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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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g h-1. Seven control fetuses were infused over the same period with vehicle (0.1% bovine serum albumin in 0.15 M saline).
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INTRODUCTION |
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In adult rats and humans, insulin-like growth factor I (IGF-I) causes a rapid rise in renal blood flow (RBF) and glomerular filtration rate (GFR), due to a reduction in renal vascular resistance (Hirschberg & Kopple, 1989; Giordano & DeFronzo, 1995). Despite this stimulatory effect on GFR, IGF-I causes an antinatriuresis (Giordano & DeFronzo, 1995). IGF-I also enhances proximal tubular function, stimulating reabsorption of sodium, fluid and phosphate in this region of the nephron (Guler et al. 1989; Hirschberg et al. 1993). To date, it is not known whether these renal responses to IGF-I occur before birth.
In the fetal sheep, renal function is characterized by a low RBF (only 2-3% of combined cardiac output; Rudolph & Heymann, 1970) and a low GFR per kilogram body weight (Hill & Lumbers, 1988). Proximal tubular function is immature (Lumbers et al. 1988), and thus both proximal and distal tubules cooperate to maintain glomerulotubular balance (i.e. to alter sodium reabsorption in response to changes in GFR, so that fractional sodium reabsorption remains relatively constant). At birth and over the first week of life, RBF and GFR increase (Aperia et al. 1977; Robillard et al. 1981a), and in the adult sheep glomerulotubular balance is sustained mainly by the proximal tubule (Lumbers et al. 1988).
Circulating levels of IGF-I in the sheep fetus at mid-gestation are approximately 25% of adult levels, and they rise to reach near adult levels by term (Carr et al. 1995). The fetal kidney is a major site of IGF-I production (Han et al. 1988; Dickson et al. 1991), and also expresses type I IGF receptors, which have been found in the glomerulus, cortical tubules and renal medulla (Gršne et al. 1992). Long term infusions of IGF-I into late gestation fetal sheep stimulate renal growth (Lok et al. 1996). If IGF-I also stimulated renal function in the fetus, in the same manner as it does in the adult, then the overall effect would be to shift fetal renal function to a more 'adult pattern', i.e. a higher RBF and GFR, with the proximal tubule playing a greater role in maintenance of glomerulotubular balance. These changes could have significance, in terms of the ability of the kidney to maintain fluid and electrolyte homeostasis after birth.
The present study was carried out to determine which aspects of renal function are influenced by IGF-I during fetal life. We examined its effects on renal haemodynamics and GFR, on proximal and distal tubular function and on the circulating renin-angiotensin system.
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METHODS |
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Experiments were carried out in 19 chronically catheterized pregnant ewes and their fetuses. The study was approved by the Animal Care and Ethics Committee of the University of New South Wales. Ewes were killed at the end of the study by I.V. injection of 3-3.5 g pentobarbitone sodium (Ilium Pentabarb; Troy Laboratories, Smithfield, NSW, Australia).
Surgical preparation and animal care
Surgery was performed between 109 and 117 days gestation (term = 150 days). Anaesthesia was induced by an injection of 1-2 g sodium thiopentone (Pentothal; Abbott, Kurnell, NSW, Australia) into a maternal jugular vein. After tracheal intubation it was maintained with 2-3% halothane (Fluothane; Clifford Hallam Pharmaceuticals Pty Ltd, Riverwood, NSW, Australia) in oxygen. As described previously (Lumbers & Stevens, 1983), catheters were inserted in the fetus into a femoral artery, both tarsal veins and the bladder. An amniotic catheter was sutured to the fetal skin over the flank for measurement of intra-amniotic pressure. The left renal artery was located via a paravertebral incision, and the cuff of a 20 MHz directional pulsed Doppler flowprobe (1.6 or 1.8 mm i.d. cuff; Iowa Doppler Products, Iowa City, IO, USA) was tied around it for measurement of fetal renal blood flow. Catheters were also placed into a maternal femoral artery and vein.
