Fig 2: The prevention of kidney function abnormalities through the maintenance of the filtration barriers by sActRIIB treatment of Ercc1 ?/- mice. (A) Urine protein measurements at the end of week 14. (B–D) Low and (F–H) high magnification of electron microscopy images of podocytes from control, mock-treated Ercc1 ?/-, and sActRIIB-treated Ercc1 ?/- mice. Pod indicates the podocyte. (C) Ercc1 ?/- tissue contains autophagosomes (yellow arrow) and enlarged mitochondria (yellow arrowhead). (D) sActRIIB-treated Ercc1 ?/- mice show some foot process effacement (red arrowheads) but significant number of mature foot processes (red arrow). (E) Quantification of foot process width. (F) Numerous mature foot processes in control sample (red arrows). (G) Very few foot processes in Ercc1 ?/- sample but thickened glomerular basement membrane (red arrowheads). (H) Treated Ercc1 ?/- sample showing numerous mature foot processes (red arrows). (I) Quantification of glomerular basement membrane thickness. (J) Nuclear size measurements in Nephrin-positive domain. (K) pSmad2/3 profile in control mice (red) in relation to podocytes, identified through Nephrin expression. (L) Abundant levels of pSmad2/3 (red arrows) in Ercc1 ?/- podocytes. (M) Few pSmad2/3 puncta in sActRIIB-treated Ercc1 ?/- podocytes (red arrow). n = 8 mice examined for each cohort for (A) and n = 5 mice examined for each cohort for (EM). Analysis performed using non-parametric Kruskal–Wallis test followed by the Dunn's multiple comparisons. *P < 0.05, **P < 0.01. sActRIIB, soluble activin receptor type IIB.

Fig 3: 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 4: Illustration of glomerular scoring. (A) An image of native tdTomato fluorescence (magenta, tdTomato+ axons) and immunolabeling for nephrin (green, glomeruli) and calcitonin gene related peptide (CGRP) (cyan, peptidergic axons) in a renal slice from a TRPV1:Ai14 mouse. The image represents a maximum intensity projection of 6 optical sections collected 13.7 microns apart. While this image includes CGRP+ and tdTomato+ labeling for illustrative purposes, only images immunolabeled for one neuronal marker at a time were used during glomerular scoring. (B–E) A magnified inset from panel (A) illustrating the labeling of glomeruli and axons in the renal cortex (scale bars: 250 µm). (B) Nephrin+ labeling of glomerular podocytes. (C) Native tdTomato fluorescence. Examples of labeled axons are highlighted with arrowheads. The identity of globular cell-like profiles is unknown. They appeared to be associated with tubules and were not co-labeled with a pan-immune cell marker (anti-CD45, data not shown). They were clearly distinguished from axonal profiles and, therefore, did not affect the glomerular scoring. (D) CGRP+ labeling of afferent axons (some highlighted with arrowheads). (E) Composite image illustrating CGRP labeling and tdTomato fluorescence in afferent axons (some highlighted with arrowheads) in the renal cortex. (F–I) Examples of anatomical relationships between axons and glomeruli as observed while scoring individual glomeruli (scale bars: 50 µm). (F) Both CGRP+ and tdTomato+ axons (arrowheads) approach the glomerulus. (G) A tdTomato+ axon bundle (arrowhead) approaches the glomerulus, and although the cyan channel exhibits some labeling, its morphology is not consistent with the appearance of axons and is therefore not scored as an anatomical interaction between CGRP+ axons and the glomerulus. (H) A clear anatomical relationship between a CGRP+ axon bundle (arrowhead) and the glomerulus. Although there is faint labeling in the magenta channel, the signal to noise ratio is not high enough to count as a close apposition. (I) CGRP+ axon (arrowhead) surrounds the glomerulus extend beyond it. Scale bars: (A) 1000 µm, (B–E) 250 µm, (F–I) 50 µm.