bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotransmission and viscoelasticity in the ovine fetal bladder after in utero bladder outflow obstruction
Authors: Thiruchelvam N. , Wu C. , David A. , Woolf A. S. , Cuckow P. M. , Fry C. H.
Published: March 2015

Fetal bladder outflow obstruction, predominantly caused by posterior urethral valves, results in significant urinary tract pathology; these lesions are the commonest cause of end-stage renal failure in children, and up to 50% continue to suffer from persistent postnatal bladder dysfunction. To investigate the physiological development of the fetal bladder and the response to urinary flow impairment, we performed partial urethral obstruction and complete urachal ligation in the midgestation fetal sheep for 30 days. By electrical and pharmacological stimulation of bladder strips, we found that muscarinic, purinergic, and nitrergic mechanisms exist in the developing fetal bladder at this gestation. After bladder outflow obstruction, the fetal bladder became hypocontractile, producing less force after nerve-mediated and muscarinic stimulation with suggested denervation, and also exhibited greater atropine resistance. Furthermore, fetal bladder urothelium exerted a negative inotropic effect, partly nitric oxide mediated, that was not present after obstruction. Increased compliance, reduced elasticity, and viscoelasticity were observed in the obstructed fetal bladder, but the proportion of work performed by the elastic component (a physical parameter of extracellular matrix) remained the same. In addition to denervation, hypocontractility may result from a reduction in the elastic modulus that may prevent any extramuscular components from sustaining force produced by detrusor smooth muscle.
Governance of arteriolar oscillation by ryanodine receptors
Authors: Takenaka Tsuneo , Ohno Yoichi , Hayashi Koichi , Saruta Takao , Suzuki Hiromichi
Published: March 2015

To investigate the role of ryanodine receptors in glomerular arterioles, experiments were performed using an isolated perfused hydronephrotic kidney model. In the first series of studies, BAYK-8644 (300 nM), a calcium agonist, constricted afferent (19.6 ± 0.6 to 17.6 ± 0.5 μm, n = 6, P < 0.01) but not efferent arterioles. Furthermore, BAYK-8644 elicited afferent arteriolar oscillatory movements. Subsequent administration of nifedipine (1 μM) inhibited both afferent arteriolar oscillation and constriction by BAYK-8644 (to 19.4 ± 0.5 μm). In the second group, although BAYK-8644 constricted afferent arterioles treated with 1 μM of thapsigargin (19.7 ± 0.6 to 16.8 ± 0.6 μm, n = 5, P < 0.05), it failed to induce rhythmic contraction. Removal of extracellular calcium with EGTA (2 mM) reversed BAYK-8644-induced afferent arteriolar constriction (to 20.0 ± 0.5 μm). In the third series of investigations, ryanodine (10 μM) but not 2-aminoethoxyphenyl borate (100 μM) abolished afferent arteriolar vasomotion by BAYK-8644. In the fourth series of experiments, in the presence of caffeine (1 mM), the stronger activation of voltage-dependent calcium channels by higher potassium media resulted in greater afferent arteriolar constriction and faster oscillation. Our results indicate that L-type calcium channels are rich in preglomerular but not postglomerular microvessels. Furthermore, the present findings suggest that either prolonged calcium influx through voltage-dependent calcium channels (BAYK-8644) or sensitized ryanodine receptors (caffeine) is required to trigger periodic calcium release through ryanodine receptors in afferent arterioles.
Normal and glucocorticoid-induced development of the human small intestinal xenograft
Authors: Nanthakumar N. Nanda , Klopcic Corrie E. , Fernandez Isabel , Walker W. Allan
Published: March 2015

The aim of this study was to determine whether intestinal xenografts could recapitulate human in utero development by using disaccharidases as markers. Twenty-week-old fetal intestine was transplanted into immunocompromised mice and was followed. At 20-wk of gestation, the fetal human intestine was morphologically developed with high sucrase and trehalase but had low lactase activities. By 9-wk posttransplantation, jejunal xenografts were morphologically and functionally developed and were then monitored for ≤6 mo. Both sucrase and trehalase activities remained unchanged, but lactase activity increased in a manner similar to that described in in utero development. Changes in sucrase and lactase activities were paralleled by protein levels. Cortisone acetate treatment at 20-wk posttransplantation accelerated the ontogeny of lactase but did not alter sucrase and trehalase activities. Biopsies from 1- and 2-yr-old infant intestine showed that all activities, except trehalase in the proximal intestine, corresponded to the levels found in jejunal xenografts at 24 wk posttransplantation. These studies suggest that 20-wk-old fetal intestine has the extrauterine developmental potential to follow normal intrauterine ontogeny as a xenograft.
Role of angiotensin in body fluid homeostasis of mice: fluid intake, plasma hormones, and brain Fos
Authors: Rowland Neil E. , Goldstein Bradley E. , Robertson Kimberly L.
Published: March 2015

