Irina Yu. Morina

Introduction: Uremic rats exhibit elevations of Na/K-ATPase inhibitor Marinobufagenin (MBG). The study investigated whether MBG-induced hypothalamic collagen-1 production occurs in uremic rats. Because choroid plexus cells are involved in transporting substances from the cerebrospinal fluid, the effects of MBG on claudin-1 and claudin-7 were also studied. Whether increased MBG would be associated with plasma procollagen type I C-peptide in patients with chronic kidney disease was also examined. Methods: Male Sprague-Dawley rats underwent 5/6 nephrectomy. 12 patients with chronic kidney disease undergoing hemodialysis were compared to 6 healthy controls. Results: In the hypothalamus and aorta of rats that underwent 5/6 nephrectomy, uremia was associated with a decrease in Fli1 expression and an increase in collagen 1 production. Consistent with these findings in animals, elevated plasma levels of procollagen type I C-peptide (170±7 ng/ml) in 12 patients with chronic kidney disease undergoing hemodialysis, compared to 6 healthy controls (111±10 ng/ml), were observed. In uremic rats treated with anti-MBG antibodies, Fli1 and collagen 1 expression did not change. Uremic rats develop hypothalamic fibrosis, which was associated with the inhibition of claudin-1 and claudin-7 (50%, P < 0.01 for both) in the choroid plexus of the third ventricle and inhibition of Na/K-ATPase in the brainstem; and administration of anti-MBG antibody prevents these effects. Na/K-ATPase in the brainstem was inhibited by 60% while the Na/K ratio increased. When uremic rats were pretreated with anti-MBG antibody, Na/K-ATPase activity did not change, and the Na/K ratio decreased compared to that of uremic rats. Discussion: The study’s data demonstrate that the kidney-brain axis, i.e., activation of peripheral MBG and brain fibrosis, and claudin activation contribute to the pathogenesis of renal failure. Conclusion: The main findings of this study are that the CTS (i) affects blood-brain permeability via mechanisms including inhibition of Na/K-ATPase, that (ii) the blood-CSF barrier is regulated by claudins 1 and 7, which are linked to the Na/K ratio in the brain, and (iii) that synthesis of collagen-1 in the hypothalamus comprises a central element in controlling these mechanisms. CKD in rats is associated with central production of collagen-1 and the effect of MBG on brain claudins; antibodies to MBG reverse these pathological changes. These results suggest the importance of the kidneybrain axis in the pathophysiology of kidney diseases.

BACKGROUND: Rodent models of polycystic ovary syndrome, including those induced by dehydroepiandrosterone, are widely used to study the pathogenesis of this disorder; however, ovarian steroidogenesis and ovulation markers in this model remain insufficiently characterized. There are no data on the ability of luteinizing hormone receptor agonists to stimulate ovulation in rats with this model of polycystic ovary syndrome, which is important for assisted reproductive technologies. AIM: This study aimed to investigate ovarian steroidogenesis, ovulation markers, and ovarian morphology in infantile female rats with dehydroepiandrosterone-induced polycystic ovary syndrome, and to assess the effects of the allosteric agonist TP03, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide, on these parameters. METHODS: Female Wistar rats, starting at 23–26 days of age, were treated with dehydroepiandrosterone for 6 weeks (6 mg/100 g/day, subcutaneously). The following parameters were assessed: body and ovarian weight, serum levels of testosterone, progesterone, and estradiol (enzyme-linked immunosorbent assay), ovarian gene expression (reverse-transcription polymerase chain reaction), and ovarian morphology (number of tertiary and preovulatory follicles, corpora lutea, and follicular cysts) by microscopy. Rats with polycystic ovary syndrome were stimulated with an ovulation-inducing drug at a dose of 15 IU/rat, followed by TP03 at a dose of 25 mg/kg 48 h later; the studied parameters were evaluated 16 h later. RESULTS: In dehydroepiandrosterone-induced rats, ovarian weight, the number of tertiary follicles and corpora lutea of previous cycles, progesterone levels, and the expression of genes responsible for steroidogenesis (Star, Cyp11a1, Cyp17a1) and ovulation (Adamts-1, Vegf-a) were decreased, whereas testosterone levels were increased and follicular cysts were detected in the ovaries, consistent with the pathogenesis of polycystic ovary syndrome. Treatment with ovulation-inducing drugs increased ovarian weight, progesterone levels, and Cyp17a1 gene expression, and stimulated ovulation, leading to the formation of corpora lutea. Luteinized unruptured follicles were also detected in the ovaries; this finding was associated with decreased expression of the ADAMTS-1 metalloproteinase and VEGF-A genes involved in follicular rupture. CONCLUSION: Treatment of infantile rats with dehydroepiandrosterone-induced polycystic ovary syndrome using TP03 induces ovulation; however, along with normal corpora lutea, luteinized unruptured follicles are formed as a result of decreased expression of genes involved in follicular rupture.

Polycystic ovary syndrome (PCOS) is associated with impaired ovarian steroidogenesis and ovulation, which necessitates the development of effective ovulation inducers for PCOS. The aim of the study was to evaluate the effects of allosteric luteinizing hormone receptor agonist TP03 and human chorionic gonadotropin (hCG) on ovarian steroidogenesis, as well as ovulation in prepubertal female rats with dehydroepiandrosterone(DHEA)-induced PCOS. Taking into account differences in progesterone levels, cohorts with high (PCOS(H)) and low (PCOS(L)) progesterone were formed and treated with Follimag and Cetrotide. After 48 h, TP03 (25 mg/kg) or hCG (25 IU/rat) were injected, and hormone levels, gene expression, and ovarian morphology were assessed. The PCOS(H)-cohort exhibited irregular estrous cycles, ovarian cysts, and increased ovarian mass and estradiol levels, but the number of corpora lutea (CL) was maintained. In the PCOS(L)-cohort, ovarian weight was increased, and Star, Cyp11a1, and Adamts1 gene expression as well as the CL number were decreased. In both cohorts, TP03 and hCG increased progesterone levels and the expression of steroidogenesis (Star, Cyp11a1) and ovulation (Cox2, Adamts1, Egr1) genes, as well as inducing CL formation. Thus, TP03, like hCG, stimulates steroidogenesis and ovulation in PCOS-rats with different progesterone levels, which provides the first evidence of the effectiveness of allosteric LHR agonists as ovulation triggers in PCOS.

Dopamine is one of the most important neurotransmitters for regulating retinal function and adjusting vision to the diurnal cycle. It exerts its regulatory effects, in part, through the cAMP pathway. Previous studies have demonstrated that dopamine affects phototransduction in amphibian rods, and that elevated intracellular levels of cAMP modulate the function of vertebrate rods and cones. Lamprey, the most primitive vertebrate, could be valuable for studying the evolution of dopamine regulatory loops in the retina. We examined whether the photoresponse properties of long (cone-like) and short (rod-like) photoreceptors in the river lamprey could be regulated by dopamine via the cAMP pathway. Using suction pipette recording, we demonstrated that forskolin-induced elevation of cAMP has no effect on long or short photoreceptors. At the same time, dopamine modifies the photoresponse properties of long, but not short, photoreceptors at high, potentially non-physiological concentrations. Immunohistochemical analysis of the lamprey retina revealed the expression of both D1 and D2 dopamine receptors in lamprey photoreceptors; however, their distribution differs from jawed vertebrates. Taken together, our results suggest that, in lampreys, dopamine does not regulate photoreceptor sensitivity to light in the circadian rhythm, but, rather, adjusts other retinal functions based on widespread distribution of its receptors.

Optogenetics is a promising technology for treating retinal degenerative diseases. This work aims to develop a behavioral test using the Transformer Maze to assess visual function in rodent retinal optogenetics experiments. The dark-light cross test, featuring 4 central and 8 peripheral compartments under different lighting conditions, was used. The test showed that sighted animals spent comparable time in the central and peripheral compartments under equal illumination, preferring the periphery or center when darkened. Blind animals (tunicamycin model) explored boundaries regardless of lighting. One month after intravitreal injections of AAV-7m8-CAG-Rhod-Venus, animals spent similar time in the periphery and center. This phenomenon is hypothesized to be linked to the initiation of rhodopsin expression in ganglion cells, activating the cortex. These signals are not yet recognized by the animal but alter its strategy compared to blindness. Thus, the new test enables non-invasive detection of the onset of expression.

