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Am J Physiol Renal Physiol 274: F120-F128, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 1, F120-F128, January 1998

Effects of insulin-like growth factor I on the renal juxtamedullary microvasculature

Burkhard Tönshoff, Frederick J. Kaskel, and Leon C. Moore

Departments of Pediatrics and Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10-9-10-7 M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 ± 1.2% of control (n = 16); mid-afferent arterioles, 11.6 ± 1.7% (n = 24); and juxtaglomerular afferent segments, 16.1 ± 2.8% (n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor N G-nitro-L-arginine methyl ester (10-4 M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10-5 M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (~10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.

renal microcirculation; nitric oxide; nitric oxide electrode; prostaglandins; renal autoregulation

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

INCREASED CONCENTRATION of circulating insulin-like growth factor I (IGF-I) in response to hypersecretion of growth hormone (GH) in acromegaly is associated with high glomerular filtration rate (GFR), whereas decreased IGF-I plasma levels, as in GH deficiency, are associated with low GFR, both in rats (13) and humans (22). Also, physiological changes in plasma IGF-I concentration in response to a high- and low-protein diet are associated with acute changes in GFR and renal plasma flow (RPF) (16). Furthermore, infusion of IGF-I in fasted rats (15) and humans (8, 14) causes a rapid and reversible increase in GFR and RPF and a decrease in renal vascular resistance. These data indicate that circulating and/or locally produced IGF-I is an important determinant of renal hemodynamics, independent of its action on renal growth. However, the exact mechanism and specific vascular sites of action of IGF-I in the renal microvasculature remain incompletely defined, especially in the juxtamedullary circulation.

We therefore performed the present study to characterize the responsiveness of renal arteriolar microvessels to IGF-I. Experiments were conducted using the in vitro blood-perfused rat juxtamedullary nephron preparation developed by Casellas and Navar (6, 21). This preparation allows visual access to the entire pre- and postglomerular microcirculation under physiological conditions; in particular, autoregulation responses are intact, and the tubuloglomerular feedback system is functional (5). The use of the juxtamedullary nephron preparation combined with video microscopy allowed us to directly visualize for the first time the effect of IGF-I on renal microvascular tone and to determine the specific sites of its action within the pre- and postglomerular microcirculation in juxtamedullary nephrons. In addition, the potential interference of IGF-I-induced vasodilation with renal autoregulatory capacity was studied. Furthermore, we evaluted the relative roles of nitric oxide (NO) and vasodilatory prostaglandins (PG) in mediating IGF-I-induced vasodilation. Previous studies have demonstrated the presence of specific endothelial receptors for IGF-I (1, 2, 7), and NO and PG have been implicated in the vasodilatory responses to IGF-I (11, 15, 31). In addition, we demonstrated by use of a NO-selective, voltametric microelectrode that IGF-I is able to directly stimulate NO production in intact renal preglomerular microvessels.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

For each experiment, two male Sprague-Dawley rats (Taconic Farms) were used, one as a blood donor weighing ~300-400 g and one as a kidney donor weighing ~200-250 g. Prior to experimentation, all animals were anesthetized with an injection of 110 mg/kg ip Inactin (Byk-Gulden, Constance, Germany).

Measurements of Renal Microvascular Reactivity

Assessment of microvascular reactivity in single vessels was conducted using the in vitro blood-perfused juxtamedullary nephron preparation, which has been described in detail previously (6, 21). After anesthesia, the abdomen was opened, and the left kidney was flushed with saline, using an aortic catheter advanced into the renal artery, before its removal with the catheter in place. An extensive dissection was used to expose a part of the cortex that lies under the pelvic mucosa inside the kidney. Major arteries supplying the rest of the kidney were ligated. During the dissection, the kidney was perfused with a gassed Krebs-bicarbonate-Ringer (KBR) solution containing 4% albumin. During measurements, the kidney was perfused with a blood solution prepared from fresh rat donor heparinized blood. The red blood cells were separated, washed, and resuspended in a 6% KBR-albumin solution to achieve a hematocrit of 30%. The resuspended red blood cells were oxygenated in a closed reservoir with a 95% O2-5% CO2 gas mixture. The preparation was superfused at high rates (3 ml/min) with a warmed (37°C) KBR solution containing 1% albumin and 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer with pH 7.4. The preparation was mounted in a small perfusion chamber that rests on a microscope stage. We have previously demonstrated that preglomerular reactivity is stable in this preparation for up to 4 h (5, 6).

