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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. Nephritis is a major cause of lupus morbidity. Putterman and colleagues use single-cell RNA sequencing on human renal and skin biopsies to describe the expression landscape associated with lupus nephritis.

In 2013, the Accreditation Council for Graduate Medical Education (ACGME) implemented Milestones 1.0 as a tool to assess trainee progress towards readiness for independent practice. Critiques of Milestones 1.0 suggested its complexity made the tool difficult to quickly understand and implement in a standardized fashion. This was particularly challenging among subspecialties due to inherent differences in clinical practice settings and make-up of procedural and patient care needs. In response, ACGME launched Milestones 2.0 in 2016 to harmonize competencies applicable to all subspecialties and develop new subspecialty specific competencies to facilitate precise feedback on subspecialty specific content domains. We describe how the Pediatric Nephrology Subspecialty Milestones were developed by a working group of pediatric nephrologists, fellows, and members of the ACGME. We highlight how this revision supports a growth-focused educational environment and equitable evaluation process. We describe how some institutions have used Milestones 2.0 to create just-in-time as well as summative feedback tools that quickly translate into individualized learning goals and guidance for programmatic improvements.

Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs) or bound and protected by extracellular proteins. Detection of cell- and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs) and repeated voids by the same individuals. Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs) and by sex, as well as a tendency of repeated (first and second void) samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen) were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs. miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important.

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.

IntroductionTreatment failures for lupus nephritis (LN) are high with 10%–30% of patients progressing to end-stage renal disease (ESRD) within 10 years. Interstitial fibrosis/tubular atrophy (IFTA) is a predictor of progression to ESRD. Prior studies suggest that tubulointerstitial injury secondary to proteinuria in LN is mediated by complement activation in the tubules, specifically through the membrane attack complex (MAC). This study aimed to investigate the associations between tubular MAC deposition with IFTA and proteinuria.MethodsIn this cross-sectional study, LN kidney biopsies were assessed for MAC deposition by staining for Complement C9, a component of the MAC. Chromogenic immunohistochemistry was performed on paraffin-embedded human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). Tubular C9 staining intensity was analysed as present versus absent. IFTA was defined as minimal (<10%), mild (10%–24%), moderate (25%–50%) and severe (>50%).ResultsRenal biopsies from 30 patients with LN were studied. There were 24 (80%) female sex, mean age (SD) was 33 (12) years old and 23 (77%) had pure/mixed proliferative LN. Tubular C9 staining was present in 7 (23%) biopsies. 27 patients had minimal-to-mild IFTA and 3 patients had moderate IFTA. Among the C9 + patients, 3 (43%) had moderate IFTA as compared with none in the C9- group, p=0.009. C9 + patients had higher median (IQR) proteinuria as compared with C9- patients: 6.2 g (3.3–13.1) vs 2.4 g (1.3–4.6), p=0.001 at the time of biopsy. There was no difference in estimated glomerular filtration rate (eGFR) between the C9 + and C9- groups.ConclusionThis study demonstrated that tubular MAC deposition is associated with higher degree of IFTA and proteinuria, which are predictors of progression to ESRD. These results suggest that tubular MAC deposition may be useful in classification of LN. Understanding the role of complement in tubulointerstitial injury will also identify new avenues for LN treatment.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Consensus treatment plans have been developed for induction therapy of newly diagnosed proliferative lupus nephritis (LN) in childhood-onset systemic lupus erythematosus. However, patients who do not respond to initial therapy, or who develop renal flare after remission, warrant escalation of treatment. Our objective was to assess current practices of pediatric nephrologists and rheumatologists in North America in treatment of refractory proliferative LN and flare. Methods Members of Childhood Arthritis and Rheumatology Research Alliance (CARRA) and the American Society for Pediatric Nephrology (ASPN) were surveyed in November 2015 to assess therapy choices (other than modifying steroid dosing) and level of agreement between rheumatologists and nephrologists for proliferative LN patients. Two cases were presented: (1) refractory disease after induction treatment with corticosteroid and cyclophosphamide (CYC) and (2) nephritis flare after initial response to treatment. Survey respondents chose treatments for three follow up scenarios for each case that varied by severity of presentation. Treatment options included CYC, mycophenolate mofetil (MMF), rituximab (RTX), and others, alone or in combination. Results Seventy-six respondents from ASPN and foty-one respondents from CARRA represented approximately 15 % of the eligible members from each organization. Treatment choices between nephrologists and rheumatologists were highly variable and received greater than 50 % agreement for an individual treatment choice in only the following 2 of 6 follow up scenarios: 59 % of nephrologists, but only 38 % of rheumatologists, chose increasing dose of MMF in the case of LN refractory to induction therapy with proteinuria, hematuria, and improved serum creatinine. In a follow up scenario showing severe renal flare after achieving remission with induction therapy, 58 % of rheumatologists chose CYC and RTX combination therapy, whereas the top choice for nephrologists (43 %) was CYC alone. Rheumatologists in comparison to nephrologists chose more therapy options that contained RTX in all follow up scenarios except one ( p  < 0.05). Conclusions Therapy choices for pediatric rheumatologists and nephrologists in the treatment of refractory LN or LN flare were highly variable with rheumatologists more often choosing rituximab. Further investigation is necessary to delineate the reasons behind this finding. This study highlights the importance of collaborative efforts in developing consensus treatment plans for pediatric LN.

Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined.

BackgroundCorticosteroids (CS) remain the mainstay of therapy for childhood-onset systemic lupus erythematosus (cSLE) although there are no widely accepted dosing strategies of oral (PO CS) or intravenous CS (IV CS). We aimed to (1) develop a standardized CS dosing regimen (SSR) and (2) achieve consensus for this SSR among pediatric rheumatology and nephrology providers treating cSLE complicated by lupus nephritis (LN).MethodsConsensus formation techniques were used. A Delphi questionnaire pertaining to CS use in cSLE was completed to inform formats of the Patient Profiles (PP, Step 1). Using data from 147 children with proliferative LN at 8 major cSLE treatment sites in North America PP were generated providing information about the course of LN and extra-renal cSLE (ER) at 2 subsequent visits (Step 2). PP were sent to 142 physicians (PP-raters) experienced in cSLE to adjudicate the course of ER and LN and propose the PO/IV CS dosages (Step 3). Using data from PP for which consensus was achieved, the SSR was developed (Step 4) and refined based on responses from another questionnaire and a focus group of experienced physicians (Step 5). The SSR was tested using a second type of PP that described ER and LN courses for 6 months from the time of biopsy (Step 6). Consensus was defined as agreement of the majority of PP-raters (Step 3, Step 6).ResultsFor Step 1 and Step 3, 103 physicians answered Delphi questions and filled 353 PP (response rate: 73%). Step 6 activities were completed by 18 physicians (13.4 years of average experience) who were asked to review 33 PP each. This resulted in 564 completed PP ratings, of which 437 (77.5%) and 460 (81.6%) ratings yielded consensus on POSSR-and IV SSR dosing, respectively. PO CS and/or IV CS dosages as per the SSR (SSR-dose) depend on patient weight, the course of ER activity measured by the ER-SLEDAI score (figure 1d), and the course of LN described by changes/status of 3 LN response variables (LN-RVs, figure 1a-c). The SSR mimics dosing customs agreed upon by the PP-raters. Table 1 summarizes the SSR with focus on 2 ER/LN settings (1:stable ER/various LN courses; 2:stable LN/various ER courses), with several permutation of the course of ER (much worse, mild-moderately worse, active stable/improved, inactive) and LN (flare, mild-moderately worse, active stable/improved/partial renal remission (PRR), complete renal remission (CRR)). Use of dosages of PO CS ≥ 40 mg are governed by the course of LN except in major ER flares with potential organ damage. The SSR adjusts PO CS dosages in at least monthly intervals. IV CS are used for worsening of ER or LN courses that fail to respond to increased oral CS of ≥ 40 mg up to 4 weeks. Small decreases of PO CS occur even with stable ER or stable LN activity. Achieving CRR leads to more pronounced reduction of PO CS (table 1). Beyond 6 months post kidney biopsy (maintenance therapy), the PO/IV CS dosage is informed by LN status (PRR, CRR), the course of LN and ER activity (table 1).Abstract 503 Figure 1Abstract 503 Table 1Steroid use provided by the standardized steroid regimen (SSR) INITIAL 