Collecting duct–specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance

Abstract

Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ASDN consists of the last portion of the distal convoluted tubule (late DCT), the connecting tubule (CNT), and the collecting duct (CD) (i.e., the cortical CD [CCD] and the medullary CD [MCD]). It has been proposed that the control of sodium transport in the CCD is essential for achieving sodium and potassium balance. We have tested this hypothesis by inactivating the α subunit of ENaC in the CD but leaving ENaC expression in the late DCT and CNT intact. Under salt restriction or under aldosterone infusion, whole-cell voltage clamp of principal cells of CCD showed no detectable ENaC activity, whereas large amiloride-sensitive currents were observed in control littermates. The animals survive well and are able to maintain sodium and potassium balance, even when challenged by salt restriction, water deprivation, or potassium loading. We conclude that the expression of ENaC in the CD is not a prerequisite for achieving sodium and potassium balance in mice. This stresses the importance of more proximal nephron segments (late DCT/CNT) to achieve sodium and potassium balance. Isabelle Rubera, Johannes Loffing, and Edith Hummler contributed equally to this work. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: epithelial sodium channel (ENaC); aldosterone-sensitive distal nephron (ASDN); distal convoluted tubule (DCT); connecting tubule (CNT); collecting duct (CD); cortical CD (CCD); medullary CD (MCD); outer MCD (OMCD); inner MCD (IMCD); adrenalectomized (adx); glomerular filtration rate (GFR); pseudohypoaldosteronism type 1 (PHA-1); X-galactosidase (X-gal); aquaporin-2 (AQP2); sodium/calcium exchanger (NCX); calbindin D28K (CB); sodium thiazide-sensitive chloride cotransporter (NCC).

Sodium and Potassium Balance Depends on αENaC Expression in Connecting Tubule

“…The previously described CD-specific KO mice (Scnn1a lox/lox /HoxB7::Cre) were used as positive (and negative) control. 10 In the CNT and the CCD of all control groups, a band at approximately 95 kD corresponding to full-length ␣ENaC protein was observed ( Figure 1A, lanes 1 and 2, and B, lanes 1 and 2 and lanes 5 and 6). This band was absent in the CCD of the CD-specific KO mice ( Figure 1A, lane 4) 10 and in the CCD of the CNT/CD-specific KO mice (Figure 1B, lanes 4 and 8).…”

“…10 In the CNT and the CCD of all control groups, a band at approximately 95 kD corresponding to full-length ␣ENaC protein was observed ( Figure 1A, lanes 1 and 2, and B, lanes 1 and 2 and lanes 5 and 6). This band was absent in the CCD of the CD-specific KO mice ( Figure 1A, lane 4) 10 and in the CCD of the CNT/CD-specific KO mice (Figure 1B, lanes 4 and 8). We observed a faint band corresponding to full-length ␣ENaC protein in the CNT of the CNT/CD-specific KO mice ( Figure 1B, lanes 3 and 7), suggesting that the inactivation of ␣ENaC in the CNT was not complete.…”

“…[7][8][9] Previously, a CD-specific conditional KO for ␣ENaC was generated, and, surprising, these mice were able to maintain water, sodium, and potassium balance even after 1 week of salt restriction, 23 hours of water deprivation, or 4 days of potassium loading. 10 In this study, we investigated the implication of the CNT and CD for the ENaC-mediated sodium reabsorption using mice that express the Cre recombinase from the Aqp2 promoter (Aqp2::iCre) and conditional alleles of ␣ENaC (Scnn1a loxlox ). 11,12 Aquaporin 2 (AQP2) is a water channel expressed along the CNT and CD.…”

Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system

“…At the time of the second nephrectomy and adrenalectomy, an osmotic minipump (ALZET model 2004; DURECT Corporation) containing aldosterone (A12930; Pfaltz & Bauer) and dexamethasone (D-1756; Sigma-Aldrich) was placed in a subcutaneous pouch. The doses of aldosterone (1 μg/day) and dexamethasone (1.2 μg/100 g body wt/d) were chosen to provide near-physiological replacement based on published studies (43,44). The aldosterone and dexamethasone were dissolved in polyethylene glycol 400 (Sigma-Aldrich) (45) and further diluted in normal saline.…”

Transgenic mice and their impact on kidney research

“…Finally, we used conditional gene targeting of ENaC to genetically dissect the aldosterone-induced ENaC-mediated sodium reabsorption along the nephron. When a floxed allele for the αENaC subunit (Scnn1a lox/lox mice) [38] was crossed with the HoxB7::Cre line this resulted in a complete abolishment of ENaC function in the cortical collecting duct (CCD), but not in the early segments of the aldosterone-sensitive distal nephron (ASDN), the late distal convoluted tubule (DCT) and connecting tubule (CNT) [85]. Surprisingly, we found that ENaC expression in the CCD is not a prerequisite for normal sodium and potassium balance.…”