2017 Sep 21
Essential Role of Kir5.1 Channels in Renal Salt Handling and Blood Pressure Control
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Essential Role of Kir5.1 Channels in Renal Salt Handling and Blood Pressure Control
Supplementing diets with high potassium helps reduce hypertension in humans. Inwardly rectifying K+ channels Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the basolateral membrane of distal renal tubules and contribute to Na+ reabsorption and K+ secretion through the direct control of transepithelial voltage. To define the importance of Kir5.1 in blood pressure control under conditions of salt-induced hypertension, we generated a Kcnj16 knockout in Dahl salt-sensitive (SS) rats (SSKcnj16-/-). SSKcnj16-/- rats exhibited hypokalemia and reduced blood pressure, and when fed a high-salt diet (4% NaCl), experienced 100% mortality within a few days triggered by salt wasting and severe hypokalemia. Electrophysiological recordings of basolateral K+ channels in the collecting ducts isolated from SSKcnj16-/- rats revealed activity of only homomeric Kir4.1 channels. Kir4.1 expression was upregulated in SSKcnj16-/- rats, but the protein was predominantly localized in the cytosol in SSKcnj16-/- rats. Benzamil, but not hydrochlorothiazide or furosemide, rescued this phenotype from mortality on a high-salt diet. Supplementation of high-salt diet with increased potassium (2% KCl) prevented mortality in SSKcnj16-/- rats and prevented or mitigated hypertension in SSKcnj16-/- or control SS rats, respectively. Our results demonstrate that Kir5.1 channels are key regulators of renal salt handling in SS hypertension.
Epithelial transport of ions and water; Ion channels; Nephrology; Potassium channels.
Conflict of interest statement
Conflict of interest: The authors have declared that no conflict of interest exists.
Figure 1. Kcnj16 knockout in the Dahl SS rat.
A) Immunostaining for Kcnj16 (K ir5.1) basolateral channels in salt-sensitive (SS) rats fed low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks). Note absence of protein staining in collecting duct (CD) intercalated cells. Right panel shows summary graphs of K ir5.1 expression in distal convoluted tubule (DCT) and cortical CD (CCD) on LS and HS diets. N = 5 rats; n ≥ 46 tubules for each group. ( B) A scheme of Kcnj16 gene showing the location of zinc finger nuclease–caused (ZFN-caused) deletion. Also shown is a specific position of deletion in the second transmembrane domain (TM2) of the protein. ( C) A representative section of Masson’s trichrome–stained kidney from 12-week-old SS and SS Kcnj16–/– rats fed a LS diet. Scale bar: 2 mm. ( D) An immunohistochemical analysis of the rat kidney tissues shows complete absence of Kcnj16 protein in SS Kcnj16–/– rats (right) compared with SS rats (left). Top and bottom images are at ×10 and ×40 magnification, respectively. Scale bar: 50 μm. ( E) Western blotting analysis of K ir5.1 expression in the kidney cortex of SS and SS Kcnj16–/– rats. Each line represents 1 rat. ( F) Body weight of age-matched SS and SS Kcnj16–/– rats fed a 0.4% NaCl diet. Normalized kidney per total body weight (TBW) is also shown ( N = 15). ( G) Mean arterial pressure (MAP) in SS and SS Kcnj16–/– rats when animals were fed a 0.4% NaCl diet ( N ≥ 9 rats in each group) measured with telemetry. Comparisons between groups were made using 1-way ANOVA. * P < 0.05.
Figure 2. Kidney function and electrolyte balance in SS
A) Light microscopy of Masson’s trichrome–stained sections of kidney cortex (at ×10 and ×40 magnification) of salt-sensitive (SS) and SS Kcnj16–/– rats fed a 0.4% NaCl diet. Scale bar: 50 μm. ( B) Renal injury as assessed by measuring albumin (normalized to creatinine) in urine samples collected for 24 hours in SS and SS Kcnj16–/– rats ( N ≥ 7). The averaged percentage of protein casts, glomerular injury, and the plasma aldosterone concentrations in SS and SS Kcnj16–/– rats are also shown ( N ≥ 6 rats). For glomeruli scoring, each point is an average of 80 glomeruli per rat. ( C) Biochemical analyses of electrolytes in plasma samples collected from SS and SS Kcnj16–/– rats (8–9 weeks old; 0.4% NaCl diet; N = 10). ( D) Fractional excretion (FE) of Na +, K +, and Mg 2+ over a 24-hour period in SS and SS Kcnj16–/– rats ( N = 7). Comparisons between groups were made using 1-way ANOVA. * P < 0.05.
Figure 3. Electrophysiological analysis of K
ir4.1 homotetrameric and K ir4.1/K ir5.1 heterotetrameric channels in SS and SS Kcnj16–/– rats.
A) Double-immunostaining images show Kcnj16 expression (red) in the distal convoluted tubule (DCT) and cortical collecting duct (CCD) cells. Aqp2 (green) was used as a marker of CD principal cells. Proximal tubules (PTs) and glomerulus (G) are also shown. Scale bar: 20 μm. ( B) Representative manually isolated distal tubule (note bifurcation) used for the patch-clamp analysis on basolateral membrane. ( C and D) Representative current traces ( C) and average current-voltage (I/V) relationships ( D) of the unitary current amplitude of 25.4 ± 3.9 pS (K ir4.1) and 48.1 ± 0.2 pS (K ir4.1/K ir5.1) K + channels measured in salt-sensitive (SS) rats. ( E and F) Representative current traces and average I/V relationships assessed in SS Kcnj16–/– rats ( N ≥ 5).
