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Rationale & Objective: Sparsentan is a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist (DEARA) examined in the ongoing phase 2 DUET trial for focal segmental glomerulosclerosis (FSGS). In the DUET 8-week double-blind period, sparsentan resulted in greater proteinuria reduction versus irbesartan. We report the long-term efficacy and safety of sparsentan during the open-label extension over more than 4 years. Study Design: Patients were examined from their first sparsentan dose (double-blind period or open-label extension) through 4.6 years. Setting & Participants: Patients with FSGS, excluding secondary FSGS. Intervention: Sparsentan (200, 400, and 800 mg/d). Outcomes: Urinary protein-creatinine ratio, FSGS partial remission endpoint (urinary protein-creatinine ratio ≤1.5 g/g and >40% reduction from baseline), estimated glomerular filtration rate, and blood pressure approximately every 12 weeks. Treatment-emergent adverse events by year and cases/100 patient-years. Results: 109 patients were enrolled; 108 received ≥1 sparsentan dose; 103 entered the open-label extension (68 sparsentan, 35 irbesartan during the double-blind period). Sparsentan was ongoing in 45/108 patients (41.7%); median time to treatment discontinuation was 3.9 years (95% CI, 2.6-5.2). Mean percent proteinuria reduction from baseline was sustained through follow-up. Achieving partial remission within 9 months of first sparsentan dose (52.8% of patients) versus not achieving (47.2%) was associated with significantly slower rate of estimated glomerular filtration rate decline over the entire treatment period (−2.70 vs −6.56; P = 0.03) and in the first 2 years (−1.69 vs −6.46; P = 0.03). The most common treatment-emergent adverse events (>9 cases/100 patient-years) were headache, peripheral edema, upper respiratory infection, hyperkalemia, and hypotension. Peripheral edema and hypotension declined from year 1 (13.9% and 15.7% of patients, respectively) to ≤4% in years ≥2. There were no cases of heart failure and no patient deaths. Limitations: The open-label extension does not include a comparison group. Conclusions: Long-term sparsentan treatment showed sustained proteinuria reduction and a consistent safety profile. Plain-Language Summary: There is substantial unmet clinical need for safe and effective treatments for focal segmental glomerulosclerosis (FSGS), a kidney lesion with varied causes. Sparsentan is being studied for treatment of FSGS and targets 2 important pathways (endothelin-1 and angiotensin II) that lead to the loss of kidney function. In the 8-week randomized, double-blind DUET study in patients with FSGS, sparsentan reduced the amount of protein in the urine better than irbesartan (a blood pressure medicine often used to treat FSGS). We examined long-term treatment with sparsentan over >4 years in the DUET open-label extension. We found sustained proteinuria reduction in patients who continued treatment with sparsentan and a consistent safety profile with no new or unexpected adverse effects.

Rationale & Objective: This study aimed to assess the effect of exposure to organic pollutants in adults with chronic kidney disease (CKD). Study Design: This was a cross-sectional and longitudinal analysis. Setting and Participants: Forty adults enrolled in the Chronic Renal Insufficiency Cohort (CRIC). Exposures: Exposure at baseline and longitudinally to various organic chemical pollutants. Outcomes: The outcomes were as follows: death; composite of congestive heart failure, myocardial infarction, and stroke; event-free survival from kidney failure or ≥50% decline in estimated glomerular filtration rate (eGFR); and longitudinal trajectory of eGFR. Analytical Approach: We used high-performance liquid chromatography with tandem mass spectrometry to measure urinary concentrations of bisphenols, phthalates, organophosphate pesticides, polycyclic aromatic hydrocarbons, melamine, and cyanuric acid at years 1, 3, and 5 after enrollment in the CRIC. Univariate and multivariable logistic regression were used to examine the association of individual compounds and classes of pollutants with the outcomes. The Cox proportional hazards model and Kaplan-Meier method were used to calculate hazard ratios and 95% CIs for each class of pollutants. Results: Median baseline eGFR and urinary protein-to-creatinine ratio were 33 mL/min/1.73 m2 and 0.58 mg/g, respectively. Of 52 compounds assayed, 30 were detectable in ≥50% of participants. Urinary chemical concentrations were comparable in patients with CKD and healthy individuals from contemporaneous National Health and Nutrition Examination Survey cohorts. Phthalates were the only class with a trend toward higher exposure in patients with CKD. There was an inverse relationship between exposure and the eGFR slopes for bisphenol F, mono-(3-carboxypropyl) phthalate, mono-benzyl phthalate, mono-[2-(carboxymethyl)hexyl] phthalate, and melamine. There were no associations between organic pollutant exposure and cardiovascular outcomes. Limitations: Small sample size, evaluation of single rather than combined exposures. Conclusions: Simultaneous measurement of multiple organic pollutants in adults with CKD is feasible. Exposure levels are comparable with healthy individuals. Select contaminants, especially in the phthalate class, may be associated with more rapid deterioration in kidney function. Plain-Language Summary: The effect of exposure to organic pollutants has not been studied in adults with chronic kidney disease. (CKD). To fill this gap, we measured the exposure to a wide range of chemicals that are found in plastics, personal care products, and food preparation. Overall, the exposure was similar to that noted in the healthy population living in the United States. Only select compounds, mainly phthalates, demonstrated a trend with a more rapid decline in kidney function. These findings provide a useful reference for future studies that aim to evaluate organic pollutant exposure in patients with CKD. This is significant because these exposures represent a modifiable risk factor for disease progression through alterations in diet or lifestyle.

