Insulin-like growth factor I (IGF-I) is a peptide growth factor with metabolic1, mitogenic and vasoactive properties2,3. In the kidney, IGF-I increases renal blood flow and glomerular filtration rate (GFR)4, stimulates mitogenicity5 and decreases apoptosis of tubular cells3. In the proximal tubule, IGF-I is not expressed6. However, after renal ischemic injury, IGF-I is transiently expressed in proximal tubular cells and in macrophages, suggesting an important role for IGF-I in the repair from acute tubular injury6,7. Indeed, administration of IGF-I to several animal models of ARF accelerates the recovery of renal function, improves renal histology and may also decrease the catabolic state8,9,10,11,12.
It is believed that the actions of IGF-I are regulated by one or more of six IGF-I binding proteins (IGFBPs) and also by the IGF type I receptor (IGF-IR)3. Hence, there has been considerable interest in the response of IGFBPs and the expression of IGF-IR in the kidney with ARF12,13,14. Moreover, the concentrations of IGFBPs in serum and the expression of IGFBP mRNAs and IGF-IR mRNA in nonrenal tissues after the induction of ARF have not been systematically evaluated, particularly when rats are receiving exogenous IGF-I. Since IGF-I has been administered in research protocols to animals and humans with ischemic ARF, we investigated the response of serum IGFBPs and tissue IGFBP mRNAs and IGF-IR mRNA in rats that were receiving subcutaneous injections of recombinant human IGF-I (rhIGF-I) or vehicle. The following hypotheses were tested: (1) In rats, ischemic ARF alters the concentrations of certain IGFBPs in serum and the expression of mRNA for IGFBPs and the IGF-IR in the kidney and other tissues. (2) Treatment with rhIGF-I independently causes other alterations in some of these potential regulatory factors.
METHODS
Animals
Male Sprague-Dawley rats weighing 200 to 250 g were studied. Rats were housed in metabolic cages from 24 hours before surgery until they were killed 72 to 74 hours postoperatively. Animals were randomly assigned to undergo one of two surgical procedures: creation of ARF or sham operation (SO). Rats were allowed free access to rat chow until surgery. At 9:00 a.m. to 10:00 a.m., rats were anesthetized with ketamine (87 mg/kg body wt) and xylazine (13 mg/kg body wt). ARF was produced by an anterior abdominal incision and clamping of both renal pedicles for 45 minutes with a baby Dieffenbach clamp. SO rats underwent laparotomy for a similar duration of time, abdominal incision length, and manipulation of organs, but the renal pedicles were not clamped. Only two rats died spontaneously. Both had ARF and died from abdominal bleeding within 24 hours after surgery, after one rhIGF-I injection. The ARF and SO rats were randomly assigned to receive subcutaneous injections of either rhIGF-I (Chiron Therapeutics, Emeryville, CA, USA), 50 
g/100 g body wt (that is, ARF+IGF-I or SO+IGF-I), or a similar volume of vehicle (V, 0.9% saline; that is, ARF+V or SO+V). The injections were given three times per day for a total of nine doses starting five hours after surgery and continuing at 8:00 p.m., 8:00 a.m. and 2:00 p.m. each day. Six animals were studied in each of the four groups. ARF and SO rats were fasted during the 72 to 74 hours after surgery except for free access to tap water containing 5% dextrose monohydrate. Tail blood samples were drawn before surgery and at 5, 24, 48 and 72 to 74 hours after surgery. Rats were killed by decapitation 72 to 74 hours after surgery, and liver, lung, heart, quadriceps muscle and kidney (cortex and medulla) tissues were taken and immediately processed as follows. Serum creatinine was measured using the Sigma kit (Sigma-Aldrich Corporation, St. Louis, MO, USA) according to the manufacturer's instructions.
Semiquantification of serum IGFBPs by Western blotting
Western ligand blotting was performed as previously described15,16 with modifications to improve band separation. Serum samples (2 l/lane) were diluted 1:10 in non-reducing Laemmli sample buffer17, boiled for three minutes and separated at room temperature with constant (12 mAmp) current for about 15 hours on 12% SDS-polyacrylamide gels using a Bio-Rad Protean II electrophoresis system (Richmond, CA, USA). Proteins were electrotransferred onto nitrocellulose at constant (0.4 Amp) current for 24 hours at room temperature using a Bio-Rad Trans Blot electrophoretic transfer cell. The membranes were blocked in the TBS buffer (20 mM Tris, 50 mM NaCl, pH 7.4) containing 3% wt/vol bovine serum albumin (BSA) at room temperature for 12 hours, and then incubated overnight at 4°C with 1.7 Ci of125I-labeled IGF-I (Amersham, Arlington Heights, IL, USA) in 15 ml of 3% wt/vol BSA in TTBS buffer (TBS buffer with 0.05% (vol/vol) Tween 20). The membranes were then washed in TBS, dried and exposed to Fuji RX x-ray film for three to four days at -70°C with Dupont Cromex intensifying screens.
To identify the binding proteins with a molecular weight of about 30 kD that were visualized by Western ligand blotting, Western immunoblots for serum IGFBP-1 and -2 were performed on the same membranes after completion of the Western ligand blottings16. Briefly, membranes were washed in TBS, blocked in TBSM [TBS buffer with in 5% (wt/vol) nonfat dry milk] at room temperature for one hour, and incubated overnight at 4°C with rabbit antibovine IGFBP-2 antibody (1:2,000; Upstate Biotechnology Incorporated, Lake Placid, NY, USA) or rabbit antirat IGFBP-1 antibody (1:800; gift from Dr. T. Unterman, Chicago, IL, USA) in TTBSM buffer [TTBS buffer with 3% (wt/vol) nonfat dry milk]. After washes with TTBS buffer, the membranes were incubated with biotinylated goat antirabbit IgG (1:3,000) and HRSP-Streptavidin (1:3,000) in TTBSM. Bands were visualized by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL, USA) and exposure to Fuji RX x-ray film. Semiquantification of IGFBP bands was performed by scanning densitometry (Bio-Rad GS 670 imaging densitometer).
