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Departments of Physiology & Biophysics/Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Submitted 12 May 2003 ; accepted in final form 2 September 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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renal function; glomerular filtration rate; sodium excretion; newborn; perinatal
Excluding the newborn infant, severe hemorrhage in infants and children, as in adults, most commonly results from major trauma such as in an automobile accident resulting in external and/or internal bleeding. Hemorrhagic shock can also occur in utero [fetomaternal hemorrhage, twin-to-twin transfusion, maternal trauma (11)], at delivery (umbilical cord rupture, incision of the placenta during caesarean delivery, placenta previa or abruptio placentae), or postnatally as the result of mild birth-related hemorrhage (soft tissue injury such as abrasions and bruising, or lacerations of the scalp, buttocks, and thighs by scalpel use during caesarian delivery). More severe hemorrhage occurring postnatally includes the occurrence of cephalhematomas, subgaleal hemorrhage, and blood loss from the retroperitoneal region, liver capsule, and/or ruptured spleen (11), which can result in severe hypovolemic shock leading to death if untreated (3). Despite the relatively common occurrence of hemorrhage during the perinatal period (3), physiological responses to hemorrhage in the newborn remain poorly understood.
In previous studies in conscious, chronically instrumented lambs, we have measured some of the physiological responses to hemorrhage during postnatal maturation and determined that several cardiovascular and endocrine responses to hemorrhage are developmentally regulated (19-21). For example, the renin response to hemorrhage is greater and more prolonged in newborns than that measured in older animals and occurs at a lesser degree of blood loss. Furthermore, there is an increase in heart rate after hypotensive hemorrhage (
20% hemorrhage) in conscious lambs, whereas heart rate remains relatively constant in young adult sheep. From this, we postulated that the effects of hemorrhage on renal function might also be altered during early postnatal life.
In the present experiments, renal responses to hemorrhage were measured in conscious, chronically instrumented lambs and young adult weanling sheep, to determine whether the renal responses to hemorrhage are also developmentally regulated.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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There were two groups of animals: Newborn lambs were studied at 1-2 wk of age (n = 8, males n = 3, females n = 5; 12 ± 2 days; 7.8 ± 0.8 kg body wt). Young adult weanling sheep were studied at 11-12 wk of age (n = 11, males n = 7, females n = 4; 80 ± 7 days; 21.3 ± 4.2 kg body wt). At this age, the adult splenic response is not present (26-28), thereby avoiding the necessity of performing a splenectomy, which would invalidate comparisons between the two ages.
During the days before and after surgery and throughout the study period, lambs and young adult sheep were weighed daily at 0800 and rectal temperatures were measured to monitor the health status of each experimental animal. Except during surgery and experiments, lambs were housed with their mothers in individual pens in the vivarium and suckled freely. Ewes were allowed water ad libitum as well as one flake of hay and six cups daily of the following food mixture: 18% lactating ewe ration (Na content 0.1%) and 14% lamb grow-finisher ration pellets (Na content 0.4%) in the ratio 1:2. Young adult sheep were housed separately in pens in the vivarium and allowed water ad libitum as well as one flake of hay and four cups daily of the aforementioned food mixture. Feed was obtained from a local source (Unifeed Limited, Okotoks, Alberta, Canada). Young adult sheep were fasted for 12 h before surgery. All surgical and experimental procedures were carried out in accordance with the "Guide to the Care and Use of Experimental Animals" provided by the Canadian Council on Animal Care and with the approval of the Animal Care Committee of the University of Calgary.
