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. 2023 Feb 22;6:208. doi: 10.1038/s42003-023-04563-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 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.