Extraction of total RNA from tissues
After killing each rat, about 200 mg of tissue were immediately collected in 3 ml of ice-cold 4 M guanidine thiocyanate. The tissues were homogenized immediately with an Ultra-Turrax TP10 N homogenizer (Ika Werk Janke & Kunkel, Staufen, Germany), placed in liquid nitrogen, and stored at -70°C. Extraction of total RNA from the tissue homogenate was performed according to Lowe et al18 with minor modifications as described by Ding et al19. One volume of tissue homogenate was thoroughly mixed with 0.1 volume of 2 M sodium acetate, 1 volume of phenol and 0.2 volume of chloroform-isoamylalcohol (49:1). The preparation was centrifuged at 16,000 g for 30 minutes at 4°C; the clear supernatant underwent precipitation with 1 volume of isopropanol at -70°C for 60 minutes. After centrifugation at 14,000 g for 10 minutes at 4°C, the resulting pellet was washed in cold 75% ethanol, air dried and resuspended in deionized formamide. The quantity of RNA was determined by absorbance at 260 nm. The quality of the samples was evaluated by visualizing the 18 S and 28 S ribosomal RNA after 3 g total RNA was separated in a 1% agarose/formaldehyde gel.
Solution hybridization/RNase protection assay
Rat DNA probes for the solution hybridization/RNase protection assay of mRNAs for IGFBPs 1 through 6 were kindly provided by Dr. Shimasaki (La Jolla, CA, USA). The probes for 
-actin and IGF-IR were a gift from Dr. LeRoith (NIDDK, NIH, Bethesda, MD, USA). Another 125 bp probe for rat -actin (-actin 125; Ambion, Austin, TX, USA) was also used for some of the assays. The antisense RNA probes were synthesized and labeled using 50 to 80 Ci (10 mCi/ml) [32P]UTP (ICN Pharmaceutical Inc., Costa Mesa, CA, USA) and T7 (for IGFBP-1, 2, 4, 5, 6 and -actin), Sp6 (for the IGF-IR) and T3 (for IGFBP-3) polymerases with the Riboprobe Gemini II System (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. The solution hybridization/RNase protection assay was performed as described by Ding et al19 with the following minor modifications. Briefly, 10 to 15 g of total RNA was mixed with 29 l of hybridization buffer and 1 l of probe mixture. The hybridization mixture was composed of 75% deionized formamide, 20 mM Tris pH 7.5, 1 mM EDTA pH 8.0, 0.4 M NaCl, 0.1% SDS and 1 to 5 
105 cpm of the different antisense RNA probes. After the mixture was incubated for 16 hours at 60°C, samples were digested with 12 g of RNase A and 0.6 g of RNase T1 for one hour at 30°C. The protected hybrids were extracted with phenol/chloroform, precipitated with ethanol and separated on an 8% polyacrylamide/8 M urea denaturing gel. The amount of each protected fragment was quantified by autoradiography and scanning densitometry.
Statistical analyses
Data are expressed as mean 
SEM. Differences between groups of data obtained at the same time point were compared by analysis of variance (SPSS software). For serial measurements of each of the serum IGFBPs, analysis of variance for repetitive measurements was used to test for significant changes within each individual group over time. If global statistical significance was observed (P < 0.05), the presence of statistically significant differences between any two groups, or between any two time points within the same group was determined by the Scheffe test. Serum IGFBPs were measured in five or six rats from each of the four treatment groups. Unless otherwise stated, the IGFBP mRNAs and IGF-IR mRNAs were measured in four rats from each group.
RESULTS
The mean serum creatinine concentrations in the four groups of rats are shown in Figure 1. The serum creatinine followed a pattern similar to that previously described8,10,11,12. After surgery, serum creatinine rose rapidly in the two ARF groups. Serum creatinine concentrations were similar in the two ARF groups at five hours, which was immediately before the first rhIGF-I injection. The mean serum creatinine levels in the ARF+V rats rose progressively to a maximum of 6.57 0.59 mg/dl at 72 to 74 hours, when the rats were killed. In the ARF+IGF rats, the mean values for serum creatinine tended to plateau about 24 hours after surgery, and their maximum mean serum creatinine value was 3.51 0.48 mg/dl at 48 hours after surgery. At 24, 48 and 72 to 74 hours after surgery, serum creatinine was significantly greater in the ARF+V rats than in the ARF+IGF rats. Mean serum creatinine concentrations in the ARF+IGF and ARF+V rats were also significantly greater than in either group of SO rats at each time point after surgery (5, 24, 48 and 72 to 74 hr). Body weights did not differ among the four groups of rats either immediately before surgery or 72 to 74 hours postoperatively.
Figure 1.
Serum creatinine concentrations in rats with ischemic acute renal failure (ARF) and sham operated (SO) control rats immediately before surgery (0 time) and at 5, 24, 48 and 72 to 74 hours after surgery. The ARF and SO rats were each randomized to receive subcutaneous injections of either recombinant human insulin-like growth factor-I (rhIGF-I), 50 g/100 g, or vehicle (V; 0.9% saline) three times daily starting five hours after surgery, immediately after the blood drawing. N = 5 to 6 rats per group. Triangles and squares represent mean values; brackets indicate SEM.aP < 0.05 versus SO+IGF rats,bP < 0.05 versus SO+V rats andcP < 0.05 versus each of the three other groups at the same time points. Symbols are: (
) ARG+IGF; (
) ARF+V; (
) SO+IGF; (
) SO+V.
The Western ligand blots for serum IGFBPs 1 to 6, the immunoblots for IGFBP-2, and the mean serum levels for IGFBPs 1 to 4, all obtained serially throughout the 72 to 74 hours of study, are shown in Figures 2 and 3. There was a 31 kD IGFBP that reacted with the IGFBP-1 antibody in the immunoblot and was therefore identified as IGFBP-1. Serum IGFBP-1 concentrations fell significantly between baseline (that is, immediately before surgery) and 72 to 74 hours in both the ARF and SO rats given vehicle. Serum IGFBP-1 was also significantly lower at 72 to 74 hours in these two groups of rats as compared to the ARF+IGF and SO+IGF animals Figures 2a and 3a. Serum IGFBP-2 rose above baseline values in the ARF and SO rats given rhIGF-I (P < 0.05) and was significantly greater at 48 and 72 to 74 hours in the ARF+IGF rats than in each of the other three groups Figures 2a and 3b. At 72 to 74 hours, serum IGFBP-2 was also greater in the SO+IGF rats than in the SO+V rats. The serum IGFBP-2 levels in the six rats from each group, when measured by Western immunoblots, were similar to the values obtained by Western ligand blotting. Therefore, a representative autoradiogram from only one rat from each group is shown Figure 2b.
Figure 2.
Western ligand blot (WLB) and Western immunoblot (WIB) of serum insulin-like growth factor (IGF) binding proteins (IGFBPs) over time for a representative rat from each of the four treatment groups. Sera were obtained from the four rats immediately before surgery (0 hr) and 5, 24, 48 and 72 to 74 hours after surgery, and processed as described in the methods section. WLB was used to assess the IGFBPs 1 to 6 (A). After WLB, the same nitrocellulose membrane was washed and further processed for assay of IGFBP-2 by WIB analysis (B). The bars on the right indicate the location of the various IGFBPs, and the bars on the left indicate the molecular size of the standard protein markers in kD. Six separate experiments were performed with different rats from the four groups, and similar results were obtained.
Full figure and legend (238K)Figure 3.
The time course of serum IGFBPs in the four groups of rats. Sera were drawn at the indicated times before and after surgery. Serum IGFBPs were analyzed by Western ligand blots and scanning densitometry. In order to standardize the densitometry readings, the readings of the IGFBPs in the sera obtained immediately before surgery (0 time) for each rat were arbitrarily defined as 1. The densitometry readings of the sera obtained at 5, 24, 48 and 72 to 74 hours after surgery from the same rats were expressed as a fraction of the 0 time levels. N = 5 to 6 rats per group. Data are expressed as mean SEM.aP < 0.05 versus ARF+IGF group,cP < 0.05 versus SO+IGF,dP < 0.05 versus SO+V andeP < 0.05 versus each of the three other groups at the same time points.fP < 0.05 versus values from the same group at zero time. Symbols are: () ARF+IGF; () ARF+V; () SO+IGF; () SO+V.
Serum IGFBP-3 decreased during the course of study in all groups except for the ARF+V rats Figures 2a and 3c. The fall in IGFBP-3 was more pronounced in the SO+IGF and the SO+V rats than in the ARF+IGF rats (P < 0.05). At both 48 and 72 to 74 hours after surgery, serum IGFBP-3 concentrations were greater in the ARF+V rats than in any other of the three groups, and were higher in the ARF+IGF rats than in either the SO+IGF or the SO+V animals. Serum IGFBP-4 (24 kD) rose significantly but transiently over baseline values, at five hours, in both the ARF+IGF and ARF+V groups Figures 2a and 3d. At this time, serum IGFBP-4 concentrations in each of these groups were significantly greater than in the SO+IGF or the SO+V rats. A faint band was visualized on Western ligand blotting at about 29 kD Figure 2a. The time course for the density of this band in each of the four groups of rats was similar to that for serum IGFBP-4. Because of the similar time course to IGFBP-4 and the greater molecular weight of this ligand protein, this band may represent a highly glycosylated form of IGFBP-4. No band that corresponded to IGFBP-5 or IGFBP-6 was observed by Western ligand blotting Figure 2a.
The mRNAs for IGFBPs-1 to -6 in renal cortex and medulla, obtained 72 to 74 hours after surgery, are shown in Figures 4 to 6. In renal cortex, there was a trend for the mean values for the mRNAs for IGFBPs-2 to -6 to be lower in the ARF+IGF and ARF+V rats than in the two SO groups Figure 6a. There was a significantly lower gene expression in renal cortex for IGFBP-3, IGFBP-4, and IGFBP-6 in both groups of ARF rats as compared to either the SO+IGF or the SO+V rats Figures 4 and 6a. In the ARF+IGF rats, IGFBP-5 mRNA was also lower than in either the SO+IGF or the SO+V rats. In the ARF+V rats, there was significantly less expression in kidney cortex for IGFBP-2 mRNA, as compared to the SO+V rats Figures 4 and 6a. There was no difference between the ARF+IGF versus the ARF+V groups with regard to the gene expression for the IGFBPs in renal cortex. The mRNAs for IGFBPs-4 and -6 were significantly lower in the SO+IGF rats as compared to the SO+V animals.
Figure 4.
Representative autoradiograms of hybridization/RNase protection assays of mRNAs for IGFBPs-1 to -6, and IGF-I receptor (IGF-IR) in kidney cortex obtained from the four groups of rats. Fifteen micrograms of total RNA were cohybridized with different cRNA probes and processed as described in the Methods section. The bars on the right indicate the location of different mRNAs for IGFBPs, IGF-IR and -actin. The bars on the left show the size of the mRNAs in terms of base pairs. Four rats were studied from each group.
Figure 6.
