J Am Soc Nephrol 10: 84–92, 1999 Elevated Myocardial Cytosolic Calcium Impairs Insulin-Like Growth Factor-1-Stimulated Protein Synthesis in Chronic Renal Failure DAVID PEIYUAN QING,* HU DING,* JAY VADGAMA,† YAN YUAN WU,† and JOEL D. KOPPLE* *Division of Nephrology and Hypertension and Department of Medicine, Harbor-UCLA Medical Center, Torrance, California, and UCLA Schools of Medicine and Public Health, and †Charles R. Drew University of Medicine and Science, Department of Medicine, Los Angeles, California. , 21 Abstract. Rats and humans with chronic renal failure (CRF) are tively; P 0.01). rhIGF-1 increased cardiomyocyte [Ca ]i in 21 reported to have resistance to recombinant human insulin-like all four groups of rats. The rise in [Ca ]i was significantly growth factor-1 (rhIGF-1). Because basal cytosolic calcium diminished in the CRF rats (P , 0.05) and did not differ 21 ([Ca ]i), a second messenger, may be increased in CRF, this among the CRF-PTX, CRF-F, and SO rats. Protein synthesis study was conducted to examine whether elevated basal after incubation with 0, 50, 100, 200, or 400 ng/ml rhIGF-1 21 [Ca ]i may cause resistance to IGF-1. Cardiomyocytes from was lower in cardiomyocytes from CRF rats than in each of the four groups of rats were studied: untreated CRF, CRF with other three groups (P , 0.05) and was significantly less in the parathyroidectomy (PTX), CRF with the calcium channel CRF-F rats compared with SO animals. IGF-1 receptor mRNA blocker felodipine (F), and sham operation of the kidney (SO). and IGF-1 receptor number and affinity were not different CRF was created by ligation of two-thirds of the left renal among the four groups. These findings suggest that cardio- 21 artery and contralateral nephrectomy. Rats from each group myocytes from CRF rats display elevated basal [Ca ]i and 21 21 were pair-fed the same diet for 20 to 22 d. Basal [Ca ]i in attenuated rhIGF-1-induced increase in [Ca ]i; basal protein cardiomyocytes (nM) in the CRF rats (102.0 6 2.8; SEM), was synthesis is decreased, and IGF-1-stimulated protein synthesis 21 significantly higher than in each of the CRF-PTX, CRF-F, and is impaired; elevated basal [Ca ]i seems to contribute to this SO groups (65.2 6 1.9, 63.8 6 2.6, and 63.5 6 2.0, respec- diminished response to rhIGF-1. Recent evidence has demonstrated that there is resistance to For calcium to act as such, the ratio of the calcium signal (i.e., 21 insulin-like growth factor-1 (IGF-1) in experimental animals the acute increase in [Ca ]i induced by an agonist) and the 21 and humans with chronic renal failure (CRF)(1–3). There is an background calcium (basal levels of [Ca ]i) must be suffi- 21 attenuation in the IGF-1-induced stimulation of protein syn- ciently great (8). Elevated basal levels of [Ca ]i decrease the thesis and inhibition of protein degradation in skeletal muscle signal-to-background ratio, and this is believed to attenuate the and also an impairment in the IGF-1-induced suppression of biologic response to the agonist. Indeed, several lines of evi- plasma amino acids, insulin, and C-peptide. Among the known dence provide support for the thesis that an elevation in basal 21 21 mechanisms for IGF-1 resistance in CRF are defects in the levels of [Ca ]i causes cell dysfunction in CRF. First, [Ca ]i phosphorylation and activity of the intrinsic tyrosine kinase of is elevated in many cell types in CRF, and these cells have the IGF-1 receptor (2) and plasma inhibitors of IGF-1 (4,5). altered function (6). Second, if basal [Ca21] is maintained at 21 i Basal cytosolic calcium ([Ca ]i) is elevated in a number of normal levels in CRF rats, many of their cell functions remain cell types in experimental rats or humans with CRF (6). Cy- normal (9–13). Third, animals with phosphate depletion and tosolic calcium serves as a second messenger in the signal 21 normal renal function have elevated basal levels of [Ca ]i transduction system for many biologic functions of the cell (7). (14–16). These animals display a number of metabolic and functional derangements that are similar to those observed in CRF, despite the absence of CRF (15–17). Received March 31, 1998. Accepted July 11, 1998. Because IGF-1 induces a transient increase or oscillation of This work was presented at the 29th Annual Meeting of the American Society 21 of Nephrology, New Orleans, LA, November 1996, and has been published in [Ca ]i in certain cells and because this IGF-1-induced change 21 abstract form (J Am Soc Nephrol 7: 1862, 1996). in [Ca ]i has been considered to mediate some of the actions Correspondence to Dr. Joel D. Kopple, Professor of Medicine and Public of IGF-1 (18–27), we hypothesized that elevation of basal Health, Harbor-UCLA Medical Center, 1000 West Carson Street, Box 406, [Ca21] in CRF may contribute to IGF-1 resistance. To test this Torrance, CA 90509. Phone: 310-222-3891; Fax: 310-782-1837; E-mail: i [email protected] hypothesis, we examined whether in cardiomyocytes of CRF 21 rats: 1. basal [Ca ]i is indeed elevated, and the increment in 1046-6673/1001-0084$03.00/0 21 Journal of the American Society of Nephrology [Ca ]i in response to IGF-1 is attenuated; 2. protein synthesis, Copyright © 1999 by the American Society of Nephrology with or without IGF-1 stimulation, is impaired; and 3. normal- 21 J Am Soc Nephrol 10: 84–92, 1999 [Ca ]i, IGF-1, and Protein Synthesis 85 21 ization of basal and IGF-1-stimulated [Ca ]i, by either para- single-cell cardiomyocyte suspensions (see below). Only the CRF rats thyroidectomy or treatment with a calcium channel blocker, that had a serum creatinine concentration of 1.10 mg/dl or greater and can increase the reduced level of protein synthesis toward had survived the 20 to 22 d of pair feeding were included in the normal, both without IGF-1 stimulation and in response to studies. Tail-cuff BP was measured by the IITC Mark 12 photoelec- treatment with IGF-1. tric/oscillometric tail-cuff system (IITC Life Sciences, Woodland Hills, CA) according to the manufacturer’s instructions. Serum cre- atinine was measured by using the Sigma kit (Sigma-Aldrich Corp.). Materials and Methods Serum calcium and phosphorus were measured with an AutoAnalyzer Reagents (Technicon Instrument Corp., Tarrytown, NY). Felodipine was kindly provided by Dr. Mark Page (Astra Ha¨ssle 14 AB, Mo¨lndal, Sweden). [ C]-Leucine (168 Ci/mmol) was purchased Preparation of RNA from DuPont NEN (Boston, MA). 125I-IGF-1 (2000 Ci/mmol) was Total RNA was extracted from the heart by the guanidine thiocy- obtained from Amersham Life Sciences (Arlington Heights, IL). anate isolation method of Chomczynski and Sacchi (29) with minor Reagents for electrophoresis and protein measurements were pur- modifications. The heart was removed and flushed with ice-cold BSS, chased from Bio-Rad Laboratories (Hercules, CA). Recombinant hu- pH 7.20, to wash out the blood. Approximately 150 mg of ventricular man insulin-like growth factor-1 (rhIGF-1) was kindly provided by tissue was then homogenized in 1.5 ml of ice-cold 4 M guanidine Dr. Hans-Peter Guler (Chiron Therapeutics, Emeryville, CA). Anti- thiocyanate buffer, pH 7.0, with a Polytron homogenizer (Brinkman sense RNA probes for detection of IGF-1 receptor mRNA were Instruments, Westbury, NY). One volume of the homogenate was graciously provided by Dr. Dereck LeRoith (National Institute of thoroughly mixed with 0.1 vol of 2 M sodium acetate, pH 4.0, 1 vol Diabetes and Digestive and Kidney Diseases, National Institutes of of phenol, and 0.2 vol of chloroform:isoamyl alcohol (49:1). The Health, Bethesda, MD). All other chemicals were obtained from mixture was centrifuged at 16,000 3 g for 30 min at 4°C, and the clear Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. supernatant was precipitated with 1.0 vol of isopropanol at 270°C for 60 min. After centrifugation at 14,000 3 g for 10 min at 4°C, the Animals resultant pellet was washed in ice-cold 75% ethanol, air-dried, and Male Sprague Dawley rats (Charles River Breeding Laboratories, resuspended in deionized formamide. The quantity of RNA was Wilmington, MA), each weighing 210 to 230 g, were studied. Ani- determined by ultraviolet absorbance at 260 nm; the quality of the mals were fed Purina Laboratory Rodent Diet (Purina Mills, St. Louis, samples was evaluated by visualizing the 18S and 28S ribosomal MO) containing 24% protein and tap water ad libitum for at least 3 d RNA after 3 mg of total RNA was resolved in a 1% agarose/formal- before surgery. Rats were then assigned randomly to one of four dehyde gel. groups: 1. sham-operated (SO), 2. CRF with parathyroidectomy (PTX, CRF-PTX), 3. CRF treated with the calcium channel blocker Riboprobe Preparation felodipine (CRF-F), and 4. CRF without either intervention (CRF). The rats were treated in accordance with National Institutes of Health The antisense cRNA probe used for the identification of IGF-1 Guide for the Care and Use of Laboratory Animals. receptor mRNA has been described previously (2,30). The probe for m CRF was created during a single laparotomy by right nephrectomy the IGF-1 receptor was synthesized and labeled with 50 Ci (10 a 32 and ligation of about two-thirds of the branches of the left renal artery, mCi/ml) [ - P]UTP (ICN Biomedicals, Costa Mesa, CA) and 0.48 as reported previously (2).