Renoprotection Provided by Additional Diuretic Treatment in Partially Nephrectomized Ren-2 Transgenic Rats Subjected to the Combined RAS and ETA Blockade.

OBJECTIVE: Our previous study in heterozygous Ren-2 transgenic rats (TGR) demonstrated that long-term treatment with endothelin receptor A (ETA) blocker atrasentan added to the renin-angiotensin system (RAS) blockade had renoprotective effects in a model of chronic kidney disease (CKD) induced by partial nephrectomy. Since ETA blockade is known to cause edema, we were interested whether diuretic treatment added to this therapy would be beneficial. DESIGN AND METHODS: Partial nephrectomy (NX) was performed at the age of 3 months in TGR rats which were subjected to: (i) RAS blockade alone (angiotensin receptor blocker losartan and angiotensin converting enzyme inhibitor trandolapril), (ii) combined RAS (losartan and trandolapril) and ETA receptor blockade (atrasentan), or (iii) diuretic (hydrochlorothiazide) added to the combined RAS + ETA blockade for 50 weeks following NX. RESULTS: At the end of the study systolic blood pressure and cardiac hypertrophy were similarly decreased in all treated groups. Survival was significantly improved by ETA receptor blockade added to RAS blockade with no further effects of diuretic treatment. However, additional diuretic treatment combined with RAS + ETA blockade decreased body weight and had beneficial renoprotective effects - reductions of both kidney weight and kidney damage markers. Proteinuria gradually increased in rats treated with RAS blockade alone, while it was substantially lowered by additional ETA blockade. In rats treated with additional diuretic, proteinuria was progressively reduced throughout the experiment. CONCLUSION: A diuretic added to the combined RAS and ETA blockade has late renoprotective effects in CKD induced by partial nephrectomy in Ren-2 transgenic rats. The diuretic improved: renal function (evaluated as proteinuria and creatinine clearance), renal morphology (kidney mass, glomerular volume), and histological markers of kidney damage (glomerulosclerosis index, tubulointerstitial injury).

Epoxyeicosatrienoic Acid-Based Therapy Attenuates the Progression of Postischemic Heart Failure in Normotensive Sprague-Dawley but Not in Hypertensive Ren-2 Transgenic Rats.

Epoxyeicosatrienoic acids (EETs) and their analogs have been identified as potent antihypertensive compounds with cardio- and renoprotective actions. Here, we examined the effect of EET-A, an orally active EET analog, and c-AUCB, an inhibitor of the EETs degrading enzyme soluble epoxide hydrolase, on the progression of post-myocardial infarction (MI) heart failure (HF) in normotensive Hannover Sprague-Dawley (HanSD) and in heterozygous Ren-2 transgenic rats (TGR) with angiotensin II-dependent hypertension. Adult male rats (12 weeks old) were subjected to 60-min left anterior descending (LAD) coronary artery occlusion or sham (non-MI) operation. Animals were treated with EET-A and c-AUCB (10 and 1 mg/kg/day, respectively) in drinking water, given alone or combined for 5 weeks starting 24 h after MI induction. Left ventricle (LV) function and geometry were assessed by echocardiography before MI and during the progression of HF. At the end of the study, LV function was determined by catheterization and tissue samples were collected. Ischemic mortality due to the incidence of sustained ventricular fibrillation was significantly higher in TGR than in HanSD rats (35.4 and 17.7%, respectively). MI-induced HF markedly increased LV end-diastolic pressure (Ped) and reduced fractional shortening (FS) and the peak rate of pressure development [+(dP/dt)max] in untreated HanSD compared to sham (non-MI) group [Ped: 30.5 ± 3.3 vs. 9.7 ± 1.3 mmHg; FS: 11.1 ± 1.0 vs. 40.8 ± 0.5%; +(dP/dt)max: 3890 ± 291 vs. 5947 ± 309 mmHg/s]. EET-A and c-AUCB, given alone, tended to improve LV function parameters in HanSD rats. Their combination amplified the cardioprotective effect of single therapy and reached significant differences compared to untreated HanSD controls [Ped: 19.4 ± 2.2 mmHg; FS: 14.9 ± 1.0%; +(dP/dt)max: 5278 ± 255 mmHg/s]. In TGR, MI resulted in the impairment of LV function like HanSD rats. All treatments reduced the increased level of albuminuria in TGR compared to untreated MI group, but neither single nor combined EET-based therapy improved LV function. Our results indicate that EET-based therapy attenuates the progression of post-MI HF in HanSD, but not in TGR, even though they exhibited renoprotective action in TGR hypertensive rats.

DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas.

BACKGROUND: Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

Transcript expression and genetic variability analysis of caspases in breast carcinomas suggests CASP9 as the most interesting target.

BACKGROUND: Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. METHODS: Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. RESULTS: CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). CONCLUSIONS: Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

Epoxyeicosatrienoic acid analog attenuates the development of malignant hypertension, but does not reverse it once established: a study in Cyp1a1-Ren-2 transgenic rats.

OBJECTIVE: We evaluated the therapeutic effectiveness of a new, orally active epoxyeicosatrienoic acid analog (EET-A) in rats with angiotensin II (ANG II)-dependent malignant hypertension. METHODS: Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. EET-A treatment was started either simultaneously with I3C induction process (early treatment) or 10 days later during established hypertension (late treatment). Blood pressure (BP) (radiotelemetry), indices of renal and cardiac injury, and plasma and kidney levels of the components of the renin-angiotensin system (RAS) were determined. RESULTS: In I3C-induced hypertensive rats, early EET-A treatment attenuated BP increase (to 175 ±â€Š3 versus 193 ±â€Š4 mmHg, P < 0.05, on day 13), reduced albuminuria (15 ±â€Š1 versus 28 ±â€Š2 mg/24 h, P < 0.05), and cardiac hypertrophy as compared with untreated I3C-induced rats. This was associated with suppression of plasma and kidney ANG II levels (48 ±â€Š6 versus 106 ±â€Š9 and 122 ±â€Š19 versus 346 ±â€Š11 fmol ml or g, respectively, P < 0.05) and increases in plasma and kidney angiotensin (1-7) concentrations (84 ±â€Š9 versus 37 ±â€Š6 and 199 ±â€Š12 versus 68 ±â€Š9 fmol/ml or g, respectively, P < 0.05). Remarkably, late EET-A treatment did not lower BP or improve renal and cardiac injury; indices of RAS activity were not affected. CONCLUSION: The new, orally active EET-A attenuated the development of experimental ANG II-dependent malignant hypertension, likely via suppression of the hypertensiogenic axis and augmentation of the vasodilatory/natriuretic axis of RAS.

Littoral cell angioma of the spleen: a study of 25 cases with confirmation of frequent association with visceral malignancies.

AIMS: Littoral cell angioma (LCA) is a rare primary splenic tumour that is frequently associated with internal malignancies. Immunohistochemistry can demonstrate a distinct hybrid endothelial-histiocytic phenotype of littoral cells, and is a helpful adjunct for making the correct diagnosis. The aims of this study were to present a series of 25 LCAs, with an emphasis on the frequent association of the neoplasm with visceral malignancies, and to provide a detailed immunohistochemical analysis by employing new markers. METHODS AND RESULTS: All 25 cases with available tissue blocks were immunohistochemically stained for endothelial and histiocytic markers. Clinical and follow-up data were retrieved from the respective institutions. The tumours were obtained from 16 males and nine females, whose age ranged from 32 to 86 years (mean 56.2 years). Clinical information was available for 24 of 25 patients, and follow-up for 11 of 25 patients (range 2-19 years; mean 11.6 years). Immunohistochemically, all cases were positive for LYVE-1, factor VIII, FLI-1, vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, claudin-5, ERG, LMO2, CD31, CD163, lysozyme, and CD4, but negative for D2-40, CD8, and factor XIIIa. Fifteen of 25 cases were associated with various malignancies, including epithelial, mesenchymal and haematological tumours. CONCLUSIONS: The cohort of 25 patients is the largest series of LCAs published to date. By using antibodies against recently introduced endothelial markers, we have expanded the immunoprofile of LCA. We have further highlighted the clinical significance of LCA, as more than half of the patients in this study also harboured a coexisting visceral malignancy. Therefore, we conclude that the finding of splenic LCA mandates a thorough clinical evaluation for a concomitant malignancy.

Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat.

In the inbred SHR/OlaIpcv rat colony, we identified males with small testicles and inability to reproduce. By selectively breeding their parents, we revealed the infertility to segregate as an autosomal recessive Mendelian character. No other phenotype was observed in males, and females were completely normal. By linkage using a backcross with Brown Norway strain, we mapped the locus to a 1.2Mbp segment on chromosome 7, harboring 35 genes. Sequencing of candidate genes revealed a G to A substitution in a canonical 'AG' splice site of intron 37 in Sbf1 (SET binding factor 1, alias myotubularin-related protein 5). This leads to either skipping exon 38 or shifting splicing one base downstream, invariantly resulting in frameshift, premature stop codon and truncation of the protein. Western blotting using two anti-Sbf1 antibodies revealed absence of the full-length protein in the mutant testis. Testicles of the mutant males were significantly smaller compared with SHR from 4weeks, peaked at 84% wild-type weight at 6weeks and declined afterward to 28%, reflecting massive germ cell loss. Histological examination revealed lower germ cell number; latest observed germ cell stage were round spermatids, resulting in the absence of sperm in the epididymis (azoospermia). SBF1 is a member of a phosphatase family lacking the catalytical activity. It probably modulates the activity of a phosphoinositol phosphatase MTMR2. Human homozygotes or compound heterozygotes for missense SBF1 mutations exhibit Charcot-Marie-Tooth disease (manifested mainly as progressive neuropathy), while a single mouse knockout reported in the literature identified male infertility as the only phenotype manifestation.

Progranulin Is Associated with Disease Activity in Patients with Rheumatoid Arthritis.

OBJECTIVE: Progranulin (PGRN) is implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to assess the relationship between PGRN and disease activity in RA. METHODS: PGRN levels were evaluated in patients with RA (n = 47) and OA (n = 42) and healthy controls (n = 41). Immunohistochemical analysis of PGRN in synovial tissues was performed. The association between PGRN and C-reactive protein (CRP), disease activity score (DAS28-CRP), and health assessment questionnaire (HAQ) was studied. RESULTS: Circulating PGRN was elevated in patients with RA and OA compared to healthy controls (227.1 ± 100.2 and 221.5 ± 102.5 versus 128.1 ± 34.7 ng/mL; P < 0.001). Synovial fluid levels of PGRN were higher in patients with RA compared to OA (384.5 ± 275.3 versus 241.4 ± 165.2 ng/mL; P = 0.002). PGRN expression was significantly upregulated in the synovial tissue of RA patients particularly in the inflammatory infiltrates. Serum PGRN levels correlated with DAS28 (r = 0.327, P = 0.049) and HAQ score (r = 0.323, P = 0.032), while synovial fluid PGRN correlated only with HAQ (r = 0.310, P = 0.043) in patients with RA. PGRN levels were not associated with CRP or autoantibodies. CONCLUSIONS: This study demonstrates increased PGRN expression at local sites of inflammation and association between PGRN levels, disease activity, and functional impairment in patients with RA.

Orally active epoxyeicosatrienoic acid analog does not exhibit antihypertensive and reno- or cardioprotective actions in two-kidney, one-clip Goldblatt hypertensive rats.

This study examined the effects of a novel orally active 14,15-epoxyeicosatrienoic acid analog (EET-A) on blood pressure (BP) and myocardial infarct size (IS) in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats during sustained phase of hypertension. Between days 31 and 35 after clip placement the rats were treated with EET-A and BP was monitored by radiotelemetry; sham-operated normotensive rats were used as controls. Tissue concentrations of epoxyeicosatrienoic acids served as a marker of production of epoxygenase metabolites. The rats were subjected to acute myocardial ischemia/reperfusion (I/R) injury and IS was determined. We found that EET-A treatment did not lower BP in 2K1C rats and did not alter availability of biologically active epoxygenase metabolites in 2K1C or in sham-operated rats. The myocardial IS was significantly smaller in untreated 2K1C rats as compared with normotensive controls and EET-A reduced it in controls but not in 2K1C rats. Our findings suggest that during the phase of sustained hypertension 2K1C Goldblatt hypertensive rats exhibit increased cardiac tolerance to I/R injury as compared with normotensive controls, and that in this animal model of human renovascular hypertension short-term treatment with EET-A does not induce any antihypertensive and cardioprotective actions.

Pro-inflammatory effects of interleukin-35 in rheumatoid arthritis.

OBJECTIVE: Interleukin-35 (IL-35) is a heterodimeric member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. Expressed in murine Treg cells, IL-35 controls inflammatory diseases in mouse models. However, human IL-35 is expressed in Teff cells rather than in Treg cells and is shown to be upregulated under inflammatory conditions. Our aim was to examine the involvement of IL-35 in the pathogenesis of rheumatoid arthritis (RA). METHODS: Immunohistochemical and immunofluorescence analysis was used to determine the expression and localization of IL-35 and its subunits (p35/EBI3) and IL-35 receptor (IL12Rß2/gp130) in RA, osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Expression of p35/EBI3 subunits and release of inflammatory cytokines upon stimulation with IL-35 were assessed in RA synovial fibroblasts (SFs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Both IL-35 and its subunits were upregulated in RA in comparison with OA or PsA synovium. Using cell-specific markers, p35 and EBI3 were identified in macrophages, dendritic cells, SFs, and T as well as B cells in RA synovium. Both p35 and EBI3 were induced by TNFα in RASFs and PBMCs. IL-35 dose-dependently upregulated release of pro-inflammatory mediators IL-1ß, IL-6 and MCP-1 in PBMCs. While gp130 receptor subunit was upregulated in RA synovium and was expressed in RASFs and PBMCs, there was no difference in IL12Rß2 expression subunit among tissues and its presence in RASFs was lacking. CONCLUSION: Upregulation of IL-35 at sites of inflammation in RA and its pro-inflammatory potential suggests that IL-35 might play an important role in RA pathogenesis.

Endothelin A receptor blocker atrasentan lowers blood pressure by the reduction of nifedipine-sensitive calcium influx in Ren-2 transgenic rats fed a high-salt diet.

BACKGROUND: Our previous experiments demonstrated that selective endothelin A (ETA) receptor blockade had antihypertensive effects in Ren-2 transgenic rats (TGRs), but the mechanisms responsible for this change of blood pressure (BP) have not been explored yet. METHOD: Four-week-old male heterozygous TGRs and their normotensive controls--Hannover Sprague-Dawley (HanSD) rats--were fed high-salt diet (2% NaCl) and were treated with selective ETA receptor blocker atrasentan (5 mg/kg per day) for 8 weeks. At the end of the study, the contribution of principle vasoactive systems was evaluated by the sequential blockade of the renin-angiotensin system (captopril), sympathetic nervous system (pentolinium) and nitric oxide synthase [N-nitro-L-arginine methyl ester (L-NAME)]. The role of calcium influx through L-type voltage-dependent calcium channels in BP maintenance was evaluated using nifedipine. In a separate group of animals, the efficiency of distinct vasodilator systems--prostanoids (blocked by nonselective cyclooxygenase inhibitor indomethacin) and Ca-activated K channels (inhibited by tetraethylammonium)--was also analyzed. RESULTS: Atrasentan attenuated the development of hypertension in heterozygous TGRs, but had no effects in Hannover Sprague-Dawley rats. Moreover, atrasentan moderately attenuated renin-angiotensin system-dependent vasoconstriction, whereas it had no effect on sympathetic vasoconstriction. The nifedipine-sensitive BP component was markedly decreased by atrasentan treatment. In contrast, vasodilatation mediated by nitric oxide, endogenous prostanoids or Ca-activated K channels was reduced in atrasentan-treated TGRs, indicating the absence of compensatory augmentation of endothelin B receptor-mediated vasodilation in these animals. CONCLUSION: BP-lowering effect of chronic atrasentan treatment in TGRs was mainly caused by reduced Ca influx through L-type voltage-dependent calcium channels due to missing ETA receptor-dependent vasoconstriction and attenuated angiotensin II-dependent vasoconstriction.

