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Circulating Growth Hormone Binding Protein Levels and Mononuclear Cell Growth Hormone Receptor Expression in Uremia
Circulating Growth Hormone Binding Protein Levels and Mononuclear Cell Growth Hormone Receptor Expression in Uremia Joshua Greenstein, MD,* Steven Guest, MD,*† Jane C. Tan, MD, PhD,* Padmaja Tummala, MS,* Stefan Busque, MD,‡ and Ralph Rabkin, MD*§ Background: Resistance to growth hormone (GH) in end-stage renal disease (ESRD) causes growth retardation and muscle wasting. In humans, circulating GH binding protein (GHBP), the extracellular domain of the GH receptor that is shed into the circulation and is believed to reflect tissue GH receptor levels, is reduced in uremia and suggests that cellular GH receptor levels are correspondingly reduced. If true, this could be a cause of GH resistance. We set out to establish whether serum GHBP levels reflect cellular GH receptor levels and whether changes in serum GHBP levels are related to nutritional or inflammatory status. Methods: GH receptor protein expression in peripheral blood mononuclear cells (PBMC) from 21 ESRD and 14 normal subjects were analyzed by fluorochrome flow cytometry. Results: The GH receptor density and percent total PBMCs expressing the GH receptor were similar in the 2 groups, and there was no difference in percent GH receptor positive T or B cells or monocytes. In contrast, serum GHBP levels were 80% lower in ESRD. GHBP levels did not correlate with serum albumin, body mass index, or muscle mass but seemed to be partly related to the log serum C-reactive protein levels. Conclusions: Serum GHBP levels are markedly reduced in ESRD; this seems to occur independent of nutritional status and may in part be caused by inflammation. Because GH receptor expression on PBMC of ESRD and control subjects was similar, our findings argue against a reduction in GH receptor as a cause of GH resistance and the use of serum GHBP levels as a reliable marker of specific tissue GH receptor levels. © 2006 by the National Kidney Foundation, Inc.
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DVANCED RENAL FAILURE is often complicated by the appearance of muscle wasting in adults, growth impairment in children, and abnormalities of the immune system.1–3 Many factors contribute to these complications, including anorexia, acidosis, inadequate dialysis, *Department of Medicine, Stanford University, Stanford, CA. †Santa Clara Valley Medical Center, San Jose, CA. ‡Department of Surgery, Stanford University, Stanford, CA. §Veterans Affairs Health Care System, Palo Alto, CA. Supported by a Norman S. Coplon Grant from Satellite Research and a Merit Review Grant from the Research Service of the Department of Veterans Affairs. Presented in part at the Annual Meeting of the American Society of Nephrology 2003 and the Seventh Annual Meeting of the International Pediatric Nephrology Symposium on Growth in Children with Chronic Kidney Disease, Germany 2004. Address reprint requests to Ralph Rabkin, MD, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail: [email protected] © 2006 by the National Kidney Foundation, Inc. 1051-2276/06/1602-0007$32.00/0 doi:10.1053/j.jrn.2006.01.007
Journal of Renal Nutrition, Vol 16, No 2 (April), 2006: pp 141-149
inflammation, undernutrition, abnormal vitamin D and parathyroid hormone physiology, and resistance to anabolic hormones such as growth hormone (GH), insulin-like growth factor 1 (IGF-1), and insulin. To some extent the GHresistant state can be overcome by large doses of recombinant GH, and this approach is effective in promoting growth in uremic children.4 In adults several small studies have shown that the administration of GH has a positive effect on urea kinetics, protein turnover, and lean body mass.2 Because most but not all of the anabolic and growth-promoting actions of GH are mediated through IGF-1, a hormone induced by GH, uremic resistance to GH has largely been attributed to the development of insensitivity to IGF-1 and reduced IGF-1 production.1,5–7 In addition, our recent studies in animals have shown that GH-mediated signal transduction through the Janus kinase 2-signal transducer and activator of transcription (JAK2-STAT) pathway is impaired 141
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in uremia, and this is another important cause of GH resistance.8,9 There are also reports describing decreased GH receptor levels in uremic animals, although this is not a uniform finding. Some investigators have reported that hepatic GH receptor mRNA and growth plate GH receptor protein levels are reduced in rats with chronic renal failure (CRF),10,11 whereas others have suggested that uremia per se does not alter GH receptor protein levels, but that reduced GH receptor levels are a consequence of inadequate food intake.8,12 The effect of uremia on GH receptor levels have not been examined directly in humans; instead, a surrogate marker of tissue GH receptor levels, namely circulating GH binding protein (GHBP) levels, has been measured.13 The intact GH receptor is a transmembrane glycoprotein that binds GH and through its intracellular domain mediates the growth-promoting, immune modulating, and metabolic actions of the hormone.14 –16 In humans the GHBP is generated by enzymatic cleavage of the GH receptor with release of the extracellular domain into the circulation.13,17 After being cleaved from the intact receptor, the GHBP, a 60-kDa single-chain glycoprotein with a high affinity for GH, enters the circulation, where it binds about 45% of the circulating GH and serves as a dynamic reservoir of circulating GH.13,18 Several19 –21 but not all22 studies of subjects with kidney failure have described a reduction in circulating GHBP levels. This has been taken to reflect reduced tissue GH receptor protein expression,19,20 especially as the low serum GHBP levels correlate with sensitivity to GH in uremic children.20 Indeed a positive relationship between GHBP levels and GH sensitivity has also been described in other conditions such as obesity23 and cirrhosis,24 and this provides further support for the assumption that serum GHBP levels reflect tissue GH receptor levels. However this concept has not been rigorously established, and several exceptions have been described.13,25,26 Because GHBP levels have been reported to be reduced in advanced renal failure, we set out to determine whether serum GHBP levels reflect cellular GH receptor levels and whether changes in GHBP levels, rather than being caused by uremia, are a reflection of malnutrition or inflammation, conditions that may also cause a reduc-
tion in GH receptor and GHBP levels.13,26 To this end we studied subjects with end-stage renal disease (ESRD) and normal age-matched control patients and measured their serum GHBP levels and the expression of the GH receptor protein on peripheral blood mononuclear cells (PBMC), a target of the immunoregulatory actions of GH.27 We chose to study PBMCs because of their ready availability and the facts that GH has immune regulatory actions on PBMC and that the immune system is dysregulated in uremia.3
Subjects and Methods Subjects Twenty-one patients with ESRD treated by maintenance hemodialysis for at least 8 weeks and 14 healthy subjects between the ages of 21 and 69 years were studied. Exclusion criteria included diabetes mellitus; pregnancy; vasculitis; autoimmune disease; malignancy; obesity (relative body weight ⬎130%); alcoholism or other recreational drug use; heart, lung, liver, or gastrointestinal dysfunction; sepsis and a Kt/V ⬍ 1.3 during the prior month; medically unstable subjects; and subjects who had received anabolic, catabolic, or cytotoxic medications during the prior 3 months. Blood samples from control patients were collected into ethylenediaminetetraacetic acid– containing tubes by venipuncture and from ESRD subjects from their vascular access predialysis, at least 48 hours after their last dialysis treatment. The study was approved by the Institutional Human Research Committee. All subjects signed informed consent to participate in the study. Cell Preparation Human PBMCs were prepared from anticoagulated whole blood by centrifugation on Histapaque 1079 density gradients (Sigma-Aldrich Corp, St. Louis, MO) after dilution in HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid)-balanced saline solution. The PBMC recovered from the interface were washed twice and resuspended in phosphate-buffered saline containing 2% bovine serum albumin and 0.1% sodium azide. Cells were counted, and viability was determined by trypan blue exclusion. The final PBMC concentration was adjusted to 1 ⫻ 107 cells/mL, and 100 L of each suspension was used for immunofluorescence staining.
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Immunostaining and Fluorescence Activating Cell Sorter (FACS) Analysis The PBMC surface markers were detected by direct labeling with antihuman antibodies: Cychrome conjugated anti-CD3 as a marker of mature T cells, R-phycoerythrin anti-CD19 as a B-cell marker, and allophycocyanin-conjugated anti-CD14 as a marker of monocytes. Fluorescein isothiocyanate (FITC)– conjugated mouse immunoglobulin (Ig) G1 k was used as an isotype control. All of these proteins were purchased from BD Biosciences Parmingen, San Diego, CA. Anti-GH receptor monoclonal antibody 263 was purchased from Biogenesis Inc, Kingston, NH, and custom labeled with FITC by Chromaprobe, Inc, Aptos, CA. PBMC, 1 million, were incubated with the GH receptor antibody at 4°C for 90 minutes, and then the PBMC surface marker antibodies were added and incubation continued for another 30 minutes. After washing, the resuspended cells were analyzed in a FacsCalibur flow cytometer (Beckton-Dickinson, San Jose, CA). Cells were also incubated separately with each antibody to serve for single-color compensation during FACS analysis. Sphero Rainbow calibration particles (Spherotech Inc, Libertyville, IL) were used to calibrate the flow cytometer for each run and for measuring the intensity of the fluorescent signal. Biochemical Assays Routine biochemical assays were performed in the clinical laboratories, including serum albumin by the bromcresol purple method. High-sensitivity C-reactive protein (CRP) levels were determined by Nephelometry (Quest Diagnostcs, San Juan Capistano, CA), serum GHBP, GH, and IGF-1 by enzyme-linked immunosorbent assays (Diagnostic Systems Laboratory, Webster, TX). Nutritional parameters assessed included height, edema-free adjusted body weight, skin fold thickness, mid-arm circumference and arm muscle area, body mass index (BMI), muscle mass, serum albumin, and normalized protein catabolic rate (nPCR). Skin-fold thickness was measured with a Lange skinfold caliper (Beta Technology Incorporated, Santa Cruz, CA) over the biceps, triceps, subscapular, and suprailiac regions, and with the average of these measurements the percent body fat was estimated from published tables.28 Adequacy of dialysis was assessed from the blood urea
nitrogen and serum creatinine levels and from the Kt/V. Formulae for the various anthropometric and kinetic calculations were according to Kidney Disease Outcomes and Quality Initiative guidelines.28
Statistical Analyses Results are expressed as the mean ⫾ SEM. The Student two-tailed unpaired t test was applied for comparing 2 normally distributed groups; comparisons between more than 2 normally distributed groups were made by one-way analysis of variance followed by pairwise multiple comparison with the Holms t test.29 For analyzing 2 nonnormally distributed groups, the MannWhitney rank sum test was used, and for more than 2 nonnormally distributed groups, the Kruskal-Wallis statistic was applied, followed by the Student Newman Keuls test to distinguish between groups.29 A P value ⬍.05 was considered statistically significant.
Results The anthropometric features of the 21 ESRD and 14 normal control subjects are shown in Table 1. The 2 groups were of similar age, and the ESRD subjects had received dialysis treatment for an average of 3.1 ⫾ 0.5 years with current Kt/V values of 1.92 ⫾ 0.09 and nPCR values of 1.02 ⫾ 0.18 g/kg/day. BMI, muscle and fat mass, mid-arm circumference, and arm muscle area were on average lower in the ESRD subjects but not statistically significantly so. The serum albumin levels were reduced significantly in the ESRD compared with the control group Table 1. Subject Characteristics Control
ESRD
Number 14 21 Age, y 44 ⫾ 2.2 47 ⫾ 3.0 Sex, M/F 8/6 14/7 Height, cm 166 ⫾ 2.8 165 ⫾ 2.6 Body weight, kg 72.8 ⫾ 3.87 66.4 ⫾ 3.25 Midarm circumference, cm 29.3 ⫾ 0.87 28.5 ⫾ 0.65 Arm muscle area, cm2 44.8 ⫾ 3.84 42.9 ⫾ 3.33 Muscle mass, kg 27.3 ⫾ 1.7 25.7 ⫾ 2 Fat mass, kg 72.12 ⫾ 1.18 75.43 ⫾ 1.53 Serum albumin, mg/dL 4.24 ⫾ 0.14 *3.66 ⫾ 0.18 Years on HD N/A 3.1 ⫾ 0.52 Kt/V N/A 1.92 ⫾ 0.09 nPCR, g/kg N/A 1.02 ⫾ 0.18 *P ⬍ .05.
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Figure 1. Serum insulin-like growth factor 1 (IGF-1), growth hormone (GH), and growth hormone binding protein (GHBP) levels in end stage renal disease and normal subjects. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.
