Nephrotic Syndrome Study Network Research Articles

Growth in children with nephrotic syndrome: a post hoc analysis of the NEPTUNE study.

Steroids, the mainstay of treatment for nephrotic syndrome in children, have multiple adverse effects including growth suppression. Anthropometric measurements in children < 18years enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) were collected. The longitudinal association of medication exposure and nephrotic syndrome characteristics with height z-score and growth velocity was determined using adjusted Generalized Estimating Equation regression and linear regression. A total of 318 children (57.2% males) with a baseline age of 7.64 ± 5.04years were analyzed. The cumulative steroid dose was 216.4 (IQR 61.5, 652.7) mg/kg (N = 233). Overall, height z-scores were not significantly different at the last follow-up compared to baseline (- 0.13 ± 1.21 vs. - 0.23 ± 1.71, p = 0.21). In models adjusted for age, sex, and eGFR, greater cumulative steroid exposure (β - 7.5 × 10-6, CI - 1.2 × 10-5, - 3 × 10-6, p = 0.001) and incident cases of NS (vs. prevalent) (β - 1.1, CI - 2.22, - 0.11, p = 0.03) were significantly associated with lower height z-scores over time. Rituximab exposure was associated with higher height z-scores (β 0.16, CI 0.04, 0.29, p = 0.01) over time. Steroid dose was associated with lower height z-score, while rituximab use was associated with higher height z-score.

Association of mental health-related patient reported outcomes with blood pressure in adults and children with primary proteinuric glomerulopathies.

The prevalence of mental health disorders including anxiety and depression is increasing and is linked to hypertension in healthy individuals. However, the relationship of psychosocial patient-reported outcomes on blood pressure (BP) in primary proteinuric glomerulopathies is not well characterized. This study explored longitudinal relationships between psychosocial patient-reported outcomes and BP status among individuals with proteinuric glomerulopathies. An observational cohort study was performed using data from 745 adults and children enrolled in the Nephrotic Syndrome Study Network (NEPTUNE). General Estimating Equations for linear regression and binary logistic analysis for odds ratios were performed to analyze relationships between the exposures, longitudinal Patient-Reported Outcome Measurement Information System (PROMIS) measures and BP and hypertension status as outcomes. In adults, more anxiety was longitudinally associated with higher systolic and hypertensive BP. In children, fatigue was longitudinally associated with increased odds of hypertensive BP regardless of the PROMIS report method. More stress, anxiety, and depression were longitudinally associated with higher systolic BP index, higher diastolic BP index, and increased odds of hypertensive BP index in children with parent-proxy patient-reported outcomes. Chronically poor psychosocial patient-reported outcomes may be significantly associated with higher BP and hypertension in adults and children with primary proteinuric glomerulopathies. This interaction appears strong in children but should be interpreted with caution, as multiple confounders related to glomerular disease may influence both mental health and BP independently. That said, access to mental health resources may help control BP, and proper disease and BP management may improve overall mental health.

Longitudinal analysis of blood pressure and lipids in childhood nephrotic syndrome.

In the current study, longitudinal BP and lipid measurements were examined in a NEPTUNE cohort of children with newly diagnosed nephrotic syndrome (cNEPTUNE). We hypothesized that hypertensive BP and dyslipidemia would persist in children with nephrotic syndrome, regardless of steroid treatment response. A multi-center longitudinal observational analysis of data obtained from children < 19years of age with new onset nephrotic syndrome enrolled in the Nephrotic Syndrome Study Network (cNEPTUNE) was conducted. BP and lipid data were examined over time stratified by disease activity and steroid exposure. Generalized estimating equation regressions were used to find determinants of hypertensive BP and dyslipidemia. Among 122 children, the prevalence of hypertensive BP at any visit ranged from 17.4% to 57.4%, while dyslipidemia prevalence ranged from 40.0% to 96.2% over a median of 30months of follow-up. Hypertensive BP was found in 46.2% (116/251) of study visits during active disease compared with 31.0% (84/271) of visits while in remission. Dyslipidemia was present in 88.2% (120/136) of study visits during active disease and in 66.0% (101/153) while in remission. Neither dyslipidemia nor hypertensive BP were significantly different with/without medication exposure (steroids and/or CNI). In regression analysis, male sex and urine protein:creatinine ratio (UPC) were significant determinants of hypertensive BP over time, while eGFR was found to be a determinant of dyslipidemia over time. Results demonstrate persistent hypertensive BPs and unfavorable lipid profiles in the cNEPTUNE cohort regardless of remission status or concurrent steroid or calcineurin inhibitor treatment.

