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Am J Physiol Renal Physiol 274: F490-F497, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 3, F490-F497, March 1998

The endogenous insulin-like growth factor system in radiocontrast nephropathy

Z. Symon, S. Fuchs, Y. Agmon, O. Weiss, I. Nephesh, R. Moshe, M. Brezis, A. Flyvbjerg, and I. Raz

Department of Medicine, Hadassah University Hospital, Jerusalem, Israel; and Institute of Experimental Clinical Research, Aarhus Kommunehospital, Aarhus C, Denmark

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The response of insulin-like growth factor (IGF) I in acute renal failure was evaluated in a model of radiocontrast nephropathy associated with selective necrosis of medullary thick ascending limbs. In brief, rats were administered radiocontrast medium or vehicle injections for controls after combined inhibition of prostanoids and nitric oxide. Twenty-four hours after the insult, tissue mRNAs for IGF-I, the IGF-I receptor, and IGF-binding proteins (IGFBP) 1 and 3 were assayed in cortex, medulla, and liver by solution hybridization-RNase protection assay, and IGFBPs were measured in serum and tissue by Western ligand blotting. Cortical IGF-I increased, whereas medullary IGF-I mRNA decreased. Renal IGFBPs decreased, whereas IGFBP-1 mRNA increased. The IGF system in the liver was unchanged. We conclude that general changes in renal IGFBPs in this experimental model of acute renal failure might increase the level of cortical IGF-I in a way that could modulate medullary recovery.

acute renal failure; nitric oxide; prostanoids

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

INSULIN-LIKE GROWTH FACTOR (IGF) I is a peptide of 70 amino acids synthesized in the liver and in other tissues (10). In the kidney, this peptide and its mRNA are found mainly in collecting ducts and thick ascending limbs of Henle's loop (8). Receptors for IGF-I are found both in the medulla and at the basolateral surface of the proximal tubule epithelium (20). Although their exact role is unclear, the binding proteins for IGFs (IGFBPs) may be important modulators of the biological activity of IGF-I on the kidney (13). In addition, a group of proteolytic enzymes function as IGFBP proteases, which regulate the bioavailability and alter the function of IGFs (24).

Recent reports have suggested a role for growth factors in the recovery of function after acute renal failure (ARF). However, the mechanism by which this occurs is far from clear. Some workers have found increases in renal IGF-I and IGF-I gene expression during recovery from postischemic ARF, with more widespread distribution along the nephron, especially in the regenerating cells of the outer stripe of the outer medulla (2, 23, 29). IGF-I binding was also increased in these areas (29), but it is unclear whether this is due to a change in receptor number (or affinity) or to other mechanisms, such as a local increase in IGFBPs. Others have observed a decrease in renal IGF-I mRNA in nephrotoxic gentamicin-induced ARF (34). Of interest, a decrease in renal epithelial growth factor mRNA after ARF was also noted to be associated with increased radioactive epithelial growth factor binding, suggesting decreased gene expression and increased affinity for growth factors in surviving cells (32).

We have recently shown that, in ARF induced by HgCl2, IGF-I mRNA tends to decrease in the whole kidney while IGF-I was either increased or unchanged (19). Widespread kidney necrosis induced by HgCl2 prevented, however, an accurate measurement of kidney IGF-I. We also observed an increase in IGFBP-1 mRNA, which would explain an increase in kidney IGF-I in spite of decreased IGF-I mRNA (19). Our study as well as others (2, 23, 29, 32, 34) used "single-insult" models that differ from clinical ARF, often resulting from multiple synergistic insults (4). In the present study, we used a model of radiocontrast nephropathy, reminiscent of the clinical syndrome and characterized by selective necrosis of medullary thick ascending limbs (1), to characterize the entire IGF system in damaged and nondamaged renal tissue.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Male Sprague-Dawley rats with a body weight range of 250-400 g were studied. The animals were maintained and treated in accordance with the regulations of the Hadassah committee on animal care and use. Rats were housed three per cage in a room with a 12:12-h (0600-1800) artificial light cycle, temperature 21 ± 2°C, and humidity 55 ± 2%. Until initiation of the experiments, the rats had free access to standard rat chow and tap water.

