Influence of dialysis modalities on serum AGE levels in end-stage renal disease patients.

BACKGROUND: The accumulation of advanced glycation end-products (AGEs) in end-stage renal disease (ESRD) influenced by dialysis modalities is of current interest. Highly permeable membranes in haemodialysis or haemofiltration should be able to eliminate circulating AGEs as well as their AGE precursors more efficiently. METHODS: In our study, 10 non-diabetic and 10 diabetic ESRD patients were on haemodialysis with low-flux membranes (LF) followed by a cross-over haemodialysis with high-flux or super-flux polysulfone membranes (HF, SF) for 6 months each. We measured the protein-bound pentosidine and free pentosidine serum levels by high-performance liquid chromatography (HPLC) as well as the serum AGE peptide, AGE-beta(2)-microglobulin and beta(2)-microglobulin concentrations, using ELISA assays. RESULTS: All parameters investigated were significantly higher in dialysis patients than in healthy subjects. The reduction rates during a single dialysis session were found to be higher using the SF than those obtained with the HF (free pentosidine 82.4+/-7.3 vs 76.6+/- 8.7%; AGE peptides 79.7+/-7.7 vs 62.3+/-14.7%; AGE-beta(2)-microglobulin 64.0+/-16.5 vs 45.4+/-17.7%; beta(2)-microglobulin 70.5+/-5.6 vs 58.2+/-6.0%). The protein-bound pentosidine levels remained constant over the respective dialysis sessions. In the 6-month treatment period with the SF, decreased pre-dialysis serum levels of protein-bound pentosidine, free pentosidine and AGE peptides were observed in non-diabetics and diabetics as compared with values obtained with the LF. The respective pre-dialysis AGE-beta(2)-microglobulin concentrations decreased insignificantly, whereas those of beta(2)-microglobulin were significantly lower. Using the HF dialyser, only moderate changes of the parameters measured were noted. CONCLUSION: Treatment with the biocompatible polysulfone SF dialyser seems to be better suited to lower serum AGE levels and to eliminate their precursors.     

Pentosidine, carotid atherosclerosis and alterations in left ventricular geometry in hemodialysis patients

OBJECTIVE: To assess the relationship between advanced glycation end products (AGE) and cardiovascular damage in end-stage renal diseases. METHODS: Ninety-one hemodialysis patients who had been on dialysis treatment for at least six months were recruited for the study. Each patient underwent echocardiography and an echo-color Doppler study of the carotid arteries. We measured plasma pentosidine and related it to intima media thickness, atherosclerotic plaques and parameters of left ventricular geometry. RESULTS: Pentosidine was higher in patients treated by low-flux dialysis (31.0+/-16.6 pmol/mg protein) than in those treated by high-flux dialysis (25.4+/-7.6 pmol/mg protein), but this difference was of marginal statistical significance (P=0.08). On multivariate analysis, plasma IgG (beta=0.24, P=0.02) was the only independent correlate of plasma pentosidine. Intima media thickness and the number of atherosclerotic plaques were unrelated to plasma pentosidine. Mean wall thickness (beta=0.18, P<0.05), relative wall thickness (beta=0.20, P<0.05) and left ventricular end-diastolic volume (beta= -0.23, P<0.01) were independently related to plasma pentosidine. CONCLUSIONS: Pentosidine, a reliable marker of "carbonyl stress", is unrelated to intima media thickness and to the number of atherosclerotic plaques, but it is related to alterations in heart geometry. These data suggest that the effect of carbonyl stress on the cardiovascular system is complex and that the effects of AGE on the heart may be dissociated from those on the arterial system.     

Low-molecular but not high-molecular advanced glycation end products (AGEs) are removed by high-flux dialysis

