Laura Barisoni

Design of the nephrotic syndrome study network (NEPTUNE) to evaluate primary glomerular nephropathy by a multidisciplinary approach

The Nephrotic Syndrome Study Network (NEPTUNE) is a North American multi-center collaborative consortium established to develop a translational research infrastructure for Nephrotic Syndrome. This includes a longitudinal observational cohort study, a pilot and ancillary studies program, a training program, and a patient contact registry. NEPTUNE will enroll 450 adults and children with minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy for detailed clinical, histopathologic, and molecular phenotyping at the time of clinically-indicated renal biopsy. Initial visits will include an extensive clinical history, physical examination, collection of urine, blood and renal tissue samples, and assessments of quality of life and patient-reported outcomes. Follow-up history, physical measures, urine and blood samples, and questionnaires will be obtained every 4 months in the first year and bi-annually, thereafter. Molecular profiles and gene expression data will be linked to phenotypic, genetic, and digitalized histologic data for comprehensive analyses using systems biology approaches. Analytical strategies were designed to transform descriptive information to mechanistic disease classification for Nephrotic Syndrome and to identify clinical, histological, and genomic disease predictors. Thus, understanding the complexity of the disease pathogenesis will guide further investigation for targeted therapeutic strategies.

Tissue transcriptome-driven identification of epidermal growth factor as a chronic kidney disease biomarker

Renal and urinary EGF can serve as biomarkers for prediction of outcomes in chronic kidney disease. Urine marker to the rescue Chronic kidney disease is a common medical problem worldwide, but it is difficult to predict which patients are more likely to progress to end-stage disease and need aggressive management. Ju et al. have now drawn on four independent cohorts totaling hundreds of patients from around the world to identify the expression of epidermal growth factor (EGF) in the kidneys as a marker of kidney disease progression. Moreover, the authors demonstrated that the amount of EGF in the urine is just as useful, providing a biomarker that can be easily tracked over time without requiring invasive biopsies. Chronic kidney disease (CKD) affects 8 to 16% people worldwide, with an increasing incidence and prevalence of end-stage kidney disease (ESKD). The effective management of CKD is confounded by the inability to identify patients at high risk of progression while in early stages of CKD. To address this challenge, a renal biopsy transcriptome-driven approach was applied to develop noninvasive prognostic biomarkers for CKD progression. Expression of intrarenal transcripts was correlated with the baseline estimated glomerular filtration rate (eGFR) in 261 patients. Proteins encoded by eGFR-associated transcripts were tested in urine for association with renal tissue injury and baseline eGFR. The ability to predict CKD progression, defined as the composite of ESKD or 40% reduction of baseline eGFR, was then determined in three independent CKD cohorts. A panel of intrarenal transcripts, including epidermal growth factor (EGF), a tubule-specific protein critical for cell differentiation and regeneration, predicted eGFR. The amount of EGF protein in urine (uEGF) showed significant correlation (P < 0.001) with intrarenal EGF mRNA, interstitial fibrosis/tubular atrophy, eGFR, and rate of eGFR loss. Prediction of the composite renal end point by age, gender, eGFR, and albuminuria was significantly (P < 0.001) improved by addition of uEGF, with an increase of the C-statistic from 0.75 to 0.87. Outcome predictions were replicated in two independent CKD cohorts. Our approach identified uEGF as an independent risk predictor of CKD progression. Addition of uEGF to standard clinical parameters improved the prediction of disease events in diverse CKD populations with a wide spectrum of causes and stages.

APOL1-G0 or APOL1-G2 Transgenic Models Develop Preeclampsia but Not Kidney Disease

APOL1 risk variants are associated with kidney disease in blacks, but the mechanisms of renal injury associated with APOL1 risk variants are unknown. Because APOL1 is unique to humans and some primates, we created transgenic (Tg) mice using the promoter of nephrin-encoding Nphs1 to express the APOL1 reference sequence (G0) or the G2 risk variant in podocytes, establishing Tg lines with a spectrum of APOL1 expression levels. Podocytes from Tg-G0 and Tg-G2 mice did not undergo necrosis, apoptosis, or autophagic cell death in vivo, even in lines with highly expressed transgenes. Further, Tg-G0 and Tg-G2 mice did not develop kidney pathology, proteinuria, or azotemia as of 300 days of age. However, by 200 days of age, Tg-G2 mice had significantly lower podocyte density than age-matched WT and Tg-G0 mice had, a difference that was not evident at weaning. Notably, a pregnancy-associated phenotype that encompassed eclampsia, preeclampsia, fetal/neonatal deaths, and small litter sizes occurred in some Tg-G0 mice and more severely in Tg-G2 mice. Similar to human placenta, placentas of Tg mice expressed APOL1. Overall, these results suggest podocyte depletion could predispose individuals with APOL1 risk genotypes to kidney disease in response to a second stressor, and add to other published evidence associating APOL1 expression with preeclampsia.