Introduction: Sparsentan is a novel single-molecule dual endothelin angiotensin receptor antagonist with hemodynamic and anti-inflammatory properties and is not an immunosuppressant. The ongoing phase 3 PROTECT trial examines sparsentan in adults with IgA nephropathy (IgAN). Methods: The PROTECT trial (NCT03762850) is a multicenter, international, randomized, double-blind, parallel-group, active-controlled study. The efficacy and safety of sparsentan versus the active control irbesartan is being evaluated in adults with biopsy-proven IgAN and proteinuria ≥1.0 g/d despite maximized treatment with an angiotensin-converting enzyme inhibitor (ACEi) and/or angiotensin receptor blocker (ARB) for at least 12 weeks. Blinded and aggregated baseline characteristics are reported descriptively and compared to contemporary phase 3 trials with patients with IgAN. Results: The primary analysis population includes 404 patients who were randomized and received study drug (median age, 46 years). Enrolled patients were from Europe (53%), Asia Pacific (27%), and North America (20%). Baseline median urinary protein excretion was 1.8 g/d. The range of estimated glomerular filtration rate (eGFR) was broad with the largest proportion of patients (35%) in chronic kidney disease (CKD) stage 3B. Before transitioning to study medication, mean systolic/diastolic blood pressure was 129/82 mm Hg, with the majority of patients (63.4%) receiving the maximum labeled ACEi or ARB dose. Patients in Asian versus non-Asian regions included a higher percentage of females, had lower blood pressures, and included lower proportions of patients with a history of hypertension and baseline antihypertensive treatment. Conclusions: Patient enrollment in PROTECT, with differing racial backgrounds and across CKD stages, will allow for important characterization of the treatment effect of sparsentan in patients with IgAN with proteinuria at high risk of kidney failure.

Introduction: Edema is a common manifestation of proteinuric kidney diseases but there is no consensus approach for reliably evaluating edema. The objective of this study was to develop an edema clinician-reported outcome measure for use in patients with nephrotic syndrome. Methods: A literature review was conducted to assess existing clinician-rated measures of edema. Clinical experts were recruited from internal medicine nephrology and pediatric nephrology practices to participate in concept elicitation using semi-structured interviews and cognitive debriefing. Qualitative analysis methods were used to collate expert input and inform measurement development. In addition, training and assessment modules were developed using an iterative process that also utilized expert input and cognitive debriefing to ensure interrater reliability. Results: While several clinician-rated measures of edema have been proposed, our literature review did not identify any studies to support the reliability or validity of these measures. Fourteen clinician experts participated in the concept elicitation interviews and twelve participated in cognitive debriefing. A clinician-reported outcome measure for edema was developed. The measure assesses edema severity in multiple individual body parts. An online training module and assessment tool was generated and refined using additional clinician input and investigative team expertise. Conclusion: The Edema ClinRO (V1) measure is developed specifically to measure edema in nephrotic syndrome. The tool assesses edema across multiple body parts, and it includes a training module to ensure standardized administration across raters. Future examination of this measure is ongoing to establish its reliability and validity.

Introduction: Minimal change disease in childhood can follow a frequently relapsing or steroid-dependent course in up to 40% of cases. Second-line immunosuppressive medications that are used to manage these patients are associated with significant adverse effects. There is a need for safer alternative treatments for difficult-to-treat nephrotic syndrome. Therefore, we conducted an open-label feasibility study to assess the safety and efficacy of a gluten-free diet as treatment for pediatric patients with difficult-to-treat nephrotic syndrome. As a second aim, we sought to determine if the plasma zonulin concentration can identify those who are more likely to respond to this intervention. Methods: Seventeen patients were placed on a gluten-free diet for 6 months. A positive response was defined as a 50% reduction in the relapse rate compared to the preceding 6 months or the ability to discontinue 1 immunosuppressive drug. Results: Five (29%) participants had a positive response to the dietary intervention. The gluten-free diet was well tolerated with no clinical or laboratory adverse events. Plasma zonulin concentration was elevated in patients who failed to benefit from the gluten-free diet. Discussion/Conclusion: A gluten-free diet may be a useful adjunctive intervention for patients with difficult-to-treat nephrotic syndrome that can be implemented prior to resorting to second-line immunosuppressive therapy. Development of the plasma zonulin level as a biomarker to predict efficacy would facilitate rational use of a gluten-free diet in the management of nephrotic syndrome.

Since it was first described more than 50 years ago, recurrence of FSGS in kidney allografts has frustrated the transplant community. This rare condition is associated with considerable morbidity, and it is the most common cause of graft loss in patients with CKD stage 5 due to FSGS. However, the problem remains insufficiently studied. It is an ultra-orphan disease and incidence rates at individual centers are often very low and unpredictable. The published literature contains conflicting reports in basic epidemiologic data. Progress in defining the mechanisms of disease and advancing therapeutic options has been limited. The treatment options that are currently available are limited and largely ineffective. The range in time to recurrence and variability in responsiveness to treatment suggest that recurrence is not a single entity, but rather multiple phenotypes resulting from diverse pathogenetic mechanisms grouped under a larger umbrella. There is an urgent need for innovative basic science and translational research to [1] better understand FSGS recurrence from a mechanistic perspective; [2] improve risk stratification to predict this outcome; and [3] develop effective therapies. In this conference report, we describe the work of investigators whose state-of-the-art research paves the way for innovative approaches to diagnosis and treatment of the problem and provides hope that we can achieve these objectives for affected patients.

