Faculty Bibliography

Background/Purpose: Fractures in patients with systemic lupus erythematosus (SLE) are more common than age and sex matched controls. Fracture risk is traditionally assessed by dual-energy X-ray absorptiometry (DEXA) measured BMD and refined using the Fracture Risk Assessment Tool (FRAX). Several studies have demonstrated fractures in SLE patients despite normal DEXA BMD. We hypothesize that changes in bone microarchitecture may explain fracture vulnerability in SLE. This study was initiated to characterize bone quality by evaluating measures of microarchitecture at the proximal femur via 3T MRI in patients with SLE and compare these measurements with a control group of patients with known low bone density (LBD) and osteoporosis (OP).
Method(s): 50 SLE patients and 177 controls with known LBD or OP underwent DEXA and 3T MRI of the non-dominant hip. DEXA measured BMD of the total hip, femoral neck, and spine. LBD was defined as Z score <=-2.0 in premenopausal women and men younger than fifty, and T score <=-1.0 in others. OP was defined as the presence of LBD and history of fragility fracture in premenopausal women and men younger than fifty, and T score <=-2.5 in others. MRI measured favorable microarchitectural characteristics trabecular plate width (PW), trabecular plate-to-rod ratio (PRR), plate volume fraction (PVF), trabecular bone thickness (Tb.Th), and trabecular network area (NA), as well as unfavorable characteristics rod volume fraction (RVF) and trabecular spacing (Tb.Sp). Demographics, medication use and inflammatory markers at the time of the study visit were recorded. Statistical analysis was performed using Pearson correlations, t-scores, and multivariable linear regressions as appropriate.
Result(s): 50 SLE patients and 177 patients with LBD or OP completed all imaging studies. The SLE cohort was younger, and a higher percent of black patients compared to controls (Table 1). SLE patients had lower MRI PW and PRR and higher Tb. Sp as compared to controls, while having higher DEXA BMDs at all sites after adjustment for confounders (Figure 1). SLE patients had an inverse relationship between ESR and PW, PRR, Tb.Th, and NA (Figure 2, A-D). Similar results were found with CRP, which had an inverse relationship with PW, PRR, and NA. Body mass index (BMI) in SLE patients had an inverse relationship between PW, PVF, Tb.Th, and NA (Figure 2, E-H), and a positive relationship between all measured BMDs. In the control group, similar relationships were found between BMI and BMD, but only Tb.Th was inversely associated with BMI. Age, current steroid use, and history of lupus nephritis were not associated with MRI measures of bone microarchitecture.
Conclusion(s): Compared to controls, SLE patients had decreased bone quality as measured by MRI bone microarchitecture, despite having higher DEXA BMD. Elevated inflammatory markers inversely associated with bone quality. Elevated BMI, despite its association with higher BMD, was also associated with lower measures of bone microarchitectural strength, unlike controls. Further connection of bone microarchitecture to fracture risk and change over time in patients with SLE are needed to determine clinical significance of these findings and to guide additional monitoring and potential treatments. A-C: multivariate linear regression adjusting for significant microarchitectural confounders of BMI and gender. D-F: multivariate linear regression adjusting for significant BMD confounders of age, BMI, race, and gender