Rationale and design of the Nephrotic Syndrome Study Network (NEPTUNE) Match in glomerular diseases: designing the right trial for the right patient, today

Glomerular diseases are classified using a descriptive taxonomy that is not reflective of the heterogeneous underlying molecular drivers. This limits not only diagnostic and therapeutic patient management, but also impacts clinical trials evaluating targeted interventions. The Nephrotic Syndrome Study Network (NEPTUNE) is poised to address these challenges. The study has enrolled >850 pediatric and adult patients with proteinuric glomerular diseases who have contributed to deep clinical, histologic, genetic, and molecular profiles linked to long-term outcomes. The NEPTUNE Knowledge Network, comprising combined, multiscalar data sets, captures each participant's molecular disease processes at the time of kidney biopsy. In this editorial, we describe the design and implementation of NEPTUNE Match, which bridges a basic science discovery pipeline with targeted clinical trials. Noninvasive biomarkers have been developed for real-time pathway analyses. A Molecular Nephrology Board reviews the pathway maps together with clinical, laboratory, and histopathologic data assembled for each patient to compile a Match report that estimates the fit between the specific molecular disease pathway(s) identified in an individual patient and proposed clinical trials. The NEPTUNE Match report is communicated using established protocols to the patient and the attending nephrologist for use in their selection of available clinical trials. NEPTUNE Match represents the first application of precision medicine in nephrology with the aim of developing targeted therapies and providing the right medication for each patient with primary glomerular disease.

Association of Fibroblast Growth Factor 23 with Blood Pressure in Primary Proteinuric Glomerulopathies

Introduction: Fibroblast growth factor 23 (FGF23) has direct effects on the vasculature and myocardium, and high levels of FGF23 are a risk factor for cardiovascular disease (CVD); however, the impact of FGF23 on CVD in primary proteinuric glomerulopathies has not been addressed. Methods: The associations of baseline plasma intact FGF23 levels with resting blood pressure (BP) and lipids over time among adults and children with proteinuric glomerulopathies enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) were analyzed using generalized estimating equation regression analyses. Models were adjusted for age, sex, glomerular diagnosis, follow-up time, estimated glomerular filtration rate, urine protein/creatinine ratio, obesity, and serum phosphorous levels. Results: Two hundred and four adults with median FGF23 77.5 (IQR 51.3–119.3) pg/mL and 93 children with median FGF23 62.3 (IQR 44.6–83.6) pg/mL were followed for a median of 42 (IQR 20.5–54) months. In adjusted models, each 1 µg/mL increase in FGF23 was associated with a 0.3 increase in systolic BP index at follow-up (p < 0.001). Greater baseline FGF23 was associated with greater odds of hypertensive BP (OR = 1.0003; 95% CI 1.001–1.006, p = 0.03) over time. Compared to tertile 1, tertile 2 (OR = 2.1; 95% CI 1.12–3.99, p = 0.02), and tertile 3 (OR = 3; 95% CI 1.08–8.08, p = 0.04), FGF23 levels were associated with greater odds of hypertensive BP over time. Tertile 2 was associated with greater triglycerides compared to tertile 1 (OR = 48.1; 95% CI 4.4–91.9, p = 0.03). Conclusion: Overall, higher baseline FGF23 was significantly associated with hypertensive BP over time in individuals with proteinuric glomerulopathies. Further study of FGF23 as a therapeutic target for reducing CVD in proteinuric glomerular disease is warranted.

