Renal denervation and CD161a immune ablation prevent cholinergic hypertension and renal sodium retention

Abstract

Cholinergic receptor activation leads to premature development of hypertension and infiltration of proinflammatory CD161a⁺/CD68⁺ M1 macrophages into the renal medulla. Renal inflammation is implicated in renal sodium retention and the development of hypertension. Renal denervation is known to decrease renal inflammation. The objective of this study was to determine the role of CD161a⁺/CD68⁺ macrophages and renal sympathetic nerves in cholinergic-hypertension and renal sodium retention. Bilateral renal nerve denervation (RND) and immune ablation of CD161a⁺ immune cells were performed in young prehypertensive spontaneously hypertensive rat (SHR) followed by infusion of either saline or nicotine (15 mg·kg^-1·day^-1) for 2 wk. Immune ablation was conducted by injection of unconjugated azide-free antibody targeting rat CD161a⁺. Blood pressure was monitored by tail cuff plethysmography. Tissues were harvested at the end of infusion. Nicotine induced premature hypertension, renal expression of the sodium-potassium chloride cotransporter (NKCC2), increases in renal sodium retention, and infiltration of CD161a⁺/CD68⁺ macrophages into the renal medulla. All of these effects were abrogated by RND and ablation of CD161a⁺ immune cells. Cholinergic activation of CD161a⁺ immune cells with nicotine leads to the premature development of hypertension in SHR. The effects of renal sympathetic nerves on chemotaxis of CD161a⁺ macrophages to the renal medulla, increased renal expression of NKCC2, and renal sodium retention contribute to cholinergic hypertension. The CD161a⁺ immune cells are necessary and essential for this prohypertensive nicotine-mediated inflammatory response.NEW & NOTEWORTHY This is the first study that describes a novel integrative physiological interaction between the adrenergic, cholinergic, and renal systems in the development of hypertension, describing data for the role of each in a genetic model of essential hypertension. Noteworthy findings include the prevention of nicotine-mediated hypertension following successful immune ablation of CD161a⁺ immune cells and the necessary role these cells play in the overexpression of the sodium-potassium-chloride cotransporter (NKCC2) in the renal medulla and renal sodium retention. Renal infiltration of these cells is demonstrated to be dependent on the presence of renal adrenergic innervation. These data offer a fertile ground of therapeutic potential for the treatment of hypertension as well as open the door for further investigation into the mechanism involved in inflammation-mediated renal sodium transporter expression. Taken together, these findings suggest immune therapy, renal denervation, and, possibly, other new molecular targets as having a potential role in the development and maintenance of essential hypertension.

Keywords: CD161; CD68; adrenergic; cholinergic; hypertension; inflammation; innate immunity; macrophage; nicotine; renal; renal denervation.

Fig. 1.

Bilateral renal nerve denervation (RND) prevents nicotine-induced hypertension in young spontaneously hypertensive rat (SHR). Young 3- to 4-wk-old SHR underwent either sham surgery (Sham Sx; n = 14) or bilateral renal nerve denervation (RND; n = 16), followed by subcutaneous infusion of either saline (n = 12) or nicotine (15 mg·kg⁻¹·day⁻¹) (n = 18) for 2 wk. Renal tissues, obtained at the end of infusion, were sectioned and homogenized. Blood pressure was measured by tail cuff twice weekly. Student’s t-test was conducted between Sham Sx and RND groups for saline and nicotine infusion separately. A: RND prevents nicotine-induced hypertension. SBP, systolic blood pressure. B: renal sections were stained for tyrosine hydroxylase (left, red arrows) and calcitonin gene-related peptide (right, red arrows). C: renal homogenates were analyzed by ELISA for norepinephrine (NE). Error bars represent means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001, RND compared with Sham Sx of same infusion.

Fig. 2.

