Figure 1. Inactivation of AP-2β or KCTD1 in DCT1s Results in Hypomagnesemia Independently of EGF.

(A) Conditional gene-targeting strategy depicted in the schematic. PvalbCre leads to Cre-mediated recombination of floxed alleles specifically in the DCT1, whereas Aqp2Cre mice target CTs/CDs. Six2Cre mice target the entire nephron, except the CDs. KCTD1 is expressed in the distal nephron (TALs, DCTs, CTs, and CDs). DBA (dolichos biflorus agglutinin) lectin marks CTs/CDs, PNA (peanut agglutinin) lectin marks distal nephron epithelium (discontinuous epithelial staining in TALs, DCTs, and CTs/CDs; continuous labeling of PTs), and LTL (lotus tetragonolobus lectin) marks PTs. Slc3a1 is expressed in PTs, NKCC2 and THP in TALs, NCC in DCTs, Pvalb in DCT1s, and Aqp2 in CTs/CDs. EGF is detected in DCTs and TALs.

(B) Serum Mg²⁺ levels (in mg/dL) in groups of 2- or 10-month-old PvalbCre⁺KCTD1^fl/fl or PvalbCre⁺TFAP2B^fl/fl mice and their Cre-negative control littermates (WT).

(C) Urine Mg²⁺ concentrations (in mEq/L) are increased in 2-month-old PvalbCre⁺KCTD1^fl/fl mice when compared with their controls, despite normal 24-h urine production in the same mice.

(D) Tamoxifen-induced inactivation of KCTD1 at 4–6 weeks of age results in hypomagnesemia when assessed at 5–8 months of age (β-actinCreERT2⁺KCTD1^fl/fl mice + TAM). Inactivation of KCTD1 in intestines (VilCre⁺KCTD1^fl/fl mice) or of KCTD1 or AP-2β in CTs/CDs (Aqp2Cre⁺KCTD1^fl/fl or Aqp2Cre⁺TFAP2B^fl/fl mice) does not affect serum Mg²⁺ levels.

(E) Kidneys of adult PvalbCre⁺KCTD1^fl/fl or PvalbCre⁺TFAP2B^fl/fl mice show reduced protein levels of NCC but not of TRPM6; 12-month-old mice. β-actin as the loading control.

(F) Semiquantitative RT-PCR for Trpm6 and Slc12a3 in whole kidneys of 2-month-old Six2Cre⁺KCTD1^fl/fl mice and their littermate controls.

(G) Heatmaps show expression levels of genes implicated in the regulation of Mg²⁺ homeostasis in the kidney. RNA-seq data from kidneys of mice with induced inactivation of AP-2β in the adult (4-month-old β-actinCreERT2⁺TFAP2B^fl/fl mice treated with TAM at 6 weeks of age) and their Cre controls (left) or from kidneys of P8 Six2Cre⁺KCTD1^fl/fl mice and their Cre control littermates (Marneros, 2020). Differentially expressed genes (DEGs) (>1.5-fold change [FC] in expression; false discovery rate [FDR] < 0.05) are indicated. In both mutant groups, the DCT-expressed genes EGF, Slc12a3, and Pvalb are significantly downregulated. Scale shows FC normalized to the control group average.

(H) EGF (arrow) is detected in Pvalb⁺ DCT1s at the apical cell membrane (EGF in green, Pvalb in red). No EGF is detected in kidneys of EGF^−/− mice. Scale bar, 50 µm. DAPI stains nuclei blue. Mg²⁺ serum levels are not significantly reduced in EGF^−/− mice compared with their WT littermates at 2 or 4 months of age. Graphs represent data as means ± SEM. Semiquantitative RT-PCRs performed in triplicate. p values are shown (two-tailed, unpaired t test). Related to Figure S1.