Surgical procedures. Surgery was performed on lambs 2-5 days after birth with the use of aseptic techniques, as previously described (18, 22), and on young adult sheep at least 4 days before the start of experiments. Anesthesia was induced with a mask and halothane (3-4%) in oxygen, the trachea was intubated, and anesthesia was maintained by ventilating the lungs with halothane (0.5-1%) in a mixture of nitrous oxide and oxygen (3:1). Catheters were inserted into left and right femoral veins and arteries and advanced to the inferior vena cava and aorta (PE 160 catheter, Intramedic) for later intravenous (IV) infusions, arterial sampling, measurement of blood pressure (BP), and blood withdrawal for hemorrhage. Catheters were tunneled subcutaneously to exit the lamb on right and left flanks. By means of an abdominal midline incision, the bladder was then exposed, and a catheter was inserted directly across the bladder wall by use of a specially adapted feeding tube (Medi-Craft), for later measurement of urinary flow rate (V). Through a left flank incision, the left renal artery was carefully dissected free of tissue, and a precalibrated ultrasonic flow transducer was placed around the renal artery (3-6S, Transonics Instruments) as previously described (15, 16) for measurement of renal blood flow (RBF) and calculation of renal plasma flow (RPF).
All catheters and the flow transducer cable were secured in a body jacket (Lomir) for safe storage between experiments. Antibiotics (5.0 mg/kg enrofloxacin, Baytril) were administered intramuscularly at surgery and at 12-h intervals thereafter, for 48 h. Lambs were allowed to recover from the effects of surgery and anesthesia in a critical care unit for small animals (Shor-line, Schroer Manufacturing), with adjustable oxygen supply. All lambs were able to stand within 30 min of completion of surgery, on which they were returned to their mothers in the vivarium. They suckled immediately on return to the ewe.
Young adult sheep were placed in recovery pens after surgery and returned to their pens in the vivarium on full recovery.
Experiments were not begun until a minimum of 4 days had elapsed after surgery. During this time, animals were trained to rest comfortably in a supportive sling in the laboratory environment. Two experiments were carried out in each animal at intervals of 2-5 days (experiment 1, 10% hemorrhage; experiment 2, 20% hemorrhage); the order of experiments was randomized.
Detailed experimental procedures. On the day of an experiment, the animal was removed from the vivarium and placed in the same supportive sling in the laboratory environment for at least 60 min. During this time, the bladder was allowed to drain. A priming dose of [14C]inulin (0.5 µCi/kg) in dextrose was injected IV followed by constant IV infusion at 0.25 µCi·kg-1·h-1 (0.5 ml·kg-1·h-1) for later measurement of glomerular filtration rate (GFR). An IV infusion of 5% dextrose in 0.9% sodium chloride was started at a rate of 4.17 ml·kg-1·h-1 and continued for the duration of the experiment to assist in maintaining fluid balance.
After the 60-min equilibration period, the experiment was started. Each experiment consisted of consecutive 20-min urinary collection periods during a 1-h control period (3 x 20 min). This was followed by hemorrhage over 10 min of 10 or 20% of blood volume, estimated as 75 ml/kg (19). [Previously, using T-1824 and the methods described by Parving et al. (8) and Thomsen et al. (25), we determined that blood volume was 75 ml/kg during the first 3 mo of life in conscious sheep.] Hemorrhage was achieved from the left femoral venous catheter by use of a programmable syringe pump (model 55-4143, Harvard Apparatus) (19). Urine collections were then continued at 20-min intervals for 1 h after hemorrhage (3 x 20 min). Five minutes before the start of hemorrhage, 400 U/kg body wt of heparin were injected IV to prevent coagulation during and after hemorrhage. Removed blood was maintained at 37°C for transfusion at the end of the experiment.
The arterial catheter was connected to a pressure transducer (Statham, P23Db, West Warwick, Rhode Island) for BP measurement; the flow transducer cable was connected to a flowmeter (Transonics Systems, Ithaca, NY) for measurement of RBF. These cardiovascular variables were recorded onto a polygraph (Grass Instruments, model 7, West Warwick, RI) and simultaneously to an IBM-PC at 200 Hz by use of the data-acquisition and analysis software CVSOFT (Odessa Systems, Calgary, Alberta, Canada).
