Vitamin D receptor activators upregulate and rescue podocalyxin expression in high glucose-treated human podocytes.

BACKGROUND: Vitamin D is beneficial in human and experimental chronic kidney disease, the leading cause of which is diabetic nephropathy. Vitamin D through its receptor, VDR, provides renal protection in diabetic nephropathy, but limited data exist about its effect on podocytes. Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. METHODS: We used immortalized human podocytes (human glomerular epithelial cells, HGEC) to assess podocalyxin and nephrin expression after treatment with 1,25-dihydroxyvitamin D3 (calcitriol) and its analogue paricalcitol. The involvement of VDR was investigated by silencing with hVDR-siRNA and ChIP analysis. RESULTS: HGEC exhibit high glucose-mediated downregulation of podocalyxin and nephrin, loss of which has been linked with loss of the permselective renal barrier and proteinuria. Calcitriol and paricalcitol reversed high glucose-induced decrease of nephrin and significantly enhanced podocalyxin expression in podocytes cultured in high glucose. HGEC express VDR and retinoid X receptor (RXR). In the presence of calcitriol and paricalcitol, VDR expression was upregulated and VDR colocalized with RXR in the nucleus. VDR knockdown abolished the protective action of calcitriol and paricalcitol on podocalyxin expression indicating that podocalyxin activation of expression is partly mediated by VDR. Furthermore, VDR specifically regulates podocalyxin expression by bounding to a site upstream of the podocalyxin promoter. CONCLUSION: Vitamin D analogues maintain and, furthermore, re-activate the expression of specialized components of podocytes including podocalyxin, hence they provide protection against loss of the permselective renal barrier, with molecular mechanisms elucidated herein.

Sex differences in the effects of two stress paradigms on dopaminergic neurotransmission.

Sex differences in behavioral and neurobiological responses to stress are considered to modulate the prevalence of some psychiatric disorders, including major depression. In the present study, we compared dopaminergic neurotransmission and behavior in response to two different stress paradigms, the Forced Swim Test (FST) and the Chronic Mild Stress (CMS). Male and female rats were subjected to one session of swim stress for two consecutive days (FST) or to a variety of mild stressors alternating for six weeks (CMS). Subsequently, the tissue levels of dopamine (DA) and its metabolites (HVA and DOPAC) in the hippocampus, the hypothalamus, the prefrontal cortex and the striatum were measured using high-performance liquid chromatography (HPLC). The ratios HVA/DA and DOPAC/DA were also calculated as indices of the dopaminergic activity. Results from the FST determined that males exhibited lower immobility, higher climbing duration and increased dopaminergic activity in the prefrontal cortex and the hippocampus compared to females. CMS induced alterations in sucrose intake in both sexes, while it only decreased dopaminergic activity in the prefrontal cortex of females. These findings show that FST and CMS have different effects on the dopaminergic activity of discrete brain regions depending on the sex of the animal. These data support the growing evidence that females display a differential response and adaptation to stress than males.

In vitro studies on ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid for T2 MRI contrast agent.

Ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid have been investigated as possible constituents of aqueous ferrofluids for biomedical applications and especially for MRI contrast agent. The structural characteristics and the size of the nanoparticles have been analyzed as well as the magnetic properties. In order to evaluate any possible capabilities as a contrast agent, the relaxation time, T2, of hydrogen protons in the colloidal solutions of nanoparticles have been measured in order to gain information on the relaxation behavior compared to other MRI contrast agents. The in vitro cytotoxicity of the obtained magnetic nanoparticles of iron oxide coated with gummic acid was investigated by two separate methods (MTT and FACS analysis) and by using three different normal and transformed cell lines. Our results showed that the synthesized nanoparticles had no toxic effect on any of the cell lines used.

Chronic mild stress impact: are females more vulnerable?

Despite the knowledge that women are more susceptible than men to stress-related mental illness, such as major depression, there is no comprehensive estimation of the role of gender in the detrimental effects of chronic stress that might cause depression. Sex differences regarding the association of behavioral parameters with serotonergic and hypothalamic-pituitary-adrenal axis activities were investigated in the chronic mild stress model of depression. Additionally, the impact of chronic mild stress exposure on an additional/novel short-term stressful procedure, such as the forced swim test was examined in male and female rats. Female rats were found to be more vulnerable to chronic mild stress and that was depicted with disruption of sucrose intake, decreases in open field activity, increased corticosterone levels, alteration in estrous cycle and decreased serotonergic activity in hippocampus and hypothalamus. On the contrary, in males the current chronic mild stress protocol elicited only behavioral changes, such as disruption in sucrose intake and decreased open field activity. Interestingly, in response to forced swim test, females previously subjected to chronic mild stress, were found to cope better by exhibiting increased active behavior in the second forced swim test session and higher hypothalamic serotonergic activity in comparison with corresponding males. On the other hand, males were more affected by previous chronic mild stress exposure and that was manifested by decreased active behavior in the first forced swim test session and increased corticosterone levels following second forced swim test session. These data indicate that although females are more vulnerable in the application of chronic mild stress than males, in response to an additional-novel stressor (forced swim test) they show better response. Therefore, both sex/gender and combination of stressful procedures should be carefully considered in the study of the pathophysiology of stress-related mental illnesses.

Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats.

