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Supplemental Table S1

Supplemental Table S1

Background Apoe-deficient (Apoe −/−) mice develop progressive atherosclerotic lesions with age but no severe renal pathology in the absence of additional challenges. We recently described accelerated atherosclerosis as well as marked renal injury in Apoe −/− mice deficient in the mesenchymal integrin chain Itga8 (Itga8 −/−). Here, we used this Apoe −/−, Itga8 −/− mouse model to investigate the sex differences in the development of atherosclerosis and concomitant renal injury. We hypothesized that aging female mice are protected from vascular and renal damage in this mouse model. Methods Apoe −/− mice were backcrossed with Itga8 −/− mice. Mice were kept on a normal diet. At the age of 12 months, the aortae and kidneys of male and female Apoe −/− Itga8 +/+ mice or Apoe −/− Itga8 −/− mice were studied. En face preparations of the aorta were stained with Sudan IV (lipid deposition) or von Kossa (calcification). In kidney tissue, immunostaining for collagen IV, CD3, F4/80, and PCNA and real-time PCR analyses for Il6, Vegfa, Col1a1 (collagen I), and Ssp1 (secreted phosphoprotein 1, synonym osteopontin) as well as ER stress markers were performed. Results When compared to male mice, Apoe −/− Itga8 +/+ female mice had a lower body weight, equal serum cholesterol levels, and lower triglyceride levels. However, female mice had increased aortic lipid deposition and more aortic calcifications than males. Male Apoe −/− mice with the additional deficiency of Itga8 developed increased serum urea, glomerulosclerosis, renal immune cell infiltration, and reduced glomerular cell proliferation. In females of the same genotype, these renal changes were less pronounced and were accompanied by lower expression of interleukin-6 and collagen I, while osteopontin expression was higher and markers of ER stress were not different. Conclusions In this model of atherosclerosis, the female sex is a risk factor to develop more severe atherosclerotic lesions, even though serum fat levels are higher in males. In contrast, female mice are protected from renal damage, which is accompanied by attenuated inflammation and matrix deposition. Thus, sex affects vascular and renal injury in a differential manner.

In humans, retinoic acid receptor responders (RARRES) have been shown to be altered in third trimester placentas complicated by the pathologies preeclampsia (PE) and PE with intrauterine growth restriction (IUGR). Currently, little is known about the role of placental Rarres in rodents. Therefore, we examined the localization and expression of Rarres1 and 2 in placentas obtained from a Wistar rat model of isocaloric maternal protein restriction (E18.5, IUGR-like features) and from an eNOS-knockout mouse model (E15 and E18.5, PE-like features). In both rodent models, Rarres1 and 2 were mainly localized in the placental spongiotrophoblast and giant cells. Their placental expression, as well as the expression of the Rarres2 receptor chemokine-like receptor 1 (CmklR1), was largely unaltered at the examined gestational ages in both animal models. Our results have shown that RARRES1 and 2 may have different expression and roles in human and rodent placentas, thereby underlining immanent limitations of comparative interspecies placentology. Further functional studies are required to elucidate the potential involvement of these proteins in early placentogenesis.

Objectives: Placental steroid metabolism is linked to the fetal hypothalamus-pituitary-adrenal axis. Intrauterine growth restriction (IUGR) might alter this cross-talk and lead to maternal stress, in turn contributing to the pathogenesis of anxiety-related disorders of the offspring, which might be mediated by fetal overexposure to, or a reduced local enzymatic protection against maternal glucocorticoids. So far, direct evidence of altered levels of circulating/local glucocorticoids is scarce. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) allows quantitative endocrine assessment of blood and tissue. Using a rat model of maternal protein restriction (low protein [LP] vs. normal protein [NP]) to induce IUGR, we analyzed fetal and maternal steroid levels via LC-MS/MS along with the local expression of 11beta-hydroxysteroid-dehydrogenase (Hsd11b).Methods: Pregnant Wistar dams were fed a low protein (8%, LP; IUGR) or an isocaloric normal protein diet (17%, NP; controls). At E18.5, the expression of Hsd11b1 and 2 was determined by RT-PCR in fetal placenta and brain. Steroid profiling of maternal and fetal whole blood, fetal brain, and placenta was performed via LC-MS/MS.Results: In animals with LP-induced reduced body (p < 0.001) and placental weights (p < 0.05) we did not observe any difference in the expressional Hsd11b1/2-ratio in brain or placenta. Moreover, LP diet did not alter corticosterone (Cort) or 11-dehydrocorticosterone (DH-Cort) levels in dams, while fetal whole blood levels of Cort were significantly lower in the LP group (p < 0.001) and concomitantly in LP brain (p = 0.003) and LP placenta (p = 0.002). Maternal and fetal progesterone levels (whole blood and tissue) were not influenced by LP diet.Conclusion: Various rat models of intrauterine stress show profound alterations in placental Hsd11b2 gatekeeper function and fetal overexposure to corticosterone. In contrast, LP diet in our model induced IUGR without altering maternal steroid levels or placental enzymatic glucocorticoid barrier function. In fact, IUGR offspring showed significantly reduced levels of circulating and local corticosterone. Thus, our LP model might not represent a genuine model of intrauterine stress. Hypothetically, the observed changes might reflect a fetal attempt to maintain anabolic conditions in the light of protein restriction to sustain regular brain development. This may contribute to fetal origins of later neurodevelopmental sequelae.

