Interaction of the diabetes insipidus locus alleles with the renal 120 kDa protein-encoding gene in rat development

Age-dependant dynamics of the kidney inner medullary 120-kDa protein content in vasopressin-deficient Brattleboro rats with di/di mutant genotype was studied in comparison with WAG rats with genotype and normal vasopressin expression. Age-dependant dynamics of vasopressin content in neurohypophysis of WAG rats was also examined. It was shown that 10-day-old WAG rats were unable to elevate the synthesis of the 120 kDa protein in respond to long-term dehydration, while this tendency was clearly observed in the 15-day-old rats and later in the development. In WAG rats the onset of this specific feature was time correlated with the development of the respond to hydration by the elevation of vasopressin synthesis and release from neurohypophysis into blood. In the di/di rats dehydration had no effect on the kidney 120-kDa protein synthesis in all ages examined. These results point to the interaction between the di alleles and the 120-kDa protein-encoding gene in the course of development.     

Concordance between vasopressin gene expression and growth of walker 256 carcinosarcoma in rats

The growth features of Walker 256 carcinosarcoma in rats of different genotypes were investigated. The experiments has been carried out on rats of the inbred Brattleboro and WAG lines, as well as on their hybrids segregated during congenic translocation of the normal vasopressin gene to the genotype of the Brattleboro rats. Brattleboro rats do not express the vasopressin gene. It has been found that there are only two types of tumor growth dynamics. In rats of the inbred Brattleboro line and in homozygotes di/di, that were segregated by backcrossings of heterozygous offsprings from the original crossbreeding between (WAG × Brattleboro) F₁ × Brattleboro and the individuals with parental Brattleboro genotype, having grown to some extent the tumor regresses and disappears. In hybrid heterozygous siblings of di/+ genotype tumor grows linearly with time and always leads to fatal outcome. It has been found that, in the congenic procedure, the tumor regression trait is stably maintained and persistently inherited in lineage concordantly with the di/di genotype and, in rats with at least one allele of a normally expressed vasopressin gene, continuous and lethal tumor growth is always observed.     

Enhanced glucose tolerance in the Brattleboro rat

[Arg⁸]-vasopressin (AVP) plays a crucial role in regulating body fluid retention, which is mediated through the vasopressin V₂ receptor in the kidney. In addition, AVP is involved in the regulation of glucose homeostasis via vasopressin V_1A and vasopressin V_1B receptors. Our previous studies demonstrated that vasopressin V_1A receptor-deficient (V_1AR−/−) and V_1B receptor-deficient (V_1BR−/−) mice exhibited hyperglycemia and hypoglycemia with hypoinsulinemia, respectively. These findings indicate that vasopressin V_1A receptor deficiency results in decreased insulin sensitivity whereas vasopressin V_1B receptor deficiency results in increased insulin sensitivity. In addition, vasopressin V_1A and vasopressin V_1B receptor double-deficient (V_1ABR−/−) mice exhibited impaired glucose tolerance, suggesting that the effects of vasopressin V_1B receptor deficiency do not influence the development of hyperglycemia promoted by vasopressin V_1A receptor deficiency, and that the blockage of both receptors could lead to impaired glucose tolerance. However, the contributions of the entire AVP/vasopressin receptors system to the regulation of blood glucose have not yet been clarified. In this study, to further understand the role of AVP/vasopressin receptors signaling in blood glucose regulation, we assessed the glucose tolerance of AVP-deficient homozygous Brattleboro (di/di) rats using an oral glucose tolerance test (GTT). Plasma glucose and insulin levels were consistently lower in homozygous di/di rats than in heterozygous di/+ rats during the GTT, suggesting that the blockage of all AVP/vasopressin receptors resulting from the AVP deficiency could lead to enhanced glucose tolerance.     

Changes in the level of specific proteins in the rat kidney during long-term dehydration]

A significant amount of specific proteins are involved in kidney's response to vasopressin. A relative content of 120 kDa protein in the Wistar rat kidney medulla following a 3-day dehydration, was found to be 0.975 +/- 0.037 and after drinking 4% sucrose solution alone--0.871 +/- 0.038. The difference of protein 120 kDa content was not revealed in Brattleboro rats: in the control rats it was 0.814 +/- 0.044, and in the dehydrated ones--0.854 +/- 0.020. The findings suggest that vasopressin directly regulates the 120 kDa protein level in the kidney inner medulla.     

Specific Expression of Gene di (diabetes insipidus) in Homologous Inbred Rat Lines

The diabetes insipidus mutation is displayed in homozygotes in the form of diabetes insipidus with water consumption from 30 to 100% of body weight per day. We developed two inbred sublines of the di/di Brattleboro rats as well as the recombinant inbred subline by integrating genes of August rats into the di/di mutant genome. Changes in the genetic background proved to have no effect on the quantitative parameters of the diabetes insipidus. The intensity of the secondary immune response and the content of tropomyosin in the medulla of the rat kidney can serve as additional marker traits of the di/di genotype.     

