Mary E. Wlodek

Normal Lactational Environment Restores Nephron Endowment and Prevents Hypertension after Placental Restriction in the Rat

Uteroplacental insufficiency in the rat restricts fetal growth, impairs mammary development, compromising postnatal growth; and increases adult BP. The roles of prenatal and postnatal nutritional restraint on later BP and nephron endowment in offspring from mothers that underwent bilateral uterine vessel ligation (restricted) on day 18 of pregnancy were examined. Sham surgery (control) and a group of rats with reduced litter size (reduced; litter size reduced at birth to five, equivalent to restricted group) were used as controls. Offspring (control, reduced, and restricted) were cross-fostered on postnatal day 1 onto a control (normal lactation) or restricted (impaired lactation) mother. BP in male offspring was determined by tail cuff at 8, 12, and 20 wk of age, with glomerular number and volume (Cavalieri/Physical Dissector method) and renal angiotensin II type 1 receptor (AT(1)R) mRNA expression (real-time PCR) determined at 6 mo. Restricted-on-restricted male offspring developed hypertension (+16 mmHg) by 20 wk together with a nephron deficit (-26%) and glomerular hypertrophy (P < 0.05). In contrast, providing a normal lactational environment to restricted offspring improved postnatal growth and prevented the nephron deficit and hypertension. Reduced-on-restricted pups that were born of normal weight but with impaired growth during lactation subsequently grew faster, developed hypertension (+16 mmHg), had increased AT(1A)R and AT(1B)R mRNA expression (P < 0.05), but had no nephron deficit. Our study identifies the prenatal and postnatal nutritional environments in the programming of adult hypertension, associated with distinct renal changes. It is shown for the first time that a prenatally induced nephron deficit can be restored by correcting growth restriction during lactation.

Growth restriction before or after birth reduces nephron number and increases blood pressure in male rats

Impaired growth in utero predicts a low nephron number and high blood pressure later in life as does slowed or accelerated growth after a normal birth weight. We measured the effects of early postnatal growth restriction, with or without prenatal growth restriction, on blood pressure and nephron number in male rat offspring. Bilateral uterine artery and vein ligation were performed to induce uteroplacental insufficiency (Restricted) on day 18 of pregnancy. Postnatal growth restriction was induced in a subset of sham operated control animals by reducing the number of pups at birth to that of the Restricted group (Reduced Litter). Compared to Controls, Restricted pups were born smaller while Reduced Litter pups weighed less by postnatal day 3 and both groups remained lighter throughout lactation. By 10 weeks of age all animals were of similar weight but the Reduced Litter rats had elevated blood pressure. At 22 weeks, Restricted but not Reduced Litter offspring were smaller and the blood pressure was increased in both groups. Restricted and Reduced Litter groups had fewer glomeruli and greater left ventricular mass than Controls. These results suggest that restriction of both perinatal and early postnatal growth increase blood pressure in male offspring. This study also demonstrates that the early postnatal period is a critical time for nephron endowment in the rat.

Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats

In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross‐fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted‐on‐Restricted offspring were born small but were of similar weight to Control‐on‐Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted‐on‐Restricted offspring at 6 months without glomerular hypertrophy. Cross‐fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control‐on‐Control and Restricted‐on‐Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control‐on‐Control and Restricted‐on‐Restricted females at 18 months. Restricted‐on‐Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor‐β1, collagen IV (α1) and matrix matelloproteinase‐9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.

Uteroplacental insufficiency programs regional vascular dysfunction and alters arterial stiffness in female offspring

Intrauterine growth restriction caused by uteroplacental insufficiency increases the risk of cardiovascular disease in adulthood. Vascular mechanisms in female offspring are poorly understood. The aim of this study was to investigate the effects of uteroplacental insufficiency on blood pressure, vascular reactivity and arterial stiffness in four vascular beds in female offspring born growth restricted. Uteroplacental insufficiency was induced on day 18 of gestation in Wistar Kyoto rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Controls). Wire and pressure myography were used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in uterine, mesenteric, renal and femoral arteries of 18‐month‐old female offspring. Collagen and elastin fibres were quantified using circular crossed‐polarized light microscopy and quantitative real time polymerase chain reaction. Restricted female offspring were born 10–15% smaller. Restricted females were normotensive, had plasma triglycerides 2‐fold elevated and had uterine endothelial dysfunction, attributed to a 23% reduction in the maximal relaxation produced by endothelium‐derived hyperpolarizing factor. Uterine artery stiffness was increased, with an augmented proportion of thick and decreased proportion of thin collagen fibres. Vascular reactivity and mechanical wall properties were preserved in mesenteric, renal and femoral arteries in growth restricted females. Female offspring born growth restricted have selective uterine artery endothelial dysfunction and increased wall stiffness. The preserved vascular function in other arteries may explain the lack of hypertension in these females. The uterine artery specific dysfunction has potential implications for impaired pregnancy adaptations and a compromised intrauterine environment of the next generation.

Improved lactational nutrition and postnatal growth ameliorates impairment of glucose tolerance by uteroplacental insufficiency in male rat offspring

Intrauterine growth restriction and accelerated postnatal growth predict increased risk of diabetes. Uteroplacental insufficiency in the rat restricts fetal growth but also impairs mammary development and postnatal growth. We used cross fostering to compare the influence of prenatal and postnatal nutritional restraint on adult glucose tolerance, insulin secretion, insulin sensitivity, and hypothalamic neuropeptide Y content in Wistar Kyoto rats at 6 months of age. Bilateral uterine vessel ligation (restricted) to induce uteroplacental insufficiency or sham surgery (control) was performed on d-18 gestation. Control, restricted, and reduced (reducing litter size of controls to match restricted) pups were cross fostered onto a control or restricted mother 1 d after birth. Restricted pups were born small compared with controls. Restricted males, but not females, remained lighter up to 6 months, regardless of postnatal environment. By 10 wk, restricted-on-restricted males ate more than controls. At 6 months restricted-on-restricted males had increased hypothalamic neuropeptide Y content compared with other groups, and together with reduced-on-restricted males had increased retroperitoneal fat weight (percent body weight) compared with control-on-controls. Restricted-on-restricted males had impaired glucose tolerance, reduced first-phase insulin secretion, but unaltered insulin sensitivity, compared with control-on-controls. In males, being born small and exposed to an impaired lactational environment adversely affects adult glucose tolerance and first-phase insulin secretion, but improving lactation partially ameliorates this condition. This study identifies early life as a target for intervention to prevent later diabetes after prenatal restraint.

Localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment

Relaxin is a potent vasodilator of small resistance arteries and modifies arterial compliance in some systemic vascular beds, yet receptors for relaxin, such as RXFP1, have only been localized to vascular smooth muscle. This study first aimed to localize RXFP1 in rat arteries and veins from different organ beds and determine whether receptors are present in endothelial cells. We then tested the hypothesis that region‐specific vascular effects of relaxin may be influenced by the cellular localization of RXFP1 within different blood vessels. The aorta, vena cava, mesenteric artery, and vein had significantly higher (P<0.05) RXFP1 immunostaining in endothelial cells compared with vascular smooth muscle, whereas the femoral artery and vein and small pulmonary arteries had higher (P<0.01) RXFP1 immunostaining in the vascular smooth muscle. Male rats were treated subcutaneously with recombinant human relaxin‐2 (serelaxin; 4 μg/h) for 5 d; vasodilation and compliance in mesenteric and femoral arteries and veins were compared with placebo controls. Serelaxin significantly (P=0.04) reduced wall stiffness and increased volume compliance in mesenteric arteries but not in the other vessels examined. This was associated with changes in geometrical properties, and not compositional changes in the extracellular matrix. Serelaxin treatment had no effect on acetylcholine‐mediated relaxation but significantly (P< 0.001) enhanced bradykinin (BK)‐mediated relaxation in mesenteric arteries, involving enhanced nitric oxide but not endothelium‐derived hyperpolarization or vasodilatory prostanoids. In conclusion, there is differential distribution of RXFP1 on endothelial and smooth muscle across the vasculature. In rats, mesenteric arteries exhibit the greatest functional response to chronic serelaxin treatment.—Jelinic, M., Leo, C‐H., Post Uiterweer, E. P., Sandow, S. L., Gooi, J. H., Wlodek, M. E., Conrad, K. P., Parkington, H., Tare, M., Parry, L. J. Localization of relaxin receptors in arteries and veins, and region‐specific increases in compliance and bradykinin‐mediated relaxation after in vivo serelaxin treatment. FASEB J. 28, 275–287 (2014). www.fasebj.org

Uteroplacental insufficiency and reducing litter size alters skeletal muscle mitochondrial biogenesis in a sex-specific manner in the adult rat

Uteroplacental insufficiency has been shown to impair insulin action and glucose homeostasis in adult offspring and may act in part via altered mitochondrial biogenesis and lipid balance in skeletal muscle. Bilateral uterine vessel ligation to induce uteroplacental insufficiency in offspring (Restricted) or sham surgery was performed on day 18 of gestation in rats. To match the litter size of Restricted offspring, a separate cohort of sham litters had litter size reduced to five at birth (Reduced Litter), which also restricted postnatal growth. Remaining litters from sham mothers were unaltered (Control). Offspring were studied at 6 mo of age. In males, both Restricted and Reduced Litter offspring had reduced gastrocnemius PPARgamma coactivator-1alpha (PGC-1alpha) mRNA and protein, and mitochondrial transcription factor A (mtTFA) and cytochrome oxidase (COX) III mRNA (P < 0.05), whereas only Restricted had reduced skeletal muscle COX IV mRNA and protein and glycogen (P < 0.05), despite unaltered glucose tolerance, homeostasis model assessment (HOMA) and intramuscular triglycerides. In females, only gastrocnemius mtTFA mRNA was lower in Reduced Litter offspring (P < 0.05). Furthermore, glucose tolerance was not altered in any female offspring, although HOMA and intramuscular triglycerides increased in Restricted offspring (P < 0.05). It is concluded that restriction of growth due to uteroplacental insufficiency alters skeletal muscle mitochondrial biogenesis and metabolic characteristics, such as glycogen and lipid levels, in a sex-specific manner in the adult rat in the absence of impaired glucose tolerance. Furthermore, an adverse postnatal environment induced by reducing litter size also restricts growth and alters skeletal muscle mitochondrial biogenesis and metabolic characteristics in the adult rat.

Short-term exercise training early in life restores deficits in pancreatic β-cell mass associated with growth restriction in adult male rats

Fetal growth restriction is associated with reduced pancreatic β-cell mass, contributing to impaired glucose tolerance and diabetes. Exercise training increases β-cell mass in animals with diabetes and has long-lasting metabolic benefits in rodents and humans. We studied the effect of exercise training on islet and β-cell morphology and plasma insulin and glucose, following an intraperitoneal glucose tolerance test (IPGTT) in juvenile and adult male Wistar-Kyoto rats born small. Bilateral uterine vessel ligation performed on day 18 of pregnancy resulted in Restricted offspring born small compared with sham-operated Controls and also sham-operated Reduced litter offspring that had their litter size reduced to five pups at birth. Restricted, Control, and Reduced litter offspring remained sedentary or underwent treadmill running from 5 to 9 or 20 to 24 wk of age. Early life exercise increased relative islet surface area and β-cell mass across all groups at 9 wk, partially restoring the 60-68% deficit (P < 0.05) in Restricted offspring. Remarkably, despite no further exercise training after 9 wk, β-cell mass was restored in Restricted at 24 wk, while sedentary littermates retained a 45% deficit (P = 0.05) in relative β-cell mass. Later exercise training also restored Restricted β-cell mass to Control levels. In conclusion, early life exercise training in rats born small restored β-cell mass in adulthood and may have beneficial consequences for later metabolic health and disease.

PLASMA ADRENOCORTICOTROPIC HORMONE AND CORTISOL AND ADRENAL BLOOD-FLOW DURING SUSTAINED HYPOXEMIA IN FETAL SHEEP

We examined the effect of sustained hypoxemia with progressive acidemia on pituitary-adrenal endocrine function (adrenocorticotropic hormone, cortisol) and on adrenal blood flow in fetal sheep. Hypoxemia was induced by the maternal sheep breathing a gas mixture containing 9% oxygen, with 3% carbon dioxide added. Induced hypoxemia resulted in a progressive fetal metabolic acidosis but with little change in maternal pH. During induced hypoxemia there was little change in maternal plasma adrenocorticotropic hormone or cortisol level. Fetal adrenocorticotropic hormone and cortisol increased to peak values within 2.8 hours of induced hypoxia but by 7.2 hours had begun to fall to values that were not significantly different from those at 1.4 hours. Fetal adrenal blood flow (microsphere technique) also increased significantly and remained elevated throughout the duration (7.2 hours) of hypoxemia. The maximum fetal adrenal blood flow achieved during hypoxemia was significantly correlated with the basal (prehypoxemia) flow to the adrenals. We conclude that the changes in fetal adrenocorticotropic hormone, cortisol, and adrenal blood flow seen in short-term hypoxemia are reproduced during sustained hypoxemia with acidemia. Furthermore, the noted rise in the fetal adrenocorticotropic hormone level may be an important factor contributing to the increase in adrenal blood flow during hypoxemia.

Cardio‐renal and metabolic adaptations during pregnancy in female rats born small: implications for maternal health and second generation fetal growth

Non‐technical summary  Low weight at birth, or being born small for gestational age, is associated with increased risk of a number of adult diseases, including cardiovascular and kidney disease and diabetes. Generally, low birth weight males have a greater risk of developing such diseases but females do present with subtle changes in organ structure and function that might render them susceptible to lifestyle challenges. We show, for the first time, that low birth weight females have largely normal cardiovascular and kidney adaptations to pregnancy but they do develop altered glucose control. We have shown that their own fetuses are growth restricted suggesting that low birth weight and risk of disease development can be passed on to subsequent generations. These results warrant close monitoring of pregnant women who were born small and shape future studies to focus on therapeutic strategies to minimize the transmission of low birth weight and adult disease risk.