A Preliminary Study On The Mechanism Of The Sodium And Chloride Cotransporters In The Distal Tubules Of The Kidney To Maintain The Balance Of Water, Salt And Blood Pressure And Participate In The Feedback Of The Bulb

Background and objectives:Water and electrolytes balance in body is dependent on the functional integrity of glomerulus and renal tubules,and the tubuloglomerular feedback(TGF)between them.The sodium-chloride symporter(also known as Na+-Cl-cotransporter,NCC)is a transmembrane protein encoded by the gene SLC12A3.It contributes to the balance of water and electrolytes as same as the the Na-K-Cl cotransporter 2(NKCC2)in thick ascending limb of the loop of Henle and the macula densa in nephrons,and the Epithelial Sodium Channel(ENaC)in the collecting tubule.NKCC2 and ENaC are proved to be an important inductor for TGF,while the role of NCC in TGF is still unclear.Gitelman syndrome(GS),a rare inherited salt-losing tubulopathy mainly caused by loss-of-function mutations in the SLC12A3 gene,is a natural disease modal to study the function of NCC.However,there is no related research about clinical and pathophysiology of GS,and no practical biomarker and functional imageology methods for evaluating the severity and guiding the treatment of GS.This study aimed to observe the clinical and pathological features,quality of life and gender difference in a GS cohort with a fairly large sample size.Compare the diagnostic value of funcitonal test of NCC.Evaluate the correlation between PGE2 level or its metabolin in urine and the clinical manifestations.Explore the the diagnosing value of exosome and functional MRI for GS patients.Establish two different salt-sensitive hypertension mice model,i.e.the deoxycorticosterone acetate(DOCA)-salt hypertensive model and hypeluricemia induced hypertensive model,and evaluate the role of NCC in the development of hypertension and kidney damage.Furthermore,investigate the relationship of tubuloglomerular feedback and NCC in the Adenosine receptor type 1 knockout(A1AR-/-)model.Methods:Part 1.Study about the characteristics of clinical manifestations,biomarkers and imageology in the congenital NCC dysfuncion patients with GS1.Collect the clinical data of the suspicious GS patients,extract the DNA of peripheral blood for exome sequencing,taking the gene test as gold standard,evaluate the function of NCC in vivo by using hydrochlorothiazide.Compare the diagnostic value of hydrochlorothiazide test to hypomagnesemia and hypocalcinuria for GS patients.2.Observe the feature of blood pressue and fluid-electrolyte metabolism in GS patient.Gather the plasma and 24h urine for testing PGE2 and its metabolin levels by ELISA,analysis the correlation between PGE2 with clinical manifestations and NCC function.Compare the gender difference in the manifestations of GS,summarize the clinical data of pregnancy and the SF-36 scale for the quality of life in GS patients.Measure the PGE2 and its metabolites both in blood and urine,to test the gender difference and the correlation of clinical manifestation.Collect the data of female patients with pregnancy,observe the elctrolytes level and complications during pregnancy.3.Collect the renal pathological data of GS patients,compare the expression of COX-2.Extract the exosome of urine,measure the expression of NCC,COX-2 and COX-1.Collect the medical imagology data of GS patients using the functional-MRI(DKI and I VIM)of kidney.Part 2.The establishment of salt sensitive hypertension mice model and the change of NCC associated water electrolyte balance.1.Establish the uninephrectomized and deoxycorticosterone acetate(DOCA)-salt hypertensive mice model,and establish the early-stage hypertensive model induced by mild hyperuricemia.Monitor the variation of blood pressure,together with the change of uric acid and electrolytes levels.Evaluate the relation between salt-intake with BP.Evaluate the renal tubulointerstitial damage by PAS and Masson staining.2.In two mice model,measure the expression of NKCC/NKCC2/ENaC including protein and mRNA,quantify the expression of SGK-1,and test the caspase-1/IL-18/IL1? in inflamation-pyroptosis pathway and adenosine receptors.3.Measure the levels of renin,angiotensin,aldosterone by radio-immunity assay.Locate the expression of renin in juxtaglomerular complex by immunohistochemistry.Observe the expression of adenosine by immunoblottingPart 3.The possible effect of NCC on blood pressure and water electrolyte metabolism when lacking tubuloglomerular feedback.1.By furosemide test,calculate teh clearance rate of creatinine(CCR),speculate the state of tubuloglomerular feedback.And compare the CCR of GS and non-GS patients to evaluate the activity of tubuloglomerular feedback.2.In A1AR knockout mice model,observe the difference of BP,water,adn electrolytes in DS model and HUA model.measure the expression of NKCC/NKCC2/ENaC including protein and mRNA and test the caspase-1/IL-18/IL1? in inflamation-pyroptosis pathway and adenosine receptors.3.Transect the afferent arterioles of wild or A1AR knockout mice,observe the effect of different UA levels on afferent arterioles by mircoperfusion.And evalutae the chronic remodelling effect of UA to afferent arterioles by semi-quantitative immunohistochemistry analysisResults:Part 1.study about the characteristics of clinical manifestations,biomarkers and imageology in the congenital NCC dysfuncion patients with GS1.Totally 105 hypokalemia patients detected SLC12A3 gene mutation and diagnosed as Gitelman syndrome,and totally 69 types of mutation were found.The mean blood pressure of GS patients was 110.0±12.4/72.1±9.8mmHg combined with Renin-Angiotension?