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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Hypertension. 2016 Aug 22;68(4):929–936. doi: 10.1161/HYPERTENSIONAHA.116.07993

RENAL DENERVATION NORMALIZES ARTERIAL PRESSURE WITH NO EFFECT ON GLUCOSE METABOLISM OR RENAL INFLAMMATION IN OBESE HYPERTENSIVE MICE

Ninitha Asirvatham-Jeyaraj 1, Jessica K Fiege 2, Ruijun Han 1, Jason Foss 1, Christopher T Banek 1, Brandon J Burbach 2, Maria Razzoli 1, Alessandro Bartolomucci 1, Yoji Shimizu 2, Angela Panoskaltsis-Mortari 3, John W Osborn 1
PMCID: PMC5016252  NIHMSID: NIHMS807611  PMID: 27550916

Abstract

Hypertension (HTN) often occurs in concurrence with obesity and diabetes, commonly referred to as metabolic syndrome. Renal denervation (RDNx) lowers arterial pressure (AP) and improves glucose metabolism in drug resistant hypertensive patients with high body mass index. In addition, RDNx has been shown to reduce renal inflammation in the mouse model of angiotensin II hypertension. The present study tested the hypothesis that RDNx reduces AP, renal inflammation and improves glucose metabolism in obesity-induced HTN. Eight-week old C57BL/6J mice were fed either a low fat diet (LFD; 10 KCal%) or a high fat diet (HFD; 45 KCal%) for 10 weeks. Body weight, food intake, fasting blood glucose and glucose metabolism (glucose tolerance test) were measured. In a parallel study, radiotelemeters were implanted in mice for AP measurement. High fat fed C57BL/6J mice exhibited an inflammatory and metabolic syndrome phenotype including increased fat mass, increased AP and hyperglycemia compared to LFD mice. Renal denervation, but not Sham surgery, normalized AP in HFD mice (115.8 ± 1.5 in sham vs. 96.6± 6.7 mmHg in RDNx). Renal denervation had no significant effect on AP in LFD mice. Also, RDNx had no significant effect on glucose metabolism or renal inflammation as measured by the number of CD8, CD4 and T helper cells or levels of inflammatory cytokines in the kidneys. These results indicate that while renal nerves play a role in obesity-induced hypertension, they do not contribute to impaired glucose metabolism or renal inflammation in this model.

Keywords: Renal denervation, metabolic syndrome, glucose, T cell, cytokines, hypertension

Introduction

Obesity is often associated with hypertension and type 2 diabetes; a condition commonly referred to as metabolic syndrome (1). Obesity is also linked with inflammation in general (2) and renal inflammation specifically (3, 4). However, the extent to which obesity-associated inflammation is primary or secondary to hypertension and type 2 diabetes is not known.

It is now widely accepted that obesity is correlated with increased activity of sympathetic nervous system (SNA) (1, 5, 6) with the growing consensus that SNA to the kidneys is a major contributor to the pathogenesis and maintenance of obesity-induced hypertension. This concept is supported by reports that renal nerve ablation prevents and reverses obesity-induced hypertension in experimental animals (7–9) and improves glucose metabolism (10). Interestingly, catheter based renal nerve ablation in drug resistant hypertensive humans, many of whom are obese, has been reported to not only reduce arterial pressure, but improve glucose metabolism as well (11, 12). It has been hypothesized that this response is secondary to ablation of sympathoexcitatory renal sensory nerves and subsequent reduction of SNA to skeletal muscle, a key glucose regulatory tissue (10, 13, 14). Finally, recent studies suggest that renal nerve ablation may also reduce renal inflammation associated with hypertension. Angiotensin II (AngII) induced hypertension in mice has been shown to be dependent on trafficking of T cells into the kidney (15) and this response is dependent on brain sites that regulate the activity of the sympathetic nervous system (16). Moreover, renal denervation (RDNx) ameliorates renal inflammation in mice with AngII-induced hypertension (17) as well as a model of experimental glomerulonephritis (18) suggesting an important role of renal nerves in trafficking of T cells and cytokines into the kidney (16, 19, 20).

