Endothelial histone deacetylase 1 activity impairs kidney microvascular NO signaling in rats fed a high-salt diet

Abstract

Aim: We aimed to test the hypothesis that a high-salt diet (HS) impairs NO signaling in kidney microvascular endothelial cells through a histone deacetylase 1 (HDAC1)-dependent mechanism.

Methods: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or HS (4% NaCl) for 2 weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured.

Results: We found HS-induced impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS-fed rats to be sufficient to induce impaired NO signaling. This indicates the presence of a humoral factor we termed plasma-derived endothelial dysfunction mediator (PDEM). Pretreatment with the antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling.

Conclusion: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.

Keywords: HDAC1; endothelium; high‐salt diet; kidney; microvasculature; nitric oxide.

Figure 1.. HDAC1 inhibition restores L-NAME constriction in the afferent arterioles of high salt fed rats.

L-N^G-Nitro arginine methyl ester (L-NAME) constricts afferent arterioles from normal salt (NS) fed rats in a concentration-dependent manner. High salt diet (HS) significantly blunts constriction to L-NAME. MS-275 (300 nM) restores constriction to L-NAME in arterioles from (HS). All arterioles were perfused with blood from a rat on a matching diet. Arteriolar diameters are presented as measured (left panel) and normalized to baseline (Con; right panel). Data are plotted as mean±SE. a and b notate groups with statistically different areas under the curve (AUC). Differences in baseline diameters and AUC were assessed by two-way ANOVA and Holms-Sidak post hoc test.

Figure 2.. High salt diet increases kidney endothelial HDAC1 activity.

(a) HDAC1 activity but not total HDAC activity increased with high salt diet (HS) in rat endothelial cells assayed with the in situ HDAC activity assay. HDAC1 activity was assessed as the portion of total HDAC activity inhibited by 300 nM MS-275. (b) HS decreases the activity of immunoprecipitated HDAC1. HDAC1 was immunoprecipitated from isolated kidney endothelial cells and assayed. (c) HS does not change the abundance of class I HDACs in isolated endothelial cells. The arrow notes HDAC2. The large nonspecific band above HDAC2 is bovine serum albumin (BSA) which is present in the endothelial cell isolation buffer. REVERT total protein stain was used to assess equal loading. (d) HS does not change HDAC activity or HDAC1 activity detected in kidney vessel lysate. HDAC1 activity was assessed as the portion of total HDAC activity inhibited by 300 nM MS-275. (e) HS does not change kidney vascular abundance of class I HDACs as measured by Western blot. Coomassie was used to assess equal loading. All data are plotted as mean±SE. Unpaired, 2-tailed Student’s t-test used for all comparisons.

Figure 3.. Plasma from high salt diet fed rats is sufficient to impair NO signaling.

(A) Kidneys from normal salt diet (NS) or high salt diet (HS) fed rats were perfused with blood from either NS or HS fed rats for 30 min. Arterioles perfused with blood from NS fed rats constricted similarly regardless of the diet of the kidney donor. Arterioles from NS fed rats perfused with blood from HS rats had blunted constriction to L-NAME, but not to the level of arterioles from HS rats perfused with blood from HS rats. (B) Kidneys from NS fed rats were perfused with reconstituted blood made of erythrocytes from either NS or HS fed rats and plasma from either NS or HS fed rats. Plasma from HS fed rats is sufficient to blunt constriction to L-NAME. (C) Arterioles from NS fed rats were perfused with erythrocytes from NS fed rats and plasma from either NS or HS fed rats. 300 nM MS-275 restores constriction to L-NAME in arterioles perfused with plasma from HS fed rats. Arteriolar diameters are presented as measured (left panels) and normalized to baseline (Con; right panels). All data are plotted as mean±SE. a, b, and c denote groups with statistically different areas under the curve (AUC). Differences in baseline diameters and AUC were assessed by two-way ANOVA and Holms-Sidak post hoc test.

Figure 4.. Antioxidants restore L-NAME constriction in the afferent arterioles of high salt fed rats.

(A) Recombinant HDAC1 was assayed in the presence of increasing doses of plasma from normal salt (NS) or high salt (HS) fed rats. Differences were assessed with repeated measures two-way ANOVA and Holms-Sidak post hoc test. (B-D) Treatment with 100 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 3 μM tetrahydrobiopterin (BH4), and 1000 U/ml PEG-catalase restore or partially restore constriction to L-NAME in HS fed rats similar to NS fed rats. Arteriolar diameters are presented as measured (b-d left panels) and normalized to baseline (Con; b-d right panels). All data are plotted as mean±SE. a, b, and c represent groups with statistically different areas under the curve (AUC). Differences in baseline diameters and AUC were assessed by one-way or two-way ANOVA and Holms-Sidak post hoc test. RXN: reaction

Figure 5.. Hypothetical Model.

High salt diet promotes the presence of a plasma derived endothelial dysfunction mediator (PDEM) which activates kidney endothelial HDAC1 through an intracellular signaling mechanism. Both increased HDAC1 activity and reactive oxygen species (O₂- and H₂O₂) contribute to the loss of NO signaling in the juxtamedullary afferent arteriole. Created with BioRender.com.