to the MRL/lpr mouse model of LN. Compared to dose equivalent daily dexamethasone sodium phosphate (Dex) treatment, monthly P-Dex or ZSJ-0228 treatments were more effective in reducing proteinuria and extending the lifespan of MRL/lpr mice. Unlike the daily Dex treatment, ZSJ-0228 was not associated with measurable GC-associated adverse effects. In contrast, adrenal gland atrophy was observed in P-Dex treated mice.

MiR-145 participates in the development of lupus nephritis by targeting CSF1 to regulate the JAK/STAT signaling pathway.

Lupus nephritis (LN) is a chronic autoimmune disease, leading to progressive renal dysfunction. MicroRNAs (miRNAs) contribute to LN pathophysiology. Nevertheless, the potential mechanisms of miR-145 in LN remain unclear. Here, we investigated the contribution of miR-145 to LN progression. qRT-PCR analysis determined miR-145 and CSF1 expression. Western blot tested CSF1, JAK2, p-JAK2, STAT3, p-STAT3, cleaved caspase3, Bax and Bcl-2 expression. Dual luciferase reporter assay confirmed the interaction between miR-145 and CSF1. ELISA assay detected the secretion of inflammatory molecules. Flow cytometric analysis determined cell cycle and apoptosis. MTT was conducted to test cell viability. The LN mouse model was constructed for in vivo experiments. HE and Masson staining examined the kidney pathologic changes. MiR-145 was down-regulated in LN patients and LPS-induced HRMCS, whereas CSF1 was up-regulated. Moreover, miR-145 overexpression inhibited HRMCS cell apoptosis and inflammatory damage. Besides, miR-145 was found to directly target CSF1. Additionally, knockdown of CSF1 inhibited HRMCS cell apoptosis and inflammatory damage by inactivating the JAK/STAT signaling pathway. Furthermore, miR-145 inhibited inflammatory damage and cell apoptosis of HRMCS by down-regulating CSF1. Finally, we verified that miR-145 suppressed LN development in vivo. Our data reveals that miR-145 regulates LN progression via CSF1 mediated JAK/STAT signaling pathway, and miR-145 may be a new therapeutic target for LN treatment.

Low-Molecular-Weight Heparin Enhanced Therapeutic Effects of Human Adipose-Derived Stem Cell Administration in a Mouse Model of Lupus Nephritis.

BackgroundLupus nephritis is a life-threatening complication in systemic lupus erythematosus (SLE), but the efficiency of current therapies involving corticosteroids, immunosuppressants, and biological agents is limited. Adipose-derived mesenchymal stem cells (ASCs) are gaining attention as a novel treatment for inflammation in SLE. Low-molecular-weight heparin (LMWH) exhibits multiple functions including anti-inflammatory, anti-fibrotic, and cell function-promoting effects. LMWH stimulation is expected to increase the therapeutic effect of ASCs by promoting cellular functions. In this study, we investigated the effects of LMWH on ASC functions and the therapeutic effect of LMWH-activated human-ASCs (hep-hASCs) in an SLE mouse model.MethodsThe cellular functions of human-derived ASCs stimulated with different LMWH concentrations were observed, and the optimum LMWH dose was selected. The mice were assigned to control, human-ASC, and hep-hASC groups; treatments were performed on week 20. Twenty-six week-old mice were sacrificed, and urine protein score, serum blood urea nitrogen, creatinine (Cr), anti-ds DNA IgG antibody, and serum IL-6 levels were analyzed in each group. Mice kidneys were evaluated via histological examination, immunohistochemical staining, and gene expression levels.ResultsLMWH significantly promoted ASC migration and proliferation and hepatocyte growth factor production and upregulated immunomodulatory factors in vitro. Hep-hASC administration resulted in significant disease activity improvement including proteinuria, serum Cr and IL-6 levels, anti-ds DNA IgG antibody, glomerulonephritis, and immune complex in mice. Inflammation and fibrosis in kidneys was significantly suppressed in the hep-hASC group; the gene expression levels of TNF-alpha, TIMP-2, and MMP-2 was significantly downregulated in the hep-hASC group compared with the control group.ConclusionsHep-hASC exhibited higher anti-inflammatory and anti-fibrotic effects than hASCs and may be a candidate tool for SLE treatment in future.

Reversible dysregulation of renal circadian rhythm in lupus nephritis

BackgroundWe have found disruption of expression of major transcriptional regulators of circadian rhythm in the kidneys of several mouse models of lupus nephritis. Here we define the consequence of this disturbance with respect to circadian gene expression and renal homeostatic function in a mouse model of lupus nephritis.MethodsMolecular profiling of kidneys from 47 young and 41 nephritic female NZB/W F1 mice was performed at 4 hourly intervals over a 24 h period. Disruption of major circadian transcriptional regulators was confirmed by qPCR. Molecular data was normalized and analyzed for rhythmicity using RAIN analysis. Serum aldosterone and glucose and urine sodium and potassium were measured at 4 hourly intervals in pre-nephritic and nephritic mice and blood pressure was measured every 4 h. Analyses were repeated after induction of complete remission of nephritis using combination cyclophosphamide and costimulatory blockade.ResultsWe show a profound alteration of renal circadian rhythms in mice with lupus nephritis affecting multiple renal pathways. Using Cosinor analysis we identified consequent alterations of renal homeostasis and metabolism as well as blood pressure dipper status. This circadian dysregulation was partially reversed by remission induction therapy.ConclusionsOur studies indicate the role of inflammation in causing the circadian disruption and suggest that screening for loss of normal blood pressure dipping should be incorporated into LN management. The data also suggest a potential role for circadian agonists in the treatment of lupus nephritis.

Research Highlights

Research Highlights