Arcuate NPY is involved in salt-induced hypertension via modulation of paraventricular vasopressin and brain-derived neurotrophic factor

Abstract

Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt-induced hypertension. Here, we demonstrate that wild-type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY-GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain-derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV-Cre recombinase into the Arc only of the NPY-targeted mutant mice carrying a loxP-flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt-dependent blood pressure. In summary, our study uncovers an important Arc NPY-originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.

Keywords: BDNF; hypertension; hypothalamic arcuate nucleus (Arc); neuropeptide Y (NPY); salt (NaCl); vasopressin.

Figure 1

Salt water‐induced hypertension in C57BL/6 wild‐type (WT) mice. (a) Serum Na⁺ was significantly increased in salt water‐treated mice relative to normal water‐given mice at the end of the experiment. (b) Serum Cl^‐ did not differ between the two groups at the end of the experiment. (c) Serum K⁺ was decreased in salt water‐treated mice. (d) Salt water‐treated mice showed a marked increase in water consumption compared to control mice which was significant after 3 weeks and every week from the 4th week to the end of the study (n = 5). (e) Daily urine output measured after 1 and 4 weeks. (f) There was no significant difference in body weight between the two groups at any point over the experimental period. (g) Daily food intake measured at the 1st week and the 4th week. (h) Weekly systolic blood pressure profile and (i) diastolic blood pressure of normal water‐ and salt water‐treated mice. The salt water‐treated mice had significantly higher systolic blood pressure after 3 weeks of stimulation through to the end of the experimental period. Values are expressed as means ± SEM (n = 5). (*p < 0.05, **p < 0.01, ***p < 0.001, salt water vs. normal water)

Figure 2

The effects of salt water drinking on NPY expression in the hypothalamic Arcuate nucleus (Arc) in the brain of WT mice. The number of positive NPY‐GFP neurons in the Arc of NPY‐GFP mice administered with normal water or 2% salt water was observed by representative images (a) and quantification (b). (a) Salt loading decreased the expression of Arc green fluorescent protein (GFP). (b) The number of NPY‐GFP neurons in the Arc of salt water‐treated mice was lower compared to normal water‐treated mice (n = 4). (c) Representative images of in situ hybridization (ISH) using RNAscope technology showing that salt water drinking decreased mRNA expression of NPY in the Arc. (d) The integral optical density (IOD) value of NPY ISH staining in salt water‐treated mice was significantly lower compared to normal water‐treated mice. (e) qPCR results of dissected Arc tissues showing that salt water drinking decreased the expression of NPY in the Arc of WT mice. (f) Schematic diagram of the arcuate nucleus (Arc) in the hypothalamus. Values are expressed as means ± SEM (n = 4). (*p < 0.05, **p < 0.01, salt water vs. normal water). 3V, third ventricle

Figure 3

c‐fos expression in the PVN of WT mice detected by immunohistochemical analysis. (a) The number of c‐fos immunopositive neurons in salt water‐treated mice was higher than in normal water‐treated mice. (b) Salt water drinking increased c‐fos expression in the paraventricular nucleus (PVN.) (c) Schematic diagram of the PVN in the hypothalamus (red boxed area adapted from Franklin 2017. 3V, third ventricle. Values are expressed as means ± SEM (n = 4). (**p < 0.01, salt water vs. normal water)

Figure 4

The effects of high salt intake on the level of circulating vasopressin and on vasopressin mRNA expression in the PVN. (a) Serum copeptin (CPP) was measured at the end of the experiment. High salt intake increased the circulating CPP level (n = 4). (b) The paraventricular nucleus (PVN) vasopressin mRNA expression level measured by in situ hybridization (ISH) using RNAscope technology. (c) Integral optical density (IOD) value comparison of amount of vasopressin observed in ISH. Salt water‐treated mice had a greater value of vasopressin mRNA observed. (d) Quantification of vasopressin mRNA level using qPCR. Salt water‐treated mice had a higher vasopressin mRNA expression level. (e) Salt intake increased the qPCR measured mRNA expression level of brain‐derived neurotrophic factor (BDNF) and decreased the mRNA level of K⁺/Cl^‐ co‐transporter 2 (KCC2) in the PVN. (f) Schematic diagram of the PVN in the hypothalamus (red boxed area adapted from Franklin 2017). Values are expressed as means ± SEM (n = 4) (*p < 0.05, ***p < 0.001, salt water vs. normal water). 3V, third ventricle

Figure 5

mRNA expression of Arc NPY in NPY targeted mutant NPY^LSL/LSLmice. (a) Schematic diagram of a targeted allele of stop cassette was cloned to the upstream of NPY coding sequence in NPY^LSL/LSL mice. Using Cre‐recombinase (Cre) can excise the stop cassette and restore NPY expression at a specific position. (b) The mRNA expression of NPY in the Arc of NPY^LSL/LSL mice determined by RNAscope in situ hybridization analysis was absent compared to the control mice, n = 3. (c) Schematic drawing of procedure to induce NPY expression in the Arc in NPY^LSL/LSL mice. For selective re‐expression of NPY in arcuate nucleus (Arc) neurons, AAV‐Cre was bilaterally injected into the Arc of NPY^LSL/LSL mice. (d) The NPY mRNA level in AAV‐Cre‐injected NPY^LSL/LSL mice determined by RNAscope in situ hybridization analysis was present in the hypothalamic Arc compared to the AAV‐Empty‐injected NPY^LSL/LSL mice, n = 3. (e) Schematic diagram of the arcuate hypothalamic nucleus dorsal part (ArcD) and arcuate hypothalamic nucleus lateral part (ArcL) adapted from Franklin (2017)

Figure 6

Effects of Arc NPY on the control of salt water drinking‐induced hypertension in NPY^LSL/LSLmice. (a) The mRNA levels of NPY and AgRP in AAV‐Cre‐injected NPY^LSL/LSL mice administered with normal water or salt water were determined by RNAscope in situ hybridization analysis, n = 3. (b) Systolic blood pressure (SBP) of Empty‐injected and Cre‐injected NPY^LSL/LSL mice in response to normal water or 2% salt water administration. SBPs of the four groups were similar in the first week. (c) AAV‐Empty‐treated mice on salt water showed a higher blood pressure compared to the other three groups, while AAV‐Cre‐induced restoration of Arc NPY significantly attenuated salt water‐induced hypertension at the eighth week of the experiment. (d) qPCR results showed that the restoration of Arc NPY in AAV‐Cre‐injected mice blunted salt water drinking‐induced vasopressin mRNA expression in the hypothalamic paraventricular nucleus (PVN) compared to AAV‐Empty‐injected mice. (e) qPCR results showed that the restoration of Arc NPY in AAV‐Cre‐injected mice blunted salt water‐induced mRNA expression of brain‐derived neurotrophic factor (BDNF) in the hypothalamic paraventricular nucleus (PVN) compared to AAV‐Empty‐injected mice. Values are expressed as means ± SEM (n = 4–5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to AAV‐Empty normal water or comparisons indicated