Fig 2: Generation of functional kidney spheroids using immortalized mouse primary cells. (a) Schematic diagram of SFU protocol (combination of hanging drop and rotation) and maturation factors for generation of kidney spheroids for structural maturation. (b) Quantitative RT-PCR analysis of kidney-specific segment markers in mouse kidney spheroids (mKidS) compared with 2D controls (2D CTL) and mouse kidney (mKidney). n = 9 for 2D CTL and mKidS, n = 3 for mKidney. (c) Western blotting analysis for renal multi-lineage markers in mouse kidney spheroids compared with 2D CTL and mKidney. The grouping of gels/blots cropped from different parts of the same gel. (d) Immunostaining images for cell polarity of Zo-1, an apical membrane, and Na/K+ ATPase, a basolateral membrane marker in mKidS. Scale bar, 10 μm. (e) Representative images of mKidS at day 5 of maturation stained with hematoxylin and eosin (H&E) or immunostained for the kidney-specific markers LTL, PNA, AQP2, and nephrin; transmission electron microscopy of kidney spheroids displaying long (black arrowhead) and short (red arrowhead) villi with tight junctions (black arrow) and foot processes (asterisks in the middle panel). Scale bar of TEM image, 1 μm. Mouse kidney from 12 weeks-old mice was used as a positive control for immunostaining of mKidS. (f) Response to parathyroid hormone (PTH) compared with 2D cells and 3D control spheroids (without maturation factors, CTL). n = 6 for 2D CTL, 2D PTH treated, n = 10 for 3D CTL and mKidS. (g) GGT activity in mKidS compared to 3D CTL after 2 and 4 days of maturation. n = 8 for CTL and mKidS, respectively. (h) Temperature-dependent albumin-fluorescein uptake compare to 3D CTL. n = 12 for 3D CTL and mKidS, respectively. (I,j) Relative mRNA expression of apical (i) and basolateral (j) transporters in mKidS compared with 2D cultured cells, TCMK-1 spheroids generated using the same protocol for mKidS (TCMK-3D) and mKidney. n = 8 for 2D CTL and mKidS and n = 4 for TCMK-3D. (k) Representative images of mKidS immunostained for p-GP, OCT2, and PTHR. All data are shown as the means ± s.e.m. *p < 0.05 compared with 2D or 3D control by unpaired Student’s t-test. Scale bar, 20 μm.

Fig 3: Reproducibility and maturity of kidney spheroids. (a) FACS analysis of tubule segment markers in mouse kidney spheroid generated by immortalized cells at the 4th and 20th passages. As a positive control, dissociated mouse whole kidney (at 6 weeks of age) was used. Mouse kidney spheroid at the 4th and 20th passages and dissociated whole mouse kidney were stained with anti-AQP1, PNA, nephrin, or AQP2 (open histograms), or isotype control mAb (grey histograms). (b) Quantitative RT-PCR analysis of kidney-specific segment markers in mKidS generated by cells at the 4th and 20th passages compared with 2D controls (2D CTL). n = 8 for 2D CTL and n = 6 for mKidS at the 4th and 20th passages. (c,d) Relative mRNA expression of apical (c) and basolateral (d) transporters in mKidS at the 4th and 20th passages compared with 2D cultured cells. n = 8 for 2D CTL and n = 6 for mKidS at the 4th and 20th passages. (e) Responses to parathyroid hormone (PTH) of mKidS at the 4th and 20th passages compared to 3D CTL. (f) GGT activity in mKidS at the 4th and 20th passages compared to 3D CTL. (g) Alexa Fluor 488-conjugated dextran uptake of mKidS at the 4th and 20th passages compared with 3D CTL. n = 6 for 3D CTL and mKidS at the 4th and 20th passages for E-G. All data are shown as the means ± s.e.m. *p < 0.05 compared with 2D or 3D control by unpaired Student’s t-test.

Fig 4: Generation of functional kidney spheroids using immortalized human primary cells. (a) Quantitative RT-PCR analysis of kidney-specific segment markers in hKidS compared with 2D controls (2D CTL) and human kidney lysates (hKidney). n = 8 for 2D CTL and hKidS and n = 3 for hKidney. (b) Western blotting analysis for renal multi-lineage marker in mouse kidney spheroids compared to 2D CTL and hKidney. The grouping of gels/blots cropped from different parts of the same gel. (c) Representative images for cell polarity of Zo-1, an apical membrane, and Na/K ATPase, a basolateral membrane marker in hKidS. Scale bar, 10 μm. (d) Representative images of human kidney spheroids (hKidS) at day 5 of maturation stained for the kidney-specific markers LTL, PNA, AQP2, and Nephrin. Human kidney was used as positive control for immunostaining of hKidS. (e) Responses to parathyroid hormone (PTH) of hKidS compared with 3D control spheroids (without maturation factors, CTL) and 2D cells. (f) GGT activity in hKidS compared with 3D CTL. (g) Temperature-dependent albumin-fluorescein uptake of hKidS compared to 3D CTL. n = 10 for 3D CTL and hKidS. (h,i) Relative mRNA expression of apical (h) and basolateral (i) transporters in hKidS compared to 2D cultured cells, hPTECs spheroids generated using the same protocol for hKidS (hPTECs-3D), and hKidney. n = 9 for 2D CTL and hKidS and n = 3 for hPTECs-3D and hKidney. (j) Representative images of hKidS immunostained for p-GP, OCT2, and PTHR. All data are shown as the means ± s.e.m. *p < 0.05 compared with 2D or 3D control by unpaired Student’s t-test. Scale bar, 20 μm.

Fig 5: Pax2 lineage-positive cells regenerate long tubule segments. a Representative image of a kidney section showing single-colored clones in S1–S2 segments of proximal tubule as demonstrated by staining with anti-megalin antibody (white) in Pax2/Confetti mice at day 30 after IRI (n = 5). Scale bar 20 µm. b, c Representative images of a kidney section showing single-colored clones in S3 segment of proximal tubule as demonstrated by staining with anti-AQP1 antibody (white) in OSOM of Pax2/Confetti mice at day 30 after IRI (n = 5). Scale bars 20 µm. d Representative image of a kidney section showing single-colored clones in thick ascending limb as demonstrated by staining with anti-THP antibody (white) in Pax2/Confetti mice at day 30 after IRI (n = 5). Scale bar 20 µm. e Frequency of Pax2+ clones in S1–S2 segment of proximal tubules (Pax2+ Megalin+ clones, n = 5), in S3 segment of proximal tubules (Pax2+AQP1+ clones, n = 7), in thick ascending limbs (TAL, Pax2+THP+ clones, n = 7) and in the collecting ducts (Pax2+AQP2+ clones, n = 7) at day 30 after IRI. Mann–Whitney test. **p < 0.01 S3 vs. S1–S2 and collecting ducts, *p < 0.05 S3 vs. TAL and TAL vs. collecting ducts. f Percentage of clonogenic cells per field in 2D vs. 3D analysis in Pax2/Confetti and in Pax8/Confetti mice at day 30 after IRI vs. T0. (n = 5 Pax2/Confetti mice and n = 5 Pax8/Confetti mice). Mann–Whitney test NS. g–i 3D reconstruction of single-colored clones in Pax2/Confetti mice at day 30 after IRI (n = 5). DAPI counterstains nuclei (white). Scale bars 50 µm. Data are mean ± SEM. Pax2+ = Pax2 lineage-positive cells, Pax8+ = Pax8 lineage-positive cells