Relationship between progesterone, oestrone sulphate and cortisol and the components of renin angiotensin aldosterone system in Spanish purebred broodmares during pregnancy

The coordinated interaction of the components of the renin angiotensin aldosterone system (RAAS) with reproductive hormones such as progesterone, oestrogens and cortisol during pregnancy has been widely reported to play a vital role in foetal and placental development in various species, significantly influencing the proper achievement of pregnancy and foetal viability at birth. These interactions have not yet been clarified in mares. Thus, the purpose of the present research was to analyse the relationship between cortisol (CORT), progesterone (P4) and oestrone sulphate (OESTRONE), and the components of the RAAS, renin (REN), angiotensin II (ANG-II) and aldosterone (ALD) concentrations in Spanish broodmares during pregnancy.Venous blood samples were obtained monthly from a total of 31 Purebred Spanish broodmares aged between 5 and 15 years during the 11 months of pregnancy. Plasma and serum REN, ANG-II, ALD, P4, OESTRONE and CORT concentrations were analysed by competitive immunoassay. Pregnancy in Purebred Spanish broodmares is characterised by a progressive increase in REN and ALD, a decrease in CORT levels, variable fluctuations in P4 and OESTRONE and no variations in ANG-II concentrations (P < 0.05). Serum P4 was not correlated with either ALD or CORT. The OESTRONE and REN levels were not correlated, while OESTRONE and ALD showed a positive correlation (r = 0.16; P < 0.05). These results suggest that the sustained stimulation of the RAAS in normal pregnancy in Spanish broodmares is not totally dependent on the changes in P4 and CORT concentrations, despite the involvement of OESTRONE in the secretion of ALD. This brings into question the possible involvement of oestrogen in the secretion of ALD by a mechanism which is not exclusively dependent on REN. Consequently, at physiological levels, OESTRONE is not the only stimulus for REN synthesis, and the mineralocorticoids ALD and CORT do not show a competitive mechanism with P4 during pregnancy in mares. Other mechanisms which do not depend on these hormones should be considered in the modification of the RAAS during pregnancy in Spanish mares.     

The retinal renin-angiotensin system: roles of angiotensin II and aldosterone

In the present review we examine the experimental and clinical evidence for the presence of a local renin-angiotensin system within the retina. Interest in a pathogenic role for the renin-angiotensin system in retinal disease originally stemmed from observations that components of the pathway were elevated in retina during the development of certain retinal pathologies. Since then, our knowledge about the contribution of the RAS to retinal disease has greatly expanded. We discuss the known functions of the renin-angiotensin system in retinopathy of prematurity and diabetic retinopathy. This includes the promotion of retinal neovascularization, inflammation, oxidative stress and neuronal and glial dysfunction. The contribution of specific components of the renin-angiotensin system is evaluated with a particular focus on angiotensin II and aldosterone and their cognate receptors. The therapeutic utility of inhibiting key components of the renin-angiotensin system is complex, but may hold promise for the prevention and improvement of vision threatening diseases.     

Angiotensin III: A physiological relevant peptide of the renin angiotensin system

The renin angiotensin system (RAS) is a peptide hormone system that plays an important role in the pathophysiology of various diseases, including congestive heart failure, hypertension, myocardial infarction, and diabetic nephropathy. This has led researchers to focus extensively on this system, leading to the discovery of various peptides, peptidases, receptors and signal transduction mechanisms intrinsic to the RAS. Angiotensinogen (AGT), angiotensin (Ang) II, Ang III, Ang IV, and Ang-(1–7) are the main biologically active peptides of RAS. However, most of the available studies have focused on Ang II as the likely key peptide from the RAS that directly and indirectly regulates physiological functions leading to pathological conditions. However, data from recent studies suggest that Ang III may produce physiologically relevant effects that are similar to those produced by Ang II. Hence, this review focuses on Ang III and the myriad of physiological effects that it produces in the body.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.     