(3.7 ⫾ 0.18 versus 4.2 ⫾ 0.14 mg/dL). The serum hormone levels measured in these subjects are shown in Figure 1. Total IGF-1 levels were significantly elevated in the ESRD group (147 ⫾ 14 versus 99 ⫾ 14 ng/mL, P ⬍ .01), and this is likely a consequence of the elevation in lowmolecular-weight serum IGF binding proteins that consistently occurs in uremia.5 Serum GH levels were also elevated in ESRD (1.5 ⫾ 0.59 versus 0.8 ⫾ 0.03 ng/mL, P ⬍ .01), and this can be attributed to a large extent to the loss of renal GH clearance30 and to a lesser extent to increased GH secretion.31 It should be recognized, however, that measurements of single serum GH levels are limited in value because of the pulsatile nature of GH secretion. In contrast to the elevated serum GH levels, serum GHBP levels were markedly depressed in ESRD subjects compared with control patients (34 ⫾ 4 versus 165 ⫾ 34 pmol/L, P ⬍ .01). Because GHBP levels are affected by the nutritional state,32 inflammation,33 and fat mass,23 we examined the relationship between the GHBP levels and selected nutritional and inflammatory parameters in all of the subjects. In respect to the serum albumin, although the levels were significantly lower in the ESRD subjects compared with the normal control patients (Table 1), over a wide range of values there was no significant relationship between serum GHBP and albumin levels (Fig 2). In those ESRD subjects with serum albumin levels below 3.5 g/dL, the GHBP levels were similar to the values obtained in those with albumin levels ⱖ 3.5 g/dL (32 ⫾ 5.9 versus 32 ⫾ 4.8 pmol/L, respectively).
There was also no correlation between the serum GHBP levels and BMI (Fig 3), fat mass (r ⫽ 0.18), muscle mass (r ⫽ 0.12), or mid-arm circumference (r ⫽ 0.09). In the ESRD group alone, there was no significant relationship between GHBP and nPCR. Taken together, these findings indicate that the low serum GHBP levels in ESRD cannot be accounted for by differences in the nutritional status between the 2 groups. High-sensitivity serum CRP levels were elevated in 10 of the 21 ESRD subjects (Fig 4), and the average CRP level of the ESRD group was significantly higher than that of the control patients (7.6 ⫾ 2.61 versus 1.1 ⫾ 0.14 mg/L, P ⬍ .01).
Figure 2. Relationship between serum growth hormone binding protein (GHBP) and serum albumin levels. r ⫽ 0.322, P ⫽ not significant.
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Figure 3. The relationship between serum growth hormone binding protein and body mass index (BMI). r ⫽ 0.1021, P ⫽ not significant.
Regression analysis of all of the data showed a significant relationship between the serum GHBP levels and the log serum CRP levels (Fig 3, P ⬍ 0.01). This suggests that inflammation may be a cause of the reduced GHBP levels in those subjects with elevated serum CRP levels. Turning to the GH receptor, we found by double immunofluorescence antibody staining and FACS analysis that in normal control subjects about 20% of all PBMCs express GH receptors
Figure 5. (A) Proportion of peripheral blood mononuclear cells (PBMC) expressing the GH receptor protein in normal and ESRD subjects. (B) Intensity of expression of GH receptors on PBMC in normal and ESRD subjects.
with low expression in T cells, 7%, whereas most B cells, 87%, and monocytes, 99%, are GH receptor positive (Fig 5A). These findings are consistent with those of others.34 Interestingly, in the ESRD group we found a near-identical distribution of GH receptor protein expression on PBMCs as observed in the control patients (Fig 5A). Furthermore, analysis of the intensity of GH receptor fluorescence staining showed that the intensity values among total PBMCs, B cells, T cells, and monocytes were similar in the ESRD and control groups (Fig 5B). Thus, although serum GHBP levels were markedly reduced in ESRD, the distribution and intensity of PBMC GH receptor expression were unchanged. Figure 4. Relationship between serum growth hormone binding protein and log serum high-sensitivity C-reactive protein (CRP) levels. r ⫽ ⫺0.552, P ⬍ .05.
Discussion Because GH resistance occurs commonly in ESRD and has significant clinical consequences,
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we set out to determine whether the resistant state could be attributed to a reduction in GH receptor levels as described in some animal studies.10,11 To this end we measured serum GHBP levels, often used as a surrogate marker of tissue GH receptor levels in humans,13 and also GH receptor protein expression in PBMC obtained from normal control and ESRD adult subjects maintained on regular hemodialysis. PBMCs, a target for the immunoregulatory actions of GH, were studied because this tissue is easy to access and the immune system is compromised in uremia. Serum GHBP levels were reduced significantly in the ESRD subjects, a finding consistent with that of most published studies.19 –21,35,36 In contrast to the reduction in serum GHBP levels in ESRD, we found that the PBMC GH receptor expression measured by double antibody immunofluorescence staining and FACS analyses did not differ between ESRD and normal subjects. In both groups of subjects, about 20% of all of the PBMCs expressed GH receptors, most of the B cells (87%) and monocytes (99%) were GH receptor positive, whereas receptor expression was low in T cells (7%). A similar distribution of GH receptors has been described by others in normal subjects.34,37 The intensity of immunofluorescence, taken to reflect GH receptor protein levels, was also similar in our 2 study groups. There was no relationship between serum GHBP levels and PBMC GH receptor levels. Although there are several descriptions of GH receptor expression in uremic animals, to our knowledge this article is the first describing the direct measurements of GH receptor protein levels in a tissue from humans with renal failure. In some animal studies, a decrease in GH receptor expression was observed in uremia, including a reduction in hepatic GH receptor mRNA levels and growth plate GH receptor protein levels.10,11 On the other hand, other animal studies have indicated that the hepatic, skeletal muscle, and myocardial GH receptor protein levels are unaltered by uremia per se,8,38,39 but suggest that reduced food intake is the main cause of the GH receptor changes.8,12 These conflicting animal studies may be explained by differences in the tissue examined, the severity of the experimental renal failure, sex or strain differences, and the presence or absence of acidosis. Nevertheless, even when GH receptor protein levels are unaffected, resistance to GH does develop in uremic
