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. 2018 May 2;8:6917. doi: 10.1038/s41598-018-25087-3

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© The Author(s) 2018

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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Proximal tubule-specific expression of Ndrg1-CreERT2 and its recombination efficiency in Ndrg1-CreERT2;αKlotho^flox/flox mice. (a) Proximal tubule-specific expression of Cre in B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J(tdTomato) mice mated with Ndrg1-CreERT2 mice. Tamoxifen (3 mg/20 g body weight/day) was injected intraperitoneally for 5 consecutive days at the age of 5–8 weeks, and the mice were sacrificed 3 weeks later. Left panels: Kidney sections were co-immunostained for red fluorescent protein (RFP; red) and calbindinD28K (a marker for distal convoluted tubules shown in blue, arrows) with affinity marking by FITC-conjugated lotus tetragonolobus lectin (LTL, green) of the brush border membrane of proximal tubules (arrowheads). Right panels: The same experiments without any primary antibody (anti-RFP or anti-calbindinD28K antibody). Cre expression shown by positive RFP staining was specific for proximal tubules. GL: glomerulus. Scale bars: 50 μm. (b) Renal αKlotho expression at 1 and 3 weeks after tamoxifen treatment in αKlotho^flox/flox, Ndrg1-CreERT2;αKlotho^flox/flox, and Ndrg1-CreERT2;αKlotho^flox/+ mice. Tamoxifen was injected to induce Cre as described above. The period after tamoxifen treatment was counted from the date of the initial tamoxifen injection. Kidney sections were immunostained for αKlotho (red) and LTL (green), and counterstained for cell nuclei (DAPI, blue). Arrows, distal convoluted tubules; arrowheads, proximal tubules. Scale bars: 50 μm. (c) Co-staining of αKlotho and calbindinD28K in the kidney cortex of αKlotho^flox/flox and Ndrg1-CreERT2;αKlotho^flox/flox mice. Kidney sections treated as in (b) were stained for αKlotho (red), LTL (green), and calbindinD28K (blue). αKlotho expression was almost completely abolished in proximal tubules at 1 week after the initial tamoxifen injection. Distal convoluted tubules started to disassemble and their αKlotho expression intensity began to decrease at 2 weeks after tamoxifen injection. Arrows, distal convoluted tubules; arrowheads, proximal tubules. Scale bars: 50 μm. These experiments were performed in paired littermates with and without Ndrg1-CreERT2 transgene under the C57BL/6 J αKlotho^flox/flox background. Similar findings were reproduced in at least 3 different litters. See also Supplementary Figures S1 and S2.