Morphological, Morphometrical and Histochemical Study of the Lining and Glandular Epithelia of the Tongue of the Bullfrog Rana catesbeiana

The dorsal surface of the tongue of the bullfrog, Rana catesbeiana, has simple columnar epithelium with a few ciliated cells and goblet cells. The entire surface is covered with numerous filiform papillae and few fungiform. Filiform papillae have a simple columnar epithelium with secretory cells, while the fungiform have a sensory disc on their upper surface the lined by a stratified columnar epithelium with basal, peripheral, glandular and receptor cells. Over the dorsal lingual surface there are numerous winding tubular glands, which penetrate deeply into the muscle of the tongue, mingling with the fibers. The gland epithelium is cylindrical with secretory and supporting cells. The first are absolute on the basis of the gland and the latter are rare in the upper third. The ventral surface of the tongue is lined by a stratified epithelium, with the presence of goblet cells, with ciliated cells among them. Morphometrically, lingual glands varies in length, according to their location: shorter in the anterior region of the tongue (330 mu m) than in the posterior region (450 mu m). Secretory cells of the anterior lingual glands are smaller (1457.7 mm(3)) than the posterior ones (2645.9 mu m(3)). The same can be said of the cell nuclei, 130.0 mu m(3) for the anterior glands and 202.3 mu m(3) for the posterior ones. Secretory cells of the lingual glands contain substances rich in protein and neutral mucopolysaccharides, which characterize the seromucous type. Goblet cells of the dorsal and ventral surface epithelia secrete neutral mucopolysaccharides and proteins, and can be characterized as type G1 cells, and the supporting cells of the superficial glands of the fungiform papillae secrete a mucus rich in neutral mucopolysaccharides, sulfomucins and sialomucins.

Persistent Impairment of Testicular Histology and Sperm Motility in Adult Rats Treated with Cisplatin at Peri-Puberty

Cisplatin is one of the most widely used and effective chemotherapeutic agents for the treatment of several human malignancies. This study evaluated the effects of peri-pubertal cisplatin administration on several reproductive end-points and the reversibility of these effects in adulthood. Peri-pubertal Wistar male rats (45 days old) were divided into two groups: control (saline 0.9%) and cisplatin (1 mg/kg/day, 5 days/week, for 3 weeks, i.p.). The study was conducted in two steps and evaluations were performed at ages of 66 (post-pubertal age) and 140 (adult age) days on: (i) organ weights, serum gonadotropins and testosterone levels, sperm counts, motility and morphology, testicular histomorphometry, spermatogenesis kinetics, Sertoli cell number and in situ detection of apoptotic germ cells and (ii) sexual behaviour, fertility and intratesticular testosterone. At the end of cisplatin therapy, rats showed reductions in sperm production and reserves, sperm with progressive movement, tubular diameter, intratesticular testosterone and fertility potential, but increased numbers of TUNEL-positive seminiferous tubules, immotile sperm and pre-implantation losses compared with control. Moreover, cisplatin-treated post-pubertal rats displayed impaired testicular histopathology and sexual behaviour. Serum gonadotropins and testosterone levels, sperm morphology, spermatogenesis kinetics and Sertoli cell number were comparable between experimental groups at both ages. Alterations found in post-puberty were recovered at adulthood, except for sperm motility and damage to testicular histology. The persistence of these cisplatin effects, despite the unaltered fertility after natural mating in rats, may have implications for reproductive function of young boys undergoing cancer therapy, given the lower reproductive efficiency in human beings compared with rats.

Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days. which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V-max of brush-border membrane (BBM) Na/Pi cotransport activity (1943 95 in control vs. 1183 +/- 99 pmol/5 sec/mg BBM protein in K deficiency. P < 0.02). Surprisingly. the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1). and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin. glucosylceramide. and ganglioside GM, content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficit nt rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 11.52 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Analytical validation for a series of marker compounds used to assess renal drug elimination processes

