The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

A randomized, controlled double-blind study comparing the efficacy and safety of dose-ranging voclosporin with placebo in achieving remission in patients with active lupus nephritis.

Calcineurin inhibitors added to standard-of-care induction therapy for lupus nephritis (LN) may increase complete renal remission (CRR) rates. The AURA-LV study tested the novel calcineurin inhibitor voclosporin for efficacy and safety in active LN. AURA-LV was a Phase 2, multicenter, randomized, double-blind, placebo-controlled trial of two doses of voclosporin (23.7 mg or 39.5 mg, each twice daily) versus placebo in combination with mycophenolate mofetil (2 g/d) and rapidly tapered low-dose oral corticosteroids for induction of remission in LN. The primary endpoint was CRR at 24 weeks; the secondary endpoint was CRR at 48 weeks. Two hundred sixty-five subjects from 79 centers in 20 countries were recruited and randomized to treatment for 48 weeks. CRR at week 24 was achieved by 29 (32.6%) subjects in the low-dose voclosporin group, 24 (27.3%) subjects in the high-dose voclosporin group, and 17 (19.3%) subjects in the placebo group (OR=2.03 for low-dose voclosporin versus placebo). The significantly greater CRR rate in the low-dose voclosporin group persisted at 48 weeks, and CRRs were also significantly more common in the high-dose voclosporin group compared to placebo at 48 weeks. There were more serious adverse events in both voclosporin groups, and more deaths in the low-dose group compared to placebo and high-dose voclosporin groups (11.2%, 1.1%, and 2.3%, respectively). These results suggest that the addition of low-dose voclosporin to mycophenolate mofetil and corticosteroids for induction therapy of active LN results in a superior renal response compared to mycophenolate mofetil and corticosteroids alone, but higher rates of adverse events including death were observed.

A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.

Combination Therapy With Rituximab and Cyclophosphamide for Remission Induction in ANCA Vasculitis.

INTRODUCTION: Remission induction in antineutrophil cytoplasmic autoantibody (ANCA) vasculitis may be complicated by slow response to treatment and toxicity from glucocorticoids. We describe outcomes with a novel remission induction regimen combining rituximab with a short course of low-dose, oral cyclophosphamide and an accelerated prednisone taper. METHODS: Patients were included in this retrospective study if they had newly diagnosed or relapsing ANCA vasculitis with a Birmingham Vasculitis Activity Score for Wegener Granulomatosis (BVAS-WG) ≥3 and received a standardized remission induction regimen. The primary outcome was complete remission, defined as a BVAS-WG of 0 and a prednisone dose of ≤7.5 mg/d. RESULTS: We identified 129 patients who met the inclusion criteria, 31% of whom also received plasma exchange (PLEX) for rapidly progressive glomerulonephritis (RPGN) or diffuse alveolar hemorrhage. Seventy percent of patients had myeloperoxidase (MPO)-ANCA and 9% had relapsing disease. Median time to complete remission was 4 months (interquartile range [IQR] 3.9-4.4), and by 5 months 84% of patients were in complete remission. Prednisone was tapered to discontinuation as tolerated, such that the median prednisone dose at 8 months was 0 mg/d (IQR 0-2.5). In patients with RPGN, proteinase 3-ANCA was associated with a greater increase in eGFR at 6 months compared with MPO-ANCA (16 vs. 5.6 ml/min per 1.73m2; P = 0.028). During the year following remission, 1 major relapse occurred over 122 patient-years. Serious infections occurred more frequently in patients receiving PLEX and were associated with increasing age and diffuse alveolar hemorrhage. Four deaths occurred, 3 of which were associated with serious infections. CONCLUSION: Combination therapy was efficacious, allowed for rapid tapering of high-dose glucocorticoids and was well tolerated.

Pregnancy Outcomes in Patients with Glomerular Disease Attending a Single Academic Center in North Carolina.

BACKGROUND: Contemporary data regarding pregnancy outcomes in US patients with primary glomerular diseases are lacking. We aimed to report fetal and maternal outcomes among women with biopsy-proven primary glomerular disease who received obstetric care at a single large academic US center. METHODS: All women with a biopsy-confirmed primary glomerular disease diagnosis and without end-stage kidney disease who received obstetric care at the University of North Carolina (UNC) Hospitals (1996-2015) were identified using the Glomerular Disease Collaborative Network registry and the UNC Hospitals Perinatal Database. The primary study outcome was perinatal death (stillbirth at >20 weeks or neonatal death). Secondary outcomes included premature birth (<37 weeks), birth weight, preeclampsia, and kidney function changes (postpartum vs. baseline). Demographics, clinical characteristics, and outcomes were compared across glomerular disease subtypes. RESULTS: Among 48 pregnancies in 43 women (IgA nephropathy n = 17, focal segmental glomerulosclerosis [FSGS] n = 16, membranous nephropathy n = 6, minimal change disease n = 4), 13% of pregnancies resulted in perinatal death and 48% of babies were born prematurely. From a maternal perspective, 33% of pregnancies were complicated by preeclampsia, 39% by a doubling of urinary protein, and 27% by a ≥50% increase in serum creatinine. Outcome differences across glomerular disease subtypes were not statistically significant, although decline in kidney function appeared most frequent in FSGS. CONCLUSION: Adverse pregnancy outcomes are frequently observed in women with glomerular disease. The independent influence of glomerular disease subtype on outcomes requires further study. More widespread reporting and analysis of pregnancy outcomes in women with glomerular disease are urgently needed.