The present study was designed to test the hypothesis that renal nerves serve as a nexus point linking obesity, hypertension, impaired glucose metabolism, and renal inflammation. Specifically, we hypothesized that trafficking of cytokine-producing T cells into the kidney, and subsequent inflammation, is dependent on renal nerves in obesity-induced hypertension. Furthermore, we predicted that these inflammatory signals activate renal sensory nerves resulting in global activation of the sympathetic nervous system.

We tested this hypothesis in C57BL/6J mice maintained on a high fat diet since this model has been reported to exhibit hypertension, hyperglycemia, and renal inflammation (4). Specifically, we measured the effects of renal denervation on arterial pressure, glucose metabolism and renal inflammation in obese, hypertensive C57BL/6J mice and lean normotensive controls.

Methods

Animals

See Figure 1 for details of the protocol timeline. Seven-week-old C57BL/6J mice weighing 20–25 grams from Jackson laboratories (Bar Harbor, Maine) were maintained on a 12h light and 12h dark cycle in a temperature-controlled room with free access to normal chow diet and distilled water. A week later, mice were switched to a low fat (LFD, 10Kcal% fat) or high fat (HFD, 45Kcal % fat) diet (Research Diets, New Brunswick, NJ) and assigned to enter either a Cardiovascular or a Metabolic Protocol as described below. Separate protocols were necessary to avoid damage to the telemeters by EchoMRI. All experiments were performed in compliance with National Institute of Health Laboratory Animal Care and Use guidelines and IACUC approval at the University of Minnesota.

Figure 1. Experimental protocol.

Figure 1

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A) In the Cardiovascular Protocol, mice on low fat diet (LFD) or high fat diet (HFD) underwent telemetry surgery. After 12 weeks of diet and a 3 day control (C) recording of mean arterial pressure (MAP) and heart rate (HR), mice in LFD-Sham (n=7), LFD-RDNx (n=5), HFD-Sham (n=6), HFD-RDNx (n=5) groups underwent renal denervation (RDNx) or sham surgery. At 17 weeks of diet, kidneys were collected for norepinephrine analysis. B) In the Metabolic Protocol body weight (weekly), food intake (weekly) and body composition (monthly) were measured in LFD-Sham, LFD-RDNx, HFD-Sham, HFD-RDNx, (n=9/group). Fasting glucose was measured one week before RDNx/Sham surgery, a glucose tolerance test (GTT) and indirect calorimetry were conducted 3 and 4 weeks after RDNx/Sham respectively. Tissues were collected at the end of the protocol for norepinephrine analysis and for inflammatory profile. C) Diagram of the kidney sections used for norepinephrine analysis, flow cytometry, cytokine analysis and histopathology.

Effect of RDNx on arterial pressure in obesity induced hypertension

See figure 1A for Cardiovascular Protocol timeline. Body weight was measured weekly. Ten to eleven weeks after switching to LFD or HFD, mice were anesthetized (1.5–2% isoflurane) for implantation of radio-telemeters (HD-X11, Data Science International, St Paul, MN) using aseptic technique (figure 1A). The protocol is described in the supplement methods section. Mean arterial pressure (MAP) and heart rate (HR) were measured continuously for 7 days post surgery. To assess the contribution of autonomic nerve activity to MAP and HR, a ganglionic blocker (hexamethonium; 10mg/kg IP) was administered on control day 2 and post-surgery (sham/RDNx) day 5. After 17 weeks of HFD and LFD to match the Metabolic Protocol (described below), mice were euthanized and RDNx was confirmed by analysis of tissue norepinephrine by high performance liquid chromatography (21).

Effect of RDNx on glucose metabolism in obesity induced hypertension

See figure 1B for the timeline of the Metabolic Protocol.

Body weight and composition

Body weight and food intake were measured weekly. Body composition was measured by placing the mice inside the Echo MRI chamber for 45–60 seconds (Echo MRI 3-in-1, Echo Medical Systems). Percent body fat was measured 2, 6, 10 and 17 weeks after switching the mice to a HFD or LFD.

Glucose metabolism

One week prior to RDNx or sham surgery, mice were fasted for 14-hours, the tail was cleaned with 70% ethanol and 1mm was snipped once from the tip using a sterile sharp scalpel. Fasting blood glucose was measured with a glucometer (Accu-chek Aviva). Three weeks after sham/RDNx, a glucose tolerance test (GTT) was conducted. Mice were fasted for 14-hours, and 2g/kg of D-glucose solution (Sigma-Aldrich) in sterile saline was administered intraperitoneally. Blood samples were collected at 0, 15, 30, 60, 120 and 180 minutes post injection for glucose analysis.

