Mechanism Of High Salt Involved In Salt-sensitive Hypertension Via CD4~+T Lympho Ion Transporter NKCC1 Regulating NOS

Background:Hypertension is a major risk factor for stroke,myocardial infarction,kidney failure,and heart failure.Salt-sensitive hypertension has become a major subtype of essential hypertension in our country due to our dietary habits.Therefore,studying the pathogenesis of salt-sensitive hypertension is of great significance to control the prevalence of hypertension and the risk of cardiovascular adverse events in our country.Studies have found that the abnormal number and function of CD4~+T lymphocytes are involved in the occurrence and development of salt-sensitive hypertension,and changes in the expression of ion transporters on the surface of lymphocytes can affect the pathogenesis of hypertension.For example,high salt stimulation can induce the up-regulation of Na~+-K~+-2Cl-cotransporter(Nkcc1)expression in mouse Th17 cells,thereby promoting Th17 cells to secrete interleukin-17A(IL-17A);IL-17A reduces the production of NO in vascular endothelial cells and reduces vascular compliance by inducing the phosphorylation of threonine site 495 on endothelial nitric oxide synthase(e NOS)in endothelial cells,ultimately leading to increased blood pressure.Conversely,phosphorylation of serine(Ser)site 1177 of e NOS(p-e NOS)induces increased NO synthesis and maintains vasomotor properties by upregulating its enzymatic activity,thus reducing the risk of hypertension.On the other hand,inducible NO synthase(i NOS),through its function of promoting NO synthesis in vascular endothelial cells,has also been proved to be an important molecule in the pathogenesis of hypertension.At this stage,the specific mechanism of the involvement of CD4~+T lymphocytes in the regulation of the pathogenesis of salt-sensitive hypertension is still not fully understood.Whether CD4~+T lymphocytes can participate in the development of salt-sensitive hypertension by regulating the protein expression and modification of e NOS and i NOS as well as the role of ion transporters in this process remains to be further investigated.Objectives:1.To explore lymphocyte cell types associated with hypertension by single-cell sequencing2.To analyze the human lymphocyte ion transporters involved in the pathogenesis of hypertension3.To explore the mechanism of different ion concentrations regulating NO production in lymphocyte-dependent/independent endothelial cells pathway at the cellular level4.To explore the mechanism of lymphocytes involved in the pathogenesis of salt-sensitive hypertension at the level of animal models5.To clarify the correlation between lymphocyte count and cardiovascular adverse events during long-term follow-up in patients with acute myocardial infarctionMethods and materials:1.Basic research part1.1 To explore lymphocyte cell types associated with hypertension by single-cell sequencingBy single-cell sequencing of peripheral blood mononuclear cell(PBMC)of spontaneously hypertensive rats(SHR)and Wistar-Kyoto(WKY)rats,the proportion of different cell populations in PBMCs to total PBMCs was determined.Differential genes of T lymphocytes in rat PBMC and their enriched pathways,as well as Nkcc1,Kcc3,e Nos,i Nos expression in T lymphocytes were further detected.1.2 To analyze the human lymphocyte ion transporters involved in the pathogenesis of hypertensionPeripheral blood CD4~+and CD8~+T lymphocytes were isolated from normotensive and hypertensive patients.The m RNA expression levels of Na~+-K~+-Cl~-cotransporter NKCC1,K~+-Cl~-cotransporter KCC1,KCC3,KCC4 and Na~+-K~+-ATPase ATP1A1 were detected by q PCR.1.3 To explore the mechanism of different ion concentrations regulating NO production in lymphocyte-dependent/independent endothelial cells pathway at the cellular levelWestern Blot and q PCR were used to detect the expression levels of NKCC1,KCC3,p-e NOS,p-AKT and i NOS in T lymphocytes under different ion concentrations.Griess method to detect the extracellular NO concentration,flow cytometry to detect the intracellular NO content,CCK8 Cell kit to detect cell proliferation.The expression of NKCC1 in T lymphocytes was down-regulated by CRISPR-Cas9 technology,the expression levels of KCC3,p-e NOS,p-AKT and i NOS were detected by Western Blot and q PCR under different ion concentrations,and the concentration of extracellular NO was detected by Griess method,the intracellular NO content was detected by cytometry.T lymphocytes with down-regulated NKCC1(Lv-NKCC1)were co-cultured with immortalized human brain microvascular endothelial cells(h CMEC)endothelial cells,i NOS,p-AKT and p-e NOS expression level in endothelial cells were detected