Christian Rosenberger

Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.

Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF‐1 and HIF‐2. Their regulation occurs through oxygen‐dependent proteolysis of the alpha subunits HIF‐1α and HIF‐2α, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF‐1α was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF‐2α expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF‐2α expression in control and hypoxic rats. Although HIF‐2α was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non‐parenchymal in some organs (kidney, pancreas, and brain), predominately parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF‐2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF‐1.

Expression of Hypoxia-Inducible Factor-1α and -2α in Hypoxic and Ischemic Rat Kidneys

Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations. HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation.

Reactive Oxygen Species and the Pathogenesis of Radiocontrast-Induced Nephropathy

Experimental findings in vitro and in vivo illustrate enhanced hypoxia and the formation of reactive oxygen species (ROS) within the kidney following the administration of iodinated contrast media, which may play a role in the development of contrast media-induced nephropathy. Clinical studies indeed support this possibility, suggesting a protective effect of ROS scavenging or reduced ROS formation with the administration of N-acetyl cysteine and bicarbonate infusion, respectively. Furthermore, most risk factors, predisposing to contrast-induced nephropathy are prone to enhanced renal parenchymal hypoxia and ROS formation.In this review, the association of renal hypoxia and ROS-mediated injury is outlined. Generated during contrast-induced renal parenchymal hypoxia, ROS may exert direct tubular and vascular endothelial injury and might further intensify renal parenchymal hypoxia by virtue of endothelial dysfunction and dysregulation of tubular transport. Preventive strategies conceivably should include inhibition of ROS generation or ROS scavenging.

Renal Parenchymal Hypoxia, Hypoxia Response and the Progression of Chronic Kidney Disease

Renal parenchymal hypoxia, documented under a variety of clinical conditions, conceivably contributes to the progression chronic kidney disease. In this review, normal physiologic medullary hypoxia and abnormal profiles of renal pO(2) in chronic kidney diseases are presented, and the mechanisms leading to anomalous renal tissue oxygenation are discussed. Direct measurements of pO(2) with oxygen electrodes, immunostaining with pimonidazole (which binds to regions with very low pO(2)), or the detection of hypoxia-inducible factor (HIF)-alpha (which accumulates in hypoxic regions, initiating hypoxia-adaptive responses), all serve to detect the distribution and extent of renal parenchymal hypoxia under experimental settings. The use of BOLD MRI as a noninvasive tool, detecting deoxygenated hemoglobin in hypoxic renal tissues, has evolved from experimental settings to human studies. All these modalities indicate that abnormal renal oxygenation develops under conditions such as chronic glomerular, tubulointerstitial or renovascular disease, in diabetes, hypertension, aging, renal hypertrophy, anemia or obstructive uropathy. Abnormal renal tissue hypoxia modifies the pattern of regional gene expression, evoking a host of adaptive and renoprotective pathways (such as HIF-mediated erythropoietin or heme-oxygenase-1), in parallel with the induction of potentially harmful mediators that participate in the progression of chronic kidney injury. Slowing the progression of chronic kidney disease may be achieved by a better understanding of these parallel processes and the accomplishment of a selective control of such protective and maladaptive responses.

Renal Parenchymal Hypoxia, Hypoxia Adaptation, and the Pathogenesis of Radiocontrast Nephropathy

BACKGROUND AND OBJECTIVES Renal parenchymal Po(2) declines after the administration of iodinated radiocontrast agents, reaching critically low levels of approximately 10 mmHg in medullary structures. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In this review, the causes of renal parenchymal hypoxia and its potential role in the pathogenesis of contrast nephropathy are appraised. RESULTS Commonly associated predisposing factors are associated with a propensity to enhance renal hypoxia. Indeed, animal models of radiocontrast nephropathy require the induction of such predisposing factors, mimicking clinical scenarios that lead to contrast nephropathy in high-risk individuals. In these models, in association with medullary hypoxic damage, a transient local cellular hypoxia response is noted, initiated at least in part by hypoxia-inducible factors. Some predisposing conditions that are distinguished by chronically aggravated medullary hypoxia, such as tubulointerstitial disease and diabetes, are characterized by a priori upregulation of hypoxia-inducible factors, which seems to confer tolerance against radiocontrast-related hypoxic tubular damage. Renal dysfunction under such circumstances likely reflects to some extent altered intrarenal hemodynamics, rather than acute tubular injury. CONCLUSIONS Real-time, noninvasive novel methods may help to differentiate between evolving tubular damage and altered hemodynamics and in the design of appropriate preventive interventions.

Experimental ischemia-reperfusion: biases and myths-the proximal vs. distal hypoxic tubular injury debate revisited.

Although the understanding of processes associated with hypoxic tubular cell injury has remarkably improved, controversies remain regarding the appropriateness of various animal models to the human syndrome of acute kidney injury (AKI). We herein compare available experimental models of hypoxic acute kidney damage, which differ both conceptually and morphologically in the distribution of tubular cell injury. Tubular segment types differ in their capacity to mount hypoxia-adaptive responses, mediated by hypoxia-inducible factors (HIFs), and in cell type-specific molecules shed into the urine, which may serve as early biomarkers for renal damage. These differences may be of value in the perception of the human AKI, its detection, and prevention.

Hypoxia-inducible factor-2α-expressing interstitial fibroblasts are the only renal cells that express erythropoietin under hypoxia-inducible factor stabilization

The adaptation of erythropoietin production to oxygen supply is determined by the abundance of hypoxia-inducible factor (HIF), a regulation that is induced by a prolyl hydroxylase. To identify cells that express HIF subunits (HIF-1alpha and HIF-2alpha) and erythropoietin, we treated Sprague-Dawley rats with the prolyl hydroxylase inhibitor FG-4497 for 6 h to induce HIF-dependent erythropoietin transcription. The kidneys were analyzed for colocalization of erythropoietin mRNA with HIF-1alpha and/or HIF-2alpha protein along with cell-specific identification markers. FG-4497 treatment strongly induced erythropoietin mRNA exclusively in cortical interstitial fibroblasts. Accumulation of HIF-2alpha was observed in these fibroblasts and in endothelial and glomerular cells, whereas HIF-1alpha was induced only in tubular epithelia. A large proportion (over 90% in the juxtamedullary cortex) of erythropoietin-expressing cells coexpressed HIF-2alpha. No colocalization of erythropoietin and HIF-1alpha was found. Hence, we conclude that in the adult kidney, HIF-2alpha and erythropoietin mRNA colocalize only in cortical interstitial fibroblasts, which makes them the key cell type for renal erythropoietin synthesis as regulated by HIF-2alpha.

RENAL PARENCHYMAL OXYGENATION AND HYPOXIA ADAPTATION IN ACUTE KIDNEY INJURY

1 The pathogenesis of acute kidney injury (AKI), formally termed acute tubular necrosis, is complex and, phenotypically, may range from functional dysregulation without overt morphological features to literal tubular destruction. 2 Hypoxia results from imbalanced oxygen supply and consumption. Increasing evidence supports the view that regional renal hypoxia occurs in AKI irrespective of the underlying condition, even under circumstances basically believed to reflect ‘direct’ tubulotoxicity. However, at present, it is remains unclear whether hypoxia per se or, rather, re‐oxygenation (possibly through reactive oxygen species) causes AKI. 3 Data regarding renal hypoxia in the clinical situation of AKI are lacking and our current concepts regarding renal oxygenation during acute renal failure are presumptive and largely derived from experimental studies. 4 There is robust experimental evidence that AKI is often associated with altered intrarenal microcirculation and oxygenation. Furthermore, renal parenchymal oxygen deprivation seems to participate in the pathogenesis of experimental AKI, induced by exogenous nephrotoxins (such as contrast media, non‐steroidal anti‐inflammatory drugs or amphotericin), sepsis, pigment and obstructive nephropathies. 5 Sub‐lethal cellular hypoxia engenders adaptational responses through hypoxia‐inducible factors (HIF). Forthcoming technologies to modulate the HIF system form a novel potential therapeutic approach for AKI.

Persistent induction of HIF-1α and -2α in cardiomyocytes and stromal cells of ischemic myocardium

Hypoxia‐inducible factor (HIF)‐1α and ‐2α are key regulators of the transcriptional response to hypoxia and pivotal in mediating the consequences of many disease states. In the present work, we define their temporo‐spatial accumulation after myocardial infarction and systemic hypoxia. Rats were exposed to hypoxia or underwent coronary artery ligation. Immunohistochemistry was used for detection of HIF‐1α and ‐2α proteins and target genes, and mRNA levels were determined by RNase protection. Marked nuclear accumulation of HIF‐1α and ‐2α occurred after both systemic hypoxia and coronary ligation in cardiomyocytes as well as interstitial and endothelial cells (EC) without pronounced changes in HIF mRNA levels. While systemic hypoxia led to widespread induction of HIF, expression after coronary occlusion occurred primarily at the border of infarcted tissue. This expression persisted for 4 wk, included infiltrating macrophages, and colocalized with target gene expression. Subsets of cells simultaneously expressed both HIF‐α subunits, but EC more frequently induced HIF‐2α. A progressive increase of HIF‐2α but not HIF‐1α occurred in areas remote from the infarct, including the interventricular septum. Cardiomyocytes and cardiac stromal cells exhibit a marked potential for a prolonged transcriptional response to ischemia mediated by HIF. The induction of HIF‐1α and ‐2α appears to be complementary rather than solely redundant.

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.