The vasopressor and oxytocic activities of the hypothalamus and neurohypophysis influenced by stimulated alpha-adrenergic transmission during dehydration and subsequent rehydration in the white rat

Under conditions of equilibrated water metabolism a single dose of methoxamine increased the content of vasopressin in the hypothalamus as well as that of oxytocin both in the hypothalamus and neurohypophysis. During dehydration the depletion of hypothalamic and neurohypophysial vasopressin was more marked in methoxamine-treated animals; this effect, however, was absent in the neurohypophysis on the 2nd day and in the hypothalamus on the 8th day of water deprivation. After two days of dehydration methoxamine inhibited the decrease of oxytocin content in the hypothalamus; simultaneously (2nd and 4th day of dehydration) it intensified this process in the neurohypophysis. During rehydration methoxamine impaired the renewal of vasopressin both in the hypothalamus and neurohypophysis; this effect was most marked on the 8th day of rehydration. On the contrary, it favoured somewhat the renewal of hypothalamic oxytocin in rehydrated rats (such an event was not found on the 8th day of rehydration). Moreover, methoxamine restrained initially (on the 2nd and 4th day of rehydration) the restoration of neurohypophysial oxytocin stores; following eight days of rehydration an opposite effect was here found. It is concluded that the response of the vasopressinergic and oxytocinergic neurons to alpha-adrenergic stimulation, brought about by using methoxamine as pharmacological tool, seems to be depended on the actual state of water metabolism. Impulses from the osmoreceptors may be therefore of some importance in modifying the change in vasopressin and oxytocin synthesis, transport and release resulting from stimulation of alpha-adrenergic transmission through neural chains including units susceptible to methoxamine.     

Urea transporter expression in aging kidney and brain during dehydration

Aging is commonly associated with defective urine-concentrating ability. The present study examined how the kidney and the brain of senescent (30-mo-old) female WAG/Rij rats respond to dehydration induced by 2 days of water deprivation in terms of urea transporter (UT) regulation. In euhydrated situation, senescent rats exhibited similar vasopressin plasma level but lower urine osmolality and papillary urea concentration and markedly reduced kidney UT-A1, UT-A3, and UT-B1 abundances compared with adult (10-mo-old) rats. Senescent rats responded to dehydration similarly to adult rats by a sixfold increase in vasopressin plasma level. Their papillary urea concentration was doubled, without, however, attaining that of dehydrated adult rats. Such an enhanced papillary urea sequestration occurred with a great fall of both UT-A1 and UT-A3 abundances in the tip of inner medulla and an increased UT-A1 abundance in the base of inner medulla. UT-A2 and UT-B1 were unchanged. These data suggest that the inability of control and thirsted senescent rats to concentrate urine as much as their younger counterparts derives from lower papillary urea concentration. In aging brain, UT-B1 abundance was increased twofold together with a fourfold increase in aquaporin-4 abundance. Dehydration did not alter the abundance of these transporters.     

The neurohypophysial vasopressin content as influenced by modified cholinergic or adrenergic transmission during long-term dehydration in the white rat.

In rats dehydrated up to 12 days the neurohypophysial vasopressin content was determined by Dekański's method. Carbamylcholine inhibited somewhat the vasopressin depletion in the neurohypophysis, but not earlier than under severe dehydration (8th and 12th day). A single dose of atropine given 24 h prior to sacrifice to not dehydrated animals resulted in a diminution of the vasopressin content in the neurohypophysis; in animals dehydrated for four days and parallely atropinized the decrease of the neurohypophyseal vasopressin content was, on the contrary, considerably inhibited. Under severe dehydration, the treatment with atropine did not change the vasopressin stores in the neural lobe. Phenoxybenzamine inhibited the vasopressin depletion in the neural lobe following four days of dehydration. Under severe dehydration, amphetamine potentiated the effect of osmoreceptor stimulation. It is supposed that impulses of osmoreceptor origin are of some importance in determining the vasopressin release following changes of cholinergic or adrenergic transmission.     