Hemostatic profile during late pregnancy and early postpartum period in mares

Hemostasis is a physiological process that prevents excessive blood loss and represents a protective mechanism at the time of delivery. Peripartum hemorrhage is a recurring hazardous condition to mare's health; therefore, we aimed to study mares' hemostatic profile to investigate whether physiological adjustments occur during late pregnancy and early postpartum. Fifteen pregnant mares have been monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares were used as control group. Jugular blood samples were analyzed for platelet count (Plt), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fb). Platelet count showed significant changes at foaling (P < 0.05) and a negative correlation (r = −0.968; P = 0.032) with postpartum. Prothrombin time changed (P < 0.05) showing a significant correlation (r = 0.675; P = 0.016) with late pregnancy. Fibrinogen concentrations changed throughout the experimental period (P < 0.0001). The linear regression model revealed a positive correlation (r = 0.9210; P < 0.0001) between Fb and late pregnancy and a negative correlation (r = −0.9583; P = 0.042) between Fb and early postpartum. The shortening in PT recorded in the imminence of parturition along with the increase in Plt and Fb at foaling might reflect a physiological hypercoagulable state that constrains excessive bleeding, enhancing mares' odds of surviving. Our research improves the knowledge about blood coagulation in periparturient mares providing specific information on routine coagulation tests that may support in monitoring mare's hemostatic profile during late pregnancy and early postpartum.     

Role of brain renin angiotensin system in neurodegeneration: An update

Renin angiotensin system (RAS) is an endocrine system widely known for its physiological roles in electrolyte homeostasis, body fluid volume regulation and cardiovascular control in peripheral circulation. However, brain RAS is an independent form of RAS expressed locally in the brain, which is known to be involved in brain functions and disorders. There is strong evidence for a major involvement of excessive brain angiotensin converting enzyme (ACE)/Angiotensin II (Ang II)/Angiotensin type-1 receptor (AT-1R) axis in increased activation of oxidative stress, apoptosis and neuroinflammation causing neurodegeneration in several brain disorders. Numerous studies have demonstrated strong neuroprotective effects by blocking AT1R in these brain disorders. Additionally, the angiotensin converting enzyme 2 (ACE2)/Angiotensin (1–7)/Mas receptor (MASR), is another axis of brain RAS which counteracts the damaging effects of ACE/Ang II/AT1R axis on neurons in the brain. Thus, angiotensin II receptor blockers (ARBs) and activation of ACE2/Angiotensin (1–7)/MASR axis may serve as an exciting and novel method for neuroprotection in several neurodegenerative diseases. Here in this review article, we discuss the expression of RAS in the brain and highlight how altered RAS level may cause neurodegeneration. Understanding the pathophysiology of RAS and their links to neurodegeneration has enormous potential to identify potentially effective pharmacological tools to treat neurodegenerative diseases in the brain.     

Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney

In our recent study using Wnk4^D561A/+ knockin mice, we determined that the WNK-OSR1/SPAK-NaCl cotransporter (NCC) phosphorylation cascade is important for regulating NCC function in vivo. Phosphorylation of NCC was necessary for its plasma membrane localization. Previously, angiotensin II infusion was shown to increase apical membrane expression of NCC in rats. Therefore, we investigated whether angiotensin II was an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in cultured cells and in vivo kidney. In mpkDCT cells, the phosphorylation of OSR1 and NCC was increased 30 min after the addition of angiotensin II (10^-9-10⁻⁷ M) but returned to baseline after 18 h. In mice, a 5-min infusion of angiotensin II (5 ng/g/min) increased NCC phosphorylation in the kidney at 30 min and 2 h after the injection but returned to baseline 24 h later. This increase was inhibited by angiotensin II receptor blocker (valsartan) but not by aldosterone receptor blocker (eplerenone). Ten-day infusions of angiotensin II (720 ng/day) also increased phosphorylation of OSR1 and NCC in the mouse kidney, and both valsartan and eplerenone inhibited the increased phosphorylation. Although angiotensin II is identified as an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in vivo, aldosterone appears to be the major regulator of this signal cascade in the long-term regulation by angiotensin II.     

