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Background: Cardiovascular diseases were with 39% the leading cause of death in Germany in 2015 and a worldwide severe health problem. Atherosclerosis has an enormous significant value in that kind of pathologies. In the inflammatory etiology of this chronic vascular disease, lipid metabolism and macrophages play a key role. The ApoE-/- mouse model is a well-established model of atherosclerosis. PACAP (Pituitary Adenylate Cyclase-Activating Peptide) is part of the glucagon-PACAP-VIP protein-superfamily. It acts antinflammatory, antiapoptotic and antioxidative has nephroprotective effects. In this study the effect of the deficiency of PACAP and its receptor PAC1 is investigated on inflammatory and morphological changes of the murine kidney of ApoE-/- mice. Additionally, the impact of cholesterol-enriched diet (CED) on these parameters was examined.
Methods and Materials: The following four genotypes of mice were reviewed: WT, ApoE-/-, PACAP-/-/ApoE-/- and PAC1-/-/ApoE-/-. The ten-week-old knockout-mice were fed for 20 weeks either a standard diet (SD) or a cholesterol-enriched diet (CED). Of WT mice we reviewed only a CED-fed group, so there are seven groups of mice in total. Body weight, plasma-cholesterol and -triglycerides were measured. After euthanizing, organs were collected and shockfrozen in liquid nitrogen-cooled isopentane. Kidney cryo-sections (6μm) were analyzed by using Azan-, ORO-staining and the antibodies MOMA-2 (monocytes and macrophages), F4/80 (resident macrophages), IL-6 and TNFα (proinflammatory cytokines). Based on Azan-staining the number, area and diameter of the glomeruli were measured as well as the relative percentage of connective tissue in the kidney sections to analyze possibly existing fibrosis.
Results: Application of the CED resulted in significant heavier PACAP-/-/ApoE-/- mice compared to SD. PACAP-/-/ApoE-/- mice fed with CED were heavier than ApoE-/- mice (control). After SD, PACAP-/-/ApoE-/- mice were not so heavy as ApoE-/- mice. Relating to the bodyweight, there was an interaction between genotype and nutrition.
After application of the CED, plasma cholesterol levels were higher in ApoE-/-, PACAP-/-/ApoE-/- and PAC1-/-/ApoE-/- compared to SD and plasma triglyceride levels of PACAP-/-/ApoE-/- mice were significantly higher than in ApoE-/- mice.
After quantification of morphology in PACAP-/-/ApoE-/- mice after CED the number of glomeruli was increased in comparison with ApoE-/- mice. After application of CED, in kidneys of PAC1-/-/ApoE-/- and WT mice the glomerular area was smaller about 22% and 21% in comparison to ApoE-/- mice. After both, SD or CED, in kidneys of PAC1-/-/ApoE-/- mice the glomeruli were smaller related to diameter than in ApoE-/- mice. Regarding the relative percentage of connective tissue and hence a possible renal fibrosis/sclerosis in the kidneys, we could not show any difference between the types of nutrition or genotypes. After application of CED, ApoE-/- mice showed a twofold higher density of MOMA-2+ macrophages in the renal cortex than WT. PAC1-/-/ApoE-/- mice showed four times less density of F4/80+ macrophages in renal cortex than ApoE-/- mice (control). In ApoE-/- mice, application of CED resulted in a 2,5-fold higher density of F4/80+ macrophages than SD. The type of nutrition influenced the density of IL-6+ cells in renal cortex: After CED, PACAP-/-/ApoE-/- mice showed a higher density of IL-6+ cells in glomeruli and tubuli than after SD. After CED, in tubuli of ApoE-/- mice density of IL-6+ cells was higher than after SD.
After CED, in tubuli of ApoE-/- mice density of TNFα+ cells was less than after SD. After application of SD, in the tubular cells of PACAP-/-/ApoE-/- mice the density of TNFα+ cells was less than in ApoE-/- mice.
Conclusion: Summed up, PAC1-/-/ApoE-/- mice fed a CED show smaller glomeruli and a reduced density of resident macrophages (F4/80) than ApoE-/- mice (control) in renal cortex. After CED, PACAP-/-/ApoE-/- mice showed significant higher density of IL-6+ cells in glomerular and tubular cells than after SD.