by Western Blot and q PCR,Griess method to detect the concentration of extracellular NO,flow cytometry to detect the content of intracellular NO.1.4 To explore the mechanism of lymphocytes involved in the pathogenesis of salt-sensitive hypertension at the level of animal modelsEight-week-old male C57BL/6 mice were divided into a normal sodium diet(NSD)group and a high sodium diet(HSD)group.After 5 months of continuous feeding,blood pressure changes were detected by tail artery sphygmomanometer.Mice in the HSD group with significantly increased systolic blood pressure were included in the high sodium diet to hypertension(HSD-H)group,while mice with no significant change in systolic blood pressure from baseline were included in the high sodium diet to normal blood pressure(HSD-N)group.Serum NO concentration was detected by Griess method,the aorta and left ventricle were stained with hematoxylin-eosin(HE),and Masson staining.q PCR was used to detect the expression levels of Nkcc1,Kcc1,Kcc3and Kcc4 in the spleen and PBMC.Western Blot was used to detect the expression levels of p-e NOS,p-AKT and i NOS in the left ventricle.q PCR was used to detect the transcript levels of the left ventricular e Nos,soluble of soluble guanylate cyclase(s GC),B-type natriuretic peptide(BNP),and c GMP-dependent protein kinase I(Prkg1).C57 mice were infused with Lv-NKCC1 Jurkat T lymphocytes and lentiviral vector control(Lv-ct)Jurkat T lymphocytes by tail vein to detect changes in blood pressure,peripheral serum NO concentration,p-e NOS,p-AKT,i NOS protein expression levels of left ventricle and aorta.1.Clinical research partWe consecutively enrolled 2399 patients who admitted for AMI at the coronary care unit between February 2014 and March 2018.After excluding 107 individuals who met the exclusion criteria,a total of 2292 patients were included in the final analysis.The primary outcome was MACE which was defined as a composite of all-cause death,heart failure(HF)rehospitalization,reinfarction,and ischemic stroke.The secondary outcomes included all-cause death,cardiac death,HF rehospitalization,reinfarction and ischemic stroke.All included patients were divided into Q1 to Q4 groups based on the quartiles of PLC.We performed the Cox regression analysis to explore the association between PLC and the primary and secondary outcomes.To validate the clinical usefulness of PLC in the risk stratification of AMI,we calculated the discrimination,calibration,net reclassification index(NRI),and integrated discrimination improvement(IDI)of the conventional and PLC-combined predictive models,respectively,to evaluate the diagnostic performance improvement of PLC on the top of conventional models.Results1.Basic research partAt the PMBC single-cell level,the average expression of Kcc3 and Icam-1 m RNA in the SHR group was higher than that in the WKY group;the average expression of Nkcc1,i Nos,and e Nos m RNA in the SHR group was lower than that in the WKY group;the proportion of CD4~+T lymphocytes in SHR group increased and the level of chemokine increased.The NKCC1,KCC1,KCC3 m RNA levels of CD8~+T lymphocytes in hypertensive patients were significantly higher than those in normotensive patients.The KCC1 and KCC3 m RNA levels of CD4~+T lymphocytes in hypertensive patients were significantly higher than those in normotensive patients.At the cellular level,the protein expression level of NKCC1 in Jurkat T lymphocytes in the high sodium(HS)and high mannitol(HM)group was lower than that in the normal(N)group;the protein expression level of NKCC1 in the high potassium chloride(HK)(7m M KCl)group was significantly higher than that in the N group.N,HS,HK and high sodium high KCl(HSHK)(180m M Na Cl+7m M KCl)can up-regulate the protein expression level of KCC3 in Jurkat T lymphocytes.The transcription level of i NOS in Jurkat T lymphocytes in HS and HK groups was significantly higher than that in other groups;the protein expression levels of p-AKT and p-e NOS in Jurkat T lymphocytes in N group were lower than those in other groups;the concentration of extracellular NO in HS,HM,HK and HSHK groups was significantly higher than that in other groups;the concentration of intracellular NO in HS,HM,HK and HSHK groups was significantly lower than that in the N group.The KCC3 protein expression level of Lv-NKCC1 Jurkat T lymphocytes was significantly lower than that of the Lv-ct group.When stimulated by normal ion concentration,the intracellular and extracellular NO contents in Lv-NKCC1 Jurkat T lymphocytes were significantly higher than those in the Lv-ct group;the transcription level of e NOS in Lv-NKCC1 Jurkat T lymphocytes was lower than that in the Lv-ct group;the