Relative mRNA levels for IGFBPs 1 to 6 from kidney cortex (A) and medulla (B). mRNA levels were detected by hybridization/RNase protection assays and adjusted for the -actin signal. The autoradiograms presented in Figure 4 and 5 were analyzed by scanning densitometry. The average of the SO+V values for each mRNA was then assigned an arbitrary unit of one, and the values from the other three groups were expressed relative to this unit. Results are from 4 rats per group. Data are expressed as mean SEM.aP < 0.05 versus SO+V,bP < 0.05 versus SO+IGF,cP < 0.05 versus each of the other three groups.
In the renal medulla, the gene expression for IGFBPs-4 and -6 were significantly lower in the ARF+IGF and ARF+V rats as compared to the SO+V animals Figures 5 and 6b. ARF+V rats displayed a lower IGFBP-6 mRNA than did the SO+IGF animals. Also, ARF+IGF and SO+IGF rats each demonstrated a lower IGFBP-2 mRNA than did the SO+V rats Figures 5 and 6b.
Figure 5.
Autoradiograms of mRNAs for IGFBPs-1 to -6 and IGF-IR from kidney medulla detected by hybridization/RNase protection assays. The methods and symbols are as described in Figure 4.
Full figure and legend (91K)There were no differences among the four groups of rats in the mRNA for IGFBPs-1, -3 to -6 in the liver, obtained 72 to 74 hours after surgery Figure 7. On the other hand, liver IGFBP-2 mRNA was significantly greater in both the ARF+IGF rats and the SO+IGF animals than in either the ARF or the SO rats that received the vehicle Figures 7 and 8. IGFBP-2 mRNA values were approximately twice as great in the two groups of rhIGF-I treated rats as compared to the two groups of vehicle injected animals. Thus, the increased liver IGFBP-2 mRNA levels parallel the elevated serum IGFBP-2 concentrations in the ARF and SO rats given rhIGF-I.
Figure 7.
Representative autoradiogram of mRNAs for IGFBPs-1 to -6 from liver detected by the hybridization/RNase protection assay. The methods and symbols are as described in Figure 4.
Full figure and legend (82K)Figure 8.
Relative mRNA levels for IGFBP-2 from liver. mRNA levels were detected by hybridization/RNase protection assays and adjusted for the 
-actin signal. The autoradiograms presented in Figure 7 were analyzed by scanning densitometry. The average of the SO+V values for IGFBP-2 mRNA was then assigned an arbitrary unit of one, and the values from the other three groups were expressed relative to this unit. Results are from four rats per group. Data are expressed as mean SEM.aP < 0.05 versus ARF+V,bP < 0.05 versus SO+V.
At 72 to 74 hours, the mRNAs for IGFBPs-3 to -6 were not different in heart, lung and quadriceps muscle among the four groups of rats data for lung are shown in Figure. 9. IGFBP-1 and -2 mRNAs were examined (and detected) in heart, lung and quadriceps muscle from only one or two rats per group and therefore could not be evaluated for differences between the four treatment groups.
Figure 9.
Representative autoradiograms of mRNAs for IGFBPs-2 to -6 and IGF-IR from lung determined by hybridization/RNase protection assays. For IGFBPs-3 to -6, four rats were analyzed from each group; for IGFBP-2 and IGF-IR, only two rats from each group were analyzed.
Full figure and legend (40K)The mRNA values for the IGF-IR in both renal cortex and renal medulla, obtained 72 to 74 hours after surgery, are shown in Figures 4 and 5. In both renal cortex and medulla, the IGF-IR mRNA was significantly lower in both the ARF+IGF and the ARF+V rats as compared to each of the two SO groups Figure 10. In both renal cortex and medulla, there was no difference between the two ARF groups or between the two SO groups with regard to IGF-IR mRNA. The reduction in IGF-IR mRNA in the ARF+IGF and ARF+V rats appeared to be more marked in the renal medulla than in the cortex. Although IGF-IR mRNA was examined in the liver in four rats from each of the four treatment groups, no IGF-IR mRNA was detected in any rat. IGF-IR was also detected in heart, lung and quadriceps muscle from each of the one or two rats studied in each of the four treatment groups.
Figure 10.
Relative IGF-IR mRNA levels from kidney cortex (A) and medulla (B). The autoradiograms presented in Figures 4 and 5 were analyzed by scanning densitometry and adjusted for the -actin signal. The average of the mRNA values in each SO+V rat was then assigned an arbitrary unit of one, and the values from the other three groups were expressed relative to this unit. Results are from four rats per group. Data are expressed as mean SEM.aP < 0.05 versus SO+IGF,bP < 0.05 versus SO+V.
DISCUSSION
IGF-I is a growth factor that stimulates replication and differentiation of renal tubular cells5. In rats with ARF caused by ischemia or certain toxins, the renal concentrations of the IGF-I peptide and IGF-I mRNA are reduced12,13,14. However, immunohistochemical and in situ hybridization studies indicate that the IGF-I peptide and IGF-I mRNA are transiently expressed in regenerating tubular cells that normally may not express this peptide7. Moreover, in rats with ARF caused by ischemia or certain toxins, rhIGF-I administration accelerates recovery of renal function8,9,10,11,12. Taken together, these observations suggest that IGF-I may play a role in the recovery of ARF caused by ischemia or nephrotoxicity.
It has been suggested that the increase in the abundance and binding affinity of the IGF-IR in the injured regenerating kidney may influence the actions of IGF-I7. Despite much research, the role of the IGFBPs in the regulation of IGF-I activity is not well defined. It has been proposed that, under certain circumstances, these binding proteins may reduce the availability of IGF-I to the target cell, increase the half-life of serum IGF-I, serve as a reservoir for this peptide, and facilitate the delivery of IGF-I to the target cells3. In some conditions, IGFBPs may potentiate the actions of IGF-I20,21. Furthermore, some IGFBPs may have direct cellular effects that are independent of IGF-I22,23. These observations suggest that it is important to elucidate the gene expression of the six IGFBPs and of the IGF-IR in the acutely ischemic kidney in order to assess the potential actions of these proteins as well as the role of IGF-I.