The Evolving Role of Rituximab in Adult Minimal Change Glomerulopathy.

BACKGROUND: Minimal-change glomerulopathy is defined histologically by the presence of normal glomeruli on light microscopy and diffuse podocyte effacement on electron microscopy. Although effective in children, corticosteroid treatment in adults is more variable and time to response can be prolonged. Data to support rituximab use in adults with corticosteroid-dependent or resistant minimal-change glomerulopathy are limited. Here, we describe the clinical course of adults with corticosteroid-dependent or -resistant minimal-change glomerulopathy who received rituximab. METHODS: Demographic and clinical data were collected and analyzed from all adult patients with native kidney, biopsy-proven, minimal-change glomerulopathy who were administered rituximab between 2009 and 2014 and cared for at the UNC Kidney Center. RESULTS: Ten patients with corticosteroid-resistant (n = 5) or corticosteroid-dependent (n = 5) idiopathic minimal-change glomerulopathy were treated with rituximab between 2009 and 2014. Rituximab treatment induced remission in all 10 patients with a median time to remission of 2 months. The median time from rituximab to corticosteroid discontinuation was 3.5 months. The median remission time was 29 months and follow-up time was 39.5 months. No serious adverse events attributable to rituximab were observed. CONCLUSION: Rituximab induced remission in all patients with corticosteroid-dependent or -resistant minimal-change glomerulopathy, and may hold great therapeutic potential with good efficacy and minimal toxicity. Mounting evidence implies that a well-conducted randomized controlled clinical trial using rituximab in adults with minimal-change glomerulopathy in both corticosteroid-resistant and corticosteroid-dependent patients is warranted.

Defining Glomerular Disease in Mechanistic Terms: Implementing an Integrative Biology Approach in Nephrology.

Advances in biomedical research allow for the capture of an unprecedented level of genetic, molecular, and clinical information from large patient cohorts, where the quest for precision medicine can be pursued. An overarching goal of precision medicine is to integrate the large-scale genetic and molecular data with deep phenotypic information to identify a new mechanistic disease classification. This classification can ideally be used to meet the clinical goal of the right medication for the right patient at the right time. Glomerular disease presents a formidable challenge for precision medicine. Patients present with similar signs and symptoms, which cross the current disease categories. The diseases are grouped by shared histopathologic features, but individual patients have dramatic variability in presentation, progression, and response to therapy, reflecting the underlying biologic heterogeneity within each glomerular disease category. Despite the clinical challenge, glomerular disease has several unique advantages to building multilayered datasets connecting genetic, molecular, and structural information needed to address the goals of precision medicine in this population. Kidney biopsy tissue, obtained during routine clinical care, provides a direct window into the molecular mechanisms active in the affected organ. In addition, urine is a biofluid ideally suited for repeated measurement from the diseased organ as a liquid biopsy with potential to reflect the dynamic state of renal tissue. In our review, current approaches for large-scale data generation and integration along the genotype-phenotype continuum in glomerular disease will be summarized. Several successful examples of this integrative biology approach within glomerular disease will be highlighted along with an outlook on how achieving a mechanistic disease classification could help to shape glomerular disease research and care in the future.

Clinical Features of Reported Ethylene Glycol Exposures in the United States.

BACKGROUND: Ethylene glycol is highly toxic and represents an important cause of poisonings worldwide. Toxicity can result in central nervous system dysfunction, cardiovascular compromise, elevated anion gap metabolic acidosis and acute kidney injury. Many states have passed laws requiring addition of the bittering agent, denatonium benzoate, to ethylene glycol solutions to reduce severity of exposures. The objectives of this study were to identify differences between unintentional and intentional exposures and to evaluate the utility of denatonium benzoate as a deterrent. METHODS AND FINDINGS: Using the National Poison Data System, we performed a retrospective analysis of reported cases of ethylene glycol exposures from January 2006 to December 2013. Outcome classification was summed for intentionality and used as a basis for comparison of effect groups. There were 45,097 cases of ethylene glycol exposures resulting in 154 deaths. Individuals more likely to experience major effects or death were older, male, and presented with more severe symptoms requiring higher levels of care. Latitude and season did not correlate with increased exposures; however, there were more exposures in rural areas. Denatonium benzoate use appeared to have no effect on exposure severity or number. CONCLUSION: Deaths due to ethylene glycol exposure were uncommon; however, there were major clinical effects and more exposures in rural areas. Addition of denatonium benzoate was not associated with a reduction in exposures. Alternative means to deter ingestion are needed. These findings suggest the need to consider replacing ethylene glycol with alternative and less toxic agents.

