Increased Soluble Leptin Receptor in Children with Nephrotic Syndrome

0021-972X/03/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 88(11):5497–5501 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2003-030539

Increased Soluble Leptin Receptor in Children with Nephrotic Syndrome ¨ RGEN KRATZSCH, MICHAEL GRO ¨ SCHL, MANFRED RAUH, MICHAEL SCHROTH, JU ¨ RG DO ¨ TSCH WOLFGANG RASCHER, AND JO Klinik mit Poliklinik fu¨r Kinder und Jugendliche (M.S., M.G., M.R., W.R., J.D.), Friedrich-Alexander-Universita¨t ErlangenNu¨rnberg, D-91054 Erlangen, Germany; and Institute of Laboratory Medicine (J.K.), Clinical Chemistry and Molecular Diagnostics, Hospital for Children and Adolescents, University of Leipzig, D-04103 Leipzig, Germany In patients with nephrotic syndrome, severe proteinuria is related to significant leptinuria; serum leptin levels remain unchanged. The goal of this study was to elucidate the role of the soluble leptin receptor (sOB-R) in maintaining serum leptin levels in nephrotic patients. Patients with proteinuria were compared with patients in remission of nephrotic syndrome. In this group proteinuria did not exceed 100 mg/m2 of body surface area per day. The period of remission was at least 6 months and was equal in all patients included. The sOB-R level (mean ⴞ SD) in serum of patients with nephrotic syndrome was significantly higher during proteinuria (61.0 ⴞ 17.8

L

EPTIN, A 167-AMINO acid polypeptide with a molecular size of 16 kDa, is mainly synthesized in adipose tissue (1, 2) but also in other tissues, such as the placenta (3), the gastrointestinal tract (4 – 6), and neuronal tissues (7). The product of the ob gene plays an important role in the regulation of appetite and food intake in mice and humans (8 –10). Mutations of the leptin gene or its receptor gene lead to obesity in mice and humans (1, 11). Leptin circulates in murine serum in a free and bound form (11–16). In human blood, it is bound to a high affinity binding protein, which is the soluble leptin receptor (sOB-R), modulating the effects of its ligand (17, 18). The sOB-R represents the major leptin binding protein in the circulation. The presumed biologically active form of leptin is determined by the free leptin index (FLI), the ratio between leptin and sOB-R levels (15, 17). The idiopathic nephrotic syndrome (INS) is an albuminloosing nephropathy in childhood often due to minimal change lesions of the kidneys. Severe proteinuria (urinary protein level exceeding 1 g/m2 of body surface area per day) leads to hypoproteinemia (hypalbuminemia, albumin in serum ⬍ 25 g/liter) and chronically to a catabolic nutritional state (19). Our previous data revealed a renal urinary leptin loss in prepubertal and early pubertal children suffering from active nephrotic syndrome with proteinuria greater than 1 g/m2 (20). Urinary leptin loss disappeared after remission. However, despite an up to 100-fold increment in leptin excretion in proteinuria, serum leptin levels are similar in both proteinuric and nonproteinuric children (20). These findings suggest that the renal loss of leptin is counterregulated. Abbreviations: BMI, Body mass index; FLI, free leptin index; NS, nephrotic syndrome; sOB-R, soluble leptin receptor.

ng/ml) than those in remission or in control patients (36.7 ⴞ 7.0 ng/ml, 36.6 ⴞ 12.0 ng/ml, respectively, P < 0.0001). The ratio between serum leptin levels and the sOB-R (free leptin index) was significantly lower in the proteinuric group (0.012 ⴞ 0.005 vs. 0.06 ⴞ 0.03 and 0.07 ⴞ 0.03 in remission and control group, respectively) (P < 0.001). Urinary sOB-R excretion was similar in all groups. Our data suggest that the counteracting pathway in case of leptin loss in parallel to severe proteinuria in nephrotic syndrome is the up-regulation of its soluble binding protein in serum, which can keep total serum leptin levels constant. (J Clin Endocrinol Metab 88: 5497–5501, 2003)

We, therefore, hypothesized that the sOB-R protein binding capacity counteracts the urinary losses of leptin. The FLI, representing the biologically active form of leptin, is expected to be altered in proteinuric children. Patients and Methods Pediatric patients with nephrotic syndrome (NS) were studied. The patients’ characteristics are shown in Table 1. In 17 patients (group I), proteinuria (urinary protein level exceeding 1 g/m2 of body surface area per day) was present. Twenty patients were in remission (group II, no significant proteinuria). In this study group, proteinuria did not exceed 100 mg/m2 of body surface area per day. The period of remission was at least 6 months and was equal in all patients included. The patients’ groups were compared with 10 healthy children (group III). Concentration of leptin in serum and urine and the levels of the soluble leptin receptor in serum and urine were measured by RIA. After centrifugation of blood samples, which were all drawn between 1000 and 1200 h, serum and urine samples were kept frozen for up to 8 wk at ⫺20 C and were analyzed when all specimens had been obtained. In four patients with NS, sequential samples were obtained over a period of 1 yr in different states of disease. In all patients, body weight and body height were measured to obtain the body mass index (BMI). Subscapular and triceps skinfold thickness was determined with a caliper by the same trained investigator (M.S.). No patient was fasting. All clinical and auxologic data were obtained during routine visits and recorded using standard data sheets. Details are shown in Table 1. The study was approved by the local ethics committee of the University of Erlangen. Informed, written consent was obtained from all parents and the older patients.

Materials and methods Hormone measurements. Serum levels of the sOB-R have been measured by a sensitive ligand-immunofunctional assay (17, 18). For the determination of sOB-R in urine, samples of 1 ml were 25-fold concentrated and assayed by the same method. Serum levels of leptin were measured using a specific RIA as described in detail elsewhere (21). Leptin in urine was measured by a highly sensitive adaptation of the assay. Tracer activity was 8000 c/m䡠25

5497

5498

J Clin Endocrinol Metab, November 2003, 88(11):5497–5501

Schroth et al. • Increased Soluble Leptin Receptor

TABLE 1. Patients’ characteristics

Number (n) Age (yr) Gender (m/f) BMI (kg/m2) Tanner 1 or 2 Diagnosis

a

Group I

Group II

Group III

Patients with NS and with proteinuria (⬎1 g/m2䡠d)

Patients with NS without proteinuria

Control group (healthy children)

17 8.8 ⫾ 3.8; 8 8/9 18.1 ⫾ 2.9; 18.1 17 INS (8), focal global glomerulosclerosisa (1), focal segmental glomerulosclerosisa (3), MCGN (3), membranoproliferative glomerulonephritisa (1), congenital nephrotic syndromea (1)

20 9.9 ⫾ 3.6; 10 17/3 18.3 ⫾ 3.6; 17.45 20 INS (15), MCGNa (5)

10 10.5 ⫾ 4.2; 9.5 5/5 17.2 ⫾ 2.4; 17.15 10 Healthy

Diagnosis confirmed by histological examination.

FIG. 1. Soluble leptin receptor concentrations in group I (patients with NS with severe proteinuria, urinary protein level exceeding 1 g/m2 of body surface area per day), group II (patients with NS without proteinuria), and group III (control group, healthy children). Data are shown as scattergram and box and whiskers. Significant higher levels in group I are evaluated by using one-way ANOVA; P values were corrected according to Bonferroni. ***, P ⫽ 0.0005.

␮l. The antiserum had a final dilution of 1:8000 in 25 ␮l. The standard raw expanded from 1250 to 10 pg/ml. Statistical analysis. Data with Gaussian distribution were correlated by linear regression. Parametric data were compared by two-tailed t test. In case of multiple tests, data were compared using one-way ANOVA and, in case of significance, post hoc t test. P values were corrected according to Bonferroni. A P value ⬍ 0.05 was considered statistically significant.

Results

The sOB-R levels in serum (mean ⫾ sd) of patients with NS presenting with proteinuria (group I) were significantly higher (61.0 ⫾ 17.8 ng/ml) than in patients with remission or control patients (36.7 ⫾ 7.0 ng/ml, 36.6 ⫾ 12.0 ng/ml, P ⬍ 0.0001), respectively (Fig. 1). The ratio between serum leptin levels and the sOB-R (FLI) was significantly lower in the proteinuric group (0.012 ⫾ 0.005 vs. 0.06 ⫾ 0.03 and 0.07 ⫾ 0.03 in remission and control group, respectively) (P ⬍ 0.001) (Fig. 2). The sOB-R was also measured in urine and did not show any significant difference among the three study groups. There was no correlation between urinary sOB-R excretion and urine albumin or urine IgG in any of the groups. There was no difference in urinary sOB-R between boys and girls or between Tanner stages 1 and 2. The patients’ medication (cyclosporine, tacrolimus, cyclophosphamide, ␤-blocking agents, prednisone) did not significantly influ-

FIG. 2. FLI in group I (patients with NS with severe proteinuria, urinary protein level exceeding 1 g/m2 of body surface area per day), group II (patients with NS without proteinuria), and group III (control group, healthy children). Data are shown as scattergram and box and whiskers. Significantly higher levels in group I are evaluated by using one-way ANOVA; P values were corrected according to Bonferroni. ***, P ⫽ 0.0005.

ence serum or urinary concentrations of sOB-R when children receiving one of the medications were compared with nontreated patients. A positive relationship was obtained between BMI and serum sOB-R in nephrotic patients with proteinuria (P ⫽ 0.008; r2 ⫽ 0.29), patients with remission (P ⬍ 0.0023; r2 ⫽ 0.37), and controls (P ⫽ 0.012; r2 ⫽ 0.38). No significant correlations were found between BMI or subscapular skinfold thickness and urinary sOB-R concentrations in any group. Also, a positive relationship was obtained between BMI and FLI in nephrotic patients with proteinuria (P ⫽ 0.036; r2 ⫽ 0.44), patients with remission (P ⬍ 0.026; r2 ⫽ 0.49), and controls (P ⫽ 0.027; r2 ⫽ 0.32). No significant correlation was found between BMI or subscapular skinfold thickness and FLI in any group. In four patients, long-term observations were evaluated, and the courses of urinary leptin excretion and the sOB-R in serum were parallel in all patients. When urinary leptin losses showed a decrease a parallel decrease of sOB-R concentration in serum was seen. With increasing proteinuria, concentrations of urinary leptin and serum sOB-R were elevated. In all patients, proteinuric or not, the levels of serum leptin and urinary sOB-R were parallel and did not show any changes during the period of observation (Fig. 3). Discussion

We could recently demonstrate a renal urinary leptin loss in prepubertal and early pubertal children suffering from


J Clin Endocrinol Metab, November 2003, 88(11):5497–5501 5499

FIG. 3. Data courses of patients 1– 4. Shown are the data for sOB-R in serum, sOB-R in urine, leptin in serum, and urinary leptin to four different points of examination (f, sOB-R serum; ⽧, leptin urine; , leptin serum; Œ, sOB-R urine).

active nephrotic syndrome with proteinuria greater than 1 g/m2 (20). Urinary leptin loss disappeared after remission and was minimal in the control group. However, despite an up to 100-fold increment in leptin excretion in the proteinwasting group, serum leptin levels were similar in all three groups (20). This finding suggests that the renal loss of leptin must be compensated. One mechanism might be the upregulation of leptin synthesis. Up to now these mechanisms still remain unclear (21–27). Elevated secretion of stored leptin, stimulated mRNA synthesis, or an increased fraction of protein-bound leptin appear possible (28). An increase in leptin binding capacity could maintain total serum leptin concentration and prevent the urinary loss of leptin. Alternatively, bioactivity might be altered by nutritional state, BMI, or gender, rather than by its soluble binding protein (29, 30). We, therefore, studied the sOB-R, which is known to be a binding protein with high affinity to leptin in serum (17, 18). Children with massive proteinuria who reveal an enormous urinary leptin loss have significantly elevated levels of the

sOB-R in serum. In all the other patients, either in state of remission of NS (i.e. without significant proteinuria) or in control patients, we could not find elevated levels of the sOB-R in serum. Both proteinuric and nonproteinuric patients had similar leptin levels in serum (20). To estimate the biologically active form of leptin, Kratzsch et al. (17) defined the ratio between serum leptin levels and its soluble receptor as FLI. These data are confirmed by others (15). We could measure a significantly decreased level of FLI in children with massive proteinuria. Reflecting these data, we postulated that the loss of leptin excreted into the urine of nephrotic patients is compensated by elevation of its sOB-R in serum. Elevated levels of the leptin binding protein are then capable to maintain the concentration of total leptin in serum. However, the biologically active form of leptin, as suggested by decreased FLI, is significantly lower in case of renal leptin loss. The courses of four patients, all suffering from NS, emphasize the sOB-R up-regulation. In all of them, in case of proteinuria, high urinary leptin losses were found, which are

5500

J Clin Endocrinol Metab, November 2003, 88(11):5497–5501

paralleled by highly elevated concentrations of sOB-R in serum. In case of remission or reduction of proteinuria, urinary leptin loss decreased, and in parallel, sOB-R decreased. At all times in patients with remission of NS or massive proteinuria, serum leptin levels were kept stable. These data suggest that the counteracting mechanism in case of leptin loss might be the up-regulation of its sOB-R in serum. Alternatively, leptin loss in NS may result in an increased leptin synthesis, leading to an increase in the binding of the soluble receptor. Huang et al. (28) demonstrated that high levels of leptin can be caused by the delayed clearance of leptin from circulation due to binding to its soluble receptor. They concluded that the soluble receptor is up-regulated and an overexpression of the sOB-R results in an increase of circulating leptin. In our study we did not find increased serum leptin levels. However, despite an enormous leptin excretion into the urine, we did not find a decrease in serum leptin levels either. Therefore, we hypothesized that the increase in serum sOB-R compensates the urinary loss of leptin from the circulating blood to keep serum leptin levels stable. However, another reason for increased serum sOB-R levels appears possible in cases of the increased urinary loss of leptin, the shedding of the ectodomain of membrane bound, functional receptor isoforms (31). Target cells could counterregulate the density of membrane bound leptin receptor molecules based on the number of ligand-receptor interaction, which should be reduced with a decreased FLI. One important mechanism by which the increased sOB-R could be generated might be that the sOB-R is predominantly derived from the truncated leptin receptor isoform (32). Several studies have shown a relationship among BMI, subscapular skinfold thickness measurements, and concentrations of sOB-R (15, 29, 33). These observations could be confirmed in the present study. However, our data did not reveal any relationship between concentrations of urinary sOB-R and BMI or skinfold thickness, suggesting that the sOB-R clearance is regulated by a kidney-specific mechanism. NS is often associated with increased proteolytic activity, leading to degraded proteins with decreased bioactivity as shown for the IGF-binding proteins. Therefore, it could be argued that an altered size of the sOB-R molecule may be related with a different bioactivity in our patients. However, the use of a ligand-immunofunctional assay for sOB-R implicates the determination of the immunological and biological activity of this protein. Therefore, we measure degraded and nondegraded sOB-R molecules that are capable of binding leptin independently on their molecular size. On the other hand, the method does not recognize bioinactive degraded receptor proteins. Additionally, we compared the size of the sOB-R of patients with NS to normal controls using a Western blot analysis. We did not find any difference between healthy subjects and patients with NS, suggesting that most of the receptor protein is not degraded (data not shown). However, it cannot be totally excluded that a certain, minor fraction might be degraded. Taking both arguments together, we can conclude that our finding of increased sOB-R levels in patients with NS should be of biological relevance. In conclusion, we can demonstrate with our data that the


counteracting pathway in case of leptin loss in parallel with severe proteinuria in NS is the up-regulation of its soluble binding protein in serum, which can keep serum leptin levels equal. Acknowledgments Received March 27, 2003. Accepted August 7, 2003. Address all correspondence and requests for reprints to: Michael Schroth, M.D., Klinik mit Poliklinik fu¨ r Kinder und Jugendliche Friedrich-Alexander-Universita¨ t Erlangen-Nu¨ rnberg, Loschgestrasse 15, D-91054 Erlangen, Germany. E-mail: [email protected].

References 1. Halaas JL, Gajiwala KS, Maffei M 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546 2. Kiess W, Anil M, Blum WF 1998 Serum leptin levels in children and adolescents with insulin-dependent diabetes mellitus in relation to metabolic control and body mass index. Eur J Endocrinol 138:501–509 3. Do¨tsch J, Nusken KD, Knerr I, Kirschbaum M, Repp R, Rascher W 1999 Leptin and neuropeptide Y gene expression in human placenta: ontogeny and evidence for similarities to hypothalamic regulation. J Clin Endocrinol Metab 84:2755–2758 4. Breidert M, Miehlke S, Glasow A, Orban Z, Stolte M, Ehninger G, Bayerdorffer E, Nettesheim O, Halm U, Haidan A, Bornstein SR 1999 Leptin and its receptor in normal human gastric mucosa and in Helicobacter pylori-associated gastritis. Scand J Gastroenterol 34:954 –961 5. Morton NM, Emilsson V, Liu YL, Cawthorne MA 1998 Leptin action in intestinal cells. J Biol Chem 273:26194 –26201 6. Gro¨schl M, Rauh M, Wagner R, Neuhuber W, Metzler M, Tamgu¨ney G, Zenk J, Schoof E, Do¨rr HG, Blum WF, Rascher W, Do¨tsch J 2001 Identification of leptin in human saliva. J Clin Endocrinol Metab 86:5234 –5239 7. Morash B, Li A, Murphy PR, Wilkinson M, Ur E 1999 Leptin gene expression in the brain and pituitary gland. Endocrinology 140:5995–5998 8. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425– 432 9. Bergendahl M, Evans WS, Pastor C, Patel A, Iranmanesh A, Veldhuis JD 1999 Short-term fasting suppresses leptin and (conversely) activates disorderly growth hormone secretion in midluteal phase women—a clinical research center study. J Clin Endocrinol Metab 84:883– 894 10. Dallongeville J, Hecquet B, Lebel P 1998 Short term response of circulating leptin to feeding and fasting in man: influence of circadian cycle. Int J Obes Relat Metab Disord 22:728 –733 11. Pelleymounter MA, Cullen MJ, Baker MB 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540 –543 12. Lammert A, Brockmann G, Renne U, Kiess W, Bottner A, Thiery J, Kratzsch J 2002 Different isoforms of the soluble leptin receptor in non-pregnant and pregnant mice. Biochem Biophys Res Commun 298:798 – 804 13. Laimer M, Ebenbichler CF, Kaser S, Sandhofer A, Weiss H, Nehoda H, Aigner F, Patsch JR 2002 Weight loss increases soluble leptin receptor levels and the soluble receptor bound fraction of leptin. Obes Res 10:597– 601 14. Brabant G, Nave H, Mayr B, Behrend M, van Hermelen V, Arner P 2002 Secretion of free and protein-bound leptin from subcutaneous adipose tissue of lean and obese women. J Clin Endocrinol Metab 87:3966 –3970 15. Chan JL, Bluher S, Yiannakouris N, Suchard MA, Kratzsch J, Mantzoros CS 2002 Regulation of circulating soluble leptin receptor levels by gender, adiposity, sex steroids, and leptin: observational and interventional studies in humans. Diabetes 51:2105–2112 16. Voegeling S, Fantuzzi G 2001 Regulation of free and bound leptin and soluble leptin receptors during inflammation in mice. Cytokine 14:97–103 17. Kratzsch J, Lammert A, Bottner A, Seidel B, Mueller G, Thiery J, Hebebrand J, Kiess W 2002 Circulating soluble leptin receptor and free leptin index durcing childhood, puberty, and adolescence. J Clin Endocrinol Metab 87: 4587– 4594 18. Lammert A, Kiess W, Bottner A, Glasow A, Kratzsch J 2001 Soluble leptin receptor represents the main leptin binding activity in human blood. Biochem Biophys Res Commun 283:982–988 19. Orth SR, Ritz E 1998 The nephrotic syndrome. N Engl J Med 338:1202–1211 20. Schroth M, Gro¨schl M, Do¨rr HG, Blum WF, Rascher W, Do¨tsch J 2001 Renal loss of leptin in patients with nephrotic syndrome. Eur J Endocrinol 145:463– 468 21. Gro¨schl M, Wagner R, Do¨rr HG, Blum WF, Rascher W, Do¨tsch J 2000 Variability of leptin values measured from different sample matrices. Horm Res 54:26 –31 22. Kiess W, Englaro P, Hanitsch S, Rascher W, Attanasio A, Blum WF 1996 High leptin concentrations in serum of very obese children are further stimulated by dexamethasone. Horm Metab Res 28:708 –710


23. Wabitsch M, Blum WF, Muche R 1997 Contribution of androgens to the gender difference in leptin production in obese children and adolescents. J Clin Invest 100:808 – 813 24. Wabitsch M, Jensen PB, Blum WF 1996 Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes 45:1435–1438 25. Jockenhovel F, Blum WF, Vogel E 1997 Testosterone substitution normalizes elevated serum leptin levels in hypogonadal men. J Clin Endocrinol Metab 82:2510 –2513 26. Fritsche A, Wahl HG, Metzinger E 1998 Evidence for inhibition of leptin secretion by catecholamines in man. Exp Clin Endocrinol Diabetes 106:415– 418 27. Mantzoros CS, Qu D, Frederich RC 1996 Activation of beta(3)adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 45:909 –914 28. Huang L, Wang Z, Li C 2001 Modulation of circulating leptin levels by ist soluble receptor. J Biol Chem 276:6343– 6349

J Clin Endocrinol Metab, November 2003, 88(11):5497–5501 5501

29. Houseknecht KL, Mantzoros CS, Luliawat R, Hadro E, Flier JS, Kahn BB 1996 Evidence for leptin binding to proteins in serum of rodents and humans: modulation with obesity. Diabetes 45:1638 –1643 30. Gavrilova O, Barr V, Marcus-Samuels B, Reitman M 1997 Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor. J Biol Chem 272:30546 –30551 31. Ge H, Huang L, Pourbahrami T, Li C 2002 Generation of soluble leptin receptor by ectodomain shedding of membrane-spanning receptors in vitro and in vivo. J Biol Chem 277:45898 – 45903 32. Maamra M, Bidlingmaier M, Postel-Vinay MC, Wu Z, Strasburger CJ, Ross RJ 2001 Generation of human soluble leptin receptor by proteolytic cleavage of membrane-anchored receptors. Endocrinology 142:4389 – 4393 33. Van Dielen FM, van’t Veer C, Buurman WA, Greve JW 2002 Leptin and soluble leptin receptor levels in obese and weight-losing individuals. J Clin Endocrinol Metab 87:1708 –1716