Long-term viable chimeric nephrons generated from progenitor cells are a reliable model in cisplatin-induced toxicity

Abstract

Kidney organoids have shown promise as evaluation tools, but their in vitro maturity remains limited. Transplantation into adult mice has aided in maturation; however, their lack of urinary tract connection limits long-term viability. Thus, long-term viable generated nephrons have not been demonstrated. In this study, we present an approachable method in which mouse and rat renal progenitor cells are injected into the developing kidneys of neonatal mice, resulting in the generation of chimeric nephrons integrated with the host urinary tracts. These chimeric nephrons exhibit similar maturation to the host nephrons, long-term viability with excretion and reabsorption functions, and cisplatin-induced renal injury in both acute and chronic phases, as confirmed by single-cell RNA-sequencing. Additionally, induced human nephron progenitor cells differentiate into nephrons within the neonatal kidneys. Collectively, neonatal injection represents a promising approach for in vivo nephron generation, with potential applications in kidney regeneration, drug screening, and pathological analysis.

Fig. 1. Neonatal niche injection (NNI) method.

a Residual Six2-positive nephron progenitor cells (NPCs) in the kidney of fetal (E14.5) and neonatal (P1.5, P3.5, and P4.5) mice. In neonates, NPCs are located in the outermost layer, whereas the differentiated nephrons are in the medulla. b Procedures of the NNI method. (1) Under anesthesia, a vertical incision is made on the left back. (2) Light pressure is applied to expose the left kidney out of the incision. (3) Approximately 1 μL of suspension (1.0 × 10⁶ cells) of renal progenitor cells (RPCs) is injected under the renal capsule with a 34G Hamilton syringe. (4) The incision is sutured with 8-0 nylon. All procedures were performed under the stereomicroscope. c Fluorescence stereomicroscopic images of the host neonatal mouse kidney after the injection. GFP-positive exogenous RPCs are confirmed to spread on the surface. d A schematic of the NNI method. e Bodyweight changes in the host neonates that received the injection on P0 and P1 compared with non-surgical controls. No significant differences were found between groups (n = 4 biologically independent samples). Error bars represent the mean ± SEM. Data were analyzed using the two-tailed unpaired t-test. ns, not significant. Scale bars, 500 μm in (a) and 2 mm in (c). CK8, cytokeratin 8; GFP, green fluorescent protein.

Fig. 2. Successful integration and differentiation of donor cells in the host neonatal nephrogenic niche.

a, b Immunostaining of the chimeric niche in the neonatal kidney 2 days after injection of renal progenitor cells (RPCs) (P3.5). In the outermost layer, the cap mesenchyme consisted of a mixture of host nephron progenitor cells (NPCs, Six2+, GFP −) and donor NPCs (Six2 +, GFP +) surrounding the host ureteric bud (CK8 +, GFP −). c A schematic of (a) and (b). d Fluorescence stereomicroscopic images of the surface and the longitudinal section of host kidney 2 weeks after injection. The yellow dotted lines encircle the host renal cortex. e–h Immunostaining of (d). e, f Chimeric glomeruli containing exogenous podocytes (Neph +, GFP +), nourished by the host endothelial cells (CD31 +, GFP−). g, h Chimeric proximal tubules (LTL +) and distal tubules (ECAD +) indicated by yellow arrows. Scale bars, 500 μm in (a), 50 μm in (b), 2 mm in (d), 100 μm in (e), 10 μm in (f), 200 μm in (g), and 20 μm in (h). CK8, cytokeratin 8; DAPI, 4’,6-diamidino-2-phenylindole; ECAD, E-cadherin; Endo, endothelial cell; GFP, green fluorescent protein; LTL, lotus tetragonolobus lectin; Neph, Nephrin; Podo, podocyte; Six2, sine oculis homeobox homolog 2.