At the end of the protocol, kidneys were harvested for confirmation of RDNx and for quantification of renal inflammation (see below). Kidneys were cut transversely (figure 1C) and into four sections. One section was frozen in liquid nitrogen and stored at −80°C for norepinephrine analysis. Second and third sections were used for flow cytometry and cytokine analysis respectively. Finally, 2–3 mm thick section was used for hematoxylin and eosin staining.

Effect of RDNx on renal inflammation in obesity induced HTN

Tissue harvested in the Metabolic Protocol was used to assess renal inflammation as follows.

Flow cytometry

Mice were euthanized and the rostral part (as shown in figure 1C) of both kidneys were cut into small pieces (2–3mm), and incubated in collagenase solution (100U/ml collagenase in Roswell Park Memorial Institute (RPMI) buffer with 2% calf serum, 2mM MgCl2 and 2mM CaCl2) for 45 min at 37°C. Samples were homogenized gently with MACS C-tubes and poured through a 70μm filter. A 44%/67% percoll was used to purify the lymphocytes from the kidney. The leukocyte interface was transferred to a new tube and washed with FACS buffer to prepare for cell staining. The single cell kidney suspension was then stained with fluorochrome labeled antibodies: anti-CD8 (Clone 53–6.7 Tonbo Biosciences, CA), anti-CD4 (Clone RM4-5, Biolegend, CA), anti-FoxP3 (Clone FJK-16S eBioscience, CA), anti-Helios (Clone 22F6, Biolegend, CA), anti-T-bet (Clone 4B10, Biolegend, CA) and anti-CD44 (Clone IM7, Biolegend, CA). Spleen and mesenteric lymph nodes were harvested into FACS buffer and a single cell suspension was obtained by mashing though a 70μm filter and stained as described above. Intracellular staining with anti-FoxP3 was performed using the FoxP3 kit as per the manufacturer’s directions (eBioscience). Events collected with LSRFortessa flow cytometer (BD Pharmingen, CA) were analyzed by FlowJo software (Tree Star, San Carlos, CA).

Renal histopathology

Hematoxylin and eosin staining was used to determine structural changes in the kidney and score for infiltration of mononuclear cells. Detail is presented in the supplement.

Renal cytokine analysis

Multiplex kits (R&D Systems, Minneapolis, MN) were used on the Luminex 200 platform (with Bioplex software) to determine renal cytokine levels. Renal tissue was collected and homogenized in PBS with protease inhibitors. Analysis of renal interleukin (IL) 1β, IL-2, IL-6, IL-17, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were performed following the protocol provided by the manufacturer and expressed as renal cytokine per mg of renal protein. Protein levels were quantified by Bradford assay.

Statistical Analysis

An unpaired t-test was used for comparisons between two groups. When more than two groups were analyzed (i.e. cardiovascular and metabolic parameters) a two-way repeated measures in 1 dimension (time) ANOVA followed by post-hoc Bonferroni’s multiple comparison between all groups (GraphPad Prism 6, La Jolla, CA) was used. Two-tailed analysis was performed for all tests. A p-value of <0.05 was considered statistically significant. Results are presented as means ± standard error (means ± SE).

Results

Effect of high fat diet on caloric intake and body composition

Figure 2 shows caloric (figure 2A) and food (figure 2B) intake throughout the protocol for the 4 experimental groups: LFD-Sham, LFD-RDNx, HFD-Sham and HFD-RDNx. Although food intake between groups was similar (figure 2B), caloric intake was significantly higher in HFD compared to LFD mice (figure 2A). Body weight (figure 2C) and fat mass (figure 2D) were higher in HFD compared to lean normotensive LFD mice. The rate of rise of body weight and fat mass plateaued following RDNx and Sham surgeries, but the difference between HFD and LFD groups remained. Finally, RDNx had no significant effect on these variables compared to sham controls for both HFD and LFD groups.

Figure 2. Metabolic Protocol: Effect of HFD on food and caloric intake, body weight and percent body fat.

