Pinelopi P. Kapitsinou

Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia

The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2α in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis.

The VHL tumor suppressor and HIF: insights from genetic studies in mice

The von Hippel–Lindau tumor suppressor gene product, pVHL, functions as the substrate recognition component of an E3-ubiquitin ligase, which targets the oxygen-sensitive α-subunit of hypoxia-inducible factor (HIF) for rapid proteasomal degradation under normoxic conditions and as such plays a central role in molecular oxygen sensing. Mutations in pVHL can be found in familial and sporadic clear cell carcinomas of the kidney, hemangioblastomas of the retina and central nervous system, and pheochromocytomas, underscoring its gatekeeper function in the pathogenesis of these tumors. Tissue-specific gene targeting of VHL in mice has demonstrated that efficient execution of pVHL-mediated HIF proteolysis under normoxia is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types, and has provided novel insights into the biological function of individual HIF transcription factors. In this review, we discuss the role of HIF in the development of the VHL phenotype.

Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Myeloid Cell-Derived Hypoxia-Inducible Factor Attenuates Inflammation in Unilateral Ureteral Obstruction-Induced Kidney Injury

Renal fibrosis and inflammation are associated with hypoxia, and tissue pO2 plays a central role in modulating the progression of chronic kidney disease. Key mediators of cellular adaptation to hypoxia are hypoxia-inducible factor (HIF)-1 and -2. In the kidney, they are expressed in a cell type-specific manner; to what degree activation of each homolog modulates renal fibrogenesis and inflammation has not been established. To address this issue, we used Cre-loxP recombination to activate or to delete both Hif-1 and Hif-2 either globally or cell type specifically in myeloid cells. Global activation of Hif suppressed inflammation and fibrogenesis in mice subjected to unilateral ureteral obstruction, whereas activation of Hif in myeloid cells suppressed inflammation only. Suppression of inflammatory cell infiltration was associated with downregulation of CC chemokine receptors in renal macrophages. Conversely, global deletion or myeloid-specific inactivation of Hif promoted inflammation. Furthermore, prolonged hypoxia suppressed the expression of multiple inflammatory molecules in noninjured kidneys. Collectively, we provide experimental evidence that hypoxia and/or myeloid cell-specific HIF activation attenuates renal inflammation associated with chronic kidney injury.

Preischemic targeting of HIF prolyl hydroxylation inhibits fibrosis associated with acute kidney injury

Acute kidney injury (AKI) due to ischemia is an important contributor to the progression of chronic kidney disease (CKD). Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia-inducible factors (HIF), which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. While activation of HIF protects from ischemic cell death, HIF has been shown to promote fibrosis in experimental models of CKD. The impact of HIF activation on AKI-induced fibrosis has not been defined. Here, we investigated the role of pharmacologic HIF activation in AKI-associated fibrosis and inflammation. We found that pharmacologic inhibition of HIF prolyl hydroxylation before AKI ameliorated fibrosis and prevented anemia, while inhibition of HIF prolyl hydroxylation in the early recovery phase of AKI did not affect short- or long-term clinical outcome. Therefore, preischemic targeting of the PHD/HIF pathway represents an effective therapeutic strategy for the prevention of CKD resulting from AKI, and it warrants further investigation in clinical trials.

The Endothelial Prolyl-4-Hydroxylase Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in Mice

ABSTRACT Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (∼118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2α but not HIF-1α. Furthermore, endothelial HIF-2α was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension.

Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

Molecular mechanisms of ischemic preconditioning in the kidney

More effective therapeutic strategies for the prevention and treatment of acute kidney injury (AKI) are needed to improve the high morbidity and mortality associated with this frequently encountered clinical condition. Ischemic and/or hypoxic preconditioning attenuates susceptibility to ischemic injury, which results from both oxygen and nutrient deprivation and accounts for most cases of AKI. While multiple signaling pathways have been implicated in renoprotection, this review will focus on oxygen-regulated cellular and molecular responses that enhance the kidneys tolerance to ischemia and promote renal repair. Central mediators of cellular adaptation to hypoxia are hypoxia-inducible factors (HIFs). HIFs play a crucial role in ischemic/hypoxic preconditioning through the reprogramming of cellular energy metabolism, and by coordinating adenosine and nitric oxide signaling with antiapoptotic, oxidative stress, and immune responses. The therapeutic potential of HIF activation for the treatment and prevention of ischemic injuries will be critically examined in this review.

HO-1 in Control of a Self-Eating Kidney

Autophagy, from Greek meaning self-eating, refers to the degradation of macromolecules and organelles by the lysosomal machinery of cells and is a tightly regulated and normally occurring process that helps maintain a balance among synthesis, degradation, and recycling of cellular components. Recent studies have uncovered fascinating links to human physiology and disease, such as aging and neurodegeneration, cancer, infection, and renal diseases.1-6 Three types of autophagy, micro, macro, and chaperone-mediated, have been described.1,2,7 The morphologic hallmark of macroautophagy (hereinafter referred to as autophagy) is the de novo formation of a double or multimembrane-bound structure known as the autophagosome. In mammalian cells, the autophagosome fuses with the lysosome, resulting in the degradation of cellular components and the recycling of nucleotides, amino acids, and free fatty acids that now can be reused for multiple biosynthetic processes. Whereas the mammalian target of rapamycin complex 1 coordinates autophagy with nutrient-sensing signaling pathways, the AuTophaGy (Atg)-related genes encode evolutionarily conserved proteins that are critically important for autophagosome generation.1,7 For instance, beclin-1, the mammalian homolog of yeast Atg6, contributes to the formation of autophagosomes,8 whereas the microtubule-associated protein 1 light chain 3 (LC3; mammalian homolog of yeast Atg8) is essential for autophagosome elongation and expansion in its membrane-bound form LC3-II.8 Autophagy is induced under various conditions of cellular stress, including nutrient deprivation, hypoxia, and oxidative stress, but also occurs during normal development and cell growth and is vital for the maintenance of cellular homeostasis in postmitotic cells, such as neurons, myocytes, and podocytes.2,3 The role of autophagy in apoptosis or programmed cell death after severe stress conditions is unclear and subject to intense investigation.1 Recent studies demonstrated a protective role for autophagy in experimental models of kidney disease.3-5 Inhibition of autophagy by pharmacologic or genetic means exacerbates injury, partially by promoting apoptosis in both ischemia-reperfusion and cisplatin-induced renal injury.4,5 Furthermore, autophagy is essential for podocyte integrity after glomerular injury.3 However, the regulation of autophagy in renal cells is poorly understood. In this issue of JASN, Bolisetty et al.9 identify heme oxygenase 1 (HO-1) as an important modulator of autophagosome formation in proximal renal tubular epithelial cells and propose a novel mechanism by which HO-1 protects kidneys from cisplatin (stress)-induced injury. HO-1 is expressed ubiquitously, induced by oxidative stress, and catalyzes the rate-limiting step in heme degradation, which produces biliverdin, carbon monoxide (CO), and iron. Clearance of excess free heme by HO-1 is critical in preventing heme-induced membrane oxidation and production of reactive oxygen species (ROS). Furthermore, the products of this reaction—CO and biliverdin and its derivative, bilirubin—have antioxidant, antiapoptotic, and anti-inflammatory properties, resulting in cytoprotection.10 Thus, HO-1 protects from ischemia-reperfusion injury in multiple tissues, including lung, heart, and kidney.10 HO-1 also protects from cisplatin-induced acute renal failure by attenuating renal tubular cell apoptosis and necrosis by decreasing intracellular ROS, which are thought to be key mediators of nephrotoxicity in this setting.9,11 Bolisetty et al.9 find increased numbers of autophagic vesicles in proximal nephron segments of HO-1–deficient mice under baseline conditions and increased expression of autophagosome markers LC3-II and beclin-1 in HO-1–deficient primary proximal renal epithelial cells, suggesting that loss of renal epithelial HO-1 activity increases autophagy.9 Although it is likely that ablation of HO-1 associates with increased macromolecular and organelle degradation, future studies are warranted to investigate the possibility of impaired autophagosome/lysosome fusion or altered lysosomal activity leading to the accumulation of autophagic vacuoles. On the basis of the expression of autophagosome markers Atg5, beclin-1, and LC3-II, Bolisetty et al.9 then show that treatment with cisplatin, which normally induces autophagy and apoptosis in renal epithelium, does not lead to additional autophagosome formation in HO-1–deficient cells, whereas apoptosis is enhanced compared with wild-type. This finding emphasizes a functional link between autophagy and apoptosis in renal cells and highlights an important association between lack of inducibility of autophagosome and increased susceptibility to cellular injury and apoptosis. It is also consistent with recent studies in mouse embryonic fibroblasts, which demonstrated functional cross-talk between autophagy and apoptosis: Deletion of BCL-2 family members Bax and Bak result in massive autophagy and beclin-1 and Atg5-dependent cell death after exposure to etoposide.12 Because the authors show that autophagy is induced before the onset of apoptosis in cisplatin-treated tubular epithelial cells, it is plausible that the extent of autophagy induction generates a signal, which then determines whether cells undergo apoptosis. This hypothesis is supported by in vitro and in vivo studies in which inhibition of autophagy led to apoptosis as a result of failed stress adaptation.13 A major question that remains unanswered in the study by Bolisetty et al.9 concerns the degree to which regulation of HO-1–dependent autophagy contributes to HO-1–mediated cytoprotection. Pharmacologic or genetic manipulation of autophagic activity in HO-1–competent and – defective backgrounds would be required to dissect antioxidant from autophagy regulatory effects. Although the effects of HO-1 activity on autophagy may be cell-type specific, as overexpression of HO-1 promotes mitochondrial autophagy in astroglia,14 the observation of increased renal autophagy in HO-1–deficient mice provokes fundamental questions regarding underlying molecular mechanisms. One possible explanation for this finding is the existence of a pro-oxidant intracellular milieu that has been observed in HO-1–deficient mice.15 Indeed, Bolisetty et al. provide evidence that increased ROS production associates with increased autophagosome formation in HO-1–deficient renal epithelial cells: HO-1 overexpression reduced ROS levels and delayed autophagy after cisplatin treatment. This is consistent with previous reports that implicated ROS in the regulation of autophagy16; however, the exact mechanism by which HO-1 controls autophagy—that is, which HO-1 reaction products are involved and to what extent altered heme clearance plays a role in its regulation—remains unclear. CO, for example, which has both pro- and antioxidant properties,10 functions as a modulator of autophagy in respiratory epithelial cells, where it induces autophagy by increasing mitochondrial-derived ROS.17 In summary, the findings by Bolisetty et al.9 provide a novel link between HO-1 signaling and the regulation of renal autophagy and highlight an important functional association between autophagosome inducibility and sensitivity to nephrotoxic renal injury. This study will certainly stimulate further research into the role of autophagy in renal physiology and disease.

ID: 113: THE ENDOTHELIAL PHD2/HIF-2 AXIS REGULATES PULMONARY ARTERY PRESSURE IN MICE

Background Pulmonary hypertension (PH), a common clinical problem characterized by increased pulmonary artery (PA) pressure, is frequently triggered by hypoxia. Key mediators of cellular hypoxia responses are hypoxia-inducible factors (HIF)-1 and -2, the activity of which is regulated by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. Although both transcription factors are expressed in the lung, little is known about their cell type-specific roles in the pathogenesis of PH. Methods and Results Here we used a genetic approach to investigate the role of endothelial PHD2/HIF axis in the regulation of PA pressure. Endothelial cell specific HIF activation was achieved by crossing Vecadherin (Cdh5)-Cre transgenics to Phd2 floxed mice (ePhd2), while the contribution of each HIF isoform was assessed by generating double mutants lacking Phd2 and Hif-2 (ePhd2Hif2) or Phd2 and Hif-1 (Phd2Hif1). Right ventricular systolic pressure (RVSP) was measured via insertion of a 1.4F Mikro-tip catheter transducer into a surgically exposed right internal jugular vein. ePhd2 mice showed activation of HIF-signaling as shown by immunoblot analysis of lung tissue for HIF-1 and HIF-2. These mice developed spontaneous PH (RVSP, ePhd2: 54.3±6.9 vs Cre-: 24.8±2.2 mm Hg, P=0.005), which was associated with right ventricular hypertrophy (RVH) (Fulton Index, ePhd2: 0.52 vs Cre-: 0.28, P=0.0004) and early mortality. While morphologic analysis of ePhd2 lungs did not demonstrate plexiform or lumen-obliterating lesions, enhanced muscularization of peripheral PAs was detected in mutants compared to controls, as indicated by an increase in the number of arteries with diameters <100 µm that stained positive for αSMA (22.1±1.6 vs. 7.6±1.5 muscularized vessels/10 hpf, P<0.0001). The PH phenotype was maintained in ePhd2Hif1 mutants but was reversed in ePhd2Hif2 mutants. To assess the contribution of endothelial HIF-2 in hypoxia induced PH, endothelial Hif2 single mutants or Cre-littermates were exposed to normobaric hypoxia (10% O2) for 4 weeks. In contrast to controls, eHif2 mutants were protected from development of PH and RVH. Bone marrow transplantation studies showed no contribution from hematopoietic HIF-2 in hypoxia induced PH. Because hypoxia regulates endothelin 1 (EDN1), a potent vasoconstrictor but also apelin (APLN), a vasodilatory peptide acting through binding to the apelin G-protein-coupled receptor (APLNR), we assessed the role of endothelial HIF-2 axis in the regulation of these molecules. Endothelial deletion of Phd2 resulted in 6.4-fold induction of pulmonary Edn1 mRNA (P=0.029), but not Apln mRNA. In contrast, Aplnr was downregulated by 2.5-fold in ePhd2 mutants (P=0.037). A similar pattern of expression was detected in ePhd2Hif1 mice, whereas simultaneous deletion of Hif2a and Phd2 reversed these changes. To investigate the differences between acute and chronic hypoxia, we examined the effects of acute HIF activation on Edn1 and Apln/Aplnr gene expression in vivo. To model acute hypoxia, we subjected WT mice to 8% O2 for 48 hrs and maintained controls in room air. Acute hypoxia resulted in a 4.3-fold and 1.6-fold up-regulation of Edn1 and Apln transcripts respectively (P=0.0011 for Edn1, P=0.08 for Apln) while Aplnr was reduced by 4.3-fold (P=0.0005). We observed similar gene expression changes in mice treated with a prolyl-4-hydroxylase inhibitor (PHI) that results in global HIF activation. Conclusions Our studies identify endothelial HIF-2 as a key transcription factor in the pathogenesis of PH and suggest that HIF-2 regulates PA pressure by modulating the expression of vasoactive molecules. Our findings identify the PHD2/HIF2 axis as a potential target for PH therapies.