Exploring complement activation as a therapeutic target in lupus nephritis

To discover novel serum biomarkers that have diagnostic or predictive value in lupus nephritis (LN). Using a quantitative protein microarray, we screened for high-abundant proteome expression in the serum of patients with LN compared to healthy controls. Top candidates from this screening were validated using a larger cohort of patients with LN compared to a disease control cohort (subjects with other chronic kidney diseases) and a healthy control cohort. Promising markers were then selected using a machine-learning model and further validated with a larger patient cohort. The corresponding autoantibodies and immune complexes containing these proteins were also examined. In total, 13 proteins were found to be significantly elevated in LN patient serum in the screening, among which 8 proteins were validated by enzyme-linked immunosorbent assay using 81 serum samples from LN patients and control subjects. Three serum markers with LN diagnostic potential were identified using feature importance analysis and further validated using 155 serum samples from LN patients and control subjects. V-set immunoglobulin domain-containing protein 4 (VSIG4) appeared to be the most promising marker in distinguishing LN from healthy controls, with an area under the curve of 0.93. VSIG4 could also discriminate active LN from inactive LN. Furthermore, serum VSIG4 levels were positively correlated with all of the following clinical parameters: the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (Spearman's rank correlation rs = 0.42, P < 0.001), the renal domain score of the SLEDAI (rs = 0.46, P < 0.001), the urinary protein-to-creatinine ratio (rs = 0.56, P < 0.001), and the serum creatinine level (rs = 0.41, P < 0.001). Importantly, we found that serum VSIG4 levels tracked with LN disease activity longitudinally, and that serum VSIG4 levels reflected the renal pathology activity index (AI), particularly the AI components of crescent formation and hyaline deposits. VSIG4 may be a promising novel serum biomarker and therapeutic target in patients with LN.

Proteomics has been a very informative tool in uncovering several biomarkers that may have clinical utility in managing patients with lupus nephritis (LN). Both non-targeted (mass spectrometry-based) and targeted (ligand-based) proteomics point to promising biomarkers in the urine of LN patients. Aptamer-based interrogation of 1129 proteins using the Somascan platform, followed by ELISA-validation in two independent cohorts has indicated that the urine proteins that best distinguish active LN from controls were ALCAM, PF-4, properdin, and VCAM-1 among African Americans, E-selectin, VCAM-1, BFL-1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians (PMID: 32366845). Most of these urine proteins correlate significantly with disease activity indices and surpass conventional metrics in identifying active LN. Several elevated urinary molecules are also expressed within LN kidneys, based on single-cell RNA-seq analysis. Of these, ALCAM has been validated in a large multi-ethnic, multi-national cohort of LN subjects, and has also been shown to be a pathogenic driver and therapeutic target in LN (PMID: 34981775). Antibody array based screening of 1000 proteins using the Raybiotech Kiloplex platform has identified urine L-selectin, Angptl4, TGF[Formula: see text] 1, TPP1, TSP-1, FOLR2, PDGFR[Formula: see text], and PRX2, as markers of active renal lupus, outperforming conventional metrics, with L-selectin, Angptl4 and TGF[Formula: see text] 1 tracking well with concurrent or pending disease flares in LN (PMID: 32651195). Of these, urine L-selectin has been validated in large multi-ethnic/national cohorts of LN subjects (under review). Proteomic screens of LN urine using an electrochemiluminescence platform (Mesoscale/MSD) has identified IL-7, IL-12p40, IL-15, IP-10, and TARC as markers of active renal lupus that correlated significantly with disease activity (PMID: 30618193). Very recent global proteomic screens of LN urine using a proximity ligation assay platform from Olink has added several additional biomarker candidates that have been successfully ELISA-validated (under review). Given the plethora of urine biomarkers that have been identified using proteomics, it now becomes important to validate the clinical utility of these urine proteins, using multiple well-annotated patient cohorts, across ethnicities. Among the completed studies, we now know that some urine proteins are diagnostic of concurrent renal pathology LN Class or Activity Index or Chronicity index (e.g. CD163: PMID: 32351512; VCAM-1: PMID: 29076253; Angiostatin: PMID: 29076253; Hemopexin, Kim-1 and Lipoxcalin-2/NGAL; Renin and SAP; PMID: 20065116), while others exhibit prognostic value (e.g., ALCAM and VCAM-1; PMID: 31722419). Ongoing studies also show that most of the urine proteins interrogated in LN are not elevated in circulation (PMID: 36091001), but are expressed within LN kidneys, as ascertained by immunohistochemistry (Clinical Immunology, 2022) and Imaging Mass Cytometry (Tissue CyTOF). To facilitate monitoring of urinary biomarkers in outpatient clinics or to enable self-monitoring of disease status by the patient from the comfort of one’s home, we have recently engineered a smartphone-readable nanophosphor-based lateral flow assay for point of care monitoring of urine ALCAM (Frontiers in Immunology, 2022). The long-term goal is to decipher the clinical utility of different urine biomarkers in managing lupus and LN in the clinics, to empower LN patients with easy-to-use tools to monitor their own disease status, and to understand the mechanistic significance of the uncovered molecules in renal disease pathogenesis. Ongoing research on this front is summarized at: https://mohanlab.bme.uh.edu/ .