Microvessel density of mantle cell lymphoma. A retrospective study of its prognostic role and the correlation with the Ki-67 and the mantle cell lymphoma international prognostic index in 177 cases.

The clinical course and therapy of mantle cell lymphoma (MCL) are heterogeneous and often unsatisfactory. Prognostic factors are needed to stratify the patients. Microvessel density (MVD) has prognostic significance in some malignancies. There is little information about the vasculature of MCL, although some antiangiogenic drugs are in use. We studied MVD using systematic uniform random sampling and unbiased counting frames in immunohistochemical reactions with anti-CD34 antibody in pre-therapeutic extramedullary MCL samples of 177 patients. We analyzed the relationship of MVD to overall survival (OS) and progression-free survival (PFS), as well as to proliferative activity (Ki-67), mantle cell lymphoma prognostic index (MIPI), morphological variant, pattern of growth, and localization. MVD varied widely: range 54.6-503.6 vessels/mm(2), median 158.2 vessels/mm(2). Higher MVD was associated with bone marrow infiltration at the time of diagnosis (P = 0.001). High MVD was associated with significantly worse OS (P = 0.04) only in patients treated with non-intensive (conventional) therapy. MVD correlated positively with MIPI scores but not with the proliferation, morphological variant, growth pattern, or localization. Univariate analysis identified a prognostic influence of morphological variant, MIPI, and proliferative activity on OS and PFS and a prognostic influence of bone marrow infiltration at the time of diagnosis on PFS. Multivariate analysis showed prognostic influence of MIPI and proliferative activity on OS and PFS only. In conclusion, this is the first clinicopathological study of MVD of MCL with long-term follow-up showing negative prognostic trends of high MVD in MCL and positive correlation of MVD and MIPI.

The occurrence of fetal microchimeric cells in endometrial tissues is a very common phenomenon in benign uterine disorders, and the lower prevalence of fetal microchimerism is associated with better uterine cancer prognoses.

This is the first study carried out to describe the role of fetal microchimerism (FM) in the pathogenesis of uterine cancer. The prevalence and concentration of male fetal microchimeric cells (FMCs) were examined in endometrial tissues in relation to subtypes of uterine cancer, and the histological grade and stage of the tumor. FM occurrence was analyzed in relation to risk factors, including hypertension, obesity, type 2 diabetes, dyslipidemia, age at cancer diagnosis, and patient pregnancy history. The prevalence and concentration of FMCs were examined in endometrial tissues using real-time polymerase chain reaction, SRY and ß-globin sequences as markers for male fetal FMCs and total DNA. The studied group involved 47 type 1 endometrial cancers, 28 type 2 endometrial cancers, and 41 benign uterine diseases. While the prevalence of FM was decreased only in type 1 endometrial cancer, compared with benign uterine disorders (38.3% vs.70.7%; odds ratio [OR]=0.257, 95% confidence interval [CI]: 0.105 to 0.628, p=0.003), FMC concentrations did not differ within examined groups. The lower FM prevalence was detected in low-grade (grade 1 and grade 2) endometrioid cancer (38.3% vs. 70.7%, OR=0.256, 95% CI: 0.105 to 0.627, p=0.003) and in FIGO 1 tumors (40.7% vs. 70.7%, OR=0.285, 95% CI: 0.120 to 0.675, p=0.004). No correlation between FM prevalence or FMC concentrations and risk factors was demonstrated. A lower prevalence of male FM seemed to be associated with better prognoses in uterine cancer based on tumor subtype, histological grade, and stage of the tumor.

Addition of ET(A) receptor blockade increases renoprotection provided by renin-angiotensin system blockade in 5/6 nephrectomized Ren-2 transgenic rats.

