Effects Of Prenatal Lipopolysaccharide Exposure On Sodium Excretion And Mesenteric Vascular Function In Offspring Rats

1. Effects of prenatal lipopolysaccharide exposure on renal dopamine D1 receptor in offspring rats1.1 Background and PurposeHypertension is a heterogeneous disorder which is may be caused by an interaction of genetic and environmental factors. In recent years, many studies have revealed that fetal programming is also an important factor for the formation of hypertension. David Barker and colleagues have shown that adverse environmental stimuli, experienced during a critical period of development in utero, could lead to long-term structural and functional effects in the developing organism.Maternal infection are common events during pregnancy, and many studies support the relationship between maternal infection and adverse pregnancy outcomes. Intrauterine infection is regarded as one of the major maternal insults during pregnancy. Lipopolysaccharide(LPS) is a toxic component of cell walls of gram-negative bacteria that is present in the digestive tracts of humans and animals. Peripheral administration of LPS from Escherichia coli is a well-characterized model of sepsis in rodents. Maternal exposure to LPS alters proinflammatory cytokine levels, including IL-1, IL-6, and TNF-α, in the placenta, amniotic fluid, and fetal brain, that may have a significant impact on fetal development. Studies have shown that prenatal exposure to LPS results in increased blood pressure in the offspring that is associated with alterations in the renin-angiotensin system in the kidney and increased circulating leptin which can increase sympathetic nerve activity. However, hypertension is caused not only by increased activity of pro-hypertensive factors, for example, renin angiotensin systems and sympathetic nervous system, but also caused by decreased activity of anti-hypertensive mechanisms, such as the renal dopaminergic system.Dopamine, produced in the kidney independent of renal nerves, is now recognized to play an important role in the regulation of sodium balance and blood pressure. Dopamine receptors are classified into D1-like and D2-like subtypes based on their structure and pharmacology. Under moderate volume expansion, dopamine, especially via D1-like receptors, acts to increase sodium excretion and keep the blood pressure in normal. Abnormal responses to dopamine and D1-like receptor function have been shown in hypertensive patients and rodent models of genetic and salt-sensitive hypertension. Studies have shown that dysfunction of D1 R could be an result of its hyper-phosphorylation that caused by G protein-coupled receptor kinase type 2(GRK2) and type 4(GRK4), which is linked to increased oxidative stress. Antioxidant supplementation with TEMPOL could decrease oxidative stress, restore D1 R signaling.We investigated whether or not dysfunction of renal D1 R signaling is involved in the fetal programming of hypertension and whether or not inhibition of ROS production would ameliorate the renal D1 R dysfunction and normalize blood pressure.1.2 MethodsPregnant Sprague–Dawley(SD) rats were intraperitoneally injected with LPS(0.79mg/kg) or saline(0.5ml) at gestation day 8, 10 and 12. After birth, the blood pressure is measeured by tail-cuff method. At postnatal 12 weeks, the offspring were treated with or without antioxidant TEMPOL in tap water for 3 weeks.Then, the following measurements were performed:1.2.1 The systolic blood pressure(SBP) and difference of SBP between two groups in 5 week- and 3-, 6-, 9-, and 12-month old offspring.1.2.2 Renal histopathology examined by H&E and Masson’s trichrome staining in 12-month-old offspring.1.2.3 Measurement of 24 h urine volume, 24 h urine sodium excretion, and blood pressure in offspring.1.2.4 The oxidative stress markers urine MDA, plasma SOD, renal SOD and GSH levels, were quantified in offspring.1.2.5 The effects of renal D1R-induced urine flow and urine sodium excretion rates in offspring.1.2.6 The protein expressions of renal GRK2, GRK4, and D1 R in offspring.1.2.7 The level of D1 R serine-phosphorylation in renal cortical membranes in offspring.1.3 Results1.3.1 Prenatal LPS exposure leads to increased blood pressure in offspring as compared with control rats.1.3.2 The total 24 h urine volume and sodium excretion decreased in LPS-treated offspring rats under basal conditions.1.3.3 The LPS-treated rats offspring had decreased D1R-mediated natriuresis and diuresis, lower renal D1 R expression and higher D1 R phosphorylation.1.3.4 As the important kinases of D1 R phosphorylation, GRK2 and GRK4 expression were increased in LPS rats.1.3.5 Prenatal LPS results in increased ROS, such as urine MDA, and decreased antioxidant capacity, sucha as SOD and GSH, and all of those reversed to normal after TEMPOL treatment for 3 week.1.3.6 Antioxidant TEMPOL treatment also reversed D1R-mediated natriuresis and diuresis, GRK2, GRK4, D1 R expressions and D1 R phosphorylation, and the increased blood pressure