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Introduction: We previously reported that malignant hypertension is associated with impaired capillary density of target organs. Here, we tested the hypothesis that stabilization of hypoxia-inducible factor (HIF) in a modified “preconditioning” approach prevents the development of malignant hypertension. To stabilize HIF, we employed pharmacological inhibition of HIF prolyl hydroxylases (PHD), that profoundly affect HIF metabolism. Methods: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in rats; controls were sham operated. 2K1C rats received either intermittent injections of the PHD inhibitor ICA (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate) or placebo. Thirty-five days after clipping, the frequency of malignant hypertension was assessed (based on weight loss and the occurrence of characteristic vascular lesions). In addition, kidney injury was compared between all ICA treated versus all placebo treated 2K1C, regardless of the occurrence of malignant hypertension. HIF stabilization was evaluated by immunohistochemistry, and HIF target gene expression by RT-PCR. Results: Blood pressure was elevated to the same degree in ICA- and placebo-treated 2K1C compared to control rats. ICA treatment did not affect the frequency of malignant hypertension or the extent of kidney tissue fibrosis, inflammation, or capillary density. There was a trend towards higher mortality and worse kidney function in ICA-treated 2K1C rats. ICA increased the number of HIF-1α-positive renal tubular cell nuclei and induced several HIF-1 target genes. In contrast, expression of HIF-2α protein as well as HIF-2 target genes were markedly enhanced by 2K1C hypertension, irrespective of ICA treatment. Discussion: We conclude that intermittent PHD inhibition did not ameliorate severe renovascular hypertension in rats. We speculate that the unexpected strong renal accumulation of HIF-2α in renovascular hypertension, which could not be further augmented by ICA, may contribute to the lack of a benefit from PHD inhibition.

Funder: Friedrich-Alexander-Universität Erlangen-Nürnberg (1041), BACKGROUND: Chronic kidney disease (CKD) is a global burden affecting both children and adults. Novel imaging modalities hold great promise to visualize and quantify structural, functional, and molecular organ damage. The aim of the study was to visualize and quantify murine renal vasculature using label-free raster scanning optoacoustic mesoscopy (RSOM) in explanted organs from mice with renal injury. MATERIAL AND METHODS: For the experiments, freshly bisected kidneys of alpha 8 integrin knock-out (KO) and wildtype mice (WT) were used. A total of n=7 female (n=4 KO, n=3 WT) and n=6 male animals (n=2 KO, n=4 WT) aged 6 weeks were examined with RSOM optoacoustic imaging systems (RSOM Explorer P50 at SWL 532nm and/or ms-P50 imaging system at 532 nm, 555 nm, 579 nm, and 606 nm). Images were reconstructed using a dedicated software, analyzed for size and vascular area and compared to standard histologic sections. RESULTS: RSOM enabled mapping of murine kidney size and vascular area, revealing differences between kidney sizes of male (m) and female (f) mice (merged frequencies (MF) f vs. m: 52.42±6.24 mm2 vs. 69.18±15.96 mm2, p=0.0156) and absolute vascular area (MF f vs. m: 35.67±4.22 mm2 vs. 49.07±13.48 mm2, p=0.0036). Without respect to sex, the absolute kidney area was found to be smaller in knock-out (KO) than in wildtype (WT) mice (WT vs. KO: MF: p=0.0255) and showed a similar trend for the relative vessel area (WT vs. KO: MF p=0.0031). Also the absolute vessel areas of KO compared to WT were found significantly different (MF p=0.0089). A significant decrease in absolute vessel area was found in KO compared to WT male mice (MF WT vs. KO: 54.37±9.35 mm2 vs. 34.93±13.82 mm2, p=0.0232). In addition, multispectral RSOM allowed visualization of oxygenated and deoxygenated parenchymal regions by spectral unmixing. CONCLUSION: This study demonstrates the capability of RSOM for label-free visualization of differences in vascular morphology in ex vivo murine renal tissue at high resolution. Due to its scalability optoacoustic imaging provides an emerging modality with potential for further preclinical and clinical imaging applications.

Additional file 1: Supplementary Table 1. Further information on RSOM Explorer P50 (RSOM P50). Adapted from user manual (RSOM Explorer P50, iThera Medical GmbH, München). Supplementary Table 2. Further information on RSOM Explorer ms-P50 (RSOM ms-P50). Adapted from user manual (RSOM Explorer P50, iThera Medical GmbH, München). Supplementary Table 3. Sex effects on kidney vascularization, independent of genotype. Supplement Table 4. Comparison of the different genotypes, independent of sex. Supplementary Table 5. Genotype effect on kidney vascularization for female sex. Supplementary Table 6. Genotype effect on kidney vascularization for male sex. Supplementary Figure 1. Kidney side and sex distinction, independent of genotype. Supplementary Figure 2. Sex effects on kidney vascularization in WT kidneys. Supplementary Figure 3. Genotype distinction, independent of sex. Supplementary Figure 4. Genotype effect on vascularization for different sexes. Supplementary Figure 5. msRSOM.

Glomerulonephritis results in a dysregulation of glomerular cells and may end up in chronic alterations and subsequent loss of renal function. Therefore, understanding mechanisms, which contribute to maintain glomerular integrity, is a pivotal prerequisite for therapeutic interventions. The alpha-8 integrin chain seems to be an important player to maintain glomerular homeostasis by conferring mechanical stability and functional support for the renal capillary tuft.

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.

Lung development involves epithelial–mesenchymal interactions and integrins represent one of the key elements. These extracellular matrix receptors form hetero-dimers of alpha and beta subunits. The integrin α8β1 is highly expressed in mouse tissues, including lung. It forms a cellular receptor for fibronectin, vitronectin, osteopontin, nephronectin, and tenascin-C. This study aims to investigate the role of the integrin α8-subunit (α8) during lung development. Wild type and α8-deficient lungs were explanted at embryonic days 11.5/12.5. After 24–73 h in culture α8-deficient lung explants displayed reduced growth, reduced branching, enlarged endbuds, altered branching patterns, and faster spontaneous contractions of the airways as compared to wild type. Postnatally, a stereological investigation revealed that lung volume, alveolar surface area, and the length of the free septal edge were significantly reduced in α8-deficient lungs at postnatal days P4 and P7. An increased formation of new septa in α8-deficient lungs rescued the phenotype. At day P90 α8-deficient lungs were comparable to wild type. We conclude that α8β1 takes not only part in the control of branching, but also possesses a morphogenic effect on the pattern and size of the future airways. Furthermore, we conclude that the phenotype observed at day P4 is caused by reduced branching and is rescued by a pronounced formation of the new septa throughout alveolarization. More studies are needed to understand the mechanism responsible for the formation of new septa in the absence of α8β1 in order to be of potential therapeutic benefit for patients suffering from structural lung diseases.

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)

Suppl_Material_Table_1 for Expression and Regulation of Retinoic Acid Receptor Responders in the Human Placenta by Hanna Huebner, Andrea Hartner, Wolfgang Rascher, Reiner R. Strick, Sven Kehl, Felix Heindl, David L. Wachter, Matthias W. Beckmann, MD, Fabian B. Fahlbusch, and Matthias Ruebner in Reproductive Sciences

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)

Suppl_Material_Table_2 for Expression and Regulation of Retinoic Acid Receptor Responders in the Human Placenta by Hanna Huebner, Andrea Hartner, Wolfgang Rascher, Reiner R. Strick, Sven Kehl, Felix Heindl, David L. Wachter, Matthias W. Beckmann, MD, Fabian B. Fahlbusch, and Matthias Ruebner in Reproductive Sciences