CD1 mice injected peripherally with either ANG I or ANG II failed to drink substantial amounts of water or NaCl, yet showed strong Fos immunoreactivity (ir) in subfornical organ (SFO). Mice injected with furosemide showed modest stimulation of NaCl intake either 3 or 24 h later, were hypovolemic, and showed elevated plasma renin activity (PRA). The pattern of Fos-ir in the brain after furosemide was similar to that seen after peripheral injection of ANG II. Mice became hypovolemic after subcutaneous injection of polyethylene glycol (PEG), showed large increases in PRA, aldosterone, and water intake, but did not show sodium appetite. PEG-treated mice had strong activation of SFO as well as other brain regions previously shown to be related to ANG-associated drinking in rats. ANG II appears to have a modified role in the behavioral response to fluid loss in mice compared with rats.
Obesity by choice: the powerful influence of nutrient availability on nutrient intake
Authors: Tordoff Michael G.
Published: March 2015

The consumption of nutrients by rodents is markedly influenced by the number of containers of each nutrient provided. Most rats given a choice from separate sources of protein, carbohydrate, and fat thrived if given one cup of each but half failed to thrive if given one cup of each and three extra cups of carbohydrate or fat. Rats given five bottles of sucrose solution and one bottle of water became fatter than rats given five bottles of water and one of sucrose. These studies in rats may point to a model for human obesity, in which the availability of food can override physiological controls of ingestion.
Spatial and developmental regulation of leptin in fetal sheep
Authors: Ehrhardt Richard A. , Bell Alan W. , Boisclair Yves R.
Published: March 2015

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue after day 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was ∼10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth.
The organum vasculosum laminae terminalis in immune-to-brain febrigenic signaling: a reappraisal of lesion experiments
Authors: Romanovsky Andrej A. , Sugimoto Naotoshi , Simons Christopher T. , Hunter William S.
Published: March 2015

The organum vasculosum laminae terminalis (OVLT) has been proposed to serve as the interface for blood-to-brain febrigenic signaling, because ablation of this structure affects the febrile response. However, lesioning the OVLT causes many “side effects” not fully accounted for in the fever literature. By placing OVLT-lesioned rats on intensive rehydration therapy, we attempted to prevent these side effects and to evaluate the febrile response in their absence. After the OVLT of Sprague-Dawley rats was lesioned electrolytically, the rats were given access to 5% sucrose for 1 wk to stimulate drinking. Sucrose consumption and body mass were monitored. The animals were examined twice a day for signs of dehydration and treated with isotonic saline (50 ml/kg sc) when indicated. This protocol eliminated mortality but not several acute and chronic side effects stemming from the lesion. The acute effects included adipsia and gross (14% of body weight) emaciation; chronic effects included hypernatremia, hyperosmolality, a suppressed drinking response to hypertonic saline, and previously unrecognized marked (by ∼2°C) and long-lasting (>3 wk) hyperthermia. Because the hyperthermia was not accompanied by tail skin vasoconstriction, it likely reflected increased thermogenesis. After the rats recovered from the acute (but not chronic) side effects, their febrile response to IL-1β (500 ng/kg iv) was tested. The sham-operated rats developed typical monophasic fevers (∼0.5°C), the lesioned rats did not. However, the absence of the febrile response in the OVLT-lesioned rats likely resulted from the untreatable side effects. For example, hyperthermia at the time of pyrogen injection was high enough (39–40°C) to solely prevent fever from developing. Hence, the changed febrile responsiveness of OVLT-lesioned animals is given an alternative interpretation, unrelated to febrigenic signaling to the brain.
A practical guide to evaluating cardiovascular, renal, and pulmonary function in mice
Authors: Lorenz John N.
Published: March 2015

The development and widespread use of genetically altered mice to study the role of various proteins in biological control systems have led to a renewed interest in methodologies and approaches for evaluating physiological phenotypes. As a result, cross-disciplinary approaches have become essential for fully realizing the potential of these new and powerful animal models. The combination of classical physiological approaches and modern innovative technology has given rise to an impressive arsenal for evaluating the functional results of genetic manipulation in the mouse. This review attempts to summarize some of the techniques currently being used for measuring cardiovascular, renal, and pulmonary variables in the intact mouse, with specific attention to practical considerations useful for their successful implementation.
Phosphate transport in the duodenum and jejunum of goats and its adaptation by dietary phosphate and calcium
Authors: Huber Korinna , Walter Christina , Schröder Bernd , Breves Gerhard
Published: March 2015

