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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

Oleg Palygin et al. JCI Insight.

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.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Kcnj16 knockout in the Dahl SS rat.
(A) Immunostaining for Kcnj16 (Kir5.1) 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 Kir5.1 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 SSKcnj16–/– 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 SSKcnj16–/– 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 Kir5.1 expression in the kidney cortex of SS and SSKcnj16–/– rats. Each line represents 1 rat. (F) Body weight of age-matched SS and SSKcnj16–/– 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 SSKcnj16–/– 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
Figure 2. Kidney function and electrolyte balance in SSKcnj16–/– rats.
(A) Light microscopy of Masson’s trichrome–stained sections of kidney cortex (at ×10 and ×40 magnification) of salt-sensitive (SS) and SSKcnj16–/– 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 SSKcnj16–/– rats (N ≥ 7). The averaged percentage of protein casts, glomerular injury, and the plasma aldosterone concentrations in SS and SSKcnj16–/– 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 SSKcnj16–/– 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 SSKcnj16–/– rats (N = 7). Comparisons between groups were made using 1-way ANOVA. *P < 0.05.
Figure 3
Figure 3. Electrophysiological analysis of Kir4.1 homotetrameric and Kir4.1/Kir5.1 heterotetrameric channels in SS and SSKcnj16–/– 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 (Kir4.1) and 48.1 ± 0.2 pS (Kir4.1/Kir5.1) K+ channels measured in salt-sensitive (SS) rats. (E and F) Representative current traces and average I/V relationships assessed in SSKcnj16–/– rats (N ≥ 5).
Figure 4
Figure 4. Expression and localization of Kcnj10 in SS and SSKcnj16–/– rats.
(A and B) Western blotting analysis of renal cortex tissues from salt-sensitive (SS) and SSKcnj16–/– 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 SSKcnj16–/– 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 SSKcnj16–/– 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
Figure 5. Differences in NCC and NKCC2 expression in SSKcnj16–/– rats.
Western blotting analysis of NCC (A) and NKCC2 (B) from the kidney cortex lysates of salt-sensitive (SS) and SSKcnj16–/– 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
Figure 6. High salt intake triggers rapid mortality of SSKcnj16–/– rats.
(A) Survival rate of salt-sensitive (SS) and SSKcnj16–/– 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 SSKcnj16–/– 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 SSKcnj16–/– 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
Figure 7. The combination of a high-potassium diet and Kir5.1 channel deletion mediate the protective effects on the development of SS hypertension.
(A) Mean arterial pressure (MAP) in salt-sensitive (SS) and SSKcnj16–/– 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). SSKcnj16–/– 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
Figure 8. Changes in ENaC, NCC, and NKCC2 expression in SS and SSKcnj16–/– 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 SSKcnj16–/– 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
Figure 9. High-potassium diet supplement restores the development of SSKcnj16–/– rats.
(A) The effect of a high-potassium diet (2% KCl) on body weight in SSKcnj16–/– rats. (B) Changes in kidney mass of salt-sensitive (SS) and SSKcnj16–/– 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
Figure 10. Summary of the proposed role of Kir4.1/5.1 in the kidney function and blood pressure control based on the SSKcnj16–/– model.

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