Rationale & Objective: Focal segmental glomerulosclerosis (FSGS) is a rare condition that can lead to kidney function decline and chronic kidney failure. Immunosuppressants are used to treat primary FSGS. However, their efficacy and safety in FSGS are not clearly established. We assessed current knowledge on clinical effectiveness and safety of immunosuppressants for primary FSGS. Study Design: Systematic review of randomized controlled trials, interventional nonrandomized controlled trials, observational studies, retrospective studies, and registries. Setting & Participants: Patients with primary and genetic FSGS. Selection Criteria for Studies: Medline, EMBASE, and the Cochrane Central Register of Controlled Trials were searched for English-language, primary-FSGS studies from inception to 2019. Clinical outcomes were changes from baseline in proteinuria, kidney function, and kidney survival. Data Extraction: 2 investigators independently screened studies and extracted data. Analytical Approach: Study results were summarized using random-effects models either as ratios of means between follow-up and baseline measurements or as HRs. Results: We included 98 articles. Substantial heterogeneity was observed in patient baseline characteristics and study designs. Most studies assessed treatment with corticosteroids alone or combined with other drugs, mainly immunosuppressants. Patients treated with immunosuppressants showed reduced proteinuria (14 studies; ratio of means, 0.36; 95% CI, 0.20-0.47), decreased creatinine clearance (mean difference, −25.03; 95% CI, −59.33 to −9.27) and (significantly) lower estimated glomerular filtration rates (mean difference, −7.61 mL/min/1.73 m2; 95% CI, −14.98 to 0.25 mL/min/1.73 m2). Immunosuppressant therapy had an uncertain effect on reducing the chronic kidney failure risk. Hypertension and infections were the most commonly reported adverse events. Limitations: Heterogeneity in study designs, patient populations, and treatment regimens; no access to individual patient–level data. Conclusions: This systematic review supports proteinuria reduction with immunosuppressant therapy in primary FSGS over varying follow-up periods. The effects of immunosuppressants on kidney survival remain uncertain. This review underscores the need for better-designed and adequately controlled studies to assess immunosuppressant therapy in patients with primary FSGS.

Rationale and Objective: Angiotensin-converting enzyme inhibitor or angiotensin receptor blocker therapy (renin-angiotensin-aldosterone system [RAAS] inhibitor) to control proteinuria in primary and genetic focal segmental glomerulosclerosis (FSGS) follows guidelines based on other proteinuria-related kidney diseases. There is no consensus on the efficacy and safety of RAAS inhibitor therapies in primary and genetic FSGS. This systematic review assessed the effects of RAAS inhibitor therapy on kidney outcomes in these patients. Study Design: Systematic review of randomized controlled trials, interventional nonrandomized studies, observational studies, and retrospective studies. Setting & Study Populations: Patients with primary and genetic FSGS. Selection Criteria for Studies: PubMed, Cochrane Library, and Embase. Data Extraction: 2 investigators independently screened studies and extracted data. Analytical Approach: Results were summarized as the ratio of means (ROM) between baseline and follow-up measurements or as the hazard ratio using random-effects models. Results: 30 publications were selected; 5 were controlled trials (4 randomized controlled trials). 8 assessed RAAS inhibitor monotherapy, while the rest studied RAAS inhibitors in combination with other drugs, mainly immunosuppressants. On average, a 32% proteinuria reduction (ROM, 0.68; 95% CI, 0.47-0.98) and no change in creatinine clearance (ROM, 0.95; 95% CI, 0.77-1.16) from baseline to the last reported follow-up was observed in patients treated with RAAS inhibitor monotherapy. When a RAAS inhibitor was combined with other drugs, a 72% proteinuria reduction was observed from baseline to the last reported follow-up (ROM, 0.24; 95% CI, 0.08-0.75). The published data did not allow for the assessment of the effects of RAAS inhibitor monotherapy on estimated glomerular filtration rate and end-stage kidney disease risks. Limitations: Large study heterogeneity in design, patient populations, and treatment regimens. No access to individual patient-level data. Conclusions: This review supports the tendency to observe a proteinuria reduction with RAAS inhibitors in patients with primary FSGS. RAAS inhibitor monotherapy was associated with maintained kidney function, as shown by no change in creatinine clearance. Strong evidence to quantify the effects of RAAS inhibitor monotherapy on end-stage kidney disease and glomerular filtration rate was lacking. Larger, well-designed clinical trials are needed to better understand the effects of RAAS inhibitors on primary FSGS.

Introduction: Prior cross-sectional studies suggest that health-related quality of life (HRQOL) worsens with more severe glomerular disease. This longitudinal analysis was conducted to assess changes in HRQOL with changing disease status. Methods: Cure Glomerulonephropathy (CureGN) is a cohort of patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA vasculitis, or IgA nephropathy. HRQOL was assessed at enrollment and follow-up visits 1 to 3 times annually for up to 5 years with the Patient-Reported Outcomes Measurement Information System (PROMIS). Global health, anxiety, and fatigue domains were measured in all; mobility was measured in children; and sleep-related impairment was measured in adults. Linear mixed effects models were used to evaluate HRQOL responsiveness to changes in disease status. Results: A total of 469 children and 1146 adults with PROMIS scores were included in the analysis. HRQOL improved over time in nearly all domains, though group-level changes were modest. Edema was most consistently associated with worse HRQOL across domains among children and adults. A greater number of symptoms also predicted worse HRQOL in all domains. Sex, age, obesity, and serum albumin were associated with some HRQOL domains. The estimated glomerular filtration rate (eGFR) was only associated with fatigue and adult physical health; proteinuria was not associated with any HRQOL domain in adjusted models. Conclusion: HRQOL measures were responsive to changes in disease activity, as indicated by edema. HRQOL over time was not predicted by laboratory-based markers of disease. Patient-reported edema and number of symptoms were the strongest predictors of HRQOL, highlighting the importance of the patient experience in glomerular disease. HRQOL outcomes inform understanding of the patient experience for children and adults with glomerular diseases.

