Fig 2: Nephrin activation results in increased active Rap1 in human podocytes. (A) A nephrin clustering assay was performed by adding anti-CD16 antibody and secondary anti-IgG antibody to the media of cultured podocytes, which induces clustering of nephrin proteins followed by recruitment of Src kinases and consecutive phosphorylation of nephrin on tyrosine residues. (B) Immunoblot with antibodies specific for Rap1A/B following immunoprecipitation (IP Rap1-GTP) with an antibody specific for active Rap1A/B of podocyte lysates. Employed were human podocyte lines that inducibly express either chimeric CD16-CD7-nephrin cytoplasmic domain (NCD) or CD16-CD7-HA (HA) as a control following doxycycline treatment followed by clustering of nephrin. Immunoblots of these lysates were also performed with antibodies specific for Rap1A/B, nephrin, phospho-nephrin (pNephrin), HA, and ß-tubulin (Input), n = 3. (C) Schematic of the hypothesis that the small GTPase Rap1 functions downstream of nephrin in signaling to integrin ß at focal adhesions and in signaling at the slit diaphragm.

Fig 3: Hepatocyte-specific overexpression of Klotho ameliorates renal injury and proteinuria in a mouse model of NTS-induced glomerulonephritis.a Left panel: Urinary albumin/creatinine ratio (U-ACR) in WT, Alb-hKL, and Pod-hKL, 0, 1, 3, and 7 days after injection of NTS. $$$$p < 0.0001 vs. Pod-hKL and @@@@p < 0.0001 vs. WT. Right panel: Blood urea nitrogen in the different groups without injury (control mice) or 7 days after injection of NTS (NTS mice). ####p < 0.0001 vs. WT-control; @p < 0.05 vs. WT-NTS. b Representative images of PAS (left panels) and TEM (right panels) of kidney cortex from WT-control and WT, Alb-hKL, Pod-hKL 7 days after NTS injection. c Percentage of affected and crescentic glomeruli were quantified from 30 randomly selected glomeruli in the corresponding PAS staining. Tubular casts were assessed over the whole cross-section giving a score between 0, no cast to 5, >80% of the tubules are affected (n = 11 WT-control; n = 10 WT-NTS; n = 5 Alb-hKL-NTS; n = 11 Pod-hKL-NTS). Quantification of mean glomerular basement membrane (GBM) thickness and the number of slits per µm GBM (n = 5 WT-control; n = 10 WT-NTS; n = 6 Alb-hKL-NTS; n = 8 Pod-hKL-NTS). ###p < 0.001, ####p < 0.0001 vs. WT-control; @p < 0.05, @@p < 0.01 vs. WT-NTS and *p < 0.05, ***p < 0.001 vs. Alb-hKL-NTS. d Representative images of immunofluorescent stainings of Nephrin, WT-1, and a-SMA in the different groups. e Quantification of the number of WT-1 positive podocytes in glomeruli (left panel), glomerular area (middle panel), and WT-1 positive podocytes/glomerular area (right panel) (n = 7 WT-control; n = 9 WT-NTS; n = 5 Alb-hKL-NTS; n = 9 Pod-hKL-NTS). a-SMA positive area was counted per cortical cross section (10 sections per sample; n = 6 WT-control; n = 10 WT-NTS; n = 5 Alb-hKL-NTS; n = 9 Pod-hKL-NTS). ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. WT-control; @p < 0.05 vs. WT vs. WT-NTS and *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. Alb-hKL-NTS. Scale bars: 50 µm, 30 µm, 5 µm (b), 10 µm, 50 µm (d). Statistical significance between groups was determined using one-way or two-way repeated measures ANOVA followed by multiple comparisons.

Fig 4: Expression of Klotho in normal human and mouse kidneys.a, b Human and mouse single-cell RNA-sequencing data extracted from Kidney glomerular single-cell atlas (data available at https://patrakkalab.se/kidney/). The left panels show a t-SNE plot of cell clusters based on the identification of highly expressed specific cell markers (left panels in supplementary Fig. 1). The t-SNE plot (right panels) shows that Klotho expression in the different clusters. The podocytes circled in red. DTL: descending thin limb of Henle’s loop; GEC: glomerular endothelial cells; MLC: Mesangial-like cells; PTC: Proximal tubular cells; T + NK: T + natural killer lymphocytes; cTAL + CD: cortical thick ascending limb of Henle’s loop + collecting duct. CD: collecting ducts; DCT: distal convoluted tubules; EC: endothelial cells; PEC: glomerular parietal epithelial cells; cTAL: cortical thick ascending limb of Henle’s loop. c In situ hybridization of human kidney cortex where Klotho (red) was co-labeled with Lotus tetragonolobus lectin (LTL, green) and Hoechst (blue). d In situ hybridization of mouse kidney cortex from 3 different strains (129S2, CD1 ICR, C57BL6/J) where Klotho (blue) and Nephrin (pink) were labeled. e, f RT-qPCR for Klotho in rest of kidney (ROK), glomerular (glom) fraction and in (f) FAC sorted mouse podocytes (Pod). 28S (e) and GAPDH (f) genes were used as a housekeeping gene and nephrin to validate the purity of glomerular fraction and isolated podocytes. Values are expressed as mean ± SD (n = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001. g, h Dual labeling with a podocyte marker nephrin (green) and Klotho (purple) in both human (g) and mouse (h). i, j Western blotting in the rest of kidney (ROK) and glomeruli fraction (Glom). Calnexin was used as a loading control and podocin antibody validates the purity of isolated glomeruli. Data are expressed as mean ± SD (n = 3). ****p < 0.0001. Scale bars: 100 µm (c); 20 µm (d), 50 µm (g), 20 µm (h). Statistical significance between groups was determined using unpaired t-test; more than 2 groups were compared with one-way ANOVA.