animals, reflecting the complexity of this condition. Indeed several factors have been implicated, including a postreceptor defect in GH-stimulated signal transduction through the JAK2-STAT (Janus kinase 2 signal transducer and activator of transcription) signaling pathway,8,38,39 impaired IGF-1 production,20,40 reduced IGF-1 bioavailability because of increased binding to the IGF binding proteins that accumulate in uremia,5,9 and finally to end-organ insensitivity to IGF-1.7 It will be interesting to determine whether GHmediated signaling and action are impaired in uremia despite a lack of change in GH receptor levels. The GH binding protein has been used as an indirect marker of tissue GH receptor number because in humans this protein is generated by incomplete proteolysis of the GH receptor with release of the extracellular domain into the circulation.13 Furthermore, several studies have described a positive relationship between GHBP levels and GH sensitivity.13,20,23,32 However, the true value of the serum GHBP levels as a measure of tissue GH receptor levels remain to be established because there are instances when the serum GHBP levels have not been representative of tissue GH receptor levels.26 Most studies of patients with renal failure have described a reduction in the serum GHBP levels,19 –21,35,36 and this has been taken to mean that tissue GH receptor levels are reduced. This assumption has been supported by the report that in children with CRF, GHBP levels correlated both with the spontaneous growth pattern and the growth response to recombinant GH.20 On the other hand, in a study of 69 children with CRF, GHBP levels were described as falling within the normal range.22 However, this finding needs verification because the control data used for comparison were historical. Our own finding that PBMC GH receptor protein levels are unaffected in adults with ESRD despite the presence of low serum GHBP levels indicates that the serum GHBP levels are not representative of PBMC GH receptor density. This argues against the use of GHBP in uremic subjects as a marker of local tissue GH receptor levels, at least in PBMC. Nevertheless, it is conceivable that the low serum GHBP levels may reflect reduced GH receptor levels in tissues other than peripheral blood mononuclear cells such as liver, which is rich in GH receptors, and perhaps adipose tissue. It is
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also possible that the low serum GHBP levels could arise because of reduced GH receptor cleavage, or alternatively from increased GHBP degradation. Nutritional status is an important determinant of serum GHBP levels, and in anorexia nervosa and other wasting states, GHBP levels are reduced.33,41 Presumably this reflects a change in tissue GH receptor levels42 because animal studies have established that GH receptor levels decrease during fasting and recover after refeeding.43 In normal healthy adults and in children with CRF, a positive relationship between GHBP and BMI has been noted.20,23 Because the BMI is significantly correlated with body fat content, this has led to the suggestion that adipose tissue is a significant source of serum GHBP levels,23 although more recent studies have seriously challenged this notion.44,45 Because tissue GH receptor protein levels were not determined in any of the above studies and as the source of GHBP release into the circulation is unknown, the exact relationship between serum GHBP and tissue GH receptor protein levels remains to be defined. In our current study there was no significant relationship between the serum GHBP levels and the BMI nor with serum albumin levels, which were modestly reduced, or estimated muscle or fat mass. Thus the low serum GHBP levels cannot be attributed to nutritional causes in these subjects. The lack of relationship between undernutrition, as evidenced by the low serum albumin, and the serum GHBP levels and PBMC GH receptor levels in this study may reflect the relative mildness of the nutritional deficit. GH receptor levels are also influenced by the presence of inflammation, and tissue GH receptor levels in liver and muscle are reduced in septic and endotoxemic rats,46 although this is not a uniform finding.47 Similarly, serum GHBP levels are decreased in elderly subjects when inflammation is present.33 In the ESRD group that we studied, there was evidence of inflammation in about half of the subjects as judged by elevated serum CRP levels. Overall there was a significant negative correlation between the serum GHBP levels and the log serum CRP levels. This suggests that inflammation may contribute to the reduced GHBP levels in those ESRD subjects with elevated CRP levels. In contrast, inflammation seemed to have no impact on PBMC GH receptor protein expression. Because the inflam-
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matory state was mild, it is possible that PBMC GH receptor expression is less sensitive to inflammation than GHBP production or metabolism. Although we found that the PBMC GH receptor levels were unchanged in ESRD, it is conceivable that GH signal transduction may well be impaired in these cells as occurs in liver, muscle, and heart of uremic rats.8,38,39 If true, then this might contribute to the compromised immunity that occurs in uremia. It turns out that in addition to its growth-promoting and metabolic properties, GH has immunoregulatory actions and plays a role in the regulation of thymus growth and function, as well as affecting mononuclear cell function including stimulation of lymphocyte proliferation, migration, and T-cell adhesion.15,16 Interestingly, although mononuclear cells express GH receptors and are a target for the hormone, these cells also produce GH and IGF-1.15,48 Because of its immunoregulatory properties, it has been suggested that GH might have a place in the treatment of immune disorders, including the abnormalities that arise in uremia.49 In conclusion, we have found that serum GHBP concentrations were markedly reduced in a population of well-dialyzed ESRD patients, and that in about half of these adults the reduction may have been partly induced by a low-grade subclinical inflammatory process. Although GHBP levels are known to be affected by nutritional status, we could not attribute the low GHBP levels to malnutrition in these subjects. Taken together, our findings suggest that the reduced GHBP levels are a consequence of other perturbations arising because of loss of renal function. Because the GH receptor protein levels on PBMCs of ESRD subjects were not different from those in normal control patients, our findings argue against a reduction in GH receptor levels as a cause of GH resistance and the use of GHBP levels as a reliable surrogate marker of specific tissue GH receptor levels. Accordingly, further studies examining the relationship between serum GHBP levels and local GH receptor levels in other GH target tissues such as liver, fat, and muscle are required before assuming that uremic GH resistance is secondary to reduced GH receptor levels and that serum GHBP levels reflect the level of GH receptors in any particular tissue.
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Acknowledgment We thank Luisijah Rott and Cathy Crumpton for their assistance in the use of flow cytometry.
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CIRCULATING GHBP LEVELS AND GHR EXPRESSION 36. Zadik Z, Frishberg Y, Drukker A, et al: Excessive dietary protein and suboptimal caloric intake have a negative effect on the growth of children with chronic renal disease before and during growth hormone therapy. Metabolism 47: 264-268, 1998 37. Rapaport R, Sills IN, Green L, et al: Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: Correlation with growth hormone-binding protein levels. J Clin Endocrinol Metab 80:2612-2619, 1995 38. Sun DF, Zheng Z, Tummala P, et al: Chronic uremia attenuates growth hormone-induced signal transduction in skeletal muscle. J Am Soc Nephrol 15:2630-2636, 2004 39. Zheng Z, Sun DF, Tummala P, et al: Cardiac resistance to growth hormone in uremia. Kidney Int 67:858-866, 2005 40. Blum WF: Insulin-like growth factors (IGFs) and IGF binding proteins in chronic renal failure: Evidence for reduced secretion of IGFs. Acta Paediatr Scand Suppl 379:24-31, discussion 32, 1991 41. Doehner W, Pflaum CD, Rauchhaus M, et al: Leptin, insulin sensitivity and growth hormone binding protein in chronic heart failure with and without cardiac cachexia. Eur J Endocrinol 145:727-735, 2001 42. Shuto Y, Nakano T, Sanno N, et al: Reduced growth hormone receptor messenger ribonucleic acid in an aged man with chronic malnutrition and growth hormone resistance. J Clin Endocrinol Metab 84:2320-2323, 1999
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43. Postel-Vinay MC, Cohen-Tanugi E, Charrier J: Growth hormone receptors in rat liver membranes: effects of fasting and refeeding, and correlation with plasma somatomedin activity. Mol Cell Endocrinol 28:657-669, 1982 44. Fisker S, Hansen B, Fuglsang J, et al: Gene expression of the GH receptor in subcutaneous and intraabdominal fat in healthy females: Relationship to GH-binding protein. Eur J Endocrinol 150:773-777, 2004 45. Fisker S, Kristensen K, Rosenfalck AM, et al: Gene expression of a truncated and the full-length growth hormone (GH) receptor in subcutaneous fat and skeletal muscle in GHdeficient adults: Impact of GH treatment. J Clin Endocrinol Metab 86:792-796, 2001 46. Hong-Brown LQ, Brown CR, Cooney RN, et al: Sepsisinduced muscle growth hormone resistance occurs independently of STAT5 phosphorylation. Am J Physiol Endocrinol Metab 285:E63-72, 2003 47. Mao Y, Ling PR, Fitzgibbons TP, et al: Endotoxininduced inhibition of growth hormone receptor signaling in rat liver in vivo. Endocrinology 140:5505-5515, 1999 48. Dixit VD, Mielenz M, Taub DD, et al: Leptin induces growth hormone secretion from peripheral blood mononuclear cells via a protein kinase C- and nitric oxide-dependent mechanism. Endocrinology 144:5595-5603, 2003 49. Derfalvi B, Nemet K, Szalai C, et al: In vitro effect of human recombinant growth hormone on lymphocyte and granulocyte function of healthy and uremic children. Immunol Lett 63:41-47, 1998
A
DVANCED RENAL FAILURE is often complicated by the appearance of muscle wasting in adults, growth impairment in children, and abnormalities of the immune system.1–3 Many factors contribute to these complications, including anorexia, acidosis, inadequate dialysis, *Department of Medicine, Stanford University, Stanford, CA. †Santa Clara Valley Medical Center, San Jose, CA. ‡Department of Surgery, Stanford University, Stanford, CA. §Veterans Affairs Health Care System, Palo Alto, CA. Supported by a Norman S. Coplon Grant from Satellite Research and a Merit Review Grant from the Research Service of the Department of Veterans Affairs. Presented in part at the Annual Meeting of the American Society of Nephrology 2003 and the Seventh Annual Meeting of the International Pediatric Nephrology Symposium on Growth in Children with Chronic Kidney Disease, Germany 2004. Address reprint requests to Ralph Rabkin, MD, 3801 Miranda Avenue, Palo Alto, CA 94304. E-mail: [email protected] © 2006 by the National Kidney Foundation, Inc. 1051-2276/06/1602-0007$32.00/0 doi:10.1053/j.jrn.2006.01.007
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inflammation, undernutrition, abnormal vitamin D and parathyroid hormone physiology, and resistance to anabolic hormones such as growth hormone (GH), insulin-like growth factor 1 (IGF-1), and insulin. To some extent the GHresistant state can be overcome by large doses of recombinant GH, and this approach is effective in promoting growth in uremic children.4 In adults several small studies have shown that the administration of GH has a positive effect on urea kinetics, protein turnover, and lean body mass.2 Because most but not all of the anabolic and growth-promoting actions of GH are mediated through IGF-1, a hormone induced by GH, uremic resistance to GH has largely been attributed to the development of insensitivity to IGF-1 and reduced IGF-1 production.1,5–7 In addition, our recent studies in animals have shown that GH-mediated signal transduction through the Janus kinase 2-signal transducer and activator of transcription (JAK2-STAT) pathway is impaired 141
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in uremia, and this is another important cause of GH resistance.8,9 There are also reports describing decreased GH receptor levels in uremic animals, although this is not a uniform finding. Some investigators have reported that hepatic GH receptor mRNA and growth plate GH receptor protein levels are reduced in rats with chronic renal failure (CRF),10,11 whereas others have suggested that uremia per se does not alter GH receptor protein levels, but that reduced GH receptor levels are a consequence of inadequate food intake.8,12 The effect of uremia on GH receptor levels have not been examined directly in humans; instead, a surrogate marker of tissue GH receptor levels, namely circulating GH binding protein (GHBP) levels, has been measured.13 The intact GH receptor is a transmembrane glycoprotein that binds GH and through its intracellular domain mediates the growth-promoting, immune modulating, and metabolic actions of the hormone.14 –16 In humans the GHBP is generated by enzymatic cleavage of the GH receptor with release of the extracellular domain into the circulation.13,17 After being cleaved from the intact receptor, the GHBP, a 60-kDa single-chain glycoprotein with a high affinity for GH, enters the circulation, where it binds about 45% of the circulating GH and serves as a dynamic reservoir of circulating GH.13,18 Several19 –21 but not all22 studies of subjects with kidney failure have described a reduction in circulating GHBP levels. This has been taken to reflect reduced tissue GH receptor protein expression,19,20 especially as the low serum GHBP levels correlate with sensitivity to GH in uremic children.20 Indeed a positive relationship between GHBP levels and GH sensitivity has also been described in other conditions such as obesity23 and cirrhosis,24 and this provides further support for the assumption that serum GHBP levels reflect tissue GH receptor levels. However this concept has not been rigorously established, and several exceptions have been described.13,25,26 Because GHBP levels have been reported to be reduced in advanced renal failure, we set out to determine whether serum GHBP levels reflect cellular GH receptor levels and whether changes in GHBP levels, rather than being caused by uremia, are a reflection of malnutrition or inflammation, conditions that may also cause a reduc-