BACKGROUND: Patients with end-stage renal disease (ESRD) display very high levels of advanced glycation end products (AGEs). These compounds are suspected to play a pathophysiological role in diabetic nephropathy and late diabetic cardiovascular complications. We investigated to what extent AGE levels can be reduced by high-flux dialysis. PATIENTS AND METHODS: Ten ESRD patients were treated three times each with DIAPES and HF60, two different synthetic, high-flux hemodialysis membranes. The kinetics of AGE removal was studied by fluorescence spectroscopy (excitation 370 nm/ emission 440 nm) and by ELISA of serum samples and the removal of beta2-m was studied by immunonephelometry of plasma samples. Samples were taken during dialysis sessions at t = 0, 30 and 180 min. In addition, molecular weight distribution of AGE products in serum of three patients was analyzed by gel filtration and fluorescence detection. RESULTS: A significant difference could be found when AGE levels in serum of controls (n = 10) were compared with serum AGE levels of ESRD patients (p < 0.01/fluorescence; p < 0.0001/ ELISA). After 3 h of dialysis AGE-related fluorescence in serum decreased by 25.5 +/- 6.8% for HF60 (p < 0.0001) and 24.3 +/- 6.9% tor DIAPES (p < 0.0001). The corresponding decline measured by ELISA was 23.3 +/- 8.9% for HF60 (p < 0.0001) and 26.1 +/- 7.0% for DIAPES (p < 0.0001). Both methods showed no significant differences for both types of dialysis membranes. Gel filtration revealed that the decrease of fluorescence can be attributed to the removal of AGE peptides with a molecular mass < 12 kDa, only. In the high molecular range (> 12 kDa) no removal but hemoconcentration was observed independent of the dialyzer type used. The reduction of beta2-m during 3 hours of dialysis was 61.8 +/- 6.9% for HF60 (p < 0.0001) and 161.7 +/- 7.0% for DIAPES (p < 0.0001). CONCLUSION: Both high-flux dialyzers were equally effective to remove low-molecular AGE products, while AGE-modified proteins of higher molecular weight were only marginally affected. On the basis of our data we suggest the study of molecular mass-dependent uremic toxicity of AGEs and the examination of the influence of other treatment modalities on the level of high-molecular AGEs.     

Renal accumulation of pentosidine in non-diabetic proteinuria-induced renal damage in rats.

BACKGROUND: Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetic glomerulopathy. The role of AGEs in non-diabetic renal damage is not well characterized. First, we studied whether renal AGE accumulation occurs in non-diabetic proteinuria-induced renal damage and whether this is ameliorated by renoprotective treatment. Secondly, we investigated whether renal AGE accumulation was due to intrarenal effects of local protein trafficking. METHODS: Pentosidine was measured (by high-performance liquid chromatography) in rats with chronic bilateral adriamycin nephropathy (AN), untreated and treated with lisinopril. Age-matched healthy rats served as negative controls. Secondly, we compared renal pentosidine in mild proteinuric and non-proteinuric kidneys of unilateral AN and in age-matched controls at 12 and 30 weeks. Intrarenal localization of pentosidine was studied by immunohistochemistry. RESULTS: Renal pentosidine was elevated in untreated AN (0.14+/-0.04 micromol/mol valine) vs healthy controls (0.04+/-0.01 micromol/mol valine, P<0.01). In lisinopril-treated AN, pentosidine was lower (0.09+/-0.02 micromol/mol valine) than in untreated AN (P<0.05). In unilateral proteinuria, pentosidine was similar in non-proteinuric and proteinuric kidneys. After 30 weeks of unilateral proteinuria, pentosidine was increased in both kidneys (0.26+/-0.10 micromol/mol valine) compared with controls (0.18+/-0.06 micromol/mol valine, P<0.05). Pentosidine (AN, week 30) was also increased compared with AN at week 12 (0.16+/-0.06 micromol/mol valine, P<0.01). In control and diseased kidneys, pentosidine was present in the collecting ducts. In proteinuric kidneys, in addition, pentosidine was present in the brush border and cytoplasm of dilated tubular structures, i.e. at sites of proteinuria-induced tubular damage. CONCLUSION: Pentosidine accumulates in non-diabetic proteinuric kidneys in damaged tubules, and renoprotective treatment by angiotensin-converting enzyme (ACE) inhibitors inhibits AGE accumulation, supporting a relationship between abnormal renal protein trafficking, proteinuria-induced tubular damage and tubular pentosidine accumulation. Future studies, applying specific AGE inhibitors, should be conducted to provide insight into the pathophysiological significance of renal AGEs in non-diabetic renal disease.     

A sensitive and specific ELISA for plasma pentosidine.

BACKGROUND: Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. METHODS: We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. RESULTS: The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. CONCLUSIONS: The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients.     

Plasma pentosidine and total homocysteine levels in relation to change in common carotid intima-media area in the first year of dialysis therapy

