Obstruction In Fetal Sheep Research Articles

Changes to the contractile function of ureter smooth muscle after partial infravesical obstruction in fetal sheep

To measure spontaneous contractile activity, and the responses to agonists using in vitro preparations of sheep ureter after a period of bladder outlet obstruction (BOO) initiated at mid-gestation. Date-mated Romney Marsh ewes, bearing fetuses of 70-75 days of gestation (term in this breed is 145 days) were used. Five fetuses underwent urachal obstruction and partial urethral constriction for 30 days. Six fetuses at the same gestational time underwent a sham operation (control group). Small strips of mid-ureter were cut in the longitudinal axis, and in the cross-sectional (transverse) plane of the ureter. Spontaneous contractile activity and the response to carbachol, high-K(+) (120 mM) solution and alpha,beta-methylene ATP (ABMA) were characterized by measuring the magnitude of evoked responses and the magnitude and frequency of spontaneous activity. The ureters from fetuses with BOO were significantly larger in diameter and had expanded lumens. The proportion of smooth muscle was not significantly different between the BOO and control groups. Spontaneous contractile activity in all preparations was resistant to atropine and the neurotoxin, tetrodotoxin. With transverse sections, the magnitude of spontaneous contractions was smaller in the BOO group, but the frequency was greater. The response to carbachol was also smaller in the BOO group, but the response to high-K(+) solution was similar to that of the control group. ABMA did not generate a response in any preparation. With longitudinal ureteric preparations, spontaneous or agonist-induced activity was negligible in the control group, while preparations from the BOO sheep had spontaneous activity and responded to carbachol and high-K(+) solutions. These results show that in fetuses with BOO and dilated ureters absolute ureteric contractile activity is diminished. However, there is functional reorganization of the muscle layers that generates more force in the longitudinal rather than the transverse axis. This would contribute to a reduced ability of the ureter to propel urine and contribute to the development of raised upper tract pressures.

Electrophysiological changes of ureter smooth muscle after partial infravesical urinary tract obstruction in fetal sheep

Electrophysiological changes of ureter smooth muscle after partial infravesical urinary tract obstruction in fetal sheep

The effects of overcoming experimental bladder outflow obstruction in fetal sheep

Objective: To develop an ovine model of fetal bladder outflow obstruction and to investigate the effect on the kidney of surgical relief of the obstruction in the prenatal period.Methods:Ultrasound examination and amniocentesis were performed on 68 date-bred pregnant ewes at day 57 of pregnancy (term = 150 days). Fetal gender was determined using a molecular technique to identify singlemale fetuses. The urethra and urachus were ligated at hysterotomy on 20 of these fetuses at 75 days' gestation. Comparisons were made with six controls that did not undergo operation. Changes that occurredin fetal urinary tract appearance were detected using serial ultrasound examinations. Seven obstructed cases chosen at random had further prenatal surgery on day 94 to decompress the obstructed urinarytract by vesicostomy. The animals were killed at 110 days' gestation and post-mortem studies were performed.Results: Fourteen days after surgical obstruction, there were increases in the summedrenal lengths (33 mm vs. 28 mm, p = 0.003) and renal pelvis anteroposterior (A-P) diameters (8 mm vs. 5.5 mm, p = 0.02). In the group allocated to receive surgical decompression, 9 days' reliefof obstruction resulted in significant reductions in summed renal lengths (30 mm vs. 41 mm, p = 0.024; controls 31 mm) and renal pelvis A-P diameters (5.8 mm vs. 8.9 mm, p = 0.012; controls< 2 mm). Post-mortem histological examination in the surgical decompression group revealed an estimated number of glomeruli similar to controls and greater than in the obstructed cases.Conclusion:Surgical relief of fetal bladder outflow obstruction in ovine mid-pregnancy results in improved renal appearance on ultrasonic and histopathological examinations.