Experimental Model of ARF

We used a model of radiocontrast nephropathy combining an intra-arterial injection of the radiocontrast iothalamate with prior inhibition of prostanoids and nitric oxide synthesis as previously reported (1).

Rats with ARF (n = 23). Rats were kept in metabolic cages (Nalge, Rochester, NY) starting 24 h before the acute insults, with free access to tap water and standard rat chow. After a baseline 24-h urinary collection, the rats were anesthetized with an intraperitoneal injection of 100 mg/kg body wt ketamine (Parke-Davis, Pontypool, Gwent, UK). The left femoral vein and artery were cannulated (PE-50; Clay-Adams, Parsippany, NJ), and a baseline blood sample was drawn (1 ml).

The rats were preconditioned by inhibition of the synthesis of prostaglandin and nitric oxide. Indomethacin (Sigma Chemical, St. Louis, MO) was dissolved in phosphate buffer (pH 8.4) and administered intravenously at the dose of 10 mg/kg. The competitive inhibitor of nitric oxide synthesis, N omega -nitro-L-arginine methyl ester (L-NAME; Sigma Chemical) was dissolved in 0.9% saline and administered intravenously at the dose of 10 mg/kg, 15 min after the administration of indomethacin. The radiological contrast material, sodium iothalamate (80%; Angio-Conray; Mallinckrodt, St. Louis, MO), was injected through the arterial cannula over 2-3 min at the dosage of 6 ml/kg body wt 15 min after the administration of L-NAME.

The rats were then returned to the metabolic cages for another 24-h urine collection, without access to water. At the end of this period, rats were anesthetized with an intraperitoneal injection of Inactin (100 mg/kg; Byk-Gulden, Konstanz, Germany), and a blood sample was drawn from the inferior vena cava. The kidneys and ~100 mg of liver were removed. Kidneys were not perfused in situ, since the amount of IGFBPs entrapped in kidney tissue during the procedures is below the detection limit of the method used previously (16). The kidneys were decapsulated. The cortex and medulla were separated surgically and immediately frozen in liquid nitrogen. Proper separation was validated by the measurement of trace levels of IGF-I mRNA in the cortex in contrast to high expression of IGF-I mRNA in the medulla.

Control rats injected with vehicle fluids (n = 17). Control rats underwent the same anesthesia and surgical preparation. Vehicle fluids (used for indomethacin and L-NAME) were injected at the same time and volume as for experimental animals. Normal saline (2 ml/kg) was injected instead of radiocontrast. The rats were then returned to the metabolic cages for another 24-h urine collection without access to water.

Rats injected with L-NAME and indomethacin (n = 8). Rats underwent anesthesia and surgical preparation. L-NAME and indomethacin were injected at the same time and dose as for experimental animals. Normal saline (2 ml/kg) was injected instead of radiocontrast.

Serum and Tissue IGF-I Extraction and IGF-I Radioimmunoassay

IGF-I extraction was performed according to D'Ercole et al. (10) as previously described (15). Briefly, the kidneys were homogenized on ice in 1 M acetic acid (5 ml/g tissue) with an Ultra Turrex TD25 and further disrupted using a Potter Elvehjelm homogenizer. The tissues were extracted two times, and, after lyophilization, the samples were redissolved in 40 mmol/l phosphate buffer (pH 8.0). Tissue extracts were kept at -80°C until IGF-I assay was performed in diluted extracts. A linear relationship was found between biosynthetic IGF-I and the IGF-I immunoreactivity of kidney and liver extracts at multiple concentrations, indicating antigen similarity and that no binding proteins or receptors from the extracts intefered in the immunoassay. Furthermore, when biosynthetic IGF-I or an aliquot of the tissue extracts was subjected to Ultragel (IBF) Aca 200 filtration, peaks in immunoreative IGF-I occurred in the same fractions. When extracts from kidney and liver were lyophilized and assayed in the presence of biosynthetic IGF-I, the mean recovery of the added IGF-I was 101% (range 84-115) and 87% (range 75-93), respectively. Finally, when 125I-IGF-I was incubated with tissue extracts under assay conditions (48 h at 4°C), identical degradation (<4%) measured by chromatography was found compared with tracer incubated with buffer alone, excluding the possibility of extract proteases degrading the tracer. The measured kidney IGF-I concentrations were all corrected for serum IGF-I entrapped in the kidney tissue as previously described (16).