Introduction/BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease that can affect almost any organ system but gastrointestinal (GI) involvement is very rare. Obinutuzumab is a humanised type II anti-CD20 monoclonal antibody that is licensed to treat haematological malignancies. It has been used in a small number of SLE patients with secondary non-depletion non-response (2NDNR) to Rituximab and appears to be effective in lupus nephritis (LN) as outlined in the NOBILITY trial. We present a case of refractory lupus enteritis (LEn) and LN in a patient with 2NDNR to repeated Rituximab cycles that achieved remission with Obinutuzumab.Description/MethodA 19-year-old African woman was diagnosed with SLE in 2018 after presenting with polyarthritis without systemic involvement. Blood tests showed leucopenia, positive antinuclear antibody, anti-double-stranded DNA >200 IU/ml and hypocomplementaemia. Prednisolone and Methotrexate were commenced. However, one month later she presented to hospital with generalised abdominal pain, vomiting and diarrhoea. CT abdomen demonstrated significant small bowel wall thickening with mucosal hyperenhancement. Multidisciplinary team (MDT) input confirmed likely LEn. She was treated successfully with intravenous Methylprednisolone and Rituximab. Total parenteral nutrition was required for two weeks.Unfortunately, four further hospital admissions followed over the next six months due to GI symptoms, transient acute kidney injury and moderate proteinuria. Small bowel oedema persisted on MRI, three months after the CT. Renal biopsy demonstrated class 2 LN. Intravenous methylprednisolone helped and further Rituximab was given but she had infusion reactions. Six months later she re-presented with abdominal pain, synovitis and had nephrotic range proteinuria. She was treated again for LEn after gastroenterology input. Further Rituximab was given (MabThera instead of Truxima) but reactions still occurred and her B cells were non-deplete. Repeat renal biopsy confirmed class 4 LN and intravenous Cyclophosphamide (Euro-Lupus regimen) was commenced. This was repeated seven months later due to persisting high disease activity and proteinuria despite a short trial of intravenous Belimumab. However, she was admitted to hospital with colitis on CT after completing the second course of Cyclophosphamide. MDT decision was to treat with Obinutuzumab. Since commencing treatment eighteen months ago, her SLE has dramatically improved and she feels well for the first time. SLEDAI-2K score is 2 (16 pre-treatment), renal function has recovered with mild proteinuria. Anti-double-stranded DNA has reduced from >200 to 52.9 IU/ml and complements normalised. She remains on Prednisolone 5mg daily and Tacrolimus and a third maintenance dose of Obinutuzumab is planned.Discussion/ResultsLEn has been reported in only 0.2-5.8% of SLE cases. It is defined by the British Isles Lupus Assessment Group (BILAG 2004) by either vasculitis or inflammation of the small bowel supported by imaging or histology. Its pathogenesis is poorly understood but thought to be related to immune complex deposition and complement activation resulting in intestinal capillary leak and submucosal oedema. Symptoms are non-specific and a review by Janssens et al. (2013) suggested abdominal pain (97%), ascites (78%), nausea (49%), vomiting (42%) and diarrhoea (32%) were the most common symptoms in patients with LEn. They reported the ileum and jejunum were most commonly affected in LE (84% and 83% respectively), followed by colon (19%), duodenum (17%) and rectum (4%).Abdominal CT is considered to be the first line investigation and ‘classic’ radiological findings are bowel wall oedema (“target sign”), engorgement/increased number of mesenteric vessels (“comb sign”) and increased attenuation of the mesenteric fat (Ha et al. 2000, and Kim et al. 2006). These were all demonstrated on our patient’s initial CT abdomen which showed engorgement of the mesenteric vessels from the proximal jejunum to the proximal ileum.LEn seldom occurs in isolation and usually presents in the context of high disease activity and other systemic involvement. It is often responsive to glucocorticoids and bowel rest but further immunosuppression with Cyclophosphamide or Rituximab might be warranted in refractory cases as in our patient’s case.Obinutuzumab has been shown to be safe, effective and steroid-sparing in renal and non-renal SLE patients with 2NDNR to Rituximab from a case series of 9 patients by Arnold et al. (2022). This applied to LN in our patient and we believe this to be the first case where refractory LEn was treated successfully with Obinutuzumab.Key learning points/ConclusionAlthough very rare, have a high index of suspicion of LEn in SLE patients who present with GI symptoms and typical radiological findings.LEn often occurs with other systemic manifestations including LN and it is important to assess for these at the time of diagnosis of LEn.Obinutuzumab is an emerging treatment for severe SLE particularly 2NDNR to Rituximab and can improve both LN and LEn. We eagerly await results from the current REGENCY and ALLEGORY clinical trials which are assessing Obinutuzumab in renal and non-renal SLE patients respectively.

Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) that involves the kidneys and severely affects renal function or even leads to renal failure. The aim of this study was to quantify the expressions of nuclear factor kappa B (NF-κ B) and heat shock protein 90 (HSP90) in patients with LN and investigate the pathological mechanisms involved. We collected fifty-two renal needle biopsy tissues and complete clinical data from patients diagnosed with LN. We also selected thirty-two renal specimens from hydronephrosis surgery and autopsy served as the control group. The expressions of NF-κ B and HSP90 in kidney histology were detected. In addition, we collected Fasting peripheral venous blood samples from 16 patients with SLE, 12 patients with LN, and 16 healthy controls diagnosed in our hospital. And the gene expression levels of NF-κ B and HSP90 in peripheral blood mononuclear cells (PBMCs) of the subjects were determined. The expression levels of NF-κ B and HSP90 in kidney histology of patients with LN were significantly higher than those in the control group. High NF-κ B and HSP90 protein expressions were associated with severe LN activity (SLEDAI >15 points) (p<0.05). Compared with the control group, the expression levels of NF-κ B and HSP90 mRNA were significantly up regulated in PBMCs of the SLE patient group (p<0.05). Moreover, the expression levels of NF-κ B and HSP90 mRNA in PBMCs were significantly up regulated in the LN patient group compared with the SLE patient group (p<0.05). In addition, the levels of NF-κ B and HSP90 mRNA expression in PBMCs of the LN patient group were positively correlated (p<0.05). The area under the receiver operating characteristic (ROC) curve for NF-κ B mRNA expression in PBMCs from patients with LN to diagnose SLE kidney damage was 0.929 (95% confidence interval [CI] 0.833-1.000, p<0.001), and the AUC for HSP90 mRNA expression was 0.742 (95% CI 0.556-0.927, p<0.05). NF-κ B and HSP90 may play a synergistic role in the pathogenesis of LN. Their elevated expression has a high diagnostic accuracy for LN or SLE without renal damage. Hence, NF-κ B and HSP90 can be used as therapeutic targets and biomarkers for the diagnosis of LN.