Reactivity of juxtamedullary afferent arterioles (JAA) to perfusion pressure, IGF-I, NG-nitro-L-arginine methyl ester (L-NAME), and indomethacin was assessed using videomicroscopy to measure luminal diameters. Low-angle-incident light and ×10 and ×25 long-working-distance objectives fitted to a Leitz intravital compound microscope were used. Video images were obtained with a charge-coupled device camera (CCD-72, Dage), enhanced with a real-time image processing system (Argus-10, Hamamatsu), displayed on a high resolution RGB monitor, and recorded with an S-VHS VCR recorder. Vessel inside diameters were measured on video prints at a single measurement site with a recognizable landmark with a digitizing tablet. The reproducibility of this measurement technique lies within 1 µm. When significant vasomotion (>1 µm spontaneous change in lumen diameter) was present (which occurred in 10% of the vessels studied), the average of the minimum and maximum values were recorded.

The vessels were allowed to equilibrate for 15 min before one of the protocols outlined below was applied. Only arterioles with rapid flow of erythrocytes and clearly visible vascular walls were included in the study. Vessels that were nonresponsive to an increase of perfusion pressure from 60 to 120 mmHg were disregarded. If flow stopped during the course of the experiment, the vessel was excluded from the analysis. Preglomerular afferent vessels were monitored at three locations: a JAA site 25-50 µm upstream from the point of entrance into Bowman's capsule, a mid-AA (MAA) site >50 µm upstream from the glomerulus, and an interlobular artery (ILA) site >100 µm away from any AA. Efferent arterioles (EA) were studied within 50 µm from their exit from Bowman's capsule. Unless otherwise indicated, responses were measured at 60 mmHg perfusion pressure. We chose to work at a low perfusion pressure to conserve perfusate in these long experiments. In addition, IGF-I did not alter autoregulatory responses, so that the vasodilatory responses to IGF-I at 60 mmHg were similar to those seen at normotensive pressure levels (see below).

Monitoring of NO Release from Perfused Renal Microvessels

Changes in the local concentration of NO in glomerular filtrate were measured with an NO-selective voltametric microelectrode. It is identical to the electrode developed by Shibuki and Okada (24) and is sold commercially, but it is much smaller (tip diameter, 30-50 µm vs. ~500 µm). The electrode detects the current produced by the oxidation of NO to NO<SUP>−</SUP><SUB>2</SUB> at the surface of a polarized (0.8 V), inert electrode (25-µm platinum wire). The oxidation current increases linearly with concentration (24). The selectivity of the electrode arises from the positive redox potential of NO and the fact that the tip of the electrode is covered by a very thin (1 µm) gas-permeable rubber membrane that is impermeable to ions, solutes, and water. Electrode current was measured with a simple voltage clamp/current monitor with picoampere resolution. The output is filtered and displayed on a storage oscilloscope. The integrity of the rubber membrane was verified periodically by measuring the resistance through the tip to an external Ag-AgCl reference. The electrodes were calibrated in vitro by exposing the tip of the electrode to NO-free and 10-6 M NO isotonic saline solutions in a continuous flow perfusion system that prevents exposure to air. The NO-free solution was prepared from saline deoxygenated by bubbling with ultrapure N2, followed by boiling. The NO standard solution was prepared by dissolving NO gas in deoxygenated saline, as previously described (24). For measurements of NO in renal microvessels, the electrode was placed in gentle contact with Bowman's capsule. As the preparation is superfused with an NO-free solution and these superficial glomeruli are covered only by a thin epithelial barrier, the major local NO source was Bowman's space, which is filled with an ultrafiltrate of plasma that has just left the preglomerular vasculature. Hence, we assume that the changes in NO concentration we detect in glomerular filtrate parallel changes in tissue NO levels in the preglomerular vasculature, although the measured absolute NO concentrations may be lower than those within the vascular wall. Furthermore, because it was not possible in our preparation to verify electrode calibration in situ, the results were quantified as the change in estimated NO concentration.

Experimental Protocols

Experimental series 1 (dose response). The tissue was exposed to the following sequence of perfusate and superfusate solutions: 1) control, perfusate (erythrocyte suspension), superfusate (1% albumin-KBR solution); 2) 10-9 M IGF-I (a gift from Genentech, South San Francisco, CA) in the perfusate. A 10-min equilibration period was allowed following initiation of IGF-I perfusion. Perfusion rather than superfusion with IGF-I was performed, because this mode of administration more closely imitates the effect of circulating IGF-I on the vasculature; 3) recovery from IGF-I for 10 min; 4) 10-8 M IGF-I. This dose of IGF-I corresponds approximately to the calculated amount of biologically available IGF-I, i.e., 5% of total IGF-I concentration circulating in a 200-g Sprague-Dawley rat (26); 5) recovery from IGF-I; 6) 10-7 M IGF-I; and 7) recovery from IGF-I.

Experimental series 2 (time control). Tissue was exposed in following sequence: 1) control; 2) 10-8 M IGF-I; 3) recovery from IGF-I; 4) to test the integrity of the renal L-arginine/NO system, 10-5 M acetylcholine (ACh) (Sigma Chemical, St. Louis, MO) was added to the superfusate for 10 min. This amount of ACh is a nonsaturating dose with abluminal application; 5) recovery from ACh; 6) control for 30 min; 7) 10-8 M IGF-I; 8) recovery from IGF-I; 9) 10-5 M ACh; and 10) recovery from ACh.

Experimental series 3. Tissue was exposed in the following sequence: 1) control; 2) autoregulatory response. This was assessed by measuring perfusion pressure-dependent vasoconstriction at the above mentioned vascular sites. Perfusion pressure was raised from 60 to 120 mmHg over a time period of 30 s and then returned to 60 mmHg over the same time period. Three minutes were allowed for equilibration after each change in perfusion pressure; 3) 10-8 M IGF-I; 4) autoregulatory response during IGF-I; 5) recovery from IGF-I: control perfusate for 10 min; 6) 10-5 M ACh; 7) autoregulatory response during ACh; 8) recovery from ACh for 10 min; 9) to achieve maximal vasodilation of renal microvessels, 10-2 M manganese chloride (MnCl2) (Sigma Chemical, St. Louis, MO) as a calcium channel blocker was added to the superfusate for 10 min; and 10) autoregulatory response during MnCl2.

Experimental series 4. The respective time period, dose, and mode of administration for each drug were the same as in experimental series 3. The tissue was exposed to the following sequence of perfusate and superfusate solutions: 1) control; 2) IGF-I; 3) recovery from IGF-I; 4) ACh; 5) recovery from ACh; 6) addition of L-NAME (10-4 M) (Bachem, Torrance, CA) to the superfusate for the remaining time of the experiment. An equilibration period of 10 min was allowed; 7) L-NAME plus IGF-I; 8) recovery from IGF-I during L-NAME; 9) L-NAME plus ACh; and 10) recovery from ACh during L-NAME.

Experimental series 5. The respective time period, dose, and mode of administration for each drug were the same as in experimental series 3 and 4. The tissue was exposed to the following sequence of perfusate and superfusate solutions: 1) control; 2) IGF-I; 3) recovery from IGF-I; 4) addition of indomethacin (10-5 M) (Sigma Chemical) to the perfusate and superfusate for the remaining time of the experiment. An equilibration period of 30 min was allowed; 5) indomethacin plus IGF-I; and 6) recovery from IGF-I during indomethacin.

Experimental series 6. To verify that IGF-I increases endothelial NO production, glomerular NO levels were measured before and after exposure to 10-8 M IGF-I added to the perfusate. A baseline measurement of NO was obtained, after which the IGF-I perfusion solution was applied. After the NO reading stabilized at the new level for a minimum of 2 min, the IGF-I perfusion solution was replaced with the control perfusate, and the resultant change in NO concentration was monitored. To verify the ability of the electrode to detect changes in NO levels, the effect of brief abluminal exposure to 10-5 M ACh was measured, as was the effect of the presence of RBC in the perfusate. These measurements were discontinued if there was evidence of excessive drift of the background electrode current or an abrupt, large increase in electrode current and noise, which was usually associated with failure of the rubber membrane on the electrode tip.

Statistical Analysis

Within each experimental series, the response of each arteriole to the respective stimulation (drug or autoregulatory response) was compared with the mean of baseline vessel diameter before stimulation and recovery vessel diameter after stimulation. For the L-NAME and indomethacin responses, only the baseline vessel diameter was taken for comparison, because the effects of these drugs are only slowly reversible. The absolute response for each pharmacological stimulation was evaluated using a two-tailed t-test for paired data. The fractional changes within each experimental series were compared by analysis of variance for repeated measures, followed by all pairwise multiple comparison (Student-Newman-Keuls method). Data from different experimental series were analyzed by a two-tailed t-test for unpaired data or by analysis of variance where appropriate. P < 0.05 was accepted for statistical significance. All values are means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Addition of IGF-I to the perfusate resulted in a dose-dependent vasodilation of preglomerular microvasculature, as illustrated in Fig. 1. The response patterns were qualitatively similar in the 2 ILA, 8 MAA, and 3 JAA segments studied. The respective percent dilation responses elicited by 10-9, 10-8, and 10-7 M IGF-I for the individual segments were ILA, -0.4 ± 0.5, 14.4 ± 3.3, and 14.7 ± 3.6%; MAA, 1.2 ± 1.1, 11.0 ± 1.7, and 15.9 ± 3.1%; and JAA, 0.3 ± 3.7, 19.0 ± 5.5, and 21.1 ± 5.1%. These results show that a dose of 10-8 M IGF-I in this preparation is sufficient to stimulate a near maximal vasodilatory response. The stability of the preparation was verified in the second series of experiments. The pooled results, illustrated in Fig. 2, show statistically similar vasodilatory responses to repeated applications of either ACh or IGF-I 30 min apart, a time period approximately equal to the average time between IGF-I challenges before and after L-NAME or indomethacin treatment (see below). Measurements were made in 2 ILA, 8 MAA, and 2 JAA segments. The respective initial and final percent dilation responses elicited by 10-8 M IGF-I for the individual segments were ILA, 13.8 ± 2.3 and 13.2 ± 2.4%; MAA, 10.6 ± 1.6 and 11.7 ± 2.3%; and JAA, 16.7 ± 5.0 and 21.8 ± 4.4%. The respective initial and final percent dilation responses elicited by 10-5 M ACh for the individual segments were ILA, 22.5 ± 2.6 and 17.5 ± 6.1%; MAA, 21.9 ± 2.5 and 17.2 ± 2.0%; and JAA, 36.7 ± 10.7 and 42.2 ± 8.1%. These measurements were made at 60 mmHg perfusion pressure.


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  		Fig. 1.   Dose-dependent vasodilation of preglomerular microvasculature by insulin-like growth factor I (IGF-I) at 60 mmHg perfusion pressure. Two interlobular artery, eight midafferent arteriole, and three juxtaglomerular afferent arteriole segments were studied, and pooled results are shown. *P < 0.05, significant vs. baseline.


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  		Fig. 2.   Fractional increase in lumen diameter of preglomerular microvessels in response to IGF-I (10-8 M) and acetylcholine (ACh) (10-5 M) after perfusion of the juxtamedullary vessel preparation for 15 and 45 min. Two interlobular artery, eight midafferent, and two juxtaglomerular afferent arteriole segments were studied. There was no significant difference between the respective responses after 15 and 45 min. Perfusion pressure was 60 mmHg. N.S., not significant.

In the third series of experiments, the mean lumen diameter of ILA segments (n = 6) under control conditions averaged 24.0 ± 1.1 µm, that of MAA segments (n = 9) averaged 20.1 ± 1.1 µm, and that of JAA segments (n = 8) averaged 13.2 ± 1.3 µm. Administration of IGF-I (10-8 M) to the perfusate at 60 mmHg perfusion pressure caused a reversible increase in vessel lumen diameter in all three preglomerular arteriole segments within 3 min, a period of time which approximates the transient time of our perfusion apparatus. The mean fractional increase in vessel caliber in the ILA segment in response to IGF-I was 8.2 ± 1.2% of control diameter; in the MAA segment, it was 9.9 ± 2.6%; and in the JAA segment, it was 16.1 ± 4.9% (Fig. 3). When the data from series 3-5 were combined, the mean fractional increase in lumen diameter in response to IGF-I in preglomerular afferent arterioles (ILA, 11.5 ± 1.2% of control, n = 16; MAA, 11.6 ± 1.7% of control, n = 24; and JAA, 16.1 ± 2.8% of control, n = 19) was statistically not different between the segments.


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  		Fig. 3.   Effects of IGF-I (10-8 M), ACh (10-5 M), and MnCl2 (10-2 M) on fractional changes in vessel lumen diameter at 60 mmHg perfusion pressure. AA, afferent arterioles. * P < 0.05, significant vs. baseline diameter before drug treatment. # Responses are not statistically different, whereas values without common # are statistically different.

To demonstrate that the renal L-arginine/NO system, a putative mediator of IGF-I action on renal hemodynamics, was intact in this renal microvessel preparation, the effect of ACh on vessel lumen diameter was studied concomitantly in the same series of experiments. The vasodilatory action of ACh is known to be mediated principally by endothelium-derived NO. Abluminal exposure of the vessels at 60 mmHg perfusion pressure to ACh resulted in a significantly greater vasodilation in all three vessel segments compared with the stimulation with IGF-I (Fig. 3). In these same vessels, maximal vasodilatory capacity was quantified by calcium channel blockade via superfusion with MnCl2 (10-2 M). The maximal vasodilatory response in the ILA segment was 30.3 ± 4.7% of control; in the MAA segment, it was 27.7 ± 6.0% of control; and in the JAA segment, it was 47.7 ± 11.9% of control (Fig. 3). In comparison to the maximum vasodilation achieved by calcium channel blockade, the vasodilatory response of preglomerular microvessels to IGF-I was moderate.