At the end of surgery all maternal wounds were infiltrated with 0.5% bupivacaine HCl (Marcain; Astra Pharmaceuticals Pty Ltd, North Ryde, NSW, Australia). Six hundred milligrams of procaine penicillin and 750 mg dihydrostreptomycin (3 ml Ilium Penstrep; Troy Laboratories Pty Ltd, Smithfield, NSW, Australia) were given I.M. to the ewe and into the amniotic cavity. The same dose was injected into the amniotic cavity for 2 days after surgery. Ewes were housed in individual metabolic cages in a room maintained between 18 and 22°C. They were fed 1200 g lucerne chaff, 300 g oats and 6 g sodium chloride daily, and had free access to water. Maternal and fetal vascular catheters were flushed daily with heparinized 0.15 M saline (100 i.u. heparin ml-1, Heparin Injection BP; David Bull Laboratories, Mulgrave, VIC, Australia). At least 5 days were allowed for recovery before experimentation.
Experimental protocol
On the day of the experiment, the fetal bladder was opened and drained under gravity for at least 45 min. The ewe was given a loading dose of lithium chloride (150 mol kg-1 I.V.). The fetus was given I.V. loading doses of lithium chloride (250 mol kg-1) and 125I-labelled sodium iothalamate (1.8 Ci kg-1, Amersham UK), followed by a continuous I.V. infusion for the duration of the experiment of 10 mol kg-1 h-1 and 0.3 Ci kg-1 h-1, respectively, in 0.15 M saline at 0.95 ml min-1.
Twelve consecutive 30 min urine collections were made, of which the first four were control periods. Blood samples were taken at the midpoint of the second, fourth, eighth and final collection period. Urine was collected anaerobically for acid-base measurements. After the first four collections, 12 fetuses aged 120 ± 1 days received an I.V. infusion of recombinant human IGF-I (rhIGF-I) at 80 g h-1 for 4 h (Lok et al. 1996). We chose this dose because, in fetal sheep of the same gestational age as those used in the present study, it led to an approximate twofold increase in circulating IGF-I levels after 4 h of infusion (Lok, 1998), and it stimulated renal growth when infused for 10 days (Lok et al. 1996). The IGF-I (GroPep; Adelaide, SA, Australia) was dissolved at a concentration of 0.05 mg ml-1 in 0.15 M saline containing 0.1% bovine serum albumin (BSA; Sigma Chemical Co., St Louis, MO, USA) and following a bolus injection of 80 g was infused at 1.65 ml h-1. Seven control fetuses aged 119 ± 0.4 days received a 4 h infusion of vehicle. Infusions were prepared immediately prior to use and delivered through a filter (0.2 m pore size, Minisart; Sartorius, Gottingen, Germany). Data from the four control collections were averaged to give baseline data.
Arterial pressure, heart rate and renal blood flow
Arterial pressure, heart rate and intra-amniotic pressure were monitored continuously using pressure transducers (EasyVent Deadender Cap; Ohmeda, BOC) and a polygraph (Model 79D; Grass Instrument Co., Quincy, MA, USA). RBF was monitored continuously using the flow probe placed at surgery, and a 545C-4 Directional Pulsed Doppler Flowmeter (Bioengineering, University of Iowa, IO, USA) connected to the polygraph. All output from the polygraph was interfaced to an IBM-compatible computer using a Metrabyte DAS16 interface card (Keithley, MA, USA). Intra-amniotic pressure was subtracted from the recorded blood pressure to obtain true fetal arterial pressure. Mean values for fetal arterial pressure, heart rate and RBF were obtained during the control period and during each of the infusion periods. As the probe measured relative blood flow only, mean values for RBF for each infusion period were expressed as a percentage of control RBF.