BACKGROUND: Homocysteine and advanced glycation end-products (AGEs), which accumulate in chronic kidney disease (CKD), are recently proposed cardiovascular risk factors. In this study, we evaluated the association between changes in calculated intima media (cIM) area of the common carotid artery during the first year of dialysis therapy and plasma total homocysteine (tHcy) level as well as circulating AGEs such as plasma pentosidine level. METHODS: We studied 63 CKD patients (38 males) aged 52 +/- 12 years at a time-point close to start of dialysis treatment and after 12 months of dialysis treatment (41 on peritoneal and 22 on hemodialysis). The tHcy and plasma pentosidine levels were measured by HPLC. Change in cIM area was evaluated by non-invasive B mode ultrasonography. Malnutrition was assessed by subjective global assessment (SGA). RESULTS: At basal, 70% of the patients had carotid plaques, 32% had symptomatic CVD, 38% had malnutrition, 30% had inflammation (CRP > or = 1 mg/dl) and 23% had diabetes mellitus, respectively. At baseline, the mean plasma pentosidine levels were similar in the patients with and without carotid plaques (36 +/- 21 vs 36 +/- 19 pmol/mg albumin, respectively), whereas the median plasma tHcy was significantly lower in the patients with carotid plaques than in the patients without carotid plaques (32 +/- 21 vs 52 +/- 42 pmol/l, p < 0.01, respectively). The prevalence of hyperhomocysteinemia (tHcy level > 13.7 micromol/l) was 95%. In univariate analysis, the change in cIM area during the first year of dialysis was significantly correlated with basal plasma pentosidine level (p = 0.31, p = 0.01), but not with basal tHcy (p = -0.11). However, neither pentosidine nor tHcy levels were correlated with cIM area at basal or at 12 months. In a stepwise multiple regression model, age and plasma pentosidine content, but not the tHcy level, associated with changes in the cIM area. CONCLUSION: Progression of atherosclerosis, as indicated by changes in carotid intima-media area during the course of dialysis treatment, was associated with pentosidine, but not with tHcy, levels at baseline in these CKD patients. This suggests that the accumulation of AGEs in CKD patients may have a role in the pathogenesis of CVD in these patients. Since almost all CKD patients have hyperhomocysteinemia, this finding, however, does not exclude a role ofhomocysteine as a risk factor for CVD in CKD patients.     

Daily haemodialysis improves indices of protein glycation.

BACKGROUND: Advanced glycation end-products (AGEs) accumulate in uraemia, regardless of hyperglycaemic conditions, and may contribute to the onset of some long-term complications, such as atherosclerosis, amyloidosis, and neurodegenerative processes. In this study, we compare a daily with a standard 3 times/week dialysis rhythm (DHD and SHD, respectively) in correcting some protein glycation indices in end-stage renal disease (ESRD) patients. METHODS: Twenty-one normoglycaemic and 11 diabetic patients on chronic haemodialysis (HD) with low-flux dialysers were studied in a prospective protocol to compare two different dialysis schedules, namely: 4 h, 3 times/week (SHD) and 2 h, 6 times/week (DHD). The patients were studied before and after 6 months of DHD. To further check the effect of DHD on glycation parameters, 4 normoglycaemic HD patients were studied in a third step in which they returned for 3 months to the SHD rhythm. Also, 11 chronic renal failure (CRF) patients not yet on HD and 11 age- and sex-matched healthy controls were studied. A new HPLC method was used to measure the following glycation indexes on plasma: the early product furosine and the advanced products protein-bound and free pentosidine, and two heterogeneous classes of low molecular mass (LMM) AGE peptides. RESULTS: All the parameters studied showed an accumulation that worsened with the progression of renal failure (controls 相似文献   

Involvement of oxidative stress in the accelerated formation of pentosidine in patients with end-stage renal failure

SUMMARY: Advanced glycation end products (AGEs) have been found to accumulate in the amyloid deposits, skin and plasma of haemodialysis patients (HD), implicating the possible involvement of AGE-modified protein in pathogenesis in dialysis-related amyloidosis. Pentosidine, an AGE cross-link, is a specific marker for AGEs. Plasma pentosidine levels in HD patients were increased dramatically. In the present study, plasma pentosidine, fructoselysine, advanced oxidation protein products (AOPP) and glutathione peroxidase (GSHPx) levels were measured to elucidate the role of oxidative stress in pentosidine formation in nondiabetic HD patients. Plasma pentosidine did not correlate with fructoselysine; plasma AOPP levels were significantly higher than those in normal subjects (201.45 ± 57.93 vs. 55.91 ± 6.57 μmol/L, P <0.001) and correlated positively with plasma pentosidine in HD patients ( r =0.52, P <0.005); plasma GSHPx levels were significantly lower than those in normal subjects (168.40 ± 65.08 vs. 348.87 ± 86.10 U/I, P <0.001) and correlated negatively with plasma pentosidine ( r =0.54, P <0.001) in HD patients. Decreased GSHPx levels may lead to the accumulation of hydrogen peroxide. These findings implicate the involvement of oxidative stress in the accelerated formation of pentosidine in uraemia and suggest that pentosidine could be considered as an oxidative stress biomarker to estimate the degree of oxidative-stress-mediated protein damage.     