<h3>Purpose</h3> In systemic lupus erythematosus (SLE), lupus nephritis (LN) represents one of the most severe organ complications. LN is associated with persistent inflammation and perpetuated fibroblast activation, both determined by epigenetic mechanisms involving aberrant CpG DNA promoter methylation. During SLE progression, global methylation patterns are commonly lost. CpG DNA promoter methylation patterns are not limited to the kidney, circulating CpG-rich DNA is also detectable in the blood. However, little is known about its specific contribution to determining disease progression. In the kidney, CpG-rich DNA activates TLR9 signalling mechanisms involved in inflammation and fibrogenesis. Based on these observations, we hypothesised that CpG-rich DNA promoter fragments potentially accelerate renal inflammation and fibrogenesis in SLE-associated LN. <h3>Methods</h3> First, CPG-rich DNA from blood samples of SLE patients with and without LN were collected. Then, we tested how these DNA promoter fragments influenced the LN phenotype in a TMPD ('pristane')-induced mouse model. The renal response to the administration of either human or synthetic methylated/unmethylated CpG-rich DNA oligodinucleotides (ODN) was observed. Downstream effects of the administration of circuclating CpG-rich DNA fragments on TLR9-signalling were analysed in endothelial cell cultures. <h3>Results</h3> Circulating CpG-rich DNA promoter fragments are detectable in SLE patients' blood. LN was associated with accumulation of unmethylated CpG-rich DNA promoter fragments, implicating a mechanistic link. In a rodent model of pristane-induced lupus, administration of CpG-rich DNA (isolated from LN patients or synthetic unmethylated CpG-rich DNA ODN) worsened the renal phenotype. TLR9-mediated intrarenal inflammation can be therapeutically targeted by administration of synthetic methylated CpG-rich DNA oligonucleotides, ultimately associated with suppression of TLR9-mediated signalling responses and renal injury in experimental LN. <h3>Conclusions</h3> Our results implicate accumulation of unmethylated CpG-rich promoter DNA fragments in LN. Furthermore. these unmethylated CpG-rich promoter DNA fragments causally contribute to TLR9-mediated inflammation and renal fibrogenesis and administration of methylated CpG-rich ODN attenuated intrarenal TLR9-mediated inflammatory signalling responses. Therefore, biomonitoring of CpG-rich promoter DNA fragments and modulation of intrarenal TLR9 signalling might be a promising therapeutical target in LN.

Reactive intermediate production is an essential component of the innate immune response that is induced during disease activity in murine lupus. This study was undertaken to determine whether a marker of systemic nitric oxide (NO) production correlates with prospectively studied disease activity in human systemic lupus erythematosus (SLE) and lupus nephritis patients. Eighty-three SLE patients and 40 control subjects were studied longitudinally. The SLE group included 23 patients with lupus nephritis documented by renal biopsy and 26 with a history of lupus nephritis. During each visit, following a 24-hour low-nitrate diet, traditional markers of disease activity and damage were determined. Serum nitrate plus nitrite (NOx) levels were determined by chemiluminescence detection. NOx levels were higher in SLE patients than in controls during the first visit. In univariate longitudinal analyses, NOx levels were associated with SLE Disease Activity Index scores. In multivariate analyses, NOx levels were associated with serum levels of C3 and creatinine and the urinary protein:creatinine ratio. Among patients with lupus nephritis, those with proliferative lesions had higher NOx levels, and higher NOx levels were associated with accumulation of renal damage and lack of response to therapy. This is the first study to prospectively demonstrate longitudinal associations between serum NOx levels and markers of SLE and lupus nephritis disease activity. The more pronounced association with proliferative lupus nephritis and with longitudinal response to lupus nephritis therapy provides a rationale for the study of reactive intermediates as biomarkers of disease activity and therapeutic targets in proliferative lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting mostly women of child-bearing age. Immune dysfunction in SLE results from disrupted apoptosis which lead to an unregulated interferon (IFN) stimulation and the production of autoantibodies, leading to immune complex formation, complement activation, and organ damage. Lupus nephritis (LN) is a common and severe complication of SLE, impacting approximately 30% to 40% of SLE patients. Recent studies have demonstrated an alteration in mitochondrial homeostasis in SLE patients. Mitochondrial dysfunction contributes significantly to SLE pathogenesis by enhancing type 1 IFN production through various pathways involving neutrophils, platelets, and T cells. Defective mitophagy, the process of clearing damaged mitochondria, exacerbates this cycle, leading to increased immune dysregulation. In this review, we aim to detail the physiopathological link between mitochondrial dysfunction and disease activity in SLE. Additionally, we will explore the potential role of mitochondria as biomarkers and therapeutic targets in SLE, with a specific focus on LN. In LN, mitochondrial abnormalities are observed in renal cells, correlating with disease progression and renal fibrosis. Studies exploring cell-free mitochondrial DNA as a biomarker in SLE and LN have shown promising but preliminary results, necessitating further validation and standardization. Therapeutically targeting mitochondrial dysfunction in SLE, using drugs like metformin or mTOR inhibitors, shows potential in modulating immune responses and improving clinical outcomes. The interplay between mitochondria, immune dysregulation, and renal involvement in SLE and LN underscores the need for comprehensive research and innovative therapeutic strategies. Understanding mitochondrial dynamics and their impact on immune responses offers promising avenues for developing personalized treatments and non-invasive biomarkers, ultimately improving outcomes for LN patients.

