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.

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential characteristics which link the protein to the injured organ have not yet been described. We generated an NGAL-reporter mouse by inserting a di-fusion reporter gene, Luciferase2(Luc2)/mCherry(mC) into the Ngal locus. The Ngal-Luc2/mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real-time. In the kidney, Ngal-Luc2/mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ and cellular specific relationship with tubular stress, which quantitatively paralleled urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2/mC mice could also track both the onset and the resolution of the injury, and monitor the actions of NF-κB inhibitors and antibiotics in the case of infection. Accordingly, the imaging of Ngal-Luc2/mC mice and cells identified injurious and reparative agents which effect kidney damage.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal [known as Neutrophil Gelatinase Associated Lipocalin, Siderocalin, Lipocalin 2] sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography, and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH sensitive mechanism. Since catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal:catechol:iron complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal:siderophore interactions, but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Summary Developing organs require iron for a myriad of functions, but embryos deleted of the major adult transport protein, transferrin or its receptor transferrin receptor1 (TfR1−/−) initiate organogenesis, suggesting that non-transferrin pathways are important. To examine these pathways, we developed chimeras composed of fluorescence-tagged TfR1−/− cells and untagged wild type cells. In the kidney, TfR1−/− cells populated capsule and stroma, mesenchyme and nephron, but were underrepresented in ureteric bud tips. Consistently, TfR1 provided transferrin to the ureteric bud, but not to the capsule or the stroma. Instead of transferrin, we found that the capsule internalized ferritin. Since the capsule expressed a novel receptor called Scara5, we tested its role in ferritin uptake and found that Scara5 bound serum ferritin and stimulated its endocytosis from the cell surface with consequent iron delivery. These data implicate cell type-specific mechanisms of iron traffic in organogenesis, which alternatively utilize transferrin or non-transferrin iron delivery pathways.

Neutrophil gelatinase-associated lipocalin (NGAL), a 25 kDa protein produced by injured nephron epithelia, is one of the most promising new markers of renal epithelial injury. In contrast to serum creatinine and urinary output, which are measures of kidney function, NGAL is specifically induced in the damaged nephron and then released into blood and urine, where it can be readily measured. Careful proof-of-concept studies using defined animal models have uncovered the sources and trafficking of NGAL in acute kidney injury (AKI) and have addressed the contributions of renal and non-renal sources. Clinical studies indicate that NGAL, unlike creatinine, is a marker responsive to tissue stress and nephron injury, but less so to adaptive hemodynamic responses. In certain clinical settings, NGAL is an earlier marker compared with serum creatinine. In addition, clinical studies have shown that NGAL is a powerful predictor of poor clinical outcomes, which can be used to risk stratify patients when combined with serum creatinine. NGAL has important limitations, including its responsiveness in systemic inflammation, which is partially uncoupled from its response to kidney injury and which needs to be considered when interpreting NGAL results clinically. This review covers the biology and pathophysiology of NGAL and summarizes the results of the growing body of clinical studies that have addressed the utility of NGAL in the early diagnosis of AKI, in the distinction of intrinsic AKI and in the prognostic assessment of broad patient populations.