Background/Purpose: SLE is characterized by autoantibody production. The most common lupus-specific serology is the anti-dsDNA antibody (anti-DNA). Traditionally, anti-DNA is measured by an enzyme immunoassay or equivalent such as multiplex flow immunoassay (EIA), which is considered sensitive. In contrast, the crithidia luciliae immunofluorescence test (CLIFT) is considered more specific. Serial measurement of anti-DNA is often used to monitor lupus disease activity. With NYU's extensive multi-race/ethnic lupus registry, we studied the relationship between these two methods, their association with lupus nephritis (LN), and their ability to predict subsequent flares.
Method(s): Using the NYU Lupus Registry of patients who meet ACR, SLICC, or EULAR criteria, we identified patients who had one or more simultaneous anti-DNA results by multiplex EIA and CLIFT. We report on their concordance (e.g., always, never, or fluctuating), association with LN, and ability to predict flare within 90 days using the SELENA-SLEDAI Flare Index. To account for degree of positivity, we defined tertiles for EIA and CLIFT as low positive [11-50 and 1:10-1:40], mild positive [51-200 and 1:80-1:320], and high positive [> 200 and > 1:640]. Table 1. 586 total visits with paired EIA and CLIFT. H = high positive M = mild positive L = low positive defined in Methods section Table 2. Relationship between EIA, CLIFT and hypocomplementemia with lupus nephritis. Chi-square test comparing significance between 60/100 v 72/100 (EIA/CLIFT) p = 0.07 Table 3. Relationship between EIA, CLIFT, hypocomplementemia and flare within 90 days Results: 207 patients had one or more paired anti-DNA results generating 586 paired results (Table 1). Cohort demographics: 92% Female, 22% Hispanic ethnicity, 24% Black, 16% Asian, 49% White, 10% Other. Overall, 377 pairs were always concordant, and 209 were never concordant. 236 pairs demonstrated titer concordance and 350 with titer discordance. Of the 207 patients, 64 patients had only one and 143 patients had two or more paired tests. Of the 64 patients, 46 were always concordant: 18 had positive EIA/CLIFT and 28 had negative EIA/CLIFT. The remaining 18 patients were never concordant: 8 had +EIA/-CLIFT and 10 had-EIA/+CLIFT. The concordance of the 143 patients with multiple paired results: 73 always, 23 never, and 47 fluctuating. Whether by one or multiple paired tests, 41/207 patients were never concordant. 100 of the 207 patients had LN associated with +EIA in 60 and +CLIFT in 72 (Table 2). Hypocomplementemia was present in 88% of +EIA and 89% of +CLIFT patients with LN. 51 visits in 42 patients had paired anti-DNA results and a SELENA-SLEDAI Flare Index assessment within 90 days. 7 patients had mild flares with +EIA in 4 and +CLIFT in 3. 4 patients had severe flares with +EIA and +CLIFT in 3 (Table 3). Low C3 and or C4 occurred in 1 of 7 (14%) mild flares and in 4 of 4 (100%) severe flares.
Conclusion(s): Our data demonstrate that discordance of positivity between two assays for anti-DNA occurred in 41/207 (20%) patients, in 207/586 (36%) visits and in 350/586 (60%) visits magnitude of positivity nonconcordant. EIA positivity is associated with LN less often than CLIFT positivity. Flares were infrequent and associated with either EIA or CLIFT positivity, with severe flares more likely if accompanied by hypocomplementemia. We recommend the utility of more than one anti-DNA assay in routine monitoring for lupus disease activity

Background/Purpose: A decline of urine protein-to-creatinine ratio (UPCR) to < 0.5 is associated with better long-term preservation of kidney function in lupus nephritis (LN). UPCR < 0.5 defines complete response in guidelines and clinical trials when achieved after 1 or 2 years. Biomarkers of early response are needed to guide early treatment changes. We studied longitudinal urine proteomic profiles in LN to identify early predictors of proteinuric response.
Method(s): We quantified 1200 biomarkers (Kiloplex, RayBiotech) in urine samples collected on the day of (73%) or within 3 weeks (27%) of kidney biopsy and week 12, 24, or 52 in LN patients (ISN class III, IV, V, or mixed) with proteinuria > 1 g/d. Response was defined at one year from renal biopsy: Complete = UPCR < 0.5, serum creatinine (sCr) < 125% of baseline, prednisone <= 10mg/d; Partial = UPCR < 50% from baseline but >0.5, sCr < 125% of baseline, but prednisone allowed to 15 mg/d; Non responder = not meeting previous definitions.
Result(s): A total of 127 patients were included: 48 (38%) with pure proliferative LN (class III or IV), 41 (32%) with mixed LN (III or IV +/-V), and 38% (30%) with pure membranous LN. Response was complete in 34 (27%), partial in 29 (23%), and none in 64 (50%). There were no urinary biomarkers at baseline that predicted response. We then analyzed the changes in urinary proteins at 3 months compared to baseline. Patients who responded at 1 year showed an early decline in 51 urinary proteins led by CD163, IL-16, and macrophage mannose receptor (MMR, CD206) (Figure 1A) which matched the proteomic signature associated with histological activity (Figure 1B). No changes were observed in nonresponders. The decline of several urinary biomarkers at 3 months outperformed a decline in UPCR (clinical standard) in predicting the 1 year response (-Figure 2A). In particular, a decline of CD163 predicted 1 year response in ROC analysis with an area under the curve (AUC) of 83% compared to an AUC of 75% for UPCR decline (Figure 2B). In proliferative LN, urinary biomarkers displayed superior performance with an AUC of 91%, 86%, and 78% for the decline of MMR, CD163, and UPCR, respectively (-Figure 2C-D). Pathway enrichment analysis identified leukocyte activation, neutrophil degranulation, and matrix degradation as the main pathways reduced at 3 months in responders.
Conclusion(s): An early decline in urinary biomarkers of histological activity is associated with proteinuric response at 1 year. These findings indicate that effective immunosuppression induces by three months an immunological response in the kidney that can be noninvasively monitored in the urine. Biomarkers of immunological response outperformed early decline of UPCR, the standard of care, in predicting 1-year proteinuric response, especially in proliferative LN. Because biomarkers of immunological response parallel intrarenal activity, they could detect early treatment response/failure and allow early treatment changes. They could serve as surrogate endpoints in clinical trials. Longitudinal studies are needed to confirm that this immunological response is a better predictor of long-term kidney function preservation than proteinuric responses. Figure 1. Proteins linked to intrarenal activity (NIH activity index) decrease in treatment responders. Volcano plot of the changes of the urinary proteomic profile of treatment responders at 3 months (A) after kidney biopsy/treatment compared to baseline at time of biopsy. (B) Volcano plot displaying the Spearman's correlation coefficients of urinary biomarkers at time of biopsy with histological activity defined as the NIH activity index. Blue arrows indicate 4 examples of shared urinary biomarkers that indicate both intrarenal activity and response to treatment. FDR, false discovery rate; q, adjusted p value

