Skip to main content
. 2018 Feb 2;7:e33432. doi: 10.7554/eLife.33432

Figure 4. Inhibition of CFTR activity in the efferent ductules pheno-copied the activity in Adgrg2-/Y mice.

(A) qRT-PCR analysis of the mRNA transcription profiles of potential osmotic drivers including selective ion channels and transporters in ADGRG2 promoter-labeled cells, non-ADGRG2 promoter-labeled cells and brain tissues of WT (n = 3) male mice. Expression levels were normalized to GAPDH levels. *p<0.05, **p<0.01, ***p<0.001, ADGRG2 promoter-labeled cells were compared with brain tissues. #p<0.05, ##p<0.01, ###p<0.001, non-ADGRG2 promoter-labeled cells were compared with brain tissues. (B–M) Effects of different channel blockers on the diameters of luminal ductules derived from WT or Adgrg2-/Y mice. (B) Bumetanide (10 μM), an NKCC blocker, WT (n = 9) or Adgrg2-/Y (n = 10); (C) Ani9 (150 nM), an ANO1 inhibitor, WT (n = 9) or Adgrg2-/Y (n = 9); (D) NFA (20 μM), a CaCC inhibitor, WT (n = 9) or Adgrg2-/Y (n = 10); (E) ruthenium red (10 μM), a non-specific TRP channel blocker, WT (n = 12) or Adgrg2-/Y (n = 12); (F) SKF96365 (10 μM), a TRPC channel inhibitor, WT (n = 12) or Adgrg2-/Y (n = 9); (G) nicardipine (20 μM), an L-type calcium channel blocker, WT (n = 12) or Adgrg2-/Y (n = 12); (H) EGTA (5 mM), an extracellular calcium chelator, WT (n = 9) or Adgrg2-/Y (n = 9); (I) DIDS (20 μM), a chloride-bicarbonate exchanger blocker, WT (n = 9) or Adgrg2-/Y (n = 10); (J) GlyH-101 (25 μM), a non-specific CFTR inhibitor, WT (n = 17) or Adgrg2-/Y (n = 15); (K) CFTRinh-172(10 μM), a specific CFTR inhibitor, WT (n = 12) or Adgrg2-/Y (n = 10). (L) Effects of angiotensin II (100 nM, an angiotensin receptor agonist) and PD123319 (1 μM, an AT2 receptor antagonist) on the diameters of luminal ductules derived from WT or Adgrg2-/Y mice (n ≥ 12). (M) Effects of angiotensin II (100 nM) and candesartan (1 μM, an AT1 receptor antagonist) on the diameters of luminal ductules derived from WT or Adgrg2-/Y mice (n ≥ 12). Application of GlyH-101 and CFTRinh-172 to ligated ductules derived from WT mice recapitulated the phenotype of the ductules derived from Adgrg2-/Y mice. (4A-M)*p<0.05, **p<0.01, ***p<0.001; Adgrg2-/Y mice compared with WT mice. #p<0.05, ##p<0.01, ###p<0.001. Treatment with selective inhibitors or stimulators was compared with control vehicles. n.s., no significant difference. At least three independent biological replicates were performed for Figure 4A–M.

Figure 4.

Figure 4—figure supplement 1. Expression and functional analysis of potential osmotic drivers in efferent ductules.

Figure 4—figure supplement 1.

(A) Quantitative RT-PCR (qRT-PCR) analysis of mRNA transcription profiles of potential osmotic drivers including selective ion channels and transporters in efferent ductules, brain and liver of wild-type (WT) (N = 3) male mice. Expression levels were normalized with GAPDH levels. *p<0.05, **p<0.01, ***p<0.001, brain were compared with efferent ductules. #p<0.05, ##p<0.01, ###p<0.001. liver were compared with efferent ductules. n.s., no significant difference. (B–C) Effects of different channel or transporter blockers on the diameters of luminal ductules derived from WT or Adgrg2-/Y mice. (B) LaCl3 (100 μM), a non-selective TRPC3/6/7 blocker, WT(n = 12) or Adgrg2-/Y(n = 12); (C) Amiloride(1 mM), a sodium/hydrogen antiporter NHE1 inhibitor, WT(n = 10)or Adgrg2-/Y(n = 12). (D) Quantitative RT-PCR (qRT-PCR) analysis of the ADGRG2,CFTR, Gαs, Gαq, β-arrestin-1 and β-arrestin-2 expression level in ADGRG2 promoter-labeled efferent ductule cells derived from Adgrg2-/Y mice(n = 3) and their WT littermates(n = 3). Expression levels were normalized with GAPDH levels.(B–D) **p<0.01, ***p<0.001; Adgrg2-/Y mice compared with WT mice. ##p<0.01, Selective inhibitors or stimulators treated were compared with control vehicles. n.s., no significant difference.