The vertebrate retina uses neurotransmitters to regulate its various functions and adjust vision according to the day/night cycle. Dopamine is probably one of the most important of these neurotransmitters. It is released by dopaminergic amacrine cells in the retina and exerts its regulatory effects, in part, through the cAMP pathway. It has been demonstrated that dopamine affects the phototransduction cascade in isolated amphibian rods. Furthermore, elevated intracellular levels of cAMP increase the light sensitivity of vertebrate rods and modulate the response of vertebrate cones. These effects can be triggered by dopamine receptors and adjust vision to daily light variations. Therefore, the evolution of dopamine loops in the retina is of interest, and the lamprey, being the most primitive vertebrate, could be valuable in this regard. In the present study, we examined whether the photoresponse properties of long (cone-like) and short (rod-like) photoreceptors in the river lamprey could be regulated by dopamine or cAMP level modulation. Using suction pipette recording, we demonstrated that dopamine slightly increased short photoreceptors sensitivity and it slowed the rising and falling phases of photoresponses in long photoreceptors and increased the integration time, with no effect on the sensitivity to brief flashes. The second part of our study — an immunohistochemical analysis of the lamprey retina — revealed that both D1 and D2 dopamine receptors are expressed in both types of lamprey photoreceptors. Our results suggest that the regulation of photoreceptor functions by the neurotransmitter dopamine originated in the early stages of vertebrate evolution, specifically during the Cambrian period. Summary: The lamprey, a primitive vertebrate, is a valuable object for studying the evolution of dopamine loops in the vertebrate retina. This study shows that photoresponse properties of lamprey photoreceptors are regulated by dopamine in a different way compared to gnathostomes.

The approaches to correct thyroid deficiency include replacement therapy with thyroid hormones (THs), but such therapy causes a number of side effects. A possible alternative is thyroid-stimulating hormone (TSH) receptor activators, including allosteric agonists. The aim of this work was to study the effect of ethyl-2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate (TPY3m), a TSH receptor allosteric agonist developed by us, on basal and thyroliberin (TRH)-stimulated TH levels and the hypothalamic-pituitary-thyroid (HPT) axis in male rats with high-fat diet/low-dose streptozotocin-induced type 2 diabetes mellitus (T2DM). Single and three-day administration of TPY3m (i.p., 20 mg/kg) was studied, and the effect of TPY3m on the HPT axis was compared with that of levothyroxine. TPY3m increased TH levels when administered to both healthy and diabetic rats, normalizing thyroxine and triiodothyronine levels in T2DM and, unlike levothyroxine, without negatively affecting TSH levels or the expression of hypothalamic and pituitary genes responsible for TSH production. TPY3m pretreatment preserved the stimulatory effects of TRH on TH levels and thyroid gene expression. This indicates the absence of competition between TPY3m and endogenous TSH for TSH receptor activation and is supported by our in vitro results on TPY3m- and TSH-stimulated adenylate cyclase activity in rat thyroid membranes. Morphological analysis of thyroid glands in diabetic rats after three-day TPY3m administration shows an increase in its functional activity without destructive changes. To summarize, TPY3m, with the activity of a partial allosteric agonist of the TSH receptor, was created as a prototype of drugs to correct thyroid insufficiency in T2DM.

BACKGROUND: Gonadotropin preparations, particularly human chorionic gonadotropin (hCG), are commonly used to induce ovulation and treat reproductive disorders in women, albeit with associated side effects. Low-molecular-weight allosteric agonists of the luteinizing hormone receptor (LHR), such as thieno[2,3-d]pyrimidine derivatives, offer a potential alternative. AIM: This study aims to compare the effects of thieno[2,3-d]pyrimidine TP03 and hCG on ovarian weight, corpus luteum formation, and plasma levels of estradiol, progesterone, and luteinizing hormone in immature female rats pre-treated with Follimag®. It also examines their impact on ovarian gene expression related to LHR and steroidogenesis. MATERIALS AND METHODS: TP03 and hCG were administered 48 h after the Follimag® injection at a dose of 20 mg/kg (i.p.) and 15 IU/rat (s.c.), respectively. Parameters were assessed at 1, 2, 4, 8, 16, and 24 h after TP03 and hCG administration. Plasma hormone levels were measured via ELISA, and ovarian gene expression was analyzed using real-time PCR. RESULTS: TP03 increased ovarian weight, progesterone levels in the blood, and expression of steroidogenic genes encoding the cholesterol-transporting protein StAR and the cytochromes CYP11A1 and CYP17A1. TP03 also stimulated corpus luteum formation (16–24 h after treatment). The temporal dynamics of its stimulating effects were similar to those of hCG, although their magnitude was slightly inferior to those of gonadotropin. TP03-induced decrease in blood estradiol levels and aromatase gene expression in the ovaries was also more moderate. Unlike hCG, which suppressed LHR gene expression 8 h after treatment, TP03 maintained a high LHR gene expression, preserving ovarian sensitivity to endogenous luteinizing hormone. CONCLUSIONS: TP03 exhibits potential as an ovulation inducer with milder stimulating effects on ovarian steroidogenesis than hCG, which reduces the risks of developing ovarian hyperstimulation syndrome and resistance to gonadotropins.