Renal drug elimination is determined by glomerular filtration, tubular secretion, and tubular reabsorption. Changes in the integrity of these processes influence renal drug clearance, and these changes may not be detected by conventional measures of renal function such as creatinine clearance. The aim of the current study was to examine the analytic issues needed to develop a cocktail of marker drugs (fluconazole, rac-pindolol, para-aminohippuric acid, sinistrin) to measure simultaneously the mechanisms contributing to renal clearance. High-performance liquid chromatographic methods of analysis for fluconazole, pindolol, para-aminohippuric acid, and creatinine and an enzymatic assay for sinistrin were developed or modified and then validated to allow determination of each of the compounds in both plasma and urine in the presence of all other marker drugs. A pilot clinical study in one volunteer was conducted to ensure that the assays were suitable for quantitating all the marker drugs to the sensitivity and specificity needed to allow accurate determination of individual renal clearances. The performance of all assays (plasma and urine) complied with published validation criteria. All standard curves displayed linearity over the concentration ranges required, with coefficients of correlation greater than 0.99. The precision of the interday and intraday variabilities of quality controls for each marker in plasma and urine were all less than 11.9% for each marker. Recoveries of markers (and internal standards) in plasma and urine were all at least 90%. All markers investigated were shown to be stable when plasma or urine was frozen and thawed. For all the assays developed, there were no interferences from other markers or endogenous substances. In a pilot clinical study, concentrations of all markers could be accurately and reproducibly determined for a sufficient duration of time after administration to calculate accurate renal clearance for each marker. This article presents details of the analytic techniques developed for measuring concentrations of marker drugs for different renal elimination processes administered as a single dose to define the processes contributing to renal drug elimination.

Simultaneous administration of a cocktail of markers to measure renal drug elimination pathways: absence of a pharmacokinetic interaction between fluconazole and sinistrin, p-aminohippuric acid and pindolol

Aims Previous studies suggest that estimated creatinine clearance, the conventional measure of renal function, does not adequately reflect charges in renal drug handling in some patients, including the immunosuppressed. The aim of this study was to develop and validate a cocktail of markers. to be given in a single administration, capable of detecting alterations in the renal elimination pathways of glomerular filtration, tubular secretion and tubular reabsorption. Methods Healthy male subjects (n = 12) received intravenously infused 2500 mg sinistrin (glomerular filtration) and 440 mg p-aminohippuric acid (PAH; anion secretion), and orally administered 100 mg fluconazole (reabsorption) and 15 mg rac-pindolol (cation secretion). The potential interaction between these markers was investigated in a pharmacokinetic study where markers (M) or fluconazole (F) were administered alone or together (M + F). Validated analytical methods were used to measure plasma and urine concentrations in order to quantify the renal handling of each marker. Plasma protein binding of fluconazole was measured by ultrafiltration. All subjects had an estimated creatinine clearance within the normal range. The renal clearance of each marker (Mean +/- s.d.) was calculated as the ratio of the amount excreted in urine and thearea-under-the-concentration-time curve. Statistical comparisons were made using a paired t-test and 95% confidence intervals were reported. Results The renal clearances of sinistrin (M: 119 +/- 31 ml min(-1); M + F: 130 +/- 40 ml min(-1); P = 0.32), PAH (M: 469 +/- 145 ml min(-1); M + F: 467 +/- 146 ml min(-1); P = 0.95), R-pindolol (M: 204 +/- 41 ml min(-1); M + F: 190 +/- 41 ml min(-1); P = 0.39; n = 11), S-pindolol (M: 225 +/- 55 ml min(-1); M + F: 209 +/- 60 ml min(-1); P = 0.27; n = 11) and fluconazole (F: 14.9 +/-3.8 ml min(-1); M + F: 13.6 +/- 3.4 ml min(-1); P = 0.16) were similar when the markers or fluconazole were administered alone (M or F) or as a cocktail (M + F). Conclusions This study found no interaction between markers and fluconazole in healthy male subjects, suggesting that a single administration of this cocktail of markers of different renal processes call be used to simultaneously investigate pathways of renal drug elimination.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.

Interacting roles of myofibroblasts, apoptosis and fibrogenic growth factors in the pathogenesis of renal tubulo-interstitial fibrosis

