Aldosterone and vasopressin affect {alpha}- and {gamma}-ENaC mRNA translation.

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, ß- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC-mRNA 3'-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3'-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA-protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V(2) receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC-mRNA/HuR complexes document the significance of γ-ENaC-mRNA-3'-UTR/HuR interaction for hormonal control of ENaC synthesis.

Induction of translationally controlled tumor protein (TCTP) by transcriptional and post-transcriptional mechanisms.

Expression of the human TPT1 gene coding for translationally controlled tumor protein (TCTP) was investigated in Calu-6 and Cos-7 cells under the influence of 4beta-phorbol 12-myristate 13-acetate (PMA), forskolin, dioxin and the heavy metals copper, nickel and cobalt. Transcriptional and post-transcriptional aspects of the mechanism were analyzed by TCTP mRNA/protein quantification, luciferase reporter gene assays depending on TPT1 promoter sequences or TCTP mRNA 5'/3'-UTRs and investigation of the interaction of RNA-binding proteins with UTRs by UV-crosslinking. PMA, forskolin, dioxin, cobalt and nickel induced TCTP expression in 24 h in both cell lines about 2.2-3.2-fold at the mRNA level and 1.6-2.2-fold at the protein level. The highest induction rate, 4.5-5.0-fold at the mRNA level and 3.5-4.0-fold at the protein level, was observed with copper. TPT1 promoter assays showed transcriptional activation by PMA, forskolin and dioxin (2.0-3.1-fold) and a 7.0-8.0-fold increase by copper, whereas cobalt and nickel had no effect. Deletion analysis revealed that copper-dependent transcriptional control was transmitted by a metal-responsive element residing in the TPT1 promoter. Post-transcriptional activation of TCTP expression was associated with the action of dioxin, nickel, cobalt (1.8-2.3-fold) and copper (2.5-3.0-fold), whereas stimulation of TCTP synthesis by copper was mediated by the TCTP mRNA 3'-UTR (3.2-fold) but not by the 5'-UTR (0.5-fold). mRNA stabilization was found to mediate these effects of cobalt and nickel. Post-transcriptional regulation was associated with qualitative and quantitative changes in the binding of specific RNA-binding proteins to UTRs.

Role of nucleolin in posttranscriptional control of MMP-9 expression.

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3'UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin ( approximately 64 kDa form) to the 3'UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

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.

Establishment of a swine model for validation of perfusion measurement by dynamic contrast-enhanced magnetic resonance imaging.

The aim of the study was to develop a suitable animal model for validating dynamic contrast-enhanced magnetic resonance imaging perfusion measurements. A total of 8 pigs were investigated by DCE-MRI. Perfusion was determined on the hind leg musculature. An ultrasound flow probe placed around the femoral artery provided flow measurements independent of MRI and served as the standard of reference. Images were acquired on a 1.5 T MRI scanner using a 3D T1-weighted gradient-echo sequence. An arterial catheter for local injection was implanted in the femoral artery. Continuous injection of adenosine for vasodilation resulted in steady blood flow levels up to four times the baseline level. In this way, three different stable perfusion levels were induced and measured. A central venous catheter was used for injection of two different types of contrast media. A low-molecular weight contrast medium and a blood pool contrast medium were used. A total of 6 perfusion measurements were performed with a time interval of about 20-25 min without significant differences in the arterial input functions. In conclusion the accuracy of DCE-MRI-based perfusion measurement can be validated by comparison of the integrated perfusion signal of the hind leg musculature with the blood flow values measured with the ultrasound flow probe around the femoral artery.

Absolute quantification of regional renal blood flow in swine by dynamic contrast-enhanced magnetic resonance imaging using a blood pool contrast agent.

AIM: To evaluate for the first time in an animal model the possibility of absolute regional quantification of renal medullary and cortical perfusion by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a blood pool contrast agent. MATERIAL AND METHODS: A total of 18 adult female pigs (age, 16-22 weeks; body weight, 45-65 kg; no dietary restrictions) were investigated by DCE-MRI. Absolute renal blood flow (RBF) measured by an ultrasound transit time flow probe around the renal vein was used as the standard of reference. An inflatable stainless cuff placed around the renal artery near its origin from the abdominal aorta was used to reduce RBF to 60%, 40%, and 20% of the baseline flow. The last measurement was performed with the cuff fully reopened. Absolute RBF values during these 4 perfusion states were compared with the results of DCE-MRI performed on a 1.5-T scanner with an 8-channel phased-array surface coil. All scans were acquired in breath-hold technique in the coronal plane using a field of view of 460 mm.Each dynamic scan commenced with a set of five 3D T1-weighted gradient echo sequences with different flip angles (alpha = 2 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees): TE, 0.88 milliseconds; TR, 2.65 milliseconds; slice thickness, 8.8 mm for 4 slices; acquisition matrix, 128 x 128; and acquisitions, 4. These data served to calculate 3D intrinsic longitudinal relaxation rate maps (R10) and magnetization (M0). Immediately after these images, the dynamic 3D T1-weighted gradient echo images were acquired with the same parameters and a constant alpha = 30 degrees, half Fourier, 1 acquisition, 64 frames, a time interval of 1.65 seconds between each frame, and a total duration of 105.6. Three milliliters of an albumin-binding blood pool contrast agent (0.25 mmol/mL gadofosveset trisodium, Vasovist, Bayer Schering Pharma AG, Berlin, Germany) was injected at a rate of 3 mL/s. Perfusion was calculated using the arterial input function from the aorta, which was extracted from the dynamic relaxation rate change maps and perfusion images were calculated on a voxel-by-voxel basis using a singular value decomposition. RESULTS: In 11 pigs, 4 different perfusion states were investigated sequentially. The reduced kidney perfusion measured by ultrasound highly correlated with total renal blood flow determined by DCE-MRI, P < 0.001. The correlation coefficient between both measurements was 0.843. Regional cortical and medullary renal flow was also highly correlated (r = 0.77/0.78, P < 0.001) with the degree of flow reduction. Perfusion values smaller than 50 mL/min/100 cm were overestimated by MRI, high perfusion values slightly underestimated. CONCLUSION: DCE-MRI using a blood pool contrast agent allows absolute quantification of total kidney perfusion as well as separate determination of cortical and medullary flow. The results show that our technique has sufficient accuracy and reproducibility to be transferred to the clinical setting.

Expression of smooth muscle MyHC B in blood vessels of hypertrophied heart in experimentally hypertensive rats.

We demonstrated recently a significantly lower fraction of cardiac precapillary arterioles that expressed smooth muscle myosin heavy chain (MyHC) B (SMB) in spontaneously hypertensive rats. To clarify whether this reduction of SMB expression is of genetic origin, we investigated SMB expression in cardiac precapillary arterioles of normotensive and experimentally hypertensive rats (one clip, one kidney or ANG II minipump). We observed similar SMB expression patterns in precapillary arterioles of experimentally hypertensive rats compared with normotensive controls. These observations suggest that the downregulation of SMB in spontaneously hypertensive rats is of genetic origin rather than an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.

Low-dose nitric oxide inhibition produces a negative sodium balance in conscious dogs.

Nitric oxide modulates renal hemodynamics and salt and water handling. Studies on the latter have provided conflicting results, however. Electrolyte and water balances were therefore studied in 28 beagles for 4 d, to determine the various effects of nitric oxide synthase (NOS) inhibition on renal function. The dogs were chronically equipped with aortic occluders to reduce renal perfusion pressure (RPP), bladder catheters, and catheters for measurements of RPP and mean arterial BP. A swivel system allowed free movement within the kennels. In a first set of experiments, a nonpressor dose of L-Nomega-nitroarginine (LN) (3 microg/min per kg body wt) was administered, to prevent increases in mean arterial BP and thus pressure effects on renin release and natriuresis. Remarkably, the nonpressor dose of LN caused a negative sodium balance. The natriuretic effect may involve reduced plasma renin activity, reduced aldosterone concentrations, and increased atrial natriuretic peptide concentrations. Changes in aldosterone levels, however, were the only parameters to parallel the time course of sodium excretion. In a second set of experiments, a sodium-retaining challenge was elicited by reduction of RPP. Dogs without NOS inhibition escaped sodium retention during RPP reduction after 2 d ("pressure escape"). LN neither ameliorated nor aggravated the sodium-retaining effect of reduced RPP, nor did it compromise the accomplishment of pressure escape. In conclusion, inhibition of NOS with a low dose of LN results in a reduction of total-body sodium. This effect mainly relies on reduced aldosterone concentrations. Furthermore, LN does not change the regulatory response to long-term RPP reduction.

ETA receptor blockade induces tubular cell proliferation and cyst growth in rats with polycystic kidney disease.

Tissue concentrations of ET-1 are markedly elevated in the kidneys of Han:Sprague-Dawley (Han:SPRD) rats, a model of human autosomal dominant polycystic kidney disease (ADPKD). This study analyzed whether disease progression might be attenuated by endothelin receptor antagonists. Heterozygous Han:SPRD rats received an ETA receptor antagonist (LU 135252), a combined ETA/ETB receptor antagonist (LU 224332), or placebo for 4 mo. Glomerulosclerosis, protein excretion, and GFR remained unchanged, whereas interstitial fibrosis was enhanced by both compounds. BP was not reduced by both compounds in Han:SPRD rats. Renal blood flow (RBF) decreased in ADPKD rats treated with the ETA receptor antagonist. Long-term ETA receptor blockade furthermore increased markedly the number of renal cysts (ADPKD rats, 390 +/- 119 [cysts/kidney section +/- SD]; LU 135252-treated APKD rats, 1084 +/- 314; P < 0.001), cyst surface area (ADPKD rats, 7.97 +/- 2.04 [% of total section surface +/- SD]; LU 135252-treated ADPKD rats, 33.83 +/- 10.03; P < 0.001), and cell proliferation of tubular cells (ADPKD rats, 42.2 +/- 17.3 [BrdU-positive cells/1000 cells]; LU 135252-treated ADPKD rats, 339.4 +/- 286.9; P < 0.001). The additional blockade of the ETB receptor attenuated these effects in Han:SPRD rats. Both endothelin receptor antagonists had no effect on BP, protein excretion, GFR, and kidney morphology in Sprague-Dawley rats without renal cysts. It is concluded that ETA receptor blockade enhances tubular cell proliferation, cyst number, and size and reduces RBF in Han:SPRD rats. This is of major clinical impact because endothelin receptor antagonists are upcoming clinically used drugs.