Faculty Bibliography

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

Asian Indians are at increased risk of developing cardiometabolic diseases. We sought to determine differences between Asian Indians and other races/ethnicities in hypertension and diabetes prevalence and associated annual blood pressure (BP) and fasting blood glucose (FBG) testing. A total of 257,652 adults >=18 years from the 2011-2018 U.S. National Health Interview Surveys (NHIS) were included. BP and FBG testing in the past 12 months was defined dichotomously (yes/not yes). Racial/ethnic groups included non-Hispanic White (NHW), non-Hispanic Black (NHB), Asian Indian, Other Asians, and Hispanic/Multiracial. We used logistic regression, adjusting for covariates and the survey design. Analyses were completed from 08/2020-06/2021. Asian Indians (N = 3049) had 21% and 99% higher odds of hypertension and diabetes, respectively, than NHWs (aOR [95% CI]; hypertension: 1.21[1.04,1.40], diabetes: 1.99[1.64,2.41]). Accordingly, Asian Indians without diabetes had significantly higher odds of FBG screening than NHWs (Asian Indian: 1.41[1.25,1.59], NHB: 0.99 [0.95,1.04], Other Asian: 1.07[0.98, 1.18], Hispanic: 1.13[1.07,1.20]). Asian Indians without hypertension had a 14% insignificant increase in BP testing compared to NHWs (1.14[0.97,1.33]). Predictors of testing in Asian Indians included older age, doctor's visit, graduate-level education, insurance coverage, and history of hypertension or diabetes. NHBs with diabetes and Hispanics with hypertension had lower odds of FBG testing (0.75[0.66,0.84]) and BP testing (0.85[0.79,0.92]), respectively, than NHWs. Asian Indians have higher odds of diabetes and hypertension than NHWs and higher, but relatively lower, odds of FBG and BP testing. Increasing routine BP and FBG testing in Asian Indians in younger adults may allow for earlier detection of high-risk individuals.
Copyright

Background/Purpose: Anti-Ro autoantibody production often precedes the development of Systemic Lupus Erythematosus (SLE) or Sjogren's syndrome (SS) by years. For anti-Ro+ mothers enrolled in the Research Registry for Neonatal Lupus (RRNL), progression to SS or SLE occurs in about a quarter, while most remain asymptomatic or develop only minor rheumatic symptoms (Asym/UAS). Thus, RRNL mothers uniquely offer a promise to identify genotype-phenotype relationships that are important to preclinical autoimmunity. Since multiple SLE risk alleles from Class II HLA genes are present in anti-Ro+ mothers, we examined interactions of specific microbiome taxa with Class II HLA by independent analytic paths with the goal to identify HLA-related microbiome associations in Anti-Ro+ Mothers of Children with Neonatal Lupus.
Method(s): Subjects included 125 RRNL mothers and 23 healthy controls. Stool microbiomes of anti-Ro+ women in RRNL (Asym/UAS, SS/SLE), and healthy controls (HC) were processed using 16S ribosomal RNA sequencing. Sera/ plasma were evaluated for cytokines and autoantibody levels. Alleles from HLA Class II genes were genotyped using NextGen sequencing of HLA region or imputed (HIBAG) from GWAS data. Independent analytic paths sought to explore associations of specific taxa and class II HLA included: 1) use of a cumulative logit model to test interactions between FDR significant genera and HLA alleles and 2) assignment of SLE, SS, UAS patients and HC to molecular phenotype clusters by Random Forest (RF), an unsupervised machine learning tool using Z-score transformed cytokine soluble mediators and autoantibody values with settings and the gap statistic that were used to estimate the optimal number of patients and HC within clusters. The overlapping distribution of SS/SLE, HLA alleles and taxa at clusters were then examined.
Result(s): Findings related to DRB1*15:01 and an interaction with genera of the Ruminococcaceae family were tested. Oscillibacter, with FDR-adjusted significance was shown to exhibit evidence of an interaction (P=0.033 (OR=0.60 (0.37-0.96)). In order to authenticate that SLE HLA risk alleles modify the strength of the association, we examined the molecular phenotype clusters from RF clustering. Radar plots were used to visualize the distribution HLA alleles and the enrichment of microbiome taxa within these clinically relevant phenotypic clusters (Figure 1). DRB1*1501 shows enrichment at cluster 4. Interestingly, the distribution of Oscillibacter, but not Coprococcus 3 was nearly superimposable with the Class II HLA allele with enrichment at cluster 4. However, the distribution of DRB1*1501 was not enriched at cluster profiles representing evaluations of DRB1*0301 and SS/SLE disease classification (Figure 2) demonstrating a limitation of DRB1*1501 to predict risk for transition from benign to pathologic autoimmunity in anti-Ro+ mothers of children with neonatal lupus.
Conclusion(s): These data support the use of molecular phenotypes that are linked to genetic-environmental interactions to identify HLA-related microbiome associations