tion in GH receptor and GHBP levels.13,26 To this end we studied subjects with end-stage renal disease (ESRD) and normal age-matched control patients and measured their serum GHBP levels and the expression of the GH receptor protein on peripheral blood mononuclear cells (PBMC), a target of the immunoregulatory actions of GH.27 We chose to study PBMCs because of their ready availability and the facts that GH has immune regulatory actions on PBMC and that the immune system is dysregulated in uremia.3
Subjects and Methods Subjects Twenty-one patients with ESRD treated by maintenance hemodialysis for at least 8 weeks and 14 healthy subjects between the ages of 21 and 69 years were studied. Exclusion criteria included diabetes mellitus; pregnancy; vasculitis; autoimmune disease; malignancy; obesity (relative body weight ⬎130%); alcoholism or other recreational drug use; heart, lung, liver, or gastrointestinal dysfunction; sepsis and a Kt/V ⬍ 1.3 during the prior month; medically unstable subjects; and subjects who had received anabolic, catabolic, or cytotoxic medications during the prior 3 months. Blood samples from control patients were collected into ethylenediaminetetraacetic acid– containing tubes by venipuncture and from ESRD subjects from their vascular access predialysis, at least 48 hours after their last dialysis treatment. The study was approved by the Institutional Human Research Committee. All subjects signed informed consent to participate in the study. Cell Preparation Human PBMCs were prepared from anticoagulated whole blood by centrifugation on Histapaque 1079 density gradients (Sigma-Aldrich Corp, St. Louis, MO) after dilution in HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid)-balanced saline solution. The PBMC recovered from the interface were washed twice and resuspended in phosphate-buffered saline containing 2% bovine serum albumin and 0.1% sodium azide. Cells were counted, and viability was determined by trypan blue exclusion. The final PBMC concentration was adjusted to 1 ⫻ 107 cells/mL, and 100 L of each suspension was used for immunofluorescence staining.
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Immunostaining and Fluorescence Activating Cell Sorter (FACS) Analysis The PBMC surface markers were detected by direct labeling with antihuman antibodies: Cychrome conjugated anti-CD3 as a marker of mature T cells, R-phycoerythrin anti-CD19 as a B-cell marker, and allophycocyanin-conjugated anti-CD14 as a marker of monocytes. Fluorescein isothiocyanate (FITC)– conjugated mouse immunoglobulin (Ig) G1 k was used as an isotype control. All of these proteins were purchased from BD Biosciences Parmingen, San Diego, CA. Anti-GH receptor monoclonal antibody 263 was purchased from Biogenesis Inc, Kingston, NH, and custom labeled with FITC by Chromaprobe, Inc, Aptos, CA. PBMC, 1 million, were incubated with the GH receptor antibody at 4°C for 90 minutes, and then the PBMC surface marker antibodies were added and incubation continued for another 30 minutes. After washing, the resuspended cells were analyzed in a FacsCalibur flow cytometer (Beckton-Dickinson, San Jose, CA). Cells were also incubated separately with each antibody to serve for single-color compensation during FACS analysis. Sphero Rainbow calibration particles (Spherotech Inc, Libertyville, IL) were used to calibrate the flow cytometer for each run and for measuring the intensity of the fluorescent signal. Biochemical Assays Routine biochemical assays were performed in the clinical laboratories, including serum albumin by the bromcresol purple method. High-sensitivity C-reactive protein (CRP) levels were determined by Nephelometry (Quest Diagnostcs, San Juan Capistano, CA), serum GHBP, GH, and IGF-1 by enzyme-linked immunosorbent assays (Diagnostic Systems Laboratory, Webster, TX). Nutritional parameters assessed included height, edema-free adjusted body weight, skin fold thickness, mid-arm circumference and arm muscle area, body mass index (BMI), muscle mass, serum albumin, and normalized protein catabolic rate (nPCR). Skin-fold thickness was measured with a Lange skinfold caliper (Beta Technology Incorporated, Santa Cruz, CA) over the biceps, triceps, subscapular, and suprailiac regions, and with the average of these measurements the percent body fat was estimated from published tables.28 Adequacy of dialysis was assessed from the blood urea
nitrogen and serum creatinine levels and from the Kt/V. Formulae for the various anthropometric and kinetic calculations were according to Kidney Disease Outcomes and Quality Initiative guidelines.28
Statistical Analyses Results are expressed as the mean ⫾ SEM. The Student two-tailed unpaired t test was applied for comparing 2 normally distributed groups; comparisons between more than 2 normally distributed groups were made by one-way analysis of variance followed by pairwise multiple comparison with the Holms t test.29 For analyzing 2 nonnormally distributed groups, the MannWhitney rank sum test was used, and for more than 2 nonnormally distributed groups, the Kruskal-Wallis statistic was applied, followed by the Student Newman Keuls test to distinguish between groups.29 A P value ⬍.05 was considered statistically significant.
Results The anthropometric features of the 21 ESRD and 14 normal control subjects are shown in Table 1. The 2 groups were of similar age, and the ESRD subjects had received dialysis treatment for an average of 3.1 ⫾ 0.5 years with current Kt/V values of 1.92 ⫾ 0.09 and nPCR values of 1.02 ⫾ 0.18 g/kg/day. BMI, muscle and fat mass, mid-arm circumference, and arm muscle area were on average lower in the ESRD subjects but not statistically significantly so. The serum albumin levels were reduced significantly in the ESRD compared with the control group Table 1. Subject Characteristics Control
ESRD
Number 14 21 Age, y 44 ⫾ 2.2 47 ⫾ 3.0 Sex, M/F 8/6 14/7 Height, cm 166 ⫾ 2.8 165 ⫾ 2.6 Body weight, kg 72.8 ⫾ 3.87 66.4 ⫾ 3.25 Midarm circumference, cm 29.3 ⫾ 0.87 28.5 ⫾ 0.65 Arm muscle area, cm2 44.8 ⫾ 3.84 42.9 ⫾ 3.33 Muscle mass, kg 27.3 ⫾ 1.7 25.7 ⫾ 2 Fat mass, kg 72.12 ⫾ 1.18 75.43 ⫾ 1.53 Serum albumin, mg/dL 4.24 ⫾ 0.14 *3.66 ⫾ 0.18 Years on HD N/A 3.1 ⫾ 0.52 Kt/V N/A 1.92 ⫾ 0.09 nPCR, g/kg N/A 1.02 ⫾ 0.18 *P ⬍ .05.