Gonadotropins, including human chorionic gonadotropin (hCG), are used to induce ovulation, but they have a number of side effects, including ovarian hyperstimulation syndrome (OHSS). A possible alternative is allosteric luteinizing hormone (LH)/hCG receptor agonists, including the compound TP4/2 we developed, which remains active when administered orally. The aim was to study the effectiveness of TP4/2 (orally, 40 mg/kg) as an ovulation inducer in FSH-stimulated immature female rats, compared with hCG (s.c., 15 IU/rat). TP4/2 stimulated progesterone production and corpus luteum formation; time-dependently increased the ovarian expression of steroidogenic genes (Star, Cyp11a1, Cyp17a1) and genes involved in ovulation regulation (Adamts-1, Cox-2, Egr-1, Mt-1); and increased the content of metalloproteinase ADAMTS-1 in the ovaries. These effects were similar to those of hCG, although in some cases they were less pronounced. TP4/2, in contrast to hCG, maintained normal LH levels and increased the ovarian expression of the LH/hCG receptor gene, indicating preservation of ovarian sensitivity to LH, and did not cause a sustained increase in expression of vascular endothelial growth factor-A involved in OHSS. Thus, TP4/2 is an effective ovulation inducer that, unlike hCG, has a lower risk of OHSS and ovarian LH resistance due to its moderate stimulating effect on steroidogenesis.

BACKGROUND: In men, obesity is accompanied by a complex of metabolic and hormonal disorders, which leads to androgen deficiency and impaired spermatogenesis. Antidiabetic drugs, including metformin (MF), and luteinizing hormone receptor (LHR) agonists, which activate testicular steroidogenesis, can be used to correct reproductive dysfunctions. However, in diet-induced obesity (DIO), their effectiveness and mechanisms of action are poorly understood.AIM: In men, obesity is accompanied by a complex of metabolic and hormonal disorders, which leads to androgen deficiency and impaired spermatogenesis. Antidiabetic drugs, including metformin (MF), and luteinizing hormone receptor (LHR) agonists, which activate testicular steroidogenesis, can be used to correct reproductive dysfunctions. However, in dietinduced obesity (DIO), their effectiveness and mechanisms of action are poorly understood.MATERIALS AND METHODS: Obesity in male Wistar rats was induced by a 23-week diet enriched with saturated fats. MF treatment was carried out for 5 weeks at a dose of 120 mg/kg/day (orally), and the treatment with hCG and TP03 was carried out for 5 days at daily doses of 20 IU/rat (s.c.) and 15 mg/kg (i.p.), respectively. Using microscopy and histochemical analysis, the number and motility of spermatozoa (SP), the number of their defective forms and the morphology of the seminiferous tubules were assessed, and the levels of testosterone and other hormones in the blood were measured using ELISA.RESULTS: MF, hCG, and TP03 to varying degrees increased the number of SP and the proportion of their mobile forms, including those with forward movement, which were reduced in DIO rats, and also normalized the thickness of the epithelium of the seminiferous tubules and the number of spermatogonia and pachytene spermatocytes in them, but did not reduced the proportion of defective forms of SP, increased in DIO. In the case of MF, this was associated with the drug-induced normalization of body weight, glucose tolerance, and the insulin and leptin levels in DIO rats. The positive effect of hCG and TP03 on spermatogenesis was due to their stimulating effect on testosterone production.CONCLUSION: The use of long-term MF therapy and short-term courses of LHR-agonists normalizes impaired spermatogenesis in DIO, which indicates the prospects for their use to improve male fertility in obesity, and in the case of MF therapy, normalization of the metabolic and hormonal status is of great importance, while in the case of LHR-agonists the most important factor is their steroidogenic effect.