August Heidland

Determination of Advanced Glycation End Products in Serum by Fluorescence Spectroscopy and Competitive ELISA

Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation. Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy. Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.

Cancer in End-Stage Renal Disease: Potential Factors Involved

Increased incidence of cancer at various sites is observed in patients with end-stage renal disease (ESRD). In particular, lymphomas and carcinomas of the kidney, prostate, liver and uterus show an enhanced prevalence in these subjects compared with the general population. A multitude of factors directly or indirectly associated with the renal disease and the treatment regimens may contribute to the increased tumor formation in these patients. Impaired function of the immune system and of DNA repair mechanisms as well as reduced antioxidant defense, accumulation of carcinogenic compounds partly due to reduced renal elimination as well as chronic infections and inflammations are found more frequently in patients with ESRD compared with the general population and may act in concert to accelerate malignant transformation and tumor formation.

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.

Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1.

Advanced glycation endproducts (AGEs) are suggested to play an important role in diabetic nephropathy. They induce specific cellular responses such as the release of cytokines in different cell lines. The effect of AGEs on signal transduction pathways was investigated in the renal tubulus cell line LLC‐PK1. Using a serine‐phosphate‐specific antibody AGE‐induced cellular responses associated with phosphorylation/dephosphorylation events were demonstrated. In particular, the p42MAP kinase and its downstream target, the AP‐1 complex, are shown to be activated by AGE‐BSA but not by BSA. In contrast, only partial phosphorylation is observed for the p70S6‐kinase. Thus, AGEs appear to induce specific signal transduction pathways.

Norepinephrine-Induced Acute Renal Failure: Beneficial Effects of Atrial Natriuretic Factor

The effect of the atrial natriuretic factor (ANF) on early norepinephrine-induced acute renal failure (ARF) was investigated. In anaesthetized female Sprague-Dawley rats, weighing 247 +/- 36 g, ARF of the left kidneys was induced by 40-min intrarenal arterial infusion of norepinephrine (NE; 0.75 micrograms/kg body weight). In each case the right kidney served as a control organ. Inulin clearance was used as an estimate of glomerular filtration rate (GFR). Urine volume (V), GFR and fractional excretion rates of sodium, potassium and chloride were studies in both kidneys. Immediately after NE-induced ARF one group received ANF (alpha-hANaP) and the other groups 5% glucose or isotonic saline; all infusions were administered intrarenally. In the experimental kidney NE-infusion induced anuria. ANF infusion was able to induce complete reversal of the NE effect, and GFR and diuresis improved markedly in the experimental kidney. In addition, a tremendous rise in fractional excretion rates of sodium and potassium after administration of ANF was observed. In the control kidney an additional increase of GFR and diuresis was seen. Following 5% glucose or isotonic sodium chloride infusion, no profound effects were obtained in both kidneys. The results of the present study demonstrate that ANF provides beneficial effects on the functional damage of NE-induced ARF. We conclude that the NE-antagonistic effect of ANF and the consecutive amelioration of GFR may play an important role in the recovery of impaired renal function.

Release of Granulocyte Proteinases during Hemodialysis

Neutral proteinases of neutrophilic polymorphonuclear leukocytes were followed up cytochemically in blood smears of 12 patients submitted to regular hemodialysis treatment (RDT). Halo formation (ring-shaped area around each neutrophil due to protein degradation) was reduced in all patients with end-stage renal disease under RDT. Concomitant to the development of leukopenia, a maximal increase of the plasma levels of the granulocytic elastase in complex with alpha 1-proteinase inhibitor was observed 3 h after starting hemodialysis (+409%; p less than 0.001). On the other hand, the proteolytic activity of the plasma samples against azocasein as substrate, being significantly higher (+244%; p less than 0.001) in RDT patients compared with healthy controls, decreased permanently during therapy (-71%; p less than 0.001; 3 h after initiation of the treatment). The mechanisms of release as well as of elimination of proteolytic activity due to RDT are discussed.

Increased genomic damage in lymphocytes of patients before and after long-term maintenance hemodialysis therapy

This study investigates spontaneous genomic damage in peripheral lymphocytes of 19 patients with severe end-stage renal disease not enrolled onto a maintenance hemodialysis (MHD) program (creatinine level, 5.4 to 10.5 mg/dL) and 16 long-term MHD patients (111 to 282 months on MHD) and the possible association of genomic damage with the degree of renal insufficiency and duration of MHD. Genomic damage was assessed by evaluating the numbers of micronuclei (MN), which are cytoplasmic DNA-containing structures. The average number of MN in the control group of 23 healthy subjects was 15.3 +/- 4.7 MN/1,000 binucleate (BN) cells. The MN frequencies in the long-term MHD group were significantly greater (44.3 +/- 13.7 MN/1,000 BN) than the control frequencies. A significant increase in MN frequencies (28.2 +/- 9.4 MN/1,000 BN) was also seen in patients with advanced renal failure. The major findings of the study, high MN frequencies in long-term hemodialysis and advanced chronic renal failure patients, may result from decreased DNA repair previously shown and may contribute to the increased cancer incidence in these patients.

Blood Rheology and Hypertension in Hemodialysis Patients Treated with Erythropoietin

Fifteen hemodialysis patients suffering from stable anemia were treated with recombinant human erythropoietin (r-HuEPO). Within 16 weeks, hematocrit values increased from 23.7 +/- 1.2 to 35.7 +/- 0.2%. Simultaneously, mean predialytic blood pressure rose significantly from 131/79 to 139/85 mm Hg. Three out of 15 patients developed frank hypertension and had to be put on antihypertensive therapy. When the hematocrit was lowered again from 36.3 +/- 1.8 to 30.5 +/- 1.2% in these 3 patients, blood pressure was attenuated and the antihypertensive medication could be reduced or abolished. With rising hematocrit values, whole blood viscosity increased at both low (+42%) and high shear rates (+33%) without reaching the values seen in healthy subjects. By contrast, plasma viscosity was already elevated in hemodialysis patients prior to r-HuEPO treatment and showed only a slight, but insignificant increase during r-HuEPO treatment. Since whole blood viscosity is one factor that determines vascular resistance, it is conceivable that the development of hypertension during correction of the renal anemia is, at least partly, due to an increment of blood viscosity.

Benfotiamine exhibits direct antioxidative capacity and prevents induction of DNA damage in vitro.

Complications in diabetes mellitus are partially mediated by enhanced formation of reactive oxygen species. Among the factors involved in reactive oxygen species formation, advanced glycation end products play a key role. Owing to a reduced activity of the enzyme transketolase, which requires diphosphorylated thiamine (vitamin B1) as cofactor, an accumulation of those deleterious glucose metabolites especially in diabetic patients can be observed. Benfotiamine, a lipophilic thiamine diphosphate prodrug, prevented early renal and retinal changes in animal studies, and reduced neuropathic pain in clinical studies. Several mechanisms for these activities have been described. We investigated for the first time direct antioxidant abilities of benfotiamine. Additionally, a potential DNA protective effect of benfotiamine was analysed.