Background/Purpose: Diagnosis of lupus nephritis (LN) relies on a kidney biopsy obtained in SLE with proteinuria. Delayed access to kidney biopsies may delay diagnosis and treatment, and can be limited by rapid access to biopsy, antithrombotic and anticoagulation treatments, thrombocytopenia, and in resource poor settings. Here, we employed urine proteomics to develop a non-invasive biomarker to predict proliferative LN.
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 in SLE patients with proteinuria >= 500mg/d and compared their abundance between patients with or without a subsequent biopsy with proliferative LN (ISN class III or IV +/- V). Prospective urine proteomic profiles were obtained in patients with class III, IV, or V at baseline and week 12, 24, or 52.
Result(s): A total of 237 patients were included: 138 (58%) with proliferative LN, 57 (24%) pure membranous LN, 21 (9%) ISN class I or II LN, 9 (4%) ISN class VI, and 12 (5%) did not have LN. Forty urinary proteins were differentially abundant in patients with proliferative LN, topped by CD163 (Figure 1). Urinary CD163 (uCD163) was significantly elevated in proliferative LN compared to all other groups (Figure 2). Longitudinal analysis revealed that uCD163 selectively declined in patients that achieved renal response at 12 months (Figure 3).
Conclusion(s): Urinary CD163, a cleaved M2c macrophage receptor, can help to identify proliferative LN in SLE patients with proteinuria. Noninvasive monitoring of uCD163 may lead to early diagnosis and treatment of proliferative LN, thus reducing irreversible kidney damage

Background/Purpose: Lupus nephritis (LN) is a leading cause of morbidity and mortality in systemic lupus erythematosus (SLE) patients. While LN pathogenesis has yet to be fully elucidated, T cells have been strongly implicated in mechanisms of disease. CD6 is a co-stimulatory receptor on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presenting cells and epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation and trafficking and is central to immune-mediated inflammation. Previously, we reported that soluble urine ALCAM is a potential biomarker of disease in LN. Here, we evaluated the correlation of serum and urine ALCAM and CD6 with disease activity over time.
Method(s): Patient samples were acquired through the Accelerating Medicines Partnership (AMP), a public-private partnership to accelerate development of therapeutics for diseases such as LN. Serum and urine samples were obtained from patients with biopsy proven LN (n=345) and living kidney donor controls (n=68). Follow-up longitudinal sampling (3, 6, and 12 months) was available for 143 LN patients. ALCAM levels were quantified by ELISA, while CD6 levels were quantified by an electrochemiluminescent assay. Levels were analyzed cross-sectionally (first visit) and longitudinally against disease measures that included proteinuria, SLEDAI, the renal components of the SLEDAI score (R-SLEDAI), ISN-RPS histological class of the lesion, serological parameters, and patient characteristics.
Result(s): Consistent with our previous findings, cross-sectional analysis showed that urinary ALCAM was significantly elevated in LN patients (mean 4333.5 pg/mL, 95% CI [3614.0, 5053.0]) compared to control subjects (mean 214.4 pg/ mL, 95% CI [152.9, 276.0]) (p< 0.001), but that there were no differences in serum ALCAM levels. Urinary ALCAM levels significantly correlated with SLEDAI and R-SLEDAI scores (but did not correlate to the non-renal portion of SLEDAI (SLEDAI -R-SLEDAI) (Figure 1), suggesting that ALCAM level is associated with the renal activity. This was supported by near-significant correlations with C3 (p=0.07) and C4 levels (p=0.05). Serum and urine levels of CD6 were similar between cases and control subjects and did not change with disease activity, suggesting that the differences observed in urinary ALCAM levels are not due to hemodynamic changes or non-specific loss of glomerular permeability. In patients followed with longitudinal sampling, urinary ALCAM reflected changes in SLEDAI and R-SLEDAI. Furthermore, in preliminary analysis of a subset of patients who exhibited significant changes in R-SLEDAI across visits, intra-patient comparison of the respective timepoints reflected concomitant significant changes in urinary ALCAM levels (Figure 2).
Conclusion(s): Here, we expand upon previous studies and provide additional support in a large multi-center cohort, by showing that urinary ALCAM levels are elevated in SLE patients with active LN and decline with clinical improvement. Studies in progress are evaluating the implications of these findings in predicting therapeutic responses in LN, as well as longer term disease outcomes and prognosis

Background/Purpose: The Accelerating Medicines Partnership (AMP) Lupus Network was established with the goal of applying novel technologies to the interrogation of blood and tissue samples from patients with lupus nephritis (LN). In contrast to global LN clinical trials, the AMP LN cohort affords an opportunity to generate outcome data representative of a US multicenter multi-ethnic real-world experience. In this analysis, the AMP clinical dataset was investigated to determine the percentages of patients who attained prespecified definitions of partial or complete responses at 52 weeks. In addition, incorporation of response rates at weeks 12 and 26 to the analysis provided longitudinal patterns of response to standard of care.
Method(s): Patients with LN who were undergoing kidney biopsies as part of standard of care were eligible to enroll in the AMP LN study. Response definitions were only applied to cases whose baseline spot urine protein/creatinine (UPCR) ratios were > 1.0. Complete response (CR) required: 1) UPCR < 0.5; and 2) normal creatinine (< 1.3 mg/dL) or, if abnormal at baseline, < 125% of baseline; and 3) prednisone < 10 mg/day at the time of the study visit. Partial response required: 1) >50% reduction in UPCR without meeting UPCR criterion for CR; and 2) normal creatinine (< 1.3 mg/dL) or, if abnormal, < 125% of baseline; and 3) prednisone dose < 15 mg/day at the time of the study visit. Patients who did not achieve a CR or PR at the specific timepoints were considered non-responders (NR). Only patients with renal biopsies that demonstrated ISN/RPS classes III, IV, V or combined III or IV with V and data available at all four timepoints (baseline, weeks 12, 26 and 52) were included in this analysis. Cross-sectional and longitudinal analyses of responses were performed, and heat maps were generated to graphically display response patterns.
Result(s): Data on 121 patients with LN enrolled in AMP were included in this analysis. Cross-sectional response rates at 52 weeks were: CR: 28.1%; PR: 23.1%; NR: 48.8% (Table 1). Response rates at weeks 12 and 26 are additionally displayed in Table 1, and Figure 1 is a heat map demonstrating longitudinal responses of our patients. All patients were considered NR at baseline. Only 7.4% of patients had week 12 CR responses sustained through week 52, whereas 19% had attained PR or CR at all 3 visits. An additional 14.9% achieved a PR or CR at 26 weeks which was sustained at 52 weeks. Overall, 36.4% of patients were NR at all time points.
Conclusion(s): Clinical data from the AMP Lupus Network revealed rates of 52-week CR and PR that were consistent with placebo response data from recently conducted LN trials. Low sustained CR rates not only underscore the need for more efficacious therapies but highlight how critically important it is to understand the molecular pathways that are associated with response and non-response. (Figure Presented)