Expression of Tropomyosin-Encoding Gene in the Kidney Depends on Functioning of Vasopressin Gene

Brattleboro diabetes insipidus mutant rats and normal WAG rats were subjected to water loading or thirst during 3 days. It was found that tropomyosin-encoding gene expression has a tissue-specific pattern in the kidney. Northern blot and western blot analysis had shown that the main expression of Tpm3(3) takes place in the renal medulla, and its intensity differs in normal and mutant rats. The differences between mutants with an ineffective vasopressin synthesizing system and the rats having an intact vasopressin gene were more distinct under long-lasting dehydration. The ratio between renal medullary tropomyosin of Brattleboro and WAG lines of rats was 39.76 +/- 0.90 versus 18.29 +/- 0.86 under water loading, and 46.12 +/- 2.14 versus 13.83 +/- 0.66 in thirst.     

Are opioid peptides co-localized with vasopressin or oxytocin in the neural lobe of the rat?

Summary The content of vasopressin, oxytocin, neurophysin, leucine-enkephalin, methionine-enkephalin, dynorphin-(1–13), and -neoendorphin in the rat neurohypophysis was measured after different periods of dehydration and after depolarisation of isolated neural lobes and of neurosecretory nerve endings. The rates at which the amount of neurohypophysial hormone and opioid peptides decreased, and the changes in the ratios between the amount of vasopressin or oxytocin and opioid peptide in the neurohypophysis after dehydration and in the incubation medium after depolarization in vitro cast some doubt on, and can be explained by mechanisms other than co-localisation of the different peptides.     

The hypothalamic and neurohypophysial vasopressin and oxytocin content under various states of adrenergic transmission in dehydrated and subsequently rehydrated rats

Rats dehydrated for 8 days and subsequently rehydrated were given intracerebroventricularly (i.c.v.) methoxamine hydrochloride (MX) or dihydroergotamine methanosulphonate (DHE), each in a daily dose of 10 micrograms dissolved in 10 microliter of 0.9% sodium chloride. A single dose of MX injected to normally hydrated animals increased the release of hypothalamic and neurohypophysial vasopressin but did not affect significantly the oxytocic activity in the hypothalamus as well as in the neurohypophysis. Under conditions of dehydration MX did not influence the hypothalamic vasopressin content but it stimulated the neurohypophysial vasopressin depletion. On the contrary, MX distinctly inhibited the decrease of hypothalamic and neurohypophysial oxytocin content in dehydrated animals. In rehydrated animals MX restrained some what the renewal of hypothalamic vasopressin and oxytocin storage but intensified this process in the neurohypophysis. A single dose of DHE decreased the vasopressin content in the hypothalamus as well as the oxytocin content both in the hypothalamus and neurohypophysis. Under conditions of dehydration DHE stimulated the depletion of hypothalamic vasopressin and oxytocin. On the contrary, DHE strongly inhibited the depletion of oxytocin in the neurohypophysis of dehydrated rats. DHE restrained the renewal of hypothalamic vasopressin and oxytocin stores as well as intensified this process in the neurohypophysis of subsequently rehydrated rats.     

Expression of the renin angiotensin system genes in the kidney and heart of ISIAH hypertensive rats

The content of mRNA of renin-angiotensin system (RAS) genes was measured in the kidney and heart of hypertensive ISIAH and normotensive WAG rats using the real-time PCR. A statistically significant decrease in the mRNA level of RAS genes was registered in the kidney of ISIAH rats, including Ren (by 45%), Ace (43%), AT1A (34%), COX-2 (50%). The level of myocardial expression of AT1A decreased by 28% while Ace expression increased by 80%. These results suggest reduction of renal RAS basal activity in the hypertensive ISIAH rats, and therefore this strain of rats may be referred to the group of models of low-renin hypertension. The ISIAH rats were also characterized by a two-fold increase in the connective tissue sodium concentration and also by a small (but statistically significant) increase in plasma sodium concentration (139 ± 0.3 mmol/l versus 136 ± 0.25 mmol/l in WAG rats). These results together with a tendency to a decrease of plasma aldosterone level also support existence of a classical low-renin hypertension in the ISIAH rats. It is suggested that altered function of renal ion channels represents a basis for the development of low renin hypertension in the ISIAH rats. In addition, impairments in renal system of NO synthesis may also contribute to the pathogenesis of arterial hypertension in the ISIAH rats.     

Phenotypic Expression of the Mutant Gene diabetes insipidus in Rats and Criteria of Genotyping by Phenotype

The autosomal semidominant mutant gene di(diabetes insipidus) is manifested in homozygotes in the form of diabetes insipidus with water consumption from 25 to 100% of body weight per day. The heterozygotesdi/+ drink water at a rate higher than 5% but lower than 25%. The level of water consumption in rats with +/+ genotype does not exceed 5% of body weight per day. Segregation analysis of F₁ animals yielded by various crosses showed that genotyping of di/di homozygotes is absolutely reliable at 30% and higher level of the water consumption per day.     