Endogenous Pirecycling is a characteristic feature of the P homeostasis in ruminants. A pronounced salivary Pi secretion into the rumen is balanced by a high intestinal Pi absorption and an almost complete renal Pi reabsorption. In monogastric animals, the major Pi transport mechanism across the apical membrane of the enterocyte is an Na+-dependent transport mediated by NaPi cotransporter type IIb. In ruminants, an Na+-, as well as an H+-dependent, Pi transport system seems to exist in the small intestines. Therefore, morphological localization, type of ionic dependence, and ability to adapt to dietary P or Ca restriction of duodenal and jejunal Pi transport were characterized in goats. In the duodenum, there was an H+-dependent, Na+-sensitive Pi transport system that did not belong to the NaPi type II family and was not influenced by dietary P or Ca restriction. In contrast, in the jejunum, there was an Na+-dependent, H+-sensitive Pi transport mainly mediated by NaPi IIb. P restriction stimulated the NaPi IIb protein expression, resulting in higher Pi transport capacity.
Site-specific modulation of LPS-induced fever and interleukin-1β expression in rats by interleukin-10
Authors: Ledeboer Annemarie , Binnekade Rob , Brevé John J. P. , Bol John G. J. M. , Tilders Fred J. H. , Van Dam Anne-Marie
Published: March 2015

Bacterial lipopolysaccharide (LPS) induces fever that is mediated by pyrogenic cytokines such as interleukin (IL)-1β. We hypothesized that the anti-inflammatory cytokine IL-10 modulates the febrile response to LPS by suppressing the production of pyrogenic cytokines. In rats, intravenous but not intracerebroventricular infusion of IL-10 was found to attenuate fever induced by peripheral administration of LPS (10 μg/kg iv). IL-10 also suppressed LPS-induced IL-1β production in peripheral tissues and in the brain stem. In contrast, central administration of IL-10 attenuated the febrile response to central LPS (60 ng/rat icv) and decreased IL-1β production in the hypothalamus and brain stem but not in peripheral tissues and plasma. Furthermore, intravenous LPS upregulated expression of IL-10 receptor (IL-10R1) mRNA in the liver, whereas intracerebroventricular LPS enhanced IL-10R1 mRNA in the hypothalamus. We conclude that IL-10 modulates the febrile response by acting in the periphery or in the brain dependent on the primary site of inflammation and that its mechanism of action most likely involves inhibition of local IL-1β production.
Dynamics and contribution of mechanisms mediating renal blood flow autoregulation
Authors: Just Armin , Arendshorst William J.
Published: March 2015

We investigated dynamic characteristics of renal blood flow (RBF) autoregulation and relative contribution of underlying mechanisms within the autoregulatory pressure range in rats. Renal arterial pressure (RAP) was reduced by suprarenal aortic constriction for 60 s and then rapidly released. Changes in renal vascular resistance (RVR) were assessed following rapid step reduction and RAP rise. In response to rise, RVR initially fell 5-10% and subsequently increased ∼20%, reflecting 93% autoregulatory efficiency (AE). Within the initial 7-9 s, RVR rose to 55% of total response providing 37% AE, reaching maximum speed at 2.2 s. A secondary RVR increase began at 7-9 s and reached maximum speed at 10-15 s. Response times suggest that the initial RVR reflects the myogenic response and the secondary tubuloglomerular feedback (TGF). During TGF inhibition by furosemide, AE was 64%. The initial RVR rise was accelerated and enhanced, providing 49% AE, but it represented only 88% of total. The remaining 12% indicates a third regulatory component. The latter contributed up to 50% when the RAP increase began below the autoregulatory range. TGF augmentation by acetazolamide affected neither AE nor relative myogenic contribution. Diltiazem infusion markedly inhibited AE and the primary and secondary RVR increases but left a slow component. In response to RAP reduction, initial vasodilation constituted 73% of total response but was not affected by furosemide. The third component's contribution was 9%. Therefore, RBF autoregulation is primarily due to myogenic response and TGF, contributing 55% and 33-45% in response to RAP rise and 73% and 18-27% to RAP reduction. The data imply interaction between TGF and myogenic response affecting strength and speed of myogenic response during RAP rises. The data suggest a third regulatory system contributing <12% normally but up to 50% at low RAP; its nature awaits further investigation.
Loss of circadian organization of sleep and wakefulness during hibernation
Authors: Larkin Jennie E. , Franken Paul , Heller H. Craig
Published: March 2015

We investigated circadian and homeostatic regulation of nonrapid eye movement (NREM) sleep in golden-mantled ground squirrels during euthermic intervals between torpor bouts. Slow-wave activity (SWA; 1–4 Hz) and sigma activity (10–15 Hz) represent the two dominant electroencephalographic (EEG) frequency components of NREM sleep. EEG sigma activity has a strong circadian component in addition to a sleep homeostatic component, whereas SWA mainly reflects sleep homeostasis [Dijk DJ and Czeisler CA. J Neurosci 15: 3526–3538, 1995; Dijk DJ, Shanahan TL, Duffy JF, Ronda JM, and Czeisler CA. J Physiol (Lond) 505: 851–858, 1997]. Animals maintained under constant conditions continued to display circadian rhythms in both sigma activity and brain temperature throughout euthermic intervals, whereas sleep and wakefulness showed no circadian organization. Instead, sleep and wakefulness were distributed according to a 6-h ultradian rhythm. SWA, NREM sleep bout length, and sigma activity responded homeostatically to the ultradian sleep-wake pattern. We suggest that the loss of sleep-wake consolidation in ground squirrels during the hibernation season may be related to the greatly decreased locomotor activity during the hibernation season and may be necessary for maintenance of multiday torpor bouts characteristic of hibernating species.
Period gene expression in mouse endocrine tissues
Authors: Bittman Eric L. , Doherty Leo , Huang Liyue , Paroskie Allison
Published: March 2015

