The Role Of PGE₂ Receptor EP3 In Hypertensive Nephropathy And Its Effect On Calcium Concentration

Background:Recently it is reported that the incidence of developing hypertension and hypertensive nephropathy has gradually increased. Control of blood pressure is the main therapeutic strategy for treating end stage hypertensive nephropathy. Renin-angiotensin-aldosterone (RAAS) system plays major roles in the regulation of blood pressure, thus its role in chronic kidney damage especially hypertensive kidney damage has been paid serious attention. AngiotensinⅡ(AngⅡ), as the important active mediator in RASS system, not only regulates blood pressure through hemodynamic effect, but also plays significant roles in causing kidney diseases. AngⅡmainly interact two types of G protein coupled receptors AT1 and AT2, activating distinct signaling transduction pathways. AngⅡactivated AT1 triggers the increase of intracellular Ca2+ concentration, activation of cPLA2, MAPK, and PKC, which are all important mediators of PG synthesis. It has been reported that inhibition one or more PG receptors achieves effective control of high blood pressure. At least one PGE2 receptor plays critical roles in RAAS regulation of blood pressure. PGs are produced in the metabolism of arachidonic acid and are important regulators of vascular tone. To date five types of biological active PG families have been found. Among them, PGE2 is the predominant one. PGE2 receptors, G protein coupled receptor, are genetically divided into four subtypes, EP1, EP2, EP3 and EP4. PGE2 can interact with different receptors, activates distinct signaling pathways and exerts diverse biological effects including regulating the blood flow of the body and the kidney, and modulating renal sodium/water balance. Although the downstream signaling pathways of EP1, EP2 and EP4 have already been understood, the molecular mechanisms of EP3 are largely unknown due to the existence of multiple splicing variants. However, among the four receptors of PGE2, the affinity between PGE2 and EP3 is the strongest. In addition, EP3 is widely expressed in the tissues of the body with high levels in the kidney, pancreas and uterus. Therefore, exploration of the biological functions of EP3, as well as the molecular basis of its roles in physiology and pathophysiology, is recently paid a wealth of attention.Objective:To develop a new model of Hypertensive nephropathy in both wild type mice and EP3 knockout mice on C57BL/6 background. To find the role of PGE2 receptor EP3 in regulating blood pressure and renal injury in hypertensive nephropathy model in vivo, by assaying systemic blood pressure, ACR, BUN, GFR, renal Col-Ⅰ, Col-Ⅳ, TGF-β, PAI-1, and AT1 mRNA expression level. And tissue damage by histopathology was also measured. The further study would focus on vitro experiments. LVIP2.0Zc cells were transfected with plasmids overexpressing AT1 receptor and EP3 receptor, and the role of EP3 in AngⅡ-AT1 signaling transduction pathway were expected. All the studies may provide experimental evidence for exploring new drug targets for curing kidney disease. Methods:1. The contribution of the Prostaglandin E2 EP3 receptor was assessed in a model of hypertensive renal damage. C57BL/6 mice including wild type and EP3 knockout mice, were subjected to uninephr-ectomy (right side) on day -14. On day 0,50 mg deoxycor- ticosterone acetate (DOCA) releasing pellets were implanted subcutaneously into mice, and mice were switched to drinking water containing 1% sodium. On day 7, mice were further implanted with minipump, which released AngⅡat 1.5ng/min/g body weight. Animals were euthanized 19 days post initiation of deoxycorticosterone acetate treatment. Spot urines and tail cuff blood pressure measurements were taken weekly. BUN, GFR and ACR were measured as well. Kidneys were either fixed and examined by histopa-thology, or homogenized for measuring mRNA expression of fibrotic factors by q-RT-PCR.2. LVIP2.0Zc cells were cultured and transfected with plasmids overexpressing AT1 receptor or EP3 receptor. Different concen- trations of AT1 agonist AngⅡor EP3 agonist sulprostore were used to stimulate the cells. Intracellular Ca2+ concentration stained with Fluo-4AM were measured by Flex station.Results:1.(1) During experiments, after implantation of AngiotensinⅡminipump, significant mortality was observed in EP3 +/+ animals and EP3-/- animals. In 23 wild type mice,16 mice died, therefore the mortality was 70%. In 19 EP3 knockout mice,7 mice died, therefore the mortality was 37%. By Chi-Square statistics, the difference in the mortality between two groups was 0.0376. In addition, in wild type groups, mice displayed severe ascites, even anasarca, while EP3 -/- rarely developed anasarca even in the subset of mice that died. This is reflected in the body weight change in two groups after AngⅡminipump implantation. While EP3 +/+ experience a marked increase in body weight, the EP3 -/- mice have a slight loss in body weight. The weight change patten over time after AngⅡwas different between two groups after adjusing baseline weight (p<0.001).(2) Blood pressure significantly increased in both groups. In wild type mice, blood pressure increased from 108.0±3 mmHg to 190.9±3.9 mmHg. In EP3 knockout mice, blood pressure increased from 105.7±1.6 mmHg to 199.1±4.0 mmHg. However, the different between wild type mice and EP3 knockout mice were not statistically different (P=0.1939).(3) GFR in both groups was not different at the beginning stage of the experiments. Following the progressing of the experiments, GFR in wild type mice dramatically decreased to 2.1±0.2 ul/min/g body weight (P<0.01), and GFR in EP3 knockout mice decreased to 5.6±0.7 ul/min/g body weight. However, no significant difference was observed. At the end point of the experiments, GFR between two groups were statistically different. The decrease of GFR in wild type mice was significantly greater. Consistently, BUN increased in both groups. In wild type mice, BUN increased from 17.6±2.7 mg/dl to 63.7±10.3 mg/dl. In EP3 knockout mice, BUN increased from 23.1±1.6 mg/dl to 39.0±2.1 mg/dl. The BUN in two groups at the end point of the experiments was statistically different (P<0.0352). Following AngⅡminipump implantation, ACR in wild type mice increased to 2393±446.7 mg/g, and ACR in EP3 knockout mice increased to 704.0±136.1 mg/g, which were statistically different (P<0.001).(4) At the beginning of the experiments, the expression of fibrotic factors in both groups were not different and at very low level. At the end point of the experiments, the mRNA expression of COL-Ⅰ, COL-Ⅳ, TGF-βand PAI-1 increased. Greater increase was observed in wild type group when compared to EP3 knockout group (P<0.01).(5) Histopathology:By light microscope, Mesangial matrix hyperplasia, glomerular sclerosis, tubular dilation, protein casts, tubular epithelial cell degeneration and focal tubular necrosis, fibrosis, lots of inflammatory cells infiltration in renal interstitium with segmental interstitial fibrosis, were found in two groups. But kidney damage in WT mice showed more serious than EP3-/- mice. Compared with EP3-/- group, glomerular sclerosis(GS) index and tubulointerstitial lesion(TIL) score were significantly increased (P<0.05).2. LVIP2.0Zc cells were transfected with plasmids overexpressing AT1 receptor and EP3, and stimulation with different concentrations of AT1 receptor agonist AngⅡand EP3 agonist sulprostore. When cells were only transfected AT1 receptor overexpressing plasmid and stimulated with AngⅡ, intracellular Ca2+ concentration remarkably increased in a dose dependent manner. When cells were co-transfected with AT1 receptor DNA and EP3 receptor DNA overexpressing plasmids, intracellular Ca2+ concentration increased with the dosage of AngⅡ. When sulprostore was further added, EP3 receptor activity increased, and intracellular Ca2+ concentration continued to rise, which was statistically different from without sulprostore.Conclusion:1.Hypertensive nephropathy model was successfully built on C57BL/6 mice background with increased albuminuria, hypertensive glomerulus damage, renal tubule damage and'fibrotic factors high expression.2.Targeted disruption of EP3 receptor does not cause significant differences in blood pressure.3.PGE2 receptor EP3 can worse renal damage in hypertensive nephropathy.4.AngⅡactivation of AT1 receptor triggered the release of intracellular Ca2+.5.The AngⅡ-AT1 receptor mediated signaling transduction that increased intracellular Ca2+ was modulated by PGE2 receptor EP3, activation of which further increased the release of intracellular Ca2+.