Blood samples
Arterial blood samples (4.5-5 ml) were withdrawn anaerobically into syringes containing 50 i.u. heparin, and replaced with equivalent volumes of heparinized saline. Arterial PO2, PCO2 and pH were measured at 37°C and corrected to 39.5°C using a Ciba-Corning Blood Gas System (Model 288; Medfield, MA, USA). Haematocrit was determined in duplicate using a microhaematocrit centrifuge and reader (Hettich, Tuttlingen, Germany). Plasma was separated by centrifuging for 10 min at 1083 g, 4°C in tubes containing an additional 50 i.u. heparin, and stored at -20°C for biochemical analysis.
Biochemical analysis
IGF-I concentrations in fetal plasma were measured by specific radioimmunoassays (RIAs; Gatford et al. 1997) calibrated with ovine IGF-I (Francis et al. 1989) after removal of IGF-binding proteins by high performance size exclusion liquid chromatography of plasma at pH 2.5 (Owens et al. 1990b). The intra- and interassay coefficients of variation were 3 and 2.9%, respectively. Plasma glucose and lactate were measured using a Glucose/L-Lactate Analyser 2300 Stat (J Morris Scientific Pty Ltd, Chatswood, NSW, Australia). Osmolality was measured by freezing point depression using a Fiske One-Ten osmometer (Needham Heights, MA, USA). Concentrations of sodium, chloride, potassium and phosphate were measured on a Beckman Synchron CX3 Clinical System (Beckman Instruments (Aust) Pty Ltd, Gladesville, NSW, Australia). GFR was measured as the renal clearance of 125I-labelled sodium iothalamate. The concentration of 125I-labelled sodium iothalamate in plasma and urine was determined from the activity of 125I using a Packard Auto Gamma counter (model 5650; Downers Grove, IL, USA). Lithium concentrations in plasma and urine were measured using a Perkin-Elmer 272 Atomic Absorption Spectrophotometer (Norwalk, CT, USA). Fractional reabsorption of sodium by the proximal and distal tubules were calculated from the renal clearance of lithium, where the fractional reabsorption of sodium by the proximal tubule (FRNa,P) was calculated from the formula:
FRNa,P = (1 - clearance of lithium/GFR) X 100% (1)
(Lumbers et al. 1988).
Fetal urinary acid-base measurements were made using methods described by Gyory & Edwards (1967) and Gyory et al. (1974).
Measurement of plasma renin activity, concentration and angiotensinogen levels
Plasma renin activity (PRA) was measured as the rate of formation of angiotensin I (Ang I) in nanograms per millilitre per hour when 200 l of plasma was incubated for 2 h at pH 7.5 and 37°C. Because the Ang I measured is formed by endogenous renin acting on endogenous substrate, PRA is an estimate of the capacity of plasma to generate angiotensin II (Ang II). Plasma renin concentration (PRC) was determined as the rate of formation of Ang I in nanograms per millilitre per hour when 100 l of fetal plasma was incubated with an excess of sheep angiotensinogen (100 l of nephrectomized sheep plasma (NSP)) at pH 7.5 and 37°C for 2 h. In this assay, the activity of renin is not limited by substrate availability, and so PRC is a measure of the amount of active renin in the circulation. The concentration of angiotensinogen in fetal plasma was measured by determining how much Ang I had been generated in 20 l of plasma by an excess of human renin (0.5 mu; Calbiochem-Novabiochem, Alexandria, NSW, Australia), during a 1 h incubation at pH 7.5 and 37°C. The total volume in each reaction tube for measurement of angiotensinogen was 200 l, following the addition of renin, phosphate buffer, and angiotensinase and converting enzyme inhibitors. This final volume, and the incubation time used, were determined in preliminary experiments. All measurements were made in duplicate, and averaged to give a single result for each plasma sample. Ang I levels were measured by RIA using methods previously described (Lumbers & Lee Lewes, 1979).
Derived variables and statistical methods
At the time of each experiment fetal body weight was estimated from gestational age using a formula derived from the body weights and ages of 84 fetuses in this laboratory (Gibson & Lumbers, 1995). Plasma bicarbonate concentrations were calculated from the measured arterial PCO2 and pH, using a formula derived from the Henderson-Hasselbach equation (Armentrout et al. 1977). Filtration fraction relative to control was calculated from the formula:
Filtration fraction = GFRc/RBFc, (2)
where GFRc and RBFc are expressed as a percentage of their respective control values. Filtration fraction therefore had a value of 1 during the control period.