Fig. 3. Maturation of exogenous nephrons (EGFP+) comparable with host nephrons (EGFP − ) evaluated with single-cell RNA-sequencing (scRNA-seq).

a A schematic of the sample collection. b Uniform manifold approximation and projection (UMAP) displaying unsupervised clustering of all 24,682 cells into 15 distinct, with annotation based on the expressions of previously reported marker genes. c Distribution of the four samples in the UMAP. d Distribution of EGFP (+) donor cells in the UMAP. The color scale indicates log-normalized EGFP expressions in each cell. e Pearson correlation analysis of the 2 weeks, CIS (−) sample, illustrating the correlation between host (EGFP −, n = 6141 cells) and donor (EGFP +, n = 64 cells) cells regarding the expression patterns of each cell type. f Violin plots depicting normalized expression levels of transporters involved in CIS uptake (Slc22a2, Slc31a1, and Slc22a1) and excretion (Slc47a1 and Abcc2) in host (n = 1291 cells) and donor (n = 20 cells) proximal tubule cells of the 2 weeks, CIS (−) sample. Data were analyzed using Welch’s t-test. g Immunostaining displaying the expressions of CTR1 and MATE1 in chimeric nephrons, indicated by yellow arrows. Scale bars, 20 μm in (g). CIS, cisplatin; DAPI, 4’,6-diamidino-2-phenylindole; DCT, distal convoluted tubule; EGFP, enhanced green fluorescent protein; Endo, endothelial cell; Epi, epithelial cell; FB, fibroblast; GFP, green fluorescent protein; IC, collecting duct intercalated cell; LOH, loop of Henle; Macro, macrophage; Mes, mesangial cell; NK, natural killer cell, ns, not significant; PC, collecting duct principal cell; Podo, podocyte; PT, proximal tubule; PT-S1 ~ 3, PT-segments 1–3; RBC, red blood cell.

Fig. 4. CIS-induced acute kidney injury model using chimeric nephrons.

a A schematic of the CIS-induced acute kidney injury model. b, c Immunostaining images of chimeric nephrons. The expression of kidney injury molecule 1 (Kim1) in proximal tubule cells (PTCs, LTL+) of host (GFP−) and donor (GFP+) origin increases in a CIS dose-dependent manner. d Kim1 positivity of host and donor PTCs (n = 6 sections from 3 biologically independent samples) after CIS treatment. Error bars represent means ± SEM. Data were analyzed using the two-tailed unpaired t-test. ns, not significant. *p < 0.05; **p < 0.01; ****p < 0.0001. e–g Comparisons between the samples at 2 weeks, CIS (−) and 2 weeks, CIS (+) (Fig. 3a) for both host and donor PTCs. e A comparison of the extent of individual gene expression changes upon CIS administration, shown by log2 fold change (log2FC), between host PTCs (n = 1291 cells in 2 weeks, CIS (−) vs. n = 1698 cells in 2 weeks, CIS (+)) and donor PTCs (n = 20 cells in 2 weeks, CIS (−) vs. n = 29 cells in 2 weeks, CIS (+)). f Heatmaps displaying genes with high variability, both up- and downregulated upon CIS administration, in host and donor PTCs. Genes with significant expression changes (log2FC > 1 and p < 0.05) in both host and donor PTCs are highlighted in red (upregulated) and blue (downregulated). g Violin plots showing normalized expression levels of representative variable genes in host and donor PTCs without (blue) and with (orange) CIS treatment. Scale bars, 100 μm in (b) and 20 μm in (c). CIS, cisplatin; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; LTL, lotus tetragonolobus lectin.

Fig. 5. Long-term viability and excretion function of chimeric nephrons.

a, b Fluorescence stereomicroscopic images (a) and immunostaining (b) of renal spheroids from EGFP mouse renal progenitor cells transplanted under the kidney capsule of adult NOD/Shi-scid, IL-2RgKO Jic mice (NOG mice) at 2 weeks, 1 month, and 2 months after transplantation. c, d Immunostaining of chimeric nephrons 2 months after injection, after systemic administration of fluorescent-labeled low-molecular-weight dextran. Yellow arrows indicate dextran excretion (d) and reabsorption (e) by the chimeric nephrons. e Fluorescence stereomicroscopic images of the host kidney 4 months after injection. f, g Immunostaining of chimeric nephrons 4 months after injection. h Pearson correlation analysis of the 2 months, CIS (−) sample showing the correlation between host (EGFP −, n = 1829 cells) and donor (EGFP +, n = 10 cells) regarding the expression patterns of each cell type. Scale bars, 1 mm in (a), 100 μm in (b), 20 μm in (c), 50 μm in (d), (f), (g), and 2 mm in (e). DAPI, 4’,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; LTL, lotus tetragonolobus lectin; Neph, Nephrin.

