Renal Uptake Of Gastrin By Inducing An Increase In Dopamine Synthesis Of L-DOPA Involved In Blood Pressure Regulation

Gastrin increases renal dopamine production by increasing the uptake of L-DOPA which play an important part in the regulation of blood pressureObjective Essential hypertension (EH) is a multi-factor disease, which is a danger to human health and the incidence trend is rising year by year. Arterial blood pressure not only depends on the vascular resistance, but also is determined by the cardiac output. Cardiac output is affected by sodium extracellular fluid volume and renal function. The total peripheral resistance is affected by the influence of the sympathetic nervous system and catecholamine hormone. Recent study found that Gastrin is a peptide hormone, which acts not only to regulate gastric acid secretion, but also to exert physiological actions such as the regulation of sodium balance. While many studies suggest that dopamine also play an important role in sodium excretion and blood pressure regulation. Dopamine receptor subtypes all directly or indirectly, interact with other blood pressure regulating system, and are involved in the regulation of renal sodium excretion and blood pressure. This article gives a possible mechanism of the interaction between gastrin and dopamine, which shows a new perspective on blood pressure regulation and provides a new target for prevention and treatment.Methods By a case (HT, n=95)-control (NT, n=82) study in Fuyang People’s Hospital, Anhui Province, China, Basic information on the subjects was collected. Blood samples were collected, and blood pressures were measured at30,60, and120min after the meal ingestion. Correlation between basal serum levels of gastrin and other hormones was calculated. In vitro study, renal proximal tubular cells were incubated with L-DOPA(100uM) and separated concentration of gastrin (50ng/ml or lOOng/ml) applied from the cell side, ELISA kit was used to measure dopamine production. Cells were pre-incubated for6min with a single concentration (1μM) of L365,260or BCH (lmmol/L) before L-DOPA (100uM) and gastrin (100ng/ml) treatment to measure dopamine production. Protein concentration was detected by BCA protein assay kit. Western blotting was performed to detect the expression of whole and plasma membrane LATl expression of cells after drug treatment. Fresh kidney pieces were cultured in DMEM medium with drug treatment as same as in vitro study. Western blotting was performed to detect the expression of whole and plasma membrane LAT1expression of tissure. The Gastrin-specific siRNA was prepared in an in vivo transfection reagent (Mirus Bio) under sterile conditions. A mini-osmotic pump was inserted and positioned subcapsularly in Uninephrectomized adult male BALB/cJ mice to continuously deliver the Gastrin-specific siRNA and silencing gastrin expression. The blood pressure was measured from femoral artery of mice before and after mock or gastrin-specific siRNA infusion. Western blotting was performed to detect gastrin expression.gastrin mRNA was measured by Real-time PCR. Western blotting was performed to detect the expression of whole and plasma membrane LAT1expression of mice kidney treated by gastrin siRNA and mock siRNA.Results Clinical study suggests that gastrin is involved in the regulation of blood pressure. There is also no difference in basal serum gastrin levels between the two groups, but fasting blood pressures are significantly higher in HT than NT (P<0.05). In the HT group, the blood pressure significantly decreases after the meal and remains low level, Food intake does not change the blood pressure in NT. The fasting and pattern of the postprandial increase (peak at30min) in serum gastrin levels are similar in HT and NT. However, the gastrin levels at30,60, and120min are significantly higher in HT than NT (P<0.05, t test). Fasting serum levels of gastrin are significantly correlated with fasting serum levels of aldosterone and angiotensin Ⅱ (P<0.05, Pearson) in HT but not NT. Mechanism research shows that gastrin can stimulate mouse proximal tubular cells(MPTCs) and human proximal tubular cells (HPTCs) uptake more L-DOPA from extracellular. Pre-incubation of L365,260decrease dopamine production indicating that gastrin via CCKBR stimulates uptake of L-DOPA. L-amino acid transporter plays an important role in the uptake of L-DOPA by treatment of BCH. Western blotting shows that gastirn can increase the number of LAT1on plasma membrane, but not the whole LAT1expression.We also show that uptake of L-DOPA in MPTCs is significantly lower than in HPTCs(12±1.5vs28±1.9P<0.05), maybe due to the stains of mice (C57BL/6J). Ex vivo study also demonstrates that gastrin plays an important role in maintaining normal dopamine synthetic and blood pressure.Conclution Based on our findings that postprandial serum gastrin levels are significantly higher in hypertensive than normaltensive, and fasting serum levels of gastrin are significantly correlated with fasting serum levels of aldosterone and angiotensin II, we conclude that gastrin is involved in the regulation of blood pressure. In vivo, in vitro and ex vivo studies indicate that gastrin can increase dopamine production by uptake of L-DOPA, in whcih LAT1may participate. Single nucleotide polymorphisms of the dopamine D2receptor increase inflammation and fibrosis in human renal proximal tubule cellsObjective The dopamine D2receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs) and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single nucleotide polymorphisms (SNPs; rs6276,6277, and1800497) in the human (h) DRD2gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory and injury markers, which could provide clues for the gene therapy of chronic renal disease and high blood pressure.Method Western blotting and Real-time PCR weres performed to detect the protein and mRNA expression of D2R in hRPTCs carrying D2R SNPs and cells carrying no D2R SNPs. After D2R agonist treatment, cyclic AMP accumulation was tested by cAMP chemiluminescent immunoassay, which results effect D2R function. We then measure the protein and mRNA expression of pro-inflammatory TNFa in hRPTCs carrying D2R SNPs and cells carrying no D2R SNPs. Real-time PCR was also used to test mRNA expression of cytokines/chemokines.The protein and mRNA expression of TGFβ1its signaling pathway facrors SMAD3, Snail1, FN-1, Col la and Vimentin were measured by Western blotting and Real-time PCR. Immunofluorescence was performed to detect TGFβ1and its signaling target, and cellular morphology. We reverse the experiment by transfected with an plasmid harboring wild-type human DRD2or empty vector in hRPTCs carrying SNPs, to make the cells have enough D2R expression. Western blotting and Real-time PCR were performed to detect the protein and mRNA expression of D2R, TGFβ1and Snail1, FN-1, Col la and Vimentin in Immunofluorescence was performed to detect TGFβ1and its signaling target, and cellular morphology, which is same as non-transfected cells. Result Human RPTCs with D2R SNPs had decreased D2R expression and function comparing with cells carrying no D2R SNPs (P<0.05, t-test). The expressions of the pro-inflammatory TNFa and the pro-fibrotic TGFβ1and its signaling targets Smad3and Snail1were increased (P<0.05, t-test) in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition (EMT) and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin-1(FN-1), and Col1a. Our results show that RPTCs from subjects carrying D2R SNPs that result in decreased D2R mRNA and protein express a pro-inflammatory and pro-fibrotic phenotype and markers of EMT. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. D2R expression was increased and those of TGFβ1, Smad3, Snail1, vimentin, fibronecti-1and Col1a were decreased (P<0.05, t-test) in hRPTC with D2R SNPs transfected with wild-type DRD2compared to hRPTC-D2R SNP transfected with empty vector. Result of Immunofluorescence shows the change of cellular morphology, cells became spindle and disorder. The result demonstrated again individuals carrying D2R polymorphisms that result in decreased D2R function could be more vulnerable to renal injury and fibrosis.Conclusion These data support the hypothesis that D2R function has protective effects in human RPTCs, which can decrease expression of pro-inflammatory and pro-fibrotic phenotype and markers of EMT. This study suggests that carriers of these SNPs may be prone to chronic renal disease and high blood pressure. Genetic testing could identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease.