Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2␣. In contrast, HIF-1␣ served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1␣ resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2␣-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2␣-dependent erythrocytosis, whereas HIF-1␣ protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia. (Blood.

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT 1-R͞cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT 2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD 4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT 2-R-and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at Ϸ60 min, were unaffected by a cysLT 1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD 4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT 2-R activation results in a proinflammatory EC phenotype. Because LTD 4 and thrombin are likely to be formed concomitantly in vivo, cysLT 2-R and PAR-1 may cooperate to augment vascular injury.cysteinyl leukotriene 2 receptor gene signature ͉ protease-activated receptor 1 gene signature ͉ vascular inflammation L eukotrienes (LTs), i.e., LTB 4 and the cysteinyl LTs (cysLT) LTC 4 , LTD 4 , and LTE 4 constitute a group of lipid mediators derived from the 5-lipoxygenase (5-LO) pathway (1, 2). LTs are either produced by leukocytes at sites of inflammation or formed through transcellular metabolism after uptake and metabolism of leukocyte-derived LTA 4 by downstream enzymes of the 5-LO pathway (LTA 4 hydrolase and LTC 4 synthase) in cells that normally do not express 5-LO, such as endothelial cells (ECs) (3, 4). LTs act through G protein-coupled surface receptors (GPCRs), i.e., the LTB 4 receptors and the cysLT receptors (LT-Rs) (cysLT 1 -R and cysLT 2 -R) (5-10). LT-Rs are expressed on multiple target cells, including leukocytes, smooth muscle cells, and ECs (1). Recent studies implicate the 5-LO pathway in cardiovascular disease (11)(12)(13)(14)(15)(16)(17).Considerable information is available on cysLT 1 -R, whereas little is known about cysLT 2 -R. We have used human umibilical vein (HUV)ECs as a model of vascular cells to study cysLT 2 -R activation by demonstrating that cysLTs exclusively signal through cysLT 2 -R in this cell type (18): In fact, HUVECs are the first primary cell type that s...

Intracellular cAMP, the production of which is catalyzed by the -subunit of the stimulatory G protein (Gs), controls renin synthesis and release by juxtaglomerular (JG) cells of the kidney, but may also have relevance for the physiologic integrity of the kidney. To investigate this possibility, we generated mice with inducible knockout of Gs in JG cells and monitored them for 6 months after induction at 6 weeks of age. The knockout mapped exclusively to the JG cells of the Gs-deficient animals. Progressive albuminuria occurred in Gs-deficient mice. Compared with controls expressing wild-type Gs alleles, the Gs-deficient mice had enlarged glomeruli with mesangial expansion, injury, and FSGS at study end. Ultrastructurally, the glomerular filtration barrier of the Gs-deficient animals featured endothelial gaps, thickened basement membrane, and fibrin-like intraluminal deposits, which are classic signs of thrombotic microangiopathy. Additionally, we found endothelial damage in peritubular capillaries and vasa recta. Because deficiency of vascular endothelial growth factor (VEGF) results in thrombotic microangiopathy, we addressed the possibility that Gs knockout may result in impaired VEGF production. We detected VEGF expression in JG cells of control mice, and cAMP agonists regulated VEGF expression in cultured renin-producing cells. Our data demonstrate that Gs deficiency in JG cells of adult mice results in kidney injury, and suggest that JG cells are critically involved in the maintenance and protection of the renal microvascular endothelium.

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