Effect of suramin on urinary excretion of diabetes-induced glomerular and tubular injury parameters in rats.

Diabetes mellitus causes changes in metabolism of extracellular nucleotides acting through P2 receptors (P2Rs). This affects renal function and may lead to glomerular and tubular disturbances. We measured urinary excretion of nucleotides (ATP, ADP, AMP, UTP, UDP, UMP) in streptozotocin-induced diabetic rats (65 mg/kg, i.p., day 0) and the effects of P2Rs' blockade by suramin (10 mg/kg, i.p., days +7, +14) on glomerular P2×7R expression and urinary excretion of glomerular (albumin, nephrin) and tubular (KIM-1, NGAL) injury markers, electrolytes, and oxidative stress markers (TBARS, 8-OHdG). Concentrations of nucleotides, specific proteins, electrolytes, and oxidative stress markers in 24-h urine samples collected in metabolic cages at days -1, +6 and +20 were measured using ion-paired reversed-phase HPLC, immunoenzymatic and fluorometric methods, and flame photometry, respectively. Expression of KIM-1 and P2×7R was examined by immunohistochemistry or immunoblotting. Diabetes was associated with increased urinary excretion of ATP, ADP, UTP, UDP and glomerular P2×7R expression. Suramin attenuated P2×7R expression but did not affect urinary excretion of nucleotides. Urinary excretion of albumin, nephrin, NGAL, and 8-OHdG were increased in diabetic rats and were not affected by suramin. TBARS was higher in diabetic rats and suramin attenuated the excretion dynamics in this group. KIM-1 excretion was higher in diabetic rats and suramin further increased excretion of KIM-1 in both diabetic and non-diabetic rats. Furthermore, suramin attenuated the diabetes-induced natriuresis and kaliuresis. It is possible that suramin affects both glomerular and tubular functions in diabetic rats.

Molecular machinery providing copper bioavailability for spermatozoa along the epididymial tubule in mouse.

Progressive functional maturation of spermatozoa is completed during the transit of these cells through the epididymis, a tubule structure connecting a testicle to a vas deferens. Epididymal epithelial cells by means of their secretory and absorptive functions determine a highly specialized luminal microenvironment containing multiple organic and inorganic components. The latter include copper ions, which due to their redox properties are indispensable for critical homeostatic processes occurring in spermatozoa floating in different part of epididymis but can be potentially toxic. Main purpose of our study was to determine epididymal region-dependent expression and localization of copper transporters ensuring a tight control of copper concentration in epididymal fluid. We also aimed at identifying proteins responsible for copper uptake by spermatozoa and verifying whether this process is coordinated with copper supply to superoxide dismutase 1 (SOD1), a copper-dependent antioxidant enzyme. Our study identifies two ATPases-ATP7A, ATP7B and Slc31a1, major copper importers/exporters depending on their differential expression on epididymal polarized epithelial cells of the caput, corpus, and cauda. Next, ceruloplasmin seems to be a chief protein transporting copper in the epididymal fluid and providing this biometal to spermatozoa. The entry of copper to germ cells is mediated by Slc31a1 and is correlated with both expressions of copper chaperone for superoxide dismutase (CCS), copper chaperone directly providing copper ions to SOD1 and with the expression and activity of the latter. Our results outline a network of cooperating copper binding proteins expressed in epididymal epithelium and in spermatozoa that orchestrate bioavailability of this microelement for gametes and protect them against copper toxicity.

The key involvement of estrogen receptor ß and G-protein-coupled receptor 30 in the neuroprotective action of daidzein.

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1-10µM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor ß (ERß) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS(∗),4R(∗),9bR(∗))-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR(∗),4S(∗),9bS(∗))-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERß- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERß and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERß without significant changing of the GPR30 protein level. Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERß. These studies uncover key roles of the ERß and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.

Are expression and localization of tight and adherens junction proteins in testes of adult boar affected by foetal and neonatal exposure to flutamide?