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Figure 1. Serum insulin-like growth factor 1 (IGF-1), growth hormone (GH), and growth hormone binding protein (GHBP) levels in end stage renal disease and normal subjects. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001.
(3.7 ⫾ 0.18 versus 4.2 ⫾ 0.14 mg/dL). The serum hormone levels measured in these subjects are shown in Figure 1. Total IGF-1 levels were significantly elevated in the ESRD group (147 ⫾ 14 versus 99 ⫾ 14 ng/mL, P ⬍ .01), and this is likely a consequence of the elevation in lowmolecular-weight serum IGF binding proteins that consistently occurs in uremia.5 Serum GH levels were also elevated in ESRD (1.5 ⫾ 0.59 versus 0.8 ⫾ 0.03 ng/mL, P ⬍ .01), and this can be attributed to a large extent to the loss of renal GH clearance30 and to a lesser extent to increased GH secretion.31 It should be recognized, however, that measurements of single serum GH levels are limited in value because of the pulsatile nature of GH secretion. In contrast to the elevated serum GH levels, serum GHBP levels were markedly depressed in ESRD subjects compared with control patients (34 ⫾ 4 versus 165 ⫾ 34 pmol/L, P ⬍ .01). Because GHBP levels are affected by the nutritional state,32 inflammation,33 and fat mass,23 we examined the relationship between the GHBP levels and selected nutritional and inflammatory parameters in all of the subjects. In respect to the serum albumin, although the levels were significantly lower in the ESRD subjects compared with the normal control patients (Table 1), over a wide range of values there was no significant relationship between serum GHBP and albumin levels (Fig 2). In those ESRD subjects with serum albumin levels below 3.5 g/dL, the GHBP levels were similar to the values obtained in those with albumin levels ⱖ 3.5 g/dL (32 ⫾ 5.9 versus 32 ⫾ 4.8 pmol/L, respectively).
There was also no correlation between the serum GHBP levels and BMI (Fig 3), fat mass (r ⫽ 0.18), muscle mass (r ⫽ 0.12), or mid-arm circumference (r ⫽ 0.09). In the ESRD group alone, there was no significant relationship between GHBP and nPCR. Taken together, these findings indicate that the low serum GHBP levels in ESRD cannot be accounted for by differences in the nutritional status between the 2 groups. High-sensitivity serum CRP levels were elevated in 10 of the 21 ESRD subjects (Fig 4), and the average CRP level of the ESRD group was significantly higher than that of the control patients (7.6 ⫾ 2.61 versus 1.1 ⫾ 0.14 mg/L, P ⬍ .01).
Figure 2. Relationship between serum growth hormone binding protein (GHBP) and serum albumin levels. r ⫽ 0.322, P ⫽ not significant.
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Figure 3. The relationship between serum growth hormone binding protein and body mass index (BMI). r ⫽ 0.1021, P ⫽ not significant.
Regression analysis of all of the data showed a significant relationship between the serum GHBP levels and the log serum CRP levels (Fig 3, P ⬍ 0.01). This suggests that inflammation may be a cause of the reduced GHBP levels in those subjects with elevated serum CRP levels. Turning to the GH receptor, we found by double immunofluorescence antibody staining and FACS analysis that in normal control subjects about 20% of all PBMCs express GH receptors
Figure 5. (A) Proportion of peripheral blood mononuclear cells (PBMC) expressing the GH receptor protein in normal and ESRD subjects. (B) Intensity of expression of GH receptors on PBMC in normal and ESRD subjects.
with low expression in T cells, 7%, whereas most B cells, 87%, and monocytes, 99%, are GH receptor positive (Fig 5A). These findings are consistent with those of others.34 Interestingly, in the ESRD group we found a near-identical distribution of GH receptor protein expression on PBMCs as observed in the control patients (Fig 5A). Furthermore, analysis of the intensity of GH receptor fluorescence staining showed that the intensity values among total PBMCs, B cells, T cells, and monocytes were similar in the ESRD and control groups (Fig 5B). Thus, although serum GHBP levels were markedly reduced in ESRD, the distribution and intensity of PBMC GH receptor expression were unchanged. Figure 4. Relationship between serum growth hormone binding protein and log serum high-sensitivity C-reactive protein (CRP) levels. r ⫽ ⫺0.552, P ⬍ .05.
