Extended Data Fig. 10: NKD2 is a potential target in kidney fibrosis.

a, b, GO biological process terms for genes that correlate or anti-correlate with NKD2⁺ expression across single cells in pericytes fibroblasts and myofibroblasts in mouse PDGFRα⁺ PDGFRβ⁺ data (a) and human PDGFRβ⁺ data (b). Genes correlated with NKD2⁺ expression are related to ECM expression, integrin signalling and focal adhesion. c, Pathway activity as estimated by the PROGENy algorithm in NKD2⁺ versus NKD2⁻ cells from the human PDGFRβ⁺ dataset. P > 0.05 n.s., *P < 0.05, **P < 0.01, ***P < 0.001, P values were adjusted for multiple testing using Benjamin and Hochberg FDR method. d, Scaled gene expression of top 100 genes whose expression is correlated or anti-correlated with NKD2 expression across single cells in human PDGFRβ⁺ data (see b). e, Gene regulatory network predicted based on the expression of cells and genes depicted in l. using the GRNBoost2⁺ algorithm. Connection between genes indicate putative direct or indirect regulatory interactions. Colours indicate clustering of the gene regulatory network using the Louvain algorithm and highlights the regulatory network of ECM expression (module 2, NKD2⁺) and fibroblast and pericyte maintenance (module 4 and 3) f, Module 2 from l. Depicted separately, connections of NKD2 are highlighted in red. g, Expression of genes highlighted in e and f, including Etv1 transcription factor and LAMP5 which are both directly connected to Nkd2 in e and f. h, Expression of COL1A1, FN1 and ACTA2 by qPCR after NKD2 overexpression in human immortalized PDGFRβ⁺ cells treated with TGFβ or vehicle (PBS). n = 3 per group. P values determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are mean ± s.d. i, Expression of NKD2 by RNA qPCR in NKD2-knockout cells. ****P < 0.0001, one-way ANOVA followed by Bonferroni’s post hoc test. Data are mean ± s.d. j, Expression of COL1A1, FN1 and ACTA2 by RNA qPCR after NKD2 knockout in the same clones depicted in h. n = 3 per group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 (versus control NTG); ****P < 0.0001 (versus TGFβ NTG), two-way ANOVA followed by Sidak’s post hoc test. Data are mean ± s.d. k, PID signalling pathways enriched in PDGFRβ⁺ NKD2-KO clones and overexpression (up indicates upregulated genes in indicated condition, and down indicates down regulated genes). l, GO biological process terms enriched in PDGFRβ⁺ NKD2-KO clones (up indicates upregulated genes in KO condition, and down indicates down regulated genes). m, Scaled gene expression of WNT pathway receptors and ligands in NKD2-perturbed human kidney PDGFRβ⁺ cells. *P < 0.05, **P < 0.01, ***P < 0.001, empirical Bayes from the test for differential expression after adjusting P values for multiple testing correction (Benjamini and Hochberg). n, Representative image of multiplex RNA ISH of PDGFRα, PDGFRβ and NKD2 in human iPS cell-derived kidney organoids. o, Immunofluorescence staining of human iPS cell-derived kidney organoids (day 7 + 18, 2D + 3D culture, respectively). LTA and HNF4α mark proximal tubular like-cells. Pan-CK (cytokeratin) marks epithelial-like cells. ERG (ETS regulated-gene) marks endothelial-like cells. DACH1 and NEPHS1 mark podocyte-like cells. COL1α1 marks fibroblast/myofibroblasts. p, Immunofluorescence staining of COL1α1 in IL-1β-treated kidney organoids. Scale bars, 40 μm (n, o) 50 μm (p). For details on statistics and reproducibility, see Methods.