Data are reported as means ± standard errors of the mean (S.E.M.). All statistical analyses were performed using a statistical software package (SPSS/PC; SPSS Inc., Chicago, IL, USA). Within each treatment group, means were compared using Student's paired t test or analysis of variance (ANOVA) for repeated measures. When differences were detected by ANOVA, Dunnett's test was used to determine which period means were different. Regression analysis (method of least squares) was used to examine relationships between variables. Statistical significance was set at P < 0.05; n = number of animals studied.
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RESULTS |
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Fetal plasma IGF-I levels
After 4 h of rhIGF-I infusion, fetal plasma IGF-I concentrations were increased by ~80% (from 153 ± 23 g l-1 during control to 279 ± 34 g l-1, n = 12, P < 0.01). Plasma IGF-I levels were not different from control levels of 109 ± 29 g l-1 following a 4 h infusion of vehicle (n = 5).
Effects of IGF-I on fetal arterial pressure and heart rate
Infusions of IGF-I had no sustained effect on arterial pressure; diastolic pressure did not change (control value, 31.3 ± 0.6 mmHg) and there were only two small, transient increases in fetal systolic pressure (from 54.7 ± 0.8 mmHg during control to 56.7 ± 1.2 and 56.9 ± 1.3 mmHg after 1.5 h and 2.5 h of infusion, respectively; n = 12, P < 0.05). These are probably of no biological significance. Heart rate did not change from control values of 191 ± 3 beats min-1 (n = 12). During vehicle infusion there were no changes in systolic, diastolic or mean arterial pressure, or heart rate (control values: 52.3 ± 1.6 mmHg, 31.0 ± 1.3 mmHg, 39.6 ± 1.4 mmHg and 187 ± 5 beats min-1, respectively, n = 5).
Fetal arterial blood gases, pH and plasma composition (Table 1)
Arterial PO2 fell in 11 of the 12 fetuses infused with IGF-I. Arterial pH also fell, in association with an increase in PCO2 (Table 1). There was a transient fall in plasma bicarbonate concentration. In fetuses infused with vehicle, arterial PO2, PCO2, pH and plasma bicarbonate concentrations did not change from control values (Table 1).
Haematocrit did not change in fetuses infused with either vehicle or IGF-I (from control values of 33 ± 1%, n = 6 and 31 ± 1%, n = 12, respectively). Plasma osmolality also remained similar to control values in both treatment groups (Table 1). Plasma glucose levels were unchanged in both IGF-I- and vehicle-infused fetuses, but during IGF-I infusion plasma lactate levels rose progressively as arterial pH fell. Plasma sodium and chloride levels increased after 4 h of IGF-I infusion, while plasma potassium levels had fallen by 2 h and remained low. During vehicle infusion, there was also a small, transient fall in plasma potassium levels after 2 h, but plasma sodium and chloride concentrations remained at control levels (Table 1).
Fetal renal function (Figs 1 and 2, Tables 2 and 3)
Fetal RBF fell during infusion of IGF-I, but not during vehicle infusion (Fig. 1). Glomerular filtration rate did not change with IGF-I infusion (Table 3). As renal blood flow fell, this meant that the relative filtration fraction was increased after 4 h (Table 3).
There was a transient diuresis during the first 30 min of IGF-I infusion, which was followed by an antidiuresis and rise in urinary osmolality (Fig. 2). Free water clearance was also markedly lowered after 2 h of IGF-I and remained low at 4 h (Table 2). A transient rise in urine flow rate also occurred in control fetuses during the first 30 min of infusion (Fig. 2). Free water clearance did not change (Table 2). Urinary osmolar excretion and excretion rates of sodium, chloride, bicarbonate and phosphate were all reduced after 4 h of IGF-I, while the excretion of potassium and the urinary sodium to potassium ratio did not change (Tables 2 and 4). None of these variables changed during infusion of vehicle.