Figure 4. Expression and localization of Kcnj10 in SS and SS
A and B) Western blotting analysis of renal cortex tissues from salt-sensitive (SS) and SS Kcnj16–/– rats. The blot was probed with Kcnj10 antibodies. Equal loading was verified by blotting with GAPDH. ( C) Representative IHC staining of kidney cortical sections for detection of Kcnj10 protein in SS and SS Kcnj16–/– rats at magnification ×40. ( D) Summary graph of the analysis of Kcnj10 protein distribution on apical/basolateral sides in the distal tubules of SS and SS Kcnj16–/– rats ( N ≥ 5 rats, n = 58 tubules for each group). Comparisons between groups were made using 1-way ANOVA. * P < 0.05. NS, not significant
Figure 5. Differences in NCC and NKCC2 expression in SS
Western blotting analysis of NCC (
A) and NKCC2 ( B) from the kidney cortex lysates of salt-sensitive (SS) and SS Kcnj16–/– rats. Active phosphorylated forms were also analyzed. Each line represents 1 rat. ( C and D) Summary graphs showing the average relative density of the bands (normalized to loading controls) in the studied groups. * P < 0.05 versus SS rats. Comparisons between groups were made using 1-way ANOVA.
Figure 6. High salt intake triggers rapid mortality of SS
A) Survival rate of salt-sensitive (SS) and SS Kcnj16–/– rats on a 4% NaCl diet ( N = 20 and 5 for male and female rats, respectively). ( B) Na + and K + concentrations in urine (normalized to creatinine) and plasma in SS Kcnj16–/– rats before and 24 hours after the diet switch from 0.4% to 4% NaCl ( N ≥ 5 rats for each group). Comparisons between groups were made using 1-way ANOVA. * P < 0.05. HS, high salt. ( C) Survival rate of SS Kcnj16–/– rats when a high-salt diet was supplemented with water containing diuretics. Benzamil and furosemide were added to drinking water at a concentration of 15 mg/l, and hydrochlorothiazide (HCTZ) at 75 mg/l. N = 7/6, 8/6, and 8/7 (male/female rats) for experiments with benzamil, furosemide, and HCTZ, respectively.
Figure 7. The combination of a high-potassium diet and K
ir5.1 channel deletion mediate the protective effects on the development of SS hypertension.
A) Mean arterial pressure (MAP) in salt-sensitive (SS) and SS Kcnj16–/– rats. Blood pressure was measured with radiotelemetry (see also Supplemental Figure 3 for circadian rhythms and heart rate analyses). Animals were switched from a 0.4% to a 4% NaCl diet at day 0. Then, SS rats were fed either a standard 4% NaCl diet (black, N = 10 rats) or a 4% NaCl diet supplemented with high K + (2% KCl; red, N = 14). SS Kcnj16–/– rats were fed a 4% NaCl diet supplemented with high K + (green, N = 8). Comparisons between groups were made using repeated-measures ANOVA. * P < 0.05 versus SS rats fed a low-K + diet. ( B) Development of albuminuria (albumin to creatinine ratio) in the same groups of animals ( N ≥ 8 rats). ( C) Urinary electrolyte analysis of rats used in the experimental protocol shown in A; bars indicate electrolyte concentrations in control (0.4% NaCl [LS] or before diet change) and at the end of the experiment (4% NaCl [HS] and with or without K + supplement) for all groups of animals ( N = 8–13 rats in each group; see also Supplemental Figure 4). Comparisons between groups were made using 1-way ANOVA. * P < 0.05 versus SS rats fed a 0.4% NaCl diet.
Figure 8. Changes in ENaC, NCC, and NKCC2 expression in SS and SS
Kcnj16–/– rats fed a high-potassium diet.
Western blotting analysis of NCC, p-NCC, NKCC2, and p-NKCC (
A) and α-, β-, and γ-ENaC subunits (truncated forms of α- and γ-ENaC subunits are also shown) ( B) from the kidney cortex lysates of salt-sensitive (SS) rats were fed either a standard 4% NaCl diet or a 4% NaCl diet supplemented with high K + (2% KCl) and SS Kcnj16–/– rats fed a 4% NaCl diet supplemented with high K +. Each line represents 1 rat. ( C) Summary graphs represent the average relative density of the bands (normalized to loading controls) in the groups. Comparisons between groups were made using 1-way ANOVA. * P < 0.05.
Figure 9. High-potassium diet supplement restores the development of SS
A) The effect of a high-potassium diet (2% KCl) on body weight in SS Kcnj16–/– rats. ( B) Changes in kidney mass of salt-sensitive (SS) and SS Kcnj16–/– rats on low (0.36% K +) and high (1.41% K +) potassium–containing diets. Comparisons between groups were made using 1-way ANOVA. * P < 0.05.
Figure 10. Summary of the proposed role of K
ir4.1/5.1 in the kidney function and blood pressure control based on the SS Kcnj16–/– model.
All figures (10)
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Amiloride / analogs & derivatives
Blood Pressure / physiology
Furosemide / pharmacology
Hydrochlorothiazide / pharmacology
Kidney Tubules, Distal / metabolism
Potassium Channels, Inwardly Rectifying / genetics
Potassium Channels, Inwardly Rectifying / physiology
Sodium Chloride / metabolism
Sodium Chloride, Dietary / administration & dosage
Potassium Channels, Inwardly Rectifying
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