Serum IGF-I was measured after extraction with ethanol (30 µl serum and 750 µl ethanol). The mixture was incubated for 2 h at room temperature and centifuged, and 25 µl of the supernatant were diluted 1:200 before analysis. Serum IGF-I was measured by radioimmunoassay (RIA) using a polyclonal rabbit antibody (Nichols Institute Diagnostics, San Capistrano, CA) and recombinant human IGF-I as standard (Amersham International, Amersham, Bucks, UK). Mono-idionated IGF-I (125I-[Tyr31]IGF-I) was obtained from Novo-Nordisk (Bagsvaerd, Denmark). When exposing the serum extract to Western ligand blot (WLB), no IGFBPs could be identified; furthermore, semilog linearity of biosynthetic IGF-I and serum extracts was seen, indicating antigen similarity and that no IGFBPs intefered in the RIA. Intra- and interassay coefficients of variation were <5 and 10%, respectively.

Gene Expression of IGF-I, IGF-I Receptor, IGFBP-1, and IGFBP-3 in Kidney and Liver Tissue

These were performed by solution hybridization-ribonuclease (RNase) protection assay as follows: total RNA was prepared from renal tissue of individual rats by TriReagent (Molecular Research Center) according to the method of Chomczynski (9) and quantified by absorbance at 260 nm. The integrity of the RNA and the accuracy of the spectrophotometric determinations were assessed by visual inspection of the ethidium bromide-stained 28S and 18S ribosomal RNA bands after agarose formaldehyde gel electrophoresis of 10-mg aliquots, as described previously (27). The IGF-I, IGF-I receptor (IGF-IR), IGFBP-1, and IGFBP-3 riboprobes were generous gifts from Derek LeRoith [National Institutes of Health (NIH), Bethesda, MD].

The antisense RNA probe used to detect IGF-IR mRNA has been previously described (35). This transcript contains 40 bases of vector sequence and 265 bases complementary to a region encompassing 15 bases of 5'-untranslated sequence, the signal peptide, and the first 53 amino acids of the IGF-IR alpha -subunit. On hybridization of this RNA probe with IGF-IR RNA and subsequent RNase digestion, a protected band of 265 bases was obtained.

The riboprobe employed to measure the levels of IGF-I mRNA has been described (26). This probe allows the detection of both IGF-I mRNA species encoding the IGF-Ia and IGF-Ib prohormones. Only the levels of IGF-Ia mRNA that constitute >90% of the total IGF-I message and that correlate with the levels of IGF-Ib mRNA were measured in this study.

The IGFBP-1 mRNA was measured using an antisense probe derived from a rat IGFBP-1 cDNA clone isolated from a dexamethasone-treated H-4-11-E-C3 hepatoma cell library (G. T. Ooi, NIH; unpublished observations). The size of the protected band obtained by hybridizing this antisense RNA probe with IGFBP-1 mRNA was 203 bases.

The IGFBP-3 mRNA measurement has been previously described (12). The size of the protected band obtained by hybridizing this antisense RNA probe with IGFBP-3 mRNA was 493 bases.

Solution hybridization-RNase protection assays were performed as described (35). Briefly, 20 µg of total RNA were hybridized with 1 × 106 disintegrations/min 32P-labeled antisense RNA probes. The hybridization was carried out at 45°C for 16 h in a buffer containing 80% formamide. After hybridization, RNA samples were digested with RNase A and T1, and the protected hybrids were extracted with phenol-chloroform, ethanol precipitated, and electrophoresed on 8% polyacrylamide-8 M urea denaturing gel. Multiple autoradiograms from each gel were scanned by a densitometer connected to an Apple Macintosh computer. Changes in the signals were expressed as the ratio between experimental and control values.