In contrast to preglomerular afferent microvessels, the EA in perfused juxtamedullary nephrons were not responsive to stimulation with IGF-I. At 60 mmHg perfusion pressure, the mean lumen diameter of EA (n = 8) under baseline conditions was 21.4 ± 1.7 µm. No significant change (Delta ) in lumen diameter on exposure with IGF-I was detected [Delta of lumen diameter, +0.1 ± 0.9% of control, not significant (NS)]. The mean fractional increase of EA caliber upon superfusion with ACh (n = 4) was 3.3 ± 3.6% (NS) and upon superfusion with MnCl2 was 9.5 ± 5.0% (NS). Because juxtamedullary EA segments were not responsive to IGF-I in this series of experiments, they were not further examined in the following series of experiments that studied the mechanism of IGF-I-induced vasodilation.

In the same series of experiments, the renal autoregulatory response to changes in perfusion pressure was studied. Under control conditions, a rise of perfusion pressure from 60 to 120 mmHg induced a rapid decrease of preglomerular arteriole lumen diameter. The mean decrease in diameter relative to control observations was 23.8 ± 5.1% in the ILA segments, 23.3 ± 5.1% in the MAA segments, and 14.4 ± 2.2% in the JAA segments (Fig. 4). This autoregulatory response was not significantly altered by concomitant IGF-I perfusion in any of the preglomerular vessel segments (Fig. 4). In this respect, the IGF-I-induced vasodilation did not differ from that induced by ACh, which also did not affect autoregulatory capacity at the dose applied (results not shown). In contrast, calcium channel blockade by MnCl2 completely inhibited the renal autoregulatory response and, instead, allowed a passive vasodilation in response to an increased perfusion pressure by 11.2 ± 1.8% of control in the ILA, by 10.7 ± 2.3% of control in the MAA, and by 6.1 ± 3.4% of control in the JAA segment, respectively (Fig. 4).


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  		Fig. 4.   Autoregulatory responses of renal microvessels in response to an increase in perfusion pressure from 60 to 120 mmHg under control conditions during IGF-I perfusion (10-8 M) and during superfusion with MnCl2 (10-2 M). 100%, individual baseline diameter before the respective change in perfusion pressure. * P < 0.05, significant vs. baseline diameter. # Responses are not statistically different, whereas values without common # are statistically different.

In the following two series of experiments, we sought to determine whether the IGF-I-induced vasodilation is mediated by or dependent on an intact endothelial synthesis of NO and vasodilatory PGs. First, the effect of the competitive inhibitor of NO biosynthesis, L-NAME, on lumen diameter of preglomerular microvessels before and during stimulation with IGF-I was studied at 60 mmHg perfusion pressure (experimental series 4). In this series, the lumen diameter of ILA segments (n = 3) under baseline conditions averaged 27.3 ± 1.4 µm; of MAA segments (n = 7), 25.1 ± 1.8 µm; and of JAA segments (n = 5), 16.3 ± 2.3 µm (not significantly different from experimental series 3). These preglomerular vessels responded to IGF-I administration with an increase in lumen diameter 17.6 ± 1.6% of control in the ILA, 10.4 ± 3.4% of control in the MAA, and 11.4 ± 3.2% of control in the JAA segment, respectively (Fig. 5). This response was similar to that observed in the first three series of experiments. Addition of L-NAME (10-4 M) to the superfusate induced a sustained vasoconstriction of 14.4 ± 2.6% of basal lumen diameter in the ILA (P < 0.05), 11.5 ± 2.0% in the MAA (P < 0.005), and 6.2 ± 4.4% in the JAA segment (NS), respectively. In the continued presence of L-NAME, the vasodilatory response to abluminal exposure with ACh (10-5 M) was completely suppressed (Fig. 5). This indicates that the amount and mode of L-NAME administration was sufficient to inhibit both endogenous and agonist-induced NO release. Pretreatment with L-NAME also completely abolished the vasodilatory response to IGF-I (Fig. 5). Furthermore, in the presence of L-NAME, IGF-I tended to induce a slight vasoconstriction, which achieved statistical significance in the MAA segment (fractional decrease in lumen diameter, 6.3 ± 4.5% of control, P < 0.05, paired t-test). It should be noted that this value was calculated using average of the pre- and post-IGF-I baseline diameters, and recovery from IGF-I was only partial during L-NAME treatment (recovery diameter was 4.7 ± 2% smaller than the pre-IGF-I diameter).


