G. Li Volti

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal α-intercalated cell’s apical H+-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in “sporadic” cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

Cyanidin and cyanidin 3-O-β-D-glucoside as DNA cleavage protectors and antioxidants

Anthocyanins, colored flavonoids, are water-soluble pigments present in the plant kingdom; in fact they are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. Present in beans, fruits, vegetables and red wines, considerable amounts of anthocyanins are ingested as constituents of the human diet (180–215 mg daily). There is now increasing interest in thein vivo protective function of natural antioxidants contained in dietary plants against oxidative damage caused by free radical species. Recently, the antioxidant activity of phenolic phytochemicals, has been investigated. Since the antioxidant mechanism of anthocyanin pigments is still controversial, in the present study we evaluated the effects of cyanidin and cyanidin 3-O-β-D-glucoside on DNA cleavage, on their free radical scavenging capacity and on xanthine oxidase activity. Cyanidin and cyanidin 3-O-β-D-glucoside showed a protective effect on DNA cleavage, a dose-dependent free radical scavenging activity and significant inhibition of XO activity. These effects suggest that anthocyanins exhibit interesting antioxidant properties, and could therefore represent a promising class of compounds useful in the treatment of pathologies where free radical production plays a key role.

Expression of pannexin2 protein in healthy and ischemized brain of adult rats

The expression pattern of the pannexin2 protein (Px2) in healthy and ischemized brains of adult rats was investigated. A polyclonal antibody for rat Px2 was generated in chicken and purified for affinity. This antibody was used to study by Western blot, Enzyme-Linked Immunosorbent Assay, and immunohistochemistry, the expression pattern of Px2 in healthy brain of adult rats and in the hippocampus of rats submitted to bilateral clamping of carotid arteries for 20 min, followed by different times of reperfusion (I/R) (8 h, 24 h, 48 h, 72 h, 14 days and 30 days). Immunohistochemical studies visualized the wide and complex expression pattern of Px2 in the healthy brain. All Px2(+) positive cells were neurons which also showed no puncta on their cellular membranes. Both pyramidal cells and interneurons, the majority of which were positive to parvalbumin, were stained in healthy hippocampus. The number of Px2 interneurons in the hippocampus showed a progressive reduction at successive time intervals after I/R, with a negative peak of about -40% after 72 h from I/R. Interneurons which were positive for both Px2 and parvalbumin, represented about the 85% of all parvalbumin cells stained in the hippocampus. This percentage rested grossly unmodified at different time intervals after I/R in spite of the progressive neuronal depletion. Concomitantly, an intense astrogliosis occurred in the hippocampus. Most of the astroglial cells expressed de novo and for a transient time (from 24 h to 14 days from I/R), Px2. Primary co-cultures of hippocampal neurons and astrocytes were submitted to transient ischemia-like injury. This set of experiments further confirmed the in vivo results by showing that Px2 is de novo and transiently expressed in astroglial cells following a transient ischemia-like injury. These results suggested the expression of Px2 in the astrocytes may be induced either from injured neurons or by biochemical pathways internal to the astrocyte itself. In conclusion, our results showed the transient expression of Px2 in astrocytes of reactive gliosis occurring in the hippocampus following I/R injury. We hypothesize that Px2 expression in astrocytes following an ischemic insult is principally involved in the formation of hemichannels for the release of signaling molecules devoted to influence the cellular metabolism and the redox status of the surrounding environment.

Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells.

Although the precise role of transglutaminase in cell death is unknown, several findings demonstrate that tissue transglutaminase selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Calcium‐dependent transglutaminase reactions are also implicated in several neurodegenerative diseases, including alterations in the release of excitatory amino acids. One prevalent theme in cell damage induced by excitotoxic stimuli in different regions of the CNS is that apoptosis may be executed by intracellular caspase proteases. Furthermore, the presence of functional ion channel‐gated receptors in glial cells suggests that also astrocytes can be susceptible to glutamates toxic effects. In this study, we demonstrated that prolonged exposure to glutamate (100 μM) of cultured astrocytes caused an increase in the expression of tissue transglutaminase (tTG). This effect was prevented by preincubation with GYKI 52466, an antagonist of AMPA/KA receptors. Glutamate exposure also promoted an increase in caspase‐3 compared with control cultures. Confocal laser microscopy analysis demonstrated the presence of activated caspase‐3 in the cytoplasm as well as in the nucleus. The inhibition of TG‐catalyzed reactions by cystamine (1 mM) blocked the activation pathway of caspase‐3, with an evident reduction of enzyme cleavage. These results suggest that glutamate increased both TG and caspase‐3 in astroglial cells early in the excitotoxin‐induced events.