Histologic and Molecular Correlates in Patients with AL Amyloidosis in Hematologic Remission but with Persistent Renal Disease

ABSTRACT: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2b randomized placebo-controlled, clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. Before drug administration, renal biopsies were taken and analyzed. OBJECTIVE: Identifying amyloid-specific molecular signatures in kidney biopsy tissue. MATERIALS &amp; METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Clinical data was collected and correlated to histologic and molecular findings. Histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial (TI) compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined . RESULTS: Ten patients were enrolled, randomized and treated in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and TI compartments ( See Figure 1). Key clinical data pertaining to kidney function and biopsy morphology by compartment and cluster type were documented. Despite similar eGFR and degree of proteinuria, there was a significantly higher AS score in the TI compartment (6.5 vs. 4.5; p=0.0290) of G2 which was driven most entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI compartment. Other enriched ingenuity canonical pathways to note in the TI included ‘coagulation system’, ‘GADD45 signaling’, ‘Wnt/Ca+ pathway’, ‘intrinsic prothrombin activation pathway’ among others. Of note, genes like BRCA and CDKN1A were enriched between G1 and G2, both of which have been associated with molecular mechanisms that underly certain neoplasms ( See Figure 2). Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli, there was increased signaling of TNF, NFKB, IL33, IL-8 and IL15, consistent with an important inflammatory response. There was also an enrichment in pathways related to migration, adhesion and integrin signaling. PI3K/Akt signaling was also noted. Tubules showed increased activation of transcriptional regulators TP53, TP73 and PI3K/AKT signaling. Some gene regulators of cellular proliferation such as ABL1, BCL2 and AKT1 were found to be enriched in the amyloid group. CONCLUSIONS: While the RAIN trial closed prematurely and NEOD001 cannot be evaluated, the molecular data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in of themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Based on this early work, targeting the PI3K/Akt pathway may be a worthy pursuit in this disease.

The Significance of Hematuria in Podocytopathies.

Hematuria is frequently present in podocytopathies, but its significance and prognostic value is not well described in these proteinuric kidney diseases. This study describes the prevalence and association between hematuria and kidney-related outcomes in these disorders. Hematuria was assessed at the initial urinalysis in participants with the following podocytopathies-membranous nephropathy, minimal change disease, and FSGS-in the Nephrotic Syndrome Study Network and Cure Glomerulonephropathy cohorts with >24 months of follow-up. Multivariable Cox proportional hazards models were fit for time to composite outcome (kidney failure or 40% decline in eGFR and eGFR <60 ml/min per 1.73 m 2 ) and proteinuria remission (urine protein-to-creatinine ratio [UPCR] <0.3 mg/mg). Among the 1516 adults and children in the study, 528 participants (35%) had FSGS, 499 (33%) had minimal change disease, and 489 (32%) had membranous nephropathy. Median (interquartile range) time from biopsy until the initial study urinalysis was 260 (49-750) days, and 498 participants (33%) were positive for hematuria. Participants with hematuria compared with those without were older (37 [16-55] versus 33 [12-55] years), more likely to have an underlying diagnosis of membranous nephropathy (44% versus 27%), had shorter time since biopsy (139 [27-477] versus 325 [89-878] days), and had higher UPCR (3.8 [1.4-8.0] versus 0.9 [0.1-3.1] g/g). After adjusting for diagnosis, age, sex, UPCR, eGFR, time since biopsy, and study cohort, hematuria was associated with a higher risk of reaching the composite outcome (hazard ratio, 1.31; 95% confidence interval, 1.04 to 1.65; P value, 0.02) and lower rate of reaching proteinuria remission (hazard ratio, 0.80; 95% confidence interval, 0.65 to 0.98; P value, 0.03). Hematuria is prevalent among participants with the three podocytopathies and is significantly and independently associated with worse kidney-related outcomes, including both progressive loss of kidney function and remission of proteinuria.