Pentosidine and its deposition in renal tissue in renal transplantation

BACKGROUND: Advanced glycation end products (AGEs) accumulate in lesions of arteriosclerosis, Alzheimer's disease, rheumatoid arthritis, diabetic retinopathy, and diabetic nephropathy. Among AGEs, chemical quantification and immunohistologic methods for pentosidine have been established. Free pentosidine-eliminated by renal excretion- is mainly affected by renal function. In this study, we measured concentrations of plasma free and total pentosidine and immunohistologically investigated kidney graft biopsy specimens in patients after renal transplantation to investigate the renal function, plasma free and total pentosidine, and its relationship with deposition in the renal tissue. PATIENTS AND METHODS: In 28 patients who underwent renal transplantation from 1996 to 2003, we measured the time course of plasma concentrations of free pentosidine, total pentosidine, and serum creatinine starting right after renal transplantation. Thirty-four graft biopsy specimens were immunohistologically investigated using anti-pentosidine antibody. Plasma free and total pentosidine, and serum creatinine were measured at the same time. RESULTS: Plasma free and total pentosidine were positively correlated with serum creatinine. Plasma free pentosidine and serum creatinine reached nadir values on day 34.2 +/- 14.2, when the blood concentrations were 5.1 +/- 1.6 pmol/mL and 1.7 +/- 0.7 mg/dL, respectively. Plasma total pentosidine reached a nadir on day 116.5 +/- 39.7 when the plasma concentration was 4.0 +/- 1.5 pmol/mg. We correlated the time required to reach the nadir of plasma free and total pentosidine concentrations. However, neither the concentration of plasma free nor plasma total pentosidine at nadir correlated with serum creatinine. The intensity of immunostaining with anti-pentosidine antibody in proximal tubular cells was graded as weakly positive, positive, or strongly positive. Significant differences were obtained among plasma free pentosidine values between the weakly positive and strongly positive groups. CONCLUSIONS: Renal transplantation improves renal function and decreases renal excretion of free pentosidine. Accordingly, total pentosidine also decreases. However, the concentrations of plasma free and total pentosidine at nadir varied among individuals; the blood concentrations were not determined by renal function alone. It was suggested that deposition of pentosidine in proximal tubular cells was more severe among patients with higher plasma free pentosidine and serum creatinine values.     

Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions

Advanced glycation end products (AGE) include a variety of protein adducts whose accumulation has been implicated in tissue damage associated with diabetic nephropathy (DN). It was recently demonstrated that among AGE, glycoxidation products, whose formation is closely linked to oxidation, such as carboxymethyllysine (CML) and pentosidine, accumulate in expanded mesangial matrix and nodular lesions in DN, in colocalization with malondialdehyde-lysine (MDA-lysine), a lipoxidation product, whereas pyrraline, another AGE structure whose deposition is rather independent from oxidative stress, was not found within diabetic glomeruli. Because CML, pentosidine, and MDA-lysine are all formed under oxidative stress by carbonyl amine chemistry between protein amino group and carbonyl compounds, their colocalization suggests a local oxidative stress and increased protein carbonyl modification in diabetic glomerular lesions. To address this hypothesis, human renal tissues from patients with DN or IgA nephropathy were examined with specific antibodies to characterize most, if not all, carbonyl modifications of proteins by autoxidation products of carbohydrates, lipids, and amino acids: CML (derived from carbohydrates, lipids, and amino acid), pentosidine (derived from carbohydrates), MDA-lysine (derived from lipids), 4-hydroxynonenal-protein adduct (derived from lipids), and acrolein-protein adduct (derived from lipids and amino acid). All of the protein adducts were identified in expanded mesangial matrix and nodular lesions in DN. In IgA nephropathy, another primary glomerular disease leading to end-stage renal failure, despite positive staining for MDA-lysine and 4-hydroxynonenal-protein adduct in the expanded mesangial area, CML, pentosidine, and acrolein-protein adduct immunoreactivities were only faint in glomeruli. These data suggest a broad derangement in nonenzymatic biochemistry in diabetic glomerular lesions, and implicate an increased local oxidative stress and carbonyl modification of proteins in diabetic glomerular tissue damage ("carbonyl stress").     