Bilateral renal denervation (RND) prevents renal infiltration of inflammatory CD161a⁺/CD68⁺ M1 macrophages. Young 3- to 4-wk-old spontaneously hypertensive rats underwent either sham surgery (Sham Sx; n = 6) or bilateral RND (n = 6), followed by subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for 2 wk. Renal tissues, obtained at the end of infusion, were sectioned and stained with antibodies targeting CD161a (FITC) and CD68 (PE). A: images of the renal cortex (left) and renal medulla (right) were captured using confocal microscopy for CD161a (FITC) and CD68 (Alexa fluor 555). B manual counts from 3 random fields from each animal of CD68⁺ macrophages (left), CD161a⁺ immune cells (middle), and CD161a⁺/CD68⁺ macrophages (right). C: flow cytometry for CD161a⁺ immune cells from the spleen, represents peripheral circulation. Two-way ANOVA with Tukey posttest was used to determine significance for renal immune cells between the renal cortex and medulla in the Sham Sx and RND groups. Student’s t-test was used to analyze splenic CD161a⁺ immune cells between the Sham Sx and RND groups. Error bars represent means ± SE. *P < 0.05 and ***P < 0.001.

Fig. 3.

Renal nerve denervation (RND) decreases inflammatory renal cytokines. Renal tissue and serum were collected from young 3- to 4-wk-old spontaneously hypertensive rats that underwent sham surgery (Sham Sx; n = 8) or bilateral RND (n = 10), followed by subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for 2 wk. A and B: levels of renal cytokines (A) and serum cytokines (B) were assessed by Luminex. Cytokines levels are reported as fold change above Sham Sx. Student’s t-test was performed to determine significance between RND and Sham Sx groups. GM-CSF, granulocyte-macrophage colony-stimulating factor; MIP-1a, macrophage inflammatory protein-1α. Error bars represent means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.

Renal nerve denervation (RND) decreases nicotine-induced renal toxicity and activates anti-inflammatory pathways. Renal tissue and serum were collected from young 3- to 4-wk-old spontaneously hypertensive rats that underwent sham surgery (Sham Sx) or bilateral RND, followed by subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for 2 wk. Sample sizes as indicated. A: levels of renal toxicity markers [kidney injury molecule-1 (Kim-1) and trefoil factor 3 (TFF3)]. Levels of toxicity markers were assessed by Luminex assay. B: anti-inflammatory M2-macrophage markers CD163, KLF9, and arginase II (Arg II) were assessed by RT-PCR. Sample sizes: **P < 0.01, compared with Sham Sx treated with saline. #P < 0.05, and ##P < 0.01, compared with Sham Sx treated with nicotine. Groups were analyzed by two-way ANOVA followed by Tukey post hoc test (A) and Student’s t-test between the RND ⁺ nicotine and Sham Sx + nicotine groups (B). Error bars represent means ± SD.

Fig. 5.

Renal nerve denervation (RND) significant reduces the expression of cellular adhesion/chemotaxis markers. Renal homogenates were prepared from kidneys isolated from young 3- to 4-wk-old spontaneously hypertensive rats (SHR) that underwent sham surgery (Sham Sx, n = 4) or bilateral RND (n = 4), followed by subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for 2 wk. MCP-1, monocyte chemoattractant protein-1; VLA-4, very-late antigen-4; LLT-1, lectin-like transcript-1. Protein levels were determined by Western blot analysis. Student’s t-test was used to determine significance between RND + nicotine and Sham Sx + nicotine groups. Error bars represent means ± SD. *P < 0.05.

Fig. 6.

Renal nerve denervation (RND) decreases cholinergic-induced renal sodium retention and expression of sodium-potassium chloride cotransporter (NKCC2). Young 3- to 4-wk-old spontaneously hypertensive rat underwent either sham surgery (Sham Sx, n = 14) or bilateral RND (n = 16), followed by subcutaneous infusion of either saline (n = 12) or nicotine (15 mg·kg⁻¹·day⁻¹; n = 18) for 2 wk (similar to Fig. 1). Renal tissues and urine were collected at the end of infusion. A–D: spot urine sodium was determined by colorimetric assay (A), fractional excretion of sodium (FeNa) was calculated (B), urine protein to creatinine ratios were calculated as a measure of renal function (C), and renal homogenates (D) were assayed for the presence of sodium-potassium chloride cotransporter (NKCC2) by Western blot analysis. Error bars represent means ± SE. P values are as indicated based on Student’s t-test. #P = 0.16, compared with Sham Sx + nicotine, suggesting trend toward increase in spot urinary sodium.

