Fig 2: AP-2a regulates medullary CD structure in the adult but is not required for CD development.a RT-PCR of whole kidney lysates of Aqp2Cre+Tfap2afl/fl mice and Cre-negative littermates (WT) with two different PCR primer pairs that span exons 5/6 of Tfap2a. Kidneys of Aqp2Cre+Tfap2afl/fl mice show efficient inactivation of Tfap2a. 36b4 housekeeping gene. DNA ladder band size is indicated. b Aqp2Cre+Tfap2afl/fl mice and Aqp2Cre+Tfap2bfl/fl mice show a normal Mendelian ratio at 4-weeks of age. c Serum BUN levels are normal in 1-month-old Aqp2Cre+Tfap2afl/fl mice but they are increased at 2–4 months of age, whereas at that age BUN levels remain normal in Aqp2Cre+Tfap2bfl/fl mice or Aqp2Cre+KCTD1fl/fl mice. Adjusted p values are shown (one-way ANOVA with Tukey’s multiple comparisons test). Graphs represent data as mean ± SEM. Number of mice per group is indicated in brackets. Source data are provided as a Source Data File. d Dilated medullary CDs with flattened epithelial cells (black arrows) in 6-months-old Aqp2Cre+Tfap2afl/fl mice, but not in WT littermates (Tfap2afl/fl mice) or Aqp2Cre+Tfap2bfl/flKctd1fl/fl mice (green arrows). Scale bars, 250 µm left, and 50 µm right. e Immunolabeling for Aqp2 and Aqp3 show dilated medullary CDs in 6-months-old Aqp2Cre+Tfap2afl/fl mice (white arrow) compared to WT littermates (yellow arrow), whereas cortical CDs/CTs appear normal. Scale bars, 100 µm. f TCF/Lef:H2B-GFP mice serve as reporter mice for cellular ß-catenin activity (GFP+ cells): GFP+ cells (yellow arrows) are detected in CDs (Aqp2+) in the medulla as well as in TALs (NKCC2+), whereas DCTs (NCC+) show no GFP+ cells (white arrows). 23-months-old TCF/Lef:H2B-GFP mouse kidney. Scale bars, 50 µm.

Fig 3: 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).

Fig 4: Effect of Ang II and/or losartan on immunofluorescence and protein expression levels of kidney cortical Na-K-2Cl cotransporter (NKCC2). Immunofluorescence images showing NKCC2 staining in the cortical area (indicated by arrows), and the basolateral α1Na+K+ pump is indicated by asterisks in the thin ascending limb (TAL). Images were captured using a Zeiss LSM 510 confocal microscope equipped with a 40 × objective. α1Na+K+ pump staining was used as the basolateral membrane integrity control, and NKCC2 and α1Na– K pump staining were merged. The enlarged images are also shown. Bar = 50 μm (A). The mean fluorescence intensity of NKCC2 is also presented (B; n = 5–7 per group). For immunoblot analysis, 50 μg cortical protein samples were resolved by 9% SDS-PAGE. The values are expressed as the mean ± SEM (n = 4–6 per group), and β-actin was used as the loading control (C). Sham (control) group; AII, angiotensin II; Los, losartan; and AII/Los, angiotensin II/losartan. ++p < 0.01 for the Ang II and losartan interaction, as indicated by two-way ANOVA. **p < 0.01, ***p < 0.001 for Ang II vs sham or #p < 0.05, ####p < 0.0001 for AII/Los vs AII, as indicated by multiple comparisons.

Fig 5: Deficiency of AP-2ß in Six2+ NPCs results in severe cystic renal degeneration, kidney fibrosis, and renal failure. a, b Whereas most Six2Cre+Tfap2bfl/fl mice died perinatally due to lack of DCTs, a few mice survived to 1–2 months of age, when they succumbed to renal failure with a very high BUN (b). These mice showed cystic degeneration of their renal cortex and tubulointerstitial fibrosis. Renal cysts were lined by either flattened epithelium (green arrow) or epithelial cells with a hobnail-like morphology (black arrow). H&E sections. Top: 1 month-old WT; middle and bottom panels: 1 month-old Six2Cre+Tfap2bfl/fl mouse. Scale bars: 1,4: 1 mm; 2,3,5,6: 250 µm; 7-9: 50 µm. P value determined by a two-tailed Mann–Whitney test (b). BUN in mg/dl. Graph represents data as mean ± SEM. Source data are provided as a Source Data File. b N = 9 WT, n = 3 Six2Cre+Tfap2bfl/fl mice. c H&E images show renal pathologies in 1–2 months-old Six2Cre+Tfap2bfl/fl mice (1–7) compared to an age-matched littermate WT mouse (8). 1: Bluish calcified intratubular deposits are observed (red arrows). 2: Hobnail-like epithelial cells (green arrow) lining renal cysts with extrusion of cells into the lumen (red arrow). Some cells show clear cell-like appearance of their cytoplasm (black arrows). 3: Subset of dilated cortical distal nephron tubules show cuboidal epithelium (yellow arrow) with apical blebbing (green arrows). Protein casts are seen in some dilated tubules (black arrow). 4: In some dilated tubules, a multilayered epithelium (black arrows) and formation of micropapillae (green arrows) were observed. Inflammatory cells and extruded epithelial cells are seen in the lumen. Some tubules are filled with protein casts (yellow arrow). 5: In addition, atrophic tubules with diminished tubular lumen were observed (green arrows). Glomeruli appeared normal (white arrow). 6: A subset of epithelial cells of dilated distal tubules had a clear-cell-like appearance with cytoplasmic vacuoles (green arrows). 7: Some distal tubule cysts are lined by flattened epithelium (green arrow), whereas proximal tubules do not show epithelial flattening (white arrow). 8: WT littermates showed none of these pathologies. Scale bars, 100 µm. d Western blotting with whole kidney lysates of 1- and 2-months-old Six2Cre+Tfap2bfl/fl mice, Six2Cre+Tfap2bfl/WT mice (3, 5–6, and 14-months old), and WT littermate controls. Lack of AP-2ß function results in absence of differentiated DCTs (the DCT marker NCC is not detected), whereas heterozygosity of AP-2ß function leads to loss of NCC with age progression. This loss of NCC is accompanied by an increase in GSK-3ß phosphorylation, in active non-phospho Ser33/37/Thr41 ß-catenin, and in an increase in mTOR signaling (increased Thr37/46 phosphorylation of 4E-BP1 and total 4E-BP1; increased phosphorylation of p70 S6 kinase; increased TFEB phosphorylation [low and high exposure shown to demonstrate upper phospho-TFEB bands (red stars); lower TFEB bands marked with green stars]). Kidneys of Six2Cre+Tfap2bfl/fl mice have diminished NKCC2, whereas aged Six2Cre+Tfap2bfl/WT mice have increased NKCC2. Both have decreased protein levels of the PT protein Slc3a1. ß-actin as a loading control. Densitometric values for Western blot bands normalized to ß-actin are shown. Size markers are indicated by arrowheads.