| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-0582fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Nephrology and Hypertension, University of Berne, 3010 Berne, Switzerland
2Correspondence: Division of Nephrology/Hypertension, University, Inselspital, Freiburgstrasse 15, 3010 Berne, Switzerland. E-mail: felix.frey{at}insel.ch
SPECIFIC AIMS
Subjects acclimatized to hypoxia exhibit elevated arterial blood pressure with intravascular volume expansion despite a decreased production of aldosterone, indicating that in hypoxic conditions another mechanism for maintaining sodium and potassium balance in the presence of the missing mineralocorticoid receptor (MR) ligand aldosterone has to be postulated. We addressed the hypothesis that hypoxia reduces the activity of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), the prereceptor gatekeeper for cortisol-mediated mineralocorticoid action, and showed that hypoxia diminishes the activity of 11ß-HSD2 by a MAPK-dependent induction of Egr-1.
PRINCIPAL FINDINGS
1. Chemical hypoxia down-regulates 11ß-HSD2 activity in LLC-PK1 cells by inducing the early growth response gene Egr-1
Antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, decreased the activity of 11ß-HSD2 concentration dependently by > 70% in a porcine renal epithelial cell line with characteristics of the proximal and distal convoluted tubule (LLC-PK1 cells) (Fig. 1
). The reduced activity of 11ß-HSD2 was not attributable to cell death.
|
To elucidate the molecular mechanism of this down-regulation, we focused on the early growth response gene Egr-1, known to be stimulated by hypoxia. Analysis of the promoter region of 11ß-HSD2 contains several GC-rich regions such as CGCCCCCGC, which can be regarded as a consensus sequence for Egr-1 binding. Messenger RNA expression, Western blots, and electrophoretic mobility shift assays (EMSA) revealed a transient Egr-1 expression as response to hypoxia in LLC-PK1 cells. The transfection of LLC-PK1 cells with increasing amounts of pcDNA3-Egr-1 plasmid resulted in a decreased amount of 11ß-HSD2 activity. Exposing LLC-PK1 cells transfected with a hEgr-prom-luciferase plasmid to antimycin A showed an increase in luciferase activity, an effect reversed when cells were incubated with PD 98059. PD 98059 is an inhibitor of the MAPK Mek, which is a known inducer of Egr-1. Thus, under hypoxic conditions Egr-1 is induced by a Mek-dependent pathway.
The direct effect of Egr-1 on the expression of 11ß-HSD2 was assessed by transient transfection with increasing amounts of the Egr-1 expression plasmid pcDNA3-Egr-1 and measuring the activity of 11ß-HSD2. In these experiments a down-regulation of 11ß-HSD2 by Egr-1 expression was observed, an interaction confirmed by a > 50% reduced luciferase activity when LLC-PK1cells were cotransfected with the Egr-1 expression plasmid together with the human 11ß-HSD2 promoter linked to a luciferase gene. Thus, hypoxia decreases the activity of 11ß-HSD2 most likely by inducing Egr-1.
2. Unilateral renal ischemia induces Egr-1 expression and down-regulates 11ß-HSD2 mRNA in the ischemic but not in the contralateral kidney
To demonstrate that ischemia affects both Egr-1 and 11ß-HSD2 in the principal organ where the 11ß-HSD2 enzyme deploys its main action, the kidney, unilateral renal ischemia was induced by clamping one renal artery for 60 min after open laparotomy. Messenger RNA levels, Western blots, and EMSA analyses of kidney tissue homogenates revealed a marked induction of Egr-1 protein in the ischemic but not in the contralateral nonischemic kidney of all rats. The 11ß-HSD2 mRNA levels analyzed by RT-PCR in hypoxic compared with normoxemic kidneys were reduced in all nine animals investigated by an average of 58% 24 h after ischemic injury was induced.
3. High altitude-associated hypoxia reduces 11ßHSD2 activity in vivo in humans
To assess the effect of hypoxia on the 11ß-HSD2 activity, 12 young healthy men, members of a high-altitude expedition to the Shisha Pagma (8046 m above sea level) in Nepal, were investigated. In these subjects average oxygen saturation decreased from 96% at 500 m altitude to 72% at 7000 m altitude (Fig. 2
). An estimate of the activity of the renal 11ß-HSD2 enzyme was obtained by measuring the urinary ratio of excreted cortisol to cortisone and the corresponding ratio of their tetrahydro-metabolites (THF+5
THF)/THE (THF=tetrahydrocortisol, 5THF=5-tetrahydrocortisol, THE=tetrahydrocortisone) by gas chromatography-mass spectrometry. The apparent activity of the 11ß-HSD2 enzyme estimated by measuring these metabolites declined by 60%. The urinary steroid metabolite ratios were independent of the total urinary excretion of cortisol metabolites, indicating that the increased ratios were not explained by the concentration-dependent kinetics of the 11ß-hydroxy-glucocorticoid. In the one member of the expedition who developed high altitude pulmonary edema before reaching 7000 m, aldosterone levels increased with increasing altitude; in all other volunteers these values declined (Fig. 2)
.
|
CONCLUSIONS
Hypoxia represents one of the most life-threatening stress stimuli for mammalians. Adrenocorticosteroids are part of the stress response system. Intracellular access of the adrenal hormone cortisol to the cytoplasmic mineralocorticoid receptor is regulated by the 11ß-HSD2 enzyme. This enzyme converts cortisol in humans and corticosterone in rodents to their receptor inactive ketoforms cortisone and dehydrocorticosterone, thus protecting the nonselective mineralocorticoid receptor from occupation by glucocorticoids. A reduced activity of 11ß-HSD2 causes an enhanced activation of the mineralocorticoid receptor by glucocorticoids. Thus, the present observation of a down-regulation of 11ß-HSD2 by hypoxemia in cell cultures, rat kidney, and humans is in line with the general concept of an enhanced adrenocorticosteroid effect in stress.
Synthesis of the adrenal mineralocorticoid aldosterone declined during hypoxia. Such a decline of the principal hormone for renal sodium retention in the presence of the known increased release of atrial natriuretic peptide during hypoxia is expected to cause renal sodium wasting with low blood pressure, which apparently does not occur. Based on the results from the present investigation, we propose that the hypoxemia-induced diminished activity of 11ß-HSD2 increases the access of cortisol to the mineralocorticoid receptor and offsets at least in part the reduced aldosterone secretion at high altitude.
Ligands for the mineralocorticoid receptor are only one of many factors determining the complex renal tubular sodium handling. Therefore, it is difficult to define quantitatively the functional importance of the decreased apparent activity of the 11ß-HSD2 enzyme by considering the 60% increase of the urinary ratio of (THF+5THF)/THE at high altitude. Based on observations in normal volunteers given high doses of liquorice, the model compound for inhibition of 11ß-HSD2 in humans, it is reasonable to conclude that these changes are biologically relevant.
The cellular response to oxygen deprivation is complex and only partly unveiled. Some of these responses to hypoxia depend on an early growth response (Egr-1). The present investigation describes that the crucial prereceptor gatekeeper for corticosteroid hormone action, the enzyme 11ß-HSD2 is down-regulated by the hypoxia/Egr-1 pathway. Egr-1 expression in the kidney after an ischemic insult has been localized to the nuclei of thick ascending limbs and principal cells of the collecting duct. The 11ß-HSD2 enzyme and the mineralocorticoid receptor are also localized to these cells within the kidney.
In conclusion, the present investigation demonstrates that hypoxemia induces a down-regulation of the 11ß-HSD2 enzyme in vitro and in vivo, an effect due to a MAPK-dependent induction of Egr-1 (Fig. 3
); we suggest that this effect is relevant for cortisol-mediated mineralocorticoid action in hypoxic disease states.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0582fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0582fje 
This article has been cited by other articles:
|
|
 |
|
  R. Alikhani-Koupaei, F. Fouladkou, P. Fustier, B. Cenni, A. M. Sharma, H.-C. Deter, B. M. Frey, and F. J. Frey Identification of polymorphisms in the human 11beta-hydroxysteroid dehydrogenase type 2 gene promoter: functional characterization and relevance for salt sensitivity FASEB J, November 1, 2007; 21(13): 3618 - 3628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Baserga, M. A. Hale, Z. M. Wang, X. Yu, C. W. Callaway, R. A. McKnight, and R. H. Lane Uteroplacental insufficiency alters nephrogenesis and downregulates cyclooxygenase-2 expression in a model of IUGR with adult-onset hypertension Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2007; 292(5): R1943 - R1955. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-H. Zhang, Y.-M. Kang, Y. Yu, S.-G. Wei, T. J. Schmidt, A. K. Johnson, and R. B. Felder 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Activity in Hypothalamic Paraventricular Nucleus Modulates Sympathetic Excitation Hypertension, July 1, 2006; 48(1): 127 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Khomenko, S. Szabo, X. Deng, M. R. Jadus, H. Ishikawa, K. Osapay, Z. Sandor, and L. Chen Suppression of early growth response factor-1 with egr-1 antisense oligodeoxynucleotide aggravates experimental duodenal ulcers Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1211 - G1218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Khachigian Early Growth Response-1 in Cardiovascular Pathobiology Circ. Res., February 3, 2006; 98(2): 186 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Kadereit, P. Fustier, K. Shojaati, B. M. Frey, F. J. Frey, and M. G. Mohaupt Extracellular ATP Determines 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Activity via Purinergic Receptors J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3507 - 3516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vengellur, J. M. Phillips, J. B. Hogenesch, and J. J. LaPres Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells Physiol Genomics, August 11, 2005; 22(3): 308 - 318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Quinkler, D. Zehnder, J. Lepenies, M. D Petrelli, J. S Moore, S. V Hughes, P. Cockwell, M. Hewison, and P. M Stewart Expression of renal 11{beta}-hydroxysteroid dehydrogenase type 2 is decreased in patients with impaired renal function Eur. J. Endocrinol., August 1, 2005; 153(2): 291 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O. Leonard, C. Godson, H. R. Brady, and C. T. Taylor Potentiation of Glucocorticoid Activity in Hypoxia through Induction of the Glucocorticoid Receptor J. Immunol., February 15, 2005; 174(4): 2250 - 2257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Xiao, X. Huang, S. Bae, C. A. Ducsay, L. D. Longo, and L. Zhang Cortisol-mediated regulation of uterine artery contractility: effect of chronic hypoxia Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H716 - H722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Pitt, C. T Stier Jr, and S. Rajagopalan Mineralocorticoid receptor blockade: new insights into the mechanism of action in patients with cardiovascular disease Journal of Renin-Angiotensin-Aldosterone System, September 1, 2003; 4(3): 164 - 168. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P<0.01, 
P<0.0001 by Kruskal-Wallis vs. control. The full circles represent values of the 12th member who suffered from high altitude pulmonary edema at 7000 m.