Introduction: Childhood-onset nephrotic syndrome has a variable clinical course. Improved predictive markers of long-term outcomes in children with nephrotic syndrome are needed. This study tests the association between baseline urinary epidermal growth factor (uEGF) excretion and longitudinal kidney function in children with nephrotic syndrome. Methods: The study evaluated 191 participants younger than 18 years enrolled in the Nephrotic Syndrome Study Network, including 118 with their first clinically indicated kidney biopsy (68 minimal change disease; 50 focal segmental glomerulosclerosis) and 73 with incident nephrotic syndrome without a biopsy. uEGF was measured at baseline for all participants and normalized by the urine creatinine (Cr) concentration. Renal epidermal growth factor (EGF) mRNA was measured in the tubular compartment microdissected from kidney biopsy cores from a subset of patients. Linear mixed models were used to test if baseline uEGF/Cr and EGF mRNA expression were associated with change in estimated glomerular filtration rate (eGFR) over time. Results: Higher uEGF/Cr at baseline was associated with slower eGFR decline during follow-up (median follow-up = 30 months). Halving of uEGF/Cr was associated with a decrease in eGFR slope of 2.0 ml/min per 1.73 m2 per year (P < 0.001) adjusted for age, race, diagnosis, baseline eGFR and proteinuria, and APOL1 genotype. In the biopsied subgroup, uEGF/Cr was correlated with EGF mRNA expression (r = 0.74; P < 0.001), but uEGF/Cr was retained over mRNA expression as the stronger predictor of eGFR slope after multivariable adjustment (decrease in eGFR slope of 1.7 ml/min per 1.73 m2 per year per log2 decrease in uEGF/Cr; P < 0.001). Conclusion: uEGF/Cr may be a useful noninvasive biomarker that can assist in predicting the long-term course of kidney function in children with incident nephrotic syndrome. Keywords: disease progression, epidermal growth factor, focal segmental glomerulosclerosis, nephrotic syndrome, pediatrics

Introduction: Focal segmental glomerulosclerosis (FSGS), a histologic lesion in the kidney caused by varied pathophysiological processes, leads to end-stage kidney disease in a large proportion of patients. Sparsentan is a first-in-class orally active compound combining endothelin type A (ETA) receptor blockade with angiotensin II type 1 (AT1) receptor antagonism in a single molecule. A Randomized, Multicenter, Double-Blind, Parallel, Active-Control Study of the Effects of Sparsentan, a Dual Endothelin Receptor and Angiotensin Receptor Blocker, on Renal Outcomes in Patients With Primary FSGS (DUPLEX) study evaluates the long-term antiproteinuric efficacy, nephroprotective potential, and safety profile of sparsentan compared with an AT1 receptor blocker alone in patients with FSGS. Methods: DUPLEX is a multicenter, international, phase 3, randomized, double-blind, active-controlled study of sparsentan in patients with FSGS. Approximately 300 patients aged 8 to 75 years, inclusive (United States), and 18 to 75 years, inclusive (outside United States) will be randomized 1:1 to daily treatment with sparsentan or irbesartan. After renin-angiotensin-aldosterone system inhibitor washout, treatment will be administered for 108 weeks, with the final assessment at week 112, four weeks after withdrawal of study drug. Results: The primary endpoint will be the slope of estimated glomerular filtration rate from week 6 to week 108. A novel surrogate efficacy endpoint, the proportion of patients achieving urinary protein-to-creatinine (UP/C) ratio of ≤1.5 g/g and >40% reduction from baseline in UP/C (FSGS partial remission endpoint: FPRE), will be evaluated at a planned interim analysis at week 36. Safety and tolerability of sparsentan will also be assessed. Conclusion: The phase 3 DUPLEX study will characterize the long-term antiproteinuric efficacy and nephroprotective potential of dual ETA and AT1 receptor blockade with sparsentan in patients with FSGS. Keywords: angiotensin II type 1 receptor blockade, endothelin type A receptor blockade, focal segmental glomerulosclerosis, irbesartan, proteinuria, sparsentan

In 2017, the NYU Clinical and Translational Science Institute’s Recruitment and Retention Unit created a Patient Advisory Council for Research (PACR) to provide feedback on clinical trials and health research studies. We collaborated with our clinical research informatics team to generate a random sample of patients, based on the International Classification of Diseases, Tenth Revision codes and demographic factors, for invitation via the patient portal. This approach yielded in a group that was diverse with regard to age, race/ethnicity, sex, and health conditions. This report highlights the benefits and limitations of using an electronic health record-based strategy to identify and recruit members for a PACR.

Background: Growing evidence suggests that exposure to environmental chemicals, such as pesticides, impacts renal function and chronic kidney disease (CKD). However, it is not clear if pesticides may affect CKD progression and no studies exist in children. Objectives: The objective of this study was to examine associations between serially measured urinary OP pesticide metabolites and clinical and laboratory measures of kidney function over time among children with CKD. Methods: This study used data on 618 participants enrolled in the CKD in Children study (CKiD), a cohort study of pediatric CKD patients from the US and Canada. Children were followed over an average of 3.0 years (standard deviation (SD) = 1.6) between 2005 and 2015. In serially collected urine samples over time, six nonspecific dialkyl phosphate (DAP) metabolites of OP pesticides were measured. Biomarkers of tubular injury (kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)) and oxidant stress (8-hydroxy-2′-deoxyguanosine (8-OHdG) and F2-isoprostane) were determined in the same specimens. Estimated glomerular filtration rate (eGFR), proteinuria, and blood pressure were assessed annually. Results: DAPs were associated with increased KIM-1 and 8-OHdG throughout follow-up. A standard deviation increase in ∑diethyl metabolites was associated with increases of 11.9% (95% Confidence Interval (CI): 4.8%, 19.4%) and 13.2% (95% CI: 9.3%, 17.2%) in KIM-1 and 8-OHdG over time, respectively. DAPs were associated with lower eGFR at baseline and higher eGFR over subsequent years. Conclusions: These findings provide preliminary evidence suggesting that urinary DAP metabolites are associated with subclinical kidney injury among children with CKD, which may signal the potential for clinical events to manifest in the future. The results from this study are significant from both a clinical and public health perspective, given that OP pesticide exposure is a modifiable risk factor.