Circadian rhythms are generated by the oscillating expression of the Per1 and Per2 genes, which are expressed not only in the central brain pacemaker but also in peripheral tissues. Hormones are likely to coordinate physiological function in time. We performed in situ hybridization to localize mPer1 and mPer2 mRNA to particular cell types and tissue compartments in adrenal, thyroid, and testis. BALB/c mice maintained in a 12:12-h light-dark cycle expressed mPer1 in adrenal medulla, particularly in late afternoon and early night. mPer2 mRNA was more intensely expressed in adrenal cortex, especially in afternoon and evening. mPer1 mRNA was detected in thyroid. mPer1 was found in some but not all seminiferous tubules of each mouse at all times of day. Quantitation in C57BL/6 mice revealed a significant increase in the number of heavily labeled seminiferous tubules early in the night. Consistent with in situ hybridization, immunocytochemistry showed PER1 protein in spermatocytes and spermatids (spermatogenic stages VII-XII). Staining in spermatogonia and interstitial cells was inconsistent. Double labeling with 5′-bromodeoxyuridine showed PER1 expression first occurring 5 days after DNA replication. We conclude that mPeriod genes are expressed in peripheral endocrine glands. Central regulation, adenohypophyseal control, and functional importance of expression and phase remain to be elucidated.
Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice
Authors: D'Arbe M. , Einstein R. , Lavidis N. A.
Published: March 2015

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.
Baroreflex control of heart rate by oxytocin in the solitary-vagal complex
Authors: Higa Keila T. , Mori Eliana , Viana Fabiano F. , Morris Mariana , Michelini Lisete C.
Published: March 2015

Previous work demonstrated that oxytocinergic projections to the solitary vagal complex are involved in the restraint of exercise-induced tachycardia (2). In the present study, we tested the idea that oxytocin (OT) terminals in the solitary vagal complex [nucleus of the solitary tract (NTS)/dorsal motor nucleus of the vagus (DMV)] are involved in baroreceptor reflex control of heart rate (HR). Studies were conducted in male rats instrumented for chronic cardiovascular monitoring with a cannula in the NTS/DMV for brain injections. Basal mean arterial pressure and HR and reflex HR responses during loading and unloading of the baroreceptors (phenylephrine/sodium nitroprusside intravenously) were recorded after administration of a selective OT antagonist (OTant) or OT into the NTS/DMV. The NTS/DMV was selected for study because this region contains such a specific and dense concentration of OT-immunoreactive terminals. Vehicle injections served as a control. OT and OTant changed baroreflex control of HR in opposite directions. OT (20 pmol) increased the maximal bradycardic response (from −56 ± 9 to −75 ± 11 beats/min), whereas receptor blockade decreased the bradycardia (from −61 ± 13 to −35 ± 2 beats/min). OTant also reduced the operating range of the reflex, thus decreasing baroreflex gain (from −5.68 ± 1.62 to −2.83 ± 1.05 beats · min−1 · mmHg−1). OT injected into the NTS/DMV of atenolol-treated rats still potentiated the bradycardic responses to pressor challenges, whereas OT injections had no effect in atropine-treated rats. The brain stem effect was specific because neither vehicle administration nor injection of OT or OTant into the fourth cerebral ventricle had any effect. Our data suggest that OT terminals in the solitary vagal complex modulate reflex control of the heart, acting to facilitate vagal outflow and the slowdown of the heart.
Development of an in situ perfused kidney preparation for elasmobranch fish: action of arginine vasotocin
Authors: Wells Alan , Anderson W. Gary , Hazon Neil
Published: March 2015

Acclimation of the European lesser-spotted dogfish Scyliorhinus canicula to reduced environmental salinity [85–70% seawater (SW)] induced a significant diuresis in addition to a significant decrease in plasma osmolality in vivo. The threshold for this diuresis was determined to be 85% SW. Therefore, S. canicula acclimated to 85% SW was selected for further study as a diuretic model in the development of an in situ perfused kidney preparation. The renal role of arginine vasotocin (AVT) in the in situ perfused trunk preparation was investigated. In SW, perfusion of 10−9and 10−10M AVT resulted in a glomerular antidiuresis and decreases in tubular transport maxima for glucose and perfusate flow. In 85% SW, 10−10M AVT had no significant effect on these renal parameters with the exception of transport maxima for glucose and perfusate flow. Tubular parameters remained unchanged by either 10−9or 10−10M AVT. The results demonstrate that the perfused kidney preparation was a viable tool for the investigation of renal parameters in elasmobranch fish and that AVT induced a glomerular antidiuresis.
Upregulation of NHE3 is associated with compensatory cell growth response in young uninephrectomized rats
Authors: Girardi Adriana C. C. , Rocha Roberto O. , Britto Luiz R. G. , Rebouças Nancy A.
Published: March 2015