At the end of each 20-min urinary collection period, urine volume was recorded and samples were stored at -70°C for later determination of urinary electrolytes (Na+, K+, Cl-) and urinary osmolality (UOsm). Arterial blood (2.5 ml) was also removed at the end of each urinary collection period for immediate measurement of hematocrit (Hct) and blood gas status; the remaining blood was centrifuged and supernatant was removed and stored at -70°C for later determination of plasma electrolytes (Na+, K+, Cl-) and plasma osmolality. Additional blood (3 ml) was removed at the end of the control period and at 20 and 60 min after hemorrhage; blood was centrifuged and supernatant was removed for measurement of plasma levels of [14C]inulin. At the end of each experiment, blood removed during the 10-min hemorrhage period was returned to the animal by use of the same programmable syringe pump.
At the end of the two experiments, animals were killed with a lethal dose of sodium pentobarbitone and, on postmortem examination, placement of catheters was verified and the zero offset of the flow transducer was measured. Right and left kidneys were removed and immediately weighed.
Analytical procedures. Hct was determined in duplicate by use of a microhematocrit centrifuge (Clay Adams, Parsippany, NJ) and careful measurements using calipers and the methods of Brace (2). Blood-gas status was evaluated by measuring pH and arterial PCO2 and PO2 with the use of a blood-gas analyzer (NOVA, Stat 3). Urinary and plasma [14C]inulin levels were measured after each experiment by liquid scintillation (Wallace 1410). Urine and plasma samples were later thawed to room temperature, and urinary and plasma electrolytes (Na+, K+, Cl-) and osmolalities were measured by using a flame photometer (IL-943), chloridometer, and micro-osmometer (Advanced Instruments model 3MO).
Computations. GFR was calculated as the clearance of [14C]-inulin. Fractional reabsorption (FR) of electrolytes (x) was determined from the ratio of electrolyte clearance (Cx) to GFR as follows: FRx(%) = (1 - Cx/GFR) x 100. Free water clearance (CH2O) was calculated as the difference between V and osmolar clearance. Filtration fraction (FF) was determined as GFR/RPF where RPF = [1 - (RBF x Hct)]. Parameters of renal function were corrected for kidney weight to normalize data between the two age groups.
Statistical analyses. Because values obtained during the first three urinary collections (3 x 20 min) were similar, these were averaged and presented as a single value (Control). To evaluate changes from control after hemorrhage, ANOVA procedures for repeated measures over time were applied to all measured and calculated variables, factors being degree of hemorrhage (10%, 20%) and age (lambs, young adult sheep). Where the F value was significant, Newman-Keuls tests were applied to determine where the significant differences occurred. All data are expressed as means ± SD; significance was accepted at the 95% confidence interval.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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There were no effects of 10% hemorrhage on BP. In response to 20% hemorrhage, BP decreased in lambs from 85 ± 7 to 74 ± 7 mmHg within 20 min and returned to 78 ± 8 mmHg by 60 min. Similarly, in young adult sheep, BP decreased from 85 ± 6 to 77 ± 8 mmHg at 20 min and returned to 83 ± 5 mmHg by 60 min.
There were no effects of 10-20% hemorrhage on RPF in newborns or adults (Fig. 1). There were no effects of 10% hemorrhage on GFR or FF. GFR and FF decreased after 20% hemorrhage in both age groups, the decrease being greater in lambs than young adult sheep (Fig. 1).
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V and urinary Na+ excretion rate (UNaV) remained constant after 10% hemorrhage. V and UNaV decreased by 40 min after 20% hemorrhage in young adult sheep but not lambs (Fig. 2) and remained decreased for 60 min; urinary Cl- excretion rate showed a similar response. There were no effects of 10-20% hemorrhage on urinary K+ excretion rate in either age group.
