Role of collecting duct principal cell NOS1β in sodium and potassium homeostasis

Figures

• 

  FIGURE 1. Effects of NOS1β inhibition with L‐NAME on Kir4.1/Kir5.1 single‐channel activity in CHO cells overexpressing Kir4.1, Kir5.1, and NOS1β. (a) A representative time control current trace of 40‐pS K+ channel (Kir4.1/Kir5.1) single‐channel activity over the course of the experiment. Patches were clamped to –Vp = −60 mV. (b) Examples of single‐channel activity on an expanded time scale. The black and white bars represent the expanded time scale. Each dashed line in the magnified images represents a single‐channel opening state (number of active channels). (c) A representative current trace of Kir4.1/Kir5.1 single‐channel activity taken before and following administration of 1 mM L‐NAME. (d) Examples of single‐channel activity on an expanded time scale. The black bar in (b) represents the expanded time scale during baseline. The white bar represents the expanded time scale at the end of the L‐NAME treatment. (e) Summary for Kir4.1/Kir5.1 activity (NPo ), (f) number of channels (N), and (g) open probability (Po ) are reported (N = 5 independent experiments). p values from a Student's paired, two‐tailed, t‐test are listed in the figure

• 

  FIGURE 2. Kir4.1/Kir5.1 single‐channel activity in the cortical collecting duct (CCD) of control (con) and CDNOS1KO (KO) mice on a 7‐day high‐salt diet (4% NaCl). (a) Representative image of a CCD isolated by the vibrodissociation technique. Arrow points to basolateral patch. Scale bar is 20 µm. (b) Representative current traces of Kir4.1/Kir5.1 channel recorded from the basolateral membrane of CCD principal cells at different membrane potentials. Solid lines represent the closed state. Examples of single‐channel potassium currents recorded from CCD cells of (c) control and (d) CDNOS1KO mice on high‐sodium/normal potassium diet. Patches were clamped to –Vp = −60 mV. Summary graphs of (e) Kir4.1/Kir5.1 activity (NPo ), (f) active channels (N), and (g) open probability (Po). p values reported from the Mann–Whitney nonparametric test. N = 6–7 independent experiments

• 

  FIGURE 3. Representative immunofluorescent images of CCD principal cells (anti‐aquaporin‐2, positive, AQP2) and distal convoluted tubules (anti‐sodium chloride cotransporter positive, NCC) expressing Kir4.1, Kir5.1, or ROMK. Control (Con) and CDNOS1KO (KO) mice were on a 7‐day high‐NaCl diet. Mean intensity normalized to control is reported. Nuclei were stained blue with Hoechst 33342 in the merged image. (a) Kir4.1 and (b) Kir5.1 were localized to the basolateral membrane of the CCD. (c) ROMK was localized to the apical membrane of the CCD. Co‐localization of NCC and the K+ channels: (d) Kir4.1, (e) Kir5.1, and (f) ROMK. Mean intensity of individual CCDs or DCTs (2–3 each) from N = 6 control and CDNOS1KO mice were plotted. Mann–Whitney test p values provided. Scale bar = 10 µm

• 

  FIGURE 4. Plasma K+ and western blot analysis of cortical lysates from male control and CDNOS1KO mice on day 7 of a high Na+, normal K+ (HSNK; 1.6% Na+/0.3% K+) diet with either vehicle (Veh.) or spironolactone (Spirono.). (a) Plasma K+, N = 6 per genotype. Two‐ Factor ANOVA reported and * represents p < 0.05 Tukey's post hoc multiple comparison test. See Table 1 for means ± SEM. (b, c) Renal cortical abundance as determined by western blot of Kir4.1 (48 kDa) and Kir5.1 (60 kDa) channels from control (C) and CDNOS1KO (K) mice. Total Protein (pr.) stained with Coomassie to demonstrate equal loading of samples. (d) Relative densitometry of the Kir4.1 or (e) Kir5.1 abundance normalized to the vehicle, control group. N = 6 mice in each group. Two‐Factor ANOVA reported and Tukey's post hoc analysis failed to detect any further statistical significance among the groups