Several recent studies have indicated that androgen disruption induced by the administration of anti-androgen flutamide during critical developmental stages results in various reproductive abnormalities, mainly in rodents. However, scarce data are available regarding the alterations caused by this toxicant on cell-cell adhesion molecules. Of note, there is no report on the expression and regulation of tight and adherens junction proteins in the boar. Therefore, the purpose of this study was to analyse whether foetal and neonatal exposure to flutamide affects the expression and distribution of ZO-1, occludin, ß-catenin, and N-cadherin in testes of adult pigs. Moreover, to evaluate whether androgen signal was altered in the boar, testicular levels of testosterone and oestradiol and the expression of androgen receptor were examined. Flutamide (50 mg/kg bw) was injected into pregnant gilts during gestational days 20-28 and 80-88 (GD20, GD80), and into male piglets on postnatal days 2-10 (PD2). In the testes of all flutamide-exposed boars, expressions of ZO-1, N-cadherin and ß-catenin were significantly decreased at mRNA and protein level, whereas expression of occludin was unchanged when compared with the controls. In addition, in severely damaged seminiferous tubules of PD2 pigs, mislocalization of ZO-1, N-cadherin and ß-catenin was observed. Changes in junction protein expressions were accompanied by disturbed intratesticular androgen-oestrogen balance, although androgen receptor expression was not altered. Taken together, these results demonstrate that blockade of androgen action by flutamide during both gestational and neonatal periods affects the expression of ZO-1, N-cadherin and ß-catenin in adult pig testes. Of concern, neonatal window seems to be most critical for the organization of BTB and consequently for normal spermatogenesis in the boar. It is likely that altered expression of junction proteins is related to insufficient testosterone production and/or excessive oestradiol synthesis, which may result from impaired Leydig cell function.

Pathological structure of the kidney from adult mice with mosaic mutation.

The mosaic (Atp7a(mo-ms)) is an X-linked, lethal mutation in mice. In mosaic mutant males, many clinical features characteristic of defective copper metabolism have been observed and they die at the age of 15 days, exhibiting strong similarities to the brindled and macular mutants. About 4% of the mutant males live to sexual maturity and some of them are fertile. In this paper, alterations in the structure of the kidney from adult mutants are described. Owing to an inherited defect of efflux, copper is accumulated in the kidney of the mutants up to a toxic level and this leads to severe damage of the renal cortex. Pathological changes in the kidney mostly affected the structure of the renal corpuscle and renal tubules.

Alterations in kidney morphology in mice with mosaic mutation.

The mosaic (Atp7a(mo-ms)) is an X-linked, lethal mutation in mice. Hemizygous males die at the age of 15 days and they exhibit strong similarities to the brindled and macular mutants. Injection of cupric chloride to mossaic mutants prolongs their life and diminishes the pathological results of mutation. Histochemical analysis of the kidneys from 14-day-old mutant males showed accumulation of copper in the renal cortex of the investigated animals leading to damage of the kidney architecture. A histological profile of the kidneys was defined for four groups of 14-day-old animals: mosaic males ms/-, control males +/-, mosaic males injected with cupric chloride ms/- (Cu), and control males injected with cupric chloride +/- (Cu). Pathological changes were observed in the cortex and in the medulla of the kidneys in both groups of mutants and control males injected with cupric chloride (50 microg of CuCl2 per each individual).

Preferential segregation of marker chromosomes 14 and 18 in mouse recombinant inbred strains derived from the KE and CBA/Kw strains.

The segregation pattern of chromosomes 14 and 18 were analyzed in recombinant inbred strains of mice developed from KE and CBA/Kw strains. The analysis was possible owing to the fact that the C-band on chromosomes 14 of the CBA/Kw strain and that of chromosome 18 of the KE strain show size polymorphism: while the CBA/Kw mice have a small sized C-band on chromosome 14, the KE mice show small C-bands on chromosome 18. Chromosomes were identified by G-banding and FISH. The results show that the chromosomes with small centromeric chromatin segregate preferentially.

[Mice with mottled mutation--a model for defective copper metabolism in humans]. / Myszy z mutacja mottled--model zaburzen metabolizmu miedzi u ludzi.

The group of X-linked mottled (Atp7aMo) mutations in mice is described. A normal gene encodes a copper-binding P-type ATPase. Mutant animals have the disturbance in copper metabolism, hemizygous males (Mo/y) die between 14-18 days of life, heterozygous females (Mo/+) are normal and fertile. This kind of copper metabolic defect is observed also in other animal and in human. In human Menkes disease caused by X-linked Atp7a mutant gene leads to death in early childhood. Because of is 89% of homology between Atp7aMo gene and Atp7a locus in human, mottled mutations are an excellent model for Menkes disease.

The comparison of primordial germ cell populations in embryos of KE and CBA inbred strains of mice.

The changes in primordial germ cell (PGC) populations were compared in embryos of two inbred strains of mice-KE and CBA/Kw on 11, 12, 13 and 14 day of foetal life. The number of PGCs did not differ in 11-day embryos of both strains. In 12-day CBA/Kw embryos the proliferation rate of PGCs was higher and the number of PGC was significantly higher than in 12-day KE embryos. However, on subsequent days the PGC proliferation rate decreased in CBA/Kw but rapidly increased in KE embryos, leading to significantly higher number of PGCs in 14-day KE embryos. These differences in proliferation rate between KE and CBA/Kw PGCs can be regarded as the reason of strong selection against CBA/Kw derived gametes observed in KE<-->CBA aggregation chimaeras.