Advanced Glycation End Products in Hemodialyzed Patients with Diabetes Mellitus Correlate with Leptin and Leptin/Body Fat Ratio

Abstract

Advanced glycation end products (AGEs) and other carbonyl and oxidative stress compounds are supposed to play a critical role in the pathogenesis of several diseases and their complications, i.e., diabetes mellitus, diabetic retinopathy, atherosclerosis, and chronic renal failure. In the present investigation, we were interested in the relationship of AGEs in plasma to other prominent factors in the patients on chronic hemodialysis treatment—27 patients with diabetes mellitus, 35 patients without diabetes mellitus. AGE-group reactivity was estimated using a spectrofluorometric method (excitation 350 nm, emission 430 nm) and is expressed in arbitrary units (AU). We found significantly higher AGEs levels in diabetics than in non-diabetics on regular hemodialysis treatment both before (2.7 ± 0.7 × 10⁴ AU vs. 2.2 ± 0.6 × 10⁴ AU, p<0.001) and after the dialysis session (2.3 ± 0.5 × 10⁴ AU vs. 1.8 ± 0.7 × 10⁴ AU, p<0.005). AGEs were significantly reduced during hemodialysis in both groups of patients—by 15.4 % in the diabetic go (p<0.001) and by 17.3% in non-diabetics (p<0.005). In the patients with diabetes mellitus, AGEs did not correlate with parameters of the glucose metabolism correction (blood glucose, HbA1c). We observed a significant correlation between AGEs and leptin (r = 0.48, p < 0.05) as well as the leptin/body fat ratio (r = 0.56, p < 0.05) only in hemodialyzed patients with diabetes mellitus. These findings suggest more detailed studies to identify the molecular links between carbonyl stress, i.e., advanced glycation end products, and leptin metabolism, sign of microinflammation and hypertension.

• Advanced glycation end products
• Leptin; Hemodialysis
• Diabetes mellitus
• Oxidative stress
• Carbonyl stress

Introduction

Advanced glycation end products (AGEs) are shown to play a role in the pathogenesis of several diseases and their complications, i.e., diabetes mellitus, diabetic retinopathy, atherosclerosis, and chronic renal failure.[[1]], [[2]], [[3]] Various biochemical pathways can contribute to the formation and increase of AGE species in the organism, i.e., non-enzymatic glycation (Maillard reaction),[[4]] increase of oxidative and carbonyl stress compounds,[[2]], [[5]] decreased renal function and AGE-precursors from the diet and tobacco smoke.[[6]]

By the reaction sequence, peptides and proteins undergo structural and conformational modification, i.e., change of the biochemical reaction pattern, which causes damage to the extracellular matrix. AGEs (among them the best characterized are carboxymethyllysine and pentosidin) can circulate in a large variety of chemical heterogeneity in body fluids and accumulate in tissues. They can also act via their specific receptors (e.g., RAGE—receptor for advanced glycation end products). AGE-RAGE interaction activates nuclear factor NF-κB, stimulates transcription of genes for cytokines, growth factors, and adhesive molecules, induces migration of macrophages and has further toxic effects.[[7]], [[8]]

Advanced glycation end products take a considerable part in the pathogenesis of diabetic complications—nephropathy, angiopathy, neuropathy, and retinopathy. They can modify several important proteins and so change their function (e.g., fibrinogen, antithrombin III),[[9]] cause damage to biological membranes and endothelium, modify LDL particles, and so, together with the vascular damage, are involved in the acceleration of atherosclerosis.[[1]]

In end stage renal disease patients, AGEs are elevated more than in diabetic patients, which indicates a specific role of reduced renal function on the AGE- and carbonyl compounds metabolism. AGEs play a key role in the development of accelerated atherosclerosis and dialysis related amyloidosis,[[10]] and can participate in the damage to peritoneum in the patients treated with continuous ambulatory peritoneal dialysis.[[11]]

The aim of this cross-sectional study was to compare AGEs in hemodialyzed patients with and without diabetes mellitus before and after the dialysis session and to find out their relationship to other parameters (age, duration of the dialysis treatment, parameters of compensation of diabetes mellitus—blood glucose and HbA1c, leptin and body fat mass).

Patients and Methods

Patients

The study was performed cross-sectionally in a non-selected study population consisting of 62 patients on regular hemodialysis treatment, i.e., 27 patients with diabetes mellitus (13 males and 14 females), mean age 72 ± 11 years (37–89 years), dialyzed for 3 ± 2 years (1–7 years), blood glucose concentration 10.5 ± 5.0 mmol/L, glycated hemoglobin 6.9 ± 1.5% (HPLC), and 35 patients without diabetes mellitus (15 males and 20 females), mean age 64 ± 13 years (33–94 years), dialyzed for 4 ± 4 years (1–15 years). The patients were dialyzed three times a weak for 4 h using dialyzers with synthetic or hemicellulosic membranes. The blood flow was 250–300 mL/min and the dialysate flow 500 mL/min. All patients received heparin during the dialysis session (2000–4000 IU) and were treated with erythropoietin at a mean dose of 60 U/kg body weigh/week. All patients were in stabilized status without acute infection and were not taking any antioxidants or trace elements. They gave their informed consent prior to entering this study.

Blood Samples

Blood samples from each patient were collected via the arteriovenous shunt at the beginning and at the end of the hemodialysis session. Blood was centrifuged at 1450 g, 4°C for 10 min and heparin-plasma was stored at −80°C.

AGEs Quantification

A spectrofluorometric method according to Henle et al.[[12]] was used for AGEs determination. Plasma was diluted 1:50 with PBS and emission at 430 nm upon excitation at 350 nm was recorded (spectrofluorometer Perkin, Elmer, Ueberlingen, Germany). Fluorescence intensity is expressed in arbitrary (fluorescence) units (AU).

Leptin

Serum leptin concentrations were determined with ELISA kit (Biovendor, CZ) according to the instructions of the manufacturer.

Body Fat

The amount of the body fat was determined according to Allen et al. (measurement of the thickness of the skinfold).[[13]]

Statistics

All results are expressed as mean ± standard deviation (SD). The statistical significance was evaluated using paired and unpaired Student's t-test and correlation coefficient r for regression analysis. All results were considered as statistically significant at p<0.05.

Results

We found significantly higher AGEs levels in the patients with diabetes mellitus than in the patients without diabetes mellitus on regular hemodialysis treatment both before (2.7 ± 0.7 × 10⁴ vs. 2.2 ± 0.6 × 10⁴, respectively, p<0.001) and after the dialysis session (2.3 ± 0.5 × 10⁴ vs. 1.8 ± 0.7 × 10⁴, respectively, p<0.005) (Table 1). Advanced glycation end products were significantly reduced during hemodialysis in both groups of patients—by 15.4% in diabetics (p<0.001) and by 17.3% in non-diabetics (p<0.005). We did not find any significant difference in leptin levels, in the amount of body fat, and in the ratio leptin/fat in both studied groups. There was no correlation between AGEs levels and age and AGEs levels and the length of the dialysis treatment (neither in diabetics nor in non-diabetics). In the patients with diabetes mellitus, AGEs did not correlate with parameters describing correction of the glucose metabolism, i.e., blood glucose and HbA1c. Interestingly, we observed significant correlation between AGEs and leptin (r = 0.48, p<0.05) (Fig. 1) as well as AGEs and the ratio leptin/body fat (r = 0.56, p<0.05, Fig. 3) only in hemodialyzed patients with diabetes mellitus. No such a correlation was observed in the group of patients without diabetes mellitus (Figs. 2 and 4).

Table 1. Comparison of hemodialyzed patients with and without diabetes mellitus

	Hemodialyzed patients with diabetes mellitus (N = 27)	Hemodialyzed patients without diabetes mellitus (N = 35)
AGEs (AU)
Before dialysis	2.7 ± 0.7 × 10^4 a	2.2 ± 0.6 × 10^4
After dialysis	2.3 ± 0.5 × 10^4 bc	1.8 ± 0.7 × 10^4 d
Leptin (ng/mL)	25.8 ± 23.3	21.4 ± 19.8
Body fat (%)	25.4 ± 8.6	23.2 ± 9.2
Leptin/body fat	0.9 ± 0.8	0.9 ± 0.7

^ap<0.001;

^bp<0.005 vs. hemodialyzed patients without diabetes mellitus;

^cp<0.001;

^dp<0.005 vs. before dialysis.

Figure 1. Correlation of AGEs with leptin in hemodialyzed patients with diabetes mellitus. r = 0.48, p < 0.05, y = −20.92 + 0.0017 × x.

Figure 2. Correlation of AGEs with leptin in hemodialyzed patients without diabetes mellitus. r = −0.24, not significant, y = 38.728 − 0.008 × x.

Figure 3. Correlation of AGEs with leptin/body fat ration in hemodialyzed patients with diabetes mellitus. r = 0.56, p < 0.05, y = −0.9159 + 0.00007 × x.

Figure 4. Correlation of AGEs with leptin/body fat in hemodialyzed patients without diabetes mellitus. r = −0.0187, not significant, y = 0.89711 − 0.0000 × x.

Discussion

Patients with diabetes mellitus on regular dialysis treatment have usually more complications and higher mortality than hemodialyzed patients without diabetes mellitus. Several articles have reported about AGEs in diabetic and dialyzed patients. However, the studies were performed mainly on smaller number of patients (e.g., 10 dialyzed patients with diabetes mellitus and 10 without), various methods were used for AGEs determination (thus measuring only a part of the heterogenous group of AGEs) and also the results differ. Several investigators refer no differences between dialyzed diabetics and non-diabetics (Refs.,[[14]], [[15]], [[16]], [[17]] 12-total AGE-fluorescence). On the other hand, Makita et al.[[18]] and Vlassara[[19]] describe higher AGE-levels in hemodialyzed patients with diabetes mellitus. Determination of fluorescent advanced glycation end products in hemodialyzed patients with and without diabetes mellitus in the presented study performed in a large cohort of patients describes significantly higher levels in diabetics. This enables the hypothesis of possible contribution of AGEs to higher morbidity and mortality of diabetic patients on regular dialysis treatment, as they take a considerable part in both diabetic and uremic complications. Thus AGEs could be considered as potential confounding factors certainly present in diabetic as well as non-diabetic dialysis patient population.

We did not find any significant correlation of AGEs with parameters of correction of the glucose metabolism in diabetes mellitus, i.e., blood glucose and HbA1c. However, formation of AGEs, apart of non-enzymatic glycation, is possible also via other pathways (oxidative stress, carbonyl stress, contribution of the diet, and of decreased renal function to AGEs elevation) which might play a more important role. Nevetherless, there is a difference in the composition of the fluorescence fraction in the serum of diabetic and non-diabetic patients on hemodialysis treatment. Henle et al. described a significantly higher fluorescence of the low molecular weigh fraction (bellow 2 kDa) and higher predialysis plasma concentrations of the Amadori product fructoselysine (a major precursor of CML) in diabetics.[[12]] Similarly, Stein et al. observed higher pre-dialysis AGE-peptides concentrations in the diabetic group (contrary to pentosidine).[[20]]

An interesting finding is the relationship between AGEs and leptin and AGEs and the ratio leptin/body fat only in hemodialyzed patients with diabetes mellitus thus enabling a hypothesis of nutritional effects in combination with altered carbohydrate metabolism on the AGEs level in these patients. However, patients with renal failure have usually hyperleptinemia along with anorexia and a tendency to low body fat.[[21]] Elevation of leptin in hemodialyzed patients has several reasons, including decreased renal excretion[[22]] (also a possible reason for AGEs elevation) and possible increased activation of proinflammatory cytokines.[[23]] Additionally, transcription of genes for cytokines may be activated as a result of interaction of AGEs with their specific receptor RAGE.[[24]], [[25]] Other authors refer no relationship between leptin and serum TNFα, soluble TNF receptor and IL-6[[26]] and describe leptin as a negative acute phase protein, which correlates negatively with CRP and positively with albumin.[[27]] Moreover, serum leptin correlates with the atherosclerotic index both in hemodialyzed men and women[[26]] and AGEs are known to play a significant role in atherosclerosis as a complication of several chronic diseases as well. Another link is that to hypertension. Hypertensive patients have higher leptin than controls and leptin levels correlate with blood pressure.[[28]] Additionally, elevated methylglyoxal and advanced glycation end products in cells from hypertensive rats were demonstrated recently and therefore it is suggested, that elevated AGEs and associated oxidative stress might possibly contribute to the pathogenesis of hypertension.[[29]]

We can conclude, that AGEs levels in hemodialyzed patients with diabetes mellitus are significantly higher than in hemodialyzed without diabetes mellitus. This finding enables the hypothesis of potential, probably synergistic, contribution of AGEs to higher morbidity and mortality of diabetics on regular dialysis treatment owing to their contribution both to diabetic and uremic complications. Nevertheless, advanced glycation end products, their formation, metabolic pathways, and adverse biological effects seem to be a multifactorial problem, which requires further investigation before definite therapeutic measures can be proposed.

Acknowledgment

This study was supported by grant IGA MH CZ No NB/7035-3.