External Validation of a Urinary Biomarker Risk Score for the Prediction of Steroid Responsiveness in Adults With Nephrotic Syndrome

In idiopathic nephrotic syndrome, response to corticosteroids remains the best indicator of prognosis. Noninvasive markers to predict a patient's response to steroids would allow improved prognostication and a more personalized approach to management. We have previously derived a urinary biomarker risk score which can differentiate steroid sensitive nephrotic syndrome (SSNS) from steroid resistant nephrotic syndrome (SRNS) in children. The goal of this study was to validate this previously derived biomarker risk score in a cohort of steroid-naïve adult patients, to determine whether the panel could be used to predict steroid responsiveness at the time of initial diagnosis. In this external validation study, clinical data, and urinary specimens (obtained before initiation of steroid treatment) from adult patients were used in the Nephrotic Syndrome Study Network (NEPTUNE) cohort. A panel of 5 previously identified and validated urinary biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), vitamin D binding protein (VDBP), Fetuin-A (FetA), Transthyretin (TTR), and alpha-1 acid glycoprotein 2 (AGP2) was measured. A summary risk score for steroid resistance was calculated based on biomarker concentrations. Receiver operating characteristic curves were created for each log-transformed biomarker concentration and for the individual and combined biomarker risk score. The urine biomarker risk score predicted development of steroid resistance, with optimal sensitivity and specificity of 0.74, and area under the receiver operating characteristic curve (AUC) of 0.79 using both absolute and creatinine-corrected concentrations. This study validates the previously derived urinary biomarker risk score to predict steroid resistance in adult patients with nephrotic syndrome at initial diagnosis.

Assessment of the Needs of Nephrology Divisions to Implement Return of Clinically Significant Research Genetic Results: A Survey of Nephrotic Syndrome Study Network (NEPTUNE) Investigators

Introduction: There is an increasing need to return genetic testing results to patients with kidney disease who were first genotyped on a research basis. Operationalizing this process in nephrology clinics is challenged by a limited number of genetic providers with whom to partner and a general lack of support services for all clinicians. Methods: We administered a survey in March 2022 to assess the current ability and ongoing needs of nephrology divisions to return clinically significant research genetic results to patients and to implement clinical genetic testing. This survey was distributed to institutions within the Nephrotic Syndrome Study Network (NEPTUNE) as part of the planning process for return of research genetic results to participants with pathogenic variants in Mendelian nephrotic syndrome genes. Results: Twenty-seven of 28 sites (96%) completed the survey. 59% (n = 16) of sites said they could handle return of research genetic results independently, with the rest expressing hesitation about the volume and complexity of patients and the limited resources and access to genetics services. 81% (n = 22) of these institutions did have a genetics clinic and 26% (n = 7) have a nephrology genetics clinic. However, 70% (n = 10) of these clinics have a waiting time over 1 month. 89% of divisions (n = 24) were conducting genetic testing and 96% of those (n = 23) used a kidney multi-gene panel. In 46% of divisions (n = 11), nephrologists were handling logistics of obtaining genetic testing samples themselves. Conclusion: We identified specific areas of support needed for return of clinically significant genetic results from research studies. While the surveyed nephrologists were conducting genetic testing, there were limitations in the support services available. This survey will help guide other research studies that wish to return genetic results to participants and also highlight the need for increasing support to effectively operationalize genetic testing in nephrology clinics.

The association of low birthweight and prematurity on outcomes in children and adults with nephrotic syndrome-a NEPTUNE cohort study.

In single-center studies, both preterm birth and low birth weight (LBW) are associated with worse outcomes in childhood nephrotic syndrome. Using the Nephrotic Syndrome Study Network (NEPTUNE) observational cohort, we tested the hypothesis that in patients with nephrotic syndrome, hypertension, proteinuria status, and disease progression would be more prevalent and more severe in subjects with LBW and prematurity singly or in combination (LBW/prematurity). Three hundred fifty-nine adults and children with focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD) and available birth history were included. Estimated glomerular filtration rate (eGFR) decline and remission status were primary outcomes, and secondary outcomes were kidney histopathology, kidney gene expression, and urinary biomarkers. Logistic regression was used to identify associations with LBW/prematurity and these outcomes. We did not find an association between LBW/prematurity and remission of proteinuria. However, LBW/prematurity was associated with greater decline in eGFR. This decline in eGFR was partially explained by the association of LBW/prematurity with APOL1 high-risk alleles, but the association remained after adjustment. There were no differences in kidney histopathology or gene expression in the LBW/prematurity group compared to normal birth weight/term birth. LBW and premature babies who develop nephrotic syndrome have a more rapid decline in kidney function. We did not identify clinical or laboratory features that distinguished the groups. Additional studies in larger groups are needed to fully ascertain the effects of (LBW) and prematurity alone or in combination on kidney function in the setting of nephrotic syndrome.