ARF engenders a catabolic response in rats that can be ameliorated by the administration of rhIGF-I8. Hence, it was also considered important to examine the gene expression for IGFBPs and IGF-IR in other tissues of rats with ischemic ARF. Finally, since the exogenous administration of rhIGF-I has beneficial effects on the course of ARF in the rat, we believed that it is important to examine the effects of rhIGF-I on the gene expression of these potentially regulatory proteins. We have previously shown that in ARF and SO rats treated by the current protocol, serum IGF-I levels do not change when the animals are injected with vehicle and rise to about twice baseline values when they are given rhIGF-I8.
The results of the present study indicate that ARF and treatment with rhIGF-I each affected the concentrations of certain IGFBPs in serum and IGFBP mRNAs or IGF-IR mRNA in kidney or liver, and that for certain proteins there was an interaction between ARF and rhIGF-I. At 72 to 74 hours after surgery, treatment with rhIGF-I had a greater influence on serum IGFBP-1 and -2 levels than did the presence of ARF. Serum levels of these binding proteins were higher in the ARF and SO rats given rhIGF-I than in the two groups given vehicle Figure 3 a and b. Moreover, serum IGFBP-2 levels at 48 and 72 to 74 hours were significantly greater in the ARF rats given rhIGF-I than in the SO animals receiving rhIGF-I. IGFBP-3 values were greater in the two groups of ARF rats and were higher in the ARF+V animals. Serum IGFBP-3 concentrations were also affected by both ARF and rhIGF-I. Serum IGFBP-4 levels rose transiently only in the two groups of rats with ARF, independently of whether the rats received rhIGF-I or vehicle.
In renal cortex 72 to 74 hours after surgery, most of the IGFBP mRNAs were reduced in the ARF groups as compared with both groups of SO animals Figures 4 and 6a. In renal medulla as compared to the SO+V animals, both groups of ARF rats displayed decreased mRNAs for IGFBPs-4 and -6 and the ARF+IGF and SO+IGF rats showed reduced mRNA for IGFBP-2 Figures 5 and 6b. ARF was associated with lower levels of IGF-IR mRNA in both renal cortex and medulla Figures 4, 5 and 10, whereas treatment with rhIGF-I had no effect on gene transcription for this receptor. In the extrarenal tissues studied (liver, lung, heart and quadriceps muscle), neither ARF nor rhIGF-I treatment appeared to affect the mRNA for the six IGFBPs with the exception of IGFBP-2 mRNA in liver. Moreover, IGF-IR mRNA was not detected in liver in any rat from any of the four treatment groups. Since the mRNA for IGFBP-I, IGFBP-2 and for IGF-IR were only measured in heart, lung and quadriceps muscle from one or two rats in each group, the values for these mRNAs cannot be interpreted.
To our knowledge these are the first studies to describe the effects of either experimental ARF or the combination of experimental ARF and rhIGF-I treatment in rats on serum IGFBPs, IGFBP mRNAs in several extrarenal tissues, and IGF-IR mRNA in liver. These are also the first studies of the effects of rhIGF-I on gene expression in the kidney for IGFBPs and IGF-IR in ischemic ARF.
It is intriguing that at 72 to 74 hours after surgery, liver IGFBP-2 mRNA levels were increased in the two groups of rats receiving rhIGF-I and were not affected by ARF Figures 7 and 8. The increased IGFBP-2 gene expression in the rhIGF-I treated rats may account for the rise in serum IGFBP-2 in these two groups of rats Figures 2 and 3. It is pertinent that injection of healthy adults with rhIGF-I increases circulating IGFBP-2 levels24. The mechanism for the increased hepatic IGFBP-2 mRNA may involve the effects of rhIGF-I on growth hormone, and insulin. These two hormones decrease the hepatic gene expression and serum concentrations of IGFBP-225. RhIGF-I suppresses serum concentrations of these hormones26,27,28. Thus, a decrease in serum growth hormone and insulin induced by rhIGF-I might lead to desuppression of hepatic IGFBP-2 mRNA.
The lack of decrease in serum IGFBP-1 concentrations in the ARF and SO rats given rhIGF-I, in contrast to those treated with vehicle, may also be explained by the effects of this hormone Figures 2a and 3a. In normal humans who are calorie-restricted, rhIGF-I increases serum IGFBP-129, whereas growth hormone suppresses serum IGFBP-1 concentrations29. rhIGF-I also suppresses growth hormone secretion28, which may contribute to the elevation in serum IGFBP-1.
Serum IGFBP-3 concentrations decrease with dietary protein deprivation in rats30 and calorie restriction in humans31. This may explain the reduction in serum IGFBP-3 in the two groups of SO rats that were fasted except for dextrose monohydrate added to the drinking water. It is not clear why serum IGFBP-3 did not decrease in the two groups of ARF rats that were similarly fasted. IGFBP-3 is glycosylated and has a molecular weight of about 46 to 53 kD. It binds in plasma with IGF-I and a glycoprotein referred to as the acid labile subunit to form a complex with a molecular weight of about 150 kD32. Both IGFBP-3 and the IGFBP-3 150 kD complex are too large to be excreted or catabolized significantly by the normal kidney. Thus, the preservation of the serum levels in the ARF rats, as compared to the SO animals, would not be expected to be due to a reduction in renal degradation or urinary excretion of IGFBP-3. Serum growth hormone is increased in ARF33,34, and growth hormone stimulates the secretion of IGFBP-330. Thus, elevated growth hormone levels might account for the maintenance of serum IGFBP-3 in the ARF rats. The ability of rhIGF-I to suppress growth hormone secretion28 may also explain why serum IGFBP-3 was higher in the ARF+V rats as compared to the ARF+IGF animals Figures 2 and 3c.
