Fig 1.
Primary renal proximal tubular epithelial cells (RPTECs) and normal human kidney cells form tubulocysts.
(A) Phase-contrast images: primary RPTECs and normal human kidney cells showing similar cellular morphologies in 2D cultures. (B) Phase-contrast images: spherule formation observed in primary RPTECs and normal human kidney cells. The 3D-on-top assay provides a clearer image of human normal kidney cell organoids than the 3D-embedded assay.
Fig 2.
Normal human kidney cells generate more tubulocysts than primary renal proximal tubular epithelial cells (RPTECs).
(A) Phase-contrast images: primary RPTECs and normal human kidney cells exhibiting tubulocysts formation. (B) Quantification (counts) of tubulocysts in primary RPTEC and normal human kidney cell organoids, categorized by size. Results expressed as mean (± SD) of three independent experiments (n = 3); *p<0.05 vs control. (C) Proliferation assay of primary RPTECs and normal human kidney cells using CCK8 for 72hr. Each matrigel dome containing 1x104 cells and 3x104 cells/25μl of matrigel were loaded in 48-well culture plates. All experiments were performed in triplicate for each condition and repeated at least twice. *p<0.05 vs control.
Fig 3.
Human kidney tubular organoid expression of kidney-specific molecules and formation of dome-like structures.
(A) Confocal images: immunostaining of normal human kidney cells for Tamm-Horsfall protein in 2D and 3D cultures (upper panel); quantification of fluorescence intensity of Tamm-Horsfall protein expression by normal human normal kidney cells in 2D and 3D cultures (lower panel). THP-FITC (green), FITC-conjugated anti-Tamm-Horsfall protein; DAPI (blue), 4′,6-diamidine-2′-phenylindole dihydrochloride. Results expressed as mean (± SD) of five independent experiments (n = 5); *p< 0.05 vs control. (B) 3D reconstruction of confocal z-stacks: immunostaining of mildly dissolved and re-cultured human kidney tubular organoids for Tamm-Horsfall protein. THP-FITC (green), FITC-conjugated anti-Tamm-Horsfall protein; pan-keratin (red), anti-pan-keratin antibody; DAPI (blue), 4′,6-diamidine-2′-phenylindole dihydrochloride.
Fig 4.
Human kidney tubular organoid response to nephrotoxic drugs.
(A) Light microscopy: hematoxylin and eosin staining of human kidney tubular organoid sections, untreated (Control) and cisplatin-treated (Cisplatin). (B, C) Confocal images: immunostaining of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and Tamm-Horsfall protein (THP) in untreated (Control) and cisplatin-treated (Cisplatin) human kidney tubular organoid sections. (D) Quantification of fluorescence intensity of KIM-1 and NGAL in untreated (Control) and cisplatin-treated (Cisplatin) human kidney tubular organoids. KIM-1 (red), anti-KIM-1 antibody; NGAL (red), anti-NGAL antibody; THP (green), FITC-conjugated anti-Tamm-Horsfall protein; DAPI (blue), 4′,6-diamidine-2′-phenylindole dihydrochloride. Results expressed as mean (± SD) of five independent experiments (n = 5); *p<0.05 vs control.