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  		Fig. 5.   Fractional changes in lumen diameter of afferent arterioles (AA) at 60 mmHg perfusion pressure in response to IGF-I perfusion (10-8 M) or ACh superfusion (10-5 M) without and with pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) (10-4 M) in the superfusate. 100%, individual baseline diameter before each respective drug administration. * P < 0.05, significant vs. baseline diameter. # Responses are not statistically different, whereas values without common # are statistically different.

Next, the relative contribution of vasodilatory PGs in mediating the IGF-I-induced vasodilation was examined by pharmacological inhibition of PG synthesis with indomethacin (experimental series 5). The mean baseline ILA lumen diameter in this experimental series (30.0 ± 1.5 µm, n = 7) was slightly higher (P < 0.05) than in series 1, and the mean MAA diameter (20.5 ± 1.8 µm, n = 8) and mean JAA diameter (16.1 ± 2.1 µm, n = 6) were comparable to series 3 and 4. The vasodilatory response to IGF-I perfusion in preglomerular microvessels (Fig. 6) was similar to that observed in experimental series 3 and 4. In contrast to L-NAME, superfusion with indomethacin (10-5 M) did not significantly alter baseline lumen diameter of preglomerular microvessels (change in lumen diameter in ILA segments, +6.4 ± 3.1%; in MAA segments, -1.8 ± 2.6%; in JAA segments, +1.2 ± 2.2%). Interestingly, pretreatment with indomethacin did not only inhibit the vasodilatory response to IGF-I; rather, the response was preglomerular vasoconstriction. The fractional decrease in lumen diameter in the ILA segment was 8.3 ± 1.8% (P < 0.05); in the MAA segment, 9.3 ± 2.3% (P < 0.05); and in the JAA segment, 9.2 ± 1.1% (P < 0.005), respectively (Fig. 6). After discontinuation of IGF-I perfusion, this vasoconstriction was fully reversible within 10 min (difference between pre-IGF-I and recovery diameters was -1.3 ± 2.5% in ILA, -1.7 ± 2.5% in MAA, and 3.4 ± 2.2% in JAA segments).


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  		Fig. 6.   Fractional changes in lumen diameter of afferent arterioles (AA) in response to IGF-I perfusion (10-8 M) without and with pretreatment with indomethacin (10-5 M) in the perfusate and superfusate. 100%, individual baseline before each respective drug administration. * P < 0.05, significant vs. baseline diameter. Responses were significantly different from each other. Perfusion pressure was 60 mmHg.

In experimental series 4, pharmacological inhibition of endothelial NO synthesis by L-NAME caused a persistent preglomerular vasoconstriction of microvessels, which could have masked the effect of any vasodilatory substance, regardless of the mode of action. We therefore investigated whether IGF-I directly stimulates endothelial NO production in the intact renal microvasculature. The results are summarized in Fig. 7. A typical tracing of electrode current is shown in Fig. 7A. Luminal application of 10-8 IGF-I induced a fairly rapid response (Delta  of concentration, 113 nM), given that the wash-in time for this preparation is ~1 min. Figure 7B shows the results from three preparations where complete sets of NO responses were obtained. In response to switching to a perfusate solution with RBC, NO levels decreased 179 ± 45 nM, presumably because of NO binding to hemoglobin in the perfusate. Abluminal application of 10-5 M ACh during perfusion with the blood solution increased NO concentration in glomerular filtrate by 116 ± 30 nM, whereas luminal application of 10-8 M IGF-I increased NO levels by 98 ± 10 nM.


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  		Fig. 7.   A: representative recording of NO concentration in the interstitium of renal afferent microvessels before and after addition of IGF-I (10-8 M) to the perfusate. B: changes (Delta ) in NO concentration elicited by addition of IGF-I to the perfusate, abluminal application of ACh (10-5 M), and the substitution of blood solution [30% red blood cells (RBC)] for a cell-free Krebs-bicarbonate-Ringer perfusate. Perfusion pressure was 60 mmHg.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study provides the first direct demonstration that IGF-I in a physiological concentration (10-8 M) stimulates vasodilation of renal microvessels in juxtamedullary nephrons. IGF-I induced a 10-15% fractional increase of lumen diameter in all preglomerular segments of juxtamedullary afferent microvessels. Assuming an inverse fourth-power relationship between vessel radius and resistance (based on the Hagen-Poiseuille equation), the functional significance of this change in vessel caliber is a drop of preglomerular resistance by 35%. This derived value corresponds well to experimental findings in the whole rat, where IGF-I infusion decreased renal vascular resistance by 25% (11, 15).

The site of action of IGF-I in juxtamedullary nephrons examined in the present study was exclusively preglomerular; we did not observe a vasodilatory effect of IGF-I in the EA. These results suggest the presence of type 1 IGF receptors on pre- but not postglomerular arterioles in juxtamedullary nephrons. An alternative explanation is that the postreceptor messenger system that communicates receptor activation to intracellular sites are either absent or inactivated. The lack of an effect of IGF-I on juxtamedullary postglomerular arterioles cannot be extrapolated to other nephron populations in the kidney, because EA of juxtamedullary nephrons have distinct anatomical and physiological characteristics that differ from those in superficial nephrons. Indeed, micropuncture experiments in superficial nephrons of fasting Munich-Wistar rats demonstrated that the rise in single-nephron GFR and RPF during acute IGF-I administration is mainly due to an increase in glomerular ultrafiltration coefficient and a fall in efferent arteriolar resistance, whereas the afferent resistance showed only an insignificant tendency to decrease (18). However, chronic (6-7 days) administration of IGF-I caused a significant drop of afferent arteriolar resistance in superficial nephrons (17). Our results are in general agreement with these earlier investigations, in that, in juxtamedullary nephrons, acute IGF-I treatment causes a vasodilation in preglomerular vessels.

In the present study, the vasodilatory effect of IGF-I was reversible within 10 min after ending the IGF-I perfusion, whereas in the intact rat, the effect of IGF-I on renal hemodynamics persisted for 90 min after the infusion (15). This difference is likely due to the absence of IGF binding proteins in the artificial blood solution in the present study.

Potential mediators of the hemodynamic effects of IGF-I have been the subject of investigation. In the present study, the effect of pharmacological inhibition of endothelial NO synthesis by L-NAME, a competitive inhibitor of endothelial NO synthase, on baseline vascular tone and the vasodilatory response to IGF-I was tested. L-NAME by itself evoked a 10% vasoconstriction in all three preglomerular arteriolar segments, indicating that, under baseline conditions, locally synthesized NO exerts a tonic vasodilatory effect on juxtamedullary afferent arterioles, a finding consistent with measurements in intact kidneys (4). In our study, pretreatment with L-NAME completely inhibited the vasodilatory response to IGF-I, suggesting that the vasodilatory action of IGF-I is mediated by increased endothelial NO synthesis. This finding demonstrates that the effect of NO blockade on IGF-I-induced vasodilation, which has been described previously in the whole kidney (11) and in peripheral arteries (31), is also apparent in juxtamedullary nephrons.

To exclude the possibility that blockade of IGF-I action by L-NAME is not specific and just a consequence of its persistent vasoconstrictive effect, we directly measured NO release in response to IGF-I perfusion in preglomerular microvessels with an NO-selective microelectrode. IGF-I perfusion induced a reversible increase of NO concentration ([NO]) in the intact renal microvasculature, similar in magnitude to the rise in [NO] induced by topical application of 10-5 M ACh (Fig. 7). We recognize that there may be some uncertainty in regard to the relationship between the NO levels within the vascular wall and the levels measured with a diffusion-limited microelectrode placed over Bowman's capsule. Given the limitations of this kind of electrode and the site of measurement, the measured NO levels are likely to be underestimates of the local NO concentrations within the afferent arteriole. Nevertheless, the pattern of measured changes in NO concentration is consistent with and extends our recent observations that IGF-I is able to induce NO release from cultured human umbilical vein endothelial cells and immortalized rat renal interlobar artery endothelial cells via a tyrosine kinase-dependent mechanism (28). As expected from the rapid onset of NO release in response to IGF-I in this and the present study, the constitutive rather than the inducible form of endothelial NO synthase appears to be stimulated by IGF-I (28).

In the intact rat (15) and in humans (27), the IGF-I-induced increase in GFR can be completely blocked by pretreatment with the cyclooxygenase inhibitor indomethacin. We found similar results in the juxtamedullary nephron preparation, as pretreatment with indomethacin completely abolished the vasodilatory response to IGF-I. This indicates that its vasoactive action is dependent on an intact cyclooxygenase activity. It is not known whether vasodilatory prostaglandins play a permissive or an active role, i.e., whether their endothelial synthesis is actually stimulated by IGF-I. In this regard, it is pertinent that IGF-I is able to stimulate cyclooxygenase activity and prostaglandin E2 production in a nonrenal system, namely, in rat thyroid cells in the presence of thyrotropin (25).