On the local cardiac renin angiotensin system. Basic and clinical implications

In the present review we reevaluated the experimental and clinical evidence that there is a local renin angiotensin system in the heart as well as the presence of a functional intracrine component which is activated during pathological conditions like heart failure and hypertension. The implications of these findings for cardiology were discussed. The novel finding that cell swelling impairs cell coupling and impulse propagation through activation of ionic channels with consequent generation of cardiac arrhythmias and the evidence that AT1 receptors are mechanosensors able to alter the heart function independently of Ang II were discussed. Particular attention was given to the role of salt loading on the activation of a local cardiac renin angiotensin and its consequences.     

E Prostanoid-1 receptor regulates renal medullary αENaC in rats infused with angiotensin II

E Prostanoid (EP) receptors play an important role in urinary Na⁺ excretion. In the kidney, the epithelial sodium channel (ENaC) is the rate-limiting-step for Na⁺ reabsorption. We hypothesized that activation of EP1/EP3 regulates the expression of ENaC in the face of renin-angiotensin-aldosterone-system (RAAS) activation. In primary cultures of inner medullary collecting duct (IMCD) cells, sulprostone (EP1 > EP3 agonist, 1 μM) and 17 Phenyl trinor (17 Pt, EP1 agonist, 10 μM) prevented the up-regulation of αENaC mRNA induced by aldosterone (10 nM). In Sprague-Dawley rats infused with angiotensin II (0.4 μg/kg/min), αENaC expression was up-regulated in renal cortex and medulla coincidently with high plasma aldosterone levels. Sulprostone and/or 17 Pt prevented this effect in renal medulla but not in cortex. Immunocytochemistry demonstrated that IMCD cells express EP1. Our results suggest that specific activation of EP1 receptor during RAAS activation antagonizes the action of aldosterone on αENaC expression in the renal medulla.     

Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.     

Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I

A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen-deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment.     

Studies on the presence of angiotensin II in rat brain

Abstract: Angiotensin II-like immunoreactivity was extracted from brains of bilaterally nephrectomized rats with several different extraction procedures (90% methanol, distilled water, 6 M urea, 0.1 N HCI, and 2 M acetic acid). The activity was measured with radioimmunoassays using three different antisera, two of which had been used previously for immunocytochemical studies. With none of the extraction procedures or antisera employed was more than 80 pg/g wet weight of angiotensin II-like immunoreactivity found. Analysis was undertaken with two different reverse-phase high-pressure liquid chromatography systems; in one of these the immunoreactivity did not coelute with angiotensin II or III. On the basis of its elution pattern from a molecular sieving column, the immunoreactivity seems to have a higher molecular weight than angiotensin II. It is concluded that neurons in the brain do not synthesize and store angiotensin II.     

Pharmacologic application of native GnRH in the winter anovulatory mare,II: Accelerating the timing of pregnancy