Plasma advanced glycosylation end-products in maintenance haemodialysis patients

BACKGROUND.: Pentosidine is a useful marker of advanced glycation end-products(AGE) which form cross-links between proteins and have beenfound elevated in plasma and tissues of uraemic and haemo-dialysedsubjects. The origin and fate of these molecules are not clearlyunderstood, but they might play a role in the cardiovascularcomplications of end stage renal failure. The aim of this studywas to evaluate the effect of different types of substitutivetherapy on the removal of pentosidine. METHODS.: Pentosidine was measured by a two-step HPLC methodology. Itsconcentration was evaluated in plasma before and after dialysissession, in 24-h urine, and in dialysate of subjects treatedwith three types of chronic substitutive therapy: bicarbonatehaemodialysis, acetate-free biofiltration, and haemofiltration.Pentosidine levels were compared among the three therapy modalitiesand correlated with clinical and biochemical parameters. RESULTS.: Plasma pentosidine level was extremely high (23.7±2.0pmol/mg protein) in the patients treated with the differentdialysis modalities. The dialysis session had no significanteffect on its plasma concentration, but haemofiltration seemedto be the most efficient method (300–2000 nmol of pentosidineremoved per session versus 250–700 nmol per session withthe two other approaches). An interesting correlation was foundbetween pentosidine and blood urea nitrogen (r=0.58, P<0.01)and pentosidine with uric acid (r=0.48, P<0.05). CONCLUSIONS.: These results suggest that none of the methodology showed agood removal of pentosidine, but among them haemofiltrationhas the best efficiency. The statistical relationships betweenpentosidine and urea and uric acid respectively might provideinsight into the origin of pentosidine. The accumulation ofreactive AGE in uraemic patients may be implicated in the organand tissue damage observed in uraemia.     

Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through inos or enos

Objectives

We hypothesized that advanced glycation end product (AGE) formation contributes to erectile dysfunction (ED) by quenching nitric oxide. Our first goal was to identify the specific AGE pentosidine in the diabetic human penis. Because AGE-mediated effects may involve inducible nitric oxide synthase (iNOS), we performed immunohistochemical and Western blot analysis of diabetic and nondiabetic human penile tissue for iNOS. Finally, because AGEs may act intracellularly to affect proteins, we set out to identify endothelial NOS (eNOS) in the human penis as an initial step in examining a possible intracellular interaction between eNOS and AGEs.

Methods

We performed high-performance liquid chromatographic analysis of diabetic human penile corpus cavernosum and serum for pentosidine and performed immunohistochemical, electron microscopic (EM), and Western blot analysis of the diabetic and nondiabetic penile corpus cavernosum and tunica for pyrraline, iNOS, and eNOS (and neural NOS [nNOS]for comparative purposes) via standard methods.

Results

We found a significant elevation of pentosidine in the penile tissue but not the serum of diabetic patients (average age 55.6 ± 2.3 years) compared with that of nondiabetic patients (average age 61.8 ± 3.6 years). Pentosidine was 117.06 ± 9.19 pmol/mg collagen in the diabetic tunica versus 77.58 ± 5.5 pmol/mg collagen in the nondiabetic tunica (P < 0.01) and 74.58 ± 8.49 pmol/mg collagen in the diabetic corpus cavernosum versus 46.59 ± 2.53 pmol/mg collagen in the nondiabetic corpus cavernosum (P < 0.01), suggesting a tissue-specific effect of the AGEs. We localized the site of deposition of the specific AGE pyrraline to the human penile tunica and the penile corpus cavernosum collagen. Immunohistochemical and EM analysis localized eNOS and iNOS to the cavernosal endothelium and smooth muscle. Western blot analysis in 6 patients revealed the following: iNOS, but no eNOS, in penile tissue from 1 insulin-dependent diabetic man; eNOS only in 1 man after radical prostatectomy; both eNOS and iNOS in 2 men with Peyronie's disease, as well as in 2 other men with impotence and hypertension. Finally, the specific iNOS inhibitor PNU-19451A significantly augmented relaxation of precontracted human cavernosal tissue, from 64.7% ± 5.58 to 80.03% ± 4.55 at 10 μM acetylcholine and 65.06% ± 2.84 to 86.16% ± 3.96 at 0.1 mM acetylcholine (n = 4, P < 0.002 and P < 0.02, respectively).

Conclusions

AGEs are elevated in diabetic human penile tissue, but not in serum, and are localized to the collagen of the penile tunica and corpus cavernosum. We identified eNOS and iNOS in the human penile cavernosal smooth muscle and endothelium. The augmentation of cavernosal relaxation with a specific iNOS inhibitor, combined with the identification of iNOS protein, but not eNOS, in a patient with severe diabetes and ED, allows for speculation of a pathophysiologic mechanism for AGE-mediated ED via upregulation of iNOS and downregulation of eNOS. These data provide further insight into the mechanisms of advanced glycation end product-mediated ED and provide a foundation for further study. UROLOGY 50: 1016-1026, 1997.     

