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Objective Current lupus nephritis (LN) treatments are effective in only 30% of patients, emphasizing the need for novel therapeutic strategies. We undertook this study to develop mechanistic hypotheses and explore novel biomarkers by analyzing the longitudinal urinary proteomic profiles in LN patients undergoing treatment. Methods We quantified 1,000 urinary proteins in 30 patients with LN at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single‐cell transcriptomics of renal biopsy sections from LN patients. Results Our analysis revealed multiple biologic pathways, including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization, which could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with LN, as compared to controls without systemic lupus erythematosus. Interleukin‐16 (IL‐16), CD163, and transforming growth factor β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction in urinary IL‐16, a CD4 ligand with proinflammatory and chemotactic properties. Single‐cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in LN kidneys. IL‐16–producing cells were found at key sites of kidney injury. Conclusion Urine proteomics may profoundly change the diagnosis and management of LN by noninvasively monitoring active intrarenal biologic pathways. These findings implicate IL‐16 in LN pathogenesis, designating it as a potentially treatable target and biomarker.

Objective: Chronic periaortitis (CP) is a rare fibro-inflammatory disorder that incorporates idiopathic retroperitoneal fibrosis, inflammatory abdominal aortic aneurysms, and perianeurysmal retroperitoneal fibrosis. CP is included in the spectrum of IgG4-related disease. Since CP patients rarely undergo diagnostic biopsies, serum IgG4 levels are often used to classify CP as IgG4-related. However, the clinical and prognostic significance of serum IgG4 in CP is unknown. Methods: We measured serum IgG4 in active CP patients and compared the clinical characteristics, response to therapy and outcome of patients with high and normal levels. We also tested the diagnostic significance of IgG4 by comparing its levels in CP patients, healthy and disease controls (malignancies, Erdheim-Chester disease, large-, and small-vessel vasculitis). Results: We studied 113 consecutive patients with active CP. Twenty-four (21.2%) had high serum IgG4 (>135 mg/dL). The demographic, laboratory, and clinical characteristics of patients with high and normal IgG4 were similar, and so were the rates of ureteral obstruction and the disease characteristics on CT, MRI, and 18 F-FDG-PET. Patients with high IgG4 only had a higher frequency of extra-retroperitoneal fibro-inflammatory lesions ( p = 0.005). There were no significant differences in response to therapy and relapses between the two groups. Serum IgG4 levels did not discriminate CP from controls. Conclusions: Serum IgG4 levels are high in a minority of CP patients and do not identify specific clinical or prognostic subgroups; only a higher frequency of extra-retroperitoneal lesions is found in high-IgG4 patients. Serum IgG4 levels do not help in the differential diagnosis between CP and its mimics.

Conflict of interest: KCAC, RWKC, and YMDL hold equities in DRA, Take2, and Grail/Illumina; are consultants to Grail; and receive research funding from Grail/Cirina. YMDL is a scientific cofounder and a member of the scientific advisory board for Grail. YMDL, RWKC, and KCAC are cofounders and board members of DRA. PJ holds equity in Grail/Illumina and is a director of DRA and KingMed Future. STKS, PJ, KCAC, RWKC, and YMDL filed a patent application (U.S. Application No. 63/315,468) using the data generated in this study.

PURPOSE.To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. METHODS.Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. RESULTS.We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. CONCLUSIONS.Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.

Background:Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded (ds)DNA of various origins. Here, we investigate the ALR absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context.Methods:AIM2 autoantibodies were measured by immunoprecipitation in SLE and control subjects. Neutrophil extracellular traps were induced in control neutrophils and combined with purified ALR proteins in immunofluorescence and DNase protection assays. SLE renal tissues were examined for ALR-containing NETs by confocal microscopy.Results:AIM2 autoantibodies were detected in 41/131 (31.3%) SLE patients and 2/49 (4.1%) controls. Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16, and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We found that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling.Conclusions:Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE.Funding:These studies were funded by NIH R01 DE12354 (AR), P30 AR070254, R01 GM 129342 (JS), K23AR075898 (CM), K08AR077100 (BA), the Jerome L. Greene Foundation and the Rheumatology Research Foundation. Dr. Antiochos and Dr. Mecoli are Jerome L. Greene Scholars. The Hopkins Lupus Cohort is supported by NIH grant R01 AR069572. Confocal imaging performed at the Johns Hopkins Microscopy Facility was supported by NIH Grant S10 OD016374.

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