Comparison and evaluation of lupus nephritis response criteria in lupus activity indices and clinical trials.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with diverse manifestations. Although the approval of new therapies includes only one agent in 50 years, a number of promising new drugs are in development. Lupus nephritis is a dreaded complication of SLE as it is associated with significant morbidity and mortality. Advancing the treatment of lupus nephritis requires well-designed clinical trials and this can be challenging in SLE. The major obstacles involve identifying the correct population of patients to enroll and ensuring that a clinically appropriate and patient-centered endpoint is being measured. In this review, we will first discuss the clinical utility of endpoints chosen to represent lupus nephritis in global disease activity scales. Second, we will review completed and active trials focused on lupus nephritis and discuss the endpoints chosen. There are many important lessons to be learned from existing assessment tools and clinical trials. Reviewing these points will help ensure that future efforts will yield meaningful disease activity measures and well-designed clinical trials to advance our understanding of lupus management.

The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity.

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4(-/-) mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

Meta-analysis of human papillomavirus infection concordance.

BACKGROUND: Estimates of human papillomavirus (HPV) concordance among sexual partners are important for various public health activities, from counseling individual patients to predicting the effect of HPV vaccination. METHODS: We systematically searched the PubMed and EMBASE databases for studies of HPV concordance among heterosexual couples published through 2008 in English. Two coders independently abstracted data using standardized forms. We integrated concordance data using random-effects meta-analysis. RESULTS: Thirty studies (33 study populations) that met inclusion criteria reported concordance data for 2,972 couples. Most studies were cross-sectional cohort studies conducted in Europe or Asia that used DNA hybridization to test for HPV, sometimes in conjunction with PCR. Overall, 25.5% (95% confidence interval, 17.2-36.1%) of couples were infected with 1 or more of the same HPV types. Among couples with both members HPV-positive, 63.2% (95% confidence interval, 49.1-75.3%) were infected with 1 or more of the same viral types. Positive concordance was higher for female partners of men with HPV infections than for male partners of women with HPV infections. Positive concordance was also higher for studies using PCR and for the few studies that recruited men with HPV-related disease. CONCLUSIONS: Sexual partners of HPV-infected individuals had high rates of HPV infection, suggesting a need for increased attention to this group. IMPACT: Our refined estimates of HPV concordance can inform clinical encounters and public health planning. Future HPV concordance studies should use more rigorous research designs, characterize their participants in greater detail, and study more meaningful populations.

Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis.

Antineutrophil cytoplasmic autoantibody (ANCA) causes vascular injury that leads to small-vessel vasculitis. Patients with ANCA aberrantly express neutrophil granule-encoding genes, including 2 that encode autoantigens: proteinase 3 (PR3) and myeloperoxidase (MPO). To uncover a potential transcriptional regulatory mechanism for PR3 and MPO disrupted in patients with ANCA vasculitis, we examined the PR3 and MPO loci in neutrophils from ANCA patients and healthy control individuals for epigenetic modifications associated with gene silencing. We found that levels of the chromatin modification H3K27me3, which is associated with gene silencing, were depleted at PR3 and MPO loci in ANCA patients compared with healthy controls. Interestingly, in both patients and controls, DNA was unmethylated at a CpG island in PR3, whereas in healthy controls, DNA was methylated at a CpG island in MPO. Consistent with decreased levels of H3K27me3, JMJD3, the demethylase specific for H3K27me3, was preferentially expressed in ANCA patients versus healthy controls. In addition, we describe a mechanism for recruiting the H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) to PR3 and MPO loci mediated by RUNX3. RUNX3 message was decreased in patients compared with healthy controls, and may also be under epigenetic control. DNA methylation was increased at the RUNX3 promoter in ANCA patients. These data indicate that epigenetic modifications associated with gene silencing are perturbed at ANCA autoantigen-encoding genes, potentially contributing to inappropriate expression of PR3 and MPO in ANCA patients.

A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein.

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3(+his), EK-cleaved (his-tag removed) rPR3(- his), and EK-cleaved, denatured/refolded rPR3(- his/dr) (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3(- his/dr) than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.