It is well established that after removal of renal mass, the remaining tissue undergoes compensatory growth. Several laboratories have reported that the activity of the apical membrane Na+-H+exchanger (NHE3) is increased after a reduction in renal mass. These studies were designed to determine whether NHE3 expression is altered early after loss of renal mass and to investigate the possible role of NHE3 activation in the compensatory tissue growth response. Experiments were performed in young male Wistar rats submitted to left nephrectomy or sham operation. At either 4 or 24 h after the surgery, the right kidney from each animal was removed and weighed. Significant increases in the wet weight of the remaining kidney were only observed 24 h after uninephrectomy (UNX). Western blot analysis of brush-border membranes and Northern blot analysis of cortex RNA showed that NHE3 protein abundance and NHE3 mRNA were greatly enhanced 4 and 24 h after UNX in relation to the sham kidney. To identify which growth pattern was mostly responsible for the enlargement of the remained kidney in our experimental models, we measured 5-bromo-2-deoxyuridine incorporation (BrdU) and protein-to-DNA ratio (protein/DNA ratio). The number of BrdU-positive nuclei increased and protein/DNA ratio slightly decreased, indicating that a hyperplastic response was the main component involved in the early compensatory renal growth in our animals. BrdU incorporation and protein/DNA were also assessed in rats treated with S3226, a selective blocker of NHE3. Neither the number of BrdU-positive nuclei nor the protein/DNA ratio was significantly altered 4 and 24 h after UNX in rats treated with S3226. In conclusion, UNX induced an upregulation of NHE3, which was evidenced at both functional and expression levels. The compensatory growth response in young UNX rats could be blocked by inhibiting NHE3 activity, suggesting that NHE3 activation may result in a facilitator state for the cell growth response in the renal proximal tubule.
Physiology and pathology of endothelin-1 in renal mesangium
Authors: Sorokin Andrey , Kohan Donald E.
Published: March 2015

Mesangial cells (MCs) play a central role in the physiology and pathophysiology of endothelin-1 (ET-1) in the kidney. MCs release ET-1 in response to a variety of factors, many of which are elevated in glomerular injury. MCs also express ET receptors, activation of which leads to a complex signaling cascade with resultant stimulation of MC hypertrophy, proliferation, contraction, and extracellular matrix accumulation. MC ET-1 interacts with other important regulatory factors, including arachidonate metabolites, nitric oxide, and angiotensin II. Excessive stimulation of ET-1 production by, and activity in, MC is likely of pathogenic importance in glomerular damage in the setting of diabetes, hypertension, and glomerulonephritis. The recent introduction of ET antagonists, and possibly ET-converting enzyme inhibitors, into the clinical arena establishes the potential for new therapies for those diseases characterized by increased MC ET-1 actions. This review will examine our present understanding of how ET-1 is involved in mesangial function in health and disease. In addition, we will discuss the status of clinical trials using ET antagonists, which have only been conducted in nonrenal disease, as a background for advocating their use in diseases characterized by excessive MC-derived ET-1.
Retinoid receptor-specific agonists alleviate experimental glomerulonephritis
Authors: Lehrke Ingo , Schaier Matthias , Schade Kerstin , Morath Christian , Waldherr Ruediger , Ritz Eberhard , Wagner Juergen
Published: March 2015

Retinoids are potent antiproliferative and anti-inflammatory compounds. We previously demonstrated that the natural pan-agonists all- trans retinoic acid (RA) and 13- cis RA efficiently preserve renal structure and function in rat mesangioproliferative glomerulonephritis. We examine effects of synthetic retinoid receptor-specific agonists 1) to identify common and receptor subtype-specific pathways in this model and 2) to characterize effects of retinoids on the renal endothelin (ET) system. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of agonists specific for retinoid A (Ro-137410) and retinoid X (Ro-257386) receptors and the complex anti-activator protein-1 active retinoid BMS-453 7 days after induction of anti-Thy1.1 nephritis ( n = 7–9/group). The different retinoids lowered glomerular ET-1 and ET type A and B receptor gene expression in control and nephritic rats with comparable efficacy. Reduction of glomerular c-Fos and GATA-2 mRNA expression levels suggests downregulation of transcription factors required for ET expression. The different retinoids were similar in their action on the glomerular capillary occlusion score, number of total glomerular cells, and glomerular infiltrating macrophage count. They differed in their ability to normalize blood pressure (Ro-257386 > BMS-453 > arotinoid), albuminuria (BMS-453 > Ro-257386 > arotinoid), and creatinine clearance (arotinoid > BMS-453 > Ro-257386). No signs of toxicity were observed. We conclude that all retinoid agonists with different subtype specificity are highly efficient in reducing renal damage and proliferation of mesangial cells. Retinoid X and A receptor-specific pathways are apparently involved in the antiproliferative, anti-inflammatory, and anti-ET action. Further studies are indicated to define the potential use of retinoid agonists in inflammatory renal disease.
Epidermal growth factor inhibits amiloride-sensitive sodium absorption in renal collecting duct cells
Authors: Shen Jie-Pan , Cotton Calvin U.
Published: March 2015

