Jonathan P. Troost

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 hours. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multi-center observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared to other diagnoses. Thus, these results do not support a pathological role for suPAR in FSGS.

Integrative Genomics Identifies Novel Associations with APOL1 Risk Genotypes in Black NEPTUNE Subjects

APOL1 variants have been associated with renal phenotypes in blacks. To refine clinical outcomes and discover mechanisms of APOL1-associated kidney injury, we analyzed clinical and genomic datasets derived from 90 black subjects in the Nephrotic Syndrome Study Network (NEPTUNE), stratified by APOL1 risk genotype. Ninety subjects with proteinuria ≥0.5 g/d were enrolled at first biopsy for primary nephrotic syndrome and followed. Clinical outcomes were determined, and renal histomorphometry and sequencing of Mendelian nephrotic syndrome genes were performed. APOL1 variants were genotyped, and glomerular and tubulointerstitial transcriptomes from protocol renal biopsy cores were analyzed for differential and correlative gene expression. Analyses were performed under the recessive model (high-risk genotype defined by two risk alleles). APOL1 high-risk genotype was significantly associated with a 17 ml/min per 1.73 m(2) lower eGFR and a 69% reduction in the probability of complete remission at any time, independent of histologic diagnosis. Neither APOL1 risk group was enriched for Mendelian mutations. On renal biopsy, high-risk genotype was associated with increased fractional interstitial area, interstitial fibrosis, and tubular atrophy. Risk genotype was not associated with intrarenal APOL1 mRNA expression levels. Differential expression analysis demonstrated an increased steady-state level of five genes associated with the high-risk genotype (CXCL9, CXCL11, and UBD in glomerulus; SNOR14B and MUC13 in tubulointerstitium). APOL1 tubulointerstitial coexpression analysis showed coexpression of APOL1 mRNA levels with a group of intrarenal transcripts that together were associated with increased interstitial fibrosis and tubular atrophy. These data indicate the high-risk APOL1 genotype confers renal risk across histopathologic diagnoses.

Complete Remission in the Nephrotic Syndrome Study Network

BACKGROUND AND OBJECTIVES This analysis from the Nephrotic Syndrome Study Network (NEPTUNE) assessed the phenotypic and pathology characteristics of proteinuric patients undergoing kidney biopsy and defined the frequency and factors associated with complete proteinuria remission (CRever). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We enrolled adults and children with proteinuria ≥0.5 g/d at the time of first clinically indicated renal biopsy at 21 sites in North America from April 2010 to June 2014 into a prospective cohort study. NEPTUNE central pathologists assigned participants to minimal-change disease (MCD), FSGS, membranous nephropathy, or other glomerulopathy cohorts. Outcome measures for this analysis were (1) CRever with urine protein-to-creatinine ratio (UPC) <0.3 g/g with preserved native kidney function and (2) ESRD. Continuous variables are reported as median and interquartile range (IQR; 25th, 75th percentile). Cox proportional hazards modeling was used to assess factors associated with CRever. RESULTS We enrolled 441 patients: 116 (27%) had MCD, 142 (32%) had FSGS, 66 (15%) had membranous nephropathy, and 117 (27%) had other glomerulopathy. The baseline UPC was 4.1 g/g (IQR, 1.9, 7.7) and the eGFR was 81 ml/min per 1.73 m(2) (IQR, 50, 105). Median duration of observation was 19 months (IQR, 11, 30). CRever occurred in 46% of patients, and 4.6% progressed to ESRD. Multivariate analysis demonstrated that higher prebiopsy proteinuria (hazard ratio, 0.3; 95% confidence interval, 0.2 to 0.5) and pathology diagnosis (FSGS versus MCD; hazard ratio, 0.2; 95% confidence interval, 0.1 to 0.5) were inversely associated with CRever. The effect of immunosuppressive therapy on remission varied by pathology diagnosis. CONCLUSIONS In NEPTUNE, the high frequency of other pathology in proteinuric patients affirms the value of the diagnostic kidney biopsy. Clinical factors, including level of proteinuria before biopsy, pathology diagnosis, and immunosuppression, are associated with complete remission.

The Application of Digital Pathology to Improve Accuracy in Glomerular Enumeration in Renal Biopsies.

Background In renal biopsy reporting, quantitative measurements, such as glomerular number and percentage of globally sclerotic glomeruli, is central to diagnostic accuracy and prognosis. The aim of this study is to determine the number of glomeruli and percent globally sclerotic in renal biopsies by means of registration of serial tissue sections and manual enumeration, compared to the numbers in pathology reports from routine light microscopic assessment. Design We reviewed 277 biopsies from the Nephrotic Syndrome Study Network (NEPTUNE) digital pathology repository, enumerating 9,379 glomeruli by means of whole slide imaging. Glomerular number and the percentage of globally sclerotic glomeruli are values routinely recorded in the official renal biopsy pathology report from the 25 participating centers. Two general trends in reporting were noted: total number per biopsy or average number per level/section. Both of these approaches were assessed for their accuracy in comparison to the analogous numbers of annotated glomeruli on WSI. Results The number of glomeruli annotated was consistently higher than those reported (p<0.001); this difference was proportional to the number of glomeruli. In contrast, percent globally sclerotic were similar when calculated on total glomeruli, but greater in FSGS when calculated on average number of glomeruli (p<0.01). The difference in percent globally sclerotic between annotated and those recorded in pathology reports was significant when global sclerosis is greater than 40%. Conclusions Although glass slides were not available for direct comparison to whole slide image annotation, this study indicates that routine manual light microscopy assessment of number of glomeruli is inaccurate, and the magnitude of this error is proportional to the total number of glomeruli.