Rationale & Objective: Assessment of how patients feel and function is needed for clinical care and research for focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). The objective of this study was to develop a patient-reported outcome assessment appropriate for use in children and adults with FSGS and MCD. Study Design: Qualitative study using semi-structured interviews. Setting & Participants: 48 semi-structured interviews with children aged 8 to 17 years (n = 11) and adults (n = 10) with FSGS and children aged 8 to 17 (n = 11) and adults (n = 16) with MCD recruited from 3 academic medical centers. Analytical Approach: Latent content analysis. Results: FSGS and MCD have a pervasive and comparable impact on physical, social, and mental health-related quality of life regardless of age or diagnosis. Physical symptoms of swelling, fatigue, and pain were articulated by most participants. Disease management was also a frequent topic of discussion; participants described their experiences with medication and associated side effects, as well as lifestyle changes made to manage their disease (ie, dietary changes and frequent medical appointments). These discussions often identified a profound impact on physical abilities and life participation. In many instances, participants described the negative impact these symptoms had on their mood and sense of self, with most participants reporting feelings of anxiety. Limitations: Participants were primarily non-Hispanic White and English speaking, which may limit generalizability. Conclusions: Our results suggest that there are commonalities to the FSGS-MCD patient experience of health-related quality of life that will enable the generation of a disease-specific FSGS-MCD patient-reported outcomes instrument for use in children and adults. The development of this tool is intended to facilitate better care and support clinical research for these individuals.

Background: High data quality is of crucial importance to the integrity of research projects. In the conduct of multi-center observational cohort studies with increasing types and quantities of data, maintaining data quality is challenging, with few published guidelines. Methods: The Cure Glomerulonephropathy (CureGN) Network has established numerous quality control procedures to manage the 70 participating sites in the United States, Canada, and Europe. This effort is supported and guided by the activities of several committees, including Data Quality, Recruitment and Retention, and Central Review, that work in tandem with the Data Coordinating Center to monitor the study. We have implemented coordinator training and feedback channels, data queries of questionable or missing data, and developed performance metrics for recruitment, retention, visit completion, data entry, recording of patient-reported outcomes, collection, shipping and accessing of biological samples and pathology materials, and processing, cataloging and accessing genetic data and materials. Results: We describe the development of data queries and site Report Cards, and their use in monitoring and encouraging excellence in site performance. We demonstrate improvements in data quality and completeness over 4 years after implementing these activities. We describe quality initiatives addressing specific challenges in collecting and cataloging whole slide images and other kidney pathology data, and novel methods of data quality assessment. Conclusions: This paper reports the CureGN experience in optimizing data quality and underscores the importance of general and study-specific data quality initiatives to maintain excellence in the research measures of a multi-center observational study.

Introduction: There is limited information on effective disease monitoring for prompt interventions in childhood nephrotic syndrome. We examined the feasibility and effectiveness of a novel text messaging system (SMS) for disease monitoring in a multicenter, prospective study. Methods: A total of 127 patients

BackgroundExposure to environmental chemicals may be a modifiable risk factor for progression of chronic kidney disease (CKD). The purpose of this study was to examine the impact of serially assessed exposure to bisphenol A (BPA) and phthalates on measures of kidney function, tubular injury, and oxidative stress over time in a cohort of children with CKD.Methods and findingsSamples were collected between 2005 and 2015 from 618 children and adolescents enrolled in the Chronic Kidney Disease in Children study, an observational cohort study of pediatric CKD patients from the US and Canada. Most study participants were male (63.8%) and white (58.3%), and participants had a median age of 11.0 years (interquartile range 7.6 to 14.6) at the baseline visit. In urine samples collected serially over an average of 3.0 years (standard deviation [SD] 1.6), concentrations of BPA, phthalic acid (PA), and phthalate metabolites were measured as well as biomarkers of tubular injury (kidney injury molecule-1 [KIM-1] and neutrophil gelatinase-associated lipocalin [NGAL]) and oxidative stress (8-hydroxy-2'-deoxyguanosine [8-OHdG] and F2-isoprostane). Clinical renal function measures included estimated glomerular filtration rate (eGFR), proteinuria, and blood pressure. Linear mixed models were fit to estimate the associations between urinary concentrations of 6 chemical exposure measures (i.e., BPA, PA, and 4 phthalate metabolite groups) and clinical renal outcomes and urinary concentrations of KIM-1, NGAL, 8-OHdG, and F2-isoprostane controlling for sex, age, race/ethnicity, glomerular status, birth weight, premature birth, angiotensin-converting enzyme inhibitor use, angiotensin receptor blocker use, BMI z-score for age and sex, and urinary creatinine. Urinary concentrations of BPA, PA, and phthalate metabolites were positively associated with urinary KIM-1, NGAL, 8-OHdG, and F2-isoprostane levels over time. For example, a 1-SD increase in ∑di-n-octyl phthalate metabolites was associated with increases in NGAL (β = 0.13 [95% CI: 0.05, 0.21], p = 0.001), KIM-1 (β = 0.30 [95% CI: 0.21, 0.40], p < 0.001), 8-OHdG (β = 0.10 [95% CI: 0.06, 0.13], p < 0.001), and F2-isoprostane (β = 0.13 [95% CI: 0.01, 0.25], p = 0.04) over time. BPA and phthalate metabolites were not associated with eGFR, proteinuria, or blood pressure, but PA was associated with lower eGFR over time. For a 1-SD increase in ln-transformed PA, there was an average decrease in eGFR of 0.38 ml/min/1.73 m2 (95% CI: -0.75, -0.01; p = 0.04). Limitations of this study included utilization of spot urine samples for exposure assessment of non-persistent compounds and lack of specific information on potential sources of exposure.ConclusionsAlthough BPA and phthalate metabolites were not associated with clinical renal endpoints such as eGFR or proteinuria, there was a consistent pattern of increased tubular injury and oxidative stress over time, which have been shown to affect renal function in the long term. This raises concerns about the potential for clinically significant changes in renal function in relation to exposure to common environmental toxicants at current levels.