A neutrophil-mesangial cell axis promotes glomerular injury in lupus nephritis.

Background Lupus nephritis affects more than one-half of patients with SLE and is the most common serious manifestation of the disease. Lupus nephritis is more common in Hispanic and African-American patients than in those of European ancestry, and class III and IV nephritis progresses to end-stage renal disease in 10%–15% of patients within 15 years of diagnosis. Identification of markers and mechanisms of lupus nephritis could provide new approaches to predict and treat disease. Methods To identify blood cell transcriptome biomarkers that differentiate renal and non-renal disease we performed RNA sequencing on peripheral blood samples from 15 patients with lupus nephritis and 14 patients with non-nephritis manifestations of SLE (samples represented each patient during flaring and quiescent disease states) and from 5 healthy donors. To relate gene expression to activity of nephritis, 216 longitudinal samples from 30 patients with lupus nephritis covering a median time frame of 28 months were analyzed using the Illumina HT-V4 Bead array. Serum albumin levels were documented at the time of each visit. Results Principal component analysis of RNA sequencing data clearly differentiated SLE patients with nephritis from those without nephritis, and linear models for microarray (limma) analysis identified 153 gene transcripts differentially expressed between the two patient groups (fold change >1.5; p Conclusions Spliceosome-associated RNAs and TREML4, a TLR7-associated gene product, may represent biomarkers of lupus nephritis, and PTTG1, the product of a lupus-associated gene reported to be involved in epithelial-mesenchymal transition, may be a novel therapeutic target associated with active nephritis. These studies provide a rich data set stimulating new understanding of mechanisms contributing to lupus nephritis. Acknowledgements This work was supported by the Lupus Research Alliance, Pfizer-Centers for Therapeutic Innovation, and the Emerald Foundation.

A comprehensive bioinformatics analysis was conducted to investigate potential new diagnostic biomarkers and immune infiltration characteristics associated with tubulointerstitial injury in lupus nephritis (LN), and to examine possible correlations between key genes and infiltrating immune cells. The GSE32591, GSE113342, and GSE200306 datasets were downloaded from the Gene Expression Omnibus database and differentially expressed genes (DEGs) were identified in the pooled dataset. Support vector machine-recursive feature elimination analysis and the least absolute shrinkage and selection operator regression model were used to screen for possible markers, and the compositional patterns of the 22 types of immune cell fractions in LN were determined using CIBERSORT. Finally, Western blotting, quantitative real-time polymerase chain reaction, and multiple immunofluorescence methods were used to confirm the significance of these feature genes in MRL/lpr mice and patients with LN. Seventeen DEGs were identified, of which 11 were considerably upregulated and six were markedly downregulated. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment in pertussis, complement and coagulation cascades, systemic lupus erythematosus, and other pathways. Based on the machine learning results, we identified IFI16, EGR1 and MX1 were key diagnostic genes for tubulointerstitial injury associated with LN. Immune cell infiltration analysis revealed that IFI16, EGR1 and MX1 were associated with M1 macrophages. Finally, the association between IFI16, EGR1, MX1 and macrophages in MRL/lpr mice and patients with LN were verified. This study suggests that IFI16, EGR1 and MX1 which are highly expressed in renal tubular epithelial cells in LN and are associated with macrophage infiltration, may be a novel diagnostic and therapeutic target.

Lupus nephritis (LN) is the renal manifestation of the autoimmune disease systemic lupus erythematosus (SLE). LN is characterized by a dysregulated immune system, the presence of autoantibodies, and renal immune complex deposits, which collectively injure the kidney. However, novel nonimmune pathogenic mechanisms of human LN are continuously uncovered, presenting new challenges as well as opportunities for intervention. Iron accumulation and ferroptosis in the glomerular structure and renal tubules are relatively newly identified pathological features in LN. Ferroptosis is an iron-dependent nonapoptotic form of regulated cell death. Unlike generic oxidative stress mechanisms, ferroptosis occurs when the cellular antioxidative mechanism cannot suppress the oxidation of the cell membrane eventually leading to cell membrane rupture. Since iron absorption and recycling occur in the renal tubules, the renal tissue is particularly susceptible to ferroptosis. Ferroptosis inhibitors that reduce toxic phospholipid hydroperoxides to their corresponding nontoxic alcohols, or trap radicals in phospholipid bilayers, have improved disease outcomes in murine models of SLE/LN. In this review, we discuss mechanisms by which iron accumulation and ferroptosis perpetuate pathology in LN. These studies suggest that ferroptosis is very likely integral to parenchymal cell dysfunction in LN and a novel therapeutic target. The goal of this review is to introduce the fundamentals of iron biology and ferroptosis to clinicians and basic scientists and spur research to identify intracellular proferroptotic enzymes and their protein conjugates as potential targets to improve LN.

T follicular (TFH) and peripheral helper (TPH) cells have been increasingly recognized as a pathogenic subset of CD4 T cells in systemic lupus erythematosus (SLE). The SLAM Associated Protein (SAP) regulates TFH and TPH function by binding to the co-stimulatory signaling lymphocyte activation molecule family (SLAMF) receptors that mediate T cell - B cell interactions. SAP and SLAMF are critical for TPH-dependent B cell maturation into autoantibody-producing plasma cells that characterize SLE pathogenesis. We hypothesized that SAP-expressing TPH cells are involved in the pathogenesis of lupus nephritis (LN). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient separation from whole blood. Cells were stained for cell surface markers, followed by permeabilization and staining of intracellular SAP for spectral flow cytometry analysis. We also analyzed SAP expression from renal infiltrating LN T cells using the available single-cell RNA sequencing (scRNA seq) Accelerated Medicines Partnership (AMP) SLE dataset. PBMC from 30 patients with SLE (34 ± 10 years old, 83% female), including 10 patients with LN, were analyzed. We found an increase in total SAP-positive CD4 and CD8 T cells in SLE compared with controls (55.5 ± 2.6 vs. 41.3 ± 3.4, p=0.007, and 52.5 ± 3.0 vs. 39.2 ± 2.8, p=0.007 respectively). In CD4 T cells, the highest SAP expression was in the TPH subset. The frequency of SAP+TPH in circulation correlated with disease activity; SLE patients with renal disease had higher levels of circulating SAP+TPH that remained significant after adjusting for age, sex, race, low complements, and elevated anti-dsDNA (p=0.014). scRNA-seq data of renal infiltrating T cells in LN identified SAP expression to localize to the TFH-like CD4 cluster and GZMK+ CD8 cluster. Increased SAP expression in LN was associated with the differential expression of SLAMF3 and SLAMF7 and granzyme K and EOMES. The existence of two predominant SAP-expressing subsets, the TFH-like CD4 T cells, and GZMK+ effector CD8 T cells, was verified using scRNA-seq data from a human transcriptomic atlas of fifteen major organs. The expansion of SAP-expressing T helper cells was associated with LN in our cohort and verified using scRNA-seq data of renal infiltrating T cells. Improved SLAM and SAP signaling understanding can identify new therapeutic targets in LN.