Deregulation of Renal Transforming Growth Factor-β1 after Experimental Short-Term Ureteric Obstruction in Fetal Sheep

Renal malformations are the commonest cause of chronic renal failure in children and they are often associated with urinary tract abnormalities that impair fetal urine flow. Up-regulation of transforming growth factor-beta1 (TGF-beta1) occurs after experimental postnatal urinary tract obstruction and we recently reported increased levels of TGF-beta1 in human renal malformations (Yang SP et al, Am J Pathol 2000, 157:1633-1647). These findings led us to propose that obstruction-induced stretch of developing renal epithelia causes up-regulation of TGF-beta1, which then perturbs renal development. In this study, therefore, we examined expression of components of the TGF-beta1 signaling axis in a previously characterized ovine model of fetal short-term urine flow impairment in which complete unilateral ureteric obstruction was induced at 90 days when a few layers of glomeruli had formed. Up-regulation of TGF-beta1 mRNA and protein was observed in obstructed kidneys, compared to sham-operated control organs, after only 10 days. Increased levels of TGF-beta1 receptors I (TGF-betaR1) and II (TGF-betaR2) were also detected on Western blot, and the cytokine and TGF-betaR1 co-localized in disrupted epithelia on immunohistochemistry. De novo expression of alpha-smooth muscle actin, a structural protein up-regulated during TGF-beta1-induced phenotypic switching between human renal dysplastic epithelial and mesenchymal lineages in vitro, was also observed in these aberrant epithelia. These findings implicate increased TGF-beta1 signaling in the early biological changes generated by fetal urinary tract obstruction.

Re-expression of pulmonary surfactant proteins following tracheal obstruction in fetal sheep.

Increased fetal lung expansion, induced by tracheal obstruction (TO), is a potent stimulus for fetal lung growth, but rapidly reduces surfactant protein (SP) mRNA levels. Our aim was to determine the time course for the re-expression of the surfactant proteins in fetal lung tissue following the release of a TO and to relate these to the changes in lung liquid volume. Fetal sheep were exposed to either: (1) no treatment (controls); (2) 4 days of TO; (3) 4 days of TO, followed by release of the obstruction for 24 h; (4) 4 days of TO followed by release of the obstruction for 3 days. Four days of TO increased lung liquid volumes from 26.8 +/- 1.9 to 72.0 +/- 5.6 ml kg(-1) and reduced SP-A, SP-B and SP-C mRNA levels to 38.5 +/- 10.7, 56.8 +/- 10.3 and 18.3 +/- 5.3 % of control values, respectively. One day after TO release, lung liquid volumes were reduced to 17.4 +/- 5.3 ml kg(-1) (control 128 days, 31.0 +/- 3.8 ml kg(-1)) and SP-A and SP-B mRNA levels were not different from control levels. In contrast, SP-C mRNA levels only increased to 45.4 +/- 17.3 % of control. Three days after TO release, lung liquid volumes increased to 48.0 +/- 8.5 ml kg(-1) and SP-A and SP-B mRNA levels were reduced to 48.8 +/- 10.2 % and 71.5 +/- 19.8 % of control, respectively; SP-C mRNA levels remained at 35.3 +/- 12.3 % of control. Following the release of a TO, SP-A, SP-B and SP-C mRNA levels were closely and inversely related to the volume of lung liquid. Based on these relationships, the lung liquid volumes that equate to 100 % expression were considerably less than control lung volumes (< 10 vs. 30-40 ml kg(-1)) in fetuses of this age. Thus, the changes in fetal lung SP-A, SP-B and SP-C mRNA levels following the release of a TO are variable, differ between the proteins and are closely related to the changes in lung liquid volumes. We conclude that the re-expression of surfactant proteins following TO is variable and that the change in lung liquid volume is potentially a good indicator for surfactant protein re-expression. Experimental Physiology (2001) 86.1, 55-63.

Influence of growth hormone on the lung growth response to tracheal obstruction in fetal sheep.

Obstructing the fetal trachea is a potent stimulus for fetal lung growth, but little is known about the factors that regulate this process. Our aim was to determine the role of growth hormone (GH) in regulating the increase in lung growth induced by obstruction of the trachea in fetal sheep. Twenty chronically catheterized fetal sheep, nine of which were hypophysectomized, were divided into four experimental groups: 1) control group (n = 4), 2) a group in which the fetal trachea was obstructed for 3 days (3-day obstructed; n = 6), 3) a 3-day obstructed group in which the pituitary was removed [hypophysectomized (HX)] and the fetus was given maintenance infusions of ACTH, thyroxine, and human GH (hGH; HX hGH 3-day obstructed; n = 5), and 4) a HX 3-day obstructed group in which the fetus was given maintenance infusions of ACTH and thyroxine (n = 5). Tracheal obstruction significantly increased fetal lung liquid volumes from 37.2 +/- 3.2 ml/kg in control fetuses to 75.6 +/- 9.0 ml/kg in 3-day obstructed fetuses, and the presence or absence of GH did not affect this increase. Similarly, the presence or absence of GH did not affect the increase in lung weight or protein content induced by 3 days of tracheal obstruction. However, in the absence of GH, 3 days of tracheal obstruction failed to increase total lung DNA content above unobstructed control values (107.9 +/- 5.3 and 94. 1 +/- 7.0 mg/kg for control and HX 3-day obstructed groups, respectively). In contrast, 3 days of tracheal obstruction increased total lung DNA content to a similar extent in fetuses with an intact pituitary and HX fetuses that received GH replacement (126.0 +/- 4.4 and 126.7 +/- 4.0 mg/kg for 3-day obstructed and HX hGH 3-day obstructed groups, respectively). These data indicate that the absence of GH either abolishes or delays the acceleration in cell division caused by an increase in fetal lung expansion.

Renal renin-angiotensin system dysregulation caused by partial bladder outlet obstruction in fetal sheep

To determine whether fetal renal obstruction activates the renal renin-angiotensin system (RAS), an important mediator in normal kidney development and obstructive nephropathy, we used a model of fetal partial bladder outlet obstruction (PBOO). Total RNA and protein was extracted from kidney of sheep fetuses with partial bladder outlet obstruction created at 95 days gestation, after 2 (N = 6) and 5 weeks of obstruction (term; N = 6), and from normal fetal sheep at various time points between 60 and 135 days of gestation (total N = 19). Relative levels of mRNA for renin, angiotensinogen, type 1 and 2 angiotensin II (Ang II) receptors (AT-1 and AT-2), and transforming growth factor-beta1 (TGF-beta1) were assessed by semiquantitative reverse transcription-polymerase chain reaction. Expression levels of AT-2 receptor protein were measured by Western blot analysis. Renin mRNA expression was increased (250%) after two weeks of obstruction. In normal fetuses, AT-1 expression was low at 60 to 75 days of gestation and increased toward the end of gestation, whereas AT-2 expression showed a reversed pattern. At 109 days, PBOO caused an increased expression of AT-2 mRNA compared with normals (400%). Correspondingly, AT-2 receptor protein was more abundant in obstructed kidneys. TGF-beta1 mRNA expression was significantly increased in obstructed kidneys at 109 days gestation. These observations confirm the reciprocal developmental regulation of AT-1 and AT-2 receptors' expression, suggesting their functional role in renal development. Partial bladder outlet obstruction produces specific alterations: increased renin expression and altered balance of receptor subtypes, which may induce altered functional and vascular regulation of the obstructed fetal kidney. TGF-beta1, a mediator of Ang II-induced fibrosis, may play a role in inducing and propagating interstitial fibrosis.

Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume.

Obstruction of the fetal trachea causes liquid to accumulate within the future airways, which is a potent stimulus for lung growth. Our aim was to determine the relationship between the increase in fetal lung growth after tracheal obstruction and the increases in lung liquid volume and tracheal pressure to better understand the mechanisms involved in the growth response. The effects of 4 and 10 d of tracheal obstruction on lung DNA and protein contents and DNA synthesis rates were determined; these data were combined with data collected previously after 2 and 7 d of tracheal obstruction. Fetal lung liquid volumes and secretion rates were measured before (d 0) and on d 1, 2, 4, 7, and 10 after tracheal obstruction; fetal tracheal pressures were monitored throughout this period. Tracheal pressures increased from 2.9 +/- 0.8 mm Hg (control) to 4.3 +/- 0.4 mm Hg within 1 d of tracheal obstruction and remained at this elevated level for the duration of the obstruction period. Lung liquid volume increased progressively from 24.7 +/- 1.1 mL/kg on d 0 to 97.3 +/- 15.2 mL/kg at d 7 of tracheal obstruction, but had not increased further by d 10. Tracheal obstruction significantly increased lung DNA and protein contents above control values; over the 10-d period the increase in lung DNA content was closely related (r = 0.99) to the increase in lung liquid volume, but not to the increase in tracheal pressure. DNA synthesis rates were increased at 4 d of tracheal obstruction (by 66%) but had returned to control levels by d 10. We conclude that: 1) the mechanisms responsible for the acceleration in lung growth induced by tracheal obstruction are most active on d 2, remain active at a reduced level on d 4 and 7, and have returned to control levels by d 10; and 2) the increase in lung DNA content during tracheal obstruction (d 2-7) is closely related to the increase in lung liquid volume, but not to the increase in intraluminal pressure. Thus, we suggest that an increase in lung expansion is one of the primary factors responsible for the acceleration in fetal lung growth induced by tracheal obstruction.

Cortisol pretreatment enhances the lung growth response to tracheal obstruction in fetal sheep.

We have investigated whether cortisol pretreatment of sheep fetuses will result in a greater liquid accumulation within the lung and a greater lung growth response to obstruction of the fetal trachea. Chronically catheterized fetal sheep received either 1) a cortisol infusion at an increasing dose (1.5-4.0 mg/day) from days 118 to 127 of gestation; the fetal trachea was then obstructed from days 128 to 131 of gestation (n = 4); 2) a saline infusion from days 118 to 127 of gestation; the fetal trachea was then obstructed from days 128 to 131 of gestation (n = 4); or 3) a saline infusion from days 118 to 127 of gestation with no period of tracheal obstruction (control; n = 4). Fetal tracheal pressures were measured from days 128 to 131 of gestation, whereas lung liquid secretion rates and volumes were measured on days 118, 128, and 131 of gestation. On day 131 of gestation, all fetuses were given an intravenous injection of [3H]thymidine and were killed 8 h later. Cortisol pretreatment increased the volume of liquid that accumulated within the fetal lung from 69.5 +/- 4.1 to 96.1 +/- 14.1 ml/kg after 3 days of tracheal obstruction. Similarly, cortisol pretreatment significantly enhanced the increase in lung DNA content from 257.4 +/- 11.0 to 309.1 +/- 16.3 mg/kg after 3 days of tracheal obstruction. We conclude that pretreatment of fetuses with cortisol increases the volume of liquid that accumulates after tracheal obstruction and, as a result, increases the fetal lung growth response to tracheal obstruction.

Experimental bilateral urinary obstruction in fetal sheep: transforming growth factor-beta 1 expression.

To gain insight into the role of transforming growth factor-beta 1 (TGF-beta 1) in the development of kidney pathology following fetal obstruction, we measured TGF-beta 1 gene expression, the active peptide, and the urinary concentration in a model of fetal bilateral urinary obstruction (BUO) in sheep. Fetal lambs underwent BUO at 60 (FO-60) or 80 days (FO-80) of gestation and were studied at 120 days. Independently of the onset or duration of obstruction, all fetuses developed type IV dysplasia (IV) associated with an arrest in the nephrogenesis or hydronephrosis. Fetal glomerular filtration rate was not significantly modified, whereas sodium tubular reabsorption was significantly decreased, and urinary TGF-beta 1 concentration was elevated in hydronephrosis but not in IV. Levels of TGF-beta 1 mRNA were increased in hydronephrosis compared with normal kidneys, and active TGF-beta 1 immunoreactivity was increased in both hydronephrotic and IV kidneys. In summary, TGF-beta 1 may play a role in the development of hydronephrosis and dysplasia in kidneys following fetal BUO. Its role in the arrest of nephrogenesis observed in the IV kidneys remains to be proved.

Hormonal and hemodynamic responses to vena caval obstruction in fetal sheep.

To test the hypothesis that ACTH and vasopressin responses are quantifiable as functions of induced changes in central venous or arterial pressures, we produced various degrees of vena caval obstruction in fetal sheep (118--134 days gestation). In seven experiments, vena caval obstruction increased heart rate 18 +/- 7 beats/min and carotid arterial oxygen saturation 8.4 +/- 2.1%, but did not alter any measured vascular pressure or hormones. More severe vena caval obstruction (n = 10) decreased mean arterial pressure 13 +/- 2 mmHg, central venous pressure 1.3 +/- 0.3 mmHg, and heart rate 47 +/- 12 beats/min, and increased fetal plasma ACTH 1,047 +/- 448 pg/ml, cortisol 4.4 +/- 2.2 ng/ml, and vasopressin 47.9 +/- 24.2 pg/ml, but did not alter 11-deoxycortisol. The stimulus increased plasma cortisol (radioimmunoassay after chromatography) 100% and "corticosteroids" (radiotransinassay without chromatography) 20%, demonstrating the nonlinear relationship between these two variables. End-inflation plasma ACTH and vasopressin concentrations were significantly related to the induced decreases in mean arterial and central venous pressures, suggesting that the hormonal responses to vena caval obstruction were mediated by cardiovascular mechanoreceptors. Plasma vasopressin concentrations were linearly related to plasma ACTH concentrations (4 = 0.94; P less than 0.001), suggesting parallel release of the two hormones.