Although the filtered sodium load and total sodium reabsorption were not different from control levels, fractional reabsorption of sodium had increased by 4 h of IGF-I infusion (Table 3). At 2 h, the amount of sodium reabsorbed by the proximal tubule was transiently reduced, but this was compensated for by an increase in the fractional reabsorption of sodium by the distal tubule, so that the amount of sodium reabsorbed by the distal tubule, as a percentage of the amount delivered to it, was increased. By 4 h, proximal tubule reabsorption was similar to control values and the distal delivery of sodium had fallen below control (but this change was not significant); only distal reabsorption of sodium as a percentage of distal delivery remained different from control.
Like fractional sodium reabsorption, fractional chloride and phosphate reabsorption rates were increased after 4 h of IGF-I infusion. Total and fractional reabsorption of potassium were not altered (Table 3). In vehicle infused fetuses, the reabsorption of both potassium and phosphate were decreased at 2 h, this was transient and they were similar to control at 4 h (Table 3).
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Fetal renal blood flow was measured by Doppler ultrasound during a 4 h infusion of vehicle (open bars, n = 7) or IGF-I (filled bars, n = 10), and is expressed as a percentage of control blood flows (CON). Values are means ± S.E.M. *P < 0.05, **P < 0.01 compared with control period (Dunnett's test).
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Fetal urine flow rate (cross-hatched bars) and urinary osmolality (filled bars) were measured before and during a 4 h infusion of either vehicle (A, n = 7) or IGF-I (B, n = 12). Values are means ± S.E.M. *P < 0.05, **P < 0.01, compared with control period (Dunnett's test).
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Renal acid-base balance (Table 4)
Urinary pH did not change during IGF-I infusion. The filtered load of bicarbonate fell, as did bicarbonate reabsorption, and fractional bicarbonate reabsorption increased (Table 4). Neither titratable acid nor ammonium excretion rates were altered by IGF-I. Renal acid-base handling did not change with vehicle infusion (Table 4).
Activity of the fetal circulating renin-angiotensin system (Figs 3 and 4)
During IGF-I infusion both PRA and PRC rose above control values (P < 0.005, Fig. 3), while plasma angiotensinogen levels fell (P < 0.05). These variables did not change during vehicle infusion. There was a positive linear relationship between the increase in PRA and that of PRC in IGF-I-infused fetuses, described by the equation PRA = 0.84PRC - 26.85 (r2 = 0.65, P < 0.005, n = 12). Infusion of IGF-I caused a rise in PRC to 375 ± 52% of control (n = 12, P < 0.005; control = 100%) and a smaller rise in PRA (to 288 ± 55% of control, n = 12, P < 0.01). This lesser rise in PRA may be due to the fall in angiotensinogen levels, but no relationship was found between the percentage change in PRA and the degree to which angiotensinogen levels fell.
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Mean values ± S.E.M. for plasma renin activity (PRA) (A), plasma renin concentration (PRC) (B), and plasma angiotensinogen concentrations ([Aogen]pl) (C) during control and after 4 h of infusion of vehicle (open bars, n = 5) or IGF-I (hatched bars, n = 12). *P < 0.05, ***P < 0.005 compared with the control period (Student's paired t test).
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Data were combined from control and treated fetuses to determine whether changes in RBF were related to changes in the activity of the renin-angiotensin system. There was an inverse linear relationship between RBF and PRA (Fig. 4).
Maternal variables
Maternal arterial oxygen tension fell from 106.7 ± 1.7 to 103.1 ± 1.7 mmHg (P < 0.05) after 4 h of fetal IGF-I infusion, a fall that was probably not biologically significant. Otherwise, maternal variables (blood pressure, heart rate, blood gases and plasma composition) did not change when fetuses were infused with either vehicle or IGF-I.