Influence of hemodialysis membrane permeability on serum levels of advanced glycation end products (AGEs) and homocysteine metabolites

BACKROUND: Advanced glycation end products (AGEs), total homocysteine (tHcy) and the homocysteine metabolites cystathionine (Cysta) and dimethylglycine (DMG) are increased in serum of patients with end-stage renal disease. The aim of this prospective randomized study was to compare the efficacy of polysulfone high-flux vs. polysulfone low-flux hemodialysis (HD) treatment regarding removal of AGEs, tHcy, Cysta and DMG. PATIENTS AND METHODS: Twenty-nine patients on chronic HD treatment were randomly assigned to 2 groups in a 3-period 2-treatment design with low flux (A)--high flux (B)--low flux (A) for group I and B-A-B for group II, 6 weeks each period. The following parameters were measured in pre- and postdialytic serum samples at baseline and the end of each period: total serum fluorescence, Nepsilon-carboxymethyllysine (CML), free and protein-bound pentosidine, tHcy, Cysta and DMG. RESULTS: There was increased removal of free pentosidine during high-flux HD treatment compared to low-flux HD treatment, attaining significance between the second and third treatment periods (group 1: 86.0 +/- 4.7% vs. 79.2 +/- 8.8%, p = 0.007; group II: 84.0 +/- 6.3% vs. 79.8 +/- 9.8%, p = 0.049 for high vs. low flux). The intradialytic reduction rates for total serum fluorescence, tHcy, Cysta, DMG did not differ between high- and low-flux HD treatment. Protein-bound pentosidine and CML did not decrease during the dialysis sessions, neither with high-flux nor with low-flux HD membrane. Despite a strong decrease during single HD session, the predialytic levels of free pentosidine, tHcy, Cysta and DMG remained unchanged during the study period both for high- and low-flux HD treatment. CONCLUSION: The more pronounced effect of high-flux dialysis on the removal rate of free pentosidine, found in this randomized crossover study, could not translate into a significant difference in predialysis levels after a 6-week treatment period. We could not find any differences between polysulfone high- and low-flux membranes for lowering predialytic serum concentrations of the measured AGEs, which are mainly bound on albumin.     

Dialysis-related amyloidosis: pathogenesis focusing on AGE modification

Dialysis-related amyloidosis (DRA) is a serious complication in long-term dialysis patients, and presents with carpal tunnel syndrome, cystic bone lesions, destructive spondylarthropathy, diffuse arthritis and periarthritis, systemic organ involvement, and dialysis-related spinal canal stenosis (DSCS). Recently a new concept of DSCS has been proposed that includes both destructive spondylarthropathy and myeloradiculopathy induced by extradural thickness. beta(2)-microglobulin (beta(2)M) amyloid was demonstrated to be modified with advanced glycation end products (AGEs) such as imidazolone, N(epsilon)-(carboxymethyl)lysine (CML), and pentosidine. Imidazolone is a reaction product of arginine residue in proteins with 3-deoxyglucosone (3-DG), which is markedly accumulated in uremic serum. Imidazolone is generated under nonoxidative conditions, while CML and pentosidine are formed by oxidative processes. Immunoelectron microscopy demonstrated that AGEs were localized not only in dialysis amyloid but also in nonamyloid collagenous structures, supporting the hypothesis that AGE modification of collagen might have pathogenic relevance in the deposition of beta(2)M on collagen. Serum levels of AGEs are increased in uremic patients. The dimeric form of beta(2)M in the dialysate and urine of uremic patients is more susceptible to imidazolone modification as observed in dialysis amyloid. However, the major component of dialysis amyloid is a native form of beta(2)M, while AGE-modified beta(2)M and truncated beta(2)M are the minor components. Thus I propose that 3-DG and the other dicarbonyl compounds accumulating in uremic serum promote the modification of beta(2)M with AGEs mainly after deposition of beta(2)M as amyloid. For the prevention and treatment of DRA, beta(2)M should be efficiently eliminated from circulating blood by kidney transplantation, hemodialysis, or hemodiafiltration using high-flux membranes and an adsorbent (Lixelle) column.     

