Author Correction: Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways.

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Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

Total cortical interstitial inflammation predicts chronic kidney disease progression in patients with lupus nephritis.

BACKGROUND: End-stage kidney disease (ESKD) from lupus nephritis (LN) is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Kidney biopsy is the gold standard for diagnosis and prognostication of LN. While interstitial fibrosis and tubular atrophy (IFTA) predict progression to ESKD, the National Institutes of Health (NIH) classification of interstitial inflammation in unscarred cortical parenchyma is not predictive of chronic kidney disease (CKD) progression. The objective of this study was to determine whether total cortical interstitial inflammation that accounts for inflammation in the entire cortical parenchyma could predict CKD progression in patients with LN. Early identification of at-risk patients may improve outcomes. METHODS: This retrospective cohort study included 125 SLE patients with LN class III, IV, V or mixed (III/V, IV/V) on the index biopsy (2005-2018). Kidney biopsies were reviewed and assigned based on the 2018 NIH Activity Index (AI) and tubulointerstitial lesion categories. Total interstitial inflammation in the entire cortical parenchyma was graded as 0, 1, 2 or 3, corresponding to <10%, 10-25%, 26-50% and >50%, respectively, of the total cortical parenchyma containing an inflammatory infiltrate (similar to the definition used in the Banff total inflammation score). CKD progression was defined as an estimated glomerular filtration rate decrease of ≥30% within 5 years after the index biopsy. Kaplan-Meier survival curves and Cox proportional hazards models were performed to compare the two scoring systems, the total cortical intestinal inflammation score and the NIH interstitial inflammation score as predictors of CKD progression. RESULTS: Of 125 patients, 46 experienced CKD progression; 21 of 46 subsequently developed ESKD, 28 (22.4%) had moderate-severe total cortical interstitial inflammation and 8 (6.4%) had moderate-severe NIH interstitial inflammation. There were no differences in baseline characteristics between progressors and nonprogressors. Total cortical interstitial inflammation was associated with CKD progression in time-dependent analyses [hazard ratio 2.45 (95% confidence interval 1.2-4.97)] adjusted for age at biopsy, race, sex, LN class and hypertensive vascular change on kidney biopsy. The NIH interstitial inflammation was not associated with CKD progression. CONCLUSIONS: In contrast to the current NIH interstitial inflammation classification, accounting for interstitial inflammation in the entire cortical parenchyma allows identification of patients at risk for CKD progression in LN.

Acute kidney injury requiring kidney replacement therapy in childhood lupus nephritis: a cohort study of the Pediatric Nephrology Research Consortium and Childhood Arthritis and Rheumatology Research Alliance.

BACKGROUND: Acute kidney injury (AKI) is common in lupus nephritis (LN) and a risk factor for development of chronic kidney disease. In adults with LN, AKI severity correlates with the incidence of kidney failure and patient survival. Data on AKI outcomes in children with LN, particularly those requiring kidney replacement therapy (KRT), are limited. METHODS: A multicenter, retrospective cohort study was performed in children diagnosed between 2010 and 2019 with LN and AKI stage 3 treated with dialysis (AKI stage 3D). Descriptive statistics were used to characterize demographics, clinical data, and kidney biopsy findings; treatment data for LN were not included. Logistic regression was used to examine the association of these variables with kidney failure. RESULTS: Fifty-nine patients (mean age 14.3 years, 84.7% female) were identified. The most common KRT indications were fluid overload (86.4%) and elevated blood urea nitrogen/creatinine (74.6%). Mean follow-up duration was 3.9 ± 2.9 years. AKI recovery without progression to kidney failure occurred in 37.3% of patients. AKI recovery with later progression to kidney failure occurred in 25.4% of patients, and there was no kidney recovery from AKI in 35.6% of patients. Older age, severe (> 50%) tubular atrophy and interstitial fibrosis, and National Institutes of Health (NIH) chronicity index score > 4 on kidney biopsy were associated with kidney failure. CONCLUSIONS: Children with LN and AKI stage 3D have a high long-term risk of kidney failure. Severe tubular atrophy and interstitial fibrosis at the time of AKI, but not AKI duration, are predictive of kidney disease progression. A higher resolution version of the Graphical abstract is available as Supplementary information.