Background/Purpose: Lupus nephritis (LN) is characterized by considerable variability in its clinical manifestations and histopathological findings. Understanding the cellular and molecular mechanisms underlying this heterogeneity is key for the development of personalized treatments for LN.
Method(s): Droplet-based single-cell RNA-sequencing was applied to the analysis of dissociated kidney samples, collected from 155 LN patients with active kidney disease and 30 living donor controls as part of a large-scale, multi-center study. 73,440 immune cells passing quality control were identified, spanning 134 cell subsets, representing various populations of tissue-resident and infiltrating leukocytes, as well as the activation states these cells assume as part of their diseaserelated activation and differentiation (Fig. 1). Principal component analysis (PCA) was used to characterize the variability in cell subset frequencies across the LN patients. Relationships between the resulting principal components (PCs) and the demographic, clinical and histopathological features of the patients were then assessed. Figure 1. Single-cell RNA-sequencing was used to profile immune cells isolated from the kidneys of LN patients and healthy controls. Five main lineages of cells were identified, as shown in a Uniform Manifold Approximation and Projection (UMAP) plot: myeloid cells, T/NK cells, B cells, plasma cells and dividing cells. The cells of each lineage were further split into finer subsets of cells (color-coded).
Result(s): The first PC (PC1), explaining 33% of the total variability in cell subset frequencies, reflected the balance between lymphocytes and monocytes/macrophages. The second PC (PC2), explaining an additional 21% of the total variability, represented the degree of macrophage differentiation to an alternatively activated phagocytic profile. The third and fourth PCs, bringing the total explained variability to 74%, were related to the balance between cell-mediated and humoral immune responses. PC1 was significantly correlated with the Chronicity index, such that patients with a higher percentage of lymphocytes compared to monocytes/macrophages had a higher Chronicity score (rho =-0.439, p-value < 0.001; Fig. 2A). A high degree of macrophage differentiation, as represented by PC2, was associated with a high Activity score (rho =-0.495, p-value < 0.001; Fig. 2B), and, in addition, with proliferative or mixed histology class, compared to pure membranous nephritis (p-value = 0.001, Kruskal-Wallis test). We further identified a significant correlation of these PCs with age; specifically, older patients had a higher relative frequency of lymphocytes compared to monocytes/macrophages, a lesser degree of macrophage differentiation, and a higher representation of cells putatively involved in a humoral immune response compared to a cell-mediated one.
Conclusion(s): These results identify distinct leukocyte populations active in different LN patients and, possibly, different stages of disease, and suggest potential therapeutic targets, that must be validated in mechanistic studies. This approach may pave the way to personalized treatment of LN