Chemerin and its receptor, chemokine-like receptor 1 (CmklR1), are associated with chemotaxis, inflammation, and endothelial function, especially in metabolic syndrome, coronary heart disease, and hypertension. In humans, circulating chemerin levels and renal function show an inverse relation. So far, little is known about the potential role of chemerin in hypertensive nephropathy and renal inflammation. Therefore, we determined systemic and renal chemerin levels in 2-kidney-1-clip (2k1c) hypertensive and Thy1.1 nephritic rats, respectively, to explore the correlation between chemerin and markers of renal inflammation and fibrosis. Immunohistochemistry revealed a model-specific induction of chemerin expression at the corresponding site of renal damage (tubular vs. glomerular). In both models, renal expression of chemerin (RT-PCR, Western blot) was increased and correlated positively with markers of inflammation and fibrosis. In contrast, circulating chemerin levels remained unchanged. Taken together, these findings demonstrate that renal chemerin expression is associated with processes of inflammation and fibrosis-related to renal damage. However, its use as circulating biomarker of renal inflammation seems to be limited in our rat models.

Neonatal nephron loss may follow hypoxic-ischemic events or nephrotoxic medications. Its long-term effects on the kidney are still unclear. Unlike term infants, preterm neonates less than 36 weeks gestational age show ongoing nephrogenesis. We hypothesized that nephron loss during nephrogenesis leads to more severe renal sequelae than nephron loss shortly after the completion of nephrogenesis. Rats show nephrogenesis until day 10 of life resembling the situation of preterm infants. Animals were uninephrectomized at day 1 (UNX d1) resulting in nephron reduction during nephrogenesis and at day 14 of life (UNX d14) inducing nephron loss after the completion of nephrogenesis. 28 days after uninephrectomy the compensatory renal growth was higher in UNX d1 compared to UNX d14. Nephrin was reduced and collagen deposition increased in UNX d1. At 1 year of age, glomerulosclerosis and markers of tubulointerstitial damage were most prevalent in UNX d1. Moreover, the number of desmin-positive podocytes was higher and nephrin was reduced in UNX d1 indicating podocyte damage. Infiltration of inflammatory cells was heightened after UNX d1. Uninephrectomized animals showed no arterial hypertension. We conclude that neonatal nephron loss during active nephrogenesis leads to more severe glomerular and tubulointerstitial damage, which is not a consequence of compensatory arterial hypertension.

Osteopontin in the kidney. Exemplary photomicrographs of osteopontin-stained renal sections of male and female Apoe −/− Itga8 +/+ or Apoe −/− Itga8 −/− mice. (PDF 548 kb)

Anatomical and metabolic parameters of mice without a deletion in Apoe (Apoe +/+ Itga8 +/+ or Apoe ++ Itga8 â /â ). (DOCX 22 kb)

Atherosclerotic plaque formation in mice without a deficiency of Apoe. Exemplary en face preparations of aortae of male and female Apoe +/+ Itga8 +/+ or Apoe +/+ Itga8 −/− mice, stained with Sudan IV (left) or unstained (right). One en face preparation of the aorta of an Apoe-deficient (Apoe −/− Itga8 +/+) female mouse was stained for Sudan IV as a positive control. (PDF 257 kb)

Primer pairs used in the study. (DOCX 24 kb)

Results from two-way ANOVA of markers of vascular and renal injury. (DOCX 19 kb)

Osteopontin in the brachiocephalic trunk. Exemplary osteopontin-stained cross sections of the brachiocephalic trunk of male and female mice with a deficiency of Apoe. (PDF 303 kb)

Vascular and renal changes in 7- and 9-month-old mice. (DOCX 25 kb)

The α8 integrin (Itga8) chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration.

Background The placental syncytiotrophoblast is the major source of maternal plasma corticotropin-releasing hormone (CRH) in the second half of pregnancy. Placental CRH exerts multiple functions in the maternal organism: It induces the adrenal secretion of cortisol via the stimulation of adrenocorticotropic hormone, regulates the timing of birth via its actions in the myometrium and inhibits the invasion of extravillous trophoblast cells in vitro. However, the auto- and paracrine actions of CRH on the syncytiotrophoblast itself are unknown. Intrauterine growth restriction (IUGR) is accompanied by an increase in placental CRH, which could be of pathophysiological relevance for the dysregulation in syncytialisation seen in IUGR placentas. Methods We aimed to determine the effect of CRH on isolated primary trophoblastic cells in vitro. After CRH stimulation the trophoblast syncytialisation rate was monitored via syncytin-1 gene expression and beta-hCG (beta-human chorionic gonadotropine) ELISA in culture supernatant. The expression of the IUGR marker genes leptin and 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) was measured continuously over a period of 72 h. We hypothesized that CRH might attenuate syncytialisation, induce leptin, and reduce 11beta-HSD2 expression in primary villous trophoblasts, which are known features of IUGR. Results CRH did not influence the differentiation of isolated trophoblasts into functional syncytium as determined by beta-hCG secretion, albeit inducing syncytin-1 expression. Following syncytialisation, CRH treatment significantly increased leptin and 11beta-HSD2 expression, as well as leptin secretion into culture supernatant after 48 h. Conclusion The relevance of CRH for placental physiology is underlined by the present in vitro study. The induction of leptin and 11beta-HSD2 in the syncytiotrophoblast by CRH might promote fetal nutrient supply and placental corticosteroid metabolism in the phase before labour induction.