Effect of vasopressin gene expression on the growth of walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WAG rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG × Brattleboro) F₁ hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG × Brattleboro) F₁ rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Reaction of alpha-actinin from renal inner medulla on prolonged dehydration

A western-blot analysis using monoclonal antibodies revealed that a reduction of alpha-actinin occurred in the renal inner medulla under the long-lasting dehydration. The ratio between protein content measured in rats of WAG line being hydrated or after 3-days water deprivation consisted of 52.7+/-6.2 against 23.9+/-3.3 as evaluated in relative units. The alpha-actinin level changes similarly in mutant rats of Brattleboro line not capable of synthesizing vasopressin. It was 57.5+/-4.6 in hydrated animals, and statistically lower in rats being under 3-day water deprivation--26.4+/-5.7 in relative units of protein.     

The vasopressin and oxytocin content in the hypothalamus and neurohypophysis as influenced by reserpine treatment during long-term dehydration in the white rat.

In dehydrated rats both neurohypophysial hormones diminished in hypothalamus as well as in the neurohypophysis. Oxytocin disappearef from the hypothalamus and neurohypophysis at a more rapid rate than vasopressin did. The minimal content of vasopressin and oxytocin in the hypothalamus was observed during 3rd--4th day, but even in extreme dehydration it was found to be relatively high: 65 per cent of vasopressin and 27 per cent of oxytocin as compared with intact animals. At that time the neurohypophysial vasopressin and oxytocin content were almost fully exhausted. In dehydrated and additionally reserpinized animals (10 mg/kg intraperitoneally, then each 48 hr 5 mg/kg of initial body weight) the vasopressin and and oxytocin hypothalamus and neurohypophysis changed in a similar manner. In some experimental groups the decrease of neurohormones in both sites was more marked under reserpine treatment. The drug seems therefore rather to potentiate the effects of physiological stimulation of osmodetectors. So the existence of monoaminergic stimulatory synapses, directly involved in the neural pathway between the osmodetector and the neurosecretory cell, appears to be hardly probable.     

Dynamics of renal medullary vinculin under changing of osmoregulating function

Reduction of vinculin occurs in the renal medulla under the long-lasting dehydration. The protein content measured in inner medulla of rats of the WAG line under hydration was 92.1 +/- 6.3 in relative units, but it was only 77.6 +/- 2.3 after a 3-day water deprivation. The vinculin content in the inner medullar layer from mutant rats of Brattleboro line incapable of synthesizing vasopressin is essentially higher: it is 188.9 +/- 8.5 in hydrated conditions and drops to 148.4 +/- 7.3 under a 3-day dehydration. The same high level of vinculin is in outer medulla from rats of Brattleboro line: 222.1 +/- 11.8 in hydrated animals and 174.9 +/- 11 after a 3-day dehydration. No differences were revealed in response of vinculin to alternative osmoregulating stimulation in cortex in both rat lines.     

Histological and Immunohistochemical Studies of the Neurohypophysis of Primitive Teleosts,the Osteoglossidae

The neurohypophysis of three species of the Osteoglossidae (Osteoglossum bicirrhosum, Scleropages jardini and Heterotis niloticus) was studied histologically. The rostral part of the neurohypophysis abuts on the pars distalis of the adenohypophysis, but does not interdigitate with it. The caudal part of the neurohypophysis is stained densely with aldehyde fuchsin and extensively interdigitates with the pars intermedia, forming a neurointermediate lobe. This organization of the neurohypophysis is intermediate between that of holosteans and that of non-osteoglossomorph teleosts. The median eminence-like features of the rostral part of the neurohypophysis are distinct in the Osteoglossidae in contrast to non-osteoglossomorph teleosts. In Osteoglossum bicirrhosum, immunohistochemical study reveals the presence of somatotropin release-inhibiting factor-like substance in the rostral part of the neurohypophysis. Fibers immunoreactive to anti-arginine vasopressin occur in the neurointermediate lobe almost exclusively. These observations also may support the assumption that the rostral part of the neurohypophysis corresponds functionally to the median eminence. For comparison, the occurrence and distribution of somatotropin release-inhibiting factor-like substance, luteinizing hormone-releasing factor-like substance and arginine vasopressin-like substance in the neurohypophysis of Gymnarchus niloticus are described.     

The hypothalamic and neurohypophysial vasopressin content as influenced by diphenylhydantoin in dehydrated rats.

Rats dehydrated up to 8 days were treated with diphenylhydantoin given intraperitoneally in daily doses of 10 mg/100 g of the initial body weight. The single dose of diphenylhydantoin diminished the vasopressin content in the hypothalamus and neurohypophysis of normally hydrated rats. Under conditions of severe dehydration (8 days), DPH treatment resulted in a more marked decrease of vasopressin in the hypothalamo-neurohypophysial system.