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Total reabsorbed Na+ decreased after 20% hemorrhage but not 10% hemorrhage in both age groups, effects being greater and more prolonged in lambs than young adult sheep. Fractional Na+ reabsorption remained constant after 20% hemorrhage in lambs and increased at 60 min after hemorrhage in young adult sheep (Table 2). Total reabsorbed K+ decreased after 20% hemorrhage but not 10% hemorrhage in young adult sheep but not lambs. Fractional K+ reabsorption remained constant after 20% hemorrhage in lambs and increased at 60 min after hemorrhage in young adult sheep (Table 2). Total reabsorbed Cl- decreased after 20% hemorrhage but not 10% hemorrhage in young adult sheep but not lambs. Fractional Cl- reabsorption remained constant after 10-20% hemorrhage in both age groups (Table 2).
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There were no effects of 10% hemorrhage on CH2O or UOsm in either age group of animals. UOsm decreased at 20 min after 20% hemorrhage in young adult sheep but not lambs, returning to control levels by 40 min (Fig. 3). In young adult sheep but not lambs, CH2O increased by 20 min after 20% hemorrhage and remained above control at 60 min. In young adult sheep but not lambs, CH2O/GFR also increased by 20 min after 20% hemorrhage and remained above control at 60 min (Fig. 3).
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Blood gases, plasma electrolytes, plasma osmolality, and Hct measured during control are shown in Table 3. There were no significant effects of 10-20% hemorrhage on any of these variables.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Stone and Stahl (24) first described the renal responses to hemorrhage in human volunteers. Hemorrhage of 20% of vascular volume was associated with a 25% decrease in creatinine clearance, a 60% decrease in V, and a 45% decrease in Na+ excretion. These renal changes occurred within 30 min of hemorrhage and were reversed after reinfusion of the shed blood. Twenty years later, Sondeen et al. (23) confirmed these renal responses to hemorrhage in conscious, chronically instrumented adult pigs. These observations are similar to those observed in the present study in young adult sheep in which there was a significant decrease in GFR, V, and UNaV after hypotensive hemorrhage. Together, these studies demonstrate that in the adult (including the young adult sheep), hemorrhage results in a decrease in ultrafiltration along with avid water and electrolyte retention, thereby contributing to the restoration of vascular volume.
Renal responses to hypotensive hemorrhage were, however, substantially different in lambs. The decrease in GFR was greater than that observed in young adult sheep, and there was little effect on V or electrolyte excretion rates in lambs compared with young adult sheep. Interestingly, the blood pressure responses to hemorrhage were similar, as we have previously demonstrated (21), suggesting that the two age groups are recruiting different mechanisms to assist in restoring vascular volume. The mechanism underlying the greater decrease in GFR in lambs compared with young adult sheep is not known. It is, however, possible to speculate on the possible underlying mechanisms by evaluating the determinants of GFR: the filtration coefficient (Kf), and net filtration pressure (NFP). Age-dependent changes in GFR after hemorrhage could result from age-dependent changes in Kf. This would occur if, in response to hemorrhage, there were a greater increase in circulating vasoconstrictor agents that promote mesangial contractility in lambs than young adult sheep. In support of this postulate, the renin response to hemorrhage is greater and more prolonged in newborns lambs than older animals (21), providing evidence to suggest that circulating ANG II levels after hemorrhage would also be greater. Additional experiments are clearly necessary to determine the role of ANG II in influencing the GFR response to hemorrhage during development.
Age-dependent changes in GFR after hemorrhage could also result from age-dependent changes in NFP after age-dependent alterations in either oncotic or hydrostatic pressure differences across the glomerular capillary in response to hemorrhage. This seems less likely after hemorrhage because 1) there were similar changes in perfusion pressure in both age groups and 2) plasma osmolality remained constant in both age groups. Additional studies are, however, warranted to more fully elucidate the mechanisms governing the age-dependent effects of hypotensive hemorrhage on GFR and FF and to determine whether the effects of GFR occurred through alterations in Kf and/or NFP.