AIMS: There is evidence that in addition to hypertension and hyperactivity of the renin-angiotensin system (RAS), enhanced intrarenal activity of endothelin (ET) system contributes to the pathophysiology and progression of chronic kidney disease (CKD). This prompted us to examine if this progression would be alleviated by addition of type A ET receptor (ETA) blockade to the standard blockade of RAS. MAIN METHODS: Ren-2 transgenic rats (TGR) after 5/6 renal ablation (5/6 NX) served as a model of CKD. For RAS inhibition a combination of angiotensin-converting enzyme inhibitor (trandolapril, 6 mg/L drinking water) and angiotensin II type 1 receptor blocker (losartan, 100 mg/L drinking water) was used. Alternatively, ETA receptor blocker (atrasentan, 5 mg·kg(-1)·day(-1) in drinking water) was added to the combined RAS blockade. The follow-up period was 44 weeks after 5/6 NX, and the rats' survival rate, systolic blood pressure (SBP), proteinuria and indices of renal glomerular damage were evaluated. KEY FINDINGS: The survival rate was at first improved, by either therapeutic regime, however, the efficiency of RAS blockade alone considerably decreased 36 weeks after 5/6 NX: final survival rate of 65% was significantly lower than 91% achieved with combined RAS and ETA receptor blockade. SBP was not affected by the addition of ETA blockade while proteinuria and renal glomerular damage were further reduced. SIGNIFICANCE: Our data show that a combined RAS and ETA receptor blockade exhibits additional beneficial effects on survival rate and the progression of CKD in 5/6 NX TGR, as compared with RAS inhibition alone.

The level of fatty acid-binding protein 4, a novel adipokine, is increased in rheumatoid arthritis and correlates with serum cholesterol levels.

OBJECTIVE: To assess the expression of the novel adipokine Fatty Acid Binding Protein-4 (FABP4) in synovial tissues, serum and the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to study the relationships among FABP4, disease activity and metabolic status. METHODS: FABP4 levels were measured in the serum and synovial fluid of 40 patients with RA and 40 control patients with OA. The disease activity score (DAS28), C-reactive protein (CRP) levels and serum lipids were assessed in patients with RA. Immunohistochemical analysis and confocal microscopy were used to study the expression and cell-specific distribution of FABP4 in synovial tissues. RESULTS: The age, sex and body mass index (BMI) adjusted levels of FABP4 were significantly higher in the serum (p=0.001) and synovial fluid (p=0.005) of patients with RA when compared to OA patients. FABP4 levels were higher in females than in males and correlated positively with body mass index (BMI) in patients with RA. Independent of confounders, FABP4 levels correlated with total cholesterol and LDL cholesterol in patients with RA, but not in OA patients. FABP4 levels were not affected by disease activity. Furthermore, the increased expression of FABP4 that was otherwise restricted to synovial fibroblasts, macrophages and B-cells was noted in RA patients at levels higher than that observed in OA patients. CONCLUSIONS: The observed elevation of FABP4 levels in RA patients and the positive correlation of the adipokine to cholesterol suggest that FABP4 may represent a potential link between RA and the increased risk of atherosclerotic changes.

Antihypertensive and renoprotective actions of soluble epoxide hydrolase inhibition in ANG II-dependent malignant hypertension are abolished by pretreatment with L-NAME.