-Aldosterone system activation.Hydrochlorothiazide test(HCT)indicated different degree of NCC depression in GS patients.Compared to the gold standard of gene diagnose,the sensitivity and specificity of hydrochlorothiazide test was 93.1%and 88.9%respectively,the AUC was 0.989(95%CI 0.961-1.000),all were better than those of the common used criteria of hypomagnesemia and hypocalcinuria.2.Male GS patients seemed to have an early onset,with lower serum potassium levels,higher blood pressure and more severe polyuria,but no difference in the test of quality of life.There were 5 female patients got pregnant after disease onset,they performed a decrease of serum potassium in the first trimester,after symptomatic treatment they all had stable process to good outcome.The average PGE2 levels of GS patients were higher than the normal control group of the same gender,and male patients had a higher level than female patients.The elevated PGE2 levels might be associated with more severe NCC damage,metabolic alkalosis and electrolyte disturbance.of GS patients was worse than normal control,and there was no difference between male and female.COX-2 expression was decreased in the cortex of kidney,while a decrease of NCC expression and increase of COX-2 and COX-1 expression were detected in urine exosome.3.The renal pathological results of GS patients showed the proliferation of juxtaglomerular apparatus.While the conventional MRI showed no abnormal of kidney for GS patients,the mean kurtosis and mean diffusivity level of diffusion kurtosis imaging(DKI)had a good correlation with serum chlorine and bicarbonate,and the D value and DP value of the inteavoxel incoherent motion imaging(IVIM)had a correlation with the NCC function.Part 2.The establishment of salt sensitive hypertension mice model and the change of NCC associated water electrolyte balance.1.The DOCA-salt hypertensive mice model was established,with had a significant continuous elevation of systolic and diastolic blood pressure,together with increased urine volume and more electrolytes excretion in urine.The kidneys were magnified and significant tubulointerstitial injury and fibrosis.The expression of NCC/NKCC-2/ENaC at 4th week and the expression of caspase-1/IL1? at 8th week were both increased.2.Oxonic acid gavage(250mg/kg/d)together with 0.1%uric acid drinking water stably induced a mild hyperuricemia and the blood pressure was upregulated,without the injury of urate nephropathy.The hypertension was regulated with the change of diet salt,consistent with the feature of salt sensitive hypertension.There was no significan change in the amout of NCC/NKCC-2/ENaC and caspase-1/IL1? of inflamation-pyroptosis.3.The renin activety in the DOCA-salt hypertensive mice was suppressed and the tubuloglomerular feed back associated adenosine receptors,A2AR was decreased,while no change of A1AR.As to the hyperuricemia hypertension mice,A1AR was upregulated while A2AR was downregulated,RAS and tubuloglomerular feed back was activated.Part 3.The possible effect of NCC on blood pressure and water electrolyte metabolism when lacking tubuloglomerular feedback.1.When the macula densa NKCC2 was blocked by furosemide,patients with normal NCC function(non GS)showed a weak decrease of creatinine clearance rate,while GS patients showed a significant decreasion.2.When lacking tubuloglomerular feedback(A1AR-/-mice),after the model was established,the fibrosis happened earlier,wihle the upregulated A2bR returned to normal;the difference of NCC expression disappeared.In the latter model,the expression of NKCC-2 was up-regulated,the excretion of urine volume and urine sodium increased,and.The blood pressure descended in the first week,and ascended thereafter,but not higher than the wild type,nor was the kidney damage.3.When A1AR was knockout,the hyperuricemia at same level would not induce hypertension,nor was the lesion of afferent arterioles,with the up-regulated of A2bR.The microperfusion of afferent arterioles showed that uric acid directly stimulated the contraction of afferent arterioles,which showed to be dose dependent.Furthermore,the effect disappeared after the A1AR knockout.Conclusions:1.The congenital NCC dysfuncion patients with GS showed normal tension?hypokalemia,the excretion of potassium,sodium and chloride were increased.Compared to the gold standard of gene diagnose,the sensitivity and specificity of hydrochlorothiazide test was better than those of the common used criteria of hypomagnesemia and hypocalcinuria.2.The elevated PGE2 levels might be helpful to evaluate the degree of clinically severity and dysfunction of NCC.The exosome COX-2 expression was elevated and may be the mechanism of the elevated PGE2,also maybe the reason of activated tubuloglomerular feedback.Functional MRI of DKI and IVIM may be associated with the NCC function but further research was needed.3.The DOCA salt-sensitive hypertensive model with low renin showed an increase of NCC and NKCC2 expression.The inflammation might be secondary to the high filtration.At the absence of tubuloglomerular feedback,NCC might contribute to the balance of normal blood pressure and electrolytes.Stimulated by aldosterone and high salt-intake,the expression of NKCC-2 increased.4.In the hyperurecima-induced hypertension,the change of NCC and activation of inflammation were not obvious.The acute contraction and chronic damage of afferent arterioles played an important role of the mechanism for hypertension.