Essential role of Kir5.1 channels in renal salt handling and blood pressure control

Abstract

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.

Keywords: Epithelial transport of ions and water; Ion channels; Nephrology; Potassium channels.

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^{Kcnj16–/–} rats.

(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 Mg²⁺ 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^{Kcnj16–/–} rats.

(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^{Kcnj16–/–} rats.

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^{Kcnj16–/–} rats.

(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^{Kcnj16–/–} rats.

(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.