Collecting duct-specific gene inactivation of alphaENaC 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 alpha 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.

Figure 1

Expression of the Hoxb7 promoter using the CreRosa 26 reporter system. The activity of Hoxb7-Cre is assayed by analysis of the Rosa reporter. (a) An intact P1 kidney stained with X-gal showing that Hoxb7-Cre is active in the CCDs and ureter (magnification, ×20). (b) A stained, thick section reveals Hoxb7-Cre activity throughout the CCD and MCD system (magnification, ×50). (c) 20 μM sections of stained kidneys demonstrate that Hoxb7-Cre is active in most if not all cells of the CD system (magnification, ×400). Some additional, mosaic staining is viewed in cells of the CNT and/or DCT, most likely due to mixing of cells at the fusion point of these two lineages.

Figure 2

Immunohistochemical characterization of ENaC antibodies on cryosections of kidneys from Scnn1a^loxlox mice kept on a sodium-free diet for 7 days. Immune sera for α, β, and γENaC show a bright apical immunostaining in CNT profiles that is not seen on sections incubated with preimmune sera or with immune sera in the presence of the immunogenic fusion proteins. Each column of images represents immunofluorescence on consecutive cryosections.

Figure 3

Immunofluorescent detection of αENaC in kidney cortex of Scnn1a^loxlox and Scnn1a^loxloxCre mice kept on a sodium-free diet for 6 days. In mice of both genotypes, αENaC is highly abundant in CNT profiles grouped around cortical radial arteries and veins located in the cortical labyrinth. Late DCT profiles exhibit a weak immunostaining. CDs running in the medullary rays are stained in loxlox (control) mice but are unstained in loxloxCre (experimental) mice. A, arteries; V, veins; CL, cortical labyrinth; MR, medullary rays; P, proximal tubules. Scale bar, 50 μm.

Figure 4

Transition of CNT to CCD in the kidney of Scnn1a^loxloxCre and Scnn1a^loxlox mice kept on a sodium-free diet for 6 days. (a) Immunofluorescence on consecutive cryosections with rabbit antibodies against αENaC and AQP2. Bright apical αENaC immunofluorescence ceases abruptly at the transition from CNT to CCD (arrows). AQP2 is seen in CNT and CCD. AQP2-negative cells in CNT and CCD are intercalated cells; the weak, punctuate staining in some tubular cells was not localized at the apical membrane, was occasionally observed with the αENaC antibody, and is nonspecific. P, proximal tubule. Scale bar, ∼20 μm. (b) Immunofluorescent detection of CB, NCX, and αENaC on consecutive cryosections from kidneys of loxlox (control) and loxloxCre (experimental) mice. In mice of both genotypes, the sharp transition from CNT to CCD (arrows) is characterized by a drop of cytoplasmic CB immunostaining and a breakoff, i.e., discontinuity, of basolateral NCX abundance. In the Scnn1a^loxlox mouse, apical αENaC immunostaining continues from the CNT to the CCD, whereas in Scnn1a^loxlox mice, αENaC immunoreactivity is seen in CNT but not CCD. Scale bar, 20 μm.

Figure 5

CNT profiles of kidneys from Scnn1a^loxlox and Scnn1a^loxloxCre mice kept on a sodium-free diet for 6 days. Immunofluorescence on cryosections with rabbit antibodies against α, β, and γENaC is shown. In mice of both genotypes, a bright apical immunostaining for α, β, and γENaC is seen in CNT cells. Unstained cells in the CNT profiles are intercalated cells. Scale bar, 15 μm.

Figure 6

CCD profiles of kidneys from Scnn1a^loxlox and Scnn1a^loxloxCre mice kept on a sodium-free diet for 6 days. Immunofluorescence on consecutive cryosections with rabbit antibodies against α, β, and γENaC. CCD cells of an Scnn1a^loxlox mouse show a weak but distinct apical immunostaining for α, β, and γENaC. Note the absence of αENaC immunostaining and the diffuse granular cytoplasmic localization of β and γENaC immunostaining in CCD cells of the Scnn1a^loxloxCre mouse. Unstained cells in the CCD profiles are intercalated cells. Scale bar, 20 μm.

Figure 7

OMCD profiles of kidneys from Scnn1a^loxlox and Scnn1a^loxloxCre mice kept on a sodium-free diet for 6 days. Immunofluorescence on cryosections with rabbit antibodies against α, β, and γENaC. Some OMCD cells of Scnn1a^loxlox mice show a very faint apical immunostaining for α, β, and γENaC. OMCD cells of Scnn1a^loxloxCre mice lack αENaC immunostaining and show a diffuse cytoplasmic localization of β and γENaC immunostaining. Scale bar, 15 μm.

Figure 8

IV curves. Cells from isolated CCDs (protocol A) were voltage clamped to a holding potential of zero. The voltage was stepped from –120 to +60 mV in 10-mV steps, and whole-cell currents were measured at the end of each 50-millisecond pulse. The procedure was repeated after addition of 10 μM amiloride to the bath (open squares [I_total = total current]; open circles, current with amiloride [I_amiloride = amiloride-resistant current]; filled circles, difference [I_Na =I_total – I_amiloride = amiloride-sensitive current]). In cells from control (Scnn1a^loxlox) animals, a substantial fraction of the whole-cell conductance was inhibited and the amiloride-sensitive currents reversed at positive cell potentials. In cells from experimental (Scnn1a^loxloxCre) animals, no amiloride-sensitive conductance could be measured. I, electrical current expressed in picoamperes.

Figure 9

Body-weight loss under salt restriction. Body-weight measurements in adult Scnn1a^loxlox (n = 9, filled circles) and Scnn1a^loxloxCre (n = 9, open triangles) mice. Animals were kept on a normal-salt diet (0.23% sodium) until day 0, followed by a sodium-deficient diet (0% sodium) for 7 days. Body weights of each group are indicated as percentages of the reference weight (100% at day 0). Mice were weighed at the same time each day.

Figure 10

Salt and water restriction. Urine and plasma osmolalities and physiological measurements in adult Scnn1a^loxlox (n = 9, filled circles) and Scnn1a^loxloxCre (n = 9, open triangles) mice. Animals were kept on a normal-salt diet (0.23% sodium) until day 0, followed by a sodium-deficient diet (0% sodium) for 5 days with free access to water. Osmolalities were measured at day 0 during the normal-salt diet and during a sodium-deficient diet before and after 23 hours of water deprivation (each group, n = 8).(a) Plasma osmolality in mice of indicated genotype. (b) Urine osmolality in mice of indicated genotype. Physiological measurements were performed at day 0 during the normal-salt diet and after 5 days of a sodium-deficient diet before and after 23 hours of water deprivation (each group, n = 8). (c) Plasma sodium levels. (d) Relative values of urine sodium normalized with creatinine. (e) Plasma potassium levels. (f) Relative values of urine potassium normalized with creatinine. CRSC, creatinine measured by single-slide method.

Figure 11

Physiological measurements under potassium loading in adult Scnn1a^loxlox (n = 9, filled circles) and Scnn1a^loxloxCre (n = 9, open triangles) mice. Animals were kept on a normal diet (0.95% potassium) until day 0 and then were fed a diet containing 2.6% potassium for 2 days followed by a diet containing 6% potassium for 2 days (each group, n = 6). (a) Plasma sodium levels. (b) Plasma potassium levels. (c) Relative values of urine sodium normalized with creatinine. (d) Relative values of urine potassium normalized with creatinine.