Pediatrician Adherence to Guidelines for Diagnosis and Management of High Blood Pressure.

OBJECTIVES: To assess pediatrician adherence to the 2017 American Academy of Pediatrics' clinical practice guideline for high blood pressure (BP). STUDY DESIGN: Pediatric primary care practices (n = 59) participating in a quality improvement collaborative submitted data for patients with high BP measured between November 2018 and January 2019. Baseline data included patient demographics, BP, body mass index (BMI), and actions taken. Logistic regression was used to test associations between patient BP level and BMI with provider adherence to guidelines (BP measurement, counseling, follow-up, evaluation). RESULTS: A total of 2677 patient charts were entered for analysis. Only 2% of patients had all BP measurement steps completed correctly, with fewer undergoing 3-limb and ambulatory BP measurement. Overall, 46% of patients received appropriate weight, nutrition, and lifestyle counseling. Follow-up for high BP was recommended or scheduled in 10% of encounters, and scheduled at the appropriate interval in 5%. For patients presenting with their third high BP measurement, 10% had an appropriate diagnosis documented, 2% had appropriate screening laboratory tests conducted, and none had a renal ultrasound performed. BMI was independently associated with increased odds of counseling, but higher BP was associated with lower odds of counseling. Higher BP was independently associated with an increased likelihood of documentation of hypertension. CONCLUSIONS: In this multisite study, adherence to the 2017 American Academy of Pediatrics' guideline for high BP was low. Given the long-term health implications of high BP in childhood, it is important to improve primary care provider recognition and management. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03783650.

Ambulatory Blood Pressure Patterns in Children, Adolescents, and Young Adults With Childhood-Onset Systemic Lupus Erythematosus and Normal Office Blood Pressure.

OBJECTIVE: Cardiovascular disease is a major cause of morbidity and mortality in systemic lupus erythematosus (SLE). Hypertension (HTN) and nondipping are modifiable risk factors for cardiovascular disease. Limited studies are available for childhood-onset SLE (cSLE). We aimed to assess ambulatory blood pressure monitoring (ABPM) pattern in cSLE patients with normal office blood pressure. METHODS: This cross-sectional single-center study enrolled 25 patients with cSLE, normal office blood pressure and normal renal function. We performed 24-hour ABPM and echocardiography to assess end-organ damage. Descriptive statistics were obtained, and comparison of variables using Fisher exact test, t test, and Mann-Whitney U test was performed to identify potential risk factors for nondipping. RESULTS: Of the 25 patients, 22 were women; the median age was 18 years (interquartile range, 16-20 years). Median SLE duration was 4.2 years (interquartile range, 2.9-8.1 years); 18 patients had a history of lupus nephritis (LN). Four patients, 3 of whom had LN, had masked HTN. Fourteen patients (56%) were nondipping. The majority of patients without LN (71%) had a nondipping profile. Echocardiography was done for 15 patients. Left ventricular mass index, relative wall thickness, and ejection fraction were normal in all patients. Ambulatory blood pressure monitoring results led to changes in therapy in 5 patients. CONCLUSION: Our data provide evidence of high prevalence of nondipping and masked HTN in patients with cSLE, even in patients without LN. Identifying ABPM abnormalities in these patients could potentially improve outcomes.

Principles of pediatric lupus nephritis in a prospective contemporary multi-center cohort.

