Fig 1: Diminished Expression of NKCC2/ATP1a1/PTH1R/Claudin-16/-19 and Increased Expression of Claudin-14 Are Associated with the Urinary Ca2+ Reabsorption Defect in TALs Lacking KCTD1(A) Semiquantitative RT-PCR in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice shows a significant decrease in NKCC2 (encoded by Slc12a1) transcripts compared with their littermate controls. P3 Six2Cre+KCTD1fl/fl mice show normal NKCC2 levels, and P8 Six2Cre+KCTD1fl/fl mice show a modest reduction in NKCC2 expression. Inducible inactivation of KCTD1 with TAM at P9 in b-actinCreERT2+KCTD1fl/fl mice results in strong reduction of NKCC2 expression in the kidney. Claudin-16 and claudin-19 expression is strongly reduced in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice, whereas claudin-14 expression is increased. Expression of ATP1a1 or PTH1R is decreased in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice but not at P3 before nephron maturation. Values normalized to their controls.(B) The expression of CLC-NKB is increased in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice. This is not observed before the manifestation of the distal nephron maturation defect (normal at P8). Values are normalized to their controls.(C) Total expression levels of claudin-16 and claudin-19 in kidneys of P3, P8, and 2-month-old Six2Cre+KCTD1fl/fl mice and their littermate controls.(D) Semiquantitative RT-PCR analysis in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice shows a significant decrease in Slc34a1 and Slc34a3 transcripts. Values normalized to their controls.(E) Semiquantitative RT-PCR analysis in kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice shows a significantly increased expression of the Ca2+ channels TRPV5 and TRPV6. NCX1 and calbindin-D28K expression is not reduced in kidneys lacking KCTD1. Values normalized to their controls.(F) Summary of expression changes of key regulators of urinary Ca2+ reabsorption shows similar results when comparing kidneys of 2-month-old KCTD1-/- and Six2Cre+KCTD1fl/fl mice.(G) Immunolabeling of kidneys from 6-month-old Six2Cre+KCTD1fl/fl mice and their WT controls for active ß-catenin, claudin-14, claudin-19, and TRPV5 shows localization of these proteins in dilated distal tubules of Six2Cre+KCTD1fl/fl mice. DAPI stains nuclei blue. Scale bars, 20 µm Graphs represent data as means ± SEM. Semiquantitative RT-PCRs performed in triplicate with n > 6 samples/group. p values are shown (two-tailed, unpaired t test).