Tissue Extraction for IGFBPs

Approximately 50 mg of thawed kidney or liver tissue were placed in 1.5-ml propylene tubes and weighed. The tissue was homogenized for 2 min in 0.5 ml of 20 mmol/l tris(hydroxymethyl)aminomethane (Tris) and 2% Triton X-100 buffer (pH 7.4) using a micropestle (catalog no. 9922; Research Products International, Mount Procpect, IL). After adding 0.13 ml of Laemmli buffer, each tube was boiled for 5 min and incubated overnight at 4°C. Aliquots of extracts were stored at -80°C. Protein content of the extracts was measured using a protein assay (catalog no. 23225; Pierce, Rockford, IL) with bovine serum as standard.

WLB for IGFBPs in Serum, Kidney, and Liver

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and WLB were performed according to the method of Hossenlopp et al. (22), as previously described (17). Thawed extracts were boiled for 1 min and centrifuged for 1 min at 13,000 revolutions/min. An aliquot of the supernatant equivalent to 200 µg of tissue protein or 2 µl of serum was subjected to SDS-PAGE (10% polyacrylamide) and nonreducing conditions. The electrophoresed proteins were transferred by electroelution onto nitrocellulose paper (Schleicher & Schuell, Munich, Gemany), and the membranes were incubated overnight at 4°C with ~500,000 counts/min 125I-IGF-I [specific activity 2,000 Ci · mmol-1 · l-1 in 10 ml of 10 mmol/l Tris · HCl buffer (TBS) containing 1% bovine serum albumin and 0.1% Tween (pH 7.4)]. Membranes were washed with TBS and, after drying overnight, the nitrocellulose sheets were autoradiographed with Kodak X-AR film and exposed to NEN enhancing sceens at -80°C for 3-7 days. Specificity of the IGFBP bands was ensured by competitive coincubation with unlabeled IGF-I purchased from Bachem (Bubendorf, Switzerland). WLB were quantified by densitometry using a Shimadzu CS-9001 PC dual wavelength flying spot scanner.

Biochemistry

Plasma and urine creatinine were determined by a standard automated application of the Jaffe reaction. Plasma glucose concentration was measured by the glucose oxidase method (Boehringer, Mannheim, Germany). Plasma insulin levels were determined by RIA, using human insulin as standard (Sorin Biomedica, Saligguia, Italy). Serum rat growth hormone was measured by a commercially available RIA purchased from Amersham. Intra- and interassay coefficient of variations were <5 and 10% for all assays.

Statistics

Analyses of variance for repeated measurement was used in evaluation of differences in combination with a Bonferroni test for multiple comparisons and unpaired Student's t-test. A P value of <0.05 was regarded as significant. Values are given as means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ARF Model Data

Table 1 shows plasma creatinine, glucose, insulin, and serum IGF-I and rat growth hormone in controls and ARF rats. As previously shown (1), plasma creatinine at death was significantly higher in the rats that received the radiocontrast protocol. Plasma glucose and insulin were similar in both groups. Food intake was not significantly less in rats with ARF compared with controls (7.2 ± 1.8 g/day per rat, n = 23 vs. 8.3 ± 1.4 g/day per rat, n = 17, respectively, P = not significant).

                              
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  		Table 1.   Plasma creatinine, glucose, insulin, and serum IGF-I and rGH in control and ARF rats

GH/IGF System in Serum

Serum IGF-I was significantly reduced (714 ± 46 vs. 1167 ± 62 µg/l, P < 0.001) in rats with radiocontrast nephropathy compaired with controls. The level of rat growth hormone increased in rats with radiocontrast nephropathy (164 ± 16 vs. 46 ± 8 µg/l, P < 0.001). Serum IGFBP-3 and the 30-kDa IGFBPs were significantly decreased in rats with ARF, whereas IGFBP-4 in the serum was unchanged (see Fig. 1, top and bottom).


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  		Fig. 1.   Top: serum insulin-like growth factor (IGF) binding proteins (BPs) in control and acute renal failure (ARF) animals as measured by Western ligand blotting. ** P < 0.05. AU, arbitrary units. Bottom: Western ligand blot showing the distinct IGFBP bands in serum samples from control (C) and ARF (A) animals. Mr, realtive molecular weight.

IGF System in Kidney and Liver

Twenty-four hours after injection of radiocontrast, there was a significant increase in IGF-I content in the kidney cortex of rats with ARF compared with control animals, whereas IGF-I contents in the kidney medulla and liver were similar in both control and ARF rats, as illustrated in Fig. 2, top.


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  		Fig. 2.   Top: tissue IGF-I in renal cortex, medulla, and liver as measured by radioimmunoassay (ng/g). ** P = 0.02. Bottom: IGF-I and IGF-I receptor gene expression in kidney cortex and medulla of control rats and of rats with ARF for 24 h. Levels of IGF-I mRNA and IGF-I receptor mRNA were measured by solution hybridization-ribonuclease (RNase) protection assays. Further details of assay are described in MATERIALS AND METHODS. Shown are results obtained with 2 representative animals from each group.

Figure 2, bottom, shows a significant decrease in medullary IGF-I mRNA (4,020 ± 140 densitometric units in controls vs. 2,400 ± 134 in ARF rats, P < 0.0001). IGF-I mRNA was unchanged in the liver (data not shown) and, as expected, hardly detectable in the cortex of both experimental and control rats. IGF-IR mRNA was unchanged in the kidney cortex and medulla of rats with radiocontrast nephropathy.

IGFBP-1 mRNA increased significantly in the cortex of ARF rats compared with control animals, as illustrated in Fig. 3, top and bottom. IGFBP-1 mRNA also increased in the medulla, as illustrated in Fig. 3, middle and bottom. In contrast, IGFBP-3 mRNA remained unchanged in the cortex (Fig. 4) and medulla.


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  		Fig. 3.   Top: expression of IGFBP-1 in the renal cortex from control rats and rats with ARF. Levels of IGFBP-1 mRNA were measured by solution hybridization RNase protection assays. Shown are results obtained with 4 representative animals from each group. Middle: expression of IGFBP-1 in renal medulla of kidneys from control rats and rats with ARF. Shown are results obtained with 3 representative animals from each group. +, Yeast RNA and probe with RNase; -, yeast RNA and probe without RNase; P, native probe. Bottom: IGFBP-1 mRNA in cortex, medulla, and liver (AU/mm2). Cumulative data (ARF n = 23, control n = 17), ** P < 0.05.


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  		Fig. 4.   IGFBP-3 expression in the renal cortex from control rats and rats with ARF. Levels of IGFBP-3 mRNA were measured by solution hybridization RNase protection assays. Shown are results obtained with 4 representative animals from each group.

In the present study, IGFBPs were measured in cortical and medullary tissue by means of WLB. The WLB method yielded four different bands of IGFBPs with apparent molecular weight of 38-42 kDa (doublet), 30 kDa, and 24 kDa. The doublet band corresponded to IGFBP-3, and the 24-kDa band corresponded to IGFBP-4, whereas the 30-kDa band in the kidney and liver tissue represents either IGFBP-1, IGFBP-2, or IGFBP-5, as these have a similar molecular weight in the rat. Based on the distribution of renal IGFBP mRNAs described above, the 30-kDa IGFBPs most likely represent IGFBP-1 and IGFBP-2 in the cortex and IGFBP-1 and IGFBP-5 in the medulla.

There was a significant decrease in cortical IGFBP-3 (P < 0.001), IGFBP-4, and 30-kDa IGFBPs (P < 0.05) in rats with ARF, whereas, in the medulla, only the 30-kDa IGFBPs decreased significantly (P < 0.05), as illustrated in Fig. 5, A-C.


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  		Fig. 5.   A: IGFBP-3, IGFBP-4, and 30-kDa IGFBPs in the renal cortex from control and ARF animals as measured by Western ligand blotting. B: IGFBP-3, IGFBP-4, and 30-kDa IGFBPs in the kidney medulla from control and ARF animals as measured by Western ligand blotting. C: IGFBP-3, IGFBP-4, and 30-kDa IGFBPs in kidney cortex, medulla, and liver as measured by Western ligand blotting. * P < 0.05 and ** P < 0.001.

In rats that received indomethacin and L-NAME without radiocontrast, renal IGF-I mRNA, IGFBP-1 mRNA, IGFBP-3 mRNA, IGFBPs, IGF-I protein, and serum IGF-I and IGFBPs were unchanged after 24 h when compared with vehicle-injected rats (data not shown).

IGFBP-1 mRNA was increased significantly in the cortex and medulla of rats with ARF as early as 2 h after the injection of radiocontrast (cortex 3,393 ± 444 densitometric units in ARF vs. 2,299 ± 199 in control; medulla 3,401 ± 266 densitometric units in ARF vs. 2,268 ± 349 in control, P < 0.05; Fig. 6). Medullary IGF-I mRNA did not decrease significantly.


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  		Fig. 6.   IGFBP-1 expression in kidney cortex and medulla of control rats and rats with ARF 2 h after induction of ARF. Levels of IGFBP-1 mRNA were measured by solution hybridization RNase protection assays. Shown are results obtained with 2 representative animals from each group.

Renal beta -actin mRNA was similar for ARF and normal rats in both cortex (1,764 ± 120 densitometric units vs. 2,160 ± 77 in ARF rats vs. controls, respectively) and medulla (2,626 ± 188 densitometric units vs. 2,870 ± 235).

In the liver, IGF-I mRNA was similar in ARF and control rats (2,539 ± 57 densitometric units in ARF rats vs. 2,389 ± 112 in controls). IGF-IR gene expression was undetectable in both groups.

IGFBP-1 mRNA in the liver was also similar in both groups (Fig. 3, bottom). There was no significant difference in the level of IGFBP-3, IGFBP-4, and the 30-kDa IGFBPs in the liver of rats with radiocontrast nephropathy when compared with controls (Fig. 5C).

Table 2 presents a summary of the directional changes in the expression of the IGF-I system observed in the present model of radiocontrast nephropathy.

                              
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  		Table 2.   Summary of directional changes in expression of IGF-I system in radiocontrast nephropathy

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In both human beings and experimental animal models, ARF induced by ischemia or toxins usually recovers spontaneously within days to weeks (6). In the kidney, the outer medulla is a vulnerable area to synergistic hypoxic and toxic insults, as illustrated in a model of radiocontrast nephrotoxicity (1, 3), and renal function after ARF may depend on the recovery of the outer medulla. Because the outer medulla is a major site of IGF-I synthesis, changes in the renal IGF-I axis may play a role in the regenerative mechanisms that take place in the kidney after ARF (5), as suggested in several animal models of ARF. After toxic ARF in rats, immunohistological staining for IGF-I shows more widespread peptide staining extending into the proximal nephron (2, 29). Increased levels of IGF-I and IGF-I mRNA have been found in remnant renal tissue adjacent to acutely infarcted areas (31). In contrast, others have reported reduced intrarenal IGF-I mRNA expression after gentamicin (34)- or HgCl2-induced ARF (19). In the latter study, IGF-I peptide was unchanged. In some but not all experimental models of ARF, recovery was enhanced by exogenous administration of IGF-I (11, 19). In contrast to single insult models of experimental ARF that result in widespread kidney damage, this multi-insult model associated with predominantly outer medullary injury is reminiscent of the clinical syndrome of contrast nephropathy in humans, which is characterized by a multiplicity of concomittant risk factors. As shown in Table 1, ARF was associated with doubling of the plasma creatinine, without changes in glucose or insulin. ARF was followed by increased serum GH levels and decreased levels of serum IGF-I, IGFBP-3, and 30-kDa IGFBPs. Serum IGFBP-4 was unchanged.

As summarized in Table 2, several specific changes ocurred in the renal IGF axis during ARF, whereas no changes were seen in the liver. A significant increase in renal cortical IGF-I content was observed, whereas, in the kidney medulla, IGF-I levels were unchanged. Theoretically, the increase in cortical IGF-I may be due to increased local IGF-I production in the kidney or increased sequestration of IGF-I from the circulation or locally through mechanisms involving changes in IFG-IR number or IGFBPs in the circulation or tissue.

We did not find support for the hypothesis that increased cortical IGF-I concentration was due to increased local production of IGF-I, as cortical IGF-I mRNA was hardly detectable (demonstrating that these samples did not contain medullary tissue), and, in the medulla, the major site of renal IGF-I production, the IGF-I mRNA level was decreased in ARF animals compared with controls. The decrease in IGF-I mRNA in the medulla may have resulted from tissue damage, which is prominent in this model in the outer medulla, especially in the inner stripe (1). However, because the expression of other genes in the medulla was unchanged (IGF-IR, actin) or even increased (IGFBP-1), the reduction of IGF-I mRNA may be a specific response to outer medullary injury.

