INTRODUCTION

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RENAL INSUFFICIENCY IS THE most frequent sequelae of neonatal asphyxia (8, 37). Obviously, asphyxial-induced acute renal failure is mainly initiated by abrupt reduction of renal blood flow (RBF) due to increased renovascular resistance (RVR). Decreased blood oxygen tension usually induces renal vasoconstriction even in newborns (14, 15). The pathophysiological mechanisms leading to this net response of renal circulation on acute hypoxemia are still controversial and involve various local vascular, humoral, and chemoreceptor effects with contradictory vasoconstrictory and vasodilatory potencies. Renal changes induced by hypoxemia are presumably mediated by overstimulation of ANG II (21) and intrarenal adenosine (13, 20) that are already hyperactive during the neonatal period. Moreover, a hypoxia-induced increase of adrenergic input obviously participates in RVR increase, presumably due to an increased sensitivity to vasoconstrictor catecholamines of neonatal kidney vasculature (7, 39). These vasoconstrictor effects overwhelm responses of vasodilatory mechanisms, such as endogenous nitric oxide release, that play a crucial role in maintaining basal renal perfusion and remain effective at least during moderate hypoxemia so that the vasoconstrictory effect is blunted (1).

Intrauterine growth restriction (IUGR) is a serious clinical problem and has been associated recently with common adult diseases including hypertension, non-insulin-dependent diabetes mellitus, dyslipidemia, and ischemic heart disease (2, 23). The mechanisms underlying the epidemiological observations are unknown, but it has been proposed that the cardiovascular adaptations that lead to hypertension are initiated during fetal life and are progressively amplified after birth and throughout adult life (26). A reduced number of nephrons at birth was proposed to be a missing link in the etiology of hypertension after intrauterine malnutrition (30). Indeed, an impaired development of renal nephrons has been suggested to occur during the third trimester of gestation (18). Growth retardation is associated with impaired renal maturation, which, during the first 24 h of life, is manifest as decreased glomerular filtration rate (GFR) and increased sodium excretion (40). Recent animal studies suggest that IUGR is accompanied by nephron deficit that may not be fully compensated within the first weeks after birth, despite compensatory hypertrophy, and that overall renal function was impaired irrespective of cause of IUGR (maternal protein malnutrition or restricted placental perfusion) (32). Glucocorticoids may contribute to impaired renal development and led to arterial hypertension in offspring (9, 24). However, the role of oxygen deprivation on renal hemodynamics and excretory function in IUGR newborns remains unclear. This is of importance because IUGR is associated with an increased incidence of neonatal asphyxia (29). Therefore, the question arises whether compromised prenatal growth has a specific influence on compromised renal function due to hypoxemia in the immediate newborn period.

In this study, renal hemodynamics and excretory function in normal weight (NW) and IUGR newborn piglets were investigated at different states of renal oxygen delivery. We asked whether IUGR influences renovascular, glomerular, or tubular responses to severe normocapnic hypoxemia. We used a morphometrically well-characterized state of asymmetrical IUGR in newborn piglets (3, 17, 41) and included animals with optimal vital conditions early after birth (10).

	MATERIALS AND METHODS

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All surgical and experimental procedures were approved by the Committee of the Thuringian State Government on animal research. Animals were obtained from a breeding farm. Delivery was observed, and the viability of neonatal piglets was assessed immediately after birth so that only animals with a viability score 7 (10) were included in the study. Immediately before the onset of the experiments, animals were carried to the laboratory in a climatized transport incubator (environmental temperature 33-34°C; time for transportation 30-60 min). Animals were divided into NW piglets and IUGR piglets according to birth weight. The NW category included animals with a birth weight >40th percentile (piglets heavier than 1,220 g); the IUGR category included animals with a birth weight >5th and <10th percentiles (piglets with a birth weight between 733 and 853 g). The birth weight distribution of the breed of piglets used here (German Landrace) has been described previously (3).

Surgical procedures. Fourteen NW newborn piglets [age 12-22 h, body wt 1,497 ± 107 g: normoxic control (group 1, n = 7); severe normocapnic hypoxemia (group 3, n = 7)] and 14 IUGR piglets [age 10-25 h, body wt 795 ± 39 g: normoxic control (group 2, n = 7); severe normocapnic hypoxemia (group 4, n = 7)] were anesthetized initially with 1.5% isoflurane in 70% nitrous oxide and 30% oxygen. Cutaneous incisions were made after subcutaneous instillation of a local anesthetic (xylocitin 2%, Jenapharm Jena, Germany). A central venous catheter was introduced through the left external jugular vein and used for the administration of drugs and for volume substitution (lactated Ringer solution, 5 ml · kg body wt¹ · h¹). An appropriate endotracheal tube (12-16 French Charier) was inserted through a tracheotomy. After immobilization with pancuronium bromide (0.2 mg · kg body wt¹ · h¹ iv), the piglets were artificially ventilated (Servo Ventilator 900C, Siemens-Elema, Sweden). Anesthesia was maintained throughout the surgical interventions with 0.5-0.8% isoflurane. A polyurethane catheter (PU 3.5 Ch, Sherwood, England) was advanced through an umbilical artery into the abdominal aorta to measure blood gases and pH, record arterial blood pressure, and withdraw reference blood samples. The left ventricle was cannulated retrogradely via the right common carotid artery with a polyurethane catheter (PU 3.5 Ch, Sherwood). The ureters were exposed through diagonal incisions in the flank (of both sides) located midway between the twelfth rib and the pelvic rim. They were intersected, and the renal pelves were drained for urine sampling using retrogradely inserted polyurethane catheters (PU 3.5 Ch, Sherwood). Correct positioning of the catheter tips was checked by autopsy at the end of the experiment. Rectal temperature was maintained throughout the experiment at 38°C using a water-perfused heating pad and a feedback-controlled heating lamp. Arterial and left ventricular catheters were connected with pressure transducers (P23Db, Statham Instruments, Puerto Rico). Physiological parameters were recorded on a multichannel polygraph (MT95K2, Astro-Med).

Experimental protocol. After the surgical preparation was complete, blood exchange of 30 ml was performed (intravenous infusion of heparinized blood obtained from a donor piglet and withrawal of the same amount from an arterial line, simultaneously) to replace blood volume throughout the experiment. Then, the isoflurane concentration was adjusted to 0.25% in 70% nitrous oxide and 30% oxygen, and the piglets were allowed to rest for ~60 min. At this time, seven NW (group 3) and seven IUGR animals (group 4) were infused intravenously with isotonic saline containing 4 g/l fluorescein isothiocyanate-inulin (Bioflor, Uppsala, Sweden) at 5 ml · kg body wt¹ · h¹ for the rest of the experiment after priming with 4 ml/kg body wt infused within 2 min. Animals of groups 1 and 2 were infused intravenously with lactated Ringer solution (5 ml · kg body wt¹ · h¹ for the rest of the experiment). This discrepancy in experimental protocol between groups 1 and 2 versus 3 and 4 was without causing detectable changes in baseline RBF, RVR, renal O₂ delivery, and urine flow as well as physiological parameters (see RESULTS). Urine was then collected for ~ 60 min, and the urine samples obtained were weighed. Then, in groups 3 and 4, the inspired fraction of oxygen (FI_O2) was reduced from 0.35 to ~0.07 for 1 h to induce severe normocapnic hypoxemia (oxygen saturation of arterial blood was adjusted between 15 and 20%). Urine was collected between the 1st and 30th and the 30th and 60th min of hypoxia, and the urine samples obtained were weighed. Thereafter, ventilatory gas mixture was reestablished, and the recovery was monitored for 180 min. Urine was collected between the 1st and 30th and the 150th and 180th min of reoxygenation. At the end of baseline period (baseline), at the 50th min of hypoxia, and at the 30th and 180th min of reoxygenation, arterial blood pressure and heart rate were measured, and then RBFs were estimated (a total of 3 ml blood was withdrawn as the reference sample) immediately followed by the withdrawal of arterial blood samples (a total of 2.5 ml) to estimate blood gases and electrolytes, pH, glucose and lactate content, and catecholamines. Subsequently, the same volume of donor blood was reinfused. Immediately after reoxygenation period was started, the metabolic acidosis was corrected by an intravenous injection of sodium bicarbonate. Animals of groups 1 and 2 received all experimental procedures except change of ventilatory gas mixture and inulin infusion and served as sham-operated control animals.

Analytical procedures and calculations. Blood pH, PCO₂, and PO₂ were determined with an ABL50 blood gas analyzer (Radiometer, Copenhagen, Denmark). Blood hemoglobin content and oxygen saturation were determined using a hemoxymeter OSM2 (Radiometer). Sodium, potassium, glucose, and lactate contents were determined with an electrolyte, metabolite laboratory EML105 (Radiometer). Hematocrit was determined using the microhematocrit method. Inulin concentration in blood and urine samples was measured fluorimetrically (42). Inulin clearance has been proposed to assess GFR adequately, because the indicator substance fulfils all prerequisites for exact GFR quantification in the mature (31) as well as the immature (16, 38) kidney.

Statistical analysis. Data are reported as means ± SD. One-way ANOVA was used to determine differences between groups. One-way ANOVA with repeated measures was used to determine within effects of experimental procedures. Post hoc comparisons were made with the Student-Newman-Keuls method. Comparisons of renal excretory functions between groups 3 and 4 were made with unpaired t-tests. Differences were considered significant when P < 0.05.

	RESULTS

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Table 1 summarizes the morphometric parameters of the experimental groups. In IUGR piglets, body weight was similarly reduced by 47%. Naturally occurring growth retardation in swine is asymmetrical with an increase in the mean ratio of brain weight to liver weight from 1.0 to 1.7-1.9 (P < 0.01). There was a significant decrease in weight of all organs measured. However, the reduction in brain weight was quite small (11%). In contrast, the decrease in liver (50%) and kidney (42 to 43%) weight was proportional to that in body weight (46 to 48%). All differences in organ weight were significant (P < 0.01).

During baseline conditions (control), arterial blood pressure and blood gas values were within the physiological range and consistent with other data obtained from anesthetized and artificially ventilated newborn piglets (28). With exception of a reduced arterial glucose content in IUGR piglets, the physiological parameters were similar in NW and IUGR piglets and remained unchanged throughout the experiment in the sham-operated NW and IUGR piglets (Table 2). In addition, arterial hemoglobin content was similar in NW and IUGR piglets (group 1: 6.2 ± 0.9 mmol/l; group 2: 5.8 ± 1.5 mmol/l; group 3: 5.6 ± 1.1 mmol/l; group 4: 5.5 ± 1.3 mmol/l). Similar values were also estimated in NW and IUGR piglets for renal hemodynamics and oxygen delivery as well as urinary flow (Fig. 1). Arterial catecholamine content, however, increased slightly throughout the experimental course in sham-operated NW and IUGR piglets (P < 0.05). Furthermore, during baseline conditions, GFR and FF were significantly less in IUGR animals compared with NW animals (Fig. 2, P < 0.05). The difference in baseline GFR between NW and IUGR was more pronounced when corrected for kidney weight (NW: 0.14 ± 0.04 ml · min¹ · g kidney wt¹; IUGR: 0.084 ± 0.02 ml · min¹ · g kidney wt¹; 42% reduction of GFR in IUGR, P < 0.01). Despite this, under baseline conditions, NW and IUGR newborn piglets reabsorbed sodium very efficiently, the FSE was <1% in both groups, and osmotic clearance was similar.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effect of severe hypoxia on renal hemodynamics, oyxgen delivery, and urinary flow. Comparison between normal weight (NW) sham-operated control newborn piglets [group 1 (NW control), n = 7], intrauterine growth-restricted (IUGR) sham-operated control animals (group 2, n = 7), NW hypoxia-treated animals (group 3, n = 7), and IUGR hypoxia-treated animals (group 4, n = 7). Values are means ± SD; * comparison between sham-operated and hypoxia-treated groups; $ significant differences between normal weight and IUGR newborn piglets (P < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of severe hypoxia on renal excretory functions in NW piglets (group 3) and IUGR newborn piglets (group 4). Values are means ± SD; § significant differences within a group, compared with control values; $ significant differences between NW and IUGR newborn piglets (P < 0.05; $$ P < 0.01).

In both groups, severe hypoxia induced pronounced metabolic acidosis with increased arterial lactate content and strongly reduced RBF, renal oxygen delivery, and GFR as well as significantly increased FSE (P < 0.05). Notable was the less-decreased RBF in IUGR piglets (25 ± 15% of control) compared with NW ones (6 ± 3% of control; P < 0.05) together with a less-pronounced increase of RVR and arterial catecolamines in IUGR piglets (P < 0.05). Furthermore, the amount of metabolic acidosis was also reduced in IUGR piglets (P < 0.05). The increase of FF found in NW piglets during normocapnic hypoxemia (P < 0.05) did not occur in IUGR piglets. However, a decrease in arterial blood pressure and arterial glucose content was found only in IUGR piglets during hypoxemia (P < 0.05).

Early recovery showed a transient period of diuresis with increased osmotic clearance and elevated FSE in both groups (P < 0.05). RBF and O₂ delivery remained reduced. A partial reestablishment of renal function had occurred at the end of the recovery period observed. FSE, osmotic clearance, urine flow rate, and FF recovered fully in both groups. However, GFR and renal O₂ delivery remained reduced in NW piglets (P < 0.05).

	DISCUSSION

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Disturbed intrauterine development yields to a marked alteration of renal excretory function. As shown in a morphometrically well-characterized state of asymmetrical IUGR (3) used in this study, GFR was significantly reduced by ~37% (P < 0.05). This is presumably caused by a marked nephron deficit shown in different species (18, 25, 32), which may not be completely compensated within the first weeks after birth, despite compensatory hypertrophy and irrespective of whether IUGR was induced by partial uterine artery ligation or maternal protein deprivation (32). Otherwise, RBF was similar in NW and IUGR newborn piglets when normalized for body weight. Discrepancy between reduced GFR but unchanged RBF in IUGR piglets under normal conditions cannot be clarified in this study. Nevertheless, the difference in FF between NW and IUGR piglets indicate that there is an altered renovascular state in IUGR piglets, presumably with a reduced vascular tone of efferent glomerular arterioles. The reduced FF in the IUGR group could also be explained by an alteration of the glomerular ultrafiltration coefficient (K_f) by highly activated ANG II. Morphometric analysis and single-nephron GFR estimation in adult rats have clearly revealed that ANG II infusion caused a marked reduction of K_f by mesangial cell contraction without causing detectable changes in epithelial cell or filtration slit structure (6, 35). Nevertheless, baseline FF in 1-day-old NW piglets is comparable with FF values shown in premature lambs ~3 h after delivery (12) and in ~1-wk-old rats (19). However, there is obviously a postnatal increase in FF, presumably because of an increase in GFR disproportionately greater than the increase in RPF (19).

Severe hypoxia induced a marked reduction of RBF that was less pronounced in IUGR piglets (25 ± 15% of control) compared with NW ones (6 ± 3% of control; P < 0.05). Similarly, during hypoxemia, the extent of catecholamine increase appeared considerably more pronounced in NW than in IUGR piglets (P < 0.05). Obviously, there was a differing increase of the -adrenergic impact on RVR during severe normocapnic hypoxemia. A previous study has shown that the renal circulation of swine contains a functioning -adrenergic neuroeffector system at birth, which was, however, less sensitive to low frequencies of renal nerve stimulation and to exogenous norepinephrine than in older piglets (7). Furthermore, in contrast to older animals, cardiovascular response in newborn piglets is mainly caused by circulating catecholamines due to delayed central sympathetic maturity. Indeed, Lee et al. (27) have shown that hypoxia-induced cardiovascular responses were observed in intact and ganglionic-blockade piglets, but no changes occurred in adrenalectomized piglets. Direct stimulation of adrenal medulla and extramedullar chromaffin cells by reduced arterial PO₂ is mainly responsible for increased catecholamine release in newborn rats early after birth (33). Therefore, sympathetic activity of newborn piglets seems to be reflected by changes in circulating catecholamines and may serve as a relevant indicator for the renovascular response on increased -adrenergic effect due to systemic hypoxia.

Further vasoactive agents may participate on renovascular response at severe hypoxemia and related effects on glomerular filtration. Renal adenosine release may be partly reponsible for RVR increase, at least early after onset of hypoxia (11). In that study, however, the extent of renal vasoconstriction was <50% and completely abolished by pretreatment of adenosine-receptor blockade. The renal renin-angiotensin system, which is highly activated at birth and remains so during the period of renal development (36), has also been implicated in hypoxemia-induced renovascular changes. Huet et al. (21) showed that in newborn rabbits, ANG II modulates the renal immature microcirculation during moderate hypoxemia and that inhibition of its formation effectively prevents the hypoxemia-induced decrease in GFR (21). A blunting effect of nitric oxide, a potent renovascular dilator in immature kidneys, has been shown during moderate hypoxemia in newborn rabbits (1); however, this could not be verified due to severe hypoxemia in newborn piglets (34).

The blunted response of renal vasoconstriction due to the same extent of severe hypoxemia in IUGR piglets may be associated with a reduced humoral catecholamine response. Influence of IUGR on catecholamine release is thought to be related to the extent of O₂ deprivation, because moderate normocapnic hypoxemia was associated with enhanced catecholamine release in newborn IUGR piglets even though the extent of arterial catecholamine increase was considerably less (5). Interestingly, the markedly increased FF in NW piglets but unchanged FF in IUGR piglets suggests that a changed responsiveness of glomerular resistive vessels occurs in IUGR piglets, which resulted in a merely moderate reduction of GFR and maintained osmotic clearance. In response to severe hypoxemia, the NW piglets decreased the RBF to a greater degree than GFR, producing an increase in the FF. However, moderate hypoxemia produced a proportionate change in these parameters in newborn rabbits not altering FF (1). This may be partly caused by the augmented catecholamine response of NW piglets compared with IUGR piglets during severe hypoxia. A similar response in RBF and catecholamine response was found during moderate normoxic hypoxemia in newborn NW and IUGR piglets (Bauer et al., unpublished data). However, a marked sodium loss occurred during hypoxemia. This is obviously caused by a widely disturbed oxidative energy production. Combined effect of hypoxemia-induced reduction of RBF and arterial O₂ content led to a pronounced deprivation of renal O₂ delivery of <5% in both groups. Consequently, FSE increased markedly and remained increased during the early recovery period. Moreover, osmotic clearance was increased fivefold, indicating not only a considerable retention of substances usually eliminated with urine during hypoxemia, but also a dangerous sodium loss as a result of elevated FSE and increased urine flow early after severe hypoxemia. Within 3 h, however, a widely reestablished renal excretory function occurred.

Perspectives