Lupus nephritis (LN) is still a great burden for patients with systemic lupus erythematosus, and also one of the most severe complications of SLE. Radix Paeoniae Alba (white peony, WP) is proved with potential efficacy in treating LN. This study was to explore the effective ingredients, potential targets, and pathways of WP in treating LN based on network pharmacology and molecular docking. The active ingredients and potential protein targets of WP were gathered on Traditional Chinese Medicine Systematic Pharmacology Database and predicted by Swiss Target Prediction. LN-related therapeutic targets were acquired from multiple databases including Genecards, DisGeNET, OMIM, Drugbank, and PharmGKB. The intersection targets of WP and LN were acquired through Veeny 2.1.0. Protein-Protein Interaction (PPI) network was established by STRING. The results were then visualized by Cytoscape version 3.7.1. to study the mechanisms of WP on LN, gene ontology and functional enrichment analysis were carried out. Finally, molecular docking presented with the binding ability of key targets and major active components. We acquired a total of 13 active ingredients and 260 potential targets of WP. Among them, the intersection with targets of LN were 82 proteins. These targets were regarded as potential therapeutic targets. Through PPI network, we found that the top three proteins were RAC-alpha serine/threonine protein kinase (AKT1), vascular endothelial growth factor A (VEGFA), and transcription factor Jun (JUN), and their corresponding components were kaempferol, paeoniflorin, lactiflorin, paeoniflorgenone, etc. The results of enrichment analysis suggested that WP treatment for LN mainly involves in signaling pathways in cancer, lipid and atherosclerosis, advanced glycation end product (AGE)-receptor of AGE (RAGE) pathways, C-type lectin receptor and nuclear factor (NF)-kappa B signaling pathways. Molecular docking predicted that the above components have excellent affinity to AKT1, VEGFA, and JUN. This study gave an insight into the key target proteins and potential underlying pharmacological mechanism of WP in treating LN, which provided evidence for further researches on the mechanism of WP on LN.

Long noncoding RNAs (lncRNAs) are persistently expressed and have been described as potential biomarkers and therapeutic targets in various diseases. However, there is limited information regarding lncRNA expression in the tissue of kidney exhibiting lupus nephritis (LN)a serious complication of systemic lupus erythematosus (SLE). In this study, RNA sequencing (RNA-seq) was performed to characterize the lncRNA and mRNA expression in kidney tissues from LN (MRL/lpr) and control mice. We identified 12,979 novel lncRNAs in mouse. The expression profiles of both mRNAs and lncRNAs were differed significantly between LN and control mice. In particular, there were more upregulated lncRNAs and mRNAs than downregulated ones in the kidney tissues of LN mice. However, GO analysis showed that more downregulated genes were enriched in immune and inflammatory response-associated pathways. KEGG analysis showed that both downregulated and upregulated genes were enriched in a number of pathways, including the SLE pathway, and approximately half of these SLE-associated genes encoded inflammatory factors. Moreover, we observed that 2,181 DElncRNAs may have targeted and regulated the expression of 778 mRNAs in LN kidney tissues. The results of this study showed that 11 DElncRNAs targeted and were co-expressed with six immune and SLE-associated genes. qPCR analysis confirmed that lncRNA Gm20513 positively regulated the expression of the SLE-associated gene H2-Aa. In conclusion, the results of our study demonstrates that lncRNAs influence the progression of LN and provide some cues for further study of lncRNAs in LN. These results regarding the lncRNA-mRNAregulatory network may have important value in LN diagnosis and therapy.

Leukocytes and the kidney contribute to interstitial inflammation in lupus nephritis