Onset of the winter anovulatory period in mares is associated with a marked diminution in adenohypophyseal synthesis and release of LH. Native GnRH, unlike its synthetic agonists, stimulates the synthesis and secretion of LH in mares without pituitary refractoriness. Herein we tested the hypotheses that (1) the average Julian day of pregnancy can be accelerated by up to 2 months in winter anovulatory mares treated continuously with native GnRH beginning on February 1 and (2) mares will sustain luteal function and pregnancy after treatment withdrawal. Forty-two winter anovulatory mares were stratified by age, body condition score, and size of the largest follicle across two locations in a randomized design and assigned to one of three groups (n = 14 per group): (1) Control: untreated, (2) GnRH-14: GnRH delivered subcutaneously in saline at a rate of 100 μg/h for 8 weeks (February 1–March 29) using four consecutive 14-day pumps (Alzet 2ML2), or (3) GnRH-28: GnRH delivered as in (2), but using two 28-day pumps (Alzet 2ML4). On development of a 35-mm follicle and expression of estrus, mares were bred the following day and treated with hCG. Pregnancies were confirmed using transrectal ultrasonography on Days 14, 24, 33, and 45, with blood samples collected to assess luteal function. Mares treated with GnRH (GnRH-14 and GnRH-28) did not differ reproductively in their responses and data were pooled for statistical comparisons. Mares treated with GnRH exhibited marked increases (P ≤ 0.04) in the frequency of development of a 35-mm follicle, submission rate for live cover and/or artificial insemination, ovulation, and pregnancy compared with control mares on treatment Day 56 (March 29). Interval to the first 35-mm follicle was 51.8 ± 4.9 and 19.3 ± 3.5 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively. Interval to pregnancy was 65.3 ± 6.7 and 28.6 ± 4.8 days (least square mean ± standard error of the mean) for control and GnRH-treated mares, respectively, excluding one GnRH-14 mare that failed to become pregnant over four cycles. By the end of the treatment period (March 29), only 21% of control mares were pregnant compared with 79% of GnRH-treated mares. Furthermore, mean serum concentrations of progesterone were similar to (GnRH-28; P = 0.26) or greater than (GnRH-14; P = 0.01) that of control mares from Day 0 to 46 postbreeding. Data illustrate that continuous administration of native GnRH is a highly efficient option for managing seasonal anovulation in mares and could be effectively used in the breeding industry if a user-friendly delivery option were available.     

Early conception factor lateral flow assays for pregnancy in the mare

The ECF™ lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF™ test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.     

Intrarenal localization of angiotensin II specific binding in rat fetuses

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]-ileu-5-angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu-5-angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.     

Influence of day of postpartum breeding on pregnancy rate, pregnancy loss rate, and foaling rate in Thoroughbred mares

Records (years 2005-2007) were analyzed from a Thoroughbred stud farm in central Kentucky. Data from all breeding cycles of foaling mares were tabulated (3184 cycles of 2003 foaling mares bred between 7 and 163 days postpartum). A multiple logistic regression model employing Bayesian statistics was used to adjust for factors that significantly affected outcome; odds ratios (ORs) for pregnancy rate, pregnancy loss rate, and foaling rate were determined to examine the influence of day of postpartum breeding on these parameters. Mares bred before Day 22 (Day 0 = day of foaling) postpartum had a decreased OR for becoming pregnant (P < 0.05); the median OR for becoming pregnant (1.00) was not reached until Day 46 postpartum. Mares bred before Day 13 postpartum had an increased OR for pregnancy loss (P < 0.05). The median OR for pregnancy loss did not decline below 1.00 until Day 78 postpartum. Mares bred before Day 20 postpartum had a decreased OR for producing a foal (P < 0.05). The median OR for producing a foal (1.00) was not reached until Day 75 postpartum. We concluded that fertility (in terms of a higher OR for becoming pregnant and a lower OR for pregnancy loss, resulting in a higher OR for producing a foal) continued to improve in Thoroughbred mares for approximately 2.5 mo postpartum. These findings are of importance to management strategies directed at early postpartum breeding, and explain some of the reported drift in subsequent foaling dates of Thoroughbred mares, despite management practices that employ early postpartum breeding.     

Heterogeneity of angiotensin II receptors in membranes of developing rat metanephros

Specific and high affinity binding sites for angiotensin II were demonstrated in the membranes of the developing rat metanephros during the second half of pregnancy and in the newborn by binding studies with 125I angiotensin II. Only one type of angiotensin receptor was found during intrauterine life while after birth two classes of angiotensin receptors were present in the membranes of the cortical renal tissue.     

Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site

Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins.