Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Multiple Modifications Allow High-Titer Production of Retroviral Vectors Carrying Heterologous Regulatory Elements

Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3′ long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5′ and 3′ of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors

Pharmacological options to protect the aged heart from ischemia and reperfusion injury by targeting the PKA-BK(Ca) signaling pathway

The beneficial effects of many cardioprotective strategies including ischemic or pharmacological conditioning are reduced in the aged heart. The underlying reason(s) for the age-dependent loss of cardioprotection is unclear. Recently, we demonstrated that protein kinase A (PKA) dependent cardioprotection is lost in the aged heart. However, activation of large-conductance Ca(2+)-sensitive K(+) (BK(Ca)) channels, a putative PKA downstream target, initiated cardioprotection also in the aged heart. Therefore, we aimed to investigate whether 1) BK(Ca) channels are critically involved in PKA activation induced cardioprotection and 2) the age-dependent loss of cardioprotection is caused by differences in PKA regulation. Using an in vivo rat model with regional myocardial ischemia, we treated young (2-4 months) and aged (22-24 months) Wistar rats with PKA activator forskolin, BK(Ca) channel activator NS1619 and/or BK(Ca) channel blocker iberiotoxin. Forskolin induced infarct size reduction was 1) age-dependent and 2) prevented by iberiotoxin. The effect of forskolin on myocardial PKA activity was comparable in young and aged animals. In addition, NS1619 initiated cardioprotection also in the aged heart both when administered before ischemia and during early reperfusion phase. Activation of BK(Ca) channels is critically involved in forskolin induced cardioprotection. The age-dependency of forskolin induced cardioprotection is not caused by age-dependent differences in PKA activation. Pharmacological targeting of BK(Ca) channels before or after myocardial ischemia is a promising therapeutic strategy to protect the aged heart from ischemia and reperfusion injur

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer\u27s disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ(1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 x Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB

miR-181a Modulation of ERK-MAPK Signaling Sustains DC-SIGN Expression and Limits Activation of Monocyte-Derived Dendritic Cells.

DC-SIGN+ monocyte-derived dendritic cells (mo-DCs) play important roles in bacterial infections and inflammatory diseases, but the factors regulating their differentiation and proinflammatory status remain poorly defined. Here, we identify a microRNA, miR-181a, and a molecular mechanism that simultaneously regulate the acquisition of DC-SIGN expression and the activation state of DC-SIGN+mo-DCs. Specifically, we show that miR-181a promotes DC-SIGN expression during terminal mo-DC differentiation and limits its sensitivity and responsiveness to TLR triggering and CD40 ligation. Mechanistically, miR-181a sustains ERK-MAPK signaling in mo-DCs, thereby enabling the maintenance of high levels of DC-SIGN and a high activation threshold. Low miR-181a levels during mo-DC differentiation, induced by inflammatory signals, do not support the high phospho-ERK signal transduction required for DC-SIGNhi mo-DCs and lead to development of proinflammatory DC-SIGNlo/-mo-DCs. Collectively, our study demonstrates that high DC-SIGN expression levels and a high activation threshold in mo-DCs are linked and simultaneously maintained by miR-181a

Astaxanthin exerts protective effects similar to bexarotene in Alzheimer's disease by modulating amyloid-beta and cholesterol homeostasis in blood-brain barrier endothelial cells

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-β peptides (Aβ) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aβ production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aβ oligomers and ~80 kDa intracellular 6E10-reactive APP/Aβ species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aβ clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aβ species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aβ oligomers in mBCEC and Aβ species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aβ from cerebrovascular endothelial cells.This work was supported by the Austrian Science Fund, grants P24783-B19 (to U.P.), and W1226-B18 (to E.F.D., J.T., F.M., and U.P.; Doctoral College of Metabolic and Cardiovascular Disease, DK-MCD, co-funded by the Medical University of Graz). F.M. is also grateful to the Austrian Science Fund for grants P23490-B20, P29262, P24381, P29203, P27893, I1000, and “SFB Lipotox” (F3012), as well as the Bundesministerium für Wissenschaft, Forschung und Wirtschaft, and the Karl-Franzens University of Graz for grant “Unkonventionelle Forschung”. We acknowledge support from NAWI Graz and the BioTechMed-Graz flagship project “EPIAge”. Additional support was provided by Fundació La Marató de TV3 grant 2014-0930 (to A.C.) and an FPU fellowship from Ministerio de Economía y Competitividad (MEC) (to C.dD) and the Austrian National Bank (OeNB, 17600 to E.M.)