The effects of the ERK pathway on electrogenic transepithelial Na+absorption by renal collecting duct cells were determined. Approximately 90% of the unstimulated short-circuit current (15 ± 1 μA/cm2, n = 10) across conditionally immortalized murine collecting duct epithelial cells (mCT1) is amiloride sensitive and is likely mediated by apical epithelial Na+channels. Chronic exposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml) or transforming growth factor-α (TGF-α; 20 ng/ml) reduced amiloride-sensitive short-circuit current by >60%. The inhibitory effect of EGF on Na+absorption was not due to inhibition of basolateral Na+-K+-ATPase, because the pump current elicited by permeabilization of apical membrane with nystatin was not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70–85% decrease in epithelial Na+channel subunit mRNA levels. Exposure of mCT1 cells to either EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (an ERK kinase inhibitor; 30 μM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented the EGF- and PMA-induced inhibition of amiloride-sensitive Na+absorption. The results of these studies demonstrate that amiloride-sensitive Na+absorption by renal collecting duct cells is regulated by the ERK pathway. This pathway may play a role in alterations in ion transport that occur in polycystic kidney disease.
Expression of osmotic stress-related genes in tissues of normal and hyposmotic rats
Authors: Zhang Zheng , Ferraris Joan D. , Brooks Heddwen L. , Brisc Ioana , Burg Maurice B.
Published: March 2015

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved in organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control (plasma osmolality 294 ± 1 mosmol/kgH2O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 ± 2 mosmol/kgH2O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain, and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70–1 and Hsp70–2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are as follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues.
Leucokinin activates Ca2+-dependent signal pathway in principal cells ofAedes aegyptiMalpighian tubules
Authors: Yu Ming-Jiun , Beyenbach Klaus W.
Published: March 2015

The role of Ca2+in mediating the diuretic effects of leucokinin-VIII was studied in isolated perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Peritubular leucokinin-VIII (1 μM) decreased the transepithelial resistance from 11.2 to 2.6 kΩ · cm, lowered the transepithelial voltage from 42.8 to 2.7 mV, and increased transepithelial Cl−diffusion potentials 5.1-fold. In principal cells of the tubules, leucokinin-VIII decreased the fractional resistance of the basolateral membrane from 0.733 to 0.518. These effects were reversed by the peritubular Ca2+-channel blocker nifedipine, suggesting a role of peritubular Ca2+and basolateral Ca2+channels in signal transduction. In Ca2+-free Ringer bath, the effects of leucokinin-VIII were partial and transient but were fully restored after the bath Ca2+concentration was restored. Increasing intracellular Ca2+with thapsigargin duplicated the effects of leucokinin-VIII, provided that peritubular Ca2+was present. The kinetics of the effects of leucokinin-VIII is faster than that of thapsigargin, suggesting the activation of inositol-1,4,5-trisphosphate-receptor channels of intracellular stores. Store depletion may then bring about Ca2+entry into principal cells via nifedipine-sensitive Ca2+channels in the basolateral membrane.
Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability
Authors: Hu Ping , Meyers Susan , Liang Feng-Xia , Deng Fang-Ming , Kachar Bechara , Zeidel Mark L. , Sun Tung-Tien
Published: March 2015

Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol151: 961–972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 ± 122, knockout 2,322 ± 114 Ω · cm2; P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 ± 0.06, knockout 1.83 ± 0.14 cm/s × 10−5; P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 ± 0.24, knockout 2.93 ± 0.31 cm/s × 10−6; P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium.
Inner medullary lactate production and urine-concentrating mechanism: a flat medullary model
Authors: Hervy Stéphane , Thomas S. Randall
Published: March 2015

We used a mathematical model to explore the possibility that metabolic production of net osmoles in the renal inner medulla (IM) may participate in the urine-concentrating mechanism. Anaerobic glycolysis (AG) is an important source of energy for cells of the IM, because this region of the kidney is hypoxic. AG is also a source of net osmoles, because it splits each glucose into two lactate molecules, which are not metabolized within the IM. Furthermore, these sugars exert their full osmotic effect across the epithelia of the thin descending limb of Henle's loop and the collecting duct, so they are apt to fulfill the external osmole role previously attributed to interstitial urea (whose role is compromised by the high urea permeability of long descending limbs). The present simulations show that physiological levels of IM glycolytic lactate production could suffice to significantly amplify the IM accumulation of NaCl. The model predicts that for this to be effective, IM lactate recycling must be efficient, which requires high lactate permeability of descending vasa recta and reduced IM blood flow during antidiuresis, two conditions that are probably fulfilled under normal circumstances. The simulations also suggest that the resulting IM osmotic gradient is virtually insensitive to the urea permeability of long descending limbs, thus lifting a longstanding paradox, and that this high urea permeability may serve for independent regulation of urea balance.
Role of hβ1in activation of human mesangial BK channels by cGMP kinase
Authors: Kudlacek Patrick E. , Pluznick Jennifer L. , Ma Rong , Padanilam Babu , Sansom Steven C.
Published: March 2015

