Optimal Reference Genes for RT-qPCR Experiments in Hippocampus and Cortex of Rats Chronically Exposed to Excessive Fluoride.

Normalization of the quantitative real-time PCR (RT-qPCR) data to the stably expressed reference genes is critically important for obtaining reliable results. However, all previous studies focused on F- toxicity for brain tissues used a single, non-validated reference gene, what might be a cause of contradictory or false results. The present study was designed to analyze the expression of a series of reference genes to select optimal ones for RT-qPCR analysis in cortex and hippocampus of rats chronically exposed to excessive fluoride (F-) amounts. Six-week-old male Wistar rats randomly assigned to four groups consumed regular tap water with 0.4 (control), 5, 20, and 50 ppm F- (NaF) for 12 months. The expression of six genes (Gapdh, Pgk1, Eef1a1, Ppia, Tbp, Helz) was compared by RT-qPCR in brain tissues from control and F--exposed animals. The stability of candidate reference genes was evaluated by coefficient of variation (CV) analysis and RefFinder online program summarizing the results of four well-acknowledged statistical methods (Delta-Ct, BestKeeper, NormFinder, and GeNorm). In spite of some discrepancies in gene ranking between these algorisms, Pgk1, Eef1a1, and Ppia were found to be most valid in cortex, while Ppia, Eef1a1, and Helz showed the greatest expression stability in hippocampus. Tbp and Helz were identified as the least stable genes in cortex, whereas Gapdh and Tbp are unsuitable for hippocampus. These data indicate that reliable mRNA quantification in the cortex and hippocampus of F--poisoned rats is possible using normalization to geometric mean of Pgk1+Eef1a1 or Ppia+Eef1a1 expression, respectively.

Silencing of Fli1 Gene Mimics Effects of Preeclampsia and Induces Collagen Synthesis in Human Umbilical Arteries.

BACKGROUND: Previously we demonstrated that in patients with preeclampsia elevated levels of endogenous Na/K-ATPase inhibitor, marinobufagenin, cause inhibition of Friend leukemia virus integration 1 (Fli1), a negative regulator of collagen-1 synthesis. We hypothesized that in vitro silencing of Fli1 in healthy human umbilical arteries would be associated with an increase in collagen-1 output, similar to the effect of preeclampsia in rat and human tissues. METHODS: The isolated segments of healthy human umbilical arteries were tested for sensitivity to MBG and Fli1 silencing with Fli1 siRNA or control siRNA. RESULTS: Following 24-hour incubation of arteries with nanomolar concentrations of marinobufagenin, Fli1 expression was inhibited 5-fold (P < 0.001), and synthesis of collagen-1 increased 3 times (P < 0.01). Twenty-four-hour incubation of umbilical artery fragments with Fli1 siRNA caused a dramatic decrease of Fli1 (7-fold; P < 0.001) and cytoplasmic PKC δ (4-fold; P < 0.001) expression in comparison to control siRNA or untreated control, followed by elevation in procollagen (3-fold; P < 0.001) and collagen-1 (3-fold; P < 0.001) levels in vascular tissue. CONCLUSIONS: Our results show that after silencing the Fli1 gene in healthy human umbilical arteries a new phenotype emerges which is typical for preeclampsia and is associated with vascular fibrosis.

Canrenone Restores Vasorelaxation Impaired by Marinobufagenin in Human Preeclampsia.

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p < 0.01). Isolated rings of umbilical arteries from the subjects with PE exhibited impaired responses to the relaxant effect of sodium nitroprusside vs. control vessels (EC50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 µmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists.

Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation.

The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May.

Antibody against Na/K-ATPase Inhibitor Lowers Blood Pressure and Increases Vascular Fli1 in Experimental Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE. METHODS: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours. RESULTS: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats). CONCLUSIONS: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1.

Cognitive Decline of Rats with Chronic Fluorosis Is Associated with Alterations in Hippocampal Calpain Signaling.

The study was designed to evaluate an influence of excessive fluoride (F-) intake on cognitive capacities of adult rats and on proteins of memory-related calpain signaling in hippocampus. Control animals were given water with natural F- content of 0.4 ppm; rats from other groups consumed the same water supplemented with 5, 20, and 50 ppm F- (as NaF) for 12 months. The efficiency of learning and memory formation was evaluated by novel object recognition (NOR) and Morris water maze tests. The expression of enzymes of calpain-1 and calpain-2 signaling in hippocampus was detected by Western blotting. Excessive F- consumption had moderate impact on short-term memory, but impaired spatial learning and long-term memory of animals. Intoxication of rats with 5-50 ppm F- led to stimulation of calpain-1 in hippocampal cells and its translocation from cytosol to membranes, accompanied by activation of GTPase RhoA. Exposure to 20-50 ppm F- resulted in proteolytic cleavage of phosphatase PHLPP1 and increased expression of phospho-ERK1/2 kinase with insignificant decline of total ERK1/2 activity. In contrast, F- did not change the expression of calpain-2 and its substrates-phosphatase PTEN and kinase mTOR. However, F- intake led to downregulation of cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF). Thus, altered expression of calpain-1 and its downstream effectors at a background of stable activity of calpain-2 indicates overstimulation of signaling pathways of early LTP phase and disrupted link between early and late LTP phases, most probably due to altered activity of transcriptional and neurotrophic factors.