restored to normal ultimately.1.3.7 Dysfunction of renal D1 R could be fetal origins.1.4 Conclusion:Prenatal LPS exposure, via impairment of ROS on renal D1 R function, leads to hypertension in offspring. Reversion of renal D1 R function by alleviation of ROS might be a target for therapy of fetal programming hypertension.2. Effects of prenatal lipopolysaccharide exposure on mesenteric vascular function in Offspring2.1 Background and PurposeOur previous study showed that prenatal LPS exposure results in increased blood pressure with decreased sodium excretion, which is ascribed to impaired dopamine-induced natriuresis and diuresis, but with normal plasma creatinine and urea nitrogen levels. In addition, studies showed that prenatal exposure to LPS leads to decreased aortic reactivity with lower expression of connexin 37(cx37), which plays a role in the regulation of vascular tone and development of the vasculature, via NF-κB activation, and increased angiotensin type 1/type 2 receptor(AT1R/AT2R) ratio. However, the effect on vascular function has not been fully evaluated, especially in smaller arteries that contribute considerably to peripheral vascular resistance.Previous studies showed that the vascular endothelium plays an important role in the modulation of vascular tone and blood pressure regulation; impaired endothelial-dependent relaxant responses may be an important factor for the development of hypertension. Endothelium could release many relaxing factors, such as nitric oxide(NO), which could diffuse into smooth muscle and stimulate enzyme GC activity, leading to increased c GMP production and vasodilation.The present study examined whether or not prenatal exposure to LPS could program dysfunction of mesenteric arteries through NO-c GMP pathway in offspring and investigated the possible underlying mechanisms and strategies to restore the impaired vascular function and hypertension in the offspring of LPS-treated dams.2.2 MethodsPregnant Sprague–Dawley(SD) rats were intraperitoneally injected with LPS(0.79 mg/kg) or saline(0.5 ml) on gestation day 8, 10, and 12, the blood pressure was measured fortnightly from 5 week postnatal using the tail-cuff method, and the vascular function was tested after treatment with vehicle or TEMPOL(1.0 mmol/L in tap water) in offspring at 15 weeks.Then, the following measurements were performed.2.2.1 The histopathologic changes of mesenteric arteries in offspring.2.2.2 The PE-induced contraction in the absence or presence of L-NAME(10-4 mol/L) or ODQ(10-5 mol/L) in endothelia-intact mesenteric arteries.2.2.3 The PE-induced contraction in endothelium-intact(E+) or endothelium-denuded(E-) mesenteric arteries.2.2.4 The ACh-induced relaxation in the absence or presence of L-NAME(10-4 mol/L) or ODQ(10-5 mol/L) in mesenteric arteries.2.2.5 The endothelium-independent relaxation induced by SNP in endotheliumdenuded mesenteric arteries.2.2.6 The ACh-stimulated NO production in endothelium-intact mesenteric arteries, SNP-induced cGMP generation in endothelium-denuded arteries.2.2.7 DHE fluorescence intensity, tissue Ang II levels.2.2.8 The protein expressions of e NOS, s GC, AT1 R, Nox2, Nox4, nitrotyrosine, SOD1, and SOD2 in mesenteric arteries.2.3 Results2.3.1 The offspring of LPS-treated dams had higher blood pressure than control from 5 weeks to 19 weeks.2.3.2 The offspring of LPS-treated dams showed decreased acetylcholine(ACh)-induced relaxation and increased phenylephrine(PE)-induced contraction in endothelium-intact mesenteric arteries.2.3.3 Endothelium removal significantly enhanced the PE-induced contraction in offspring of control but not LPS-treated dams.2.3.4 The arteries pretreated with L-NAME to inhibit nitric oxide synthase(eNOS) in the endothelium or ODQ to inhibit cGMP production in the vascular smooth muscle, had attenuated ACh-induced relaxation but augmented PE-induced contraction to a larger extent in arteries from offspring of control than those from LPS-treated dams.2.3.5 The endothelium-independent relaxation caused by sodium nitroprusside(SNP) was also decreased in arteries from offspring of LPS-treated dams.2.3.6 The offspring of LPS-treated dams had reduced expressions of eNOS and soluble guanylate cyclase(s GC) and production of NO and cGMP in arteries, and higher oxidative stress and lower antioxidant capacity.2.3.7 Three-week treatment with TEMPOL, a reactive oxygen species(ROS) scavenger, normalized the alterations in the levels of ROS, e NOS, and sGC, as well as in the production of NO and cGMP and vascular function in the arteries of the offspring of LPS-treated dams.2.4 ConclusionPrenatal LPS exposure programs vascular dysfunction of mesenteric arteries through increased oxidative stress and impaired NO-cGMP signaling pathway.3. Total ConclusionPrenatal LPS exposure leads to dysfunction of renal D1 R, impaired NO-c GMP signaling in mesenteric arteries and increased blood pressure via enhanced ROS, and alleviation of ROS might be a target for therapy of fetal programming hypertension.