| Background:Hypertension is one of the most common chronic diseases in China,which has become the leading risk factor for cardiovascular disease(CVD).A high salt diet,which is a well-established risk factor for hypertension,has been a dietary characteristic of Chinese inhabitants for a long time.In view of the increasing burden of CVD in China,how to improve the dietary habit of the Chinese population so as to effectively improve the prevention and treatment of hypertension and reduce the burden of CVD,has become a social and medical problem to be solved urgently.Objective:This study was designed to analyze the effects of a long-term potassium-enriched salt diet,leading to low sodium and high potassium intake,on blood pressure and mortality in nursing homes,aiming to provide an optimizing non-drug therapy for hypertension in the Chinese elderly.Methods:This study was a cluster-randomized controlled,multi-center cohort intervention study.Twenty-nine nursing homes in northern China were cluster-randomized into two groups:the regular salt group(control),which intakes 100%sodium chloride salt;and the potassium-enriched salt group(intervention),which used the salt contained 49%potassium chloride and 49%sodium chloride.Participants living in the nursing room during the baseline survey were recruited for the study of blood pressure analysis if they met the following criteria:aged ≥ 40 years old,having meals only provided by the nursing homes,and no kidney injuries,or other limitations of potassium intake.Those who also lived in nursing homes for≥6 months were enrolled in the survival study.A baseline survey and 7 follow-up visits(3 months,6 months,9 months,12 months,24 months,36 months,60 months)were completed in this study,during which blood pressure,blood samples,and urine samples(24-hour urine and spot urine)were collected.Urinary sodium to potassium ratio,blood pressure,the hazard ratio of all-cause death,CVD death,and stroke death events were analyzed and compared between intervention and control groups.At the same time,endpoint events were also compared with different subgroups by age over 70 years old or 40-70 years old.Results:1784 residents entered the study of blood pressure analysis,with 874 in the regular salt group and 910 in the intervention group.Compared with the baseline survey,the 24h urinary sodium/potassium(Na+/K+)ratio was significantly reduced in the intervention group(3.5±2.5 vs.10.8±2.9,P<0.001).And the 24h urinary Na+/K+ratio in the intervention group was always lower than the regular salt group at all follow-up visits.Compared with the baseline survey,the intervention group showed a significant reduction in systolic(β=-5.01;95%CI-6.63 to-3.39;P<0.001)and diastolic blood pressure(β=-1.89;95%CI-2.73 to-1.07;P<0.001)after adjustment for age and sex.The survival study cohort comprised 3543 people,1887 were in the regular salt group,and 1656 were in the intervention group.During the follow-up period,all-cause death was significantly reduced in the intervention group(HR 0.88;95%CI 0.78 to 1.00;P=0.044)compared with the regular salt group,but there was no difference in CVD death and stroke death.Subgroup analysis showed that in the age group of 40-70 years old,potassium-enriched salt significantly lowered the mortality of all-cause(HR 0.78;95%CI 0.63 to 0.96;P=0.022),CVD(HR 0.66;CI 0.49 to 0.90;P=0.009),and stroke(HR 0.68;95%CI 0.47 to 0.98;P=0.039)than the regular salt.However,the mortality of all-cause,CVD,and stroke was similar between intervention and control groups for the age group over 70.Conclusions:This study demonstrated that long-term consumption of potassium-enriched salt provided a safe and effective method to decrease urinary Na+/K+ratios and blood pressure.Also,long-term potassium-enriched dietary intervention is beneficial to reducing all-cause of death,CVD death,and stroke death,especially in the 40～70-year-old group.This study provides an individualized plan in China for the prevention and treatment of hypertension.Objectives:CCKBR(cholecystokinin B receptor)serves as a receptor for gastrin,a peptide hormone that is secreted by G cells in the stomach and duodenum to stimulate HCl secretion,and to regulate sodium absorption and blood pressure as well.The present study aims to establish a salt-sensitive mouse model by conditional knockout of Cckbr in intestine,and to explore the underlying mechanism(s)of intestinal Cckbr in regulating blood pressure through sodium absorption.Methods:Two-month-old Cckbrfl/fl villin-Cre mice(conditional knockout,CKO)and control Cckbrfl/fl mice(wild type,WT)were fed with normal diet(0.4%NaCl)or high salt diet(4%NaCl),separately,for 6 weeks.Blood pressure was weekly monitored by the radio telemetry method in conscious mice.The blood pressure for these mice was finally validated by carotid artery catheterization,and the samples of urine,blood,and tissues of intestine,heart and kidney were collected at the time of experiments termination.Electrolytes(sodium,potassium and chlorine)in urine and intestine,creatinine and hypertensive hormones including renin,angiotensin II and aldosterone in serum and urine were measured by electrolyte analyzer and ELISA.Western blot was used to test the expression of MMP2(matrix metalloproteinase 2)and MMP9(matrix metalloproteinase 9)in heart and kidney.Hematoxylin-eosin(HE)staining,Masson staining,and immunohistochemistry were used to detect pathological variations,collagen deposition and renal fibrosis.The expression and localization of some intestinal sodium transporters such as NHE3,NKCC1,and ENaC were examined by Western blot and immunofluorescent staining.Furthermore,the CKO mice fed with a high salt diet were treated with hydrochlorothiazide or saline intraperitoneally daily for one