Óscar Álvarez-García

Coexpression of MT1 and RORα1 melatonin receptors in the Syrian hamster Harderian gland

Abstract:  Melatonin acts through several specific receptors, including membrane receptors (MT1 and MT2) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT1 and RORα1 in this gland was determined by nested RT‐PCR followed by amplicon sequencing and Western‐blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT1 receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal‐synthesized melatonin. On the contrary, although a RORα1‐immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORα1 on the role of this indoleamine against oxidative stress is discussed.

Melatonin neutralizes neurotoxicity induced by quinolinic acid in brain tissue culture.

Abstract:  Quinolinic acid is a well‐known excitotoxin that induces oxidative stress and damage. In the present study, oxidative damage to biomolecules was followed by measuring lipid peroxidation and protein carbonyl formation in rat brain tissue culture over a period of 24 hr of exposure to this prooxidant agent at a concentration of 0.5 mm. Quinolinic acid enhanced lipid peroxidation in an early stage of tissue culture, and protein carbonyl at a later stage. These data confirm and extend previous studies demonstrating that quinolinic acid can induce significant oxidative damage. Melatonin, an antioxidant and neuroprotective agent with multiple actions as a radical scavenger and signaling molecule, completely prevented these prooxidant actions of quinolinic acid at a concentration of 1 mm. Morphological lesions and neurotoxicity induced by quinolinic acid were evaluated by light microscopy. Quinolinic acid produced extensive apoptosis/necrosis which was significantly attenuated by melatonin. Cotreatment with melatonin exerted a profound protective effect antagonizing the neurotoxicity induced by quinolinic acid. Glutathione reductase and catalase activities were increased by quinolinic acid and these effects were antagonized by melatonin. Furthermore, melatonin induced superoxide dismutase activity. Quinolinic acid and melatonin acted independently and by different mechanisms in modulating antioxidant enzyme activities. Our findings using quinolinic acid and melatonin clearly demonstrate that such changes should always be seen in the context of oxidative neurotoxicity and antioxidant neuroprotection.

Rapamycin induces growth retardation by disrupting angiogenesis in the growth plate

Rapamycin, a potent immunosuppressant used in renal transplantation, has been reported to impair longitudinal growth in experimental studies. Rapamycin is both antiproliferative and antiangiogenic; therefore, it has the potential to disrupt vascular endothelial growth factor (VEGF) action in the growth plate and to interfere with insulin-like growth factor I (IGF-I) signaling. To further investigate the mechanisms of rapamycin action on longitudinal growth, we gave the 4-week-old rats rapamycin daily for two weeks. Compared with a vehicle-treated group, rapamycin-treated animals were severely growth retarded and had marked alterations in the growth plate. Vascular invasion was disturbed in the rapamycin group, there was a significant reduction in osteoclast cells near the chondro-osseus junction, and there was lower VEGF protein and mRNA expression in the terminal chondrocytes of the growth cartilage. Compared with the control group, the rapamycin group had higher levels of circulating IGF-I as well as the mRNAs for IGF-I and of the receptors of IGF-I and growth hormone in the liver but not in the growth cartilage. Thus our findings explain the adverse effect of rapamycin on growth plate dynamics. This should be taken into account when the drug is administered to children.

Inverse correlation between endogenous melatonin levels and oxidative damage in some tissues of SAM P8 mice

Abstract:  To assess whether oxidative damage in some tissues was related to their melatonin concentration, endogenous melatonin levels and the age‐linked protein and lipid damage in spleen, thymus and liver in 5‐month‐old SAM P8 mice were examined. The results show that high levels of melatonin in spleen and thymus correlate with lower protein and lipid damage. The liver, which had much lower melatonin concentrations than the other two tissues, had much higher levels of oxidatively damaged protein, as measured by carbonyl values. These results add new evidence concerning the protective role of endogenous melatonin as an antioxidant agent, and suggest that a treatment with this molecule might help to reduce age‐associated functional deficits in many organs, including those of the immune system.

Growth hormone improves growth retardation induced by rapamycin without blocking its antiproliferative and antiangiogenic effects on rat growth plate.

Rapamycin, an immunosuppressant agent used in renal transplantation with antitumoral properties, has been reported to impair longitudinal growth in young individuals. As growth hormone (GH) can be used to treat growth retardation in transplanted children, we aimed this study to find out the effect of GH therapy in a model of young rat with growth retardation induced by rapamycin administration. Three groups of 4-week-old rats treated with vehicle (C), daily injections of rapamycin alone (RAPA) or in combination with GH (RGH) at pharmacological doses for 1 week were compared. GH treatment caused a 20% increase in both growth velocity and body length in RGH animals when compared with RAPA group. GH treatment did not increase circulating levels of insulin-like growth factor I, a systemic mediator of GH actions. Instead, GH promoted the maturation and hypertrophy of growth plate chondrocytes, an effect likely related to AKT and ERK1/2 mediated inactivation of GSK3β, increase of glycogen deposits and stabilization of β-catenin. Interestingly, GH did not interfere with the antiproliferative and antiangiogenic activities of rapamycin in the growth plate and did not cause changes in chondrocyte autophagy markers. In summary, these findings indicate that GH administration improves longitudinal growth in rapamycin-treated rats by specifically acting on the process of growth plate chondrocyte hypertrophy but not by counteracting the effects of rapamycin on proliferation and angiogenesis.