Interstitial nephritis (IN) is a relatively rare entity in children and adolescents that can be caused by a range of disorders including infection, medications, inflammatory bowel disease, and sarcoid. There is no proven therapy for this condition. We present 2 cases of biopsy-proven interstitial nephritis, of which 1 case was with granulomatous features that presented with unusual sonographic findings of discrete mass lesions in the kidney parenchyma bilaterally. Although a precise cause could not be identified in either case, 1 patient progressed to end-stage kidney disease (ESKD) and the other is in the early stages of treatment. We suggest that recognition of the atypical imaging features of interstitial nephritis may enable early recognition of this condition and avoid confusion with neoplastic or infectious processes. Keywords: Interstitial nephritis, Granulomatous interstitial nephritis, Pediatric, Mass-like lesions, Sonography, Kidney biopsy

Treatment-resistant nephrotic syndrome is a rare form of glomerular disease that occurs in children and adults. No Food and Drug Administration−approved treatments consistently achieve remission of proteinuria and preservation of kidney function. CD80 (B7-1) can be expressed on injured podocytes, and administration of abatacept (modified CTLA4-Ig based on a natural ligand to CD80) has been associated with sustained normalization of urinary protein excretion and maintenance of glomerular filtration rate in experimental and clinical settings. Methods: In this report, we describe the rationale for and design of a randomized, placebo-controlled, clinical trial of abatacept in patients with treatment-resistant nephrotic syndrome caused by focal segmental glomerulosclerosis or minimal change disease. The design is a hybrid of a parallel-group and crossover design (switchover) with the primary objectives assessed in the first period of the study and the secondary objectives assessed using data from both periods. All participants will receive the active agent in 1 of the periods. The duration of treatment will be 4 months per period. Results: The primary outcome will be improvement in nephrotic-range proteinuria to subnephrotic range, that is, reduction from baseline to 4 months in urine protein:creatinine ratio ≥ 50% and to a level < 3. The projected sample size is 90 patients, which has 80% power to detect a treatment difference of 28%. Conclusion: This study advances efforts to validate CD80 as a therapeutic target for treatment-resistant nephrotic syndrome, and implements a precision medicine-based approach to this serious kidney condition in which the selection of a therapeutic agent is guided by the underlying disease mechanism operating in individual patients.

Primary focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease. There are no US Food and Drug Administration−approved therapies for FSGS, and treatment often fails to reduce proteinuria. Endothelin is an important factor in the pathophysiology of podocyte disorders, including FSGS. Sparsentan is a first-in-class, orally active, dual-acting angiotensin receptor blocker (ARB) and highly selective endothelin Type A receptor antagonist. This study is designed to evaluate whether sparsentan lowers proteinuria compared with an ARB alone and has a favorable safety profile in patients with FSGS. Methods: DUET is a phase 2, randomized, active-control, dose-escalation study with an 8-week, fixed-dose, double-blind period followed by 136 weeks of open-label sparsentan treatment. Patients aged 8 to 75 years with primary FSGS will be randomized to treatment with sparsentan or irbesartan for 8 weeks. Results: The primary efficacy objective is to test the hypothesis that sparsentan over the dose range (200 mg, 400 mg, or 800 mg daily) is superior to irbesartan (300 mg daily) in decreasing the urinary protein-to-creatinine ratio (UPC) from baseline to 8 weeks postrandomization. As secondary objectives, the trial will evaluate the proportion of patients who achieve prespecified targets of UPC reduction, changes in laboratory and quality-of-life indices, and detailed safety analysis. Analyses will be conducted at the end of the double-blind (week 8) and open-label (week 144) periods. Discussion: This study will provide important evidence on whether dual ARB and endothelin blockade may be an effective therapeutic strategy for FSGS and may provide the rationale for next-phase trials.

Background: Study coordinators play an essential role on study teams; however, there remains a paucity of research on the supports and services they need to effectively recruit and retain study participants. Methods: A cross-sectional survey was conducted with 147 study coordinators from a large academic medical center. Survey items assessed barriers and facilitators to recruitment and retention, anxiety about reaching enrollment numbers, confidence for talking to potential study participants about research involvement, awareness and use of CTSA resources, and PI involvement with recruitment planning. Results: Significant associations were found between anxiety about reaching target enrollment numbers and whether the study coordinator was the primary person responsible for developing a recruitment strategy. Three years or more serving as a study coordinator and levels of anxiety for reaching enrollment numbers was also significant. Conclusion: More institutional level supports and formal training opportunities are needed to enhance study coordinators’ effectiveness to recruit participants. Keywords: Research personnel, Patient selection, Clinical trials as topic, National center for advancing translational sciences (U.S.), Cross-sectional studies, Academic medical centers

There is a note of caution expressed when clinical care providers enroll their own patients into investigational trials, a concern expressed in the called dual-role consent. There is concern that this circumstance may create a conflict of interest for the physician-investigator, lead to loss of patient voluntarism, and promote the therapeutic misconceptions. In this opinion paper, I review the circumstances surrounding participation in clinical research and the conduct of standard patient care. I propose that when a patient is eligible for an institutional review board-approved clinical trial, instead of representing a potential ethical lapse, soliciting enrollment by the clinician-researcher may represent optimal care for the patient.