Since serum IGFBPs are considered to be derived, at least to a substantial degree, from the liver, it is puzzling that the mRNAs in liver for IGFBP-1 and -3 were not different in the four groups of rats at 72 to 74 hours after surgery although the serum IGFBP-1 and -3 concentrations did differ. This discrepancy might be explained by the fact that the IGFBP mRNAs were only measured at one point in time and that the hepatic IGFBP mRNA levels may not correlate precisely with the synthesis of these proteins. Moreover, serum IGFBPs concentrations also could be influenced by degradation and compartmental distribution of these compounds.
The results of this study are similar to other investigations in rats with ischemic, mercuric chloride or folic acid induced ARF or ischemic acute renal injury12,13,14,35. Most reports of renal IGFBP mRNAs -2, -3, and -5 showed decreased levels, particularly on days 2 and 3 after the onset of ARF13,14,35, although in one report, IGFBP-2 was increased on day 114. In various studies, renal IGFBP-1 mRNA was unchanged or increased in ARF12,13,14,35. We also found renal cortical and medullary IGFBP-I mRNA levels in the ARF rats to be not different from the SO animals. In contrast to our findings, Friedlander and coworkers found IGFBP-1 mRNA to be elevated to a similar degree in ARF rats given rhIGF-I as compared to ARF rats not receiving this hormone12. IGFBP-4 mRNA was reduced or not different from SO values on days 1 to 3 of ARF14,35. By day 7, the mRNA for most of these IGFBPs was not different from the SO values or was increased. Renal IGFBP-6 mRNA also was reported to be unchanged for three days after the onset of folic acid-induced renal failure14.
In these latter studies there were no changes in IGF-IR mRNA in whole kidney at different time points after the induction of ARF12,13,14,35. An exception was the study of Hise et al, who observed increased IGF-IR mRNA on day 1 after the onset of ARF and no change in the IGF-IR gene expression on days 2 and 314. In contrast, we observed a decrease in IGF-IR mRNA in both renal cortex and medulla. As with the study of Friedlander et al12, we observed that rhIGF-1 treatment for three days had no effect on gene expression for this receptor. In a previous unrelated study of ARF rats from our laboratory, Ding et al also found that renal cortical IGF-IR mRNA was decreased in comparison to preoperative levels or to SO rats studied at the same time point, on days 1, 3 and 5, and was increased on day 7 after the onset of the ischemic ARF36. Rather similar findings were observed for renal medullary IGF-IR in these ARF rats36. Interestingly, Tsao and coworkers13 and Fervenza, Tsao and Rabkin35 observed an increase in the IGF-IR number in the injured kidney, suggesting a post-translational up-regulation of the receptor protein.
The causes for the discrepant results are unclear. The times of measurements of renal IGF-IR mRNA or IGFBP mRNAs in these other studies were at one, two, three or seven days after the onset of ARF12,13,14,35, whereas in the present study the renal mRNAs were measured only at 72 to 74 hours after surgery. The methods of measurements of the IGFBP mRNAs and IGF-IR mRNA were similar in most of these studies. However, IGFBP mRNAs were measured separately in renal cortex and medulla only in our study, and IGF-IR mRNA was measured separately in the renal cortex and medulla only in the present study and also by Ding et al36 from our laboratory. In the other studies, the IGFBP mRNAs and IGF-IR were measured in the whole kidney12,13,14,35. In two of these studies, ARF was induced by a nephrotoxin12,14, whereas in the present study, ARF was induced by ischemia.
The variation in nutrient intakes after creation of ARF also might contribute to some of the differences in the results from these studies. In rats that do not have ARF, fasting or protein deprivation engenders serum concentrations and hepatic gene expressions that are increased for IGFBP-1 and reduced for IGFBP-330,37. In the normal kidney, fasting apparently does not affect mRNA for IGFBPs-2 to -538, whereas low protein diets decrease IGFBP-2 mRNA37. The effects of different nutrient intakes on the gene expression and concentrations of IGFBPs or IGF-IR in ARF are unknown. In many of the previous studies, rats with ARF were allowed to eat ad libitum7,10,11,12,13,14. In the present study, all rats were fasted for the 72 to 74 hours after surgery, except for 5% dextrose monohydrate in drinking water. We adopted this study design in order to avoid the possibility that the fasting ARF or SO rats given rhIGF-I might develop hypoglycemia and, at the same time, to give all rats in the study approximately the same nutrient intake. Rats were not given access to a complete diet because we have previously observed that rats with severe ARF caused by a surgical procedure eat very little, if at all, and have poor gastric emptying. Thus, to allow the rats to eat could have led to marked variations from rat to rat in nutrient intake and absorption. This might have made it difficult, if not impossible, to perform accurate comparisons between groups in our study or between our study and other studies.
It is also of potential importance that the magnitude of acute renal failure varied in these studies12,13,14,35. The maximum serum creatinine concentrations were greatest in the present study, which suggests that the renal injury may have been more severe. Also, in the study of Fervenza et al, only one kidney was made ischemic, and the rats did not develop uremia35. We elected to create a rather severe model of ARF, in order to more closely resemble the patient with severe ARF, because these are the individuals who tend to have the highest morbidity and mortality and for whom the clinician may have the greatest interest in understanding the pathophysiology of the ARF and in developing methods of treatment.