transcription level of i NOS transcription was higher than that in the Lv-ct group.When stimulated with different ion concentrations(including:N,HM,HS,HK,HSHK),the expression of p-e NOS protein in Lv-NKCC1 Jurkat T lymphocytes was significantly lower than that in Lv-ct group.When stimulated by N,HS and HK,the proliferation ability of Lv-NKCC1 Jurkat T lymphocytes was significantly higher than that of Lv-ct group.Under the condition of normal ion concentration,h CMEC(h CMEC with ISO,h CMEC-I)were pretreated with isoproterenol(ISO)(10μM)for 24h,and then co-cultured with Lv-NKCC1 and Lv-ct Jurkat T lymphocytes,respectively.The intracellular and extracellular NO levels of h CMEC-I showed that the NO content in the Lv-NKCC1 co-culture group was significantly higher than that in the Lv-ct co-culture group.Lv-NKCC1 Jurkat T lymphocytes co-cultured with h CMEC-I can induce the down-regulation of e NOS m RNA transcription,total e NOS(pan-e NOS)and p-e NOS protein expression,while up-regulation of i NOS m RNA transcription and protein expression levels in h CMEC-I.In addition,h CMECs were co-cultured with Lv-NKCC1and Lv-ct Jurkat T lymphocytes after pretreatment with angiotensin II(Ang II)(1μM)for 24 h,respectively.The protein expression of p-AKT and p-e NOS in h CMEC can be down-regulated after co-culture with Lv-NKCC1 Jurkat T lymphocytes.At the animal model level,the concentration of serum NO and the transcription level of Prkg1 in the left ventricle of the mice in the HSD-H group were lower than those in the NSD and HSD-N groups.The protein expression levels of i NOS,p-AKT and p-e NOS in the left ventricle,the protein expression levels of pan-e NOS and pan-AKT in the kidney,and the transcription level of BNP in the left ventricle were higher than those in the HSD-N and NSD groups.In addition,the s GC transcript level in the left ventricle in HSD-H group was significantly lower than that in HSD-N group.The blood pressure of C57BL/6 mice after infusion of Lv-NKCC1 Jurkat T lymphocytes was lower than that infused with Lv-ct Jurkat T lymphocytes.In addition,infusion of Lv-NKCC1 Jurkat T lymphocytes could significantly increase the serum NO concentration,up-regulate i NOS protein levels in the left ventricle and aorta,as well as down-regulate p-e NOS protein levels in the left ventricle and aorta.2.Clinical research partOf the included 2292 patients with AMI,the mean age was 65.8±12.4 years old,76.3%were men,and the mean value of body mass index(BMI)was 24.6±3.4kg/m~2.According to the quartiles of PLC,patients were divided into Q1(<1.13*10~9,N=567),Q2(1.13～1.57*10~9,N=575),Q3(1.57～2.09*10~9,N=576),and Q4(≥2.09*10~9,N=574)groups.Over the median follow-up of 2.7 years,a total of 146(29.7%),120(24.4%),85(16.1%),and 69(12.7%)MACE were observed in PLC Q1 to Q4 groups,respectively.When treating patients in the Q1 group as the reference,we found that those in the Q4 group experienced significantly lower risk of MACE(HR:0.61,95%CI:0.45-0.83,P=0.002),all-cause death(HR:0.49,95%CI:0.31-0.77,P=0.002),cardiac death(HR:0.40,95%CI:0.23-0.70,P=0.001),as well as HF rehospitalization(HR:0.58,95%CI:0.37-0.92,P=0.020).However,no statistical significance was achieved when comparing the risk of reinfarction and ischemic stroke across the PLC quartiles.Furthermore,we showed that the incorporation of PLC into the conventional risk prediction model could evidently improve its diagnostic performance(NRI:0.153,95%CI:0.047-0.259,p=0.005).Conclusions1.The down-regulation of NKCC1 expression in CD4~+T lymphocytes under high sodium stimulation leads to increased NO synthesis by inhibiting self-e NOS phosphorylation and up-regulating i NOS expression(endothelial cell-independent pathway).The down-regulation of NKCC1 expression in CD4~+T lymphocytes under high sodium stimulation lead to interaction with endothelial cell,as well as increase NO synthesis in endothelial cell through inhibition of its e NOS phosphorylation and up-regulation of i NOS expression,thus playing a role in antagonizing salt-sensitive hypertension.Our findings suggest that CD4~+T lymphocyte NKCC1 is involved in the pathogenesis of salt-sensitive hypertension through restricted NO synthesis mediated by endothelial cell-dependent and independent pathways,which also suggests that clinically intervening the expression of NKCC1 or limiting its function by drugs may become one of the important strategies for the treatment of salt-sensitive hypertension in the future.2.The decreased PLC is an independent predictor of MACE during the long-term follow-up period after AMI.The incorporation of PLC on top of the conventional risk prediction model could significantly improve its diagnostic performance.