Reproducibility of the NEPTUNE descriptor-based scoring system on whole-slide images and histologic and ultrastructural digital images

The multicenter Nephrotic Syndrome Study Network (NEPTUNE) digital pathology scoring system employs a novel and comprehensive methodology to document pathologic features from whole-slide images, immunofluorescence and ultrastructural digital images. To estimate inter- and intra-reader concordance of this descriptor-based approach, data from 12 pathologists (eight NEPTUNE and four non-NEPTUNE) with experience from training to 30 years were collected. A descriptor reference manual was generated and a webinar-based protocol for consensus/cross-training implemented. Intra-reader concordance for 51 glomerular descriptors was evaluated on jpeg images by seven NEPTUNE pathologists scoring 131 glomeruli three times (Tests I, II, and III), each test following a consensus webinar review. Inter-reader concordance of glomerular descriptors was evaluated in 315 glomeruli by all pathologists; interstitial fibrosis and tubular atrophy (244 cases, whole-slide images) and four ultrastructural podocyte descriptors (178 cases, jpeg images) were evaluated once by six and five pathologists, respectively. Cohens kappa for inter-reader concordance for 48/51 glomerular descriptors with sufficient observations was moderate (0.40<kappa≤0.60) for 17 and good (0.60<kappa≤0.80) for 8, for 52% with moderate or better kappas. Clustering of glomerular descriptors based on similar pathologic features improved concordance. Concordance was independent of years of experience, and increased with webinar cross-training. Excellent concordance was achieved for interstitial fibrosis and tubular atrophy. Moderate-to-excellent concordance was achieved for all ultrastructural podocyte descriptors, with good-to-excellent concordance for descriptors commonly used in clinical practice, foot process effacement, and microvillous transformation. NEPTUNE digital pathology scoring system enables novel morphologic profiling of renal structures. For all histologic and ultrastructural descriptors tested with sufficient observations, moderate-to-excellent concordance was seen for 31/54 (57%). Descriptors not sufficiently represented will require further testing. This study proffers the NEPTUNE digital pathology scoring system as a model for standardization of renal biopsy interpretation extendable outside the NEPTUNE consortium, enabling international collaborations.

Patient-Reported Outcomes in Glomerular Disease

Incorporation of the patient perspective into research and clinical practice will enrich our understanding of the status and management of patients with glomerular disease and may result in therapies that better address patient needs. In recent years, the importance of the patient experience of glomerular disease has become clear, and significant efforts have been undertaken to systematically capture and describe the patients disease experience. Patient-reported outcome instruments provide a means to assess the patients experience in a quantitative manner, thus enabling for comparisons within and between patients. Patient-reported outcome assessments are solely on the basis of a patient report about the status of their health without amendment or interpretation by a clinician or others. Patient-reported outcome assessments provide an opportunity to incorporate the patient perspective into clinical care, research, and clinical trials. Our paper provides an overview of terminology and development methods for patient-reported outcomes and reviews (1) currently available patient-reported outcome instruments appropriate for use in glomerular disease, (2) existing patient-reported outcome data in glomerular disease, and (3) opportunities for incorporating patient-reported outcome instruments into clinical care and research.

Interstitial fibrosis scored on whole-slide digital imaging of kidney biopsies is a predictor of outcome in proteinuric glomerulopathies

Background Interstitial fibrosis (IF), tubular atrophy (TA) and interstitial inflammation (II) are known determinants of progression of renal disease. Standardized quantification of these features could add value to current classification of glomerulopathies. Methods We studied 315 participants in the Nephrotic Syndrome Study Network (NEPTUNE) study, including biopsy-proven minimal change disease (MCD = 98), focal segmental glomerulosclerosis (FSGS = 121), membranous nephropathy (MN = 59) and IgA nephropathy (IgAN = 37). Cortical IF, TA and II were quantified (%) on digitized whole-slide biopsy images, by five pathologists with high inter-reader agreement (intra-class correlation coefficient >0.8). Tubulointerstitial messenger RNA expression was measured in a subset of patients. Multivariable Cox proportional hazards models were fit to assess association of IF with the composite of 40% decline in estimated glomerular filtration rate (eGFR) and end-stage renal disease (ESRD) and separately as well, and with complete remission (CR) of proteinuria. Results IF was highly correlated with TA (P < 0.001) and II (P < 0.001). Median IF varied by diagnosis: FSGS 17, IgAN 21, MN 7, MCD 1 (P < 0.001). IF was strongly correlated with baseline eGFR (P < 0.001) and proteinuria (P = 0.002). After adjusting for clinical pathologic diagnosis, age, race, global glomerulosclerosis, baseline proteinuria, eGFR and medications, each 10% increase in IF was associated with a hazard ratio of 1.29 (P < 0.03) for ESRD/40% eGFR decline, but was not significantly associated with CR. A total of 981 genes were significantly correlated with IF (|r| > 0.4, false discovery rate (FDR) < 0.01), including upstream regulators such as tumor necrosis factor, interferon gamma (IFN-gamma), and transforming growth factor beta 1 (TGF-B1), and signaling pathways for antigen presentation and hepatic fibrosis. Conclusions The degree of IF is associated with risk of eGFR decline across different types of proteinuric glomerulopathy, correlates with inflammatory and fibrotic gene expression, and may have predictive value in assessing risk of progression.

Changing the Paradigm for the Treatment and Development of New Therapies for FSGS

Focal segmental glomerulosclerosis (FSGS) is a renal pathology finding that represents a constellation of rare kidney diseases, which manifest as proteinuria, edema nephrotic syndrome, hypertension, and increased risk for kidney failure. Therapeutic options for FSGS are reviewed displaying the expected efficacy from 25 to 69% depending on specific therapy, patient characteristics, cost, and common side effects. This variability in treatment response is likely caused, in part, by the heterogeneity in the etiology and active molecular mechanisms of FSGS. Clinical trials in FSGS have been scant in number and slow to recruit, which may stem, in part, from reliance on classic clinical trial design paradigms. Traditional clinical trial designs based on the “learn and confirm” paradigm may not be appropriate for rare diseases, such as FSGS. Future drug development and testing will require novel approaches to trial designs that have the capacity to enrich study populations and adapt the trial in a planned way to gain efficiencies in trial completion timelines. A clinical trial simulation is provided that compares a classical and more modern design to determine the maximum tolerated dose in FSGS.

Global glomerulosclerosis with nephrotic syndrome; the clinical importance of age adjustment

Globally sclerotic glomeruli (GSG) occur with both normal aging and kidney disease. However, it is unknown whether any GSG or only GSG exceeding that expected for age is clinically important. To evaluate this, we identified patients with a glomerulopathy that often presents with nephrotic syndrome (focal segmental glomerulosclerosis, membranous nephropathy, or minimal change disease) in the setting of the Nephrotic Syndrome Study Network (NEPTUNE), China-Digital Kidney Pathology (DiKiP), and the Southeast Minnesota cohorts. Age-based thresholds (95th percentile) for GSG based on normotensive living kidney donors were used to classify each patient into one of three groups; no GSG, GSG normal for age, or GSG abnormal for age. The risk of end-stage renal disease or a 40% decline in glomerular filtration rate during follow-up was then compared between groups. Among the 425 patients studied, 170 had no GSG, 107 had GSG normal for age, and 148 had GSG abnormal for age. Compared to those with no GSG, the risk of kidney disease progression with GSG normal for age was similar but was significantly higher with GSG abnormal for age. This increased risk with GSG abnormal for age remained significant after adjustment for interstitial fibrosis, arteriosclerosis, age, hypertension, diabetes, body mass index, glomerulopathy type, glomerular filtration rate, and proteinuria. Thus, in patients with glomerulopathy that often presents with nephrotic syndrome, global glomerulosclerosis is clinically important only if it exceeds that expected for age.

Hypertension and health outcomes in the PICU

Objectives: Reports of the burden of hypertension in hospitalized children are emerging, but the prevalence and significance of this condition within the PICU are not well understood. The aims of this study were to validate a definition of hypertension in the PICU and assess the associations between hypertension and acute kidney injury, PICU length of stay, and mortality. Design and Setting: Single-center retrospective study using a database of PICU discharges between July 2011 and February 2013. Patients: All children discharged from the PICU with length of stay more than 6 hours, aged 1 month through 17 years. Exclusions were traumatic brain injury, incident renal transplant, or hypotension. Interventions: None. Measurements and Main Results: Potential definitions of hypertension utilizing combinations of standardized cutoff percentiles, durations, initiation or dose escalation of antihypertensives, and/or billing diagnosis codes for hypertension were compared using receiver operator characteristic curves against a manual medical record review. Multivariable logistic and linear regression analyses were conducted using the selected definition of hypertension to assess its independent association with acute kidney injury and PICU length of stay, respectively. A definition requiring three systolic and/or diastolic readings above standardized 99th percentiles plus 5 mm Hg over 1 day was selected (area under the curve, 0.91; sensitivity, 94%; specificity, 87%). Among the 1,215 patients in this analysis, the prevalence of hypertension was 25%. Hypertension was independently associated with acute kidney injury (odds ratio, 2.89; 95% CI, 1.64–5.09; p < 0.01) and increased PICU length of stay (1.50 d; 95% CI, 0.94–2.05; p < 0.01) in multivariable analyses. Deaths were rare—0 in the normotension group and 3 (1%) in the hypertension group—but were statistically different (p = 0.02). Conclusions: Hypertension is common in the PICU and is associated with worse clinical outcomes. Future studies are needed to confirm these results.