Hypoxia-inducible factor-2alpha-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.

Wilms' tumor protein (-KTS) modulates renin gene transcription.

Renin plays a crucial role in the control of various physiological processes such as blood pressure and body fluid homeostasis. Here, we show that a splice variant of the Wilms' tumor protein lacking three amino acids WT1(-KTS) suppresses renin gene transcription. Using bioinformatics tools, we initially predicted that a WT1-binding site exists in a regulatory region about 12 kb upstream of the renin promoter; this was confirmed by reporter gene assays and gel shift experiments in heterologous cells. Co-expression of Wt1 and renin proteins was found in rat kidney sections, mouse kidney blood vessels, and a cell line derived from the juxtaglomerular apparatus that produces renin. Knockdown of WT1 protein by siRNA significantly increased the cellular renin mRNA content, while overexpression of WT1(-KTS) reduced renin gene expression in stable and transiently transfected cells. A mutant WT1(-KTS) protein found in Wilms' tumors failed to suppress renin gene reporter activity and endogenous renin expression. Our findings show that renin gene transcription is regulated by the WT1(-KTS) protein and this may explain findings in patients with WT1 gene mutations of increased plasma renin and hypertension.

Oxygen-regulated expression of the Wilms' tumor suppressor Wt1 involves hypoxia-inducible factor-1 (HIF-1).

The Wilms' tumor gene Wt1 is unique among tumor suppressors because of its requirement for the development of certain organs. We recently described de novo expression of Wt1 in myocardial blood vessels of ischemic rat hearts. The purpose of this study was to analyze the mechanism(s) of hypoxic/ischemic induction of Wt1. We show here that Wt1 mRNA and protein is up-regulated in the heart and kidneys of rats exposed to normobaric hypoxia (8% O2). Ectopic Wt1 immunoreactivity was detected in renal tubules of hypoxic rats, which also expressed the antiapoptotic protein Bcl-2 and contained significantly fewer TUNEL-positive cells than in normoxic kidneys. Wt1 expression was enhanced in the osteosarcoma line U-2OS and in Reh lymphoblast cells that were grown either at 1% O2 or in the presence of CoCl2 and desferrioxamine, respectively. The promoter of the Wt1 gene was capable of mediating expression of a luciferase reporter in response to hypoxia. We identified a hypoxia-responsive element in the Wt1 sequence that bound to hypoxia-inducible factor-1 (HIF-1) and was required for activation of the Wt1 promoter by CoCl2 and HIF-1. These findings demonstrate that Wt1 expression can be stimulated by hypoxia, which involves activation of the Wt1 promoter by HIF-1.

The Wilms' tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction.

Expression of the Wilms' tumor gene Wt1 in the epicardium is critical for normal heart development. Mouse embryos with inactivated Wt1 gene have extremely thin ventricles, which can result in heart failure and death. Here, we demonstrate that Wt1 can be activated in adult hearts by local ischemia. Wt1 mRNA was increased more than twofold in the left ventricular myocardium of rats between 1 day and 9 wk after infarction. Wt1 expression was localized by means of mRNA in situ hybridization and immunohistochemistry to vascular endothelial and vascular smooth muscle cells in the border zone of the infarcted tissue. A strikingly similar distribution was seen for vascular endothelial growth factor and two different cell proliferation markers in the coronary vessels of the ischemic heart. No Wt1 could be detected in the vasculature of the noninfarcted right ventricles. Wt1 expression in the coronary vessels of the ischemic heart was mimicked by exposure of rats to normobaric hypoxia (8% O2) and 0.1% CO, respectively. These findings demonstrate that Wt1 is expressed in the vasculature of the heart in response to local ischemia and hypoxia. They suggest that Wt1 has a role in the growth of coronary vessels after myocardial infarction.

Influence of epicardial stenosis severity and central venous pressure on the index of microcirculatory resistance in a follow-up study.

AIMS: This study sought to evaluate the reproducibility of the index of microcirculatory resistance (IMR) in a follow-up model and the role of epicardial artery stenosis and central venous pressure (Pv) on IMR. METHODS AND RESULTS: Twenty-two patients with stable coronary artery disease underwent coronary catheterisation at baseline and after seven weeks. The IMR was calculated at baseline and follow-up in several ways: as IMRuncorrected=Pd·Tmn (Pd: intracoronary pressure distal to the stenosis; Tmn: transit mean time); IMRcorrected=Pa·Tmn·(Pd - Pw)/(Pa-Pw), (Pw: coronary wedge pressure; Pa: aortic pressure); and as IMRcentral venous pressure (IMRcvp)=(Pa-Pv)·Tmn·(Pd-Pw)/(Pa-Pw). By neglecting Pw, IMR was overestimated irrespective of the haemodynamic severity of the epicardial stenosis (baseline: IMRuncorrected=15.5±8.9 U vs. IMRcorrected=13.5±8 U, p<0.001; follow-up: IMRuncorrected=16.9±4.9 U vs. IMRcorrected=13.8±4.6 U, p<0.001). In the intra-individual analysis IMR did not differ between the two time points. The IMRcvp equalled the IMRcorrected at all time points. CONCLUSIONS: IMR is a reproducible index in follow-up studies, independent of any overestimation existing when collateral flow status is neglected. Pv can be neglected for calculation of the IMR.

Induction of cerebral arteriogenesis leads to early-phase expression of protease inhibitors in growing collaterals of the brain.

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68+ macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis.

Positive cardiac troponin I and T and chest pain in a patient with iatrogenic hypothyroidism and no coronary artery disease.

We report about a 41-year old male patient who presented to the emergency room with acute chest pain, exertion dyspnoea, muscle stiffness, myalgia and adynamia. There was no history of coronary artery disease but known arterial hypertension and insulin dependent diabetes mellitus. Four weeks before submission the patient had been thyroidectomized after he had been diagnosed with papillary thyroid carcinoma and was now awaiting further radioiodine therapy. The thyroid-stimulating hormone level was markedly elevated to 67 mU/l (normal range 0.27-4.20 mU/l) and fT4 significantly reduced to 0.19 ng/ml (normal range 0.9-1.9 ng/ml). CK was elevated to 328 U/l, cardiac Troponin I (Stratus CS) above the threshold with 0.13 microg/l and Elecsys third generation troponin T above the threshold with 0.04 microg/l. The electrocardiogram showed a normal sinus rhythm and did not reveal any signs of ST-elevation or -depression. During follow-up a cardiac MRI was performed, showing normal dimensions and function but a very small area of diffuse myocardial damage, atypical of ischemic injury. In coronary angiography normal coronary arteries were found. We conclude that cardiac troponins I and T may be elevated in severe hypothyroidism without coronary artery disease due to diffuse myocardial injury which can be imaged by MRI.