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Individual data points from both treatment groups are plotted for fetal RBF (ordinate) and PRA (abscissa). Data from the control period and after 4 h of infusion are taken from fetuses infused with vehicle (
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DISCUSSION |
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To our knowledge this study demonstrates for the first time that during fetal life, IGF-I alters renal function and acts as a powerful stimulus for renin secretion.
The renin-angiotensin system
This study reports the novel finding that fetal IGF-I infusions were associated with a marked increase in the activity and levels of renin in fetal plasma. The rise in PRA was due to increased renin secretion, as PRC increased but angiotensinogen levels did not. This increase in renin secretion may have been a secondary effect of IGF-I, in response to stimuli such as hypoxia (Robillard et al. 1981b), hypotension (Lumbers & Stevens, 1987), haemorrhage (Broughton Pipkin et al. 1974) or 
-adrenergic stimulation (Lumbers & Lee Lewes, 1979). These have all been reported to increase fetal PRA. However, the fall in fetal PO2 was small compared with the level of hypoxia used in the study by Robillard et al. Furthermore, Broughton Pipkin et al. (1974) showed that any rise in renin levels during hypoxia could be related to removal of blood samples and not to the hypoxia itself. In the present study, hypotension did not occur, and since heart rate did not change, it is unlikely that there was a marked increase in sympathetic tone. In addition, haemorrhage due to removal of blood was not a factor as neither PRC nor PRA changed in vehicle infused fetuses.
Although the fall in distal sodium delivery did not reach significance, it still amounted to a decrease of about 15% in sodium flow past the macula densa. This may have been a stimulus for renin release, but it is more likely that IGF-I directly stimulated the release of renin by the fetal kidney. IGF-I stimulates renin secretion from adult rat renal cortical slices (Jost-Vu et al. 1992). Acute administration of IGF-I to streptozotocin-induced diabetic rats increased renal renin mRNA levels twofold within 2 h (from < 50% of normal prior to infusion), and increased PRA (Jaffa et al. 1997). The renin-angiotensin system also has effects on IGF-I. For example, angiotensin II stimulates expression and/or levels of IGF-I and the IGF-I receptor in vascular smooth muscle cells (Delafontaine & Lou, 1993), heart (Brink et al. 1999) and adrenocortical cells (Louveau et al. 1989; Penhoat et al. 1989). Conversely, IGF-I increases angiotensin type 2 receptor numbers in R3T3 fibroblast cells (Li et al. 1998) and in cultured aortic vascular smooth muscle cells (Kambayashi et al. 1996).
The fall in plasma angiotensinogen levels during IGF-I infusion suggests that the high circulating levels of renin may have consumed angiotensinogen at a rate greater than its production and release from the fetal liver. In 7 of 12 fetuses, plasma angiotensinogen levels during control were below 2 g ml-1, the Km for the reaction between renin and angiotensinogen in sheep (Skinner et al. 1975). The less than proportionate increase in PRA compared with the rise in PRC is therefore probably due to rate limitation of renin by its substrate, especially as circulating renin levels were so high. Although we could not show that the rise in PRA was influenced directly by the degree to which angiotensinogen levels fell in IGF-I-infused fetuses, the decrease in plasma angiotensinogen levels may also have limited the rise in PRA. The question arises as to the extent to which any of the effects of IGF-I on renal function were caused by the changes in the activity of the fetal renin-angiotensin system. This question is addressed below.
Renal function
Although studies on adults describe a rapid rise in both GFR and RBF during IGF-I administration (Hirschberg & Kopple, 1989; Hirschberg et al. 1993; Giordano & DeFronzo, 1995), these effects of IGF-I did not occur in the fetal sheep in late gestation. Fetal GFR did not change, while RBF actually decreased (Table 3 and Fig. 1).