Alterations of growth plate and abnormal insulin-like growth factor I metabolism in growth-retarded hypokalemic rats: effect of growth hormone treatment.

Hypokalemic tubular disorders may lead to growth retardation which is resistant to growth hormone (GH) treatment. The mechanism of these alterations is unknown. Weaning female rats were grouped (n = 10) in control, potassium-depleted (KD), KD treated with intraperitoneal GH at 3.3 mg x kg(-1) x day(-1) during the last week (KDGH), and control pair-fed with KD (CPF). After 2 wk, KD rats were growth retarded compared with CPF rats, the osseous front advance (+/-SD) being 67.07 +/- 10.44 and 81.56 +/- 12.70 microm/day, respectively. GH treatment did not accelerate growth rate. The tibial growth plate of KD rats had marked morphological alterations: lower heights of growth cartilage (228.26 +/- 23.58 microm), hypertrophic zone (123.68 +/- 13.49 microm), and terminal chondrocytes (20.8 +/- 2.39 microm) than normokalemic CPF (264.21 +/- 21.77, 153.18 +/- 15.80, and 24.21 +/- 5.86 microm). GH administration normalized these changes except for the distal chondrocyte height. Quantitative PCR of insulin-like growth factor I (IGF-I), IGF-I receptor, and GH receptor genes in KD growth plates showed downregulation of IGF-I and upregulation of IGF-I receptor mRNAs, without changes in their distribution as analyzed by immunohistochemistry and in situ hybridization. GH did not further modify IGF-I mRNA expression. KD rats had normal hepatic IGF-I mRNA levels and low serum IGF-I values. GH increased liver IGF-I mRNA, but circulating IGF-I levels remained reduced. This study discloses the structural and molecular alterations induced by potassium depletion on the growth plate and shows that the lack of response to GH administration is associated with persistence of the disturbed process of chondrocyte hypertrophy and depressed mRNA expression of local IGF-I in the growth plate.

Differential gene expression induced by growth hormone treatment in the uremic rat growth plate

OBJECTIVES Treatment with growth hormone (GH) improves growth retardation of chronic renal failure. cDNA microarrays were used to investigate GH-induced modifications in gene expression in the tibial growth plate of young rats. DESIGN RNA was extracted from the tibial growth plate from two groups, untreated and treated with GH, of young rats made uremic by subtotal nephrectomy (n=10). To validate changes shown by the Agilent oligo microarrays, some modulated genes known to play a physiological role in growth plate metabolism were analyzed by real-time quantitative polymerase chain reaction (qPCR). RESULTS The microarrays showed that GH modified the expression of 224 genes, 195 being upregulated and 29 downregulated. qPCR results confirmed the sense of expression change found in the arrays for insulin-like growth factor I, insulin-like growth factor II, collagen V alpha 1, bone morphogenetic protein 3 and proteoglycan type II. CONCLUSIONS This study shows for the first time the profile of growth plate gene expression modifications caused by GH treatment in experimental uremia and provides a basis to further investigate selected individual genes with potential implication in the stimulating effect on the growth of GH treatment in chronic renal failure.