4 WEEKS OF INDUCTION THERAPY PO CS IV CS Patients ≥ 50 kg Prednisone* 60 mg/day divided in up to 4 doses Up to 3 doses (30 mg/kg; max 1 gram of methylprednisolone) Patients < 50 kg Prednisone 1.5 mg/kg/day Median – lowest PO CS dose at week 4** 40 mg/day - 30 mg/day WEEK 5 – 26 OF INDUCTION THERAPY (based on LN and ER trends since last visit) LN course (assumption ER is stable) Much worse Increase PO CS to 50-60mg/day; re-assess in 1-3 weeks; if response to increased PO CS is (a) Satisfactory→ No IV CS; (b) non-satisfactory→ IV pulses + PO CS; Possible change of immunosuppressive drug Mild – moderately worse Increase PO CS by about 30% (if dose < 40 mg; max 60 mg) Active stable Stable PO CS dose (if dose < 40 mg; else: slow decrease) Improved active or PRR1 Slow decrease of PO CS dose CRR2 More pronounced decrease of PO CS dose ER course (assumption LN is stable) Much worse Increase PO CS dose; Re-assess in 1-3 weeks; if response to increased PO CS dose is (a) Satisfactory→ No IV CS; (b) non-satisfactory→ IV pulses + PO CS dose; Possible change of immunosuppressive drug Mild- moderately worse Increase PO CS by 20% for doses < 40 mg; otherwise stable PO CS dose Active stable or improved active Stable PO CS dose Inactive Decrease PO CS dose Median - Lowest PO CS dose possible at week 26 12.5 - 10 mg/day BEYOND 26 WEEKS POST KIDNEY BIOPSY - MAINTENANCE THERAPY LN course (assumption ER is stable) Flare3 after PRR/CRR Prednisone ≥ 40 mg, irrespective of ER courseRe-assess in 1-3 weeks; if response to increased PO CS is (a) Satisfactory→ No IV CS; (b) non-satisfactory→ IV pulses + PO CS Worse after PRR/CRR Increase the PO CS dose FIRST PRR stable Slow decrease of the SSR-dose Inactive/CRR or PRR improved More pronounced decrease of the SSR dose ER course (assumption PRR parameters are stable) Much worse Increase PO CS dose by 30-50% (max 60 mg) ; Re-assess in 1-3 weeks; if response to increased PO CS dose is (a) Satisfactory→ No IV CS ; (b) non-satisfactory→ IV pulses + PO CS; Possible change of immunosuppressive drug Mild- moderately worse Increase PO CS dose by 25% for doses < 40 mg; otherwise stable PO CS dose Stable/Improved/Inactive Decrease of the PO CS dose ** For patients ≥ 50 kg; * or corticosteroid equivalent dose. 1 Partial renal remission (PRR): >50% improvement of ≥2 LN-RVs PLUS remaining LN-RV is NOT worse. 2 Complete renal remission (CRR): All LN-RVs are NORMAL. 3 LN flare defined by at least 1 of the LN-RV changes being persistently present on ≥2 subsequent time points ≥1week apart. LN-RV changes are defined as (a) newly abnormal GFR, (b) abnormal GFR that decreased by >10%, (c) persistent increase of UPCR to ≥0.5, after CRR, (d) persistent doubling of UPCR with values ≥1.0, after PRR, or (e) newly active or worsening glomerular hematuria.ConclusionsSSR for the treatment of cSLE complicated by LN has been developed which simulates PO/IV CS use among treating physicians. The proposed SSR may be useful for clinical care and to regulate background CS use during clinical trials of new medication for cSLE.AcknowledgmentsPresented on behalf of the LaUNCH Project Investigator and supported by PORTICO (P30AR076316).