Fig. 6. Application of chimeric nephrons in repeated-dose testing.

a A schematic of the repeated-dose testing of CIS. b, c Immunostaining demonstrating that the expression level of Kim1 in proximal tubule cells (PTCs, LTL+) of the host (GFP−) and donor (GFP+) origin increases in a dose-dependent manner with CIS treatment. d Kim1 positivity of host and donor PTCs after CIS treatment (n = 6 sections from 3 biologically independent samples). Error bars represent means ± SEM. Data were analyzed using the two-tailed unpaired t-test. ns, not significant. *p < 0.05; **p < 0.01. e–g Comparison between the samples at 2 months, CIS (−) and 2 months, CIS (+) (Fig. 3a) for both host and donor PTCs. e A comparison of the extent of individual gene expression changes upon repeated CIS administration, shown by log2 fold change (log2FC), between host PTCs (n = 1829 cells in 2 months, CIS (−) vs. n = 3559 cells in 2 months, CIS (+)) and donor PTCs (n = 10 cells in 2 months, CIS (−) vs. n = 103 cells in 2 months, CIS (+)). f Heatmaps displaying genes with high variability, both up- and downregulated upon CIS administration, in host and donor PTCs. Genes with significant expression changes (log2FC > 1 and p < 0.05) in both host and donor PTCs are highlighted in red (upregulated) and blue (downregulated). g Violin plots showing normalized expression levels of representative variable genes in host and donor PTCs without (blue) and with (orange) CIS treatment. Scale bars, 200 μm (b) and 50 μm in (c). CIS, cisplatin; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; LTL, lotus tetragonolobus lectin; Kim1, kidney injury molecule 1.

Fig. 7. Xenogeneic chimeric nephrons derived from rat renal progenitor cells (RPCs) in immunosuppressed neonatal mouse hosts.

a A schematic of the method to inject rat RPCs under the renal capsule of neonatal NOD/Shi-scid, IL-2RgKO Jic mice. b, c Immunostaining for rat-specific Kim1 in exogenous rat proximal tubules after 2 weeks, comparing samples without CIS treatment (b) and with CIS treatment (c). d Kim1 positivity of exogenous rat proximal tubule cells (n = 6 sections from 3 biologically independent samples) after CIS treatment. Error bars represent means ± SEM. Data were analyzed using the two-tailed unpaired t-test. ****p < 0.0001. e, f Immunostaining of xenogeneic chimeric nephrons after 2 months showing chimeric glomeruli (Neph+) and proximal tubules (LTL +, white dotted lines). Scale bars, 100 μm in (b) and (c), 500 μm in (e), and 20 μm in (f). CIS, cisplatin; DAPI, 4’,6-diamidino-2-phenylindole; GFP, green fluorescent protein; LTL, lotus tetragonolobus lectin; Neph, Nephrin; Kim1, kidney injury molecule 1; PTC, proximal tubule cell.

Fig. 8. Human nephrons generated in immunosuppressed neonatal mouse kidneys.

a A schematic of the protocol followed for inducing nephron progenitor cells (NPCs) from EGFP-labeled human-induced pluripotent stem cells. b Representative images of the flow cytometric analysis of NPCs (n = 12 sections from 3 biologically independent samples). c Microscopic images of the in vitro differentiation of human NPCs for 9 days. d Hematoxylin and eosin-stained section of (c) demonstrating glomeruli (yellow arrows) and tubules formation. e–h Immunostaining of human nephrons in the neonatal kidneys 2 weeks after injection. e A human glomerulus (GFP +, Neph+) supplied by host vessels (yellow arrows. GFP−, CD31+). f CTR1 expression of human tubules (red dotted lines) and host mouse tubules (a yellow dotted line). g, h HuKim1 expression in human proximal tubules (LTL+, yellow arrows), comparing samples without CIS treatment (g) and with CIS treatment (h). Scale bars, 500 μm in (c), 100 μm in (d), 50 μm in (e), (f), (g), and (h). CIS, cisplatin; DAPI, 4’,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; HuKim1, human-specific kidney injury molecule 1; LTL, lotus tetragonolobus lectin; Neph, Nephrin.