Live functional assays reveal longitudinal maturation of transepithelial transport in kidney organoids

Abstract

Kidney organoids derived from hPSCs have opened new opportunities to develop kidney models for preclinical studies and immunocompatible kidney tissues for regeneration. Organoids resemble native nephrons that consist of filtration units and tubules, yet little is known about the functional capacity of these organoid structures. Transcriptomic analyses provide insight into maturation and transporter activities that represent kidney functions. However, functional assays in organoids are necessary to demonstrate the activity of these transport proteins in live tissues. The three-dimensional (3D) architecture adds complexity to real-time assays in kidney organoids. Here, we develop a functional assay using live imaging to assess transepithelial transport of rhodamine 123 (Rh123), a fluorescent substrate of P-glycoprotein (P-gp), in organoids affixed to coverslip culture plates for accurate real-time observation. The identity of organoid structures was probed using Lotus Tetragonolobus Lectin (LTL), which binds to glycoproteins present on the surface of proximal tubules. Within 20 min of the addition of Rh123 to culture media, Rh123 accumulated in the tubular lumen of organoids. Basolateral-to-apical accumulation of the dye/marker was reduced by pharmacologic inhibition of MDR1 or OCT2, and OCT2 inhibition reduced the Rh123 uptake. The magnitude of Rh123 transport was maturation-dependent, consistent with MDR1 expression levels assessed by RNA-seq and immunohistochemistry. Specifically, organoids on day 21 exhibit less accumulation of Rh123 in the lumen unlike later-stage organoids from day 30 of differentiation. Our work establishes a live functional assessment in 3D kidney organoids, enabling the functional phenotyping of organoids in health and disease.

Keywords: functional assay; kidney; nephron; organoid; real-time imaging; tubular transport.

FIGURE 1

Kidney organoids utilized for live imaging functional assay. (A) Steps in generating kidney organoids using the 6-step differentiation protocol (Morizane et al., 2015; Morizane and Bonventre, 2017). Organoids at > d14 are used for live imaging functional assays. (B) SIX2 staining is used to confirm the quality of kidney organoid differentiation on embryonic (H9) and induced (BJFF.6) stem cell lines. (C) The d21 kidney organoids derived from both lines form a segmentation of nephrons as presented by CDH1⁺, PODXL⁺, and LTL⁺ cells, as well as containing (D) PDGRFβ⁺ stromal and CD31⁺ endothelial cells. FGF9: fibroblast growth factor 9. LTL, lotus tetragonolobus lectin; PODXL, podocalyxin; CDH1, cadherin-1; PDGFRβ, platelet-derived growth factor receptor beta.

FIGURE 2

Sample preparation and setup for kidney organoid functional assays by live imaging. (A) A schematic illustration of the live imaging protocol. Following the organoid attachment on culture plate/chamber slides, structural markers were utilized to help distinguish nephron segmentation. The orbital shaker allows the live staining dyes to penetrate into the kidney organoid. The stage-top incubator maintains the culture environment of live organoids and enables real-time functional assay. (B) Immunofluorescent images of live LTL and WGA staining in kidney organoids. WGA, wheat germ agglutinin.

FIGURE 3

Transepithelial transport in live kidney organoids. (A) Protocol for Rh123 assay using the microscope stage incubator. (B) Rh123 transport into tubular lumen after 20 min incubation. The tubular structure was later confirmed as an LTL⁺ proximal tubule after organoid PFA fixation. (C) High magnification images display a distinct Rh123 trans-cellular transport from the interstitial to luminal space of proximal tubules. The dashed lines show the Rh123 movement, and the arrowhead indicates the transport direction. (D,E) In contrast, tubular structures that transport less Rh123 are CDH1⁺ distal tubules as confirmed by immunostaining.

FIGURE 4

Rh123 transport is mediated by MDR1 and OCT2 in kidney organoids. (A) Protocol for Rh123 assay using transporter inhibitors. (B) Rh123 transport into the lumen of proximal tubules in the control samples. (C) MDR1i blocks luminal secretion of Rh123, resulting in fluorescence accumulation in the apical cytoplasmic region. (D) OCT2i decreases the Rh123 luminal secretion and cellular uptake. (E) Quantification of the Rh123 intensity in the tubular lumens and cytoplasm n = 6. ****: p < 0.0001.

FIGURE 5

Functional maturation assessed by live functional assay, immunostaining, and RNA-seq in kidney organoids. (A) Rh123 transport in proximal tubules of kidney organoids between d21 and d49. Organoids at d49 show prominent luminal accumulation which is later (B) confirmed by quantification between 0 and 20 min time points. (C) Immunostaining validates that MDR1 expression on d35/49 is greater than on the d21. MDR1 expression is localized to the apical membrane of the proximal tubules. (D) Quantification of MDR1 staining confirms a significant increase in MDR1 expression on d35/49 n = 15. (E) RNA-seq for MDR1 in kidney organoids and human fetal kidneys (hfKidney). (F) Immunostaining for OCT2 in d21, d35, and d49 kidney organoids. (G) Quantification of OCT2 staining shows stable expression from d21. n = 15. (H) RNA-seq for OCT2 in kidney organoids and hfKidney. n = 3 for organoid samples and 2 for hfKidneys.