Figure 2

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A) Calorie intake (kilocalories/week). Letters a, b, c and d represent the time at which fasting glucose, Sham/RDNx, glucose tolerance test and indirect calorimetry were conducted respectively. *p<0.05 LFD vs. HFD. Two-way repeated measures ANOVA, Bonferroni’s multiple comparison post-hoc test B) Food intake (grams/7-days). Two-way repeated measures ANOVA, Bonferroni’s multiple comparison post-hoc test C) Body weight (g). *p<0.05 LFD vs. HFD. Unpaired t test. D) % Body fat *p<0.05 LFD vs. HFD. Unpaired t test.

Effect of RDNx on arterial pressure, heart rate, and responses to ganglionic blockade in obese hypertensive and lean normotensive mice

Body weight in mice instrumented with telemeters was similar over the time course of the Cardiovascular Protocol to that observed in non-instrumented mice in the Metabolic Protocol (Supplement figure S1). As shown in figure 3A, the 3-day control average of mean arterial pressure (MAP) was higher in HFD (116 ± 2 mmHg) compared to LFD (103 ± 5 mmHg) mice. The response of MAP to Sham and RDNx was calculated as the difference on each day post surgery to the 3-day control MAP (ΔMAP; figure 3B). RDNx normalized MAP in hypertensive HFD mice with MAP falling 19 ± 8 mmHg from 116 ± 2.0 mmHg before RDNx to 97 ± 7 mmHg 7 days later. In contrast, Sham surgery had no significant effect on MAP in HFD mice over the 7-day period. Finally, RDNx and Sham surgery had no significant effect on MAP in LFD mice.

Figure 3. Mean arterial pressure (MAP) and response to ganglionic blockade.

Figure 3

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A) MAP in LFD and HFD mice before surgery *p<0.05. Unpaired t test B) Change in MAP after surgery *p<0.05 LFD-sham vs. HFD-RDNx, + p<0.05 HFD-sham vs. HFD-RDNx. Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparison post-hoc test. C) Change in MAP on control day-2 with hexamethonium *p<0.05 LFD vs. HFD. Unpaired t test and D) after 5-days of sham/RDNx in different groups *p<0.05 LFD-sham vs. HFD-sham. One-way ANOVA.

We have previously validated the acute depressor response to ganglionic blockade as an indirect measure of sympathetic pressor activity (22). As shown in figure 3C, the depressor response to hexamethonium was greater in hypertensive HFD mice compared to LFD controls. Assessment of sympathetic pressor activity was repeated on day 5 following Sham/RDNx in LFD and HFD mice (figure 3D). Sympathetic pressor activity remained higher in HFD-Sham compared to LFD-Sham mice, but RDNx abolished this difference in that LFD-RDNx and HFD-RDNx groups were nearly identical.

Heart rate (HR) was elevated in hypertensive HFD compared to normotensive LFD mice (figure 4A), however, the response of HR to RDNx and Sham was similar in the four groups (figure 4B). In contrast to the response of MAP to ganglionic blockade, which is mediated by the loss of sympathetic tone exclusively, the HR response is dependent on sympatho-vagal balance to the heart. Ganglionic blockade had no significant effect on HR in LFD mice prior to Sham/RDNx suggesting equally balanced sympatho-vagal tone (figure 4C). In contrast, HR fell in HFD mice indicating a shift in cardiac sympatho-vagal activity. More importantly, RDNx abolished the difference in cardiac sympatho-vagal activity between LFD and HFD mice 5-days later (figure 4D).

Figure 4. Heart rate (HR) and the response to ganglionic blockade.

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A) Heart rate (beats/min) in HFD and LFD mice before surgery *p<0.05. Unpaired t test B) Change in HR after surgery. C) Change in HR with hexamethonium before surgery and D) 5 days after RDNx. *p<0.05 LFD-sham and LFD-RDNx vs. HFD-sham, + p<0.05 HFD-sham vs. HFD-RDNx. One-way ANOVA.

Finally, successful denervation of the kidneys was confirmed in that renal norepinephrine content was ~88% lower in RDNx compared to Sham mice (Supplement figure S2).

Effect of RDNx on glucose metabolism

Fasting blood glucose was significantly elevated in HFD compared to LFD mice prior to Sham/RDNx (figure 5A). Further evidence of impaired glucose metabolism in HFD mice was evident from the glucose tolerance test conducted 3 weeks after RDNx or Sham surgery. HFD-fed mice exhibited significantly higher blood glucose levels than LFD mice over the 180-minute period following glucose administration (figures 5B and 5C). However, RDNx had no significant effect on the glucose tolerance test in either HFD or LFD groups.

Figure 5. Effect of HFD and RDNx on glucose metabolism.

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A) Fasting blood glucose levels in LFD and HFD mice. *p<0.05. Unpaired t test. B) Glucose tolerance test showing changes in glucose metabolism with RDNx in groups *p<0.05 LFD vs. HFD (Sham/RDNx). Two-way repeated measures ANOVA followed by Bonferroni’s multiple comparison post-hoc test. C) Area under curve for GTT *p<0.05. One-way ANOVA.

Successful denervation was confirmed in that renal norepinephrine was ~78% lower in RDNx compared to Sham mice (Supplement figure S3).

Effect of RDNx on renal inflammation in HFD and LFD mice

One approach we used to assess the effect of a HFD and RDNx on renal inflammation was to quantify the trafficking of T cells into the kidneys. We also assessed the number of T cells in the spleen and mesenteric lymph node as measures of systemic immune status. The gating parameters used to quantify populations of CD4, CD8, T helper 1 (Th1) and regulatory (Treg) cells are described in the supplement (Supplement figure S4).

HFD compared to LFD mice exhibited generalized inflammation with significant elevation of splenic CD8, CD4, Th1 and Treg cells. Note that RDNx had no significant effect on splenic (figure 6A) or mesenteric lymph node (figure 6B) T cells. In contrast to our hypothesis, with the exception of a higher number of Treg cells in the HFD-RDNx compared to LFD-Sham, we did not observe any effect of either HFD or RDNx on the number of renal T cells (figure 6C). Finally, protein levels of renal pro-inflammatory cytokines Interleukin (IL) 1β, IL2, IL6, IL17, TNF-α and IFN-γ, as well as the anti-inflammatory cytokine IL-10 (figure 6D) showed no differences between HFD and LFD groups. Similarly, renal cytokines were not different between RDNx and Sham groups.

Figure 6. Renal inflammatory T cells and cytokines with diet and RDNx.

Figure 6

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Flow cytometry analysis of samples from LFD-Sham, LFD-RDNx, HFD-Sham, HFD-RDNx, (n=9/group) in A) spleen, B) mesenteric lymph node and C) kidney *p<0.05. One-way ANOVA. D) Renal cytokine levels after sham or RDNx.

Hematoxylin and eosin (H&E) staining was used to assess renal pathology. In contrast to previous report in which HFD was associated with renal pathology [4], we observed only minor structural changes in the kidney, indicative of fatty degeneration compared to LFD (Supplement figure S5A). Indeed, the renal inflammation score (single blinded score based on inflammatory mononuclear cell infiltration) showed no significant difference between HFD vs. LFD groups and RDNx had no impact on this measure (Supplement figure S5B).

Discussion

Obesity induced hypertension (HTN) has been reported to be associated with increased SNA (23, 24), impaired glucose metabolism (25), and renal inflammation (4). Moreover, it has been reported that RDNx reverses preclinical models of obesity-induced HTN (9, 11), improves glucose metabolism in hypertensive humans (12) and an animal model of type 2 diabetes (10), and prevents trafficking of T cells into the kidney in a murine model of AngII-induced HTN (17). Collectively, these reports led us to the hypothesis that renal nerves serve as a mechanistic nexus point between obesity, hypertension, impaired glucose metabolism, and renal inflammation. We tested this hypothesis by measuring the effects of RDNx on arterial pressure, glucose metabolism, and renal inflammation in C57BL/6J mice fed a high fat diet. We found that, although RDNx reversed hypertension caused by high fat feeding, it had no significant effect on glucose metabolism or measures of renal inflammation.

Normalization of arterial pressure by renal denervation is associated with normalization of sympatho-vagal tone in obese hypertensive C57BL/6J mice

Obesity-induced hypertension is associated with increased SNA to the kidneys (1, 5, 6). This idea is supported by direct measurement of renal SNA in rabbits fed a high fat diet (26) and reports that RDNx prevents and reverses obesity-induced hypertension in experimental animals (7–9). The three classical explanations for this RDNx mediated antihypertensive response are a decrease in renin release, an increase in renal sodium excretion, and/or a reduction in renal vascular resistance (7, 27). A forth possibility is that RDNx reduces renal inflammation and therefore hypertension (17).

Several findings in the present study support the neurogenic hypothesis of obesity induced hypertension and suggest that RDNx reduces arterial pressure by restoring sympathetic tone. First, both MAP and HR were elevated in obese C57BL/6J mice. Although multiple mechanisms can mediate the HFD induced increase in MAP, HR is regulated by both the sympathetic and parasympathetic nervous systems and therefore reflects cardiac sympatho-vagal balance. Second, the acute depressor response to ganglionic blockade was greater in obese compared to lean mice, which is consistent with increased sympathetic pressor activity. Third, elevated sympathetic pressor activity in obese mice was normalized by RDNx. Finally, in contrast to lean normotensives in which HR did not change following ganglionic blockade (suggesting equally balanced sympatho-vagal input to the heart), HR decreased in obese hypertensive mice. This is consistent with increased sympathetic and/or decreased parasympathetic drive to the heart in obese mice. Although RDNx did not lower HR in obese C57BL/6J mice, as has been reported in obese dogs (28), RDNx did restore cardiac sympatho-vagal balance in obese hypertensive mice to levels observed in lean normotensive controls. Taken together these findings suggest that RDNx reverses obesity-induced hypertension secondary to a reduction in sympathetic pressor activity.

The simplest explanation for these findings is that ingestion of a HFD could increase SNA to the kidneys specifically, resulting in increased renal vascular resistance and hypertension. This is consistent with the acute reversal of hypertension by ganglionic blockade, as well as the chronic reversal by RDNx. Although we cannot rule out the effects of RDNx on renin release or renal sodium excretion, however these may not completely explain the responses to acute ganglionic blockade since these mechanisms operate on a longer time scale than neural control of renal vascular resistance. Our findings are also consistent with studies in which renal SNA was directly recorded in rabbits (26) and rats (29) consuming a HFD (30).

It is important to note that RDNx had no significant effect on long-term regulation of arterial pressure or measures of sympathetic pressor activity and cardiac sympatho-vagal balance in lean normotensive mice. Similarly, the responses of obese mice to RDNx were not secondary to changes in food intake or body weight since there were no differences between Sham or RDNx groups for these variables between groups.

Effect of RDNx on glucose metabolism in obese C57BL/6J mice

Sympathetic over activity is linked to hyperglycemia and insulin resistance in patients with drug-resistant hypertension (31, 32). In addition, RDNx has been reported to improve glucose metabolism in drug-resistant hypertensive patients (12). This unexpected finding has been hypothesized to result from ablation of sympathoexcitatory renal afferent nerves and a reduction in SNA to insulin sensitive tissues (33, 34). Based on these studies, and our observation that RDNx abolished the increase in sympathetic pressor activity in HFD mice, we predicted RDNX would also improve glucose metabolism in this model.

Obese mice were characterized by elevated fasting glucose and impaired glucose tolerance. Importantly, RDNx had no significant effect on fasting glucose or the glucose tolerance test metabolism in any of the experimental groups in spite of improved obesity-induced hypertension. This suggests that RDNx did not alter SNA to vascular beds important for glucose homeostasis such as liver, pancreas, adipose tissue, and muscle. One caveat to our study is that we did not measure the effect of RDNx on plasma insulin or insulin sensitivity as has been reported in clinical studies demonstrating that RDNx improved glucose metabolism (11, 12). Previous studies have shown that high fat induced hyperglycemia in mice is associated with hyperinsulinemia and increased insulin resistance as measured by the homeostatic model assessment (HOMA-IR) (35, 36). Although we did not measure insulin resistance in the present study, since renal denervation had no effect on the glucose tolerance test in either LFD or HFD mice we hypothesize that renal denervation did not affect insulin resistance. However, this hypothesis remains to be tested.

Effect of RDNx on renal inflammation in hypertensive obese C57BL/6J mice

We tested our hypothesis in obese C57BL/6J mice in this study since it was recently reported that HFD causes renal inflammation and hypertension in this strain (4). Moreover, our hypothesis was based on reports that, 1) renal nerves mediate trafficking of T cells into the kidney (17, 37), that 2) subsets of T cells (CD4 and CD8) contribute to renal inflammation in AngII hypertensive mice (38), and these responses are prevented by RDNx (17).

Contrary to previous reports, we did not find any differences between HFD and LFD mice in the number of CD8 or CD4 cells in the kidneys or renal inflammatory cytokines such as IL1β, IL2, IL6, IL17, TNF-α and IFN-γ. Moreover, RDNx had no significant effect on either the number of T cells, or levels of renal cytokines, in lean or obese mice. We conclude that the renal inflammatory cells do not play a role in the pathogenesis of hypertension in obese C57BL/6J mice and that the antihypertensive response to RDNx occurs independent of renal inflammation.

The reasons for the discrepancies between our findings and studies that led to our hypothesis remain to be determined but there are a number of possibilities. First, although feeding a HFD to C57BL/6J mice has been shown to result in structural and functional changes in the kidneys (4), this previous study used 60% fat diet in contrast to the 45% fat diet in the our study. Second, the emerging concept that renal nerves traffic T cells into the kidney is predominantly based on the AngII-induced model in the mouse (17). Our results suggest that this mechanism does not translate to obesity-induced hypertension, since the anti-hypertensive response to RDNx occurred independent of differences in renal T cells or cytokines. It should be noted that the AngII-induced increase in arterial pressure is on the order of 40–50 mmHg (17). In contrast, there is only a 10–15 mmHg increase in obesity-induced hypertension. Thus the difference in the magnitude of the hypertension may be a factor in the interaction of renal nerves and renal inflammation. However, recently Xiao and colleagues (17) investigated the contribution of pressure induced renal inflammation in AngII-induced hypertension in mice with unilateral renal denervation. Their study suggested a role for renal nerve dependent (i.e. pressure independent) renal inflammation in this model.

It should be noted that we did not investigate the role of renal macrophages in our study. Deji and colleagues found that a 60% HFD increased inflammatory macrophages in the kidney of C57BL/6J mice (4). Xiao et. al have also reported increased macrophages in the kidneys of AngII-induced hypertensive mice that were reduced by RDNx (17). Nonetheless, we did not observe any differences between HFD and LFD mice in the number of CD8 or CD4 cells in the kidneys or renal inflammatory cytokines such as IL1β, IL2, IL6, IL17, IL10, TNF-α and IFN-γ. Finally, RDNx had no significant effect on either the T cells and cytokines in the kidney, renal cytokines, or renal histopathology in lean or obese mice.

Perspectives

The antihypertensive effect of renal denervation in obesity-induced hypertension in C57BL/6J mice occurred independent of effect on inflammatory mediators with no improvement in glucose metabolism. The results of this study provide important new information regarding the clinical benefit of renal nerve ablation in the treatment of hypertension and associated metabolic diseases.

Supplementary Material

Clean version_Online supplement

Novelty and Significance.

What is new?

  • Renal denervation lowers mean arterial pressure in hypertensive obese C57BL/6J mice by normalizing sympathetic pressor activity.

  • Despite the normalization of sympathetic pressor activity in obese hypertensive mice, RDNx had no impact on glucose metabolism.

  • The effect of RDNx on arterial pressure in obesity-induced hypertension occurred independent of indicators of renal inflammation.

What is relevant?

  • The results of this study provide further mechanistic insight to the use of renal nerve ablation for the treatment of human hypertension and associated metabolic syndromes.

Summary

Renal nerves in obese hypertensive mice affect blood pressure but do not control changes in glucose metabolism and renal inflammation.

Acknowledgments

Sources of funding

This work was supported by NIH grants R01HL06735715 and R01HL116476-02.

We would like to thank Robert Burnett and Dr. Gregory Fink at Michigan State University for norepinephrine analysis and Michael Ehrhardt at University of Minnesota for technical expertise. The metabolic phenotyping was conducted at the Intergrative Biology and Physiology Phenotyping Core.

Footnotes

Disclosures

Angela Panoskaltsis-Mortari has a family member who works for BioTechne that owns R&D Systems.

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