Fig 2: Treatment with S3I-201 improves glomerular injury in Klf4?Pod mice.(A) Urine albumin-to-creatinine ratio of DMSO-treated and S3I-201–treated Klf4?Pod mice. n = 9 per group, ***P < 0.001, unpaired t test. (B) Representative images of PAS staining in Klf4?Pod mice treated with S3I-201 or DMSO. n = 9 per group; asterisks indicate proteinaceous casts and tubule dilatation; # indicates interstitial and periglomerular inflammation; arrows indicate glomerulosclerosis with extracapillary proliferation. Representative images and quantification (insets) of immunostaining for (C) CD44, (D) Nephrin, (E) FN1, and (F) ITGB6. n = 4 per group; 20 glomeruli per mouse; ***P < 0.001, unpaired t test. Dotted white lines indicate glomerular area; solid white lines indicate area of extracapillary proliferation. Scale bars, 20 µm (CD44, Nephrin, FN1, and Hoechst) or 40 µm (PAS).

Fig 3: Stiffness-mediated differentiation phenotype induces upregulation of podocyte-specific functional proteins.(A) Representative images of Western blots for five proteins critical for physiological function of podocytes. Images of the complete blots are shown in Figure S3. (B) Quantification of kidney podocyte protein markers. Highest upregulation of WT-1 was found on the 2 kPa gel (2.2 ± 0.3). Nephrin, podocin, and CD2AP showed similar trends. Synaptopodin did not show this trend and there was no statistical difference between softer gels and control. T-statistics showed statistically significant changes of the ratios of five protein markers after normalization by control (two-tails, a = 0.05). The average t-statistics were 4.5 for the 2 kPa gel and 4.0 for the 5 kPa gel, which were significantly different relative to the control. Those for the 0.6 kPa and 13 kPa gels were 0.8 and 1.5, and the differences were not significant.

Fig 4: snRNA-seq profiles transcriptomic changes of Klf4?Pod mice.From kidney cortex samples of six different mice, 63,868 nuclei (30,838 from Klf4fl/fl and 33,030 from Klf4?Pod) meeting proper quality control thresholds were sequenced. (A) Uniform Manifold Approximation and Projection (UMAP) plot illustrates all 63,868 nuclei, mapping to 23 clusters. (B) Cluster identities aligned to canonical cell types in the adult mouse kidney based on a variety of cell type–specific marker genes. (C) Relative cell-type abundance compared between Klf4fl/fl and Klf4?Pod mice. (D) Cd44 expression plotted in UMAP space. Gray indicates no expression and purple indicates high expression. (E) Subclustering of the PEC/Prolif.PT cluster and expression of Cd44, Slc5a12, Nphs1, and Vcam1 across different subclusters. (F) Relative cell-type abundance compared between Klf4fl/fl and Klf4?Pod mice in PEC/Prolif.PT subclusters. (G) KEGG and (H) Reactome pathway analysis with ClusterProfiler for up-regulated genes in PEC/Prolif.PT subclusters 2/3. Pod, podocyte; Endo, endothelial; Mes, mesangial; Fib, fibroblast; PT, proximal tubule; LoH, loop of Henle; DCT, distal convoluted tubule; CNT, connecting tubule; CD-PC, collecting duct principal cell; IC, intercalated cell; JGA, juxtaglomerular apparatus; Mø, macrophage; GTPase, guanosine triphosphatase.

Fig 5: Gene expression indicates podocyte mechanotransduction can induce a pro-differentiation phenotype.Expression levels of six genes associated with podocyte differentiation and/or function were measured using qRT-PCR to test podocyte phenotype, namely: WT-1, KIRREL, SYNPO, NPHS1, NPHS2 and CD2AP. Colors in the heatmap represent the ratios of differential expression versus control. Four of the six genes showed upregulation on gels with different elastic moduli. Average fold-changes for these genes were 3.2, 7.5, 6.6, and 1.7 on the gels of 0.6 kPa, 2 kPa, 5 kPa, and 13 kPa, respectively.