In response to hypotensive hemorrhage, there was a decreased production of urine in young adult sheep, confirming the early studies by Stone and Stahl (24) in humans and the more recent observations in pigs (23). Previously, we demonstrated that there is an increase in AVP levels after 20% hemorrhage at both 1 and 12 wk of life (21). Therefore, one would expect that the increase in circulating AVP levels would promote tubular water reabsorption. It appears, however, that a decrease in tubular water reabsorption may have occurred at least transiently, as assessed by an increase in CH2O. This could simply represent the dependence of the free water calculation on osmolar clearance, because the linear relationships between free water and osmolar clearances and between V and osmolar clearances were also altered 20 min after hemorrhage. On the other hand, the calculation of CH2O/GFR still revealed a significant increase after hypotensive hemorrhage, and UOsm decreased 20 min after hemorrhage, although this response was transient (see also Fig. 3). Such a paradoxical decrease in tubular water reabsorption in the face of a decreased urinary production after hypotensive hemorrhage in young adult sheep could be explained by an increase in the endogenous 
-opioid dynorphin (5). Activation of -opioid receptors in the kidney (6, 17) results in a rapid and marked diuresis accompanied by an increase in CH2O (1, 7, 9, 12). In support of this premise, we have recently shown that the specific -opioid receptor agonist, U50488H, promotes a water diuresis in conscious lambs aged 1-6 wk of postnatal life (10). The fact that this paradoxical decrease in tubular water reabsorption did not occur in lambs could reflect the fact that opioids may not participate in the physiological responses to hemorrhage early in life, although additional studies are necessary to explore this possibility more fully.
Although no other investigations have been carried out into the study of the renal responses to hemorrhage during postnatal development, there have been some investigations during fetal life. For example, in chronically instrumented fetal sheep studied at mid to late gestation, Schröder et al. (14) measured renal responses to hemorrhage and reported an 20% decrease in GFR and 75% decrease in V. Gomez et al. (4) also measured fetal renal responses to hemorrhage at mid and late gestation after consecutive hemorrhages of 10, 20, and 30% of fetoplacental volume. No changes in GFR were observed after hemorrhage, although in both age groups there was a 75-80% decrease in V after 30% hemorrhage and a 60-70% decrease in Na+ excretion. These renal responses in fetal sheep appear different both from our observed responses in lambs and from previous studies in adult animals, providing further evidence that the renal responses to hypotensive hemorrhage are developmentally regulated.
Prospective. Despite the relatively common occurrence of hemorrhage during the perinatal period, physiological responses to hemorrhage during this time remain poorly understood. Our recent findings have demonstrated that many of the cardiovascular and endocrine responses to hypotensive blood loss are developmentally regulated. To this we add new information that the renal responses to hemorrhage are also altered during postnatal maturation, effects on ultrafiltration being greater in lambs than in young adult sheep and effects on tubular function being greater in young adult sheep than in lambs. Future studies on the potential mechanisms underlying the observed age-dependent renal responses to blood loss should include the role of angiotensin II as well as endogenous -opioids in influencing the physiological responses to hemorrhage during development.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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A portion of this work was presented as a poster to the Annual Meetings of Experimental Biology, 2002, and published in the proceedings (Smith FG, Johnston L, Abu-Amarah I, Basati R, and Sener A. FASEB J 15: A436, 2002).
GRANTS
This work was supported by grants provided by the Canadian Institutes for Health Research. During the tenure of the experiments described herein, A. Sener was supported by a PMAC/MRC graduate studentship, R. Basati was supported by an Heritage Summer Studentship supported by the Alberta Heritage Foundation for Medical Research, and F. G. Smith was a Heritage Senior Medical Scholar supported by the Alberta Heritage Foundation for Medical Research.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-Opioid-induced changes in renal water and electrolyte management and endocrine secretion. Br J Pharmacol 97: 769-776, 1989.[ISI][Medline]
-Opiates and urination: pharmacological evidence for an endogenous role of the -opiate receptor in fluid and electrolyte balance. Eur J Pharmacol 107: 1-10, 1985.[CrossRef]
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P < 0.05 compared with lambs.