The forced swim test (FST) has been considered as a pharmacologically valid test of the depressive syndrome in rodents. However, few studies have focused on neurochemical and behavioral responses during FST in both male and female rats. Thus, we investigated certain behavioral and neuroendocrine responses as well as the serotonergic activity after the application of FST in both sexes. Our data show that the duration of immobility was increased in both male and female rats during the 2nd session of the FST. Sex differences are observed in some behavioral responses, such as head swinging that is mostly present in male rats. In female rats FST induced a decrease in serotonergic activity in hippocampus and hypothalamus while in male rats it induced an increase in serotonergic activity in hypothalamus. Corticosterone serum levels were elevated in both sexes. However, hippocampal GR mRNA levels tended to be increased in males and females respectively. Moreover, hypothalamic serotonin (5-HT)1A mRNA levels were decreased in female rats while in male rats hippocampal 5-HT1A mRNA levels were increased. These data have shown that FST induces "depressive like symptoms" in both sexes and provide evidence that sex differences characterize certain behavioral aspects in the FST. Notably, hippocampal and hypothalamic serotonergic activity has been differentially modified in male rats compared with female rats and these neurochemical findings could be relevant to the differentiated expression of 5-HT1A receptor. Hypothalamic-pituitary-adrenal axis activity was also affected by FST application in a sex specific manner. The present results support that FST induced behavioral, neurochemical and neurobiological alterations, which are sex dependent.

Oxotechnetium 99mTcO[SN(R)S][S] complexes as potential 5-HT1A receptor imaging agents.

A series of 99mTcO[SN(R)S][S] complexes carrying the 1-(2-methoxyphenyl)piperazine moiety on the tridentate ligand [SN(R)S] was synthesized. For structural characterization and for in vitro binding assays the analogous oxorhenium complexes were prepared. As demonstrated by appropriate competition binding tests in rat hippocampal preparations, all oxorhenium analogues showed affinity for the 5-HT(1A) receptor binding sites with IC(50) values at the nanomolar range (IC(50)= 5.8-103 nM). All 99mTcO[SN(R)S]/[S] complexes showed significant brain uptake in rats at 2 min p.i. (0.24-1.31% ID). However, a clear correlation between distribution of radioactivity in the brain and distribution of 5-HT(1A) receptors could not be established.

Characterisation of hoxa gene expression in the chick limb bud in response to FGF.

We tested a diffusion gradient model for setting up overlapping domains of Hoxa gene expression in the chick limb bud. The model is based on morphogen production at the limb bud tip where the apical ridge is located and assumes that cells respond to a series of concentration thresholds. Consistent with the model, Hoxa13 gene expression rapidly switches off when the ridge is removed from stage 21/22 buds, while Hoxa11 and Hoxa10 expression is stable; Hoxa13 expression can be initiated and maintained in absence of the ridge by FGF soaked beads; the Hoxa13 domain first expands quickly and then slows up and the size is related to the dose of FGF4. Contrary to the model, addition of FGF4 to early limb buds does not activate Hoxa13 prematurely nor extend the Hoxa13 expression domain proximally. Therefore FGF4 signalling is necessary but not sufficient for Hoxa gene expression in the limb bud.

A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling.

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.

Expression of ptc and gli genes in talpid3 suggests bifurcation in Shh pathway.

talpid3 is an embryonic-lethal chicken mutation in a molecularly un-characterised autosomal gene. The recessive, pleiotropic phenotype includes polydactylous limbs with morphologically similar digits. Previous analysis established that hox-D and bmp genes, that are normally expressed posteriorly in the limb bud in response to a localised, posterior source of Sonic Hedgehog (Shh) are expressed symmetrically across the entire anteroposterior axis in talpid3 limb buds. In contrast, Shh expression itself is unaffected. Here we examine expression of patched (ptc), which encodes a component of the Shh receptor, and is probably itself a direct target of Shh signalling, to establish whether talpid3 acts in the Shh pathway. We find that ptc expression is significantly reduced in talpid3 embryos. We also demonstrate that talpid3 function is not required for Shh signal production but is required for normal response to Shh signals, implicating talpid3 in transduction of Shh signals in responding cells. Our analysis of expression of putative components of the Shh pathway, gli1, gli3 and coupTFII shows that genes regulated by Shh are either ectopically expressed or no longer responsive to Shh signals in talpid3 limbs, suggesting possible bifurcation in the Shh pathway. We also describe genetic mapping of gli1, ptc, shh and smoothened in chickens and confirm by co-segregation analysis that none of these genes correspond to talpid3.

Inhibitory action of BMPs on Pax1 expression and on shoulder girdle formation during limb development.

Pax1 expression in vertebrate limb buds is confined to cells in a discrete anterior proximal domain (Timmons et al. [1994] Development 120:2773-2785; Ebensperger et al. [1995] Anat. Embryol. 191:297-310). In dorsoventral patterning of Drosophila, expression of pox meso, an insect gene with high sequence similarity to Pax1, is repressed by decapentaplegic (dpp) in dorsal mesoderm and, thus, is restricted to a discrete ventral domain (Staehling-Hampton et al. [1994] Nature 372:783-786). In the chick wing, cells expressing a vertebrate homolog of dpp, bone morphogenetic protein 4 (Bmp4), abut the Pax1 domain, suggesting a similar relationship between homologous genes in both vertebrates and invertebrates. Here, we show that two BMPs (BMP4, and BMP2, also highly related to dpp) can repress Pax1 in the developing chick wing. Chick wing bud cells expressing Pax1 give rise to the shoulder girdle. Cells in an equivalent position in the mouse forelimb also express Pax1, and Pax1 mutant mice display shoulder girdle defects. Similarly in chick embryos, girdle defects are produced by treatments with signalling molecules that lead to expression of BMPs, which subsequently reduce Pax1 expression in the limb bud. Recently, BMP4 has been shown to inhibit Pax1 expression in the developing trunk (Monsoro-Burq et al. [1996] Development 122:3607-3616) and Pax9 expression in developing teeth (Neubüser et al. [1997] Cell 90:247-255). Thus, a property of BMPs appears to be to regulate pox meso homologs negatively and, thus, limit their expression domains.

Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb.

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.