Lupus nephritis (LN) is a life-threatening manifestation of systemic lupus erythematosus (SLE) and is more common in children than adults. The epidemiology and management of childhood-onset SLE (cSLE) have changed over time, prompting the need to reassess expected outcomes. The purpose of this study is to use the Childhood Arthritis and Rheumatology Research Alliance (CARRA) prospective registry to validate historical principles of LN in a contemporary, real-world cohort. After an extensive literature review, six principles of LN in cSLE were identified. The CARRA registry was queried to evaluate these principles in determining the rate of LN in cSLE, median time from cSLE diagnosis to LN, short-term renal outcomes, and frequency of rituximab as an induction therapy. Of the 677 cSLE patients in the CARRA registry, 32% had documented LN. Decline in kidney function was more common in Black cSLE patients than non-Black patients (p = 0.04). Black race was associated with worse short-term renal outcomes. In short-term follow up, most children with LN had unchanged or improved kidney function, and end stage kidney disease (ESKD) was rare. Ongoing follow-up of cSLE patients in the CARRA registry will be necessary to evaluate long-term outcomes to inform risk, management, and prognosis of LN in cSLE.

Systemic lupus Erythematosus activity and Hydroxychloroquine use before and after end-stage renal disease.

BACKGROUND: SLE manifestations after ESRD may be underdiagnosed and undertreated, contributing to increased morbidity and mortality. Whether specific symptoms persist after ESRD or a shift towards new manifestations occurs has not been extensively studied, especially in the non-Caucasian patients in the United States. In addition, hydroxychloroquine (HCQ) prescribing patterns post-ESRD have not been described. The objective of this study was to assess lupus activity and HCQ prescribing before and after ESRD development. Knowledge gained from this study may aid in the identification of SLE manifestations and improve medication management post-ESRD. METHODS: We performed a retrospective cohort study of SLE patients with incident ESRD between 2010 and 2017. SLE-related symptoms, serologic markers of disease activity, and medication use were collected from medical records before and after ESRD development. RESULTS: Fifty-nine patients were included in the study. Twenty-five (43%) patients had at least one clinical (non-renal) SLE manifestation documented within 12 months before ESRD. Of them, 11/25 (44%) continued to experience lupus symptoms post-ESRD; 9 patients without clinical or serological activity pre-ESRD developed new symptoms of active SLE. At the last documented visit post-ESRD, 42/59 (71%) patients had one or more clinical or serological markers of lupus activity; only 17/59 (29%) patients achieved clinical and serological remission. Thirty-three of 59 (56%) patients had an active HCQ prescription at the time of ESRD. Twenty-six of the 42 (62%) patients with active SLE manifestations post-ESRD were on HCQ. Patients who continued HCQ post-ESRD were more likely to be followed by a rheumatologist (26 [87%] vs 17 [61%], p = 0.024), had a higher frequency of documented arthritis (10 [32%] vs 1 [4%], p = 0.005), CNS manifestations (6 [20%] vs 1 [4%], p = 0.055), and concurrent immunosuppressive medication use (22 [71%] vs 12 [43%], p = 0.029). CONCLUSIONS: Lupus activity may persist after the development of ESRD. New onset arthritis, lupus-related rash, CNS manifestations, low complement and elevated anti-dsDNA may develop. HCQ may be underutilized in patients with evidence of active disease pre- and post ESRD. Careful clinical and serological monitoring for signs of active disease and frequent rheumatology follow up is advised in SLE patients both, pre and post-ESRD.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

International validation of a urinary biomarker panel for identification of active lupus nephritis in children.

BACKGROUND: Conventional markers of juvenile-onset systemic lupus erythematosus (JSLE) disease activity fail to adequately identify lupus nephritis (LN). While individual novel urine biomarkers are good at detecting LN flares, biomarker panels may improve diagnostic accuracy. The aim of this study was to assess the performance of a biomarker panel to identify active LN in two international JSLE cohorts. METHODS: Novel urinary biomarkers, namely vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1), lipocalin-like prostaglandin D synthase (LPGDS), transferrin (TF), ceruloplasmin, alpha-1-acid glycoprotein (AGP) and neutrophil gelatinase-associated lipocalin (NGAL), were quantified in a cross-sectional study that included participants of the UK JSLE Cohort Study (Cohort 1) and validated within the Einstein Lupus Cohort (Cohort 2). Binary logistic regression modelling and receiver operating characteristic curve analysis [area under the curve (AUC)] were used to identify and assess combinations of biomarkers for diagnostic accuracy. RESULTS: A total of 91 JSLE patients were recruited across both cohorts, of whom 31 (34 %) had active LN and 60 (66 %) had no LN. Urinary AGP, ceruloplasmin, VCAM-1, MCP-1 and LPGDS levels were significantly higher in those patients with active LN than in non-LN patients [all corrected p values (p c) < 0.05] across both cohorts. Urinary TF also differed between patient groups in Cohort 2 (p c = 0.001). Within Cohort 1, the optimal biomarker panel included AGP, ceruloplasmin, LPGDS and TF (AUC 0.920 for active LN identification). These results were validated in Cohort 2, with the same markers resulting in the optimal urine biomarker panel (AUC 0.991). CONCLUSION: In two international JSLE cohorts, urinary AGP, ceruloplasmin, LPGDS and TF demonstrate an 'excellent' ability for accurately identifying active LN in children.

Serum autoantibodies in pristane induced lupus are regulated by neutrophil gelatinase associated lipocalin.

The onset of autoantibodies in systemic autoimmunity can be the result of a breakdown in tolerance at multiple checkpoints. Genetic, hormonal, and immunological factors can combine with environmental influences to accelerate the onset of disease and aggravate disease outcome. Here, we describe a novel mechanism relating to the regulatory role of Neutrophil Gelatinase Associated Lipocalin (NGAL) in modulating the levels of autoantibodies in pristane induced lupus. Following a single injection of pristane intraperitoneally, NGAL expression was induced in both the serum and spleen. Furthermore, NGAL deficient mice were more susceptible to the induction of pristane stimulated autoimmunity, and displayed higher numbers of autoantibody secreting cells and increased expression of activation induced cytidine deaminase (AID) and other inflammatory mediators in the spleen. In contrast, kidney damage was milder in NGAL deficient mice, indicating that NGAL was detrimental in autoantibody mediated kidney disease. These studies indicate that NGAL plays differential roles in different tissues in the context of lupus, and suggest a previously unrecognized role for NGAL in adaptive immunity.

The pathogenesis, diagnosis and treatment of lupus nephritis.

PURPOSE OF REVIEW: Renal involvement is a major cause of morbidity and mortality in systemic lupus erythematosus. In this review, we provide an update on recent discoveries in the pathogenesis, diagnosis, and treatment of lupus nephritis. RECENT FINDINGS: Localized long-lived plasma cells have been identified as playing an important role in lupus nephritis. In addition, the roles of aberrant expression of microRNAs and proinflammatory cytokines have been explored. Early diagnosis is important for effective treatment and multiple biomarkers have been identified; however, none has been yet validated for clinical use. Biomarker panels may turn out to be more accurate than each individual component. Biologic agents for the treatment of lupus nephritis are being studied, including belimumab which was recently approved for nonrenal systemic lupus erythematosus. Rituximab has not proven itself in large, placebo-controlled trials, although it is still being used in refractory cases of lupus nephritis. SUMMARY: Lupus nephritis is a potentially devastating complication of systemic lupus erythematosus. Immune cells, cytokines, and epigenetic factors have all been recently implicated in lupus nephritis pathogenesis. These recent discoveries may enable a paradigm shift in the treatment of this complex disease, allowing the tailoring of treatment to target specific pathogenic mediators at specific points in time in the progression of disease.

Development of systemic lupus erythematosus in NZM 2328 mice in the absence of any single BAFF receptor.

OBJECTIVE: To determine the necessity for any individual BAFF receptor in the development of systemic lupus erythematosus (SLE). METHODS: Bcma-, Taci-, and Br3-null mutations were introgressed into NZM 2328 mice. NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice were evaluated for lymphocyte phenotype and BAFF receptor expression by flow cytometry; for B cell responsiveness to BAFF by in vitro culture; for serum levels of BAFF and total IgG and IgG anti-double-stranded DNA (anti-dsDNA) by enzyme-linked immunosorbent assay; for renal immunopathology by immunofluorescence and histopathology; and for clinical disease. RESULTS: BCMA, TACI, and B lymphocyte stimulator receptor 3 (BR3) were not surface-expressed in NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice, respectively. Transitional and follicular B cells from NZM.Br3-/- mice were much less responsive to BAFF than were the corresponding cells from wild-type, NZM.Bcma-/-, or NZM.Taci-/- mice. In comparison with wild-type mice, NZM.Bcma-/- and NZM.Taci-/- mice harbored an increased number of spleen B cells, T cells, and plasma cells, whereas serum levels of total IgG and IgG anti-dsDNA were similar to those in wild-type mice. Despite their paucity of B cells, NZM.Br3-/- mice had an increased number of T cells, and the numbers of plasma cells and levels of IgG anti-dsDNA were similar to those in wild-type mice. Serum levels of BAFF were increased in NZM.Taci-/- and NZM.Br3-/- mice but were decreased in NZM.Bcma-/- mice. Despite their phenotypic differences, NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice had renal immunopathology and clinical disease that were at least as severe as that in wild-type mice. CONCLUSION: Any single BAFF receptor, including BR3, is dispensable for the development of SLE in NZM mice. Development of disease in NZM.Br3-/- mice demonstrates that BAFF-BCMA and/or BAFF-TACI interactions contribute to SLE, and that a profound, life-long reduction in the numbers of B cells does not guarantee protection against SLE.

Efficacy and Safety of Pharmacokinetically-Driven Dosing of Mycophenolate Mofetil for the Treatment of Pediatric Proliferative Lupus Nephritis-A Double-Blind Placebo Controlled Clinical Trial (The Pediatric Lupus Nephritis Mycophenolate Mofetil Study).

Background: The safety and efficacy of mycophenolate mofetil (MMF) for lupus nephritis (LN) treatment is established in adults and in some children. MMF is rapidly converted to the biologically active metabolite mycophenolic acid (MPA) whose pharmacokinetics (PK) is characterized by large inter- and intra-individual variability. Methods/Design: This randomized, double-blind, active comparator, controlled clinical trial of pediatric subjects with proliferative LN compares pharmacokinetically-guided precision-dosing of MMF (MMFPK, i.e. the dose is adjusted to the target area under the concentration-time curve (AUC0-12h) of MPA ≥ 60-70 mg*h/L) and MMF dosed per body surface area (MMFBSA, i.e. MMF dosed 600 mg/m2 body surface area), with MMF dosage taken about 12 hours apart. At baseline, subjects are randomized 1:1 to receive blinded treatment with MMFPK or MMFBSA for up to 53 weeks. The primary outcome is partial clinical remission of LN (partial renal response, PRR) at week 26, and the major secondary outcome is complete renal response (CRR) at week 26. Subjects in the MMFBSA arm with PRR at week 26 will receive MMFPK from week 26 onwards, while subjects with CRR will continue MMFBSA or MMFPK treatment until week 53. Subjects who achieve PRR at week 26 are discontinued from study intervention. Discussion: The Pediatric Lupus Nephritis Mycophenolate Mofetil (PLUMM) study will provide a thorough evaluation of the PK of MMF in pediatric LN patients, yielding a head-to-head comparison of MMFBSA and MMFPK for both safety and efficacy. This study has the potential to change current treatment recommendations for pediatric LN, thereby significantly impacting childhood-onset SLE (cSLE) disease prognosis and current clinical practice.

Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice.

OBJECTIVE: The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways. METHODS: To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis. RESULTS: We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo. CONCLUSION: We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.