The increases in GFR and RBF observed in the adult in response to IGF-I can be partly attributed to a reduction in renal vascular resistance (Hirschberg & Kopple, 1989; Hirschberg et al. 1993). In rats, IGF-I reduced both afferent arteriolar and efferent arteriolar resistance, and thus single nephron filtration fraction did not change (Hirschberg & Kopple, 1989). The vasodilator nitric oxide is thought to at least partly mediate these effects (Haylor et al. 1991). In the present study, however, relative filtration fraction increased (Table 3), suggesting that renal vasoconstriction occurred, rather than vasodilatation, and that there was preferential constriction of the efferent arteriole. This vasoconstriction may in part be due to the intense activation of the fetal renin- angiotensin system that occurred. We have previously shown that normal fetal renal function depends on the integrity of the fetal renin-angiotensin system (Lumbers et al. 1993). Blockade of the fetal renin-angiotensin system causes a rise in RBF and a fall in GFR that can be so severe that fetuses become anuric. Infusions of angiotensin II into these blocked fetuses caused a fall in RBF and a rise in GFR, with restoration of urine flow. In agreement with these effects of angiotensin II on fetal renal blood flow, in the present study we found a negative relationship between PRA and RBF in fetuses from both treatment groups combined (Fig. 4). The increased activity of the circulating renin-angiotensin system in IGF-I-infused fetuses may therefore have contributed to the progressive fall in RBF that occurred in these animals.
The effects of high renin levels on RBF may have been supplemented by increases in other circulating vasoactive substances, in particular arginine vasopressin (AVP). The marked antidiuresis, rise in urinary osmolality, fall in free water clearance and fall in PO2 are all consistent with increased secretion of this hormone in IGF-I-infused fetuses (Table 1, Table 2 and Fig. 2). Urinary osmolality is a very sensitive index of AVP levels in the fetus (Wintour et al. 1982). The hormone is released in response to fetal hypoxia (Rurak, 1978; Robillard et al. 1981b) and is a potent antidiuretic and vasoconstrictor (Gibson & Lumbers, 1993).
Fetal sheep made hypoxic in late gestation (130-140 days) exhibited a remarkably similar pattern of changes in renal haemodynamics and filtration fraction as in the present study, and also have increased PRA and plasma AVP levels (Robillard et al. 1981b). The similarity in responses of fetal GFR, RBF and filtration fraction to both hypoxia and IGF-I raises the question of whether the changes in the present study were due to the mild hypoxaemia associated with IGF-I infusion. It should, however, be noted that the fall in PO2 in the present study (~7% after 4 h) was much smaller than in the study by Robillard et al. (~40%). Furthermore, others (Broughton Pipkin et al. 1974) have been unable to show that hypoxia is a potent stimulus for renin release, and in the present study the rise in plasma renin levels that occurred was very marked.
The fall in PO2 was associated with a lactic acidaemia (Table 1), which probably reflected a reduced availability of oxygen to fetal tissues and consequent anaerobic metabolism. Placental function may also have been reduced, as there was an increase in PCO2. However, when fetal sheep aged 120 days of gestation were infused I.V. for 4 h with the same dose of IGF-I as in the present study, umbilical blood oxygen extraction increased, indicating that placental function was not compromised during the infusion (Lok, 1998). Instead, since the anabolic effects of IGF-I (Harding et al. 1994; Lok et al. 1996; Liechty et al. 1999) require oxygen, they are more likely to be responsible for the disturbance to blood gases and pH in the present study. The fetal kidney responded appropriately to this disturbance by conserving bicarbonate and by excreting more acid.
Tubular reabsorption
The biphasic response in the renal handling of sodium may be explained by changes in bicarbonate reabsorption (Table 3). In adults at least 85% of filtered bicarbonate is reabsorbed by the proximal convoluted tubule, in a process involving at least an equimolar reabsorption of sodium (Mathisen et al. 1978, 1979). Proximal sodium reabsorption will therefore fall in response to decreased bicarbonate reabsorption, as occurred after 2 h of IGF-I. The distal tubule compensated during this period.