Objective: We conducted this study to test the hypothesis that plasma zonulin levels are elevated in pediatric patients with nephrotic syndrome compared to healthy controls.Study Design: Plasma zonulin levels were measured by ELISA in 114 children enrolled in the NEPTUNE study. Clinical and laboratory data were retrieved from the NEPTUNE database.Results: The median age of the patients was 10 (IQR = 5 to 14) years, 59 were male, 64 had minimal change disease, 47 focal segmental glomerulosclerosis, median eGFR was 96 (IQR = 80 to 114) ml/min/1.73 m2, and median urine protein:creatinine ratio was 0.5 (IQR = 0.1 to 3.4) (g:g). The plasma zonulin level was 14.2 ± 5.0 vs. 10.2 ± 2.5 ng/ml in healthy adults in a report using the same assay kit, P = 0.0025. These findings were confirmed in an independent cohort of children with nephrotic syndrome compared to healthy age-matched controls, P = 0.01. Zonulin concentrations did not differ in children with minimal change disease vs. focal segmental glomerulosclerosis, frequently relapsing vs. steroid-dependent vs. steroid-resistant clinical course, and were not influenced by the immunosuppressive treatment regimen. There was no relationship between plasma zonulin levels and the absolute or percentage change in proteinuria from enrollment until the time of the zonulin assay.Conclusion: Plasma zonulin levels are elevated in childhood nephrotic syndrome regardless of level of proteinuria or specific treatment. The cause of the high plasma zonulin levels and whether zonulin contributes to glomerular injury requires further study.

Background . Restless legs syndrome (RLS) is a sleep disorder characterized by an urge to move or the presence of unpleasant sensations in the extremities. The prevalence of RLS is higher in children and adults with chronic kidney disease and in adults with glomerular disease. Objective . To determine the prevalence of RLS in children with nephrotic syndrome. Methods . We studied 50 children with nephrotic syndrome and 22 controls. The following surveys were administered: Pediatric Emory RLS questionnaire, Pediatric Daytime Sleepiness Scale, and Pediatric Sleep Questionnaire. Results . Children with nephrotic syndrome were 9.0 ± 4.4 years old, 27 were male, and 27 were in remission. The prevalence of RLS was similar in the nephrotic syndrome cases and controls, whether or not indeterminate cases were considered positive: 14.0% versus 13.6% including indeterminate cases, and 8.0% versus 9.1% excluding indeterminate cases. Conclusion . RLS is not more common in children with glomerular disease compared to healthy controls.

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1HM1549V mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca2+, the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension.

Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process.Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed.We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis.Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR).Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine.Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study.Limited number of patients with samples from all time-points.The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.

Recurrence of focal segmental glomerulosclerosis (FSGS) after renal transplantation is a complication that often leads to graft loss. There is no consensus on the optimal treatment of recurrent FSGS. Rituximab, a monoclonal antibody to CD20, may be a useful treatment of this complication. Methods. We report four pediatric cases of recurrent FSGS treated with rituximab and plasmapheresis. Results. Four children (2M/2F), age 15.3 ± 2.6, with recurrent FSGS posttransplant were identified. Four doses of rituximab were administered 171 ± 180 days posttransplant and 114 ± 169 days after the start of plasmapheresis. Three children responded with complete remission, one of whom relapsed after four months. One child had a partial response with a decrease in proteinuria that was not sustained. No adverse side effects were reported during treatment or followup (mean 22.5 months). Conclusions. Rituximab is a safe and well-tolerated ancillary treatment for recurrent FSGS in pediatric patients in conjunction with plasmapheresis.

Chromatin regulation provides a mechanism through which cells dynamically and rapidly regulate their gene expression profiles, playing a pivotal role in diverse biological processes and disease states. The Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method that enables genome-wide detection of accessible chromatin regions, providing information on nucleosome positioning and the epigenetic regulation of the chromatin structure. ATAC-seq has been used in various biological contexts, and several reports have demonstrated its application to studying infections with viral or bacterial pathogens. The ability to characterize changes in viral or bacterial genome accessibility during infections provides insights into both pathogen replication and host defense mechanisms. Viral genomes undergo dynamic changes in their structural landscape to facilitate replication and evade host immune responses. Additionally, host cells encode DNA sensors, which are specialized proteins that bind to viral genomes to initiate innate immune responses and sometimes, to suppress viral gene expression. ATAC-seq enables the systematic detection of key structural changes on the viral genome mediated by either viral or host proteins, offering mechanistic insights into virus-host interactions. Here, we describe an ATAC-seq method optimized for studying changes in chromatin accessibility in both host and viral genomes. We have previously applied this method to demonstrate a systematic decrease in the genome accessibility of herpes simplex virus type I (HSV-1) enabled by a host antiviral factor, the interferon-gamma inducible protein 16 (IFI16) during infection of human fibroblasts. This protocol can be adapted to various biological contexts involving the introduction of foreign DNA, making it a valuable tool for a broad range of research endeavors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3β, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, including DNA sensors. The interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor that recognizes double-stranded DNA from a number of viral sources, including genomes of nuclear-replicating viruses. Among these is the prevalent human pathogen herpes simplex virus 1 (HSV-1). Upon binding to the HSV-1 DNA genome, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we used a multiomics approach of DNA sequencing techniques paired with targeted mass spectrometry to obtain an extensive view of the interaction between IFI16 and the HSV-1 genome and how this binding affects the viral DNA structure and protein expression. Through chromatin immunoaffinity purification coupled with next-generation DNA sequencing (ChIP-seq), we found that IFI16 binds to the HSV-1 genome in a sequence-independent manner while simultaneously exhibiting broad enrichment at two loci: UL30, the viral DNA polymerase gene, and US1 to US7. The assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed that these two regions are among the most accessible stretches of DNA on the genome, thereby facilitating IFI16 binding. Accessibility of the entire HSV-1 genome is elevated upon IFI16 knockout, indicating that expression of IFI16 globally induces chromatinization of viral DNA. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry of viral proteins representing 80% of the HSV-1 genome. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, coordinating epigenetic silencing of the viral genome and decreasing protein expression and virus replication. IMPORTANCE Mammalian host defense against viral infection includes broad-acting cellular restriction factors, as well as effectors of intrinsic and innate immunity. IFI16 is a critical nuclear host defense factor and intrinsic immune protein involved in binding viral DNA genomes, thereby repressing the replication of nucleus-replicating viruses, including the human herpes simplex virus 1. What has remained unclear is where on the viral genome IFI16 binds and how binding affects both viral DNA structural accessibility and viral protein expression. Our study provides a global view of where and how a nuclear restriction factor of DNA viruses associates with viral genomes to exert antiviral functions during early stages of an acute virus infection. Our study can additionally serve as a systems-level model to evaluate nuclear DNA sensor interactions with viral genomes, as well as the antiviral outcomes of transcriptionally silencing pathogen-derived DNA.