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

Malignant hypertension develops in some cases of hypertension but not in others. We hypothesized that an impaired neovascularization and a reduced capillary supply characterizes the malignant course of experimental hypertension. Two-kidney, one-clip renovascular hypertension was induced in rats; controls (sham) were sham operated. To distinguish malignant hypertension from non-malignant hypertension, we considered two factors: weight loss, and the number of typical vascular lesions (onion skin lesions and fibrinoid necroses) per kidney section of the nonclipped kidney. Animals in the upper half for both criteria were defined as malignant hypertensives. After 5 weeks, mean arterial blood pressure was elevated to the same degree in malignant hypertension and non-malignant hypertension whereas plasma renin and aldosterone were significantly higher in malignant hypertensives. The expression of plasminogen activator inhibitor-1 was elevated (up to 14-fold) in non-malignant but significantly more increased (up to 36-fold) in malignant hypertensive rats, compared to sham. As a bioassay for neovascularization, the area of granulation tissue ingrowth in polyvinyl discs (implanted subcutaneously) was reduced in malignant hypertension compared to non-malignant hypertension and sham, while there was no difference between non-malignant hypertension and sham. The number of renal and left ventricular capillaries was significantly lower in malignant hypertension compared to non-malignant hypertension, as was the number of proliferating endothelial cells. We conclude that an impaired neovascularization and capillarization occurs in malignant renovascular hypertension but not in the non-malignant course of the disease despite comparable blood pressure levels. This might contribute to the unique vascular lesions and progressive target organ damage observed in malignant hypertension.

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/Aims: _ 8 _ 1 -Integrin is expressed in mesangial cells. In vitro studies suggest a role for _ 8 -integrin in the regulation of cell proliferation and apoptosis. We tested the hypothesis that _ 8 -integrin is essential for the healing process after mesangioproliferative glomerulonephritis. Methods: Mice homozygous for a deletion of the _ 8 -integrin chain were compared with wild-type mice. To study glomerular healing, we used the habu toxin model of reversible mesangioproliferative glomerulonephritis. Animals received 6 mg/ kg habu toxin intravenously; controls received saline only. Results: Early mesangiolysis occurred in wild-type and _ 8 - integrin-deficient mice. However, mesangiolysis was no longer detectable after 7 days in wild types but persisted after 14 days in _ 8 -integrin-deficient animals. Mesangial activation marker _ -smooth muscle actin was detectable only at day 7 in wild-type mice but persisted until day 14 in _ 8 -integrin- deficient mice. In wild types, glomerular cell proliferation and apoptosis peaked at day 7 and decreased thereafter but remained elevated in _ 8 -integrin-deficient mice until day 28. In cultivated mesangial cells, _ 8 -integrin expression was associated with increased cell survival. Conclusion: Interactions between _8 -integrin and the mesangial matrix may contribute to healing of glomerular injury by influencing cell proliferation and apoptosis.

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.

Liddle's syndrome is an autosomal dominant form of human hypertension, caused by gain-of-function mutations of the epithelial sodium channel (ENaC) which is expressed in aldosterone target tissues including the distal colon. We used a mouse model for Liddle's syndrome to investigate ENaC-mediated Na+ transport in late distal colon by measuring the amiloride-sensitive transepithelial short circuit current (Delta I SC-Ami) ex vivo. In Liddle mice maintained on a standard salt diet, Delta I SC-Ami was only slightly increased but plasma aldosterone (P Aldo) was severely suppressed. Liddle mice responded to a low or a high salt diet by increasing or decreasing, respectively, their P Aldo and Delta I SC-Ami. However, less aldosterone was required in Liddle animals to achieve similar or even higher Na+ transport rates than wild-type animals. Indeed, the ability of aldosterone to stimulate Delta I SC-Ami was about threefold higher in Liddle animals than in the wild-type controls. Application of aldosterone to colon tissue in vitro confirmed that ENaC stimulation by aldosterone was not only preserved but enhanced in Liddle mice. Aldosterone-induced transcriptional up-regulation of the channel's beta- and gamma-subunit (beta ENaC and gamma ENaC) and of the serum- and glucocorticoid-inducible kinase 1 (SGK1) was similar in colon tissue from Liddle and wild-type animals, while aldosterone had no transcriptional effect on the alpha-subunit (alpha ENaC). Moreover, Na+ feedback regulation was largely preserved in colon tissue of Liddle animals. In conclusion, we have demonstrated that in the colon of Liddle mice, ENaC-mediated Na+ transport is enhanced with an increased responsiveness to aldosterone. This may be pathophysiologically relevant in patients with Liddle's syndrome, in particular on a high salt diet, when suppression of P Aldo is likely to be insufficient to reduce Na+ absorption to an appropriate level.