Background/Purpose: Patients with SLE may be at increased risk for developing a venous thromboembolism (VTE), particularly in the postpartum period. The Royal College of Obstetricians and Gynaecologists (RCOG) guideline for postpartum VTE prophylaxis is unique in its inclusion of "active" SLE as an actionable risk factor. In this guideline, a score >= 3 drives a formal recommendation for a 6-week prophylactic treatment course with enoxaparin. Although not defined, "active" SLE alone scores 3 points. The inclusion of SLE raises concerns regarding appropriate attribution and subsequent management decisions. The current study applied the RCOG model to a cohort of postpartum SLE patients to determine whether these patients a) qualify as having "active" SLE b) have other risk factors for VTE c) received the recommended prophylaxis and d) had a postpartum VTE.
Method(s): The retrospective study comprised 55 pregnancies in 49 patients fulfilling criteria for classification of SLE based on ACR, SLICC or EULAR/ACR definitions consecutively seen over the last 5 years. Disease activity at delivery was assessed by the SLEPDAI using SELENA and Hybrid SELENA definitions for scoring proteinuria. Remission was assigned by applying the DORIS (Definitions of Remission in SLE) criteria. Patients not in remission were considered to have "active" SLE, even if a low level with only one clinical domain scored. RCOG scoring was calculated for each patient prior to and after delivery.
Result(s): The median age was 32 years (IQR 29-36 years) and the median BMI was 26.6 kg/m2 (IQR 23.0-30.9 kg/m2), with 49.1% African-American, 16.4% Asian, 29.1% White, 5.5% Other and 32.7% of Hispanic ethnicity. The median SELENA and Hybrid SELENA SLEPDAI scores were 2.0 (IQR 0-6) and 2.0 (IQR 0-5) respectively. The components of the RCOG model with each of its elements scored for the cohort (Table 1). 34 pregnancies (61.8%) were in DORIS remission throughout pregnancy. 21 (38.2%) were not in DORIS remission at delivery and received 3 points on the RCOG model, since by not achieving remission their SLE could be considered at least mildly active. Of these pregnancies, only 19% were recommended for VTE prophylaxis despite RCOG score >= 3. Only 35.7% of pregnancies in DORIS remission, but with 3 points for non-SLE related VTE risk factors, were recommended for VTE prophylaxis (Table 2). Of the 20 pregnancies in remission with an RCOG score < 3 after assessing all risk factors, 15% were nevertheless recommended for VTE prophylaxis. In contrast, of the 14 inactive pregnancies with RCOG score >= 3 for non-SLE activity factors, only 35.7% were recommended for VTE prophylaxis. No patients had a postpartum VTE regardless of therapy.
Conclusion(s): These data reveal that even for SLE patients in remission at the time of delivery, points for SLE alone should not automatically be assigned on the RCOG model. However, those who are in remission may still warrant VTE prophylaxis if other non-SLE related risk factors are present. Although no patient had a postpartum VTE, prophylactic anticoagulation should be instituted only when clinically appropriate. The healthcare team should carefully consider disease activity before applying 3 points for the diagnosis of SLE

BACKGROUND:Complement activation has been associated with adverse pregnancy outcomes (APO) in SLE. Pregnant women with SLE were studied to evaluate whether complement dysregulation within the first two pregnancy trimesters predicts APO. METHODS:Pregnant women fulfilled classification criteria for SLE. APO included neonatal death, preterm delivery before 36 weeks and small for gestational age newborn. Pre-eclampsia was also evaluated. Erythrocyte complement receptor 1 (ECR1) and erythrocyte-bound C4d (EC4d) were measured by flow cytometry. Complement proteins C3 and C4 were measured by immunoturbidimetry and anti-double-stranded DNA by ELISA in serum. Statistical analysis consisted of t-test, confusion matrix-derived diagnostic analysis, and multivariate logistic regression. RESULTS:Fifty-one women had 57 pregnancies and 169 visits during the study. Baseline visits occurred mainly in the first (n=32) and second trimester (n=21). Fourteen (24.6%) pregnancies resulted in 21 APO with preterm delivery being the most common (n=10). ECR1 <5.5 net mean fluorescence intensity in the first trimester predicted APO with a diagnostic OR (DOR) of 18.33 (95% CI: 2.39 to 140.4; t-test p=0.04). Other individual biomarkers did not reach statistical significance. To estimate the likelihood of APO, we developed an algorithm that included the week of pregnancy, ECR1 and EC4d. From this algorithm, a Pregnancy Adversity Index (PAI) was calculated, and a PAI >0 indicated an elevated likelihood of pregnancy complications (DOR: 20.0 (95% CI: 3.64 to 109.97)). CONCLUSIONS:Low levels of ECR1 in early or mid-pregnancy are predictive of an APO. Incorporating the weeks of gestation and both ECR1 and EC4d generated a PAI, which further predicted serious pregnancy complications.

OBJECTIVES/OBJECTIVE:Current treatments are effective only in 30% of lupus nephritis patients emphasizing the need for novel therapeutic strategies. To develop mechanistic hypotheses and explore novel biomarkers, we analyzed the longitudinal urinary proteomic profiles in patients with lupus nephritis undergoing treatment. METHODS:We quantified 1,000 urinary proteins in 30 patients with lupus nephritis 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 biopsies from lupus nephritis patients. RESULTS:Our analysis revealed multiple biological pathways including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization that could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with lupus nephritis as compared to controls without SLE. IL-16, CD163, and TGF-β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction of 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 lupus nephritis kidneys. IL-16 producing cells were found at key sites of kidney injury. CONCLUSION/CONCLUSIONS:Urine proteomics may profoundly change the diagnosis and management of lupus nephritis by noninvasively monitor active intrarenal biological pathways. These findings implicate IL-16 in lupus nephritis pathogenesis designating it as a potentially treatable target and biomarker.