| Kidney is a vital organ in the maintenance of blood pressure and water-electrolyte metabolism. The renin-angiotensin system (RAS), a key hormonal cascade in reguating arterial blood pressure and electrolyte balance, exerts a critical role in the pathophysiology of hypertension. Classical RAS refer to circulating RAS, but there is still local RAS in kidney. An intact renin-angiotensin system is essential for normal kidney development. Inadvertent use of ACE inhibitors or AT1-R blockers during pregnancy causes structural and functional developmental abnormalities of the kidney in the fetus. Angiotensin II, the major biologically active component of renin-angiotensin system, induces not only vasoconstriction, aldosterone release, and sodium reabsorption by the nephron, but is also intricately interrelated with inflammation, playing an important role in the development of hypertension and target organ damage. Growing body of evidence indicates that hypertension is an inflammatory state wherein proinflammatory cytokines, such as tumor necrosis factor (TNF)-αand interleukin-6 (IL-6), contribute to the hypertensive effect. Lipopolysaccharide (LPS) is a component of the cell wall of gram-negative bacteria and acts as a non-specific immuno-inflammatory stimulant to mimic the bacterial inflammatory response. LPS initiates a series of phosphorylation events by binding to its toll-like receptor 4, promoting the nuclear transcription factor NF-κB to translocate into the nucleus. In the nucleus, NF-κB promotes transcription of TNF-α, IL-1β, and IL-6 and ultimately induces inflammatory response. Pyrrolidine dithiocarbamate (PDTC), a synthetic antioxidant, inhibit NF-κB by preventing IκB degradation and the translocation of the active form to the nucleus. Zymosan, a component of yeast cell walls, comprised mainly ofβ-glucan and mannan, is a specific ligand for TLR2/6 and is capable of inducing inflammation. Zymosan can activate macrophages, monocytes and leucocytes to stimulate the release of inflammatory products, including TNF-α, IL-8 and hydrogen peroxide, which results in systemic inflammation. It has been shown in our laboratory that maternal exposure to LPS or zymosan on 8th, 10th, 12th day during gestation results in hypertension in offspring rats. However, the mechanisms are not well understood. The administration of LPS or zymosan on 8th, 10th, 12th day during gestation is at the critical ontogenic window early in renal development during which it is more susceptible to the inflammatory responses. Therefore we envisage that prenatal exposure to LPS or zymosan might have influenced the renal development of embryos and caused hypertension in adulthood. The present study was designed to explore the effects of LPS and zymosan, two immuno-inflammatory stimulants, on renal structure and function in offspring rats and the possible mechanism.MethodsIn the model of prenatal exposure to LPS, the pregnant rats were randomly divided into four groups (n=8 in each group), i.e. control group, LPS group, pyrrolidine dithiocarbamate (PDTC) group and LPS + PDTC group. Rats in these groups were administered intraperitoneally with vehicle, 0.79mg/kg LPS, 100mg/kg PDTC, LPS plus PDTC, respectively. LPS was given on 8th, 10th and 12th day while PDTC from 8th to 14th day during gestation. The rats in LPS group were given a vehicle injection on days 9,11,13,14 and rats in control group were given every day during d8-14.In the model of prenatal exposure to zymosan, the pregnant rats were randomly divided into two groups (n=6 in each), i.e., control group and zymosan group. The rats in these two groups were administered intraperitoneally with normal saline 0.5 ml and 2.37 mg/kg zymosan respectively, on 8th, 10th and 12th day during gestation.1. To evaluate the renal development, glomerular number was determined at 7 weeks of age with the method of acid maceration.2. To evaluate the renal founction, the creatinine clearance rate and urinary protein level were determined at 7, 16 and 25 weeks of age. Plasma and urinary creatinine concentration were measured by multichannel kinetic spectrophotometry and creatinine clearance rate was calculated. Urinary protein level was determined using spectrophotometric sequential injection method.3. The expression levels of renal cortex renin, ACE mRNA were assessed in offspring rats with real-time PCR.4. Intrarenal Ang II-positive cells, monocyte/macrophages, lymphocytes, TUNEL-positive cells and collagen I were identified by immunohistochemical staining (IHC) or TUNEL staining.5. Plasma renin activity and angiotensin II concentration were determined in offspring at 7, 16 and 25 weeks of age by radioimmunoassay determination.6. AT1-R protein expression was assessed by Western blot.7. NF-κB activation was evaluated by Electrophoretic Mobility Shift Assay (EMSA).8. The gene expressions of the components of RAS in rat fetus were assessed by microarray analysis using Rat Genome 230 2.0 Array GeneChip, and the mRNA expressions of IL-6 and TNF-αin rat fetus were assessed by real-time PCR.9. The systolic blood pressure in offspring rats was measured once every three weeks from 7 weeks of age to 25 weeks of age using the standard tail-cuff method.Results1. Prenatal exposure to LPS resulted in decrease in glomerular number in offspring rats and it was markedly reversed by PDTC treatment.2. Prenatal exposure to LPS resulted in decrease in creatinine clearance rate and increase in urinary protein level in offspring rats at 7, 16 and 25 weeks of age and they were completely reversed by PDTC treatment.3. Prenatal exposure to LPS resulted in decreased renal cortex renin mRNA expression in offspring rats at 1 day of age but increased renin mRNA expression at 7, 16 and 25 weeks of age, and it was markedly reversed by PDTC treatment.4. Prenatal exposure to LPS resulted in increase in ACE mRNA expression in offspring rats at 25 weeks of age and it was markedly reversed by PDTC treatment.5. Prenatal exposure to LPS decreased renal cortex Ang II-positive cells in new born offspring rats and increased renal cortex Ang II-positive cells in 7, 16 and 25-week-old offspring rats. These changes were markedly reversed by PDTC treatment.6. Prenatal exposure to LPS exerts no effect on plasma renin activity and Ang II level in offspring rats.7. AT1-R protein expression increased in new-born offspring of LPS-treated rats and decreased in 7, 16 and 25-week-old offspring, negatively correlated with renin mRNA expression.8. Prenatal exposure to LPS increased NF-κB activation in offspring and it was markedly reduced by PDTC treatment.9. Prenatal exposure to zymosan resulted in decrease in glomerular number and creatinine clearance rate in offspring rats at 7, 16 and 25 weeks of age.10. Prenatal exposure to zymosan resulted in increased renal cortex renin mRNA expression and increased renal cortex Ang II-positive cells in offspring rats at 7, 16 and 25 weeks of age.11. Prenatal exposure to zymosan had no effect on plasma renin activity and Ang II level in offspring rats.12. Prenatal exposure to LPS down-regulated the gene expression of AT2-R and increased mRNA expressions of IL-6 and TNF-αin rat fetus.13. Prenatal exposure to LPS increased renal TUNEL-positive cells expression, monocyte/macrophages and lymphocytes infiltration and collagen I expression in kidney of adult offspring rats.Conclusion1. Prenatal immuno-inflammatory stimulation resulted in abnormality in renal development and renal dysfunction in offspring rats, which suggests renal damage in offspring rats.2. The down-regulation of AT2-R in embryos, low expression of Ang II in kidneys of new-born offspring rats, increased apoptosis and inflammation contribute to the abnormality of renal development and renal dysfunction.3. The abnormality in renal development and renal dysfunction accompanied by the altered activity of intrarenal renin-angiotensin system are the key mechanism of hypertension induced by prenatal immuno-inflammatory stimulation.4. NF-κB activation is implicated in the hypertension and renal damage induced by prenatal immuno-inflammatory stimulation.
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