Katarína Šebeková

Markedly elevated levels of plasma advanced glycation end products in patients with liver cirrhosis – amelioration by liver transplantation

BACKGROUND/AIMS Modification by advanced glycation renders macromolecules susceptible to elimination in the liver via scavenger receptors. Thus, in advanced liver disease an accumulation of advanced glycation end products (AGEs) in circulation might occur, due to the reduction of effective liver mass. METHODS Plasma AGE levels (fluorescent AGEs-AGE-Fl and N(epsilon)-carboxymethyllysine - CML) were determined in 51 patients with liver cirrhosis (Ci) and 19 healthy controls. Five patients were followed 36 months after liver transplantation. RESULTS In cirrhotic patients, markedly elevated concentrations of AGEs were revealed (AGE-Fl: control, 0.3+/-0.01 x 10(5) AU, Ci: 1.06+/-0.06 x 10(5) AU, P<0.01; CML, control: 431.7+/-16.3 ng/ml, Ci: 647.6+/-258.5, P<0.01). CML levels correlated with the severity of liver disease, as determined by clinical score (r=0.663, P<0.001), albumin level (r=0.704, P<0.001) and monoethylglycinexylide test (r=0.852, P<0.01). Reduced renal function contributed to the rise of CML in proportion to the degree of renal impairment. Liver transplantation resulted in about 50% decline of CML levels within 3 months, while impairment of renal function still persisted, underlying the central role of the liver for AGE removal. CONCLUSIONS In liver Ci, hepatic removal of AGEs is impaired. With regard to the toxicity of AGEs, their accumulation could be of pathophysiological relevance.

Genotoxicity of advanced glycation end products in mammalian cells

In patients with chronic renal failure, cancer incidence is enhanced. Since levels of advanced glycation end products (AGEs) are markedly elevated in renal insufficiency, we investigated potential effects of various AGEs on structural DNA integrity in tubule cells. The comet-assay was employed, a method based on the computer-aided microscopic analysis of single cells after electrophoretic separation of their nuclear DNA. Incubation of pig kidney LLC-PK1-cells for 24 h with AGE-BSA (AGE-bovine serum albumin), carboxymethyllysine-BSA as well as methylglyoxal-BSA resulted in a significant increase in DNA damage. Pretreatment of the cells with the proteases trypsin and bromelain abolished the AGE-induced comet-formation. This is in agreement with the idea that the observed genotoxicity of AGEs could be receptor-mediated and that proteases inactivate the extracellular domain of the receptor for AGEs. Binding of AGEs to the RAGE receptor leads to an increased intracellular formation of active oxygen species, which are known to induce DNA damage. It is concluded that AGEs induce genotoxicity in tubule cells, which may be involved in the enhanced cancer development in advanced kidney diseases.

Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats

Advanced glycation end products (AGEs) play a key role in the pathogenesis of diabetes and its complications, including the diabetic nephropathy. The renoprotective effects of exercise are well known; however, the mechanisms remain elusive. Here we examined whether a regular moderate exercise in obese Zucker rats (OZR), a model of diabetes- and obesity-associated nephropathy, will affect the development of early renal injury in OZR possibly via alteration of AGEs formation. The OZR were left without exercise (sedentary) or subjected to 10 weeks intermittent treadmill running of moderate intensity. Compared with sedentary OZR, kidneys of running OZR had significantly less glomerular mesangial expansion and tubulointerstitial fibrosis. Running OZR had significantly lower plasma AGEs-associated fluorescence and N(epsilon)-carboxymethyllysine. Correspondingly, renal AGEs and N(epsilon)-carboxymethyllysine content were lower in running OZR. Systemically, exercise increased aerobic metabolism, as apparent from urinary metabolite profiling. No differences in plasma glucose, insulin, or lipid profile were found between the 2 groups. Apart from lower advanced oxidation protein products (a marker of myeloperoxidase activity), no other marker of inflammation was altered by exercise, either systemically or locally in kidneys. No indication of changed oxidative status was revealed between the groups. Exercise in OZR decreased advanced glycation. This might represent the early event of exercise-induced renoprotection in diabetic nephropathy in OZR. If confirmed in clinical studies, regular moderate exercise could represent an easy and effective nonpharmacologic approach to reduce advanced glycation.

Plasma Concentration and Urinary Excretion of Nɛ-(Carboxymethyl)lysine in Breast Milk– and Formula-fed Infants

Industrial processing of infant formulas (IFs) induces the formation of Maillard products, namely Nɛ‐(carboxymethyl)lysine (CML). CML content is expected to be several times higher in IFs than in fresh human breast milk. To elucidate whether CML is absorbed from IFs into the bloodstream, CML concentration in the plasma and urine were analyzed in 6‐month‐old infants (34 breast fed and 25 fed exclusively with IFs) and in 56 samples of human breast milk and 16 commercial IFs. We found that IFs contain higher amounts of CML compared to mothers milk (median: 70‐fold; range: 28‐ to 389‐fold), and CML content was higher in hydrolyzed IFs than in nonhydrolyzed IFs (P < 0.03). Plasma CML levels were 46% higher (P < 0.01) and urinary excretion of CML was 60‐fold higher (P < 0.001) in the formula‐fed infants than in the breast‐fed group. Infants fed with hydrolyzed IFs displayed significantly higher plasma CML levels than those on nonhydrolyzed formulations. We conclude that CML from IFs is absorbed into the circulatory system and is rapidly excreted in the urine.

Plasma advanced glycation end products are decreased in obese children compared with lean controls

OBJECTIVE In obesity, the combined effects of enhanced food consumption, enhanced oxidative stress and microinflammation could augment the advanced glycation end products (AGEs) accumulation in plasma. We compared the plasma concentrations of AGEs and the soluble receptor for AGEs (sRAGE) in relation to markers of oxidative stress, microinflammation and renal function in obese and lean children/adolescents. METHODS In 18 apparently healthy obese children/adolescents (7 females/11 males; age: 5-18 years; body mass index, BMI: 27.3+/-3.3 kg/m2) and 18 healthy lean controls (10 females/8 males; age: 4-17 years, BMI: 22.4+/-2.1 kg/m2) the plasma concentration of N(epsilon)-carboxymethyllysine (CML), fructoselysine (FL), AGE-associated fluorescence, sRAGE, high sensitive-C-reactive protein (hsCRP), interleukin-6 (IL-6) and urinary 8-hydroxy-2-deoxyguanosine (U-8-OHdG) excretion, plasma advanced oxidation protein products (AOPPs), renal function, and the HOMA index of insulin resistance were determined. RESULTS Obese children/adolescents had significantly lower concentrations of plasma FL (6.8+/-0.3 mmol/mol lysine vs. 7.7+/-0.3, p<0.02), CML (0.14+/-0.03 mmol/mol lysine vs. 0.22+/-0.04, p<0.001), and fluorescent AGEs (223+/-37 arbitrary units (AU) vs. 318+/-64, p<0.01) than their lean counterparts. Plasma sRAGE concentration did not differ (2.3+/-0.6 ng/ml vs. 2.6+/-0.6). Obese children/adolescents were more insulin-resistant (HOMA index: p<0.01), exhibited higher levels of markers of inflammation (hs-CRP: p<0.03; IL-6: p<0.02), of oxidative stress (AOPPs: p<0.05; 8-OHdG: p<0.04) and had a higher creatinine clearance (p<0.01) and proteinuria (p<0.01). CONCLUSIONS We present the first evidence that childhood/adolescent obesity is characterized by lower plasma AGE levels, despite lower insulin sensitivity, enhanced oxidative stress and microinflammation. An enhanced removal of AGE peptides via hyperfiltration may partially contribute to the lower plasma AGE levels.

Dietary bread crust advanced glycation end products bind to the receptor for AGEs in HEK-293 kidney cells but are rapidly excreted after oral administration to healthy and subtotally nephrectomized rats.

Abstract: In renal HEK‐293 cells, the dietary Maillard reaction compounds casein‐linked Nε‐carboxymethyllysine (CML), CML, bread crust (BC), and pronyl‐glycine (a key compound formed in association with the process‐induced heat impact applied to bread dough) all showed activation of p38‐MAP kinase. Expression of the C‐terminus truncated receptor for advanced glycation end products (RAGE) resulted in a reduction of HEK‐293‐MAP kinase activation. As these findings suggested a RAGE‐mediated activating effect of CML, BC, and pronyl‐glycine on kidney cellular signal transduction pathways, an in vivo study was performed. Male Wistar rats were subjected to a sham operation (CTRL, n= 20) or to 5/6 nephrectomy (NX, n= 20). Both groups were randomized into two subgroups and fed 20 g of a diet containing either 25% by weight BC or wheat starch (WS). GC‐MS analyses of CML, carboxyethyllysine (CEL), and pentosidine revealed increased levels of CML and CEL in the liver but decreased levels of CML in the kidneys of CTRL and NX rats fed the BC diet compared to those on the WS diet. However, urinary levels of CML were also elevated in the CTRL and NX rats on the BC diet, pointing to enhanced excretion of AGEs after BC administration. Although renal insufficiency in the NX rats was reflected by proteinuria, the renal handling of CML and, presumably, other AGEs was not impaired.

Renal effects of oral maillard reaction product load in the form of bread crusts in healthy and subtotally nephrectomized rats

Abstract: The biological consequences of chronic consumption of Maillard reaction products (MRPs) on renal function in health and renal disease are still incompletely understood. We investigated the metabolic and renal effects of a diet with varying MRP content in healthy and subtotally nephrectomized rats. Male Wistar rats were subjected to sham operation (control, C, n= 12), or to 5/6 nephrectomy (5/6NX, n= 12). Both groups were randomized into subgroups and pair‐fed with either a MRP‐poor or ‐rich diet for six weeks. The diet was prepared by replacing 5% or 25% of wheat starch by bread crust (BC). In spite of pair‐feeding, the rats on the 25% BC diet gained more body weight (C: 183 ± 6 g; C + 5% BC: 197 ± 7 g; C + 25% BC: 229 ± 6 g [P < 0.05]; 5/6NX: 165 ± 10 g; 5/6NX + 5% BC: 202 ± 3 g; 5/6NX + 25% BC: 209 ± 8 g [P < 0.05]) and had a higher organ weight (heart, liver, lung, kidney/remnant kidney). Bread crust‐enriched diet induced proteinuria (C: 15 ± 5 mg/24 h; C + 5% BC: 19 ± 4; C + 25% BC: 26 ± 3 [P < 0.05]; 5/6NX: 30 ± 7 mg/24 h; 5/6NX + 5% BC: 47 ± 9; 5/6NX + 25% BC: 87 ± 19 [P < 0.01]) and a rise in urinary transforming growth factor β1 excretion (C: 0.4 ± 0.1 ng/24 h; C + 5% BC: 0.6 ± 0.1; C + 25% BC: 1.2 ± 0.3; 5/6NX: 0.5 ± 0.1 ng/24 h; 5/6NX + 5% BC: 0.9 ± 0.1; 5/6NX + 25% BC: 1.6 ± 0.2 [P < 0.01]). Plasma creatinine or creatinine clearance were not affected significantly. In conclusion, our data suggests that long‐term consumption of a diet rich in MRPs may lead to damage of the kidneys.

Plasma levels of advanced glycation end products in children with renal disease

Abstract. In adults, advanced glycation end products (AGEs) rise slowly in tissues and circulation during aging, and accumulate at an accelerated rate both in diabetes and chronic renal insufficiency (CRI). We aimed to investigate the pattern of AGE accumulation in children/adolescents with CRI and on renal replacement therapy by dialysis and transplantation. Concentrations of fluorescent AGEs, carboxymethyllysine (CML) and lipofuscin-like substance (LFLS, a marker of lipid peroxidation) were followed. Data were obtained from 11 CRI patients on conservative treatment (age 12.6±1.7 years, serum creatinine: 205.7±17.5 µmol/l), ten patients on renal replacement therapy with dialysis (13.6±1.7 years, 698.2±48.9 µmol/l) and nine patients after kidney transplantation (15.9±1.1 years, 115.9±12.0 µmol/l) and comparison made with the data from 28 healthy controls (11.8±8.2 years, 44.1±8.2 µmol/l). In controls, an age-dependent rise of fluorescent AGE and CML levels was observed. In the CRI group, fluorescent AGEs [0.38±0.03×105 arbitrary units (AU)] and CML (369±26 ng/ml) concentrations were doubled compared with controls (0.16±0.03×105 AU and 189±42 ng/ml, respectively) and even higher levels were revealed in dialyzed patients (0.80±0.05×105 AU; 650±94 ng/ml). Successful kidney transplantation significantly reduced but did not normalize fluorescent AGE levels (0.39±0.03 ×105 AU), while the decline in CML levels (550±47 ng/ml) was insignificant. Plasma LFLS was elevated in CRI (19.6± 1.7 AU) and was even higher in dialyzed children (32.0±5.3 AU) compared with healthy controls (7.1± 1.4 AU). Kidney transplantation did not normalize LFLS levels (20.3±5.3 AU), pointing to persistently enhanced lipid peroxidation. Our study provides the first data on enhanced fluorescent AGEs and CML levels in children/adolescents with CRI and on dialysis. Successful renal transplantation decreased but did not normalize AGE levels, probably because of still-impaired renal function with enhanced oxidative stress, as well as the influence of immunosuppressive therapy.

Advanced glycation end-product levels in subtotally nephrectomized rats: beneficial effects of angiotensin II receptor 1 antagonist losartan.

The angiotensin II receptor 1 antagonist losartan (L) inhibited the advanced glycated end-products (AGEs) induced expression of transforming growth factor β1 in in vitro experiments performed on renal tubuloepithelial cells. To test the pathophysiological importance of these findings, the possible link between serum AGEs levels and angiotensin system was investigated in the model of normotensive subtotally nephrectomized rats(4/6-NX). Concentration of AGEs in serum of placebo administered 4/6-NX rats (n = 7, 1.09±0.09 U/l) increased slightly in comparison with sham-operated healthy controls (CTRL, n = 8, 0.94±0.10 U/l, p<0.02) as measured by competitive ELISA. Treatment of 4/6-NX rats with L over 12 weeks ameliorated the rise in serum AGEs concentration (1.00±0.12 U/l, n = 15 <0.005) almost to the level observed for CTRL. This effect was further corroborated by the observation, that the impaired renal excretion of AGEs in 4/6-NX-placebo rats (0.07±0.02 U/µmol creatinine) was significantly restored by L (0.09±0.02 U/µmol creatinine, <0.009) and resembled that of the CTRL (0.10±0.03 U/µmol creatinine). Administration of L to 4/6-NX rats significantly improved renal function as evaluated by a smaller rise in serum creatinine and urea concentration. In spite of the improvement in renal function, there were no differences in concentrations of transforming growth factor β1 in serum and in urine among the two groups. These effects were independent of blood pressure. Our data give first evidence, that long-term treatment with angiotensin II receptor 1 antagonist may exert salutary effects on AGEs levels in the rat remnant kidney model, probably due to improved renal function.

Renal Effects of S18886 (Terutroban), a TP Receptor Antagonist, in an Experimental Model of Type 2 Diabetes

Thromboxane A2 (TxA2) is assumed to contribute to the development of diabetes complications, including nephropathy. We investigated whether the selective thromboxane-prostanoid endoperoxide receptor antagonist, S18886, ameliorates renal damage in uninephrectomized (UNX) obese Zucker rats (OZR). S18886, at doses of 10 (S18886-10) and 30 (S18886-30) mg · kg−1 · day−1, was administered to UNX-OZR by gavage over 8 weeks (n = 8 each group). UNX lean rats (n = 12) and OZR rats that received placebo (OZR-PLAC, n = 8) served as controls. As compared with the OZR-PLAC, S18886 had no significant effect on the elevated blood pressure and the enhanced creatinine clearance, while augmented proteinuria was partially prevented (−12 and −37%, low and high dose, respectively; NS). The increased excretion of transforming growth factor β1 (TGF-β1) and of the thromboxane metabolite 2,3-dinor thromboxane B2 (TxB2) was lowered (P < 0.05). S18886 prevented both the enhanced mesangiolysis (P < 0.01) in the OZR-PLAC as well as enlargement and degeneration of podocytes. In the blood, S18886-30 augmented the antioxidant enzymes (P < 0.01) and lessened the increase of plasma advanced oxidation protein products (−25%, NS). Body weight, hyperglycemia, and dyslipidemia remained uninfluenced under both doses of treatment. S18886 has renoprotective properties in the model of UNX-OZR. It prevents mesangiolysis, reduces urinary TGF-β1 and 2,3-dinor-TxB2 excretion, and enhances the antioxidative defense.