Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.

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f First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cellspecific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public-omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.

Diabetic kidney disease is the leading cause of kidney failure. However, studies of molecular mechanisms of early kidney damage are lacking. Here we examined for possible linkage between transcriptional regulation and quantitative structural damage in early diabetic kidney disease in Pima Indians with type 2 diabetes. Tissue obtained from protocol kidney biopsies underwent genome-wide compartment-specific gene expression profiling and quantitative morphometric analysis. The ultrastructural lesion most strongly associated with transcriptional regulation was cortical interstitial fractional volume (VvInt), an index of tubule-interstitial damage. Transcriptional co-expression network analysis identified 1843 transcripts that correlated significantly with VvInt. These transcripts were enriched for pathways associated with mitochondrial dysfunction, inflammation, migratory mechanisms, and tubular metabolic functions. Pathway network analysis identified IL-1β as a key upstream regulator of the inflammatory response and five transcription factors cooperating with p53 to regulate metabolic functions. VvInt-associated transcripts showed significant correlation with the urine albumin to creatinine ratio and measured glomerular filtration rate 10 years after biopsy, establishing a link between the early molecular events and long-term disease progression. Thus, molecular mechanisms active early in diabetic kidney disease were revealed by correlating intrarenal transcripts with quantitative morphometry and long-term outcomes. This provides a starting point for identification of urgently needed therapeutic targets and non-invasive biomarkers of early diabetic kidney disease.