Development of liver fibrosis during aging: effects of caloric restriction.

Liver is the central metabolic organ of the body and diet is considered one of the main environmental factors that can impact on aging liver. In the elderly stage liver function is relatively well conserved although there are a variety of not well defined morphological changes related to liver fibrosis which is commonly associated with an inflammatory state. The aim of this paper is to study these alterations during the physiological process of aging in Wistar rats and also test if caloric restriction (CR) could ameliorate them. As fibrosis is associated to hepatic stellate cell (HSC) function we also analyzed these cells during aging. Livers from five groups of male Wistar rats (3-, 8-, 24-months old ad libitum and 8- and 24-months caloric restricted rats) were used in this study. Histological analysis, expression of genes implicated in liver fibrosis and the status of inflammatory step-pathways as p38 mitogen-activated protein kinase (p38-MAPK), c-Jun N-terminal kinase (JNK) and the nuclear factor kappa B (NFkB) isoforms, p50 and p65, in cytosolic and nuclear fractions were performed. During elderly, associated with morphological change of HSC, there is a progressive increase in collagen deposition due to an inhibition in collagen degradation. Higher expression of cytokines and the activation of inflammatory pathways are associated with aging. CR ameliorates these circumstances being more effective when it started in middle age. In conclusion elderly stage is associated to a mild fibrotic and inflammatory state in the liver which could be ameliorated after CR.

Long-term food restriction prevents ageing-associated central leptin resistance in wistar rats.

AIMS/HYPOTHESIS: Ageing is associated with insulin and leptin resistance in mammals. These alterations might be caused by the increased adiposity associated with ageing, by ageing alone or both. We studied whether leptin resistance occurs at the central level in the Wistar rat and we aimed to discriminate between the effects of ageing from those of the increased adiposity associated with ageing. METHODS: Leptin was infused intracerebroventricularly at a constant rate in young adult, old and old Wistar rats fasted for 3 months, using osmotic pumps. The effects on body weight, daily food intake, Lee index, adiposity and serum leptin values were analysed. The effect of food restriction on the expression of the long form of leptin receptor in the hypothalamus was also studied. RESULTS: Leptin decreased daily food intake and body weight in young and old Wistar rats. With a dose of 10 microg/day similar responses were obtained in young and old rats but with a dose of 0.2 microg/day, only young rats showed decreases in these parameters. Food-restriction in old rats lowered adiposity and serum leptin to values close to those of young rats, recovered responsiveness to i.c.v. administration of leptin at the dose of 0.2 microg/day and increased leptin receptor expression in the hypothalamus. CONCLUSION/INTERPRETATION: Our data show that old Wistar rats have a decreased response to leptin at the central level. Food-restriction recovers leptin responsiveness and increases leptin receptor in the hypothalamus suggesting that adiposity plays a key role in the development of leptin resistance associated with ageing.

Up-regulation of parathyroid hormone-related protein in folic acid-induced acute renal failure.

BACKGROUND: Parathyroid hormone (PTH)-related protein (PTHrP) is present in many normal tissues, including the kidney. Current evidence supports that PTHrP is involved in renal pathophysiology, although its role on the mechanisms of renal damage and/or repair is unclear. Our present study examined the changes in PTHrP and the PTH/PTHrP receptor (type 1) in folic acid-induced acute renal failure in rats. The possible role of PTHrP on the process of renal regeneration following folic acid administration, and potential interaction between angiotensin II (Ang II) and endothelin-1, and PTHrP, were examined in this animal model. METHODS: PTHrP, PTH/PTHrP receptor, ACE, and preproendothelin-1 (preproET-1) mRNA levels in the rat kidney were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and/or RNase protection assay. Immunohistochemistry also was performed for PTHrP, the PTH/PTHrP receptor, and Ang II in the renal tissue of folic acid-injected rats. The role of PTHrP on tubular cell proliferation following folic acid injury was investigated in vitro in rat renal epithelial cells (NRK 52E). PTHrP secretion in the medium conditioned by these cells was measured by an immunoradiometric assay specific for the 1-36 sequence. RESULTS: Using RT-PCR, PTHrP mRNA was rapidly (1 hour) and maximally increased (3-fold) in the rat kidney after folic acid, decreasing after six hours. At 72 hours, renal function was maximally decreased in these rats, associated with an increased PTHrP immunostaining in both renal tubules and glomeruli. In contrast, the PTH/PTHrP receptor mRNA (RNase protection assay) decreased shortly after folic acid administration. Moreover, PTH/PTHrP receptor immunostaining dramatically decreased in renal tubular cell membranes after folic acid. A single subcutaneous administration of PTHrP (1-36), 3 or 50 microg/kg body weight, shortly after folic acid injection increased the number of tubular cells staining for proliferating cell nuclear antigen by 30% (P < 0.05) or 50% (P < 0.01), respectively, in these rats at 24 hours, without significant changes in either renal function or calcemia. On the other hand, this peptide failed to modify the increase (2-fold over control) in ACE mRNA, associated with a prominent Ang II staining into tubular cell nuclei, in the kidney of folic acid-treated rats at this time period. The addition of 10 mmol/L folic acid to NRK 52E cells caused a twofold increase in PTHrP mRNA at six hours, without significant changes in the PTH/PTHrP receptor mRNA. The presence of two anti-PTHrP antibodies, with or without folic acid, in the cell-conditioned medium decreased (40%, P < 0.01) cell growth. CONCLUSIONS: Renal PTHrP was rapidly and transiently increased in rats with folic acid-induced acute renal failure, featuring as an early response gene. In addition, changes in ACE and Ang II expression were also found in these animals. PTHrP induces a mitogenic response in folic acid-damaged renal tubular cells both in vivo and in vitro. Our results support the notion that PTHrP up-regulation participates in the regenerative process in this model of acute renal failure and is a common event associated with the mechanisms of renal injury and repair.