Effect of the peritoneal dialysis prescription on pentosidine in children

Enhanced formation of advanced glycation end products (AGEs) by peritoneal dialysate containing high dextrose concentrations has been implicated as a source of peritoneal membrane toxicity and loss of viability in patients treated with peritoneal dialysis (PD). The goal of this project was to elucidate the relationship between the structurally defined AGE pentosidine accumulation on peritoneal and plasma proteins and peritoneal membrane function, and to identify clinical factors leading to alterations in these parameters. The study comprised 27 pediatric patients (14 continuous ambulatory PD, 13 chronic cycling PD) on PD for a mean duration of 37.0+/-22.8 months (range 1-120 months) and with a mean age of 13.3+/-4.4 years (range 2.4-20 years). The pentosidine contents of plasma and peritoneal proteins were significantly lower in patients with residual renal function than in patients who were anuric (plasma pentosidine 11.2+/-8.8 vs. 24.1+/-16.6, P=0.02, respectively, peritoneal pentosidine 14.9+/-11.9 vs. 31.1+/-3.7, P=0.01, respectively). There was no effect of treatment modality on plasma pentosidine (18.1+/-11.2, 18.8+/-19.3, CAPD vs. CCPD, P>0.05) or peritoneal pentosidine content (24.1+/-14.1, 24.9+/-19.6, CAPD vs. CCPD, P>0.05). There was no evidence that increased levels of pentosidine on peritoneal proteins reflect or affect peritoneal membrane function in these patients. Furthermore, there was no effect of peritonitis on the pentosidine content of peritoneal proteins or peritoneal function as measured by peritoneal equilibration test. In conclusion, PD represents a well-tolerated therapy in children with no evidence that current practice causes changes in peritoneal membrane function, or in the peritoneal clearance of plasma or peritoneal proteins rich in pentosidine.     

Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats.

BACKGROUND: Advanced glycation end products (AGEs) are involved in diabetic nephropathy (DN). The AGE formation inhibitor pyridoxamine (PM) is renoprotective in DN and in normoglycaemic obese Zucker rats. In chronic allograft nephropathy (CAN), renal AGE accumulation occurs as well. METHODS: To investigate whether inhibition of AGE formation is renoprotective in CAN, we studied the Fisher 344 to Lewis (F-L) allograft rat model of experimental CAN. Fisher to Fisher (F-F) isografts served as controls. Proteinuria, renal function and renal histology of untreated transplanted rats (F-L n = 8, F-F n = 8) were compared to rats receiving PM 2 g/l in drinking water for 20 weeks starting at transplantation (F-L n = 5, F-F n = 10). All rats received cyclosporin A (1.5 mg/kg/day) for 10 days after transplantation to prevent early acute rejection. RESULTS: Compared to untreated allografts, PM significantly decreased proteinuria (76 +/- 18 vs 29 +/- 3 mg/day), serum creatinine (130 +/- 12 vs 98 +/- 5 micromol/l), focal glomerulosclerosis (116 +/- 27 vs 16 +/- 5 AU), glomerular macrophage influx (5.6 +/- 0.6 vs 3.3 +/- 1.0), interstitial fibrosis (132 +/- 24 vs 76 +/- 2 AU) and interstitial macrophage influx (47.0 +/- 8.7 vs 15.4 +/- 5.0. Moreover, PM significantly ameliorated tubular accumulation of pentosidine, compared to untreated allografts (2.5 +/- 0.6 vs 0.3 +/- 0.3, all p < 0.05). In the isograft controls, these values did not differ between untreated and PM treated rats. CONCLUSION: PM exerts renoprotective effects and decreases renal pentosidine accumulation in experimental CAN, suggesting a detrimental role for renal AGE accumulation in the pathogenesis of renal damage in this non-diabetic model. These results indicate that inhibition of AGE formation might be a useful adjunct therapy to attenuate CAN.     

Role of glycation in modification of lens crystallins in diabetic and nondiabetic senile cataracts

To assess the significance of glycation, nonenzymatic browning, and oxidation of lens crystallins in cataract formation in elderly diabetic patients, we measured three distinct products of glycation, browning, and oxidation reactions in cataractous lens crystallins from 29 diabetic patients (mean +/- SD age 72.8 +/- 8.8 yr) and 24 nondiabetic patients (age 73.5 +/- 8.3 yr). Compounds measured included 1) fructoselysine (FL), the first stable product of glycation; 2) pentosidine, a fluorescent, carbohydrate-derived protein cross-link between lysine and arginine residues formed during nonenzymatic browning; and 3) N epsilon-(carboxymethyl)lysine (CML), a product of autoxidation of sugar adducts to protein. In diabetic compared with nondiabetic patients, there were significant increases (P less than 0.001) in HbA1 (10.2 +/- 3.1 vs. 7.1 +/- 0.7%), FL (7.6 +/- 5.4 vs. 1.7 +/- 1.2 mmol/mol lysine), and pentosidine (6.3 +/- 2.8 vs. 3.8 +/- 1.9 mumol/mol lysine). The disproportionate elevation of FL compared with HbA1 suggests a breakdown in the lens barrier to glucose in diabetes, whereas the increase in pentosidine is indicative of accelerated nonenzymatic browning of diabetic lens crystallins. CML levels were similar in the two groups (7.1 +/- 2.4 vs. 6.8 +/- 3.0 mmol/mol lysine), providing no evidence for increased oxidative stress in the diabetic cataract. Thus, although the modification of lens crystallins by autoxidation reactions was not increased in diabetes, the increase in glycation and nonenzymatic browning suggests that these processes may acclerate the development of cataracts in diabetic patients.     

Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat

BACKGROUND: Nonenzymatic reactions between sugars or lipids and protein and formation of advanced glycation and lipoxidation end products (AGE/ALEs) contribute to the chemical modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is implicated in the development of diabetic complications. In this study, we examined the effect of the AGE/ALE inhibitor pyridoxamine on chemical modification and cross-linking of collagen and development of renal disease in the streptozotocin-diabetic rat. METHODS: Diabetic rats were treated with pyridoxamine; parallel experiments were conducted with aminoguanidine, the prototype AGE inhibitor. Progression of renal disease was evaluated by measurements of albuminuria and plasma creatinine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ALEs in skin collagen by HPLC and GC-MS assays. RESULTS: Pyridoxamine significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia and plasma lactate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and cross-linking of skin collagen increased approximately twofold in diabetic versus control rats after seven months of diabetes. Pyridoxamine caused a significant (25 to 50%) decrease the AGE/ALEs, carboxymethyllysine and carboxyethyllysine, cross-linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. CONCLUSIONS: Pyridoxamine inhibits the progression of renal disease, and decreases hyperlipidemia and apparent redox imbalances in diabetic rats. Pyridoxamine and aminoguanidine had similar effects on parameters measured, supporting a mechanism of action involving AGE/ALE inhibition.     

Plasma pentosidine is associated with inflammation and malnutrition in end-stage renal disease patients starting on dialysis therapy

Pentosidine is an advanced glycation end-product (AGE), formed by glycosylation and oxidation, that accumulates markedly in end-stage renal disease (ESRD). It has been speculated that AGE and carbonyl stress contributes to long-term complications such as cardiovascular disease (CVD) in ESRD patients. This study determined plasma levels of pentosidine as well as the presence of inflammation (CRP > or = 10 mg/L), clinical CVD (CVD(clin)), and malnutrition (subjective global assessment [SGA] > 1) in a cohort of 191 ESRD patients, median age of 55 yr (range, 23 to 70 yr) and median GFR = 7 ml/min (range, 2 to 17 ml/min), close to start of renal replacement therapy. Fifty-one elderly subjects, median age of 82 yr (range, 71 to 110 yr), with mild renal impairment, median GFR = 67 ml/min (range, 38 to 113 ml/min), were also studied for comparative analysis of plasma pentosidine. The plasma pentosidine content was elevated in all patients compared with the levels in the elderly subjects and were negatively correlated with GFR both in the ESRD patients (Rho = -0.24; P < 0.01; n = 159) and in the elderly subjects (Rho = -0.31; P < 0.05). Moreover, the plasma pentosidine content was correlated with age in the ESRD patients (Rho = 0.26; P < 0.001) and in the elderly subjects (Rho = 0.44; P < 0.001). The 63 malnourished ESRD patients (35%) had a significantly higher (P < 0.05) median plasma pentosidine than the well-nourished patients (39 versus 27 pmol/mg albumin). Similarly, 73 inflamed patients (38%) had a significantly higher (P < 0.001) median pentosidine content compared with 118 non-inflamed patients (37 versus 24 pmol/mg albumin). Also, the plasma pentosidine content showed weak but significant positive correlations with CRP (Rho = 0.28; P < 0.0001), fibrinogen (Rho = 0.23; P < 0.01; n = 126), IL-6 (Rho = 0.22; P < 0.01; n = 169), and soluble vascular cellular adhesion molecule-1 (Rho = 0.38; P < 0.001; n = 74). On the other hand, no significant differences in plasma pentosidine content were noted between the patients with and those without CVD(clin) (32 versus 27 pmol/mg albumin, respectively). Analyses of all-cause mortality, by Kaplan-Meier, showed that mortality was not linked to the plasma pentosidine content. Moreover, survival analysis by the Cox regression model showed that age (P < 0.001), diabetes mellitus (P < 0.01), malnutrition (P < 0.01), and CVD(clin) (P < 0.01) independently predicted poor outcome, whereas an elevated plasma pentosidine content did not. The present study shows that an elevated plasma pentosidine content in ESRD patients is significantly associated with both inflammation and malnutrition and confirms that low residual renal function and high age further contribute to an increased plasma pentosidine content. However, in this small cohort, the plasma pentosidine content did not predict outcome. Thus, accumulation of plasma pentosidine is unlikely to be an appropriate clinically useful marker to predict mortality in ESRD patients.