Association of Increased Serum Leptin with Ameliorated Anemia and Malnutrition in Stage 5 Chronic Kidney Disease Patients after Parathyroidectomy

Leptin is an adipokine that regulates various metabolism, but its association with secondary hyperparathyroidism (SHPT), a clinical manifestation of chronic kidney disease-mineral and bone disorder (CKD-MBD), remains obscure. Parathyroidectomy (PTX) is recommended for severe SHPT patients. Here, the associations between circulating leptin and clinical characteristics in CKD patients were investigated. Effects of PTX on leptin production were analyzed in vivo and in vitro. Controls and CKD patients had approximate serum leptin levels in that a larger proportion of CKD patients with body mass index (BMI) <23 kg/m2. Serum leptin was related to anemia, albumin, and bone metabolism disorders in CKD patients. Lower intact parathyroid hormone (PTH) was related with higher leptin in PTX patients group. Severe SHPT inhibited uremia-enhanced leptin production in 3T3-L1 adipocytes, which was attenuated after PTX. High levels of PTH were found to reduce Akt phosphorylation and leptin production in vitro but high levels of calcium and phosphorus were not. Successful PTX was found to improve anemia and malnutrition in severe SHPT patients, and this was correlated with increased circulating leptin levels via up-regulated Akt signaling in adipocytes. These findings indicated the therapeutic potential of leptin and related target pathway for improving survival and quality of life in CKD.


Protocols
It has been previously shown that leptin release is a pulsatile manner, and modulated by additional short-term signals, i.e., the levels increase acutely after a meal, and are suppressed with fasting 1 . Leptin concentrations also follow a diurnal rhythm which is highest between midnight and early morning and lowest in midafternoon 2 . In our study at enrollment, venous whole blood samples were drawn in the morning from the participants with an overnight fast. For hemodialysis patients, blood samples were collected before dialysis. Clinical characteristics, medical history, and use of anti-hypertension medications were strictly recorded.

Analysis of Laboratory Values
Routine blood tests were performed using the LH-750 Hematology Analyzer from Beckman Coulter, Inc. Fullerton, CA. Biochemical indices were measured using an Automatic Biochemical Analyzer named AU5400 from Olympus Corporation, Tokyo, Japan. Serum iPTH levels were measured using a UniCel DxI800 Access Immunoassay System from Beckman Coulter, Inc. Fullerton, CA. Human serum leptin levels were measured using the Human Leptin Quantikine ELISA Kit from R&D Systems, Minneapolis, USA. The inter-assay variation coefficient of human leptin ELISA kit ranged from 3.0% to 3.3%. The lower sensitivity limit of the assay was 7.8 pg/ml.

Reagents and Chemicals
Dulbecco's Modified Eagle Medium/high glucose supplemented (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, New York, USA. 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, rat parathyroid hormone (1-34) and oil red O were obtained from Sigma-Aldrich, Louis, USA. Anti-leptin antibody was obtained from Abcam, Cambridge, UK. Antibody against Akt, phosphate-Akt (Ser473) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology, Boston, USA. LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor) was from MedChem Express, New Jersey, USA. ECL Detection Systems were from Amersham, Buckinghamshire, UK. Mouse Leptin Quantikine ELISA Kits used for measurement of leptin in cell culture media were from R&D Systems, Minneapolis, USA. The inter-assay variation coefficient of mouse leptin ELISA kit ranged from 3.3% to 4.3%. The lower sensitivity limit of the assay was 22 pg/ml.

Western Blot
The immunoblotting procedure was previously described 3 . Briefly, 30 μg protein extracts were separated by acrylamide electrophoresis and transferred to polyvinylidene fluoride membranes. After 1 hour incubation at room temperature in 5% dry milk powder, the membranes were immunoblotted with primary antibodies against Leptin (1:2000), phosphate-Akt (1:1000), Akt (1:1000) or GAPDH (1:1000), and followed by the addition of HRP-labeled secondary antibodies. The blots were visualized with Amersham ECL Detection Systems. Quantitative analysis of immunoblotting images was performed using Image Lab software from Bio-Rad Laboratories, California, USA.

Clinical Definition
Successful PTX was defined as normalization of serum calcium, serum phosphorus, alkaline phosphatase, and maintenance of no more than two to three times serum iPTH compared with normal (ranging from 10-88 pg/ml). The serum iPTH levels detected during the first postoperative week >300 pg/ml were considered indicative of persistent SHPT 4 . In these cases, SHPT was partially but not completely corrected. Patients with persistent SHPT after PTX were commonly considered to have supernumerary/ectopic parathyroid glands and remain one or two parathyroid glands while most of them had being moved during surgery 5 . The diagnosis of anemia was made at the hemoglobin concentrations: <13.5 g/dl in adult males, <12.0 g/dl in adult females 6 .