Establishment Of Gitelman Syndrome’s Diagnosis Methods And A Preliminary Study Of Yitamiu D’s Inhibition Mechanism On Renin Activation

Backgroud and objective:Gitelman syndrome (GS) is an autosomal recessive renal tubular salt-wasting disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria, renin-angiotensin Ⅱ-aldosterone system (RAAS) activation with normal blood pressure. GS results from loss-of-function mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl co-transporter (NCC) protein of the distal convoluted tubule (DCT). Easly accessible diagnosing method is still lacking, which limits the development of diagnosis and treatment of GS. This study aims to establish Gitelman syndrome (GS) patients’ clinical, genetic and physiological function (thiazide test) diagnostic methods, combine with in vitro gene mutation function test, identify Chinese GS patients’ mutation sites and it’s pathogenicity, investigate hypomagnesemic GS subtype and glucose intolerance impairment phenotype. Besides, this study tries to discover the mechanism of GS patients’ systemic and local RAAS activation, and investigate different vitamin D receptor (VDR) agonists’ inhibition effect on renin.Methods:1. Formulate clinical symptom standard questionnaire, collect clinical data, isolate peripheral blood DNA, direct sequence SLC12A3 gene exons, and estimate GS patients’ NCC protein function status by thiazide test. Conclude the frequent mutations and novel mutations of Chinese GS patients, construct wild type and mutated NCC plasmids, transcribe them into RNA in vitro, and quantitatively detect the mutations’ function by xenopus oocyte expression system.2. Detect the serum and urinary electrolyte level of GS patients,64 healthy volunteers serve as control. Evaluate GS patients’ insulin sensitivity and secretion ability by 3h oral glucose tolerance test,20 healthy volunteers and 20 DM patients serve as controls.3. Compare the clinical presentation and thiazide test results between hypomagnesemic and normomagnesemic GS patients. Analyse and the expression level of renal magnesium transporter-- transient receptor potential channel melastatin subtype 6 (TRPM6) on the renal biopsy sections of GS patients and glomerular minor lesion control.4. Detect the systemic plasma renin activity(PRA),angiotensin Ⅱ and aldosterone level ofGS patients by supine-upright RAAS test, and investigate the expression of renin and α- Actin difference between GS patients’ renal section and fetus renal section by immunohistochemistry staining and immunofluorescence double staining.5. Observe the effect of active vitamin D and it’s anologs on renin activity, renin mRNA and protein level by acute and chronic animal stimulation test, and study renin, COX2, NCC and TRPM6 expression level of VDR knockout mice. Investigate the expression of VDR on juxtaglomerular cell (JGC) and macula densa (MD) cell, stimulate MD cell by active vitamin D and observe it’s effect on low chloride induced COX2 increasement.Results:1.60 GS patients were diagnosed by clinical and SLC12A3 gene screen, the most frequent symptoms were muscle weakness (79.7%), fatigue (55.9%), carpopedal spasm/tetany (45.8%), palpitations (40.7%), paresthesias (35.6%) and nocturia (33.9%), and the clinical presentation of male GS patients were more severe than female counterparts.2. Totally,54 mutations were detected (include 14 novel mutations). Another 3 mutations (T60M, D486N and R913Q) were identified as frequent mutation (>5%) of Chinese GS patients. In vitro functional experiments found that mutated NCC protein (T60M, L215F, D486N, N534K, Q617R and R928C) presented significantly lower 22Na+ absorption ability than wild type NCC protein, and the decreased transport activity was due to the special mutation of transporter.3. Simplified thiazide test was established in this study, the normal reference range of Chinese healthy controls were obtained for the first time. The thiazide test confirmed the dysfunction of NCC protein of GS patients. Compared with the gold standard-gene secquencing, the area under ROC curve of delta FEci for the diagnosis of GS after thiazide administration is 0.983. When 2.86% was chosen as the cutoff value, the sensitivity and specificity for diagnosing hypokalemia, metabolic alkalosis GS patients were 95% and 95.2%, respectively.4. OGTT result indicated that GS patients manifested abnormal glucose metabolism and insulin function. Compared with healthy control, GS patients exhibited glucose peak and insulin peak delay, increased area under curve of glucose and decreased insulin secretion sensitivity index (ISSI). While no significant differences of the insulin resistant indexes (ISOGTT, QUICKI and HOMA-IR) were found between healthy control and GS patients.5. Normomagnesemic GS patients exhibited milder clinical presentation, better reaction to thiazide than hypomagnesemic counterparts. NCC and TRPM6 co-expressed at the DCT. Compared with glomerular minor lesion controls, the TRPM6-positive area was significantly decreased in hypomagnesemic pa-tients (4.96±1.88 vs.8.63±2.67%) while it was near normal (7.82±5.23%) in 2 normomagnesemic GS patients.6. Systemic RAAS activation was observed in 88% GS patients. Juxtaglomerular apparatus hyperplasia was observed in all 18 GS patients who accepted renal biopsy, and the renin expression of GS patients was notably higher than glomerular minor lesion patients. Additionally, renin and a-Actin co-expression was found not only at juxtaglomerular apparatus but also at micro-artery wall, and this co-expression status was also observed in fetus renal section.7. Active vitamin D and its analogs can’t inhibit the acute renin secretion. However, chronic injection of active vitamin D’s analog-RO compound was able to decrease renin mRNA, without elevating serum calcium level. VDR knockout mice presented hypocalcemia, and their renin mRNA level was about 2.9 times of wild type mice. MD cell expressed VDR. Active vitamin D didn’t affect low chloride induced COX2 expression on MD cell.Conclusion:This study established the clinical, genetic and functional diagnosis methods of GS patients. Thiazide test was an easy, cost-effective and reliable pathway to diagnose and differentiate GS.14 novel mutations were identified in this study, and some frequent mutations of Chinese GS patients were found in the study (included T60M, D486N and R913Q). Male patients exhibited more severe clinical presentation than female counterparts, and some GS patients manifested abnormal glucose metabolism and insulin function. Normomagnesemia was a clinical subtype of GS, serum magnesium level was related to GS patients’ clinical severity and NCC dysfunction extent, and decreased TRPM6 may be an important cause of hypomagnesemia. The structure foundation of GS patients’ RAAS activation was renal renin producing cell recruitment. Active vitamin D’s analog-RO compound was able to inhibit renin expression, which may provide potential treatment value for GS.