In vascular smooth muscle and glomerular mesangial cells, relaxing agents such as nitric oxide and atrial natriuretic peptide activate large-conductance Ca2+-activated K+channels (BK) via the cGMP kinase pathway. BK are composed of pore-forming α-subunits, encoded by the slopoke gene ( Slo), and one of four cell-specific accessory β-subunits (hβ1–4). We used patch-clamp analysis to determine the influence of hβ1, hβ2, and hβ4on activation of human mesangial BK by cGMP kinase. We found that HEK 293 cells, coexpressing human (h) Sloα with either hβ1or hβ2, contained single BK currents activated by db-cGMP in cell-attached patches. However, recombinant BK were not activated by db-cGMP when h Sloα was expressed alone or with hβ4. DNA-RNA hybridization revealed that mesangial cells contained mRNA for hβ1but not hβ2or hβ4. The BK response to db-cGMP was decreased when hβ1antisense but not scrambled oligonucleotides were incorporated into mesangial cells. Western blot analysis showed that hβ1antisense oligonucleotide inhibited the amount of hβ1-V5 fusion protein expressed in HEK 293 cells by ∼50%. These results show that mesangial cells contain hβ1, a BK accessory protein, which confers activation of BK by cGMP kinase.
rBAT-b0,+AT heterodimer is the main apical reabsorption system for cystine in the kidney
Authors: Fernández Esperanza , Carrascal Montserrat , Rousaud Ferran , Abián Joaquín , Zorzano Antonio , Palacín Manuel , Chillarón Josep
Published: March 2015

Mutations in the rBAT and b0,+AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b0,+AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b0,+-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b0,+AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b0,+AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b0,+AT followed by SDS-PAGE and mass spectrometry analysis established that b0,+AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b0,+AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b0,+AT protein in the kidney. Indeed, complete immunodepletion of b0,+AT did not coprecipitate >20–30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.
Phospholipid metabolite production in human urothelial cells after protease-activated receptor cleavage
Authors: Rickard Alice , McHowat Jane
Published: March 2015

Our laboratory demonstrated previously that stimulation of protease-activated receptors (PARs) on the human urothelial carcinoma cell line RT4 results in activation of a calcium-independent phospholipase A2 (iPLA2), leading to arachidonic acid and PGE2 release. In this study, we have examined PAR activation in normal human urothelial cells (HUR) leading to the production of inflammatory or cytoprotective phospholipid metabolites. The presence of both PAR-1 and PAR-2 on HUR was confirmed by immunoblotting. Stimulation of PAR-1 with thrombin or PAR-2 by tryptase leads to activation of a membrane-associated iPLA2 and the production of platelet-activating factor, arachidonic acid, and PGE2. These responses were all blocked by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. Thus stimulation of PAR-1 or PAR-2 on HUR leads to iPLA2-catalyzed phospholipid hydrolysis, resulting in the production of metabolites that may mediate inflammation or provide cytoprotection to the bladder.
Nitric oxide inhibits superoxide-stimulated urea permeability in the rat inner medullary collecting duct
Authors: Zimpelmann Joseph , Li Ningjun , Burns Kevin D.
Published: March 2015

The inner medullary collecting duct (IMCD) contains relatively high nitric oxide (NO) synthetic capacity, but the effect of NO on IMCD transport remains unclear. We determined the effect of NO on basal and vasopressin (AVP)-stimulated urea ( Purea) and water ( Pf) permeabilities in isolated, perfused rat IMCD. The NO donor S-nitroso- N-acetylpenicillamine (SNAP) increased cGMP production in IMCD, but neither SNAP (10–4M) nor 8-BrcGMP (10–4M), the cell-permeable analog of cGMP, affected basal or AVP-stimulated Purea. The free radical superoxide is produced by oxidases in the kidney and can interact with NO. To determine the effect of superoxide generation on transport, IMCDs were incubated with diethyldithiocarbamate (DETC; 10–3M), the inhibitor of superoxide dismutase (SOD). DETC significantly increased basal and AVP-stimulated Purea(control: 28.7 ± 4.5 vs. DETC: 40.9 ± 6.2 × 10–5cm/s; P < 0.001; n = 9). Preincubation of IMCD with SNAP or the SOD mimetic tempol completely inhibited DETC-stimulated Purea. DETC caused a significant increase in superoxide generation by IMCD, and this was blocked by SNAP. Incubation of IMCD with the NO synthase (NOS) substrate l-arginine blocked the stimulatory effect of DETC on Purea, and this was reversed by the neuronal NOS inhibitor 7-nitroindazole. In contrast, neither basal nor AVP-stimulated Pfwas affected by NO donors or DETC. In summary, exogenous or endogenously produced NO does not affect basal urea transport in the IMCD but inhibits superoxide-stimulated Purea. In the inner medulla, superoxide generation by local oxidases may stimulate urea transport, and the role of endogenous NO may be to dampen this effect by decreasing superoxide levels.
Blood flow-dependent changes in renal interstitial guanosine 3′,5′-cyclic monophosphate in rabbits
Authors: Nishiyama Akira , Kimura Shoji , Fukui Toshiki , Rahman Matlubur , Yoneyama Hirohito , Kosaka Hiroaki , Abe Youichi
Published: March 2015