How the interaction between vasodilatory prostaglandins and NO in renal microvessels is regulated remains to be investigated. Kinins, which stimulate both the release of NO (19) and vasodilatory prostanoids (3) from endothelial cells, have been suggested to partly mediate the renal hemodynamic response to IGF-I, because a kinin-receptor antagonist blocked the initial, but not late, GFR and RPF responses to IGF-I infusion (20). However, the rapid increase (within 1 min) in NO release in response to IGF-I observed in intact renal microvessels in the present study and in endothelial cells in culture (28) suggests that IGF-I is capable of directly stimulating the constitutive NO synthase in vascular endothelium. It is noteworthy that, in other experimental systems, there is evidence for mutual stimulation of prostaglandins and NO: in a mouse macrophage cell line and in human fetal fibroblasts, NO was able to activate both the constitutive and induced forms of cyclooxygenase (23). In rat mesangial cells, the NO release in response to a reduction of [Cl-] was blunted by indomethacin pretreatment, suggesting that metabolite(s) of cyclooxygenase may regulate the activation of NO synthase (29).

An unexpected finding in the present study was that during pretreatment with indomethacin, IGF-I induced a ~10% vasoconstriction in all three preglomerular arteriolar segments (Fig. 6). A similar tendency, although less pronounced because of a smaller baseline vessel diameter, was observed in response to IGF-I during L-NAME pretreatment (Fig. 5). This indicates that IGF-I not only stimulates the endothelial release of vasodilatory substances but also of a cyclooxygenase-independent vasoconstrictor, the action of which is unmasked when one of its vasodilatory counterparts, i.e., NO or vasodilatory prostaglandins, is inhibited. This also may account for our observations that the amounts of NO released by IGF-I and ACh are similar, whereas the net vasodilation in response to IGF-I is smaller than that elicited by ACh. The vasoconstrictor released is likely to be endothelin, as IGF-I is capable of stimulating immunoreactive endothelin-1 release from cultured porcine aortic endothelial cells in a dose-dependent manner (10). Although highly suggestive of endothelin involvement, studies with selective endothelin receptor blockers in renal vessels will be required to assess this possibility, as IGF-I may also have direct effects on vascular smooth muscle. In the intact rat, a vasoconstrictive effect of IGF-I after indomethacin pretreatment was not detectable (11, 15). This difference may be due to a differential reactivity of juxtamedullary compared with superficial nephrons in the rat.

Renal autoregulatory capacity in response to an increase in perfusion pressure was not significantly altered by IGF-I perfusion. This was important to document, because the IGF-I-induced vasodilation mediated by endothelial NO and vasodilatory prostaglandin release presumably involves guanosine 3',5'-cyclic monophosphate- and adenosine 3',5'-cyclic monophosphate-regulated reduced availability of intracellular calcium, due to enhanced sequestration of calcium into intracellular stores in vascular smooth muscle cells (12, 30). Notably, calcium channel inhibition in the present study completely inhibited autoregulatory responses and, instead, allowed afferent arteriolar segments to passively dilate with increased perfusion pressure, consistent with our previous report (5). The preserved renal autoregulatory capacity during IGF-I administration is an important safety issue in view of the therapeutic potential of this growth factor in the setting of acute and chronic renal failure (9).

In summary, the present study demonstrates that IGF-I can selectively alter intrarenal microvascular dimensions: it induces a reversible 10-15% vasodilation of preglomerular afferent but not postglomerular efferent arterioles in rat juxtamedullary nephrons. This vasodilation is mediated by an increased endothelial NO production in afferent renal microvessels and requires the presence of an intact renal cyclooxygenase system. The vasodilatory response is modulated by the concomitant release of a vasoconstrictive agent, presumably endothelin, which, in concert with the vasodilatory agonists, determines the net vasodilatory response to IGF-I.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical assistance of Aija Birzgalis. We thank Genentech (South San Francisco, CA) for providing recombinent human IGF-I.

    FOOTNOTES

B. Tönshoff supported by a Feodor Lynen grant from the Alexander von Humboldt Stiftung and by a fellowship award from the American Heart Association, New York State Affiliate.

Address for reprint requests: B. Tönshoff, Univ. Children's Hospital, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany.

Received 30 December 1996; accepted in final form 18 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Renal Physiol 274(1):F120-F128
0363-6127/98 $5.00 Copyright © 1998 the American Physiological Society



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