Decreased leptin uptake in hypothalamic nuclei with ageing in Wistar rats.

Leptin interacts with specific receptors in hypothalamic nuclei and modulates energy balance. Growing evidence has shown the association of obesity and hyperleptinaemia with non-insulin-dependent diabetes mellitus and insulin resistance. The aged Wistar rat shows peripheral insulin resistance in the absence of obesity and alterations of glucose homeostasis. However, it is not known whether, in these animals, the leptin action is altered. Here we studied the effect of ageing on plasma leptin concentration and the ability of hypothalamic nuclei to capture i.c.v.-injected digoxigenin-labelled leptin. Our data indicate that 24-month-old animals are hyperleptinaemic. However, daily food intake was greater in old animals, suggesting that they are leptin resistant. Leptin uptake in the hypothalamus was reduced in old rats. This uptake was a receptor-mediated process as demonstrated by displacement. Leptin accumulation in hypothalamic nuclei was partially colocalized with neuropeptide Y fibres. Immunohistochemical and western blot analyses showed a lower amount of the long form of leptin receptors in the hypothalamus of aged rats. Analysis by RT-PCR also demonstrated a decreased expression of leptin receptor mRNA in old animals. We conclude that the lower leptin uptake may be explained, at least in part, by a decreased amount of receptors in hypothalamic neurones of the aged rats.

Parathyroid hormone-related protein as a renal regulating factor. From vessels to glomeruli and tubular epithelium.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) produce similar biological effects through the PTH/PTHrP receptor. Less is known about the physiological role of PTHrP, which was first identified as the agent of the humoral hypercalcemia of malignancy. Despite the widespread production of PTHrP in healthy individuals, the concentration of the protein is below the detectable limit of current assays, suggesting that PTHrP normally functions locally in an autocrine or paracrine manner. Thus, some differences in their biological activities have been described and they may be related to the presence of different receptors. In this regard, a second receptor that binds selectively to PTH has also been found. Recent studies have demonstrated the expression of both PTH/PTHrP receptor and protein in the renal glomeruli. Moreover, there are convincing data that support a direct role of PTH and PTHrP in modulating renal blood flow and glomerular filtration rate. This multifunctional protein, PTHrP, also has a proliferative effect on both glomerular mesangial cells and tubular epithelial cells. Increases in the expression of PTHrP have been observed in several experimental models of nephropathies, suggesting that PTHrP upregulation is a common event associated with the mechanism of renal injury and repair.

Thyrotropin-releasing hormone and its receptor in glia.

The presence of thyrotropin-releasing hormone (Thyroliberin, TRH) and its receptor (TRH-R) in frozen coronal sections of the adult rat spinal cord and neonatal rat astroglial cultures was investigated by means of immunocytochemistry and Western blot using polyclonal antibodies generated against the hormone and monoclonal antibodies originated against discrete sequences of the type 1 rat TRH receptor (TRH-R1). TRH-R1 and TRH are present both in astroglial cells from adult rats and in cultured cells from newborn animals. The localization of TRH and TRH-R1 in nonneuronal cells in the central nervous system may reflect that some of the neurotrophic actions of TRH upon the central nervous system are mediated by glial cells.

Proteína relacionada con la hormona paratiroidea: efectos autocrinos y paracrinos en los vasos sanguíneos y en el glomérulo renal / Protein related to parathyroid hormone: autocrine and effects of blood vessels and renal glomeruli

La hormona paratiroidea (PTH) y la proteína relacionada con la PTH (PTHrP) producen efectos biológicos similares a través del receptor PTH/PTHrP. Poco se sabe del papel fisiológico de la PTHrP, que inicialmente se identificó como el agente responsable de la hipercalcemia asociada a enfermedades malignas. Aunque esta proteína se produce ampliamente en diferentes tejidos, su concentración se encuentra debajo de los límites de detección, hecho que sugiere que en circunstancias fisiológicas actúa en forma autocrina o paracrina. Se han descrito algunas diferencias en los efectos de ambas proteínas posiblemente relacionados con la existencia de diferentes receptores. En este sentido, recientemente se ha descrito un receptor específico para la PTH, el receptor PTH-2. Estudios recientes han demostrado la expresión del receptor PTH/PTHrP y de la PTHrP en el glomérulo renal. Además, existen datos que muestran un efecto directo de PTH y PTHrP sobre el flujo plasmático renal y la filtración glomerular. La PTHrP posee las características de una proteína multifuncional, incluyendo efectos proliferativos sobre las células mesangiales, y se especula que tenga un papel importante en la fisiología y fisiopatología renales (AU)

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.