Background/Purpose: The Accelerating Medicines Partnership (AMP) will use multi-omics modalities including single cell RNA sequencing to understand lupus nephritis with the ultimate goal to devise novel and personalized treatment strategies. African-Americans have more lupus nephritis and worse outcomes in terms of end stage renal disease. We report here the clinical findings to date on African-American patients in the AMP cohort.
Method(s): We included 118 patients with urine protein-to-creatinine ratio (UPCR) >= 1 and biopsy proven class III, IV, V or mixed lupus nephritis at time of enrollment. All patients met revised ACR or SLICC classification criteria. Clinical data were obtained at baseline, 12, 26, and 52 weeks after the renal biopsy. Response status at week 52 was defined as follows. Complete: UPCR <= 0.5, normal serum creatinine (sCr) or < 25% increase from baseline if abnormal, and prednisone < 10mg daily; partial: UPCR > 0.5 but <= 50% of the baseline value and same sCr and prednisone rules as complete response; no response: UPCR > 50% of baseline value or new abnormal elevation of sCr or >= 25% from baseline or prednisone >= 10mg daily.
Result(s): Table 1 shows that African Americans were more likely to have class V lupus nephritis (38% vs 22.5%, p=0.06), were less serologically active (low C3 50% vs 77.5%, p=0.002; anti-dsDNA 63% vs 79%, p=0.006), and were more likely to have elevated serum creatinine (55% vs 30%, p=0.03). Caucasians were older (47 vs 34 years, p=< 0.001) and more likely to be at their first biopsy (64% vs 31%, p=0.04). Table 2 shows the differences based on the first biopsy versus a repeat biopsy. African-Americans were significantly less likely to have a treatment response at the first biopsy. Regardless of first or later biopsy, they were less likely to have low C3. Table 3 shows multi-variate models. African-American patients at their first episode of lupus nephritis were less likely to respond to treatment (37.5% vs 75%, p=0.018) independently of histological features including class, activity and chronicity.
Conclusion(s): The AMP cohort demonstrates the current unmet clinical need to improve treatment of lupus nephritis in the United States. African-American lupus nephritis is different in terms of ISN class, serologies, first biopsy, and worse in terms of response status even after adjusting for activity and chronicity. Personalized treatments should be developed to improve outcomes in high risk populations such as African-Americans.Table 1. Patients characteristics by race/ethnicity. Data are presented as n (%) or mean (SD). Two patients identified as "Other" and are not shown in this Table. P values > 0.1 are indicated as ns

Background/Purpose: Lupus nephritis continues to be the complication with the highest standardized mortality ratio in Systemic Lupus Erythematosus (SLE), and a late diagnosis associates with worse outcomes. Clinicians traditionally rely on proteinuria to drive decisions regarding renal biopsy and subsequent management. Since threshold levels for such determinations are variable but critically important, this study leveraged the well-phenotyped multi-center multi- racial Accelerating Medicines Partnership (AMP) lupus nephritis cohort, to address whether urine protein to creatinine ratios (UPCR) between.5 and 1 differ from higher ratios with regard to clinical, serologic and histologic variables.
Method(s): 239 patients fulfilling ACR or SLICC criteria for SLE with a random or 24 hr uPCR > or =.5 and histologic biopsy Class III, IV, V, or mixed were consecutively enrolled in AMP at the time of renal biopsy and demographics, clinical history, medications, disease activity as assessed by the hybrid SELENA-SLEDAI were recorded. Patients with biopsy Classes I, II and VI were ineligible. Patients were followed at 3, 12, 26 and 52 weeks.
Result(s): At baseline, 38 patients had a UPCR < 1 (A), 113 had a UPCR 1-3 (B), and 88 had a UPCR > 3 (C). There were 14 additional patients with UPCR < 1, and 11 patients with UPCR > 1 who had biopsy class I or II. In group A, there were significantly more male patients (44% A; 23% B; 26% C, p=0.012) with no differences in age, race or ethnicity. Neither the SLEDAI nor serologic parameters (anti-dsDNA, C3, or C4) distinguished among the groups. Those in group C had a significantly increased creatinine and decreased hemoglobin and albumin compared to the other two groups (Table 1). Patients in group A trended toward having an increased frequency of proliferative histology (Table 2). This trend was not observed when considering patients for whom this was their first biopsy, but was significant for repeat biopsy patients (56% A; 41% B; 24% C, p=0.03). The activity index was independent of UPCR regardless of biopsy number. However, those in group C had a significantly higher chronicity index than those with lower UPCR. This correlation was shown for patients with a repeat biopsy (r=0.2299, p=0.003) but not first biopsy patients (r=0.0891, p=0.45). Although medications did not differ at baseline among the groups, at 12 weeks, for each group significantly more patients were taking Mycophenolate Mofetil than at the time of biopsy (Table 3).
Conclusion(s): A significant proportion of both first and recurrent biopsies in patients with a UPCR < 1 have proliferative histology and accompanying activity scores similar to that of patients with nephrotic range proteinuria. These results support renal biopsy at thresholds lower than a UPCR of 1 since histologic findings can inform therapeutic decisions