DNA sensors are critical components of innate immunity that enable cells to recognize infection by pathogens with DNA genomes. The interferon-inducible protein X (IFIX), a member of the PYHIN protein family, is a DNA sensor capable of promoting immune signaling after binding to double-stranded DNA (dsDNA) within either the nucleus or cytoplasm. Here, we investigate the impact of IFIX on the cellular proteome upon introduction of foreign DNA to the nucleus or the cytoplasm as well as regulatory hubs that control IFIX subcellular localization. Using quantitative mass spectrometry, we define the effect of CRISPR-mediated IFIX knockout on nuclear and cytoplasmic proteomes in fibroblasts. Proteomes are probed in response to either nuclear viral DNA, during herpes simplex virus 1 (HSV-1) infection, or cytoplasmic viral DNA, following transfection with dsDNA derived from vaccinia virus (VACV 70-mer). We show that IFIX broadly impacts nuclear and cytoplasmic proteomes, inducing alterations in the abundances of immune signaling, DNA damage response, and vesicle-mediated transport proteins. To characterize IFIX properties that regulate its localization during DNA sensing, we perform deletion and mutagenesis assays. We find that IFIX contains a multipartite nuclear localization signal (NLS) and highlight the main contributing motif for its nuclear localization. Using immunoaffinity purification, we identify IFIX acetylation and phosphorylation sites. Mutations to acetyl or charge mimics demonstrate that K138 acetylation, positioned within the NLS, affects nuclear localization. Altogether, our study establishes a mechanism regulating IFIX subcellular localization and contextualizes this localization with the involvement of IFIX in host cell responses to pathogenic DNA. IMPORTANCE Mammalian cells must be able to detect and respond to invading pathogens to prevent the spread of infection. DNA sensors, such as IFIX, are proteins that bind to pathogen-derived double-stranded DNA and induce antiviral cytokine expression. Here, we characterize the host proteome changes that require IFIX during both viral infection and DNA transfection. We show IFIX mobilizes numerous pathways and proteome alterations within the nucleus and the cytoplasm, pointing to a multifunctional protein with roles in immune signaling, DNA damage response, and transcriptional regulation. We next interrogate the IFIX domains required for nuclear localization, discovering its regulation via a multipartite nuclear localization motif. The acetylation of this motif promotes IFIX cytoplasmic localization, in agreement with its detection of pathogenic DNA in both the nucleus and the cytoplasm. This study established NLS acetylation as a conserved mechanism for regulating the localization of nuclear DNA sensors from the PYHIN family of proteins.

Nuclear DNA sensors are critical components of the mammalian innate immune system, recognizing the presence of pathogens and initiating immune signaling. These proteins act in the nuclei of infected cells by binding to foreign DNA, such as the viral genomes of nuclear-replicating DNA viruses herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). Upon binding to pathogenic DNA, the nuclear DNA sensors were shown to initiate antiviral cytokines, as well as to suppress viral gene expression. These host defense responses involve complex signaling processes that, through protein-protein interactions (PPIs) and post-translational modifications (PTMs), drive extensive remodeling of the cellular transcriptome, proteome, and secretome to generate an antiviral environment. As such, a holistic understanding of these changes is required to understand the mechanisms through which nuclear DNA sensors act. The advent of omics techniques has revolutionized the speed and scale at which biological research is conducted and has been used to make great strides in uncovering the molecular underpinnings of DNA sensing. Here, we review the contribution of proteomics approaches to characterizing nuclear DNA sensors via the discovery of functional PPIs and PTMs, as well as proteome and secretome changes that define a host antiviral environment. We also highlight the value of and future need for integrative multiomic efforts to gain a systems-level understanding of DNA sensors and their influence on epigenetic and transcriptomic alterations during infection.

The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication. IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens., (Copyright © 2019 Lum et al.)

DNA sensors are a core component of innate immunity in mammalian cells. In response to pathogen infection, these specialized proteins sense pathogenic DNA from bacteria or viruses and initiate immune signaling cascades. These defense mechanisms rely on the rapid formation and temporal regulation of protein-protein interactions. Similarly, protein interactions underlie virus immune evasion mechanisms, as proteins from diverse viruses associate with and inhibit DNA sensors. Here, we describe experimental protocols for identifying protein interactions of DNA sensors, and discuss considerations for optimal isolation of protein complexes when targeting either endogenous or tagged proteins. Additionally, as viral infections and immune responses are known to induce prominent changes in cellular protein abundances, we provide a workflow for investigating these protein associations in the context of proteome alterations., (© 2019 Elsevier Inc. All rights reserved.)