The uncoupling of mRNA and protein levels with decreased medullary IGF-I mRNA and increased cortical and preserved medullary IGF-I protein is in contrast to the findings of decreased renal IGF-I mRNA and IGF-I protein that was found in a rat model of acute ischemic injury induced by renal artery clamping (14). Our finding, however, can be explained by posttranslational upregulation or delayed degradation of the IGF-I protein. However, an increased renal sequestration of IGF-I by IGF-IR or IGFBPs from the circulation or from local production in the kidney can also result in increased renal IGF-I protein.

Many of the biological actions of IGF-I are thought to be mediated through the IGF-IR. The IGF-IR is a heterotetrameric glycoprotein with a primary structure highly homologous to the insulin receptor, consisting of two alpha - and beta -subunits, linked by disulfide bridges and belonging to a family of tyrosine kinases. In the kidney, IGF-IR mRNA has been localized to the glomeruli and the tubular epithelium (7). Furthemore, IGF-IR are widely distributed in the proximal tubules, present on both the luminal and basolateral aspects of the cell (18). In the present study, IGF-IR mRNA was present both in the cortex and the medulla. We describe unchanged levels of IGF-IR mRNA levels in kidneys from ARF animals, thereby supporting the notion of unchanged levels of the IGF-IR. We have not excluded the possibility that changes in receptor number may occur through changes in receptor affinity or as a result of posttranslational upregulation. Indeed, an increase in IGF-IR number without a change in IGF-IR mRNA levels was demonstrated in a rat model of acute ischemic injury to the kidney. However, in that model, renal IGF-I was decreased (14).

In the circulation and in the extracellular space, IGFs are bound to specific IGFBPs. To date, six different IGFBPs have been cloned and designated IGFBP-1 to -6 (33). Under normal circumstances, IGFBP-3 is the predominant carrier of IGFs in the circulation, and one of its roles as a carrier protein is to protect IGFs from degradation and sequestration and to facilitate delivery to target tissues (28). It seems evident today, however, that IGFBPs may also act as modulators of IGF action at a cellular level, by enhancing or inhibiting the biological actions of IGFs. IGFBP-1 to -5 are all expressed in the kidney. IGFBP-1 mRNA is localized mainly in the medulla and to a lesser extent to the cortex. IGFBP-2 mRNA is localized exclusively to the glomeruli (25), IGFBP-3 mRNA to the interstitial cells in the cortex (33), IGFBP-4 mRNA to the distal tubule in the cortex (25), and IGFBP-5 mRNA to the interstitial cells in the medulla (25).