OBJECTIVE: The present study was performed to investigate in a model of malignant hypertension if the antihypertensive actions of soluble epoxide hydrolase (sEH) inhibition are nitric oxide (NO)-dependent. METHODS: ANG II-dependent malignant hypertension was induced through dietary administration for 3 days of the natural xenobiotic indole-3-carbinol (I3C) in Cyp1a1-Ren-2 transgenic rats. Blood pressure (BP) was monitored by radiotelemetry and treatment with the sEH inhibitor [cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyl-oxy]-benzoic acid (c-AUCB)] was started 48 h before administration of the diet containing I3C. In separate groups of rats, combined administration of the sEH inhibitor and the nonspecific NO synthase inhibitor [Nω-nitro-L-arginine methyl ester (L-NAME)] on the course of BP in I3C-induced and noninduced rats were evaluated. In addition, combined blockade of renin-angiotensin system (RAS) was superimposed on L-NAME administration in separate groups of rats. After 3 days of experimental protocols, the rats were prepared for renal functional studies and renal concentrations of epoxyeicosatrienoic acids (EETs) and their inactive metabolites dihydroxyeicosatrienoic acids (DHETEs) were measured. RESULTS: Treatment with c-AUCB increased the renal EETs/DHETEs ratio, attenuated the increases in BP, and prevented the decreases in renal function and the development of renal damage in I3C-induced Cyp1a1-Ren-2 rats. The BP lowering and renoprotective actions of the treatment with the sEH inhibitor c-AUCB were completely abolished by concomitant administration of L-NAME and not fully rescued by double RAS blockade without altering the increased EETs/DHETEs ratio. CONCLUSION: Our current findings indicate that the antihypertensive actions of sEH inhibition in this ANG II-dependent malignant form of hypertension are dependent on the interactions of endogenous bioavailability of EETs and NO.

Soluble epoxide hydrolase inhibition exhibits antihypertensive actions independently of nitric oxide in mice with renovascular hypertension.

OBJECTIVE: The present study was performed to examine whether the blood pressure (BP)-lowering effects of soluble epoxide hydrolase (sEH) inhibition in two-kidney, one-clip (2K1C) Goldblatt hypertension are nitric oxide (NO) dependent. METHODS: Mice lacking the endothelial NO synthase (eNOS) gene (eNOS-/-) and their wild-type controls (eNOS+/+) underwent clipping of one renal artery. BP was monitored by radiotelemetry and the treatment with the sEH inhibitor cis-4-[4-(3-adamantan-1-yl-ureido)cyclohex-yloxy]-benzoic acid (c-AUCB) was initiated on day 25 after clipping and lasted for 14 days. Renal concentrations of epoxyeicosatrienoic acids (EETs) and their inactive metabolite dihydroxyeicosatrienoic acids (DHETs) were measured in the nonclipped kidney. Renal NO synthase (NOS) activity was determined by measuring the rate of formation of L-[(14)C]citruline from L-[(14)C]arginine. RESULTS: Treatment with the sEH inhibitor elicited similar BP decreases that were associated with increases in daily sodium excretion in 2K1C eNOS+/+ as well as 2K1C eNOS-/- mice. In addition, treatment with the sEH inhibitor increased the ratio of EETs/DHETs in the nonclipped kidney of 2K1C eNOS+/+ as well as 2K1C eNOS-/- mice. Treatment with the sEH inhibitor did not alter renal NOS activity in any of the experimental groups. CONCLUSIONS: Collectively, our present data suggest that the BP-lowering effects of chronic sEH inhibition in 2K1C mice are mainly associated with normalization of the reduced availability of biologically active EETs in the nonclipped kidney and their direct natriuretic actions.

heterogeneity of dna ploidy in endometrial carcinoma: comparison of different tissue samples obtained during diagnosis and treatment.

AIM: Comparison of DNA ploidy status of different tumour tissue samples (fresh/frozen vs. paraffin-embedded; curettage vs. hysterectomy samples) obtained during diagnosis and treatment of patients with endometrial carcinoma. PATIENTS AND METHODS: DNA ploidy status and conventional prognostic parameters were recorded for 74 patients with endometrial carcinoma prospectively. RESULTS: In 59 (79.7%) patients the DNA status was described as diploid in all analyzed tissue samples. The remaining 15 (20.3%) cases were described as DNA aneuploid in at least one of the corresponding tissue samples. The concordance between DNA ploidy status in fresh vs. paraffin-embedded hysterectomy samples as well as curettage vs. hysterectomy paraffin-embedded samples was high (kappa coefficient κ=0.6348, 95% confidence interval CI=0.3673-0.9023, and p=0.6408, 95% CI=0.3977-0.8838), however, the methods are not interchangeable. CONCLUSION: The DNA ploidy discordance observed in our study group seems to document intratumoral heterogeneity that should be expected when applying DNA ploidy status in the clinical management of endometrial carcinoma.