Mechanism Of TRPV1 Activation Promoting Kidney Sodium Excretion And Lowering Blood Pressure

Background and objective:Hypertension is one of the most common diseases in the world. The current pr evalence of hypertension is in sustained growth trend in our country. It is not only a major risk factor for stroke, coronary heart disease, heart failure, kidney failure and other dise ases, but also an important reason for disability and death, so its prevention has important practical significance.The occurrence of hypertension is the result of interaction of genetic factors and external environment, and one of the major environmental factors-high salt intake plays a key role. Salt restriction has been actively advocated in the prevention and treatment of hypertension. However, the poor compliance becomes the disadvantage of long-term adherence and promotion in halophilic crowd. Study confirmed that the normal urinary sodium secretion was crucial to the stable blood volume and maintained a stable blood pressure. Patients may be given diuretics for hypertension treatment, but long-term administration has caused water and electrolyte disorders and other side effects. Therefore, further study on the regulatory mechanisms of renal sodium reabsorption state under high-salt load and exploring new targets for economically feasible interventions has important implications for the prevention and control of hypertension.Total amount of sodium in vivo determines the extracellular fluid volume and blood volume, which is an important determinant of blood pressure. The daily original urine of a normal adult can reach up to 180 L, of which more than 99 percents of the sodium and water is reabsorbed in the renal tubules and collecting ducts. About 10% of the sodium is reabsorbed in the distal nephron(distal convoluted tubule and collecting duct), which is the target of a variety of hormones and signaling molecules, and the changes of its function can lead to severe abnormalities in blood pressure. The amiloride-sensitive epithelial sodium channels(ENaC) in the last paragraph of the distal convoluted tubule and collecting duct are the final fine-tuned site of urinary sodium. ENa C can be regulated by with-no-lysine kinase(WNK) family, which includes four types: WNK1, kidney-specific WNK1, WNK3 and WNK4. After given a high-salt diet, compared with WT, WNK1-/- mice’s renal tubular ENa C is blunted.Transient receptor potential channel vanilloid 1(TRPV1) is a non-selective cation channel, which can be activated by capsaicin. TRPV1 was first discovered in sensory neurons. Recent studies show that TRPV1 exists not only in the nerve cells but also in renal tubular epithelial cells, which participates in renal sodium and water balance regulation. However, the long-term capsaicin intervention on mice with high-salt diets and its impact on renal excretion of sodium and blood pressure is unclear. Therefore, this stud y chooses C57BL/6J(wild-type mice, WT) and TRPV1 knockout mice(TRPV1-/-) as research objects. From the overall animal perspective to cellular level, we firstly confirmed functional TRPV1 expressed in renal cortical collecting duct(CCD) of mice and vitro M1-CCD cells and mediated capsaicin-induced sodium excretion. Then we verified that long-term dietary capsaicin intervention in mice lowered the increased blood pressure caused by high salt diet by activating TRPV1 in renal cortical collecting duct. Finally we explored the molecular mechanisms of dietary capsaicin promoting urinary sodium excretion and revealed the role of TRPV1 in this process.Materials and Methods:Both in vitro and in vivo experiments were included in the present study. In vitro models included M1-CCD cells from mice. In vivo models were TRPV1-knockout(TRPV1–/–) mice and C57BL/6J wild-type(WT) mice fed with interventional diet. TRPV1–/– mice and WT mice were randomly grouped and fed with a high-salt diet(HS), normal-salt diet(ND) or high-salt plus capsaicin diet(HSC) for 10 months.1. 24-hour ambulatory systolic and diastolic pressures were measured by telemetry in conscious unrestrained mice.2. Mice treated with each diet were put into the respective metabolic cage for 48 hours. The 24-hour water consumption, urine output and urinary sodium excretion(UNaV) were measured by electrolyte analyzer.3. The cultured M1-CCD cells were loaded with Ca2+ indicator Fura-2/AM or Na+ indicator sodium-binding benzofuran isophthalate.4. The changes of WNK1, WNK4, SGK1, αENa C, βENaC expression in kidney were measured by immunoblotting analysis.5. The co-expression of TRPV1 and αENa C in renal cortical collecting duct cells were measured by immunoprecipitation.6. Immunofluorescence studies were performed on M1-CCD cells and mice renal CCDs to observe the expression of TRPV1 and αENa C.Results:1. TRPV1 and αENa C co-expressed in WT mice CCDs and M1-CCD cells. Administration of capsaicin caused a dose-dependent increase in the cytosolic freecalcium concentration([Ca]2+i) in M1-CCD cells. Capsaicin treatment significantly increased the UNaV of WT mice on a HS diet compared with the mice on a normal diet(ND) and the HS diet.2. Capsaicin significantly lowered tail systolic blood pressure in WT mice on a HS diet after the eighth month. Chronic dietary capsaicin also lowered 24-hour ambulatory arterial pressure in WT HS group mice after 10-month treatment. Importantly, the hypotensive effect of capsaicin was absent in TRPV1–/– mice. Plasma aldosterones were lower in mice on a HS and HSC diet compared with mice on a normal diet in WT and TRPV1-/- mice. The urinary chloride excretion and volume of water intake were higher in mice on a HS or HSC compared with mice on an ND in WT and TRPV1-/- mice.3. The natriuretic effect of amiloride injection of WT mice on a HS diet was significantly greater than that of the mice fed a ND. Dietary capsaicin decreased the UNaV changes in WT mice but not TRPV1–/– mice on a HS diet. The HS diet significantly increased the expressions of α and βENa C both in WT and TRPV1–/– mice CCDs. Dietary capsaicin reduced αENa C expression but no other isoforms of ENa Cs in the CCDs of WT mice but not TRPV1–/– mice on a HS diet. The expressions of ENa C regulatory kinases, WNK1 and SGK1 in CCDs were upregulated in WT mice on a HS diet and inhibited by dietary capsaicin. However, these changes were not found in CCDs of TRPV1–/– mice on a HSC diet administration.4. High concentration of Na Cl reduced TRPV1 but increased αENa C expressions in M1-CCD cells. However, capsaicin upregulated TRPV1 and reduced αENaC expressions in M1-CCD cells, which was inhibited by 5’-iodoresiniferatoxin. αENa C interacted specifically with the immobilized TRPV1 in CCDs from WT mice fed with ND, HS, and HSC diet. On the acute addition of extracellular 100 mmol/L Na Cl, the cytosolic fluorescence intensity ratio of M1-CCD cells treated with NaCl plus capsaicin(HSC) decreased compared with only Na Cl(HS) treatment for 24 hours. The 5’-iodoresiniferatoxin increased Na+ uptake in the cytosol antagonizing capsaicin effect.Conclusion:1. Functional TRPV1 expresses in WT mice CCDs and M1-CCD cells.2. Long-term dietary capsaicin intervention increases the urinary sodium excretion in WT mice, thereby reducing the increased blood pressure caused by long-term high-salt diet.3. Long-term dietary capsaicin intervention reduces urinary sodium reabsorption in WT mice by inhibiting the renal CCD αENa C and WNK1/SGK1 pathway, thus contributing to urinary sodium excretion.