Sirolimus and growth

In this issue of the journal, Hymes and Warshaw publish an interesting article on growth of 25 pediatric renal transplant recipients on sirolimus (SRL) treatment (1). They did not find any difference in the height outcome during a 24-month period between two groups of kidney-transplanted patients treated with either SRL or tacrolimus and matched for sex, age, prednisone dose, renal function, and degree of initial growth retardation. Fifty-two percent of patients treated with SRL exhibited a positive linear velocity Z-score, and the mean age of this subgroup of children being five yr less than those whose height Z-score worsened at the end of the follow-up period. The authors point out that their data indicate that SRL does not adversely affect growth. This is the second published series analyzing the effect of SRL on growth in pediatric renal transplant recipients. Recently, Gonzalez et al. (2) reported the findings of a multicenter observational study that retrospectively analyzed the growth pattern of 34 kidney-transplanted children changed to SRL treatment because of nephrotoxicity, chronic allograft nephropathy, malignancy, acute rejection, tacrolimus-induced glucose intolerance, or diabetes. This group was compared with the other group not treated with SRL matched for age, gender, renal function, and dose of corticosteroids. According to this report, SRL exerted an adverse effect on growth as demonstrated by lower growth velocity and smaller change in height SD in the SRL group after six, 12, and 24 months of treatment. In two matched subgroups of prepubertal children (n = 11 per group) with a glomerular filtration rate greater than 50 mL/min/1.73 m and prednisone dose lower than 5 mg/day, the delta height and longitudinal growth velocity were again worse in children treated with SRL. These two studies that led to conflicting results were based on a small number of patients, and the clinical and biochemical data were retrospectively obtained. Thus, definitive conclusions cannot be reached on whether or not SRL accounts for an additional adverse factor on growth of patients with a kidney transplant. A prospective well-designed multicenter study is required to clarify this issue. SRL has a unique mechanism of action that is different from classic calcineurin inhibitors. It specifically binds to FKBP12 and interferes with mammalian target of rapamycin (mTOR) signaling (3). mTOR is a serine/threonine kinase that has a central role in the regulation of cell growth and metabolism (4). In mammals, mTOR is found as two different complexes, mTORC1 (SRL-sensitive) and mTORC2 (SRL-insensitive) (5). mTORC1 activation, either by growth factors, cytokines, nutrients or high ATP/AMP ratio, results in cell proliferation, protein synthesis, and cell growth (6). On the contrary, mTORC1 inhibition results in cell cycle arrest, protein synthesis reduction, and induction of autophagy. SRL immunosuppressant effects are mainly attributed to blockade of mTORC1 in T and B lymphocytes, leading to reduced interleukin-driven proliferation and differentiation of these cells (7). In addition to its antiproliferative and immunosuppressive activities, SRL is a potent inhibitor of angiogenesis. SRL has been reported to significantly decrease protein and mRNA levels of vascular endothelial growth factor (VEGF) and also the endothelial cell response to this angiogenic factor (8). Chondrocyte proliferation and differentiation within the epiphyseal growth plate as well as vascular invasion of the growth cartilage are essential processes for physiological bone formation during endochondral ossification. SRL has the potential to alter growth plate function and impair longitudinal bone growth in young individuals as a result of its marked antiproliferative and antiangiogenic activities. In addition, the growth hormone (GH) GH – insulin-like growth Pediatr Transplantation 2011: 15: 546–547 2011 John Wiley & Sons A/S.

The impact of sirolimus on sex hormones in male adolescent kidney recipients

Sirolimus is being used in pediatric renal transplantation as a rescue agent for acute and chronic graft rejection as well as for primary immunosuppression (1). Sirolimus treatment, in combination with other immunosuppressive agents, has been reported to improve renal function and to decrease the elevated risk of developing cancer in renal transplanted patients. In comparison with calcineurin inhibitors, sirolimus also has less adverse effects with respect to neurotoxicity, arterial hypertension, and posttransplant diabetes mellitus. Reports in transplanted adults have suggested that sirolimus may affect gonadal function. Kaczmarek et al. (2) in a pair-matched analysis on heart-transplanted men showed a significant decrease in serum testosterone and a significant increase in serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in the sirolimus treated group. These effects were trough level-dependent. These findings have been confirmed in adult renal transplant recipients (3, 4). Lee et al. (5) in a cross-sectional study on 66 male kidney transplants found a significant negative correlation between sirolimus trough levels and testosterone levels. Zuber et al. (6) in an observational study on male kidney transplant between 20 and 40 yr showed that patients treated with sirolimus had a significant reduced total sperm count, motile spermatozoa, and low fathered pregnancy rate. The effect of sirolimus treatment on the gonadal function of pediatric patients is largely unknown. In the previous issue, Cavanaugh et al. (7) report the circulating concentrations of testosterone, FSH, and LH in 15 male adolescent kidney transplant patients at onset and one and two yr after starting sirolimus. Serum testosterone values physiologically increase during puberty up to achieve a normal range between 500 and 700 ng/dL in the early adulthood (8). In the group of patients studied by Cavanaugh et al., testosterone concentrations after two yr on sirolimus (349 ng/dL) were not higher than at the beginning of the treatment, and almost 50% of patients experienced a decrease in testosterone levels during the study period; in two patients, testosterone levels fell by more than 100 ng/dL (7). Time on sirolimus was found to be independently and positively correlated with the decrease in serum testosterone. Glomerular filtration rate remained stable and above 50 mL/min/1.73 m in all but two patients and did not correlate with the testosterone concentrations. The circulating levels of FSH and LH did not significantly change over the study period. The pathophysiology of the decreased testosterone production induced by sirolimus remains to be clarified. In addition to its potent antiproliferative action, various molecular events occurring intracellularly after mammalian target of rapamycin (mTOR) inhibition might be potentially responsible. Spermatogonial proliferation and differentiation requires signaling through phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which is inhibited by sirolimus. In addition, sirolimus decreases the sensibility of gonadotrophin-releasing hormone (GnRH) receptors and, thus, blunts the effect of GnRH (9). The study by Canaugh et al. (7) is retrospective and does not have a control group, and the assessment of puberty and sexual maturation is not strictly documented. However, with these important limitations in mind, it calls attention on the potential effect of sirolimus on gonadal function of kidney-transplanted adolescent male patients with quite good renal function. Hypogonadism will interfere with a normal sexual development and may contribute to the impairment of longitudinal growth reported in some of these patients (10). A prospective well-designed study addressed at analyzing the impact of sirolimus on gonadal function of pubertal men having a kidney graft is needed. Pediatr Transplantation 2012: 16: 310–311 2012 John Wiley & Sons A/S.