When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms’ tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1⁺CD133⁻ marks SIX2⁺ multipotent renal stem cells transiting to NCAM1⁺CD133⁺ differentiating segment-specific SIX2⁻ epithelial progenitors and NCAM1⁻CD133⁺ differentiated nephron cells. In tumorigenesis, NCAM1⁺CD133⁻ marks SIX2⁺ blastema that includes the ALDH1⁺ WT cancer stem/initiating cells, while NCAM1⁺CD133⁺ and NCAM1⁻CD133⁺ specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1⁺ nephron stem cells in normal and malignant nephrogenesis.

Introduction: Neutrophil extracellular traps (NETs) release (i.e., NETosis) has been recently implicated in the pathomechanism underlying severe end-organ damage in Coronavirus Disease 2019 (COVID-19) and could present a novel therapeutic target. We aimed to determine whether circulating levels of cell-free DNA (cfDNA), a surrogate for NETosis, may be associated with the development of acute kidney injury (AKI), a major contributor to poor outcomes and mortality in COVID-19. Methods: Blood samples were collected prospectively from adult patients infected with SARS-CoV-2 presenting to the emergency department (ED). Circulating levels of cfDNA were quantified from patients’ serum. Further assessment of correlations between cfDNA levels and markers of AKI (i.e., serum creatinine (SCr), cystatin C, neutrophil gelatinase–associated lipocalin (NGAL)), biomarkers of thrombotic microangiopathy and of inflammation in patients’ serum was performed. Results: Fifty-one COVID-19 patients were enrolled. cfDNA levels were found to be significantly higher in those who developed severe AKI ( ) and those needing renal replacement therapy ( ). cfDNA positively correlated with ED SCr, NGAL, cystatin C, neutrophil count, neutrophil-to-lymphocyte ratio, C3a, C5a, Scb5-9, IL-6, IL-8, IL-10, TNF-α, LDH, CRP, ferritin, and fibrinogen and negatively correlated with ADAMTS13/von-Willebrand factor ratio and lymphocyte count. In a multivariate logistic regression, a one-unit increase in cfDNA value was associated with 4.6% increased odds of severe AKI ( ; ). Finally, cfDNA significantly correlated with established NETs components, myeloperoxidase, and neutrophil elastase. Conclusion: Intravascular NETosis could be an important contributing factor in the development of microthrombosis and COVID-19-associated AKI. Further research is urgently needed to understand the role of NETosis in COVID-19 and evaluate therapeutic avenues for targeting this process.

Context Congenital Adrenal Hyperplasia (CAH) was among the first genetic disorders included in newborn screening (NBS) programs worldwide, based on 17α-hydroxyprogesterone (17-OHP) levels in dried blood spots. However, the success of NBS for CAH is hampered by high rates of false positive (FP) rates, especially in preterm and low birth-weight infants. Objective To establish a set of cutoff values adjusting for both gestational-age (GA) and birth-weight (BW), with the aim of reducing FP rates. Design This cross-sectional population-based study summarizes 10 years of experience of the Israeli NBS for diagnosis of CAH. Multi-tiered 17-OHP cutoff values were stratified based on both BW and GA. Participants A total of 1,378,132 newborns born between 2008-2017 were included in the NBS Program. Results 88 newborns were ultimately diagnosed with CAH, 84 of whom detected upon NBS. The combined parameters-adjusted approach significantly reduced the recall FP rate (0.03%) and increased the positive predictive value (PPV) (16.5%). Sensitivity among those referred for immediate attention increased significantly (94%). There were four FN cases (sensitivity, 95.4%), all ultimately diagnosed as simple-virilizing. Sensitivity and specificity were 95.4% and 99.9%, respectively, and the percentage of true positive cases from all newborns referred for evaluation following a positive NBS result was 96%. Conclusions The use of cutoff values adjusted for both GA and BW significantly reduces FP rates (0.03%), and increases overall PPV (16.5%). Based on our ten years of experience, we recommend the implementation of this novel, two parameter-adjusted approach for NBS of classical CAH, in NBS programs worldwide.

Background: Over 500 epigenetic regulators have been identified throughout the human genome. Of these, approximately 30 chromatin modifiers have been implicated thus far in human disease. Recently, variants in BRPF1, encoding a chromatin reader, have been associated with a previously unrecognized autosomal dominant syndrome manifesting with intellectual disability (ID), hypotonia, dysmorphic facial features, ptosis, and/or blepharophimosis in 22 individuals.Patients and Methods: We report a multiply affected nonconsanguineous family of mixed Jewish descent who presented due to ID in three male siblings. Molecular analysis of the family was pursued using whole exome sequencing (WES) and subsequent Sanger sequencing.Results: Whole exome sequencing analysis brought to the identification of a novel heterozygous truncating mutation (c.556C>T, p.Q186*) in the BRPF1 gene in the affected siblings and their mother. The four affected individuals showed varying degrees of intellectual disability, distinct facial features including downslanted palpebral fissures, ptosis, and/or blepharophimosis. Their clinical characteristics are discussed in the context of previously reported patients with the BRPF1‐related phenotype.Conclusion: The reported family contributes to the current knowledge regarding this unique and newly recognized genetic disorder, and further implicates the role of BRPF1 in human brain development.

Significance Statement: Kidney development is a complex process involving multiple interacting and transitioning cell types. Drop-seq single-cell technology, which measures gene expression from many thousands of individual cells, has been used to characterize these cellular differentiation changes that underlie organ development. However, the alternative splicing of many genes creates an additional layer of cellular heterogeneity that Drop-seq technology cannot measure. Therefore, in this study, full transcript length single-cell RNA sequencing was used to characterize alternative splicing in the mouse embryonic kidney, with particular attention to the identification of genes that are alternatively spliced during the transition from mesenchymal to epithelial cell states, as well as their splicing regulators. These results improve our understanding of the molecular mechanisms that underlie kidney development. Background: During mammalian kidney development, nephron progenitors undergo a mesenchymal-to-epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, Drop-seq single-cell RNA sequencing technology for measuring gene expression from thousands of individual cells identified the different cell types in the developing kidney. However, that analysis did not include the additional layer of heterogeneity that alternative mRNA splicing creates. Methods: Full transcript length single-cell RNA sequencing characterized the transcriptomes of 544 individual cells from mouse embryonic kidneys. Results: Gene expression levels measured with full transcript length single-cell RNA sequencing identified each cell type. Further analysis comprehensively characterized splice isoform switching during the transition between mesenchymal and epithelial cellular states, which is a key transitional process in kidney development. The study also identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. Conclusions: Discovery of the sets of genes that are alternatively spliced as the fetal kidney mesenchyme differentiates into tubular epithelium will improve our understanding of the molecular mechanisms that drive kidney development.

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human induced pluripotent stem cells (iPSCs) to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the demonstration that nicastrin (NCSTN)-deficient human iPSC-derived organoids differentiate into TFA2B+ distal tubule and CDH1+ connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.