We examined responses of renal interstitial guanosine 3′,5′-cyclic monophosphate (cGMP) to changes in renal perfusion pressure (RPP) within and below the range of renal blood flow (RBF) autoregulation. A microdialysis method was used to monitor renal cortical and medullary interstitial cGMP levels in anesthetized rabbits. RPP was reduced in two steps: from ambient pressure (89 ± 3 mmHg) to 70 ± 2 mmHg ( step 1) and then to 48 ± 3 mmHg ( step 2). RBF was maintained in step 1 but was significantly decreased in step 2 from 2.94 ± 0.23 to 1.47 ± 0.08 ml · min−1 · g−1. Basal interstitial concentrations of cGMP were significantly lower in the cortex than in the medulla (12.1 ± 1.4 and 19.9 ± 0.4 nmol/l, respectively). Cortical and medullary cGMP did not change in step 1 but were significantly decreased in step 2, with significantly less reduction in cGMP concentrations in the medulla than in the cortex (−25 ± 3 and −44 ± 3%, respectively). Over this pressure range, changes in cortical and medullary cGMP were highly correlated with changes in RBF ( r= 0.94, P < 0.005 for cortex; r = 0.82, P < 0.01 for medulla). Renal interstitial nitrate/nitrite was not changed in step 1 but was significantly decreased in step 2 (−38 ± 2% in cortex and −20 ± 2% in medulla). Nitric oxide synthase inhibition with N G-nitro-l-arginine methyl ester (l-NAME, 30 mg/kg bolus, 50 mg · kg−1 · h−1 iv infusion) significantly decreased RBF (by −46 ± 4%) and interstitial concentrations of cGMP (−27 ± 4% in cortex and −22 ± 4% in medulla, respectively). During l-NAME treatment, renal interstitial concentrations of cGMP in the cortex and medulla were similarly not altered in step 1. However, l-NAME significantly attenuated cGMP responses to a reduction in RPP in step 2. These results indicate that acute changes in RBF result in alterations in nitric oxide-dependent renal interstitial cGMP levels, with differential effects in the medulla compared with the cortex.
Downregulation of renal aquaporins in response to unilateral ureteral obstruction
Authors: Li Chunling , Wang Weidong , Knepper Mark A. , Nielsen Søren , Frøkiær Jørgen
Published: March 2015

The expression of aquaporin-2 (AQP2) is decreased in rats with bilateral ureteral obstruction (BUO) and unilateral ureteral obstruction (UUO). Therefore, the expression of additional renal aquaporins (AQP1–4) and phosphorylated AQP2 (p-AQP2), known to play a role in urinary concentration, was examined in a Wistar rat model with 24 h of UUO. In obstructed kidneys, immunoblotting revealed a significant decrease in the expression of inner medullary AQP2 to 42 ± 4, p-AQP2 to 23 ± 5, AQP3 to 19 ± 6, AQP4 to 11 ± 5, and AQP1 to 64 ± 8% of sham levels. AQP1 expression located in the proximal tubule decreased to 74 ± 4% of sham levels ( P < 0.05). Immunocytochemistry confirmed the downregulation of AQP3, AQP4, and p-AQP2. In contralateral nonobstructed kidneys, immunoblotting also revealed significant reductions of AQP1 in the inner medulla, outer medulla, and cortex, whereas expression of AQP2, AQP3, AQP4, and p-AQP2 was unchanged. Furthermore, we collected the urine from both obstructed and nonobstructed kidneys for 2 h, respectively, after 24 h of UUO. Urine collection from obstructed kidneys during 2 h after release of UUO revealed a significant reduction in urine osmolality and solute-free water reabsorption (TcH2O). Moreover, an increase in urine production and TcH2O was observed in contralateral kidneys. To examine whether vasopressin-independent mechanisms are involved in AQP2 regulation, vasopressin-deficient Brattleboro (BB) rats with 24 h of UUO were examined. Immunoblotting revealed downregulation of AQP2, p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation of p-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1) UUO is associated with severe downregulation of AQP2, AQP3, AQP4, and AQP1; thus all of these AQPs may play important roles in the impaired urinary concentrating capacity in the obstructed kidney; 2) the reduced levels of AQP1 in the nonobstructed kidney may contribute to the compensatory increase in urine production; and 3) downregulation of AQPs in BB rats supports the view that vasopressin-independent pathways may be involved in AQP2 and AQP3 regulation in the obstructed kidney.