Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases.

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation.

Real-world study: Escalating targeted lipid-lowering treatment with PCSK9-inhibitors and lipoprotein apheresis.

INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition with monoclonal antibodies has complemented the armamentarium of lipid-lowering therapy (LLT) before the final step of commencing chronic lipoprotein apheresis (LA). Data are scarce on patients who, after escalation of LLT with PCSK9 antibodies, have commenced chronic LA or PCSK9 antibody treatment during ongoing long-term LA. PATIENTS AND METHODS: In this study, a cohort of 110 patients with established atherosclerotic cardiovascular disease (ASCVD) due to hypercholesterolemia or concomitant lipoprotein(a)-hyperlipoproteinemia, who received PCSK9 antibodies for the first time during routine care, were consecutively identified. RESULTS: Mean LDL-C concentration prior to initiation of LA or PCSK9 antibody treatment was 5.3 ± 2.6 mmol/L (205 ± 102 mg/dL). Due to established ASCVD, the risk-adjusted LDL-C target value was <1.8 mmol/L (<70 mg/dL) in all patients. Use of PCSK9 antibodies increased the proportion of patients attaining the LDL-C target concentration by 41.8% overall. Treatment emergent adverse events (TEAE) associated with PCSK9 antibody medication were reported in 35 patients (31.8%). Discontinuation of PCSK9 antibody therapy due to TEAEs occurred in 25 patients (22.7%). CONCLUSION: Finally, 55.5% of patients received a combination of PCSK9 antibody therapy and LA at individually optimized treatment frequencies resulting in an increase of target attainment in 54.1% of patients. About 18.1% of chronic LA patients terminated LA treatment in this real-world study. The termination of long-term LA therapy, which has hitherto prevented the progression of ASCVD, requires careful individual risk assessment and cannot be recommended by the general criteria of LDL-C reduction.

The protective effect of prolyl-hydroxylase inhibition against renal ischaemia requires application prior to ischaemia but is superior to EPO treatment.

BACKGROUND: Inhibition of the HIF regulating prolyl hydroxylation domain (PHDs) proteins prior to renal injury (preconditioning) has been shown to protect the kidney via activation of hypoxia-inducible transcription factors (HIF). Application of erythropoietin (EPO), one of the HIF target genes, has also been shown to be nephroprotective, and it remains unclear to what extent the effect of HIF induction is mediated by EPO. It is also unknown whether HIF activation after the onset of ischaemia (postconditioning) is still able to protect the kidney. METHODS: Using a rat model of renal ischaemia-reperfusion injury, animals were treated with the PHD inhibitor (PHD-I) 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), vehicle (Veh) or recombinant human EPO (300 IU/kg) 6 h (ICA or Veh) or 30 min (EPO) prior to ischaemia (preconditioning) or with ICA prior to reperfusion (postconditioning). Renal function was assessed at baseline, 24 h and 72 h. After 72 h, kidneys were processed for histology and morphometric analysis. HIF immunohistochemistry and real-time polymerase chain reaction for HIF target genes, including EPO, were performed to evaluate ICA effects. RESULTS: ICA treatment resulted in stabilization of HIF-1α and -2α and up-regulation of HIF target genes in a dose-dependent manner. Preconditional activation of HIF by ICA significantly improved serum creatinine levels and renal morphology in comparison to Veh (P < 0.05), while postconditional ICA treatment was ineffective. EPO therapy improved tissue morphology but had no impact on the course of serum creatinine. CONCLUSION: These findings are in line with the concept that PHD-Is exert their protective effects through accumulation of HIF target gene products, with time requirements for increased transcription and translation of HIF-dependent genes, and suggest that their renoprotective effect is not predominately mediated by EPO.

The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568. CONCLUSIONS: PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis. These data suggest that effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts.

Inhibition of prolyl hydroxylases increases erythropoietin production in ESRD.

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing--not a loss of EPO production capacity--causes renal anemia.

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

Mutual regulation of hypoxia-inducible factor and mammalian target of rapamycin as a function of oxygen availability.

The mammalian target of rapamycin (mTOR) regulates cellular growth and proliferation, mainly by controlling cellular translation. Most tumors show constitutive activation of the mTOR pathway. In hypoxia, mTOR is inactivated, which is believed to be part of the program of the cell to maintain energy homeostasis. However, certain proteins are believed to be preferentially translated during hypoxia via 5' terminal oligopyrimidine tract mechanisms with controversial discussion about the involvement of the mTOR-dependent ribosomal protein S6 (rpS6). The hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxic adaptation and itself strongly implicated in tumor growth. HIF is translationally regulated by mTOR. The regulatory features and the involvement of molecular oxygen itself in this regulation of HIF by mTOR are poorly understood. mTOR inhibition leads to profound attenuation of HIFalpha protein in the majority of primary and cancer cells studied. Under severe hypoxia, no influence of mTOR inhibitors was observed; thus, stimulation of HIFalpha by mTOR may only be relevant under mild hypoxia or even normoxia. HIF expression and phosphorylated rpS6 negatively correlate in experimental tumors. In cell culture, prolonged hypoxia abolishes rpS6 phosphorylation, which seems to be partly independent of the upstream p70S6 kinase. We show that hypoxic repression of rpS6 is largely dependent on HIF, implicating a negative feedback loop, which may influence cellular translational rates and metabolic homeostasis. These data implicate that the hypoxic microenvironment renders tumor cells resistant to mTOR inhibition, at least concerning hypoxic gene activation, which would add to the difficulties of other established therapeutic strategies in hypoxic cancer tissues.

HIF-prolyl hydroxylases in the rat kidney: physiologic expression patterns and regulation in acute kidney injury.

Hypoxia-inducible transcription factors (HIFs) play important roles in the response of the kidney to systemic and regional hypoxia. Degradation of HIFs is mediated by three oxygen-dependent HIF-prolyl hydroxylases (PHDs), which have partially overlapping characteristics. Although PHD inhibitors, which can induce HIFs in the presence of oxygen, are already in clinical development, little is known about the expression and regulation of these enzymes in the kidney. Therefore, we investigated the expression levels of the three PHDs in both isolated tubular cells and rat kidneys. All three PHDs were present in the kidney and were expressed predominantly in three different cell populations: (a) in distal convoluted tubules and collecting ducts (PHD1,2,3), (b) in glomerular podocytes (PHD1,3), and (c) in interstitial fibroblasts (PHD1,3). Higher levels of PHDs were found in tubular segments of the inner medulla where oxygen tensions are known to be physiologically low. PHD expression levels were unchanged in HIF-positive tubular and interstitial cells after induction by systemic hypoxia. In rat models of acute renal injury, changes in PHD expression levels were variable; while cisplatin and ischemia/reperfusion led to significant decreases in PHD2 and 3 expression levels, no changes were seen in a model of contrast media-induced nephropathy. These results implicate the non-uniform expression of HIF-regulating enzymes that modify the hypoxic response in the kidney under both regional and temporal conditions.

The human HIF (hypoxia-inducible factor)-3alpha gene is a HIF-1 target gene and may modulate hypoxic gene induction.

HIF (hypoxia-inducible factor)-3alpha is the third member of the HIF transcription factor family. Whereas HIF-1alpha and -2alpha play critical roles in the cellular and systemic adaptation to hypoxia, little is known about the regulation and function of HIF-3alpha. At least five different splice variants may be expressed from the human HIF-3alpha locus that are suggested to exert primarily negative regulatory effects on hypoxic gene induction. In the present paper, we report that hypoxia induces the human HIF-3alpha gene at the transcriptional level in a HIF-1-dependent manner. HIF-3alpha2 and HIF-3alpha4 transcripts, the HIF-3alpha splice variants expressed in Caki-1 renal carcinoma cells, rapidly increased after exposure to hypoxia or chemical hypoxia mimetics. siRNA (small interfering RNA)-mediated HIF-alpha knockdown demonstrated that HIF-3alpha is a specific target gene of HIF-1alpha, but is not affected by HIF-2alpha knockdown. In contrast with HIF-1alpha and HIF-2alpha, HIF-3alpha is not regulated at the level of protein stability. HIF-3alpha protein could be detected under normoxia in the cytoplasm and nuclei, but increased under hypoxic conditions. Promoter analyses and chromatin immunoprecipitation experiments localized a functional hypoxia-responsive element 5' to the transcriptional start of HIF-3alpha2. siRNA-mediated knockdown of HIF-3alpha increased transactivation of a HIF-driven reporter construct and mRNA expression of lysyl oxidase. Immunohistochemistry revealed an overlap of HIF-1alpha-positive and HIF-3alpha-positive areas in human renal cell carcinomas. These findings shed light on a novel aspect of HIF-3alpha as a HIF-1 target gene and point to a possible role as a modulator of hypoxic gene induction.

The specific contribution of hypoxia-inducible factor-2alpha to hypoxic gene expression in vitro is limited and modulated by cell type-specific and exogenous factors.

Cellular integrity in hypoxia is dependent on molecular adaptations dominated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). The HIF complex contains one of two alternative oxygen-regulated alpha-subunits considered to play distinct roles in the hypoxia response. Although HIF-2alpha may be more important in tumour biology and erythropoiesis, the spectrum of individual target genes is still insufficiently characterized. We therefore performed an Affymetrix gene array on Hep3B cells stimulated with a hypoxia-mimetic and transfected with either HIF-1alpha or HIF-2alpha siRNA. 271 transcripts were found to be induced HIF-dependently, including most previously identified HIF targets and a number of novel genes. Most were influenced by HIF-1alpha knock-down, whereas a smaller number were regulated by HIF-2alpha. Validation of a selection of genes by RNase protection confirmed the hypoxic regulation and HIF-1alpha- or HIF-2alpha-dependency in most cases, with the latter showing a lower amplitude. Many HIF-2alpha targets also responded to HIF-1alpha knock-down. Interestingly, regulation by HIF-2alpha was markedly influenced not only by cell type, but also by cell culture conditions, features that were not shared with HIF-1alpha-regulated genes. Thus, HIF-2alpha effects are modulated by a number of intrinsic and extrinsic factors which may be most relevant in tumour cells.

HIF activation protects from acute kidney injury.

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.

Physiological basis for the use of erythropoietin in critically ill patients at risk for acute kidney injury.

PURPOSE OF REVIEW: Acute kidney injury (AKI) frequently occurs in critically ill patients and is an independent risk factor for poor outcome. The prevention of kidney injury in intensive care remains a great challenge as specific nephroprotective therapies are still lacking. The present review summarizes recent evidence for the use of erythropoietin as a promising candidate to provide protection from AKI. RECENT FINDINGS: Beyond the known hematopoietic actions of erythropoietin, a number of preclinical studies demonstrated that erythropoietin possesses pleiotropic, organ-protecting properties. Preconditional and postconditional erythropoietin treatment was shown to protect from ischemic, toxic and septic AKI. Despite heterogeneities in study design and dose, erythropoietin consistently ameliorated renal injury. The mechanisms of protection remain largely unclear but may involve reduction of apoptosis, induction of cellular proliferation and tissue repair as well as mobilization of stem cells. SUMMARY: Animal studies revealed a physiological basis for the use of erythropoietin in AKI, which may be clinically applicable to prevent AKI in critically ill patients, but clinical studies are still lacking.

Organ protection by hypoxia and hypoxia-inducible factors.

Since the first description of a protective effect of hypoxic preconditioning in the heart, the principle of reducing tissue injury in response to ischemia by prior exposure to hypoxia was confirmed in a number of cells and organs. However, despite impressive preclinical results, hypoxic preconditioning has so far failed to reach clinical application. Nevertheless, it remains of significant interest to induce genes that are normally activated during hypoxia and ischemia as part of an endogenous escape mechanism prior to or during the early phase of an ischemic insult. This approach has recently been greatly facilitated by the identification of hypoxia-inducible factors (HIFs), transcription factors that operate as a master switch in the cellular response to hypoxia. Far more than 100 target genes are regulated by HIF, including genes such as erythropoietin and hemoxygenase-1, which have been shown to be tissue-protective. The identification of small molecule inhibitors of the oxygen-sensing HIF-prolyl hydroxlases now offers the possibility to mimic the hypoxic response by pharmacological stabilization of HIF in order to achieve organ protection. Oxygen-independent activation of HIF is therefore a promising therapeutic strategy for the prevention of organ injury and failure.

Hypoxia-inducible transcription factors and their role in renal disease.

The 2 hypoxia inducible factors (HIF)-1alpha and HIF-2alpha are key mediators of cellular adaptation to hypoxia. They show a specific distribution pattern and possibly have complementary transcriptional targets in the kidney: HIF-1alpha is found mainly in tubular and HIF-2alpha in peritubular interstitial, endothelial, and glomerular cells. Both isoforms are regulated by oxygen-dependent hydroxylation of specific amino acid residues, which determines protein stability and transcriptional activity. Small molecule inhibitors of HIF hydroxylases act as pharmacologic inducers of HIF. HIF target genes are involved in cellular mechanisms that increase hypoxia tolerance or improve oxygen supply at the systemic or regional level, but also have been implicated in cellular apoptosis and profibrotic mechanisms. In experimental acute kidney injury the up-regulation of HIF either through endogenous hypoxia-sensing or after pharmacologic HIF stabilization confers tissue protection. Thus, HIF stabilization offers a promising novel and clinically feasible approach for nephroprotection. On the other hand, continuous activation of the HIF system occurs in kidney cancer and potentially promotes tumor growth. HIF therefore also is explored as a target for anticancer therapy.

Stabilization of hypoxia inducible factor rather than modulation of collagen metabolism improves cardiac function after acute myocardial infarction in rats.

UNLABELLED: Prolyl hydroxylase domain-containing enzymes (PHD) hydroxylate a proline residue that controls the degradation of hypoxia inducible factor (HIF). Hypoxia inhibits this hydroxylation thus increasing HIF levels. HIF is upregulated in ischemic tissues, growing tumors and in nonischemic, mechanically stressed myocardium. Pharmacological inhibition of prolyl 4-hydroxylase (P4-H) stabilizes HIF-protein in vitro and may modulate collagen turnover. The aims of this study were to investigate whether inhibition of P4-H protects myocardium against ischemia, and whether the observed effects are related to modulation of collagen metabolism or due to the stabilization of HIF. METHODS: Rats were treated with a specific P4-H inhibitor (P4-HI) or vehicle starting 2 days before induction of myocardial infarction (MI). Rats were investigated 7 or 30 days after MI. Induction of HIF-1alpha and -2alpha was visualized by immunohistochemistry. Expression of growth factors (connective tissue growth factor, Osteopontin) and mRNA expression and protein levels of Collagen I and III as well as HIF-2alpha were measured. RESULTS: P4-HI augments HIF in the myocardium as early as 24 h after treatment. P4-HI did not alter the MI-induced enhanced expression of growth factors and collagen. Treatment with P4-HI significantly reduced heart and lung weight, improved left ventricular contractility, prevented left ventricular enlargement and improved left ventricular ejection fraction without affecting infarct size after 30 days. CONCLUSIONS: Specific inhibition of the P4-H improved cardiac function without affecting the infarct size after experimental myocardial infarction in rats. Stabilization of HIF rather than inhibition of collagen maturation by P4-HI may prevent cardiac remodeling after MI.

Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model.

OBJECTIVES: The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. METHODS: MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). RESULTS: Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P < 0.01]. In addition, experimental groups revealed a down-regulation of E-selectin (-57%) and MCP1 (-33%) expression after ICA pretreatment compared with controls, going along with decreased T-cell [1.4% CD4+ T-cell infiltration vs 8.4% (control) and 4.9% CD8+ T-cell infiltration vs 10.7% (control)], dendritic cell (0.6% dendritic cells infiltration vs 1.9% infiltration(control)] and macrophage infiltration [4.8% macrophages (ICA) vs 10.9% (control)] within vascular grafts. CONCLUSIONS: These data of an animal transplant model show that the pharmaceutical activation of HIF with endogenous up-regulation of protective target genes leads to adaptation of the graft to low oxygen-saturation and hereby attenuates the development of transplant arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

Reduction of obliterative bronchiolitis (OB) by prolyl-hydroxylase-inhibitors activating hypoxia-inducible transcription factors in an experimental mouse model.

BACKGROUND: Obliterative bronchiolitis (OB) is the major limiting factor for long-term survival after lung transplantation. As previously shown, donor treatment with a PHD-inhibitor activating hypoxia-inducible transcription factors (HIFs) prevents graft injury both in an allogenic kidney and aortic allograft transplant model. The aim of this study was to investigate the effect of HIF activation with a PHD-inhibitor on the development of OB. METHODS: Fully MHC-mismatched C57BL/6 (H-2b) donor tracheas were orthotopically transplanted into CBA/J (H-2k) recipients. Donor animals received a single dose of PHD-inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40mg/kg i.p.) or vehicle control 4h before transplantation. Transplanted tracheas were harvested 14 or 30days after transplantation and were analyzed by histology, by immunofluorescence and by rtPCR for mRNA expression. RESULTS: Donor pre-conditioning with ICA resulted in HIF accumulation and induction of HIF target genes: HO-1, VEGF, MIF, TGFß, and EpoR, which persisted during different times of ischemia. Grafts of vehicle treated controls showed substantially more luminal obliteration on postoperative day 30 in contrast to groups pre-treated with ICA [luminal obliteration 29.2±5% (ICA) vs. 36.7±8% (control), p<0.01]. We found significantly lower expression of TNFα, PDGFß, MCP-1, E-selectin, and ICAM-1 14days after ICA premedication. In addition ICA pre-treated groups revealed decreased T-cell and macrophage infiltration in tracheal grafts on days 30 after transplantation (p<0.05). CONCLUSION: Pre-treatment with ICA effectively reduced obliterative bronchiolitis. Our data suggest that activation of hypoxia-inducible transcription factors (HIFs) and thereby adaptation to low oxygen prevents the development of OB and allograft injury. Pharmaceutical inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves further clinical evaluation.

Role of hypoxia in the pathogenesis of renal disease.

The kidney shows a remarkable discrepancy between blood supply and oxygenation. Despite high blood flow and oxygen delivery, oxygen tensions in the kidney are comparatively low, in particular in the renal medulla. The reason for this lies in the parallel arrangement of arterial and venous preglomerular and postglomerular vessels, which allows oxygen to pass from arterioles into the postcapillary venous system via shunt diffusion. The limitation in renal tissue oxygen supply renders the kidney susceptible to hypoxia and has long been recognized as an important factor in the pathogenesis of acute renal injury. In recent years, evidence has accumulated that hypoxia does also play a significant role in the pathogenesis and progression of chronic renal disease, because different types of kidney disease are usually associated with a rarefication of postglomerular capillaries. In both acute and chronic diseases, tissue hypoxia does not only imply the risk of energy deprivation but also induces regulatory mechanisms and has a profound influence on gene expression. In particular, the transcription factor hypoxia inducible factor (HIF) is involved in cellular regulation of angiogenesis, vasotone, glucose metabolism, and cell death and survival decisions. HIF has been shown to be activated in renal disease and presumably plays a major role in protective responses to oxygen deprivation. Recent insights into the regulation of HIF increase our understanding of the role of hypoxia in disease progression and open new options to improve hypoxia tolerance and to induce nephroprotection.

Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens.

BACKGROUND: Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS: Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i) in all ex vivo in biopsies of patients suffering from skin infections, (ii) in vitro using cell culture infection models and (iii) in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs) of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. CONCLUSIONS/SIGNIFICANCE: Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections.