Andrew J. Jefferies

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.

Prenatal glucocorticoid exposure in the sheep alters renal development in utero: implications for adult renal function and blood pressure control

Treatment of the pregnant ewe with glucocorticoids early in pregnancy results in offspring with hypertension. This study examined whether glucocorticoids can reduce nephron formation or alter gene expression for sodium channels in the late gestation fetus. Sodium channel expression was also examined in 2-mo-old lambs, while arterial pressure and renal function was examined in adult female offspring before and during 6 wk of increased dietary salt intake. Pregnant ewes were treated with saline (SAL), dexamethasone (DEX; 0.48 mg/h) or cortisol (CORT; 5 mg/h) over days 26-28 of gestation (term = 150 days). At 140 days of gestation, glomerular number in CORT and DEX animals was 40 and 25% less, respectively, compared with SAL controls. Real-time PCR showed greater gene expression for the epithelial sodium channel (α-, β-, γ-subunits) and Na(+)-K(+)-ATPase (α-, β-, γ-subunits) in both the DEX and CORT group fetal kidneys compared with the SAL group with some of these changes persisting in 2-mo-old female offspring. In adulthood, sheep treated with dexamethasone or cortisol in utero had elevated arterial pressure and an apparent increase in single nephron glomerular filtration rate, but global renal hemodynamics and excretory function were normal and arterial pressure was not salt sensitive. Our findings show that the nephron-deficit in sheep exposed to glucocorticoids in utero is acquired before birth, so it is a potential cause, rather than a consequence, of their elevated arterial pressure in adulthood. Upregulation of sodium channels in these animals could provide a mechanistic link to sustained increases in arterial pressure in cortisol- and dexamethasone-exposed sheep, since it would be expected to promote salt and water retention during the postnatal period.

Differential effects of prenatal exposure to dexamethasone or cortisol on circulatory control mechanisms mediated by angiotensin II in the central nervous system of adult sheep

Prenatal exposure to elevated maternal glucocorticoids (dexamethasone (DEX) or cortisol (CORT)) for 2 days early in pregnancy can ‘programme’ alterations in adult offspring of sheep, including elevated arterial pressure. DEX treatment also results in greater angiotensin II type 1 (AT1) receptor expression in the medulla oblongata in late gestation fetuses than in saline (SAL)‐ or CORT‐exposed animals. We hypothesized that this would result in functional changes in brainstem angiotensinergic control of cardiovascular function in DEX‐ but not CORT‐exposed animals. To test this hypothesis, cardiovascular responses to intracerebroventricular (i.c.v.) angiotensin II were examined in adult male offspring exposed to DEX (0.48 mg h−1; n= 7), CORT (5 mg h−1, n= 6) or SAL (n= 9) from 26 to 28 days of gestation. Increases in mean arterial pressure during i.c.v. infusion of angiotensin II (1 or 10 μg h−1) were significantly greater in the DEX group (10 ± 1 mmHg at 1 μg h−1) compared with SAL (6 ± 1 mmHg) or CORT (6 ± 1 mmHg) animals (P < 0.05). i.c.v. infusion of the AT1 antagonist losartan significantly decreased cardiac output and heart rate in DEX animals, but not in SAL or CORT animals. Thus, increased expression of brainstem AT1 receptor mRNA after prenatal DEX is associated with increased responsiveness of cardiovascular control to activation of brain AT receptors by exogenous and endogenous angiotensin II. The altered role of the brain RAS in sheep exposed prenatally to DEX was not observed in sheep exposed prenatally to cortisol, suggesting these two glucocorticoids have distinct programming actions.

Development of cardiovascular disease due to renal insufficiency in male sheep following fetal unilateral nephrectomy.

Background Renal insufficiency is associated with the development of cardiovascular disease. Objectives This study investigated whether reduced fetal renal mass resulted in renal insufficiency, hypertension, cardiac dysfunction and whether these changes progressed with age. Methods and results Fetal uninephrectomy was performed at 100-day gestation (term, 150 days) and studies performed in male sheep from 6 weeks to 24 months of age. Renal function declined with age in sham animals as demonstrated by increasing plasma creatinine levels and urinary excretion of albumin. The age-related decline in renal function was exacerbated in animals that had undergone fetal uninephrectomy. Evidence of renal insufficiency was indicated from as early as 6 weeks of age with elevations in plasma creatinine (Ptreatment < 0.001), urea (Ptreatment < 0.001) and sodium (Ptreatment < 0.05) levels in uninephrectomized lambs as compared with sham animals. At 6 months, urinary albumin excretion (P < 0.001) was increased and urinary sodium excretion (P < 0.001) decreased in the uninephrectomized animals. By 24 months, renal function had deteriorated further with significant progression of albuminuria (Ptreatment×age < 0.001). Elevation of mean arterial pressure (∼15 mmHg) was associated with significantly increased cardiac output, stroke volume and plasma volume at 6 months; arterial pressure (∼27 mmHg) had increased further in uninephrectomized animals at 24 months and was driven by increased total peripheral resistance. Cardiac functional reserve (dobutamine challenge) was reduced in uninephrectomized animals at 6 and 24 months of age (Ptreatment < 0.001), and this was associated with left ventricular enlargement (P < 0.001) and reduced fractional shortening (P < 0.01). Conclusion Fetal uninephrectomy causing a reduction in nephron endowment results in an accelerated age-related decline in renal function. This is associated with an early onset of elevated blood pressure and impairments in cardiac structure and function.

Transgenerational programming of fetal nephron deficits and sex-specific adult hypertension in rats

A developmental insult that restricts growth in the first generation has the potential to program disease in subsequent generations. The aim of this study was to ascertain transgenerational growth and cardio-renal effects, via the maternal line, in a rat model of utero-placental insufficiency. Bilateral uterine vessel ligation or sham surgery (offspring termed first generation; F1 Restricted and Control, respectively) was performed in WKY rats. F1 Restricted and Control females were mated with normal males to produce second generation (F2) offspring (Restricted and Control) studied from fetal (embryonic Day 20) to adult (12 months) life. F2 Restricted male and female fetuses had reduced (P<0.05) nephron number (down 15-22%) but this deficit was not sustained postnatally and levels were similar to Controls at Day 35. F2 Restricted males, but not females, developed elevated (+16mmHg, P<0.05) systolic blood pressure at 6 months of age, which was sustained to 9 months. This was not explained by alterations to intra-renal or plasma components of the renin-angiotensin system. In a rat model of utero-placental insufficiency, we report alterations to F2 kidney development and sex-specific adult hypertension. This study demonstrates that low birthweight can have far-reaching effects that extend into the next generation.

Reduced nephron endowment due to fetal uninephrectomy impairs renal sodium handling in male sheep

Reduced nephron endowment is associated with development of renal and cardiovascular disease. We hypothesized this may be attributable to impaired sodium homoeostasis by the remaining nephrons. The present study investigated whether a nephron deficit, induced by fetal uninephrectomy at 100 days gestation (term=150 days), resulted in (i) altered renal sodium handling both under basal conditions and in response to an acute 0.9% saline load (50 ml.kg-1 of body weight.30 min-1); (ii) hypertension and (iii) altered expression of renal channels/transporters in male sheep at 6 months of age. Uninephrectomized animals had significantly elevated arterial pressure (90.1+/-1.6 compared with 77.8+/-2.9 mmHg; P<0.001), while glomerular filtration rate and renal blood flow (per g of kidney weight) were 30% lower than that of the sham animals. Total kidney weight was similar between the groups. Renal gene expression of apical NHE3 (type 3 Na+/H+ exchanger), ENaC (epithelium Na+ channel) beta and gamma subunits and basolateral Na+/K+ ATPase beta and gamma subunits were significantly elevated in uninephrectomized animals, while ENaC alpha subunit expression was reduced. Urine flow rate and sodium excretion increased in both groups in response to salt loading, but this increase in sodium excretion was delayed by approximately 90 min in the uninephrectomized animals, while total sodium output was 12% in excess of the infused load (P<0.05). In conclusion, the present study shows that animals with a congenital nephron deficit have alterations in tubular sodium channels/transporters and cannot rapidly correct for variations in sodium intake probably contributing to the development of hypertension. This suggests that people born with a nephron deficit should be monitored for early signs of renal and cardiovascular disease.

Transgenerational metabolic outcomes associated with uteroplacental insufficiency

Intrauterine growth restriction increases adult metabolic disease risk with evidence to suggest that suboptimal conditions in utero can have transgenerational effects. We determined whether impaired glucose tolerance, reduced insulin secretion, and pancreatic deficits are evident in second-generation (F2) male and female offspring from growth-restricted mothers, in a rat model of uteroplacental insufficiency. Late gestation uteroplacental insufficiency was induced by bilateral uterine vessel ligation (restricted) or sham surgery (control) in Wistar-Kyoto rats. First-generation (F1) control and restricted females were mated with normal males and F2 offspring studied at postnatal day 35 and at 6 and 12 months. F2 glucose tolerance, insulin secretion, and sensitivity were assessed at 6 and 12 months and pancreatic morphology was quantified at all study ages. At 6 months, F2 restricted male offspring exhibited blunted first-phase insulin response (-35%), which was associated with reduced pancreatic β-cell mass (-29%). By contrast, F2 restricted females had increased β-cell mass despite reduced first-phase insulin response (-38%). This was not associated with any changes in plasma estradiol concentrations. Regardless of maternal birth weight, F2 control and restricted males had reduced homeostatic model assessment of insulin resistance and elevated plasma triglyceride concentrations at 6 months and reduced whole-body insulin sensitivity at 6 and 12 months compared with females. We report that low maternal birth weight is associated with reduced first-phase insulin response and gender-specific differences in pancreatic morphology in the F2. Further studies will define the mode(s) of disease transmission, including direct insults to developing gametes, adverse maternal responses to pregnancy, or inherited mechanisms.

Pregnancy in aged rats that were born small: cardiorenal and metabolic adaptations and second-generation fetal growth

Uteroplacental insufficiency is associated with adult cardiorenal and metabolic diseases, particularly in males. Pregnancy is the greatest physiological challenge facing women, and those born small are at increased risk of gestational hypertension and diabetes and delivering smaller babies. Increased maternal age is associated with exacerbated pregnancy complications. We hypothesized that pregnancy in aged, growth‐restricted females unmasks an underlying predisposition to cardiorenal and metabolic dysfunction and compromises fetal growth. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (restricted group) or sham surgery (control group) on d 18 of gestation in Wistar Kyoto rats. At 12 mo, growth‐restricted F1 female offspring were mated with a normal male. F1 restricted females had elevated systolic blood pressure, before and during pregnancy (+10 mmHg) but normal renal and metabolic pregnancy adaptations. F2 fetal weight was not different between groups. In control and restricted females, advanced maternal age (12 vs. 4 mo) was associated with a reduction in the hypoglycemic response to pregnancy and reduced F2 fetal litter size and body weight. Aged rats born small exhibited mostly normal pregnancy adaptations, although they had elevated blood pressure. Advanced maternal age was associated with poorer fetal outcomes that were not exacerbated by low maternal birth weight.—Gallo, L. A., Tran, M., Moritz, K. M., Jefferies, A. J., Wlodek, M. E. Pregnancy in aged rats that were born small: cardiorenal and metabolic adaptations and second‐generation fetal growth. FASEB J. 26, 4337–4347 (2012). www.fasebj.org

Long-Term Alteration in Maternal Blood Pressure and Renal Function After Pregnancy in Normal and Growth-Restricted Rats

Intrauterine growth restriction is associated with increased risk of adult cardiorenal diseases. Small birth weight females are more likely to experience complications during their own pregnancy, including pregnancy-induced hypertension, preeclampsia, and gestational diabetes. We determined whether the physiological demand of pregnancy predisposes growth-restricted females to cardiovascular and renal dysfunction later in life. Late gestation bilateral uterine vessel ligation was performed in Wistar-Kyoto rats. At 4 months, restricted and control female offspring were mated with normal males and delivered naturally (ex-pregnant). Regardless of maternal birth weight, at 13 months, ex-pregnant females developed elevated mean arterial pressure (indwelling tail-artery catheter; +6 mm Hg), reduced effective renal blood flow (14C-PAH clearance; −23%), and increased renal vascular resistance (+27%) compared with age-matched virgins. Glomerular filtration rate (3H-inulin clearance) was not different across groups. This adverse cardiorenal phenotype in ex-pregnant females was associated with elevated systemic (+57%) and altered intrarenal components of the renin-angiotensin system. After pregnancy at 13 months, coronary flow (Langendorff preparation) was halved in restricted females compared with controls, and together with reduced NO excretion, this may increase susceptibility to additional lifestyle challenges. Our results have implications for aging females who have been pregnant, suggesting long-term cardiovascular and renal alterations, with additional consequences for females who were small at birth.

Effect of Pregnancy for Females Born Small on Later Life Metabolic Disease Risk

There is a strong inverse relationship between a females own birth weight and her subsequent risk for gestational diabetes with increased risk of developing diabetes later in life. We have shown that growth restricted females develop loss of glucose tolerance during late pregnancy with normal pancreatic function. The aim of this study was to determine whether growth restricted females develop long-term impairment of metabolic control after an adverse pregnancy adaptation. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) in late pregnancy (E18) in F0 female rats. F1 Control and Restricted female offspring were mated with normal males and allowed to deliver (termed Ex-Pregnant). Age-matched Control and Restricted Virgins were also studied and glucose tolerance and insulin secretion were determined. Pancreatic morphology and hepatic glycogen and triacylglycerol content were quantified respectively. Restricted females were born lighter than Control and remained lighter at all time points studied (p<0.05). Glucose tolerance, first phase insulin secretion and liver glycogen and triacylglycerol content were not different across groups, with no changes in β-cell mass. Second phase insulin secretion was reduced in Restricted Virgins (−34%, p<0.05) compared to Control Virgins, suggestive of enhanced peripheral insulin sensitivity but this was lost after pregnancy. Growth restriction was associated with enhanced basal hepatic insulin sensitivity, which may provide compensatory benefits to prevent adverse metabolic outcomes often associated with being born small. A prior pregnancy was associated with reduced hepatic insulin sensitivity with effects more pronounced in Controls than Restricted. Our data suggests that pregnancy ameliorates the enhanced peripheral insulin sensitivity in growth restricted females and has deleterious effects for hepatic insulin sensitivity, regardless of maternal birth weight.