Role of the kidney in human leptin metabolism

Christian Meyer1, Dave Robson1, Noya Rackovsky1, Veena Nadkarni1, and John Gerich1,2

Departments of 1 Medicine and 2 Physiology and Pharmacology, University of Rochester School of Medicine, Rochester, New York 14642

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To assess the role of the human kidney in leptin metabolism, we measured renal leptin net balance and urinary leptin excretion in 16 normal postabsorptive volunteers with varying degrees of obesity. Arterial leptin concentrations (11.6 ± 2.7 ng/ml) significantly exceeded renal vein concentrations (10.3 ± 2.5 ng/ml, P < 0.001). Renal leptin fractional extraction averaged 13.1 ± 1.1%, and renal leptin net balance (uptake) averaged 1,070 ± 253 ng/min. Lineweaver-Burk analysis indicated that renal leptin uptake followed saturation kinetics with an apparent Michaelis-Menten constant of 10.9 ng/ml and maximal velocity of 1,730 ng/min. Leptin was generally undetectable in urine. Using literature values for systemic leptin clearance, we calculated that renal leptin uptake could account for ~80% of all leptin removal from plasma. These data indicate that the human kidney plays a substantial role in leptin removal from plasma by taking up and degrading the peptide.

obesity

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LEPTIN, THE 16-kDa product of the obese gene, is currently believed to be involved in the regulation of appetite and energy expenditure (3, 7, 15, 18). Although it is well established that leptin is secreted exclusively by adipocytes (12, 21), little is known regarding its metabolism in humans aside from its systemic clearance rate, half-life, and volume of distribution (8). Mouse kidney has been reported to have a substantial number of leptin receptors (19). This observation and the fact that bilateral nephrectomy reduced plasma leptin clearance ~80% in rats (5) suggest that, in these species, the kidneys may play an important role in removal of leptin from plasma. However, in humans with end-stage renal failure of various etiologies, plasma leptin has been reported to be increased only about twofold, and no correlation was found between plasma leptin levels and creatinine clearance (13). These latter observations suggest that the human kidney may play a less important role in leptin metabolism.

To date, neither renal extraction nor urinary excretion of leptin has been reported in humans. If kidneys were the major route of clearance of plasma leptin in humans and if leptin was quantitatively excreted in urine, urinary leptin could be used to assess leptin secretion. Therefore, in the present studies, we measured urinary excretion and the net balance of leptin across the kidney in normal postabsorptive volunteers with varying degrees of obesity.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Subjects. Informed written consent was obtained from 16 normal volunteers (6 men and 10 women) after the protocol had been approved by the University of Rochester Institutional Review Board. The subjects were (means ± SE) 40 ± 3 yr of age, weighed 74 ± 4 kg (body mass index 26.1 ± 1.0 kg/m2), and had normal glucose tolerance tests according to World Health Organization criteria (20) and no family history of diabetes mellitus. For 3 days before the study, all had been on a weight-maintaining diet containing at least 200 g carbohydrate and had abstained from alcohol.

Protocol. Subjects were admitted to the University of Rochester General Clinical Research Center the evening before experiments. They consumed a standard meal (10 kcal/kg, 50% carbohydrate, 35% fat, and 15% protein) between 6:00 and 7:00 PM and were fasted overnight until experiments were completed.

Between 8:00 and 9:00 AM, a renal vein was catheterized through the right femoral vein under fluoroscopy, and the position of the catheter tip was ascertained by injecting a small amount of iodinated contrast material. The catheter was then continuously infused with a saline solution (heparinized at 5.6 U/min) to maintain patency. At 9:00 AM, a dorsal hand vein was cannulated and kept in a thermoregulated Plexiglas box at 65°C for sampling arterialized venous blood (1), and an antecubital venous infusion of p-aminohippuric acid (PAH; 12 mg/min) was started for determination of renal plasma flow. After we allowed 1 h for PAH to achieve steady state, three blood samples were collected simultaneously from the dorsal hand vein and the renal vein at 30-min intervals (0, 30, and 60 min) for determination of plasma leptin and PAH. Urine was collected in sterile containers between 6:00 and ~10:00 AM for the determination of leptin and creatinine in the urine and immediately frozen at -20°C.

Analytic procedures. Blood samples for PAH and leptin concentrations were collected in oxalate-fluoride tubes immediately placed in a 4°C ice bath. Plasma was separated within 30 min by centrifugation at 4°C. Plasma PAH concentrations were measured by a colorimetric method (2). Plasma and urine leptin concentrations were measured by a commercial radioimmunoassay (RIA) (Linco Research, St. Louis, MO). The assay limits of detection and linearity were 0.5 and 100 ng/ml, respectively (10). The day-to-day precision of the assay, expressed as coefficients of variation, ranged from 3.6 to 6.2% (10). Serum and urine creatinine were measured by standard laboratory methods.

Stability of leptin in the urine and validation of urinary leptin analysis. To determine whether urine degrades leptin, 0.5 ml 125I-labeled leptin from the RIA kit containing ~60,000 disintegrations per minute was incubated in 0.5 ml urine at 37°C for 15 h and subjected to gel permeation chromatography. A 1 × 29 cm column of Sephadex G-50 coarse, equilibrated with incubation buffer for the leptin RIA (0.05 M phosphosaline, pH 7.4, containing 0.025 M EDTA, 0.05% Triton X-100, and 1% RIA-grade bovine serum albumin), was used. The flow rate was 20 ml/h, and the eluate was collected as 375-µl fractions. Application of 0.5 ml of 125I-leptin from the RIA kit directly onto the column served as a control.

To determine whether urine interferes with the leptin RIA kit, known quantities of leptin were added to urine and incubated for 15 h at 37°C. Aliquots of urine were assayed before and after addition of 25 ng leptin.

Calculations. Renal plasma flow was determined by the PAH clearance technique (6). Renal leptin uptake was calculated as renal plasma flow × (arterial leptin concentration - renal venous leptin concentration). Because the kidney does not produce leptin (12), renal fractional extraction of leptin was calculated as (arterial leptin concentration - renal venous leptin concentration) / arterial leptin concentration × 100. Renal tissue degradation was calculated as renal leptin net balance - urinary leptin excretion. The contribution of the kidney to overall systemic removal of leptin from plasma was calculated as renal leptin uptake / overall systemic removal of leptin from plasma × 100. Overall systemic removal of leptin from plasma was calculated as mean plasma leptin concentration × mean plasma leptin clearance × mean body weight. Plasma leptin clearance was assumed to be 1.50 ml · kg-1 · min-1, based on the mean value obtained by Klein et al. (8) in 14 normal volunteers.

Statistical analysis. Unless stated otherwise, data are expressed as means ± SE. Least-square regression analysis was used for correlations and determinations of Michaelis-Menten constant (Km) and maximal velocity (Vmax) for renal leptin uptake from Lineweaver-Burk plots. A P value < 0.05 was considered to be statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

To assess whether urine degrades leptin, the recovery and elution profile of 125I-leptin incubated with urine were determined. As shown in Fig. 1, 125I-leptin incubated with urine eluted as a single peak and its recovery (88%) was comparable with that of 125I-leptin not incubated with urine. These observations indicate that urine does not appreciably degrade leptin. To determine whether urine interferes with the leptin assay, measurements of known amounts of leptin added to urine were made. As shown in Table 1, the recovery of leptin added to the urine averaged 106 ± 3%. Additionally, 250 µl leptin standard were incubated in urine for 15 h at 37°C and were serially diluted with incubation buffer for the leptin RIA. Serial dilutions from 250 µl urine from the same urine to which the leptin standard had not been added served as controls. Leptin was then analyzed by RIA on all dilutions. As shown in Table 2, the samples diluted proportionately and recovery averaged 98 ± 4%.


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Fig. 1.   Gel permeation chromatography of 125I-labeled leptin without (A) and with (B) prior incubation in urine at 37°C for 15 h. dpm, Disintegrations/min.

                              
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Table 1.   Recovery of 20 ng/ml leptin incubated in urine at 37°C for 15 h and leptin concentrations of urine to which leptin had not been added

                              
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Table 2.   Serial dilutions of 25 ng leptin incubated in urine and of urine to which leptin had not been added

Renal plasma flow and creatinine clearance averaged 827 ± 55 ml/min and 113 ± 11 ml/min, respectively. Fasting arterial plasma leptin concentrations ranged from 0.75 to 36.1 ng/ml (mean 11.6 ± 2.7 ng/ml; Table 3). As expected (11, 14), there was a positive correlation between arterial plasma leptin concentrations and body mass index (r = 0.88, P < 0.001). Renal venous leptin concentrations (10.3 ± 2.5 ng/ml) were significantly lower than arterial concentrations (P < 0.001), indicating net renal uptake of leptin. Renal leptin fractional extraction averaged 13.1 ± 1.1% and was inversely related to arterial plasma leptin concentrations (r = -0.58, P < 0.03; Fig. 2). There was no correlation between renal fractional extraction of leptin and creatinine clearance (r = 0.034, P > 0.9). Renal leptin clearance averaged 110 ± 10 ml/min and was greater in men than in women (141 ± 19 vs. 91 ± 11 ml/min).

                              
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Table 3.   Renal plasma flow; arterial, renal vein, and urinary leptin concentrations; renal leptin fractional extraction; and renal leptin uptake on normal postabsorptive volunteers


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Fig. 2.   Correlation between renal leptin fractional extraction and arterial leptin concentrations.

Renal leptin uptake averaged 1,070 ± 253 ng/min. Urinary leptin was detectable in only four cases and ranged in these subjects between 0.8 and 1.0 ng/ml, which corresponded to excretion rates of 0.13-1.29 ng/min. In the other 12 subjects, urinary leptin was <0.5 ng/ml and thus below the sensitivity of the leptin RIA. With the assumption of values at the sensitivity of the assay, this could account at most for only 0.65 ± 0.09 ng/min urinary leptin excretion. Consequently, renal tissue leptin uptake (overall uptake minus urinary excretion) closely approximated overall renal uptake (1,069 ± 253 ng/min).

The decrease in renal leptin fractional extraction as a function of arterial leptin concentrations suggested that renal leptin uptake may be a saturable process subject to Michaelis-Menten kinetics. This was therefore assessed by the Lineweaver-Burk analysis (Fig. 3). There was a highly significant inverse correlation between 1/renal leptin net balance and 1/arterial plasma leptin concentration (r = -0.93, P < 0.001). The calculated apparent Km and Vmax were 10.9 ng/ml and 1,730 ng/min, respectively, consistent with Michaelis-Menten kinetics for the removal of plasma leptin by the kidney.


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Fig. 3.   Lineweaver-Burk analysis of renal leptin uptake. Km, Michaelis-Menten constant; Vmax, maximal velocity.

The mean estimated overall systemic removal of leptin from plasma in our subjects, calculated by using the mean clearance data of Klein et al. (8), was 1,288 ng/min (11.6 ng/ml × 1.50 ml · kg-1 · min-1 × 74 kg). Renal leptin uptake accounted for ~80% of this.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present studies indicate that, in postabsorptive normal human volunteers, 1) the kidneys account for a substantial proportion of overall systemic leptin removal from the circulation, 2) little or no leptin cleared by the kidneys appears in the urine, 3) thus renal removal of leptin from plasma involves tissue degradation rather than mere urinary excretion, and, finally, 4) the removal of leptin from plasma by the kidneys is a saturable process following Michaelis-Menten kinetics.

We found that the kidneys could account for ~80% of the overall systemic removal of leptin from plasma. This calculation was based on the use of a mean value for systemic leptin clearance from the data of Klein et al. (8). Such an approach assumes that systemic leptin clearance does not vary with plasma leptin concentrations and could actually underestimate the contribution of the kidney in people with high leptin levels if systemic leptin clearance decreases as plasma leptin increases. This appears to be the case, since we found that renal fractional extraction decreased as plasma leptin increased. Moreover, we found that renal leptin uptake could be ascribed to a saturable process as assessed by the Lineweaver-Burk analysis.

It is of interest that the calculated apparent Km of 10.2 ng/ml for renal leptin uptake approximates the mean arterial plasma leptin concentration in our subjects. This indicates that renal leptin uptake would be half-maximal at physiological plasma leptin concentrations and that small increases in leptin secretion or small decrements in renal uptake would result in relative disproportional increases in plasma leptin.

In general, we found urinary leptin excretion to be negligible compared with overall renal leptin uptake. We also found that renal leptin fractional extraction was unrelated to creatinine clearance. These observations strongly suggest that leptin is taken up and degraded by renal parenchymal cells independently of glomerular filtration rate and are consistent with the finding of Merabet et al. (13). Although the renal metabolic pathway for leptin is not known, there is evidence for glomerular filtration followed by tubular reabsorption and peritubular extraction of various hormones such as insulin, C-peptide, glucagon, and parathyroid hormone (16). Similar to the finding in the present study, few of these hormones are found in the urine compared with their overall clearance by the kidney (16). It is of interest to note that men had greater renal clearance of leptin than women. Because renal fractional extraction of leptin was not significantly different, this appears to be explicable on the basis of greater renal plasma flow in men (980 ± 93 vs. 736 ± 48 ml/min, P = 0.031).

From our data suggesting an important role of the human kidney in leptin metabolism, one would expect that plasma leptin levels should be markedly increased in people with end-stage renal disease. The fact that Merabet et al. (13) found only a twofold elevation of plasma leptin in such patients suggests that in end-stage renal disease, either other tissues become more important for leptin removal from plasma or there may be reduced leptin release from adipose tissue. Altered body composition with a reduction of adipose tissue and/or reduced food intake, as is commonly observed in people with end-stage renal disease (4, 9, 17), might also affect plasma leptin concentrations.

In conclusion, the results of the present study indicate that, in postabsorptive normal volunteers, the kidneys account for a substantial proportion of the overall systemic leptin removal from the circulation. Renal leptin removal from plasma is due to renal tissue leptin uptake and degradation rather than glomerular filtration and urinary excretion and is subject to a saturable process following Michaelis-Menten kinetics.

    ACKNOWLEDGEMENTS

The present work was supported in part by Grants DK-20411 and 5MOI-RR-00044 from the National Institutes of Health.

    FOOTNOTES

Address for reprint requests: J. E. Gerich, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Box MED/CRC, Rochester, NY 14642.

Received 21 April 1997; accepted in final form 23 July 1997.

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Abstract
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Discussion
References

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AJP Endocrinol Metab 273(5):E903-E907
0193-1849/97 $5.00 Copyright © 1997 the American Physiological Society