Tobacco exposure in adults and children with proteinuric glomerulopathies: a NEPTUNE cohort study

BackgroundTobacco exposure has been recognized as a risk factor for cardiovascular disease (CVD) and progression of kidney disease. Patients with proteinuric glomerulopathies are at increased risk for cardiovascular morbidity and mortality. Multiple studies have linked tobacco exposure to CVD and chronic kidney disease, but the relationships between smoking and proteinuric glomerulopathies in adults and children have not been previously explored.MethodsData from the Nephrotic Syndrome Study Network (NEPTUNE), a multi-center prospective observational study of participants with proteinuric glomerulopathies, was analyzed. 371 adults and 192 children enrolled in NEPTUNE were included in the analysis. Self-reported tobacco exposure was classified as non-smoker, active smoker, former smoker, or exclusive passive smoker. Baseline serum cotinine levels were measured in a sub-cohort of 178 participants.ResultsThe prevalence of active smokers, former smokers and exclusive passive smoking among adults at baseline was 14.6%, 29.1% and 4.9%, respectively. Passive smoke exposure was 16.7% among children. Active smoking (reference non-smoking) was significantly associated with greater total cholesterol among adults (β 17.91 95% CI 0.06, 35.76, p = 0.049) while passive smoking (reference non-smoking) was significantly associated with greater proteinuria over time among children (β 1.23 95% CI 0.13, 2.33, p = 0.03). Higher cotinine levels were associated with higher baseline eGFR (r = 0.17, p = 0.03).ConclusionTobacco exposure is associated with greater risk for CVD and worse kidney disease outcomes in adults and children with proteinuric glomerulopathies. Preventive strategies to reduce tobacco exposure may help protect against future cardiovascular and kidney morbidity and mortality in patients with proteinuric glomerulopathies.

Identification of Glomerular and Plasma Apolipoprotein M as Novel Biomarkers in Glomerular Disease

Dysregulation of sphingolipid and cholesterol metabolism contributes to the pathogenesis of glomerular diseases (GDs). Apolipoprotein M (ApoM) promotes cholesterol efflux and modulates the bioactive sphingolipid sphingosine-1-phosphate (S1P). Glomerular ApoM expression is decreased in patients with focal segmental glomerulosclerosis (FSGS). We hypothesized that glomerular ApoM deficiency occurs in GD and that ApoM expression and plasma ApoM correlate with outcomes. Patients with GD from the Nephrotic Syndrome Study Network (NEPTUNE) were studied. We compared glomerular mRNA expression of ApoM (gApoM), sphingosine kinase 1 (SPHK1), and S1P receptors 1 to 5 (S1PR1-5) in patients (n= 84) and controls (n= 6). We used correlation analyses to determine associations between gApoM, baseline plasma ApoM (pApoM), and urine ApoM (uApoM/Cr). We used linear regression to determine whether gApoM, pApoM, and uApoM/Cr were associated with baseline estimated glomerular filtration rate (eGFR) and proteinuria. Using Cox models, we determined whether gApoM, pApoM, and uApoM/Cr were associated with complete remission (CR) and the composite of end-stage kidney disease (ESKD) or≥40% eGFR decline. gApoM was reduced (P< 0.01) and SPHK1 and S1PR1 to 5 expression was increased (P< 0.05) in patients versus controls, consistent with ApoM/S1P pathway modulation. gApoM positively correlated with pApoM in the overall cohort (r= 0.34, P< 0.01) and in the FSGS (r= 0.48, P< 0.05) and minimal change disease (MCD) (r= 0.75, P< 0.05) subgroups. Every unit decrease in gApoM and pApoM (log2) was associated with a 9.77 ml/min per 1.73 m2 (95% confidence interval [CI]: 3.96-15.57) and 13.26 ml/min per 1.73 m2 (95% CI: 3.57-22.96) lower baseline eGFR, respectively (P< 0.01). From Cox models adjusted for age, sex, or race, pApoM was a significant predictor of CR (hazard ratio [HR]: 1.85; 95% CI: 1.06-3.23). pApoM is a potential noninvasive biomarker of gApoM deficiency and strongly associates with clinical outcomes in GD.

Ridge Regression for Functional Form Identification of Continuous Predictors of Clinical Outcomes in Glomerular Disease

Introduction: Penalized regression models can be used to identify and rank risk factors for poor quality of life or other outcomes. They often assume linear covariate associations, but the true associations may be nonlinear. There is no standard, automated method for determining optimal functional forms (shapes of relationships) between predictors and the outcome in high-dimensional data settings. Methods: We propose a novel algorithm, ridge regression for functional form identification of continuous predictors (RIPR) that models each continuous covariate with linear, quadratic, quartile, and cubic spline basis components in a ridge regression model to capture potential nonlinear relationships between continuous predictors and outcomes. We used a simulation study to test the performance of RIPR compared to standard and spline ridge regression models. Then, we applied RIPR to identify top predictors of Patient-Reported Outcomes Measurement Information System (PROMIS) adult global mental and physical health scores using demographic and clinical characteristics among N = 107 glomerular disease patients enrolled in the Nephrotic Syndrome Study Network (NEPTUNE). Results: RIPR resulted in better predictive accuracy than the standard and spline ridge regression methods in 56–80% of simulation repetitions under a variety of data characteristics. When applied to PROMIS scores in NEPTUNE, RIPR resulted in the lowest error for predicting physical scores, and the second-lowest error for mental scores. Further, RIPR identified hemoglobin quartiles as an important predictor of physical health that was missed by the other models. Conclusion: The RIPR algorithm can capture nonlinear functional forms of predictors that are missed by standard ridge regression models. The top predictors of PROMIS scores vary greatly across methods. RIPR should be considered alongside other machine learning models in the prediction of patient-reported outcomes and other continuous outcomes.

Gene Expression Sets and Renal Profiling from the Rain (Renal AL Amyloid Involvement and NEOD00) Trial

INTRODUCTION: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2B randomized placebo-controlled, open-label clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. OBJECTIVES: Identifying amyloid-specific molecular signatures in kidney biopsy tissue obtained from AL patients with nephropathy.MATERIALS& METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Trial clinical data was incorporated to link tissue, molecular, and kidney function data. The histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined with the R limma package based on the voom transformed log2 count data with an FDR of <=0.05. The AL and NEPTUNE datasets were merged and batch-corrected using ComBat. RESULTS: Ten patients were enrolled and randomized in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and tubulointerstitial compartments. Key data of kidney function and biopsy morphology by compartment and cluster type are shown in Table 1. Despite similar eGFR, there was a higher AS and a non-significant increase in 24-hour urine protein in the glomeruli of individuals in the G2 patient cluster. Subcomponent analysis of AS revealed that the increase in amyloid score in G2 was driven almost entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli of biopsies from the G2 cluster, there was evidence of increased TNF, IFNG, IL33 and VEGFA cytokine activity and a predicted increase in canonical PI3K/Akt signaling and innate immune responses. Tubules showed increased activation of transcriptional regulators TP53, TP73, increased cytokine signaling of IFNG, TGFb1 and VEGF. There was also a predicted activation of major canonical pathways including B cell receptor signaling, PI3K/AKT and Ephrin receptor signaling (Fig 1). Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 mediated inhibition of RXR function that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI. CONCLUSIONS: The RAIN trial closed prematurely, and thus no conclusions can be reached about the efficacy of NEOD001 in patients with AL nephropathy. However, these limited data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T-cell receptor diversity in minimal change disease in the NEPTUNE study.

Minimal change disease (MCD) is the major cause of childhood idiopathic nephrotic syndrome, which is characterized by massive proteinuria and debilitating edema. Proteinuria in MCD is typically rapidly reversible with corticosteroid therapy, but relapses are common, and children often have many adverse events from the repeated courses of immunosuppressive therapy. The pathobiology of MCD remains poorly understood. Prior clinical observations suggest that abnormal T-cell function may play a central role in MCD pathogenesis. Based on these observations, we hypothesized that T-cell responses to specific exposures or antigens lead to a clonal expansion of T-cell subsets, a restriction in the T-cell repertoire,and anelaboration of specific circulating factors that trigger disease onset and relapses. To test these hypotheses, we sequenced T-cell receptors in fourteen MCD, four focal segmental glomerulosclerosis (FSGS), and four membranous nephropathy (MN) patients with clinical data and blood samples drawn during active disease and during remission collected by the Nephrotic Syndrome Study Network(NEPTUNE). We calculated several T-cell receptor diversity metrics to assess possible differences between active disease and remission states in paired samples. Median productive clonality did not differ between MCD active disease (0.0083; range: 0.0042, 0.0397) and remission (0.0088; range: 0.0038, 0.0369). We did not identify dominant clonotypes in MCD active disease, and few clonotypes were shared with FSGS and MN patients. While these data do not support an obvious role of the adaptive immune system T-cells in MCD pathogenesis, further study is warranted given the limited sample size. A higher resolution version of the Graphical abstract is available as Supplementary information.

Kidney Biopsy Features Most Predictive of Clinical Outcomes in the Spectrum of Minimal Change Disease and Focal Segmental Glomerulosclerosis.

Heterogeneity in disease course and treatment response among patients with MCD/FSGS necessitates a granular evaluation of kidney tissue features. This study aimed to identify histologic and ultrastructural descriptors of structural changes most predictive of clinical outcomes in the Nephrotic Syndrome Study Network (NEPTUNE). Forty-eight histologic (37 glomerular, 9 tubulointerstitial, 2 vascular) and 20 ultrastructural descriptors were quantified by applying the NEPTUNE Digital Pathology Scoring System to NEPTUNE kidney biopsies. Outcomes included time from biopsy to disease progression, first complete remission of proteinuria, and treatment response. Relative importance of pathology and clinical predictors was obtained from random forest models, and predictive discrimination was assessed. Among 224 participants (34% Black, 24% Hispanic), model performance was excellent, with predictive discrimination of 0.9 for disease progression, 0.85 for complete remission, and 0.81 for treatment response. The most predictive descriptors of outcomes included both conventional-e.g., global sclerosis or segmental sclerosis and interstitial fibrosis/tubular atrophy-and novel features, including adhesion, interstitial foam cells, deflation, periglomerular fibrosis, mononuclear white blood cells, endothelial cell abnormalities, microvillous transformation, and acute tubular injury. The most predictive descriptors of clinical outcomes among MCD/FSGS patients reflected structural changes in multiple renal compartments. Reporting these descriptors should be standardized to guide prognostication of proteinuric glomerular diseases.

Molecular Characterization of Membranous Nephropathy.

Molecular characterization of nephropathies may facilitate pathophysiologic insight, development of targeted therapeutics, and transcriptome-based disease classification. Although membranous nephropathy (MN) is a common cause of adult-onset nephrotic syndrome, the molecular pathways of kidney damage in MN require further definition. We applied a machine-learning framework to predict diagnosis on the basis of gene expression from the microdissected kidney tissue of participants in the Nephrotic Syndrome Study Network (NEPTUNE) cohort. We sought to identify differentially expressed genes between participants with MN versus those of other glomerulonephropathies across the NEPTUNE and European Renal cDNA Bank (ERCB) cohorts, to find MN-specific gene modules in a kidney-specific functional network, and to identify cell-type specificity of MN-specific genes using single-cell sequencing data from reference nephrectomy tissue. Glomerular gene expression alone accurately separated participants with MN from those with other nephrotic syndrome etiologies. The top predictive classifier genes from NEPTUNE participants were also differentially expressed in the ERCB participants with MN. We identified a signature of 158 genes that are significantly differentially expressed in MN across both cohorts, finding 120 of these in a validation cohort. This signature is enriched in targets of transcription factor NF-κB. Clustering these MN-specific genes in a kidney-specific functional network uncovered modules with functional enrichments, including in ion transport, cell projection morphogenesis, regulation of adhesion, and wounding response. Expression data from reference nephrectomy tissue indicated 43% of these genes are most highly expressed by podocytes. These results suggest that, relative to other glomerulonephropathies, MN has a distinctive molecular signature that includes upregulation of many podocyte-expressed genes, provides a molecular snapshot of MN, and facilitates insight into MN's underlying pathophysiology.

POS-359 Developing targeted therapies for kidney disease in public private partnerships

Integrating public and private research efforts is key to expediting precision medicine for glomerular diseases. The Nephrotic Syndrome Study Network (NEPTUNE) has developed an innovative research and business model: “MATCH”, to enable targeted patient enrollment in clinical trials via the NEPTUNE public private partnership (NP5). The NEPTUNE community knowledge base is leveraged to identify a molecular signal matching therapeutic targets under development by NP5 industry partners. The three steps of MATCH are: (1) predictive biomarker development; (2)patient communication about precision medicine; and (3) return of results from NP5 partner clinical trials for evaluation of patient stratification effects on trial outcomes. MATCH begins with NEPTUNE patient education on risks and benefits of the study. Once enrolled in MATCH, a patient’s biosamples undergo biomarker profiling. MATCH provides individual pathway activation information to patients for consideration in their independent decision to enroll in a trial that may be more tailored to their specific disease and stage. Finally, clinical trial outcomes will be reported by NP5 partners to NEPTUNE to evaluate the MATCH impact on treatment responses. MATCH aims to assess the ability to communicate molecular information to patients, and to test precision medicine across multiple trials and partners to move science closer to the overall goal: reaching the right patient with the right medicine at the right time. MATCH is an open framework available to additional partners interested in joining NP5.

Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.

Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell-targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

Quantification of Glomerular Structural Lesions: Associations With Clinical Outcomes and Transcriptomic Profiles in Nephrotic Syndrome

The current classification system for focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) does not fully capture the complex structural changes in kidney biopsies nor the clinical and molecular heterogeneity of these diseases. Prospective observational cohort study. 221 MCD and FSGS patients enrolled in the Nephrotic Syndrome Study Network (NEPTUNE). The NEPTUNE Digital Pathology Scoring System (NDPSS) was applied to generate scores for 37 glomerular descriptors. Time from biopsy to complete proteinuria remission, time from biopsy to kidney disease progression (40% estimated glomerular filtration rate [eGFR] decline or kidney failure), and eGFR over time. Cluster analysis was used to group patients with similar morphologic characteristics. Glomerular descriptors and patient clusters were assessed for associations with outcomes using adjusted Cox models and linear mixed models. Messenger RNA from glomerular tissue was used to assess differentially expressed genes between clusters and identify genes associated with individual descriptors driving cluster membership. Three clusters were identified: X (n= 56), Y (n= 68), and Z (n= 97). Clusters Y and Z had higher probabilities of proteinuria remission (HRs of 1.95 [95% CI, 0.99-3.85] and 3.29 [95% CI, 1.52-7.13], respectively), lower hazards of disease progression (HRs of 0.22 [95% CI, 0.08-0.57] and 0.11 [95% CI, 0.03-0.45], respectively), and lower loss of eGFR over time compared with X. Cluster X had 1,920 genes that were differentially expressed compared with Y+Z; these reflected activation of pathways of immune response and inflammation. Six descriptors driving the clusters individually correlated with clinical outcomes and gene expression. Low prevalence of some descriptors and biopsy at a single time point. The NDPSS allows for categorization of FSGS/MCD patients into clinically and biologically relevant subgroups, and uncovers histologic parameters associated with clinical outcomes and molecular signatures not included in current classification systems.