Fig 2: Enhanced organoids possess functional PT transporters and appropriate injury response.a Live confocal images of standard and PT-enhanced organoids (transmitted light and fluorescence overlays) depicting (left panel) uptake of TRITC-albumin (red) into MEGALIN-positive PTs (blue) and (right panel) uptake of DAPI (blue; surrogate for organic cation transport capacity). White boxed areas on left images of left panel are shown as higher magnification on right (insets depict MEGALIN staining alone). Organic cation uptake image set (right panel) depicts organoids exposed to substrate alone (DAPI; blue, left images) or a combination of substrate/DAPI + inhibitor/Cimetidine (right images). Dead cells (right panel) are labelled with DRAQ7 (red). Right panel insets depict blue channel only without brightfield overlay. Scale bars represent 200 µm (whole organoid images in left panel) and 100 µm (right images in left panel, all images in right panel). b Confocal immunofluorescence of representative D7 + 14 (standard; top panels) and D13 + 14 (PT-enhanced; bottom panels) line-matched organoids following 24 h treatment with E6 media containing either (ii) 20 µM cisplatin or (i) an equivalent volume of PBS. Images depict KIM-1-expressing cells (red) in LTL + proximal tubules (blue) with nephron epithelium co-stained with EPCAM (green). Insets of bottom row images for standard and PT-enhanced organoids show KIM-1 staining (red channel) alone from white boxed regions in top row images. Scale bars in all images represent 100 µm. c, d qRT-PCR analyses depicting KIM-1 gene (HAVCR1) expression in standard (blue) and PT-enhanced (red) organoids from experiments shown in (b). HAVCR1 gene expression values are normalised to the expression of housekeeping gene GAPDH and depicted both with (d) and without (c) compensation for differences in PT proportion (expressed as a ratio of HNF4A). Error bars represent SEM from n = 8 (control) and n = 9 (cisplatin-treated) biological replicates across 3 replicate experiments as indicated. Statistical significance was determined using an unpaired t test. Asterisk represents two-tailed P value (*P = 0.05 [P = 0.014837]) adjusted for multiple comparisons using the Holm–Sidak method alpha = 0.05. NS non-significant. Source data are provided as a Source data file.

Fig 3: Endogenous expression of TIM-1 facilitates CHIKV infection of hepatocytes. (A) Endogenous surface expression of TIM-1 and Axl in Huh7.5 cells. (B) MTT assay-based viability of Huh7.5 cells. Cells were re-seeded in 96-well plate 48 h after treatment with targeting and non-targeting (NT) siRNA and incubated overnight. Afterwards, MTT assay was performed to compare proliferation of cells. (C) TIM-1 and MXRA8 surface expression 48 h after siRNA treatment measured by antibody staining followed by flow cytometry. One representative dataset shown. (D) Huh7.5 cell susceptibility to CHIKV after treatment with targeting and NT siRNA. Cells were inoculated with 5'GFP CHIKV (WA genotype) and infectivity determined by flow cytometry 24 hpi. The error bars represent mean ± SEM of at least three independent experiments. Statistical significance was calculated using a Dunnet’s multiple comparisons test (2way ANOVA) ns > 0.05, * p < 0.05 and *** p < 0.001.

Fig 4: Entry kinetics of CHIKV are altered by TIM-1. (A) Scheme showing binding and entry assay. CHIKV (Asian genotype) encoding nano-luciferase fused to E2 glycoprotein (nLuc-CHIKV) was added to parental and TIM-1 expressing HEK293T cells, which were subsequently incubated at 4 °C for 1 h to synchronize binding. After washing, cells were transferred to 37 °C and medium with 20 mM NH4Cl was added at indicated time points. Assay was stopped after 10 h of incubation at 37 °C and relative endosomal escape determined by luciferase assay. (B) Entry kinetics of nLuc-CHIKV in the indicated cell lines normalized to parental HEK293T cells at each time point. (C) Live cell imaging of internalized CHIKV. Cells were inoculated with fluorescent Asian mc-CHIKV at MOI of 50 and monitored by confocal microscopy. After 20 min of live imaging of fluorescent mc-CHIKV (Asian genotype) and cells at 37 °C, trypan blue was added to quench extracellular particles and only internalized viruses were imaged and counted using ImageJ. The error bars represent standard errors of the mean (SEM). Statistical significance was calculated using Dunnet’s multiple comparisons test (2way ANOVA) (B) and Welch t-test (C) with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Fig 5: Hemin injury leads to induction of inflammation and nitric oxide mediated nitrosylation. a Representative quantitative PCR (qPCR) of kidney organoids treated with hemin at increasing concentrations. Expression relative to the control (untreated) organoids. b Time course qPCR of control and hemin treated organoids from day 15 to day 20. Results show mean and standard deviation of 3 technical replicates. c Immunofluorescence of kidney organoid paraffin sections of control and hemin treated organoids, showing localization of kidney injury marker 1 (KIM1, red) to proximal tubules labelled with Lotus tetragonolobus lectin (LTL, green), apoptotic marker activated caspase 3 (Casp3, red), DNA damage marker (?H2AX, green), heme catabolizing enzyme heme oxygenase 1 (HMOX1, orange), and nephrin labelling podocytes (NPHS1, red). d Immunofluorescence of kidney organoid paraffin sections at day 16, 18 and 20 showing 3-nitrotyrosine staining (red) indicative of nitrositive stress. Cell line MANZ2-2. Scale bar = 200 µm