• 

  FIGURE 5. Plasma Na+ and K+ from male control and CDNOS1KO mice on various Na+ and K+ purified chow diets for 4 days. (a) Plasma Na+ was similar among the groups: normal Na+/normal K+ (NSNK: 0.3% Na+, 1% K+), normal Na+/deficient K+ (NSDK: 0.3% Na+, 0.0001% K+). N = 4–6. (b) Plasma K+ was significantly reduced in both genotypes after 4 days of NSDK diet and did not differ between genotypes. N = 4–6. (c) On a high‐Na+ diet with normal K+ (HSNK; 1.6% Na+, 1% K+), CDNOS1KO mice had significantly greater plasma Na+, which was reversed by K+‐deficient diet. N = 7–8. (d) Plasma K+ was significantly reduced by 4 days of high‐Na+ /deficient K+ diet (HSDK; 1.6% Na+, 0.0001% K+), without differences between genotypes. N = 8–9. Two‐Factor ANOVA reported with * represents p < 0.05 from the Tukey's multiple comparison test

• 

  FIGURE 6. Plasma aldosterone from male control and CDNOS1KO mice on various Na+ and K+ diets for 4 days. (a) Plasma aldosterone concentration is similar between the genotypes on the normal Na+/deficient K+ (NSNK: 0.3% Na+/1% K+) and normal Na+/deficient K+ (NSDK:0.3% Na+,0.0001%K+). Deficient K+ diet significantly reduced plasma aldosterone in both genotypes. N = 4–7 mice per group. (b) Plasma aldosterone from mice on a high Na+, normal K+ (HSNK, 1.6% Na+/1% K+), or high Na+, deficient K+ diet (HSDK, 1.6% Na+, 0.0001% K+). CDNOS1KO mice have greater plasma aldosterone on a HSNK diet; however, this genotype difference was abolished in the mice on a HSDK diet. HSNK N = 4–6 mice per group. Two‐Factor ANOVA reported with * represents p < 0.05 from the Tukey's multiple comparison test. (c) Agarose gel image of the PCR products of genomic DNA amplifying wildtype alleles (500 base pairs, bp) or the mutant recombined alleles (250 bp) of Nos1 from the adrenal gland and inner medulla samples from control (Con) and CDNOS1KO (KO) mice. Although there is genomic recombination in the inner medulla, there was no detectable recombination in the adrenal gland. Thus, NOS1 DNA is not mutated in the adrenal…

• 

  FIGURE 7. Renal cortical abundance of Kir4.1 and Kir5.1 channels from male control (C) and CDNOS1KO (K) mice on day 4 of a high Na+, normal K+ (HSNK; 1.6% Na+, 1% K+) diet or a high Na+, deficient K+ diet (HSDK; 1.6% Na, 0.0001% K+). (a) Western blots of Kir4.1 abundance, N = 5 animals per group. Total Protein (pr.) stained with Coomassie to demonstrate equal loading of samples. (b) Western blots of Kir5.1 abundance, N = 5 animals per group. (c) Relative to control HSNK, Kir4.1 and (d) Kir5.1 densitometry. Two‐Factor ANOVA reported, and * represents p < 0.05 from the Tukey's multiple comparison test. M‐ molecular marker, kDa‐ kilodalton

• 

  FIGURE 8. Hypothetical scheme of the proposed involvement of principal cell NOS1β in regulating Na+ and K+ balance in vivo during a chronic increase in dietary NaCl. (a) Physiologically, chronic high‐salt diet suppresses aldosterone thus reducing ENaC expression and activity and promoting natriuresis. Principal cell NOS1β is also activated leading to decreased ENaC activity and putative activation of basolateral Kir4.1/Kir5.1 to maintain fluid‐electrolyte balance. NOS1β may also stimulate other unknown pathways to maintain potassium secretion under conditions of suppressed aldosterone. (b) However, if principal cell NOS1β is not activated during high salt feeding there is a failure to appropriately suppress aldosterone, likely due in part to increased plasma K+ (that is, unmasked with spironolactone treatment), that then activates both ENaC and Kir4.1/Kir5.1, and overrides the decrease in Kir4.1/Kir5.1 activity expected from loss of NOS1β. Consequently, there is likely a resetting of the osmostat (high plasma Na+), a rightward‐shift in the pressure‐natriuresis curve (Hyndman et al., 2013), but plasma K+ is kept within the normal range. The mechanism by which plasma K+ is increased in NOS1β…