Discussion Because GH resistance occurs commonly in ESRD and has significant clinical consequences,
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we set out to determine whether the resistant state could be attributed to a reduction in GH receptor levels as described in some animal studies.10,11 To this end we measured serum GHBP levels, often used as a surrogate marker of tissue GH receptor levels in humans,13 and also GH receptor protein expression in PBMC obtained from normal control and ESRD adult subjects maintained on regular hemodialysis. PBMCs, a target for the immunoregulatory actions of GH, were studied because this tissue is easy to access and the immune system is compromised in uremia. Serum GHBP levels were reduced significantly in the ESRD subjects, a finding consistent with that of most published studies.19 –21,35,36 In contrast to the reduction in serum GHBP levels in ESRD, we found that the PBMC GH receptor expression measured by double antibody immunofluorescence staining and FACS analyses did not differ between ESRD and normal subjects. In both groups of subjects, about 20% of all of the PBMCs expressed GH receptors, most of the B cells (87%) and monocytes (99%) were GH receptor positive, whereas receptor expression was low in T cells (7%). A similar distribution of GH receptors has been described by others in normal subjects.34,37 The intensity of immunofluorescence, taken to reflect GH receptor protein levels, was also similar in our 2 study groups. There was no relationship between serum GHBP levels and PBMC GH receptor levels. Although there are several descriptions of GH receptor expression in uremic animals, to our knowledge this article is the first describing the direct measurements of GH receptor protein levels in a tissue from humans with renal failure. In some animal studies, a decrease in GH receptor expression was observed in uremia, including a reduction in hepatic GH receptor mRNA levels and growth plate GH receptor protein levels.10,11 On the other hand, other animal studies have indicated that the hepatic, skeletal muscle, and myocardial GH receptor protein levels are unaltered by uremia per se,8,38,39 but suggest that reduced food intake is the main cause of the GH receptor changes.8,12 These conflicting animal studies may be explained by differences in the tissue examined, the severity of the experimental renal failure, sex or strain differences, and the presence or absence of acidosis. Nevertheless, even when GH receptor protein levels are unaffected, resistance to GH does develop in uremic
animals, reflecting the complexity of this condition. Indeed several factors have been implicated, including a postreceptor defect in GH-stimulated signal transduction through the JAK2-STAT (Janus kinase 2 signal transducer and activator of transcription) signaling pathway,8,38,39 impaired IGF-1 production,20,40 reduced IGF-1 bioavailability because of increased binding to the IGF binding proteins that accumulate in uremia,5,9 and finally to end-organ insensitivity to IGF-1.7 It will be interesting to determine whether GHmediated signaling and action are impaired in uremia despite a lack of change in GH receptor levels. The GH binding protein has been used as an indirect marker of tissue GH receptor number because in humans this protein is generated by incomplete proteolysis of the GH receptor with release of the extracellular domain into the circulation.13 Furthermore, several studies have described a positive relationship between GHBP levels and GH sensitivity.13,20,23,32 However, the true value of the serum GHBP levels as a measure of tissue GH receptor levels remain to be established because there are instances when the serum GHBP levels have not been representative of tissue GH receptor levels.26 Most studies of patients with renal failure have described a reduction in the serum GHBP levels,19 –21,35,36 and this has been taken to mean that tissue GH receptor levels are reduced. This assumption has been supported by the report that in children with CRF, GHBP levels correlated both with the spontaneous growth pattern and the growth response to recombinant GH.20 On the other hand, in a study of 69 children with CRF, GHBP levels were described as falling within the normal range.22 However, this finding needs verification because the control data used for comparison were historical. Our own finding that PBMC GH receptor protein levels are unaffected in adults with ESRD despite the presence of low serum GHBP levels indicates that the serum GHBP levels are not representative of PBMC GH receptor density. This argues against the use of GHBP in uremic subjects as a marker of local tissue GH receptor levels, at least in PBMC. Nevertheless, it is conceivable that the low serum GHBP levels may reflect reduced GH receptor levels in tissues other than peripheral blood mononuclear cells such as liver, which is rich in GH receptors, and perhaps adipose tissue. It is
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also possible that the low serum GHBP levels could arise because of reduced GH receptor cleavage, or alternatively from increased GHBP degradation. Nutritional status is an important determinant of serum GHBP levels, and in anorexia nervosa and other wasting states, GHBP levels are reduced.33,41 Presumably this reflects a change in tissue GH receptor levels42 because animal studies have established that GH receptor levels decrease during fasting and recover after refeeding.43 In normal healthy adults and in children with CRF, a positive relationship between GHBP and BMI has been noted.20,23 Because the BMI is significantly correlated with body fat content, this has led to the suggestion that adipose tissue is a significant source of serum GHBP levels,23 although more recent studies have seriously challenged this notion.44,45 Because tissue GH receptor protein levels were not determined in any of the above studies and as the source of GHBP release into the circulation is unknown, the exact relationship between serum GHBP and tissue GH receptor protein levels remains to be defined. In our current study there was no significant relationship between the serum GHBP levels and the BMI nor with serum albumin levels, which were modestly reduced, or estimated muscle or fat mass. Thus the low serum GHBP levels cannot be attributed to nutritional causes in these subjects. The lack of relationship between undernutrition, as evidenced by the low serum albumin, and the serum GHBP levels and PBMC GH receptor levels in this study may reflect the relative mildness of the nutritional deficit. GH receptor levels are also influenced by the presence of inflammation, and tissue GH receptor levels in liver and muscle are reduced in septic and endotoxemic rats,46 although this is not a uniform finding.47 Similarly, serum GHBP levels are decreased in elderly subjects when inflammation is present.33 In the ESRD group that we studied, there was evidence of inflammation in about half of the subjects as judged by elevated serum CRP levels. Overall there was a significant negative correlation between the serum GHBP levels and the log serum CRP levels. This suggests that inflammation may contribute to the reduced GHBP levels in those ESRD subjects with elevated CRP levels. In contrast, inflammation seemed to have no impact on PBMC GH receptor protein expression. Because the inflam-
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matory state was mild, it is possible that PBMC GH receptor expression is less sensitive to inflammation than GHBP production or metabolism. Although we found that the PBMC GH receptor levels were unchanged in ESRD, it is conceivable that GH signal transduction may well be impaired in these cells as occurs in liver, muscle, and heart of uremic rats.8,38,39 If true, then this might contribute to the compromised immunity that occurs in uremia. It turns out that in addition to its growth-promoting and metabolic properties, GH has immunoregulatory actions and plays a role in the regulation of thymus growth and function, as well as affecting mononuclear cell function including stimulation of lymphocyte proliferation, migration, and T-cell adhesion.15,16 Interestingly, although mononuclear cells express GH receptors and are a target for the hormone, these cells also produce GH and IGF-1.15,48 Because of its immunoregulatory properties, it has been suggested that GH might have a place in the treatment of immune disorders, including the abnormalities that arise in uremia.49 In conclusion, we have found that serum GHBP concentrations were markedly reduced in a population of well-dialyzed ESRD patients, and that in about half of these adults the reduction may have been partly induced by a low-grade subclinical inflammatory process. Although GHBP levels are known to be affected by nutritional status, we could not attribute the low GHBP levels to malnutrition in these subjects. Taken together, our findings suggest that the reduced GHBP levels are a consequence of other perturbations arising because of loss of renal function. Because the GH receptor protein levels on PBMCs of ESRD subjects were not different from those in normal control patients, our findings argue against a reduction in GH receptor levels as a cause of GH resistance and the use of GHBP levels as a reliable surrogate marker of specific tissue GH receptor levels. Accordingly, further studies examining the relationship between serum GHBP levels and local GH receptor levels in other GH target tissues such as liver, fat, and muscle are required before assuming that uremic GH resistance is secondary to reduced GH receptor levels and that serum GHBP levels reflect the level of GH receptors in any particular tissue.
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Acknowledgment We thank Luisijah Rott and Cathy Crumpton for their assistance in the use of flow cytometry.
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