The reduction in the mRNA for several IGFBPs and for IGF-IR in renal cortex and medulla suggests that the concentrations of these binding proteins and of IGF-IR may also be reduced in these tissues. Since binding proteins and IGF-IR may have regulatory effects on the actions of IGF-I and IGF-II20,39,40, changes in binding protein and IGF-IR concentrations might affect the ability of the kidney to recover from ARF. A reduced tissue IGF binding capacity might increase the effects of these growth factors by making them more available to tissues39. Alternatively, decreased binding capacity might reduce the actions of these growth factors by trapping lesser amounts of the growth factors in kidney tissue of by reducing the facilitative effects of IGFBPs on IGF-I that have been described under some circumstances20,40. Similarly, a decrease in IGF-IR may reduce the actions of IGF-I or IGF-II in the acutely injured kidney. The results of the present study also indicate that administration of IGF-I may alter the gene expression and possibly the concentrations of certain binding proteins. Further research, including actual measurements of IGF binding proteins and the IGF-IR in kidney tissue, may be necessary to resolve these questions.
In summary, the present study indicates that ARF and rhIGF-I both independently and conjointly affect serum IGFBP concentrations. ARF affects the gene expression in kidney for IGFBPs-1 to -6 and IGF-IR, whereas rhIGF-I enhances hepatic IGFBP-2 mRNA and possibly suppresses renal medullary IGFBP-2 mRNA. This study, in conjunction with other published research, suggests the possibility that the effects of ARF or rhIGF-I may be influenced by the type and severity of the renal injury and the nutritional intake of the animal. The clinical effects of these alterations on the serum concentrations of IGFBPs and the gene expression of IGFBPs and IGF-IR remain to be elucidated.
References
| 1. | Guler HP, Zapf J & Froesch ER. Short-term metabolic effects of recombinant human insulin-like growth factor I in healthy adults. N Engl J Med 1987; 317: 137−140. | PubMed | ChemPort | |
| 2. | Feld S & Hirschberg R. Growth hormone, the insulin-like growth factor system and the kidney. Endocr Rev 1996; 17: 423−480. | Article | PubMed | ChemPort | |
| 3. | Jones JI & Clemmons DR. Insulin-like growth factors and their binding proteins: Biological actions. Endocr Rev 1995; 16: 3−34. | Article | PubMed | ISI | ChemPort | |
| 4. | Hirschberg R & Kopple JD. Evidence that insulin-like growth factor I increases renal plasma flow and glomerular filtration rate in fasted rats. J Clin Invest 1989; 83: 326−330. | PubMed | ChemPort | |
| 5. | Hirschberg R. Bioactivity of glomerular ultrafiltrate during heavy proteinuria may contribute to renal tubulo-interstitial lesions. Evidence for a role for insulin-like growth factor I. J Clin Invest 1996; 98: 116−124. | PubMed | ChemPort | |
| 6. | Andersson G & Jennische E. IGF-I immunoreactivity is expressed by regenerating renal tubular cells after ischaemic injury in the rat. Acta Physiol Scand 1988; 132: 453−457. | PubMed | ChemPort | |
| 7. | Matejka GL & Jennische E. IGF-I binding and IGF-I mRNA expression in the post-ischemic regenerating rat kidney. Kidney Int 1992; 42: 1113−1123. | PubMed | ChemPort | |
| 8. | Ding H, Kopple JD, Cohen A & Hirschberg R. Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure. J Clin Invest 1993; 91: 2281−2287. | PubMed | ChemPort | |
| 9. | Clark R, Mortensen D & Rabkin R. Recovery from acute ischemic renal failure is accelerated by des-(1−3)-insulin-like growth factor-1. Clin Sci 1994; 86: 709−714. | PubMed | ChemPort | |
| 10. | Miller SB, Martin DR, Kissane J & Hammerman MR. Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. Proc Natl Acad Sci USA 1992; 89: 11876−11880. | PubMed | ChemPort | |
| 11. | Miller SB, Martin DR, Kissane J & Hammerman MR. Rat models for clinical use of insulin-like growth factor I in acute renal failure. Am J Physiol 1994; 266: F949−F956. | PubMed | ChemPort | |
| 12. | Friedlander M, Popovtzer MM, Weiss O, Nefesh I, Kopolovic J & Raz I. Insulin-like growth factor-1 (IGF-1) enhances recovery from HgCl2-induced acute renal failure: The effects on renal IGF-1, IGF-1 receptor, and IGF-binding protein-1 mRNA. J Am Soc Nephrol 1995; 5: 1782−1791. | PubMed | |
| 13. | Tsao T, Wang J, Fervenza FC, Vu TH, Jin IH, Hoffman AR & Rabkin R. Renal growth hormone—Insulin-like growth factor-I system in acute renal failure. Kidney Int 1995; 47: 1658−1668. | PubMed | ChemPort | |
| 14. | Hise MK, Li L, Mantzouris N & Rohan RM. Differential mRNA expression of insulin-like growth factor system during renal injury and hypertrophy. Am J Physiol 1995; 269: F817−F824. | PubMed | ChemPort | |
| 15. | Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S & Binoux M. Analysis of serum insulin-like growth factor binding proteins using western blotting: Use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 1986; 154: 138−143. | Article | PubMed | ISI | ChemPort | |
| 16. | Hirschberg R & Kaysen GA. Insulin-like growth factor I and its binding proteins in the experimental nephrotic syndrome. Endocrinology 1995; 136: 1565−1571. | Article | PubMed | ChemPort | |
| 17. | Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680−685. | Article | PubMed | ChemPort | |