The interrelationship between myofibroblasts and fibrogenic growth factors in the pathogenesis of renal fibrosis is poorly defined. A temporal and spatial analysis of myofibroblasts, their proliferation and death, and presence of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-B (PDGF-B) was carried out in an established rodent model in which chronic renal scarring and fibrosis occurs after healed renal papillary necrosis (RPN), similar to that seen with analgesic nephropathy. Treated and control groups (N = 6 and 4, respectively) were compared at 2, 4, 8 and 12 weeks. A positive relationship was found between presence of tubulo-interstitial myofibroblasts and development of fibrosis. Apoptotic myofibroblasts were identified in the interstitium and their incidence peaked 2 weeks after treatment. Levels of interstitial cell apoptosis and fibrosis were negatively correlated over time (r = -0.57, p < 0.01 ), suggesting that as apoptosis progressively failed to limit myofibroblast numbers, fibrosis increased. In comparison with the diminishing apoptosis in the interstitium, the tubular epithelium had progressively increasing levels of apoptosis over time, indicative of developing atrophy of nephrons. TGF-beta1 protein expression had a close spatial and temporal association with fibrosis and myofibroblasts, whilst PDGF-B appeared to have a closer link with populations of other chronic inflammatory cells such as infiltrating lymphocytes. Peritubular myofibroblasts were often seen near apoptotic cells in the tubular epithelium, suggestive of a paracrine toxic effect of factor/s secreted by the myofibroblasts. In vitro , TGF-beta1 was found to be toxic to renal tubular epithelial cells. These findings suggest an interaction between myofibroblasts, their deletion by apoptosis, and the presence of the fibrogenic growth factor TGF-beta1 in renal fibrosis, whereby apoptotic deletion of myofibroblasts could act as a controlling factor in progression of fibrosis.

Renal pathology of polycystic kidney disease and concurrent hereditary nephritis in Bull Terriers

Objective To describe the renal lesions in Bull Terrier polycystic kidney disease (BTPKD), to confirm that the renal cysts in BTPKD arise from the nephron or collecting tubule, an to identify lesions consistent with concurrent BTPKD and Bull Terrier hereditary nephritis (BTHN). Design Renal tissue from five Bull Terriers with BTPKD and eight control dogs was examined by light and transmission electron microscopy. Clinical data were collected from all dogs, and family history of BTPKD and BTHN for all Bull Terriers. Results In BTPKD the renal cysts were lined by epithelial cells of nephron or collecting duct origin that were usually squamous or cuboidal, with few organelles. They had normal junctional complexes, and basal laminae of varying thicknesses. Glomeruli with small, atrophic tufts and dilated Bowman's capsules, tubular loss and dilation, and interstitial inflammation and fibrosis were common. Whereas the lesions seen in BTHN by light microscope were nonspecific, the presence of characteristic ultrastructural glomerular basement membrane (GMB) lesions and a family history of this disease indicated concurrent BTHN was likely in three of five cases of BTPKD. Conclusion This paper provides evidence that renal cysts in BTPKD are of nephron or collecting duct origin. In addition, GBM lesions are described that strongly suggest that BTPKD and BTHN may occur simultaneously.

Comparative sperm ultrastructure of the Baikalian endemic prosobranch gastropods

Mature euspermatozoan ultrastructure is described for seven species of the rissooidean family Baicaliidae (endemic to Lake Baikal, Russia)-Liobaicalia stiedae, Teratobaikalia ciliata, T. macrostoma, Baicalia carinata, Pseudobaikalia pulla, Maackia bythiniopsis, M. variesculpta, and M. herderiana. For comparison with these species and previously investigated Rissooidea, two species of the Lake Baikal endemic genus Benedictia (B. cf. fragilis and B. baicalensis; Hydrobiidae: Benedictiinae of some authors, Benedictiidae of other authors) in addition to Lithoglyphus naticoides (Hydrobiidae: Lithoglyphinae) and Bythinella austriaca (Hydrobiidae: Bythinellinae) were also investigated. Paraspermatozoa were not observed in any of the species examined, supporting the view that these cells are probably absent in the Rissooidea. In general, the euspermatozoa of all species examined resemble those of many other caenogastropods (basally invaginated acrosomal vesicle, mid-piece with 7-13 helical mitochondria, an annulus, glycogen piece with nine peri-axonemal tracts of granules). However, the presence of a completely flattened acrosomal vesicle and a specialized peri-axonemal membranous sheath (a scroll-like arrangement of 4-6 double membranes) at the termination of the mid-piece, clearly indicates a close relationship between the Baicaliidae and other rissooidean families possessing these features (Bithyniidae, Hydrobiidae, Pyrgulidae, and Stenothyridae). Euspermatozoa of Benedictia, Lithoglyphus, Bythinella, and Pyrgula all have a solid nucleus, which exhibits a short, posterior invagination (housing the centriolar complex and proximal portion of the axoneme). Among the Rissooidea, this form of nucleus is known to occur in the Bithyniidae, Hydrobiidae, Truncatellidae, Pyrgulidae, Iravadiidae, Pomatiopsidae, and Stenothyridae. In contrast, the euspermatozoa of the Baicaliidae all have a long, tubular nucleus, housing not only the centriolar derivative, but also a substantial portion of the axoneme. Among the Rissooidea, a tubular nuclear morphology has previously been seen in the Rissoidae, which could support the view, based on anatomical grounds, that the Baicaliidae may have arisen from a different ancestral source than the Hydrobiidae. However, the two styles of nuclear morphology (short, solid versus long, tubular) occur widely within the Caenogastropoda, and sometimes both within a single family, thereby reducing the phylogenetic importance of nuclear differences within the Rissooidea. More significantly, the occurrence of the highly unusual membranous sheath within the mid-piece region in the Baicaliidae appears to tie this family firmly to the Bithyniidae + Hydrobiidae + Stenothyridae + Pyrgulidae assemblage. Eusperm features of Benedictia spp. strongly resemble those of hydrobiids and bithyniids, and neither support recognition of a distinct family Benedictiidae (at best this is a subfamily of Hydrobiidae) nor any close connection with the hydrobiid subfamily Lithoglyphinae.

GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway

Endocytosis of cell-surface proteins via specific pathways is critical for their function. We show that multiple glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed to the recycling endosomal compartment but not to the Golgi via a nonclathrin, noncaveolae mediated pathway. GPI anchoring is a positive signal for internalization into rab5-independent tubular-vesicular endosomes also responsible for a major fraction of fluid-phase uptake; molecules merely lacking cytoplasmic extensions are not included. Unlike the internalization of detergent-resistant membrane (DRM)-associated interleukin 2 receptor, endocytosis of DRM-associated GPI-APs is unaffected by inhibition of RhoA or dynamin 2 activity. Inhibition of Rho family GTPase cdc42, but not Rac1, reduces fluid-phase uptake and redistributes GPI-APs to the clathrin-mediated pathway. These results describe a distinct constitutive pinocytic pathway, specifically regulated by cdc42.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Análise Imuno-histoquímica de hipóxia renal em modelo de isquemia/reperfusão tratado com Fator Estimulador de Colônia de Granulócitos

O rim demonstra uma capacidade singular em reparar-se após danos locais, no entanto, depois de acometido, as chances de desenvolvimento de lesões renais elevam-se. A patofisiologia da isquemia/reperfusão (IR) é complexa porque há ocorrência simultânea de danos celulares e inflamação. O decréscimo na quantidade de oxigênio requer um sistema capaz de evitar seus efeitos prejudiciais e uma maquinaria molecular HIF (Hypoxia Inducible Factor), um complexo, atua como fator de transcrição de diversos genes desde os da regulação da proliferação celular e apoptose até a sinalização para angiogênese. O Fator Estimulador de Colônia de Granulócitos (G-CSF) é uma glicoproteína conhecida pela sua capacidade de promover a sobrevivência, proliferação e diferenciação de células estimulando a recuperação aos efeitos advindos da IR. Com o intuito de observar as influências dessas proteínas foi realizada uma análise semi-quantitativa de amostras renais submetidas ou não à IR, usando-se descrições microscópicas morfológicas e imunohistoquímicas, com os cálculos e gráficos estatísticos foram feitos no software GraphPad Prism®. Das análises morfológicas, constatou-se que as lesões características de IR foram observadas em espécimes não tratados: bolhas em epitélio tubular; vacuolização citoplasmática, distalização tubular e congestão luminal. De forma análoga, foi encontrada nos tratados, contudo em estágios menos avançados e em animais controle, não foi houve esta diferença tissular. As análises de microscopia eletrônica demonstraram alteração na barreira filtrante com concomitante perda de outras características glomerulares. Aos animais controle foi observada a arquitetura típica, ao passo que para os animais tratados notou-se conservação da barreira. A presença de HIF-1α nos rins contralaterais demonstrouse significante quando comparadas às amostras isquêmicas e tratadas (p<0,05). Já a ocorrência da mesma proteína em rins isquêmicos não apresentou qualquer diferença. Analisando-se a proteína VEGF foi comprovado que em rins contralaterais não há diferença estatística, contudo nos rins esquerdos há significância entre os três grupos (p<0,05). Já a correlação entre estas duas proteínas não se mostrou estatisticamente significante. Em relação às atividades de proliferação e morte celulares, todos os três grupos foram significantes entre si (p<0,05). Ao que concerne o tratamento, foi demonstrada a atividade protetora do medicamento e uma possível interação molecular com a HIF, enquanto que a ativação desta proteína corrobora sua rota metabólica já previamente descrita.