New-Onset Diabetes Mellitus After Kidney Transplantation: The Role of Immunosuppression

BACKGROUND Complications related to posttransplantation immunosuppressive therapy remain common. New-onset diabetes mellitus after transplantation (PTDM) is a well-recognized complication associated with reduced graft and patient survival. The type of immunosuppression may be responsible for more than two thirds of PTDM. We retrospectively reviewed our experience in a population of 284 kidney transplant recipients, evaluating the incidence of PTDM with regard to the type of immunosuppression. PATIENTS AND METHODS From January 2001 to December 2005, 284 kidney transplantations were performed using tacrolimus-based (TAC) immunosuppression in 192 patients and a cyclosporine-based (CyA) regimen in 62 patients, whereas 30 patients received sirolimus-based immunosuppression. RESULTS The overall incidence of PTDM was 4.9%. Among the immunosuppression protocols, 8 patients (4.1%) received TAC and 6 patients (9.6%) received CyA, whereas no patients treated with sirolimus developed PTDM. Graft and patient survival rates were 93% and 100%, respectively. CONCLUSIONS The overall risk of PTDM with recent immunosuppressive protocols is low, but it is increased among calcineurin inhibitor (CNI)-treated kidney transplant recipients. Sirolimus did not increase the risk of PTDM, allowing potential clinical application in diabetic recipients and in patients affected by PTDM.

Glutamate-evoked redox state alterations are involved in tissue transglutaminase upregulation in primary astrocyte cultures

The aim of this study was to evaluate the involvement of oxidative stress in glutamate‐evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose‐dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor‐mediated, as demonstrated by inhibition with GYKI 52466. The pre‐incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate‐increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate.

Excitotoxic and post-ischemic neurodegeneration: Involvement of transglutaminases

Summary.Neurodegeneration induced by excitotoxicity is a common feature in various neurological disorders. This pathological condition is caused by prolonged stimulation of glutamate receptor subtypes, followed by both intracellular Ca2+ overload and activation of specific genes, resulting in synthesis of enzymes involved in cell stress response.Using experimental in vitro models of excitotoxicity, we demonstrated that glutamate exposure up-regulated tissue transglutaminase in primary cultures of both cerebellar granule cells and astrocytes. These changes were consequent to receptor-mediated Ca2+ influx, as demonstrated by the inhibition with selective antagonists, MK-801 and GYKI 52466. Early increases in different transglutaminase isoforms were also observed in global cerebral ischemia, which closely resembles neuronal damage caused by NMDA receptor activation.These findings agree with a postulated role for transglutaminases in molecular mechanisms of several neurodegenerative diseases. Indeed, increased cross-linking reactions could be of pathologic relevance, as part of biochemical changes observed in neurological disorders.

Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase‐1 (HO‐1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17β‐estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve‐ or twenty‐four‐hour epidermal growth factor (EGF) treatment significantly enhanced HO‐1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO‐1 expression was obtained after the last‐12‐hr EGF treatment in 48‐hr E2‐pretreated astrocyte cultures; this enhancement was particularly significant in 48‐hr E2‐pretreated cultures as well as in the last‐12‐hr insulin‐treated ones pretreated for 48 hr with E2. Sixty‐hour DEX‐alone pretreatment as well as the last‐12‐hr EGF treatment in 60‐hr DEX‐pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last‐12‐hr insulin‐like growth factor‐I (IGF‐1)‐treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF‐1 in 48‐hr E2‐pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO‐1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.

Effect of Choline-Containing Phospholipids on Transglutaminase Activity in Primary Astroglial Cell Cultures

The aim of the present investigation was to study the effects of choline and choline-containing phospholipids CDP-choline (CDPC) and L-alpha-glyceryl-phosphorylcholine (AGPC) on transglutaminase (TG) activity and expression in primary astrocyte cultures. TG is an important Ca2+-dependent protein that represents a normal constituent of nervous systems during fetal stages of development, playing a role in cell signal transduction, differentiation, and apoptosis. Confocal laser scanning microscopy (CLSM) analysis showed an increase of TG activity in astrocyte cultures treated with choline, CDPC, or AGPC at 0.1 μM or 1 μM concentrations. Comparatively, AGPC induced the most conspicuous effects enhancing monodansyl-cadaverine fluorescence both in cytosol and in nuclei, supporting the evidence of the important role played by AGPC throughout differentiation processes tightly correlated to nucleus-cytosol cross- talk during astroglial cells proliferation and development. Western blot analysis showed that in 24h 1 μM AGPC and choline-treated astrocytes increased TG-2, whereas no effect was observed in 24h 1 μM CDP-choline treated astrocytes. Our data suggest a crucial role of choline precursors during different stages of astroglial cell proliferation and differentiation in cultures.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures.

Type‐2 transglutaminase (TG‐2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin‐like growth factor–I (IGF‐I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron–glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17β‐estradiol (E2) on TG‐2 activity and their expression in cultured astrocytes. We observed a significant increase in TG‐2 activity and expression in astroglial cells treated for 24 hr with IGF‐I, EGF, or INS. Priming of the cells with DEX or E2, for 48 hr also led to an increase in TG‐2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG‐2 expression and activity and a different subcellular TG‐2 distribution were found. Our data indicate that steroid hormone–GF interaction may play an important role in astroglial function. The effect on TG‐2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.