Inactivation of Integrin-β1 Prevents the Development of Polycystic Kidney Disease after the Loss of Polycystin-1

Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.

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.

Morphology in the Digital Age: Integrating High-Resolution Description of Structural Alterations With Phenotypes and Genotypes

Conventional light microscopy has been used to characterize and classify renal diseases, evaluate histopathology in studies and trials, and educate renal pathologists and nephrologists. The advent of digital pathology, in which a glass slide can be scanned to create whole slide images (WSIs) for viewing and manipulating on a computer monitor, provides real and potential advantages compared with conventional light microscopy. Software tools such as annotation, morphometry, and image analysis can be applied to WSIs for studies or educational purposes, and the digital images are available globally to clinicians, pathologists, and investigators. New ways of assessing renal pathology with observational data collection may allow better morphologic correlations and integration with molecular and genetic signatures, refinements of classification schema, and understanding of disease pathogenesis. In multicenter studies, WSIs, which require additional quality assurance steps, provide efficiency by reducing slide shipping and consensus conference costs, and they allow slide viewing anytime and anywhere. Although validation studies for the routine diagnostic use of digital pathology still are needed, this is a powerful tool currently available for translational research, clinical trials, and education in renal pathology.

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.

Morphometry Predicts Early GFR Change in Primary Proteinuric Glomerulopathies: A Longitudinal Cohort Study Using Generalized Estimating Equations.

Objective Most predictive models of kidney disease progression have not incorporated structural data. If structural variables have been used in models, they have generally been only semi-quantitative. Methods We examined the predictive utility of quantitative structural parameters measured on the digital images of baseline kidney biopsies from the NEPTUNE study of primary proteinuric glomerulopathies. These variables were included in longitudinal statistical models predicting the change in estimated glomerular filtration rate (eGFR) over up to 55 months of follow-up. Results The participants were fifty-six pediatric and adult subjects from the NEPTUNE longitudinal cohort study who had measurements made on their digital biopsy images; 25% were African-American, 70% were male and 39% were children; 25 had focal segmental glomerular sclerosis, 19 had minimal change disease, and 12 had membranous nephropathy. We considered four different sets of candidate predictors, each including four quantitative structural variables (for example, mean glomerular tuft area, cortical density of patent glomeruli and two of the principal components from the correlation matrix of six fractional cortical areas–interstitium, atrophic tubule, intact tubule, blood vessel, sclerotic glomerulus, and patent glomerulus) along with 13 potentially confounding demographic and clinical variables (such as race, age, diagnosis, and baseline eGFR, quantitative proteinuria and BMI). We used longitudinal linear models based on these 17 variables to predict the change in eGFR over up to 55 months. All 4 models had a leave-one-out cross-validated R2 of about 62%. Conclusions Several combinations of quantitative structural variables were significantly and strongly associated with changes in eGFR. The structural variables were generally stronger than any of the confounding variables, other than baseline eGFR. Our findings suggest that quantitative assessment of diagnostic renal biopsies may play a role in estimating the baseline risk of succeeding loss of renal function in future clinical studies, and possibly in clinical practice.

Digital pathology imaging as a novel platform for standardization and globalization of quantitative nephropathology

Abstract The introduction of digital pathology to nephrology provides a platform for the development of new methodologies and protocols for visual, morphometric and computer-aided assessment of renal biopsies. Application of digital imaging to pathology made substantial progress over the past decade; it is now in use for education, clinical trials and translational research. Digital pathology evolved as a valuable tool to generate comprehensive structural information in digital form, a key prerequisite for achieving precision pathology for computational biology. The application of this new technology on an international scale is driving novel methods for collaborations, providing unique opportunities but also challenges. Standardization of methods needs to be rigorously evaluated and applied at each step, from specimen processing to scanning, uploading into digital repositories, morphologic, morphometric and computer-aided assessment, data collection and analysis. In this review, we discuss the status and opportunities created by the application of digital imaging to precision nephropathology, and present a vision for the near future.