Fig. 7.

CD161a immune cell ablation prevents renal infiltration of CD161a⁺ immune cells. Young (3–4 wk) spontaneously hypertensive rat underwent intraperitoneal injection of either isotype control IGG (n = 5)- or anti-CD161a-specific antibodies (n = 5) in conjunction with subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for a 2-wk period. Renal tissues, obtained at the end of infusion, were sectioned and stained antibodies targeting CD161a (FITC) and CD68 (Alexa fluor 555). A: images of the renal cortex (left) and renal medulla (right) were captured using confocal microscopy for CD161a and CD68. B: manual counts from 3 random fields from each animal of CD161a⁺ immune cells in the renal cortex (left) and renal medulla (right). C: manual counts from 3 random fields from each animal of CD68⁺ macrophages in the renal cortex (left) and renal medulla (right). Student’s t-test was used to determine significance for renal immune cells between the CD161a Ablation and Isotype IGG groups. P values are as indicated.

Fig. 8.

CD161a⁺ immune cell ablation prevents cholinergic hypertension, decreases renal sodium retention, and renal expression of sodium-potassium chloride cotransporter (NKCC2). Young 3- to 4-wk-old spontaneously hypertensive rats underwent intraperitoneal injection of either Isotype Control IGG (n = 5) or anti-CD161a specific antibodies (n = 5) in conjunction with subcutaneous infusion of nicotine (15 mg·kg⁻¹·day⁻¹) for a 2-wk period. A: systolic blood pressure (SBP) was measured throughout the infusion period. Urine, serum, and renal tissues were collected at the end of the infusion period with measurements of sodium, creatinine, and protein. BM, bone marrow. B: spot urinary sodium (left), fractional excretion of sodium (FeNa; middle), and renal function (as measured by urinary protein to creatinine ratio; right) were determined by colorimetric assay. C and D: renal expression of NKCC2 was determined by Western blot analysis on renal homogenates (C) and markers of anti-inflammatory M2 markers (D) were determined by RT-PCR on renal tissue. Student’s t-test was conducted to determine significance between the CD161a ablation and isotype IGG-treated groups. Error bars represent means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 9.

Proposed hypothesis of “neuro-immune” axis in cholinergic hypertension and effects of bilateral renal denervation (RND) and CD161a immune cell ablation in young prehypertensive spontaneously hypertensive rat (SHR). A: schematic of the interactions between the cholinergic activation of CD161a⁺/CD68⁺ inflammatory macrophages and sympathetic-mediated chemotaxis of these macrophages and their role on renal sodium retention and cholinergic hypertension. 1A: nicotine directly activates efferent central sympathetic pathways. 2A and 3A: this leads to increased renal sympathetic nerve activity (2A) and expression of cellular adhesion molecules (3A) in the renal paranchyma (e.g., monocyte chemoattractant protein-1). 1B: direct activation of nicotinic acetylcholine receptors (nAChRs) on immune cells induces the expansion of the CD161a⁺ immune cell population and its differentiation into CD161a⁺/CD68⁺ inflammatory macrophages that hone into the renal medulla. 2B and 4: infiltration of the renal medulla with CD161a⁺/CD68⁺ inflammatory macrophages (2B) induces the renal expression of the sodium-potassium chloride cotransporter 2 (NKCC2; 4) in the thick ascending limb of the loop of Henle. Increased renal expression of NKCC2 in the renal medulla increases renal sodium retention and plays a role in the development of cholinergic hypertension. Ablation of the renal nerve or CD161a⁺ immune cells in vivo abolishes the development of cholinergic hypertension in young prehypertensive SHR animals.