What is interesting is that by 4 h, proximal sodium reabsorption was restored, with a concurrent increase in both bicarbonate and phosphate reabsorption. Increased proximal bicarbonate reabsorption will be associated with a rise in both sodium and chloride reabsorption by the proximal tubule. The enhancement of proximal tubular function by IGF-I in the present study is consistent with its effects in the adult, where increases in proximal tubule sodium, fluid and phosphate reabsorption have been reported in healthy men in response to acute and chronic administration of IGF-I (Guler et al. 1989; Hirschberg et al. 1993). IGF-I also stimulates apical sodium-hydrogen exchange activity in adult human proximal tubule cells in vitro (Johnson et al. 1997).
The distal tubule also continued to reabsorb an increased percentage of the sodium delivered to it at this time (4 h). Thus, total fractional sodium and chloride reabsorption were enhanced. The continued higher rate of reabsorption of sodium by the distal tubule could account for the sustained excretion of potassium in the presence of a fall in plasma potassium levels. This fall was particularly marked considering that fetuses became mildly acidaemic, a change normally associated with a rise in plasma potassium (as protons exchange for intracellular potassium).
Increases in phosphate reabsorption of a similar magnitude to those reported here have been reported in adult humans in response to IGF-I (Hirschberg et al. 1993; Giordano & DeFronzo, 1995) and this action appears to be independent of parathyroid hormone (Hirschberg et al. 1993). IGF-I increases apical sodium-phosphate cotransport in perfused isolated rabbit proximal tubules (Quigley & Baum, 1991). It has been suggested that the increase in renal phosphate reabsorption that accompanies postnatal growth is mediated by IGF-I (Bonjour & Caverzasio, 1991). The growth-promoting actions of IGF-I during fetal life would certainly increase phosphate requirements, by enhancing fetal skeletal ossification (Liu et al. 1993) and inducing cell proliferation and DNA synthesis (Jones & Clemmons, 1995), and the large increase in fractional phosphate reabsorption seen in the present study could provide an increased supply of phosphate for use in these processes.
Circulating IGF-I levels in the present study rose by approximately 80%, a greater change than the reported gestational increase in plasma IGF-I concentrations (approximately 30% from 120 to 145 days; Carr et al. 1995). In the present study, IGF-I levels at the end of the 4 h infusion were similar to those observed in hyperglycaemic fetal sheep (Owens et al. 1990a). Therefore, while these levels are probably in the pathophysiological range, they could be achieved through stimulation of endogenous IGF-I production.
To conclude, IGF-I caused a marked increase in the secretion of renin by the fetal kidney in late gestation, perhaps by a direct action on the renin secreting cell. Infusions of IGF-I in late gestation also stimulated renal function by stimulating tubular solute reabsorption, but not by increasing GFR or RBF. The effects of IGF-I on GFR and RBF suggest that vasoconstriction of the efferent arteriole occurred, possibly as a result of the increase in circulating levels of renin. This action is in marked contrast to the effects of IGF-I in the adult kidney in which, overall, there is renal vasodilatation and hyperfiltration. The enhancement of proximal tubular function by IGF-I, if sustained, could contribute to maturation of fetal renal function in preparation for life after birth.
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REFERENCES |
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Acknowledgements
We thank Dr A. D. Stevens, Dr J. H. Burrell and Ms P. Bode for their technical assistance. We also thank the Department of Clinical Chemistry, Prince of Wales Hospital, Sydney, Australia for measurement of electrolyte levels. This work was supported by the National Health and Medical Research Council of Australia. Ms A. Marsh is supported by a Postgraduate Science Research Scholarship from the National Heart Foundation of Australia.
Corresponding author
A. Marsh: School of Physiology and Pharmacology, University of New South Wales, Sydney, NSW 2052, Australia.
Email: a.marsh{at}unsw.edu.au
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, n = 5 fetuses) or IGF-I (
, n = 10 fetuses). RBF is not corrected for control values and is expressed in arbitrary units. In the group of 15 fetuses combined, there was an inverse linear relationship between RBF and PRA (RBF = -9.39PRA + 418.97, r = -0.54, r2 = 0.29, P < 0.005, n = 30 data points, method of least squares).