Background/Purpose: Poor therapeutic response rates contribute to the increased morbidity and mortality associated with lupus nephritis. Early identification of patients likely to respond is crucial as delays in treatment associate with worse outcomes. This study evaluated response using prospectively collected data obtained from the multi-ethnic/racial, multi-center Accelerating Medicines Partnership (AMP) lupus nephritis cohort. This cohort represents a real-world clinical setting using provider chosen standard of care and uniform collection of data.
Method(s): This study included SLE patients based on ACR or SLICC classification enrolled in AMP who met the following criteria: urine protein-creatine ratio (UPCR) > 1 at entry, and histologic biopsy Class III, IV, V, or mixed. Patients were followed at 3, 12, 26 and 52 wks with demographics, history, laboratory results, disease activity, and medica-tions recorded at each visit. Follow up data were available for 136 patients at 26 wks and 118 at 52 wks. Complete response was defined as a reduction in UPCR to <.5, a normal serum creatinine or no greater than 125% of baseline, and < 10 mg prednisone at time of response assessment. Patients were partial responders if UPCR decreased > 50% but remained >.5 and nonresponders if < 50% reduction in UPCR and/or did not meet the other response criteria.
Result(s): Medications were reported at 12 wks (Table 1). The complete response rate was 26% at both 26 and 52 wks. For patients undergoing a first biopsy, the rates were 37% and 40% and for those with repeat biopsies, the rates were lower at 21% and 19% respectively (p=0.042 at 26 wks; p=0.015 at 52 wks). The complete response at 26 wks was generally sustained with only 4 of 27 patients experiencing a relapse at 52 wks. At 26 wks, patients with membranous histology were less likely to be complete responders than patients with proliferative histology. This trend was observed regardless of biopsy number and persisted for response status at 52 wks. Although baseline activity score did not predict responder status, complete responders had a significantly lower chronicity index than nonresponders (mean + SD, 2.26 + 2.22 vs 3.83 + 2.57, p=0.016) at 26 wks with similar results at 52 wks. Responder status at 26 and 52 wks whether first or repeat biopsy was independent of extrarenal disease at entry (Table 2). Complete responder status was associated with positive anti-dsDNA serology at baseline for repeat biopsy patients. Complete responders had a greater change in C3, hemoglobin, lymphocyte count, albumin, and UPCR at 12 wks compared to baseline values than nonresponders (Table 3). Similar trends were observed when considering response status at 52 wks.
Conclusion(s): The low complete response rates reported in the AMP cohort are consistent with findings in blinded controlled trials of standard-of-care therapies and support the critical need for new therapeutics particularly in patients undergoing repeat biopsies and those with increased chronicity