In the present study, a pronounced increase in IGFBP-1 mRNA was seen both in the cortex and medulla, whereas IGFBP-3 mRNA remained unchanged. In a model of renal failure induced by folic acid, IGFBP-1 mRNA increased sixfold, and IGFBP-3 mRNA decreased (21). As insulin is known to be an important regulator of IGFBP-1, the increase in kidney IGFBP-1 mRNA could be due to a change in nutritional status or insulin levels. However, ARF animals had insignificantly less food intake than controls and similar plasma insulin, glucose, and IGFBP-1 mRNA levels in the liver, thus making this possibility unlikely. Futhermore, some of the changes were already apparent 2 h after the insult. In contrast to the pronounced rise in renal IGFBP-1 mRNA, the 30-kDa IGFBP band, which partly represents IGFBP-1 in both cortex and medulla, was significantly decreased in ARF animals in comparison with controls. Our findings are in contrast to streptozocin-induced diabetes or nephrectomy in which renal IGF-I increased in concert with increased cortical IGFBP-1 mRNA and protein (25). A similar pattern of increased IGFBP-1 mRNA with a decrease in 30-kDa protein was reported in a model of acute ischemic injury in rat kidney. In this model, however, renal IGF-I was decreased (14). The discrepancy between IGFBP-1 and -3 mRNA and protein levels in the present study suggests either decreased translation or increased posttranslational degradation of these proteins. However, in the model of ARF mentioned above, increased renal IGFBP-1 mRNA with decreased 30-kDa protein was not associated with increased protease activity (14). It might be that the decrease in renal 30-kDa IGFBPs in our study reflect a major fall in the kidney of IGFBP-2 and or IGFBP-5, since it was shown that acute iscemic injury to rat kidney resulted in a decrease in renal IGFBP-2 and -5 mRNA levels parallel to an increase in IGFBP-1 mRNA (14). It is notable that, in our ARF animals, a general decrease was seen in all IGFBPs (IGFBP-3, IGFBP-4, and the 30-kDa IGFBPs) in the cortex, whereas more selctive changes involving only the 30-kDa IGFBPs were seen in the medulla. In the liver, an untargeted organ in this model, the IGFBPs were unchanged. The general decrease in renal IGFBPs, together with an increase in IGF-I in the cortex demonsrated in our experimental model of ARF, may contribute to an increase in IGF-I avaliability to the damaged medulla. The more than twofold increase in renal IGFBP-1 mRNA may represent an early response to injury, as suggested by the observation in this model of ARF of an increase in both cortical and medullary IGFBP-1 mRNA already 2 h after the acute insult. In the regenerating liver, however, IGFBP-1 was shown to be an early response gene (30). IGFBP-1 could also be a tissue-specific acute-phase reactant in response to renal injury with a very short half-life after capturing IGF-I from serum carriers such as IGFBP-3 and positioning IGF-I to interact with the IGF-IR. Alternatively, increased IGFBP-1 mRNA may be secondary to a negative feedback pathway between IGFBP-1 tissue concentration and IGFBP-1 gene transcription. IGF-I itself has been shown to be an important regulator of IGFBP availability, and the increase in IGFBP-1 mRNA may be due to increased tissue IGF-I, as recently shown in fibroblasts in which IGF-I treatment induced synthesis of IGFBP-3 and IGFBP-5 mRNA (IGFBP-1 mRNA was not detectable in these cells). The increased kidney IGF-I may also be the result of the decrease in serum IGFBP-3 and 30-kDa IGFBPs, which can facilitate increased IGF-I extraction from the serum to the kidney. Further studies are warranted to confirm these hypotheses.

Separation of cortex from medulla enabled us to measure, for the first time, IGF-I and IGFBP levels and IGF system gene expression in nonnecrotic vs. necrotic tissue in response to renal injury. IGF-I produced by the outer medulla may act as a paracrine growth factor through receptors and IGFBPs localized in the cortex, via the portal blood vessel system that constitutes the venous vasa recta ascending from the medulla to the cortex in the medullary rays. The medullary rays resemble expansions of medulla into cortex. Devoid of glomeruli, these regions are the site of the terminal, straight portions of the proximal tubules leading down to Henle's loop in the medulla; from there, they receive their blood supply (3). IGF-I produced by the renal cortical tubules may travel to the outer medulla by several other pathways; IGF-I may be secreted into the tubular lumen and move downstream to act upon the distal tubule; alternatively, IGF-I may be secreted at the contraluminal side of the tubular cells into the interstitium, from where it may diffuse directly or via the vasa recta to the medulla.

This is the first study that examines the response of the entire IGF system in reaction to radiocontrast utilizing a model reminiscent of radiocontrast nephropathy in humans. This study has shown that severe selective medullary injury results in changes in the IGF system, also in the undamaged cortex. Cortical IGF-I content increased significantly despite its reduced synthesis in the renal medulla. A general decrease in IGFBPs was observed in both cortex and medulla, which might enhance the bioavailability of cortical IGF-I to the medulla via the portal system. Further studies are warranted to elucidate whether the changes in the IGF-I system are passive events occurring in the pathogenesis of ARF or alternatively are of importance in the process of repair, recovery, and preservation of kidney function.

    FOOTNOTES

Address for reprint requests: I. Raz, Dept. of Medicine, Hadassah Univ. Hospital-Ein Kerem, PO Box 12000, Jerusalem, Israel.

Received 10 December 1996; accepted in final form 12 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Renal Physiol 274(3):F490-F497
0363-6127/98 $5.00 Copyright © 1998 the American Physiological Society



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