Role Of PM2.5in Regulation Of G Protein-coupled Receptor Kinase4Innatriuresis And Diuresis In Vitro

BackgroundEssential Hypertension (EH) is one of the most significant risk factors ofcardiovascular disease (CV). Since hypertension and its related complications areconsidered as crucial lethal factors all around the world, it is extremely momentous to makeclear and explicit the risk factors and the mechanisms of hypertension for furtherprophylaxis and treatment.Currently, cardiovascular diseases that result from environmental factors are attachedan increasing attention. Fine particulate matter (PM2.5,≤2.5μm diameter), which isconfirmed by numerous evidence-based medicine materials to relate with cardiovasculardiseases, is reported as the most hazard suspended particles in the atmosphere to humanhealth. Also, the systolic and diastolic blood pressure (SBP and DBP) of rehabilitationpatients of CV who are exposed to PM2.5will separately rise and what’s worse, populationSBP is on the increase with the total suspended particles (TSP) are augmenting.Although quantities of evidences affirm that PM2.5have something to do withpopulation hypertension, the mechanism is still a lack of clarity. As known to all, the studiesbefore have mainly focused on the impact of PM2.5on vascular function. Meanwhile, PM10has a high correlation with raised blood pressure (BP) at nighttime and reduced natriuresisand diuresis at daytime, maybe which would illustrate that renal natriuresis and diuresiscould be affected by atmospheric particles as well as PM2.5.Consequently, we designed theexperiments and found that the BP and natriuresis of WKY rats were indeed influenced byPM2.5.It is generally known that kidney which is the most important organ to regulate BPplays an irreplaceable role in the pathogenesis of hypertension. Moreover, the dopaminereceptors which belongs to G protein-coupled receptor (GPCR) are divided into2types:dopamine D1-like receptors (D1, D5) and dopamine D2-like receptors (D2, D3, D4) and the phosphorylation GPCR is attested primarily by G protein-coupled receptor kinase (GRK).In the light of several reports, the activity of G protein-coupled receptor kinase4(GRK4, GRK subtype) that basically express on kidney, vascular and heart always altersbefore the occurrence of hypertension. On the basis of previous studies, the GRK4expression and activity of hypertensive patients as well as that of spontaneouslyhypertensive rats (SHRs) is significantly enhanced, which bring about an increasephosphorylation of dopamine D1receptors (D1R) and the loss of coupling between D1R andG protein. Thus, D1Rwill be deprived of receptor function and cannot inhibitNa+-K+-ATPase causing water-sodium retention and increasing blood pressure.To sum up, hypothesis has been proposed: PM2.5could make an impact on renalnatriuretic and diuretic process by regulating GRK4so as to elevate blood pressure.Furthermore, this research has verified that PM2.5could affect the natriuresis and diuresis ofrenal proximal tubule cells (RPTc) to promote high BP of WKY rats via influencing theexpression of GRK4on RPTc to change that of D1R which impact on the inhibition ofNa+-K+-ATPase.PurposeTo investigate the abnormal regulation of PM2.5on RPTc GRK4and to verify its effecton hypertension.Content1. To use WKY rats to construct a PM2.5exposed model in order to observe the effecton BP and urinary sodium excretion;2. To study the effect of PM2.5on the expression of WKY rats’ kidney GRK4;3. To investigate the impacts of different doses of PM2.5on viability of RPTc toestablish cells’ PM2.5exposed models.4. To determine whether PM2.5regulates RPTc Na+-K+-ATPase through affectingGRK4, and observe the expression of GRK4, D1R on RPTc;5. To observe if PM2.5has an effect on oxidative stress indicators-superoxidedismutase (SOD) and malondialdehyde (MDA) of RPTc and in addition, to ascertain itsimpact on natriuresis and diuresis.Method1. WKY rats were perfused PM2.5via airway to establish PM2.5exposed model (exposure:8weeks) and HE staining of lung tissue was used to confirm whether the modelssucceed. Then, both BP among every groups and the urinary volume and biochemicalindexes-through diuresis metabolic cages method were detected;2. The total protein of WKY rats’ renal cortex was extracted so as to test the expressionof GRK4via Western-Blot (WB);3. RPTc was treated with different doses of PM2.5(10,50,100μg/ml) for24hours andcell viability was measured with CCK8kit;4. The activity of Na+-K+-ATPase of RPTc PM2.5exposed or unexposed models wasdetected with or without D1R agonist (Fenoldopam) and a small interfering (siRNA) whichspecifically silenced GRK4;5. The change of the protein expression of RPTc GRK4and D1R was determined byWB and the mRNA expression was ensured by real-time quantitative PCR (q-PCR);6.RPTc SOD and MDA were studied after PM2.5exposure for24hours with or withoutoxidative stress inhibitor-Tempol.Result1. The blood pressure of WKY rats that were exposed to PM2.5was higher than thecontrol group and simultaneously the urine volume and urinary sodium of exposed groupobviously decreased;2. The expression of renal GRK4of that WKY rats chronically exposed to PM2.5wasevidently higher than the control group;3. Compared to the control group, each dose chosen in this research makes no obviousdecrease on cell viability via CCK8kit;4. The activity of RPTc Na+-K+-ATPase of exposed group (regardless of exposure dose)was higher than the control group. After fenoldopam given, Na+-K+-ATPase activity of thecontrol group declined while the exposed group had no change. Additionally, as GRK4silenced by siRNA, fenoldopam could decrease the Na+-K+-ATPase activity of exposedgroup;5. The mRNA and protein expression of RPTc GRK4in exposed group were bothdistinctly higher and those of D1R were much lower the control group;6. RPTc SOD activity was lower and MDA activity was higher in exposed group thanthe control group. After giving Tempol, SOD activity of the exposed group rose and the latter abated compared with intervention before. Meanwhile, both the expression of RPTcGRK4and the activity ofRPTc Na+-K+-ATPase in Tempol group were much lower than thePM2.5exposed one, and also the reaction of Fenoldopam definitely recovered.ConclusionPM2.5increases the GRK4expression, decreases the D1expression and increases theNa+-K+-ATPase activity, which plays a important role in renal sodium excretion.