week,followed by measurement of blood pressure,analysis of electrolytes in urine and hypertensive hormones in serum,as well as the organ injury markers(MMP2/MMP9)and the histopathology of kidneys.Results:Compared with the control mice(Cckbrfl/fll),the mice with intestinal knockout of Cckbr(Cckbrfl/fl villin-Cre)fed with high salt diet showed high blood pressure,increased duodenal sodium absorption and urinary sodium excretion,and renal injury.The expressions of NHE3,NKCC1 and ENaC were also upregulated significantly in the intestine of CKO mice fed with high salt diet.Treatment with hydrochlorothiazide remarkably attenuated the elevated blood pressure resulted from high salt absorption in the Cckbrfl/fl villin-Cre mice,wheras there was no significant effect observed on the injured kidneys after hydrochlorothiazide treatment.Conclusion:These results support a crucial role of intestinal Cckbr deficiency on SSH development and the diuretic antihypertensive effect in Cckbrfl/fl villin-Cre mice.Thus,the Cckbrfl/fl villin-Cre mice with high salt intake may serve as a stable model of SSH induced by sodium overloading,and provide a new strategy for the clinical treatment of SSH.Objectives:The outward delayed rectifier Kv1.3 is the third member of the delayed rectifier Kvl class subfamily which belongs to the voltage-gated potassium ion channel family.It plays important roles in regulating cell resting membrane potential,apoptosis,differentiation,proliferation,cell volume,and activation of white blood cells by mediating K+ion efflux.Abnormal Kv1.3 functions are closely associated with various diseases such as obesity,rheumatoid arthritis,autoimmune diseases,hepatitis,neuropathy,and tumors.It has been considered as an important target for the treatment of inflammation-related diseases.However,whether Kv1.3-mediated potassium efflux plays a role in cardiovascular diseases such as hypertension is unclear.This study was designed to explore the effects of knockout Kv1.3 on blood pressure and its possible underlying mechanisms at molecular level by constructing Kv1.3 systemic gene knockout mice using CRISPR/Cas9 techniques.Methods:Systemic Kv1.3 gene knockout mice(KO,Kv1.3-/-)were constructed using CRISPR/Cas9 technology,with wild-type C57BL/6 mice(WT,Kv1.3+/+)as controls.Randomly selected 28-week-old mice from the experimental and control groups were placed in metabolic cages to collect urine from each mouse.The urine electrolytes(sodium,potassium,chloride),urinary protein,and creatinine were measured using an electrolyte analyzer and ELISA.After measuring the body weight,the blood pressure was measured by carotid artery cannulation under 2%isoflurane anesthesia.An electrolyte analyzer and ELISA kit were used to measure plasma electrolytes(sodium,potassium,chloride),renin,angiotensin II,and aldosterone levels in mice.RNA was extracted from the renal cortex,renal medulla,and adrenal tissues of the two groups of mice for sequencing using the Illumina Hiseq 2000 platform to identify differentially expressed molecules and/or signaling pathways after Kv1.3 knockout.Renin expression in the kidney and adrenal was measured using western blotting.Results:Compared with wild-type C57BL/6 mice(WT,Kv1.3+/+),Kv1.3 gene knockout mice(KO,Kv1.3-/-)had a lower plasma potassium concentration(KO vs.WT:2.93±0.65 mmol/L vs.3.64±0.15 mmol/L,P=0.043),while there was no significant change in the urinary potassium-to-creatinine ratio.There were no significant differences in the levels of sodium and chloride ions in blood and urine(after corrected by creatinine levels)or urinary protein-to-creatinine ratio between the two groups of mice.The body weight of Kv1.3 knockout mice decreased significantly(KO vs.WT:23.4 ± 1.2g vs.28.0 ± 1.3g,p=0.0004).Compared with the control group,Kv1.3 systemic knockout mice had significantly increased systolic blood pressure(KO vs.WT:115.3± 9.4 mm Hg vs.87.3 ± 10.2 mm Hg,P=0.0068)and diastolic blood pressure(KO vs.WT:79.5±2.8 mm Hg vs.63.4± 7.4 mm Hg,P=0.0067).Furthermore,the renin-angiotensin-aldosterone system(RAAS)was significantly activated after knockout(KO vs.WT:renin:593.5 ± 67.5 vs.409.9 ± 94 pg/ml,P=0.0075;angiotensin II:156.6± 26.5 vs.78.4 ± 23.0 pg/ml,P=0.0011;aldosterone:349.6 ± 61.6 vs.230.9± 50.9 pg/ml,P=0.0106).Interestingly,the Renl mRNA was found elevated by 2.01 times and the Cyp11b2 encoding aldosterone synthase increased by 1.87 times in the adrenal glands of KO mice compared with that in the control group,while no significant differences were found in the levels of Ren1 mRNA in the renal cortex and medulla as detected by RNA-seq.In addition,differentially expressed genes in the adrenal glands in Kv1.3 knockout mice were significantly enriched in cGMP-PKG,calcium signaling,MAPK,gonadotropin-releasing hormone(GnRH)signaling,renin secretion,aldosterone synthesis and secretion pathways.Western blotting confirmed that renin expression was significantly increased in the adrenal glands of Kv1.3 knockout mice compared with that in wild type mice.Conclusion:Kv1.3 gene knockout in mice can reduce blood potassium concentration without increasing urinary potassium excretion or absorption and excretion of sodium and chloride ions.Kv1.3 gene deficiency could decrease body weight and induce hypertension in mice.The effects of Kv1.3 knockout might be related to the activation of the renin-angiotensin-aldosterone signaling pathway in the adrenal glands,rather than in the kidneys.This study reveals the role and potential mechanism of Kv1.3 in blood pressure regulation,and suggests that enough attention should be paid to the potential risk of inducing hypertension when antagonists targeting Kv1.3 are used to prevent inflammatory. |
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