| 18. | Lowe WL, JR, Roberts CT, JR, Lasky SR & Leroith D. Differential expression of alternative 5' untranslated regions in mRNAs encoding rat insulin-like growth factor I. Proc Natl Acad Sci USA 1987; 84: 8946−8950. | PubMed | ChemPort | |
| 19. | Ding H, Gao XL, Hirschberg R, Vadgama JV & Kopple JD. Impaired actions of insulin-like growth factor 1 on protein synthesis and degradation in skeletal muscle of rats with chronic renal failure. Evidence for a postreceptor defect. J Clin Invest 1996; 97: 1064−1075. | PubMed | ChemPort | |
| 20. | Elgin RG, Busby WH, JR & Clemmons DR. An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I. Proc Natl Acad Sci USA 1987; 84: 3254−3258. | PubMed | ChemPort | |
| 21. | Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB & Bierich JR. Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 1989; 125: 766−772. | PubMed | ChemPort | |
| 22. | Jones JI, Gockerman A, Busby WH, JR, Wright G & Clemmons DR. Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 1993; 90: 10553−10557. | PubMed | ChemPort | |
| 23. | Oh Y, Muller HL, Pham H & Rosenfeld RG. Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs5787 human breast cancer cells. J Biol Chem 1993; 268: 26045−26048. | PubMed | ISI | ChemPort | |
| 24. | Zapf J, Kiefer M, Merryweather J, Masiarz F, Bauer D, Born W, Fischer JA & Froesch ER. Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF-I administration and in extrapancreatic tumor hypoglycemia. J Biol Chem 1990; 265: 14892−14898. | PubMed | ChemPort | |
| 25. | Boni-Schnetzler M, Schmid C, Mary JL, Zimmerli B, Meier PJ, Zapf J, Schwander J & Froesch ER. Insulin regulates the expression of the insulin-like growth factor binding protein 2 mRNA in rat hepatocytes. Mol Endocrinol 1990; 4: 1320−1326. | PubMed | ISI | ChemPort | |
| 26. | Jacob R, Barrett E, Plewe G, Fagin KD & Sherwin RS. Acute effects of insulin-like growth factor I on glucose and amino acid metabolism in the awake fasted rat. Comparison with insulin. J Clin Invest 1989; 83: 1717−1723. | PubMed | ChemPort | |
| 27. | Boulware SD, Tamborlane WV, Matthews LS & Sherwin RS. Diverse effects of insulin-like growth factor I on glucose, lipid and amino acid metabolism. Am J Physiol 1992; 262: E130−E133. | PubMed | ChemPort | |
| 28. | Guler HP, Schmid C, Zapf J & Froesch ER. Effects of recombinant insulin-like growth factor I on insulin secretion and renal function in normal human subjects. Proc Natl Acad Sci USA 1989; 86: 2868−2872. | PubMed | ChemPort | |
| 29. | Young SC, Underwood LE, Celniker A & Clemmons DR. Effects of recombinant insulin-like growth factor I (IGF-I) and growth hormone on serum IGF binding proteins in calorically restricted adults. J Clin Endocrinol Metab 1992; 75: 603−608. | Article | PubMed | ChemPort | |
| 30. | Clemmons DR, Thissen JP, Maes M, Ketelslegers JM & Underwood LE. Insulin-like growth factor-I (IGF-I) infusion into hypophysectomized or protein-deprived rats induces specific IGF-binding proteins in serum. Endocrinology 1989; 125: 2967−2972. | PubMed | ChemPort | |
| 31. | Kupfer SR, Underwood LE, Baxter RC & Clemmons DR. Enhancement of the metabolic effects of growth hormone and insulin-like growth factor I by use of both agents simultaneously. J Clin Invest 1993; 91: 391−396. | PubMed | ChemPort | |
| 32. | Baxter RC & Martin JL. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity-labeling. Proc Natl Acad Sci USA 1989; 86: 6898−6902. | PubMed | ChemPort | |
| 33. | Kokot V. The endocrine system in patients with acute renal failure. Proc Eur Dial Transplant 1981; 18: 617−629. |
| 34. | Massry SG & Smogorzenski M. Metabolic and endocrine abnormalities in acute renal failure,. inTextbook of Nephrology 1995; vol 2 edited by Massry Sg, Smogorzenski M, Baltimore, Williams and Wilkins pp: 1004−1013. |
| 35. | Fervenza FC, Tsao T & Rabkin R. Response of the intrarenal insulin-like growth factor-I axis to acute ischemic injury and treatment with growth hormone and epidermal growth factor. Kidney Int 1996; 49: 344−354. | PubMed | ChemPort | |
| 36. | Ding H, Qing PY, Gao XL, Hirschberg R & Kopple JD. Time dependent changes in the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) in tissues of rats with acute renal failure (ARF). (abstract). J Am Soc Nephrol 1995; 6: 461. |
| 37. | Lemozy S, Pucilowska JB & Underwood LE. Reduction of insulin-like growth factor-I (IGF-I) in protein-restricted rats is associated with differential regulation of IGF-binding protein messenger ribonucleic acids in liver and kidney, and peptides in liver and serum. Endocrinology 1994; 135: 617−623. | Article | PubMed | ChemPort | |
| 38. | Kobayashi S, Nogami H & Ikeda T. Growth hormone and nutrition interact to regulate expressions of kidney IGF-I and IGFBP mRNAs. Kidney Int 1995; 48: 65−71. | PubMed | ChemPort | |
| 39. | Feyen JH, Evans DB, Binkert C, Heinrich GF, Geisse S & Kocher HP. Recombinant human [Cys281] insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae. J Biol Chem 1991; 266: 19469−19474. | PubMed | ChemPort | |
| 40. | Jones JI, Gockerman A, Busby WH, JR, Camacho-Hubner C & Clemmons DR. Extracellular matrix contains insulin-like growth factor binding protein-5: Potentiation of the effects of IGF-I. J Cell Biol 1993; 121: 679−687. | Article | PubMed | ISI | ChemPort | |
